Characterization of Skin Immunity to Aedes Aegypti Saliva in Dengue-Endemic Participants in Cambodia
Phase: PHASE1
Conditions: Vector Borne Diseases
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

 
National Institute of Allergy and Infectious Diseases (NIAID)
National Institutes of Health (NIH)
Protocol Face Sheet
NIH Principal Investigator (PI):
Institute/Branch:
Address:
Phone:
Email:
Jessica Manning, MD, MSc 
NIAID/Laboratory of Malaria and Vector Research (LMVR)
United States (US) Embassy Phnom Penh
Unit 4540, Box 10
DPO, AE 09975
+855 61 202 473
Jessica.manning@nih.gov 
Cambodian Co-PI:
Institute/Branch:
Phone:
Rekol Huy, MD, PhD
National Malaria Center (CNM), Ministry of Health, Cambodia
+855 23 223 442
Cambodian Co-PI:
Institute/Branch:
Phone:
Rithea Leang, MD, DrPH
National Dengue Control Program, CNM, Ministry of Health, Cambodia
+855 12 523 150
Protocol Number:
20-I-N053
Version Description: 
Version 1.0
Version Date: 
13 March 2020
Protocol Title:
Characterization of skin immunity to Aedes aegypti saliva in dengue-
endemic participants in Cambodia  
Abbreviated Title:
PINCH
Institute Name: 
NIAID
Accrual Period:
3 months
Proposed Dates:
01 May 2020 – 01 May 2022
Total Subjects to be Accrued:
42
Ionizing Radiation Use: 
None
Is Tissue Being Collected for 
Research Purposes?
Yes (whole blood, sera, and skin punch biopsy)
Location of the Study
Kampong Speu Referral Hospital, Chbar Mon, Kampong Speu, 
Cambodia
Investigational New 
Drug/Device:
None  

 
Table of Contents
STATEMENT OF COMPLIANCE ......................................................................................................................4
1
PROTOCOL SUMMARY ......................................................................................................................4
1.1
Synopsis...................................................................................................................................4
1.2
Precis .......................................................................................................................................7
1.3
Schedule of Activities (SoA).....................................................................................................8
2
INTRODUCTION.................................................................................................................................9
2.1
Study Rationale........................................................................................................................9
2.2
Background..............................................................................................................................9
2.2.1
Scientific Rationale ..............................................................................................10
2.2.2
Conclusion ...........................................................................................................11
2.3
Risk/Benefit Assessment .......................................................................................................11
2.3.1
Known Potential Risks .........................................................................................11
2.3.2
Known Potential Benefits ....................................................................................12
2.3.3
Assessment of Potential Risks and Benefits ........................................................12
3
OBJECTIVES AND ENDPOINTS .........................................................................................................12
4
STUDY DESIGN.................................................................................................................................14
4.1
Site Description .....................................................................................................................14
4.2
Overall Design........................................................................................................................14
4.3
End of Study Definition..........................................................................................................15
5
STUDY POPULATION........................................................................................................................15
5.1
Inclusion Criteria....................................................................................................................15
5.2
Exclusion Criteria...................................................................................................................15
5.3
Screen Failures ......................................................................................................................16
5.4
Strategies for Recruitment and Retention ............................................................................16
5.4.1
Justification for Exclusion of Minors and pregnant women ................................17
6
PARTICIPANT DISCONTINUATION/WITHDRAWAL ..........................................................................17
6.1
Participant Discontinuation/Withdrawal from the Study......................................................17
6.2
Lost to Follow-Up ..................................................................................................................17
7
STUDY INTERVENTION.....................................................................................................................18
7.1
Arthropod Feeding ................................................................................................................18
7.2
Early Termination Visit ..........................................................................................................19
7.3
Unexpected or Incidental Medical Conditions of the Participant .........................................19
8
STUDY ASSESSMENTS AND PROCEDURES.......................................................................................19
8.1
Clinical Evaluations................................................................................................................19
8.2
Study Procedures...................................................................................................................21
8.2.1
Vital Signs.............................................................................................................21
8.2.2
Mosquito Feeding................................................................................................21
8.2.3
Skin Punch Biopsies .............................................................................................21
8.3
Laboratory Evaluations..........................................................................................................22
8.3.1
Safety Laboratory Evaluations.............................................................................22
8.3.2
Laboratory Testing of Samples for Study Endpoints............................................22
8.3.3
Specimen Collection, Preparation, Handling, and Shipping.................................23
8.4
Safety and Other Assessments..............................................................................................23
8.4.1
Adverse Event Definitions....................................................................................23
8.4.2
Adverse Event Management ...............................................................................24
8.4.3
Reporting.............................................................................................................26
8.4.4
Pregnancy ............................................................................................................26

 
8.4.5
Special Reporting Situations................................................................................27
8.4.6
Type and Duration of the Follow-up Of Participants After AEs ...........................27
8.4.7
Pausing Rules for an Individual Subject...............................................................27
8.4.8
Reporting a Pause................................................................................................27
8.4.9
Resumption of a Paused Study............................................................................28
8.4.10
Halting Rules for the Protocol..............................................................................28
8.4.11
Reporting a Study Halt.........................................................................................28
8.4.12
Resumption of a Halted Study.............................................................................28
8.4.13
Premature Withdrawal of a Participant...............................................................28
8.4.14
Replacement of Withdrawn Participants or Participants who Discontinue Study 
Intervention
..............................................................................................................29
9
STATISTICAL CONSIDERATIONS.......................................................................................................29
9.1
Statistical Hypotheses ...........................................................................................................29
9.2
Sample Size Justification and Analysis Plan...........................................................................30
10
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS.......................................31
10.1
Regulatory, Ethical, and Study Oversight Considerations .....................................................31
10.1.1
Informed Consent Process...................................................................................31
10.1.2
Study Discontinuation and Closure......................................................................31
10.1.3
Confidentiality and Privacy..................................................................................31
10.1.4
Future Use of Stored Specimens and Data..........................................................32
10.1.5
Clinical and Safety Monitoring.............................................................................33
10.1.6
Quality Assurance and Quality Control................................................................34
10.1.7
Data Handling and Record Keeping .....................................................................35
10.1.8
Protocol Deviations .............................................................................................36
10.1.9
Publication and Data Sharing Policy ....................................................................36
10.2
Abbreviations ........................................................................................................................37
11
REFERENCES ....................................................................................................................................38

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STATEMENT OF COMPLIANCE
  
The trial will be carried out in accordance with International Council on Harmonisation of Technical 
Requirements for Pharmaceuticals for Human Use (ICH) Good Clinical Practice (GCP) and the following: 

US Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 46) 
NIH-funded investigators and clinical trial site staff who are responsible for the conduct, management, 
or oversight of NIH-funded clinical trials have completed Human Subjects Protection and ICH GCP 
Training.
The protocol, informed consent form (ICF), recruitment materials, and all participant materials will be 
submitted to the NIH Intramural Institutional Review Board (IRB) and Cambodian National Ethics 
Committee for Health Research (NECHR) for review and approval. Approval of both the protocol and the 
ICF must be obtained before any participant is enrolled. Any amendment to the protocol will require 
review and approval by the NIH IRB and NECHR before the changes are implemented to the study. In 
addition, all changes to the ICF will be approved by the NIH IRB and NECHR; a determination will be 
made regarding whether a new consent needs to be obtained from participants who provided consent, 
using a previously approved ICF.
1
PROTOCOL SUMMARY
1.1
SYNOPSIS 
Title:
Characterization of skin immunity to Aedes aegypti saliva in dengue-
endemic participants in Cambodia 
Study Description:
This is a single-center study characterizing the innate and adaptive 
immune response of endemic human skin to Aedes aegypti mosquito 
bites. Participants will undergo vector feeding and have a skin punch 
biopsy taken from the site of mosquito bites (exposed) and from unbitten 
skin (unexposed). Additionally, blood will be collected after feeding to 
evaluate systemic immune response. All protocol-specified data will be 
recorded and entered in a central data management system for the 
purposes of analysis of composite data from the study. Please see Schema 
and Schedule of Activities (SoA), Sections 1.2 and 1.3. 
Study Intervention 
Description:
Controlled feeding by colony-reared Aedes aegypti mosquitoes.
Objectives:
The primary objectives of the study are to:
1.
Compare the early and late innate immune response in the 
skin of Aedes aegypti–bitten (exposed) versus unbitten 
(unexposed) skin
2.
Characterize the local skin adaptive immune response
The secondary objective is to:

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1.
Analyze systemic immune response to vector salivary proteins 
in endemic participants with lifelong Aedes aegypti exposure 
after antigen encounter
The exploratory objectives are to:
1.
Describe the development of vector saliva–specific 
tissue-resident memory responses 
2.
Validate development of reliable biomarkers of vector 
exposure
Endpoints:
The primary endpoints are:
1.
Measurement of changes in the early and late innate immune 
response and cellular recruitment in bitten skin versus 
unbitten skin by:
a.
immunohistochemistry of target proteins at Day 0 
timepoints  
b.
immunophenotyping of innate immune cell subsets in 
dissociated skin sample at Day 0 timepoints  
c.
determination of cytokine profile in dissociated skin 
sample supernatant at Day 0 timepoints  
d.
differential complementary DNA (cDNA) expression 
prepared from skin RNA and analyzed via RNA 
sequencing (RNASeq) at Day 0 timepoints  
2.
Measurement of changes in the adaptive immune response 
and cellular recruitment bitten skin versus unbitten skin by:
a.
immunohistochemistry of target proteins at Day 2 
timepoints  
b.
phenotyping of adaptive immune cell subsets in 
dissociated skin sample at Day 2 timepoints
c.
determination of cytokine profile in dissociated skin 
sample supernatant at Day 2 timepoints  
d.
differential cDNA expression prepared from skin RNA 
and analyzed via RNASeq at Day 2 timepoints 
The secondary endpoint is:
1.
Flow cytometry analysis of peripheral blood mononuclear cells 
(PBMCs) collected Day 0 (baseline) and Days 2 and 14 after 
feeding for saliva-specific T cells
The exploratory endpoints are:
1.
Comparison of T-cell receptor (TCR) sequencing of skin 
biopsies at Day 0 versus Day 2
2.
Antibody determination in sera at Day 0 and Day 14  

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Study Population:
Total Enrollment:
Healthy adults aged 18 to 45 years (at study entry)
42 subjects
Description of 
Sites/Facilities Enrolling 
Participants:
Kampong Speu District Referral Hospital, Chbar Mon town, Cambodia
Study Duration:
24 months
Participant Duration:
2 weeks 

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1.2
PRECIS
Mosquito-borne viruses continue to cause significant global morbidity and mortality, particularly in 
Southeast Asia. When mosquitoes deliver the virus into the skin of humans while probing for a blood 
meal, they deposit also saliva, which contains a myriad of pharmacologically active compounds that 
modulate the host immune system. Little is known about skin immunity to mosquito saliva, particularly 
in endemic volunteers as most clinical studies are performed in naïve individuals who have never or 
rarely been exposed to a particular mosquito vector. People living in endemic areas have had long-term 
repeated exposure to these vectors and therefore have different immune response to mosquito saliva, 
which could interfere with mosquito-borne disease vaccine effectiveness. Characterization of skin 
immunity via various technical modalities will be important in order to identify critical aspects of the 
innate and adaptive immune responses after a vector bite.   
Here, we will execute a paired study of exposed-unexposed skin to carefully examine the innate and 
adaptive immune responses in the skin and blood to exposure of the saliva of Aedes aegypti, the 
mosquito vector of dengue, Zika, and chikungunya viruses. We will enroll 42 participants to undergo 
vector feeding and give blood samples at baseline and 2 and 14 days later. Additionally, participants will 
give skin punch biopsy samples of bitten (exposed) and unbitten (unexposed) skin. For analysis, we will 
group 10-12 participants in each of 4 technical modality “cohorts” or “groups”: 
1) immunohistochemistry, 2) RNA sequencing, 3) flow cytometry, and 4) T-cell receptor sequencing. 
With the current rise of vector-borne diseases in the United States and around the world, we hope the 
results of this study contribute to future vaccine design and clinical development strategies for 
vector-borne diseases.
Schema 1: Study Flow Design
 
SCREEN
Day -90 to 0
ENROLL
Visit 1
(Day 0)
Visit 2
(Day 2)
Visit 3
(Day 14)
Total N: Screen potential participants by inclusion and exclusion criteria; obtain 
pertinent history; obtain and document informed consent.
Perform baseline assessments, focused physical exam, and skin and blood 
sampling; mosquito feeding (refer to Section 8.3 for assays).  
Blood sampling, targeted exam, and final assessment.
 STUDY EXIT.
Blood and skin sampling, targeted exam and interim assessment.

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1.3
SCHEDULE OF ACTIVITIES (SOA)
Procedures
Screen (Day -90 to 0)
Enroll/Visit 1 (Day 0)
Visit 2 (Day 2)
+ 1 day
Visit 3 (Day 14)
±3 days
Informed Consent1
X
 
Medical/Medication History
X
X
Review of Eligibility Criteria
X
X
Documentation of Usual Bite Response
X
 
Clinician Assessment
X
X
X
X
Vital Signs2
X
X
X
X
Urine Pregnancy Test3
X
X
Mosquito Feeding
X
Skin Punch Biopsy
X4
X5
Blood Draw6
X
X
SKIN BIOPSIES7
Immunohistochemistry
X
X
RNASeq
X
X
Flow Cytometry
X
X
TCR sequencing 
X
X
BLOOD SAMPLING
 
Whole blood for PBMC isolation
X
X
X
Sera for antibody determination
X
X
Abbreviations: PBMC, peripheral blood mononuclear cell; RNASeq, RNA sequencing; TCR, T-cell 
receptor.
1 Informed consent will be obtained prior to any study procedures.  
2 Vital signs include blood pressure, heart rate, respiratory rate, and temperature; height and weight are 
taken at Day 0 visit only.  
3 Females of childbearing potential only. Mosquito feeding will not be initiated until results are available.
4 Two biopsies from bitten skin will be collected: one no earlier than 30 minutes and no later than 
90 minutes after the feeding is completed, and one no earlier than 3.5 hours and no later than 4.5 hours 
after the feeding is completed. Two biopsies from unbitten skin on the opposite arm will be collected no 
earlier than 30 minutes before feeding and no later than 6 hours after the feeding is completed.
5 One biopsy will be collected from bitten skin at least 40 hours after the feeding is completed.
6 Blood volume will not exceed 21 mL per visit; each whole blood collection will be 16 mL in a heparin 
tube, and each serum collection will be 5 mL in a serum-separating tube (SST).
7 Participants will be assigned to one of the following cohorts for skin sample technical evaluation 
modality: 10 participants to immunohistochemistry; 10 participants to RNASeq; 12 participants to flow 
cytometry; 10 participants to TCR sequencing.

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2
INTRODUCTION
 
2.1
STUDY RATIONALE 
Responsible for nearly one million deaths per year,1 vector-borne diseases are on the rise.2 Vector-borne 
disease occurs when a pathogen is transmitted by the bite of an infected blood-feeding arthropod such 
as a mosquito, fly, mite, flea, bug, or tick. Effective vaccines are needed to counter this global public 
health threat. At present, research efforts are mostly focused on the pathogen-host interactions without 
acknowledging the significant contribution of vector-derived products to disease development. Yet, as 
early as the 1940s, physicians observed humans’ variable clinical responses to mosquito saliva and 
surmised that it may contain molecules with immunomodulatory capabilities.3 However, not until the 
1970s did scientists recognize the potential role of vector saliva in pathogen transmission.4 Now, some 
attribute the complications of vector-borne disease vaccine development to the pleiotropic effects of 
vector saliva on both host and pathogen.5 Pathogen-host interactions are complex in any disease setting 
but become more nuanced with the addition of the vector, a previously underappreciated disease 
determinant that is increasingly recognized as a vaccine target.6–8  
For the few vaccines licensed for arthropod vector-borne disease and for the majority of the candidates 
in the pipeline, the focus is exclusively on the pathogen. However, vaccine development for these 
diseases may lie at the unique interface of the hematophagous insect vector, the pathogen, and the 
human host. The opportunity for vaccine development to disrupt disease transmission at “the bite site,” 
where the host, pathogen, and vector initially intersect, is gaining traction.7–9 Given the growing body of 
evidence of this concept in animal models, this protocol aims to achieve broader, translational 
understanding of cutaneous host-vector interactions via characterization of downstream immune 
cascades after exposure of endemic human skin to mosquito saliva. Specifically, we will carefully 
examine the early immune response to bites of Aedes aegypti mosquitoes in humans and explore how 
intense exposure in endemic areas may modulate future local skin responses, potentially affecting 
pathogen transmission and vaccine efficacy. We hypothesize that bites of arthropod vectors of disease 
egest specific vector-derived factors, including saliva,10 that modulate the innate immune response in 
individuals and recall a saliva-specific adaptive immune response to the bite site, thereby altering the 
outcome of disease caused by pathogens transmitted by these vectors and potentially the immune 
response to vaccines in endemic populations. Results from this study will provide a better understanding 
of the role that vector-derived factors play in vector-borne infections in humans who live in endemic 
areas. Additionally, the findings will be essential in informing future vaccine design and clinical 
development for both well-established vector-borne diseases like dengue virus (DENV) as well as 
emerging threats requiring the rapid development of countermeasures like Zika virus (ZIKV) and 
chikungunya virus (CHIKV).11,12
 
2.2
BACKGROUND 
Mosquito-borne disease is increasing worldwide, particularly in Southeast Asia.1,12 Cambodia has 
multiple mosquito-borne diseases including DENV, CHIKV, ZIKV, Plasmodium vivax and falciparum 
malaria, and Japanese encephalitis virus (JEV).13 With regard to Aedes-transmitted disease, DENV is the 
most common and is highly endemic with year-round transmission that peaks from July to November 
when rainfall is at its highest.14,15 In 2019, Cambodia experienced its worst outbreak in decades with 
DENV incidence peaking at nearly 5,000 cases of symptomatic DENV per week (National Dengue Control 

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Program communication 2019).   
Preliminary evidence from our ongoing pediatric cohort in Cambodia suggests intense exposure to 
Aedes aegypti mosquitoes particularly during the rainy season in July and August as evidenced by 
antibody reactivity against whole salivary gland homogenate of Aedes aegypti as measured by total 
immunoglobulin G enzyme-linked immunosorbent assay (ELISA). Others’ retrospective studies of human 
sera have demonstrated the usefulness of anti-Aedes salivary gland extract antibodies as 1) a marker of 
Aedes exposure and 2) a general corollary of DENV transmission potential.16–20   
2.2.1
SCIENTIFIC RATIONALE
When a mosquito inserts its proboscis and probes for blood, the mosquito ejects a salivary mix of 
vasodilators, anticoagulants, and other anti-hemostatic components into both the epidermis and the 
dermis.7 In addition to components that facilitate a bloodmeal, mosquito saliva also contains proteins 
that modulate both innate and adaptive immune responses. Feeding by Aedes and Culex mosquitoes in 
C3H/HeJ mice downregulated T helper type 1 (Th1) cytokines like IFN-gamma and upregulated Th2 
cytokines such as IL-4 and IL-10, cytokine shifts that were not seen when mosquitoes fed on 
flavivirus-resistant host mice.21 Increased Th2 response is the cause of irritation and allergic reaction 
seen with mosquito bites.9 While there is decreased T-cell recruitment to the site of injury after 
mosquito feeding, there is increased recruitment of neutrophils, eosinophils, and dendritic cells.5 
However, with repeated exposures to the vector’s bites as one would expect in endemic areas, animals 
became desensitized to some components of the saliva and were able to mount a more effective 
proinflammatory Th1 response, presumably owing to the presence of anti-saliva antibodies.23,25,26 Of the 
few human studies available, the majority of endemic participants in Mali had strong Th1-polarizing 
delayed type hypersensitivity reactions to sand fly saliva, suggesting a role for the adaptive immune 
response that will be investigated in this study with a 48-hour timepoint biopsy.27
Multiple arboviral studies in mice have demonstrated that the presence of saliva or salivary gland 
extract increases pathogenesis and dissemination of viruses. When a pathogen like DENV is present, 
mosquito saliva can facilitate the infectivity of an arbovirus by dampening the host’s anti-viral Th1 
immune response.23,28–30 Further, codeposition of mosquito saliva with an offending pathogen allows the 
pathogen to enter the host and more easily replicate.22,29,30,32,33 Mice inoculated intradermally with West 
Nile virus in the presence of Culex tarsalis saliva (via a mosquito feeding or co-inoculation) developed 
higher viral loads and faster onset of neuroinvasive disease compared to mice who were inoculated 
intradermally with virus alone.34 LMVR investigators have shown that the immune profile and cellular 
recruitment at the injection/bite site of animals infected by needle compared with those infected by 
bites of infected sand flies are different.35 Animals bitten by Leishmania-infected sand flies activated the 
host inflammasome and displayed an increased recruitment of neutrophils that was sustained for more 
than 18 hours, a completely different innate response than animals who received parasite injected by 
needle.10 Pingen et al. also reported similar findings, showing that Aedes aegypti mosquito bites induced 
an inflammatory response in mice characterized by the presence of neutrophils, and this immune 
response augmented the severity of Semliki Forest and Bunyamwera virus infections.32 
Given the majority of arboviral mouse models rely on type I interferon-deficiency, it is difficult to draw 
conclusions on the effects of saliva on the development of disease. A newer humanized mouse model 
was generated by transfer of human hematopoietic stem cells into NOD/SCID/IL1-gamma chain null 
(hu-NSG) mice.36 The footpad skin of these hu-NSG mice exposed to Aedes aegypti mosquito bites 
showed changes in various T-cell populations as well as systemic effects in the blood (Table 1).36

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Table 1. Changes in T-cell populations in bitten humanized NOD/SCID/IL1-gamma chain null mice
6 Hours Post-bite
24 Hours Post-bite
7 Days Post-bite
Blood
↓ Regulatory T Cells
↑ Double-positive (DP) T 
Cells
↑ CD8+ T Cells
↑ Natural Killer T Cells
↑ Regulatory T Cells
↑ DP T Cells
Skin
No Significant Changes
↓ DP T Cells
↓ Regulatory T Cells
↑ DP T Cells
Spleen
↓ Total T Cells
↓ Regulatory T Cells
↑ CD8+ T Cells
Bone Marrow
↓ Total T Cells
↓ Regulatory T Cells
No Significant Changes
↑ DP T Cells
All these results strongly suggest that a mosquito bite alters the skin environment, and consequently, 
the establishment of the virus delivered by the mosquito vector. However, we know very little about 
saliva-mediated immune cascades in the skin, particularly in endemic populations. The majority of 
studies are animal-based, and the few retrospective studies available in endemic volunteers are focused 
on systemic antibody-mediated immunity to vector saliva.19,37,38
2.2.2
CONCLUSION
Our aim is to translate the aforementioned preliminary findings in animal models to better understand 
the immune response to Aedes mosquito saliva. Our preliminary cohort data confirm that this study 
area is endemic for biting Aedes aegypti mosquitoes and will provide a suitable adult population to 
study immune cascades in vector-experienced human skin. We hypothesize that there will be different 
innate and adaptive immune signatures in the bitten versus unbitten skin that can ultimately guide 
future rationale design of saliva-based, or vector-targeted, vaccines.
2.3
RISK/BENEFIT ASSESSMENT  
   
2.3.1
KNOWN POTENTIAL RISKS 
 
The risk of enrollment in the study is the effect of mosquito feeding, skin punch biopsy, and 
venipuncture.  
Mosquito feeding may result in itchiness, pain, swelling, and redness at the site of bites. These reactions 
resolve after a short period of time and pose little risk to the participant. Applying cold packs and 
administering over-the-counter pain medications or antihistamines (topical or oral) if necessary can 
generally treat these reactions. Mosquito feeding or scratching after feeding may result in scarring such 
as hyperpigmentation. In rare cases a more severe irritation could occur, such as anaphylactic reaction 
or secondary infection at the site of the bite. Signs of infection include pain, redness, swelling, and 
drainage at the site. Oral antibiotics or anti-inflammatory medications can be administered if necessary.
Risks of skin punch biopsy include local pain, bleeding, redness, infection, a scar and possible keloid 
formation. Oral or topical antibiotics and oral analgesics will be used to manage pain and infection as 

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needed. Any non-routine complications resulting from the procedure will be addressed in consultation 
with available dermatological specialists in Phnom Penh. Injection of local anesthetic may cause a 
minimal burning discomfort or bruising at the site of the needle puncture.
Risks of blood draw include pain, bruising, bleeding, local discomfort, lightheadedness, dizziness or 
possibly fainting and rarely infection or blood clot. The amount of blood drawn is specified in the SoA 
table (Section 1.3). The amount of blood drawn will be within the limits allowed for adult participants by 
the local Cambodian authority NECHR, which is 21 mL in a single visit. 
2.3.2
KNOWN POTENTIAL BENEFITS 
There is no direct benefit to the participant. The information collected from this study will allow a better 
understanding of the skin immune response to vector bites to inform future vaccine development.
2.3.3
ASSESSMENT OF POTENTIAL RISKS AND BENEFITS 
Although there is no direct benefit of participation in the study, study results could contribute to 
generalizable knowledge informing future vaccine development for diseases relevant to the study 
population and their communities. The study will only enroll healthy volunteers and will perform careful 
monitoring following study procedures to minimize risks and discomforts.
3
OBJECTIVES AND ENDPOINTS
OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR 
ENDPOINTS
Primary
Compare the early and late innate 
immune response in the skin of 
Aedes aegypti–bitten (exposed) 
versus unbitten (unexposed) skin  
Measurement of changes in the 
early and late innate immune 
response and cellular recruitment 
in bitten skin versus unbitten skin 
by:
a)
immunohistochemistry of 
target proteins at Day 0 
timepoints  
b)
immunophenotyping of innate 
immune cell subsets in 
dissociated skin sample at Day 
0 timepoints  
c)
determination of cytokine 
profile in dissociated skin 
sample supernatant at Day 0 
timepoints  
d)
differential cDNA expression 
prepared from skin RNA and 
analyzed via RNASeq at Day 0 
timepoints  
Characterization of the 
responsible cell 
populations of the innate 
immune system in the 
epidermal-dermal 
microenvironment from 
exposure to mosquito 
saliva will highlight 
fundamental mechanisms 
of disease pathogenesis for 
mosquito-borne diseases.   

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OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR 
ENDPOINTS
Characterize the local skin 
adaptive immune response  
Measurement of changes in the 
adaptive immune response and 
cellular recruitment in bitten skin 
versus unbitten skin by:
a)
immunohistochemistry of 
target proteins at Day 2 
timepoints  
b)
phenotyping of adaptive 
immune cell subsets in 
dissociated skin sample at Day 
2 timepoints
c)
determination of cytokine 
profile in dissociated skin 
sample supernatant at Day 2 
timepoints  
d)
differential cDNA expression 
prepared from skin RNA and 
analyzed via RNASeq at Day 2 
timepoints 
Characterization of the 
cellular changes in the 
adaptive immune system in 
the epidermal-dermal 
microenvironment after 
exposure to mosquito 
saliva will also elucidate 
the underpinnings of 
mosquito saliva–mediated 
disease pathogenesis.   
Secondary
Analyze systemic immune 
response to vector salivary 
proteins in endemic participants 
with lifelong Aedes aegypti 
exposure after antigen encounter
Flow cytometry analysis of PBMCs 
collected Day 0 (baseline) and 
Days 2 and 14 after feeding for 
saliva-specific T-cells
Describing and 
understanding cellular 
immunity to Aedes saliva in 
heavily exposed individuals 
will simulate endemic 
conditions and will inform 
vaccine design in these 
target populations.
Tertiary/Exploratory 
Describe the development of 
vector saliva–specific tissue-
resident memory responses 
Validate development of reliable 
biomarkers of vector exposure
Comparison of TCR sequencing of 
skin biopsies at Day 0 versus Day 2  
Antibody determination in sera at 
Day 0 and Day 14  
Understanding tissue-
resident skin immune 
memory to Aedes saliva 
will highlight the 
responsible tissue-resident 
cell populations and 
possibly provide new 
targets for vaccine design 
of vector-borne disease.   
Assessing the anti-saliva 
antibody response in 
endemic volunteers in a 
quantitative fashion is 
critical to a biomonitoring 
strategy. Development and 

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OBJECTIVES
ENDPOINTS
JUSTIFICATION FOR 
ENDPOINTS
refinement of biomarkers 
are essential to targeted 
vector control initiatives in 
the future, particularly in 
resource-poor settings 
where limited supplies 
exist.
4
STUDY DESIGN 
4.1
SITE DESCRIPTION
Located in Cambodia, the main town of Chbar Mon (estimated population approximately 55,000-60,000 
over 10 km2) is located 44 km from Phnom Penh city center. Chbar Mon is the largest operational district 
in Kampong Speu province and is the site of the Kampong Speu District Referral Hospital, a hospital of 
approximately 120 beds with 130 staff of medical doctors, nurses, and lab technicians. NIAID has a field 
house behind the hospital that serves as a temporary clinic in which to perform mosquito feedings, skin 
biopsies, and venipuncture. 
In pupal surveys of Aedes mosquitoes in the region, 95.5% were Aedes aegypti, and the remainder were 
Aedes albopictus. This proportion was similar in both urban (94.7%) and rural (96.1%) areas.14 In this 
part of the province, in Chbar Mon town, there is essentially no malaria transmission as Anopheles 
vectors are predominantly in the forested hills that make up the westernmost part of Kampong Speu.39 
However, Culex mosquitoes are fairly abundant throughout rural, peri-urban, and urban areas of 
Cambodia and are responsible for transmitting JEV. Our preliminary cohort data from Kampong Speu 
confirms Aedes exposure, and we have ongoing studies into cross-reactivity between Culex and 
Anopheles saliva in our cohort participants. Preliminary results via western blot with endemic human 
sera suggest little cross-reactivity between Culex and Aedes species. Mosquito trapping of adult 
specimens reveals abundant Aedes aegypti, Aedes albopictus, and Culex species, but much fewer 
anopheline vectors as would be expected. Our ongoing entomological surveys reveal that Aedes aegypti 
1) has increased abundance compared to Aedes albopictus, 2) has 3-times increased odds of being found 
indoors compared to Aedes Albopictus, and 3) is equally distributed across the dense urban, transition, 
riverine, and rural areas in Chbar Mon. 
4.2
OVERALL DESIGN
This is a single-center study of healthy skin immune response to bites of colony-reared Aedes aegypti 
mosquitoes not infected with any pathogen compared to unbitten (unexposed) skin. Healthy 
participants will undergo a single Aedes aegypti feeding on Day 0 and give blood and skin samples 
through 2 weeks following the feeding. A total of 5 skin punch biopsies will be performed: one from 
bitten skin at each of 3 different timepoints (30-90 minutes, 3.5-4.5 hours, and about 48 hours after 
feeding is completed), and two from unbitten skin collected on Day 0. For feasibility of skin sample 
analysis, each participant will be assigned to 1 of 4 technical modality cohorts: 
1) immunohistochemistry, 2) RNASeq, 3) flow cytometry, and 4) TCR sequencing (see Section 9.1). 

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Additionally, systemic immune response will be evaluated on blood collected at baseline, and 2 and 14 
days after feeding (see SoA, Section 1.3). 
4.3
END OF STUDY DEFINITION
A participant is considered to have completed the study if he or she has completed all visits of the study 
as shown in the SoA, Section 1.3.  
The end of the study is defined as completion of the last visit shown in the SoA (Visit 3).
If the participant has a complication, he or she will be followed until the PI deems that the complication 
or adverse event (AE) has resolved. In the unlikely event that an AE (such as a keloid) has not resolved by 
the end of the study at Day 14, the participant will be referred to the District Hospital for any necessary 
follow-up.
5
STUDY POPULATION
5.1
INCLUSION CRITERIA
In order to be eligible to participate in this study, an individual must meet all of the following criteria:
1.
Provision of signed and dated ICF.
2.
Stated willingness to comply with all study procedures and availability for the duration of the 
study. 
3.
Male or female, aged 18–45 years.
4.
In good general health as evidenced by medical history.  
5.
Willing to allow biological samples to be stored for future research.
6.
A female is eligible for this study if she meets 1 of the following:
a.
Of non-childbearing potential (i.e., women who have had a hysterectomy or tubal 
ligation or are postmenopausal, as defined by no menses in ≥1 year).
b.
Of childbearing potential but has negative urine pregnancy test on Day 0.
7.
Agrees to not use scented lotions, deodorants, or topical creams on any part of the body on the 
feeding day.
8.
Agrees to not take aspirin or any other nonsteroidal anti-inflammatory drug (e.g., ibuprofen) 
within 7 days of a biopsy.
9.
Agrees to not use oral or topical antihistamines or steroid creams or ointments throughout the 
study without prior permission of PI.
 
5.2
EXCLUSION CRITERIA
1.
Any underlying or current medical condition that, in the opinion of the investigator, would 
interfere with participation in the study. 

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2.
History of severe allergic reaction (including to mosquito or other insect bites) with generalized 
urticaria, angioedema, anaphylaxis, anaphylactoid reaction, or any other reaction described by 
the participant and deemed severe by the PI.
3.
Self-reported or known history of alcoholism or drug abuse within 6 months prior to enrollment. 
4.
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. 
5.
Any use of medications that affect blood clotting within 3 months or history of abnormal blood 
clotting. 
6.
History of significant scarring such as keloids after previous biopsies, lacerations, abrasions, 
surgeries, or other skin procedures (e.g., cosmetic piercings) that are deemed by the PI to be a 
contraindication to protocol participation.
7.
Pregnant or breastfeeding. 
5.3
SCREEN FAILURES
Screen failures are defined as participants who consent to participate in the clinical trial but are not 
subsequently entered into the study. A minimal set of screen failure information is required to ensure 
transparent reporting of screen failure participants, to meet the Consolidated Standards of Reporting 
Trials (CONSORT) publishing requirements and to respond to queries from regulatory authorities. 
Minimal information includes demography, screen failure details, eligibility criteria, and any serious 
adverse event (SAE).
5.4
STRATEGIES FOR RECRUITMENT AND RETENTION
Advertising and recruitment in this province will mainly be via word-of-mouth using provincial health 
staff and the local village chiefs to organize informational meetings and subsequent dates for enrollment 
for those interested. Community engagement will begin 1-2 months prior to study start and will be 
organized in conjunction with the provincial health director of Kampong Speu province.
We anticipate having to screen no more than 80 to enroll goal number of 42 participants. We have no 
pre-set male:female ratios or age structure for the participant cohort. In Cambodia, it may be more 
likely for men to participate in healthy volunteer studies because they generally have more leisure time 
than women.
The anticipated length of individual study participation is approximately 2 weeks so participant retention 
should not be difficult. We will use multiple methods of contacting participants (e.g., phone number, 
Facebook Messenger username, and house visit if necessary). Retention compensation will include $5 
US (to be paid in Cambodian Riel) for venipuncture blood draw, $10 for each biopsy, and $5 for 
mosquito feeding. The total amount for completion of all study procedures would be $70. These are 
standard compensation rates that have been approved by the local ethics boards in prior NIAID-led 
malaria studies over the last 12 years, although mosquito feedings and skin biopsies have not been 
performed and therefore a similar value was chosen.  
Transportation for study visits will be provided to the referral hospital and NIAID Field Clinic as needed 
in a NIAID-CNM vehicle or be reimbursed (usually $1 maximum).   

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Lastly, the typical cost of an adult hospitalization is $10 in Kampong Speu District Referral Hospital. If any 
of the participants require hospitalization as a result of complications from the study (e.g., intravenous 
antibiotics for superimposed infection of bite site), they will have their hospitalizations and medications 
compensated. 
5.4.1
JUSTIFICATION FOR EXCLUSION OF MINORS AND PREGNANT WOMEN
Exclusion of Pregnant Women: In this study, participants will undergo mosquito feeding. Because 
pregnant women are known to be immunosuppressed to some degree and the primary objective of the 
study is to examine immune response to vectors, they will be excluded so as not to compromise the 
scientific validity of the study. 
Exclusion of Children: This is a study of the adult immune response to insect bites. Therefore, children 
will be excluded from the study.
6
PARTICIPANT DISCONTINUATION/WITHDRAWAL
6.1
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY
 
Participants are free to withdraw from participation in the study at any time upon request. An 
investigator may discontinue or withdraw a participant from the study if the subject meets study 
withdrawal criteria, as described in Section 8.4.13. If participants withdraw or are withdrawn prior to 
end of study, then they may be replaced as described in Section 8.4.14. The reason for participant 
discontinuation or withdrawal from the study will be recorded on the case report form (CRF).  
6.2
LOST TO FOLLOW-UP
A participant will be considered lost to follow-up if he or she fails to return for a scheduled visit and 
he/she is unable to be contacted by the study site staff.  
The following actions must be taken if a participant fails to return to the clinic for a required study visit:

The site will attempt to contact the participant and reschedule the missed visit within the 
protocol window, counsel the participant on the importance of maintaining the assigned visit 
schedule, and ascertain if the participant wishes to and/or should continue in the study.

Before a participant is deemed lost to follow-up, the investigator or designee will make every 
effort to regain contact with the participant (where possible, 3 telephone calls and local instant 
messaging methods).   

Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. The PI may be able to 
replace the participant who is lost-to-follow-up if they have not followed up all procedures.

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7
STUDY INTERVENTION
7.1
ARTHROPOD FEEDING
At the Malaria and Vector Research Laboratory (MVRL), an established state-of-the-art insectary for 
mosquitoes was built in 2014 to ACL2 (arthropod containment level 2) specifications. 
The Aedes aegypti colony was first established in 2018, reared from larvae obtained in Chbar Mon, 
Kampong Speu province. The colonies have been raised in the CNM-MVRL insectaries since that time. 
The insectaries include temperature- and humidity-controlled rooms. We have experienced technicians 
that take care of the colonies on a daily basis. For our study, the mosquito pupae collected after 
emergence will be placed into clean new containers dedicated to mosquitoes for use in human studies, 
located in a separate room from the rest of the colony. The mosquitoes used in human studies never 
come into contact with human or any other blood prior to their use. 
Colony-reared, clean female arthropods from Aedes aegypti mosquitoes will be bred in the CNM-MVRL 
insectary. Participants will be asked to not use scented lotions, deodorants, or topical creams on any 
part of the body starting 24 hours before the feeding day (or from their last shower as it is not common 
to shower every day). 
Each feeding will be conducted as follows:
i.
Assessment of the skin will be documented pre-feeding. 
ii.
5 starved female Aedes aegypti mosquitoes will be aspirated or placed into a secured feeding 
device prior to feeding and brought to the Referral Hospital or NIAID Field Clinic (about 35 km 
away) from the CNM-MVRL insectary. 
iii.
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 up to 20 minutes. The insects will feed through a disposable 
mesh on the bottom of the feeding device. This device permits the evaluation of feeding on the 
human participant at the end of exposure, where the abdomens of blood-fed mosquitoes are 
clearly visible through the mesh. 
a.
In the unlikely event of no feeding or poor feeding (only 0-2 insects fed or probed as 
noted by trained staff), the participant may undergo a repeat feed on the same arm 
with 5 fresh insects once. 
b.
If a repeat feed is required, the post-feeding biopsies will be done based on the timing 
of the repeat feed.
c.
Post-feeding times will be calculated from the time the feeding device is removed.
iv.
Post-feeding, redness, swelling, and number of visible bites will be assessed and documented 
immediately (+15 minutes) and 30 minutes (+15 minutes) after the feeding device is removed. 
This may also include taking photographs of the bites.
v.
Once the mosquitoes have fed, they will be brought back to the lab for evaluation and then 
disposal.
Post-feeding, insect bites will be counted. In individuals that lack a robust response to saliva, each bite 
site will be distinguished by a red pinpoint on the skin, visible to the eye with careful inspection of the 

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skin. Other participants may be more reactive to component of saliva, such as vasodilators, and will 
exhibit clear redness and/or induration that denotes a clear site of bite.
After the feeding, participants may be offered standard treatment as needed to control reactions to 
vector bites, but we will otherwise try to avoid topical or oral antihistamine or steroid treatments. These 
treatments may include cold compresses, topical or oral antihistamines, or topical steroids. On days 
when biopsies will be performed, necessary treatment to control the reaction to the vector bite will be 
offered after the biopsy is completed. 
To reduce confounding of new mosquito bites occurring between study visits, participants will be asked 
to wear long sleeves and pants and be offered available repellents that are commonly used in Cambodia 
(e.g., diethyltoluamide, bed nets, impregnated wall hangings).
7.2
EARLY TERMINATION VISIT
Participants who withdraw before the end of study will be encouraged to attend an early termination 
visit, where they will complete as many of the following procedures and evaluations as possible: 

Discussion to review reason for termination.

Review of medical/medication history.

Review of inclusion/exclusion criteria.

Clinician exam/assessment. 

Vital signs. 

Blood draw for research labs if participant is willing.
7.3
UNEXPECTED OR INCIDENTAL MEDICAL CONDITIONS OF THE PARTICIPANT
If unexpected or incidental medical conditions are diagnosed during the medical evaluation in this 
protocol, the participant will be referred to an appropriate physician and/or hospital and encouraged to 
follow up for treatment of their condition. Standard-of-care treatment may be offered by the study 
team if necessary while the participant is being referred to appropriate outside medical care.
8
STUDY ASSESSMENTS AND PROCEDURES
8.1
CLINICAL EVALUATIONS
Prior to conducting any study-related procedures, informed consent in Khmer will be obtained from the 
participant via in-person interview with an investigator. If illiterate, a literate witness will read and sign 
the ICF on behalf of the participant, and the participant will provide a thumbprint. The participant will be 
queried regarding a history of significant medical disorders. A screening log will be kept to document 
eligibility of all participants.  
Screening (-90 to Day 0)/Enrollment (Day 0):
The vector feeding will take place in the NIAID Field Clinic or the Kampong Speu District Referral 
Hospital. The following procedures will be performed: 

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
Informed consent.

Review of medical/medication history.
-
Baseline demographics will be collected such as sex, age, mosquito-borne biting risk 
factors (e.g., number of domestic water containers at household), bed net use, past 
medical history, drug allergies, and any current or previous (within past month) use of 
over-the-counter, prescription, or traditional remedies. Other behaviors related to 
disease development may also be collected here.
-
Documentation of usual responses to mosquito or other insect bites as none (0), 
mild (1), moderate (2), or severe (3).

Review of inclusion/exclusion criteria.

Clinician targeted exam/assessment.

Vital signs. 

Urine pregnancy test (for women of childbearing potential; confirmed negative before feeding). 
After these screening and/or enrollment procedures, participants will undergo a blood draw (Section 
1.3) and an Aedes aegypti mosquito feeding. Feeding and monitoring will be conducted as described in 
section 7.1. 
After the feeding, participants will undergo the following procedures: 

Skin punch biopsy collections as follows:

1 biopsy from bitten skin collected no earlier than 30 minutes and no later than 
90 minutes after the vector feeding is completed.

1 biopsy from bitten skin collected no earlier than 3.5 hours and no later than 4.5 hours 
after the vector feeding is completed.

Any other clinical tests that are medically indicated or appropriate to ensure the safety of the 
individual participant as determined by the PI.
Additionally, at Day 0, we will collect 2 biopsies from unbitten skin on the opposite arm. The unbitten 
samples will be collected no earlier than 30 minutes before and no later than 6 hours after the vector 
feeding is completed.
Skin samples will be placed immediately and no later than 1 minute after excision into pre-labeled 
Eppendorf tubes containing either a fixative (10% buffered formalin), RNAlater, or other specified 
medium for immunohistochemical, transcriptomic, or cytometric analyses (depending on the 
participant’s cohort for sample analysis).
Following these study procedures, participants will be allowed to leave as long as there are no safety 
concerns. They will be provided with medications if needed before they leave, and they will be asked to 
notify the study team if they have any reactions or side effects.

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Day 2 (48-hour post-feeding visit +1 day):
All participants will return for a visit about 48 hours after the feeding. At this visit, they will be asked 
about interim medications, symptoms (e.g., fever), healing of previous biopsy sites and mosquito bites, 
and new mosquito bites, and the following procedures will be performed.

Vital signs.

Clinician assessment of bites and biopsy sites.

1 skin punch biopsy of bitten skin (at site of previously marked mosquito bite).

Blood draw for research labs (ELISAs and cellular response assays, Section 1.3).
Day 14 (final study contact ±3 days):
All participants will return for a 14-day post-feeding visit. At this visit, they will be asked about interim 
medications, symptoms (e.g., fever), healing of previous biopsy sites and mosquito bites, and new 
mosquito bites, and the following procedures will be performed.

Vital signs.

Clinician assessment of bites and biopsy sites.

Blood draw for research labs (ELISAs and cellular response assays) (Section 1.3)
Any participant who experiences complications due to feeding or biopsies will be examined in the clinic 
and followed until complications have resolved and/or referral to the necessary medical care has been 
made. 
After the Day 14 visit, participation will be complete unless participants experience any AEs that need 
ongoing clinical monitoring. 
8.2
STUDY PROCEDURES
8.2.1
VITAL SIGNS
Vital signs include blood pressure, heart rate, respiratory rate, and temperature. Height and weight will 
be taken at the Day 0 visit only. On feeding days, vital signs will be performed pre-feeding.
8.2.2
MOSQUITO FEEDING
The mosquito feeding procedure is described in Section 7.1.
8.2.3
SKIN PUNCH BIOPSIES
From each participant, five 3-mm skin biopsies will be taken: two at the bite site on Day 0, two at an 
unbitten site on the opposite arm (unexposed) on Day 0, and one at the bite site at 48 hours, using the 
following procedure:
1.
The participant will be placed in a relaxed position with their arm out to expose the insect bites 
or unexposed site on the ventral side of the appropriate forearm.

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2.
A time-out will take place where the participant’s name and medical record number and the site 
of the biopsy will be confirmed. The time and date of the time-out will be documented.
3.
Non-sterile gloves will be worn and pads will be laid out to protect clothing as needed.
4.
Biopsy location will be marked with small circle.
5.
2% lidocaine with epinephrine will be drawn up in a 3-mL syringe.
6.
A 30-gauge needle will be used to inject lidocaine: It will be inserted into the skin just outside 
the circle drawn around the biopsy site; 1-1.5 cc of lidocaine will be injected slowly.
7.
Anesthesia will be tested using sharp forceps. More lidocaine will be injected as needed to 
achieve proper local anesthesia.
8.
Chlorhexidine or povidone iodide swabs will be used to disinfect the biopsy site by wiping 
concentrically from the center of the site to the outside of biopsy site.
9.
A steri-drape may be applied around the biopsy site.
10. The biopsy will be performed by pulling skin taut with thumb and forefinger above and below 
the marked area. A punch biopsy tool will be used to apply pressure and turned back and forth 
360 degrees. Forceps will be used to pull out the biopsy. Scissors may also be used if necessary 
to cut the specimen from the subcutaneous tissue.
11. Pressure will be held at the site using gauze, and then the site will be cleaned and dressed 
appropriately.
12. Steri-strips will be used to close biopsies, although sutures may rarely be placed if deemed 
necessary by the clinical team. If sutures are placed, the participant will return to clinic 
approximately 10 days later for suture removal.  
Any necessary treatment to control bleeding, discomfort, or reduce scarring from the biopsy will also be 
offered. A member of the study team may contact or visit the participant after biopsies to monitor 
healing.
8.3
LABORATORY EVALUATIONS
Specific laboratory methods for each assay and instructions for handling and storage of samples will be 
maintained in a manual of procedures that includes all the standard operating procedures (SOPs) for the 
study. 
8.3.1
SAFETY LABORATORY EVALUATIONS
For women of childbearing potential, a urine pregnancy test will be performed before the vector feeding 
procedure at the Kampong Speu District Referral Hospital laboratory or NIAID Field Clinic just behind 
hospital.
8.3.2
LABORATORY TESTING OF SAMPLES FOR STUDY ENDPOINTS
Skin samples collected from each participant (timepoints specified in Section 1.3) will be analyzed by 
one of the following technical modalities according to the participant’s assigned cohort:

Immunohistochemistry of target proteins (10 participants)

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
Immunophenotyping of innate immune cell subsets in dissociated skin sample plus cytokine 
profile determination in dissociated skin sample supernatant (12 participants)

Differential cDNA expression prepared from skin RNA and analyzed via RNASeq (10 participants)

TCR sequencing prepared from skin RNA (10 participants)
Blood samples collected at each timepoint specified in Section 1.3 will be used for the following 
evaluations: 

Isolation of PBMCs to assess cellular response to salivary gland homogenate and/or salivary 
recombinant proteins 

Antibody determination in sera
Samples remaining after completion of these evaluations may be stored for future research use.
8.3.3
SPECIMEN COLLECTION, PREPARATION, HANDLING, AND SHIPPING
All human biological samples will be received, processed, aliquoted, and stored at the central 
laboratories of CNM-NIH and/or Institut Pasteur in Phnom Penh according to standard lab procedures.
Samples requiring transport from the clinical collection site to the designated central repository and, 
with host country permission, to the main NIH laboratory in the US will follow designated study sample 
shipping procedures, which will include shipment of samples with no participant identifiers. This will 
include all skin samples designated for sequencing.
8.4
SAFETY AND OTHER ASSESSMENTS
  
8.4.1
ADVERSE EVENT DEFINITIONS
AE: Any untoward medical occurrence in a human subject, including any abnormal sign (e.g., 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 subject’s participation in the 
research.
SAE: Any AE that

results in death;

is life-threatening (places the subject at immediate risk of death from the event as it occurred);

results in inpatient hospitalization or prolongation of existing hospitalization;

results in a persistent or significant disability/incapacity

results in a congenital anomaly/birth defect; or

based upon appropriate medical judgment, may jeopardize the subject’s health and may require 
medical or surgical intervention to prevent one of the other outcomes listed in this definition 
(examples of such events include allergic bronchospasm requiring intensive treatment in the 
emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse).

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Unanticipated Problem Involving Risks to Subjects or Others (UP): Any incident, experience, or 
outcome that meets all of the following criteria:
1.
unexpected (in terms of nature, severity, or frequency) given
a.
the research procedures that are described in the protocol-related documents, such 
as the IRB-approved research protocol and informed consent document; and
b.
the characteristics of the subject population being studied; and
2.
related or possibly related to participation in the research (possibly related means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by 
the procedures involved in the research); and
3.
suggests that the research places subjects or others (which may include research staff, 
family members or other individuals not directly participating in the research) at a greater 
risk of harm (including physical, psychological, economic, or social harm) related to the 
research than was previously known or expected.
Protocol Deviation: Any change, divergence, or departure from the IRB-approved research protocol. 

Major Deviations: Deviations from the IRB-approved protocol that have or may have the 
potential to negatively impact the rights, welfare, or safety of the subject, or to substantially 
negatively impact the scientific integrity or validity of the study.

Minor Deviations: Deviations that do not have the potential to negatively impact the rights, 
safety, or welfare of subjects or others, or the scientific integrity or validity of the study.
Non-compliance: Failure of investigator(s) to follow the applicable laws, regulations, or institutional 
policies governing the protection of human subjects in research, or the requirements or determinations 
of the IRB, whether intentional or not.

Serious non-compliance: Non-compliance, whether intentional or not, that results in harm or 
otherwise materially compromises the rights, welfare and/or safety of the subject. 
Non-compliance that materially affects the scientific integrity or validity of the research may be 
considered serious non-compliance, even if it does not result in direct harm to research 
subjects.

Continuing non-compliance: A pattern of recurring non-compliance that either has resulted, or, 
if continued, may result in harm to subjects or otherwise materially compromise the rights, 
welfare and/or safety of subjects, affect the scientific integrity of the study or validity of the 
results. The pattern may comprise repetition of the same non-compliant action(s), or different 
noncompliant events. Such non-compliance may be unintentional (e.g., due to lack of 
understanding, knowledge, or commitment), or intentional (e.g., due to deliberate choice to 
ignore or compromise the requirements of any applicable regulation, organizational policy, or 
determination of the IRB).
8.4.2
ADVERSE EVENT MANAGEMENT 
Subjects entered onto this protocol will only be subject to skin biopsies, mosquito feedings, and limited 
blood sampling. There will be expected events as a result of these procedures. 

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Expected Events
The following are mild (Grade 1) to moderate (Grade 2) signs or symptoms that are induced by or 
associated with vector feeding or skin biopsy. If deemed related to vector feeding or skin biopsy by the 
PI, they will not be recorded as AEs in the Research Electronic Data Capture (REDCap) system per 
protocol, unless deemed by the PI to be abnormal or greater than Grade 2.
Feeding:

Local swelling

Pruritis

Erythema

Infection
Biopsy:

Local swelling

Erythema

Bleeding at biopsy site

Scar at biopsy site

Infection
Information on AEs related to research procedures developing after the procedure will be collected by 
the investigators and entered into a computerized database (REDCap, Sections 10.1.6 and 10.1.7).
All AEs will be graded according to the following AE management guidelines, which are intended to 
ensure the safety of each subject while on the study. The AEs will be graded by severity (Table 2) and 
attributed to study procedures (unrelated, unlikely, possibly, probably, or definitely; Table 3).  
Table 2. Grading of Adverse Events
1
Mild
Symptom barely noticeable to the subject; does not influence 
performance or functioning. Prescription drug not ordinarily needed 
for relief of symptom but may be given because of personality of 
subject. 
2
Moderate
Symptom of a sufficient severity to make the subject uncomfortable; 
performance of daily activities influenced; subject is able to continue 
in study; treatment for symptom may be needed. 
3
Severe
Symptom causes severe discomfort. May be of such severity that the 
subject cannot continue. Severity may cause cessation of study 
intervention; treatment for symptom may be given and/or subject 
hospitalized. 
4
Life-threatening
Symptom places the subject at immediate risk of death from the 
reaction as it occurred; it does not include a reaction that, had it 
occurred in a more serious form, might have caused death. 
5
Death
Symptom results in the death of the subject.

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Table 3. Attribution of Adverse Events (AEs)
Relationship 
Attribution 
Description 
Unrelated 
The AE is clearly NOT related 
to the intervention 
Unrelated to intervention
Unlikely 
The AE is doubtfully related 
to the intervention 
Possibly 
The AE may be related to the 
intervention 
Probably
The AE is likely related to the 
intervention 
Related to intervention
Definitely
The AE is clearly related to 
the intervention 
8.4.3
REPORTING
The PI is responsible for reporting per NIH IRB and NECHR reporting requirements. Reportable events 
will be tracked and submitted to the NIH IRB and NECHR as outlined in NIH Human Research Protections 
Program Policy 801. 
According to Policy 801, all UPs, major protocol deviations, all non-compliance, new information that 
might affect the willingness of a subject to enroll or remain in the study, and any suspension or 
termination of research activity will be reported within 7 calendar days of investigator awareness. Any 
death of a research subject that is possibly, probably, or definitely related to the research will be 
reported within 24 hours of investigator awareness.
The following events will be reported to the NIH IRB and NECHR in summary at the time of continuing 
review:

AEs and SAEs that are not UPs, as a narrative summary indicating whether these events were 
within the expected range.

Minor and major protocol deviations.

UPs reported to the NIH IRB/NECHR.

Non-compliance reported to the NIH IRB/NECHR that is not related to a protocol deviation. 
The PI will report UPs, major protocol deviations, and deaths to the NIAID clinical director according to 
institutional timelines.
8.4.4
PREGNANCY
Although pregnancy itself is not an AE, events occurring during pregnancy, delivery, or in the neonate 
(e.g., congenital anomaly/birth defect) may be AEs or SAEs.
In the event of pregnancy, the following steps will be taken:

Discontinue the study intervention and procedures but continue to follow-up for safety.

Report to the NIH IRB and NECHR.

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
Advise research participant to notify the obstetrician of study participation and study 
intervention exposure.
8.4.5
SPECIAL REPORTING SITUATIONS
Safety events of interest that may require expedited reporting and/or safety evaluation include but are 
not limited to:

Excessive reactions to study intervention.

Inadvertent or accidental exposure to a study intervention.

Medication error involving a product (with or without participant exposure to the study 
intervention, e.g., name confusion).
Special reporting situations will be recorded. Any special reporting situation that meets the criteria of an 
SAE should be reported as described in sections 8.4.2 and 8.4.3.
8.4.6
TYPE AND DURATION OF THE FOLLOW-UP OF PARTICIPANTS AFTER AES
AEs that occur following signing the ICF will be followed until the final outcome is known or until the end 
of the study follow-up period. AEs that have not resolved by the end of the study follow-up period will 
be recorded as “not recovered/not resolved.” If a participant is lost to follow-up and AEs have not 
resolved, the outcome of these AEs will be recorded as “unknown.” Any participant who experiences 
complications due to feeding or biopsy will be followed until such complications have resolved or 
appropriate referral to the necessary medical care has been made. For SAEs, if it is not possible to obtain 
a final outcome (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 REDCap. 
8.4.7
PAUSING RULES FOR AN INDIVIDUAL SUBJECT
Pausing is the suspension of administration of study intervention to a single subject until a decision is 
made whether or not to resume administration of the study intervention.
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 
intervention;

A subject experiences two Grade 3 or greater AEs that are possibly, probably, or definitely 
related to a study intervention;

Any safety issue that the investigator determines should pause administration of a study 
intervention to a single subject.
The PI will determine if study intervention administration to an individual participant should be paused. 
The study may also be paused for an entire group if a safety concern is identified.
8.4.8
REPORTING A PAUSE

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If a pausing criterion is met, a description of the AE(s) or safety issue must be reported by the PI within 
1 business day to the NIH IRB and NECHR.
8.4.9
RESUMPTION OF A PAUSED STUDY
The PI will determine whether or not it is safe to resume administration of the study intervention to the 
participant. The PI will notify the NIH IRB and NECHR of the decision on resumption of the study 
intervention.
A subject who does not resume study interventions will continue to be followed for safety.
8.4.10 HALTING RULES FOR THE PROTOCOL
Halting the study requires immediate discontinuation of study intervention administered for all 
participants and suspension of enrollment until a decision is made whether or not to continue 
enrollment and study intervention administration.
The halting rules are:

2 or more participants experience the same or similar SAEs that are possibly, probably, or 
definitely related to the study intervention;
OR

2 or more of the same or similar AEs in different participants that are grade 3 or above and are 
possibly, probably, or definitely related to the study intervention;
OR

any safety issue that the PI determines should halt the study.
The PI will determine if the study should be halted.
8.4.11 REPORTING A STUDY HALT
If a halting rule is met, a description of the AE(s) or safety issue must be reported by the PI within 
1 business day to the NIH IRB and NECHR.
8.4.12 RESUMPTION OF A HALTED STUDY
The PI will determine if it is safe to resume the study. The PI will notify the NIH IRB and NECHR of the 
decision on resumption of the study.
Participants who do not resume study intervention will continue to be followed for safety.
8.4.13 PREMATURE WITHDRAWAL OF A PARTICIPANT
An individual participant will be withdrawn for any of the following:

An individual participant’s decision. (The investigator should attempt to determine the reason 
for the participant’s decision.)

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
Non-compliance with study procedures to the extent that it is potentially harmful to the 
participant or to the integrity of the study data.

If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation (e.g., moves outside of study area)

The investigator determines that continued participation in the study would not be in the best 
interest of the participant.
8.4.14 REPLACEMENT OF WITHDRAWN PARTICIPANTS OR PARTICIPANTS WHO 
DISCONTINUE STUDY INTERVENTION
If participants withdraw or are withdrawn prior to end of study, then they may be replaced at the 
discretion of the PI if withdrawal or loss-to-follow up rates will impact study validity. Participants who 
withdraw at any time before all biopsies may be replaced. If a participant is removed prior to the 
feeding, none of their data will be used in the analysis or publication of the study. If a participant 
completes at a minimum the first feeding, data will be included in the analysis and publication. 
However, if they miss the second biopsy, they will not contribute to the 48-hour adaptive response 
studies. 
9
STATISTICAL CONSIDERATIONS 
 
9.1
STATISTICAL HYPOTHESES
This study is highly exploratory as very little is known about skin immunity to mosquito saliva in human 
skin. The simple characterization of this skin immunity will be important, as even with a small sample 
sizes we expect to be able to identify important aspects of the innate and adaptive immune response 
after a mosquito bite by identifying cell populations and examining the transcriptomic responses of 
differentially expressed genes.  
The study hypothesis is that there will be differential phenotypic, transcriptomic, and 
immunohistochemical differences between the exposed (bitten) and unexposed (unbitten) skin with 
regard to innate immune markers at Day 0 after the mosquito feeding in an individual. We also expect to 
see differential phenotypic, transcriptomic, and immunohistochemical signals of adaptive immune 
markers at Day 2 as compared to unexposed (unbitten) skin.
To ensure sufficient sample amount to achieve study endpoints, skin samples from each participant will 
be assigned to a technical modality cohort. We are defining “cohort” as the 10-12 participants assigned 
to each technical evaluation modality. There are 4 cohorts for 4 types of evaluations: 
1) immunohistochemistry, 2) RNASeq, 3) flow cytometry, and 4) TCR sequencing. To understand the 
real-world feasibility issues in Cambodia, we plan to first proceed with 10 participants in the 
immunohistochemistry cohort in Week 1 (since there are no temperature, time-sensitive storage, or 
processing requirements for formalin-preserved biopsies). Additionally in Week 1, we will enroll 
2 participants in the flow cytometry cohort. These samples, which must be processed “fresh” in 
real-time due to time and temperature sensitivity, will be used for optimizing the flow cytometry cohort. 
The other 10 participants in the flow cytometry cohort will be sampled in Week 2.

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9.2
SAMPLE SIZE JUSTIFICATION AND ANALYSIS PLAN
Given no prior saliva skin immunity studies published in humans, feasibility and sample sizes are 
adapted from literature concerning neoplastic or autoimmune disorders of the skin versus healthy 
comparator skin. With regard to flow cytometry of skin samples, 4-mm biopsies typically yield 
2200 ±615 cells in healthy skin and 178,000 cells in lesional skin of psoriatic patients.40 Previous studies 
of skin transcriptomes have estimated approximately 13,000 genes per biopsy of healthy individuals.41
Given the high cost of many of these analyses, several published human studies typically only have 
between 2 and 10 skin samples per group or total.40,42,43 Currently, we do not know whether skin 
immune responses to bites of this vector will be uniform or how much variability will be observed. 
Therefore, it is very difficult to estimate an exact samples size. As this study is very exploratory in nature 
and has a relatively small number of individuals in each cohort, it is likely that the results will be very 
informative of future studies as it will offer more insight into the diversity or lack thereof in the skin 
responses. The cohort sizes of 10 (+2 additional participants in the flow cytometry cohort for 
optimization) will allow of us to have adequate numbers to perform these detailed and expensive 
immunohistochemical and transcriptomic techniques that could lead to future larger studies with more 
focused testing once the differential genes and important areas of immune response are identified.  
As we expect transcriptomic analyses to yield at least 13,000 genes, this large number of genes will be 
examined to find a smaller list of differentially expressed genes within cohorts. For transcriptomic 
analysis, non-normalized raw counts will be used with an analysis program (e.g., the EdgeR package) to 
perform differential gene expression analysis after quality control (QC) of samples. Our anticipated 
analysis program will utilize model-based scale normalization, estimate dispersions, and apply a negative 
binomial model for RNA transcriptomic data analysis to find differentially expressed genes. 
Single comparisons will be made within cohorts of bitten skin at a particular timepoint to the unbitten 
skin biopsy. Because our study will analyze paired samples (bitten skin and unbitten skin biopsies from 
the same individual), we can employ two different approaches. First, we can apply general linear 
modelling (GLM) using a GLM likelihood ratio test for comparing bitten versus unbitten skin. Also, we 
can employ group-wise comparisons where negative binomial fitting is followed by exact test. False 
discovery rate (FDR) adjustment will be used for multiple testing correction. An FDR threshold of 0.1 for 
statistical significance is typically applied. Because this is exploratory, an FDR or a q-value equivalent of 
0.05 will be applied. Genes with larger differential expression will be defined with a 4-fold change 
threshold between experimental conditions.
For flow cytometric analysis, data will be collected using the BDCanto software (BD Biosciences). Data 
will be analyzed using FlowJo (v10.2; FlowJo, LLC). Differences will be assessed by Mann Whitney-U test 
or Wilcoxon matched-pairs signed rank test. A p value below 0.05 is considered significant for a single 
comparison, but flow cytometry will likely need to be adjusted for multiple comparisons as we 
anticipate using a minimum of 8 markers.

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10
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS
10.1.1 INFORMED CONSENT PROCESS
10.1.1.1 CONSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO PARTICIPANTS
ICFs describing in detail the study procedures and risks are given to the participants, and written 
documentation of informed consent is required prior to starting research procedures and evaluations. 
The ICFs in English (to be translated into Khmer for submission for Cambodian ethics review and for use 
in study) are submitted with this protocol.
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION
Informed consent is a process where information is presented to enable persons to voluntarily decide 
whether or not to participate as a research subject. It is an ongoing conversation between the human 
research subject and the researchers that begins before consent is given and continues until the end of 
the subject's involvement in the research. Discussions about the research will provide essential 
information about the study in Khmer and include at the very least the purpose, duration, procedures, 
alternatives, and risks and benefits. Potential participants will be given the opportunity to ask questions 
and have them answered. Those who are literate will sign and date the ICF (written in Khmer) prior to 
undergoing any research procedures. In this case, a witness will not sign and date the ICF. If the  
potential participant is illiterate, a thumbprint will be obtained and the date will be documented by the 
investigator. In this case, an impartial witness who is literate and not involved with the study will then 
sign and date the ICF.   
Participants may withdraw consent at any time throughout the course of the study. A signed copy of the 
ICF will be given to them for their records. The investigator will document the signing of the ICF in the 
participant’s medical record. The rights and welfare of the participants 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.
10.1.2 STUDY DISCONTINUATION AND CLOSURE
This study may be temporarily suspended (paused) or prematurely terminated (halted) as described in 
Sections 8.4.7 and 8.4.10. If the PI prematurely terminates or suspends the study, then the PI will 
promptly inform study participants and the NIH IRB and NECHR in writing, and will provide the reason(s) 
for the termination or suspension. Study participants will be contacted, as applicable, and be informed 
of changes to study visit schedule. The study may resume once concerns about safety, protocol 
compliance, and data quality are addressed.
10.1.3 CONFIDENTIALITY AND PRIVACY 
Participant confidentiality and privacy is strictly held in trust by the participating investigators and their 
staff. This confidentiality is extended to cover testing of biological samples in addition to the clinical 

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information relating to participants. Therefore, the study protocol, documentation, data, and all other 
information generated will be held in strict confidence. No information concerning the study or the data 
will be released to any unauthorized third party without prior written approval of the sponsor. 
All research activities will be conducted in as private a setting as possible. 
The study monitor, other authorized representatives of the sponsor, representatives of the NIH IRB, 
NECHR, or regulatory agencies may inspect all documents and records required to be maintained by the 
investigator, including but not limited to, medical records (office, clinic, or hospital) and pharmacy 
records for the participants in this study. The clinical study site staff will permit access to such records.
The study participant’s contact information will be securely stored at each clinical site for internal use 
during the study. At the end of the study, all records will continue to be kept in a secure location for as 
long a period as dictated by the reviewing IRB and ethics committee, institutional policies, or sponsor 
requirements.
Study participant research data, which is captured on tablets for purposes of statistical analysis and 
scientific reporting, will be transmitted to and stored on a central server either in Phnom Penh and/or 
LMVR at NIH main campus. This will not include the participant’s contact or identifying information. 
Rather, individual participants and their research data will be identified by a unique study identification 
number. The study data entry and study management systems used by research staff will be secured 
and password protected. At the end of the study, the study database will be de-identified and archived 
at LMVR in Rockville, MD.
10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA 
Intended Use: Samples, specimens, and data collected under this protocol may be used to study the 
following: 

Development of new diagnostic tests to enable more sensitive and specific tests for 
vector-borne diseases.

Determination of immune response to vector saliva, vector-borne diseases, or other infectious 
diseases or immunologic phenomena.

Determination of incidence and pathogenesis of co-circulating infections (e.g., DENV, JEV, 
CHIKV, ZIKV, influenza), as it contributes to this community.

Use of samples for developing vaccines and therapeutic interventions to prevent or treat 
vector-borne infections or other infectious diseases.

Isolation or sequencing of viruses (e.g., DENV, CHIKV, ZIKV), parasites, or bacteria.
Storage: All of the stored study research samples are labeled by a code that only the investigators can 
link to the subject. Samples are stored at the Malaria Vector and Research Laboratory at the National 
Center for Parasitology, Entomology, and Malaria Control and the Institut Pasteur Cambodge, which are 
both secure facilities with limited access. During the high-volume biopsy collection, samples may be 
temporarily stored in the Kampong Speu District Referral Hospital main laboratory, which is also a 
secure facility with limited access. In the future, it will be necessary to ship a limited number of samples 
to NIH for analyses that cannot be performed in Cambodia, and these would be kept in a secure facility 

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at the LMVR in Rockville, MD. Data will be kept in password-protected computers. Only investigators or 
their designees will have access to the samples and data.
Tracking: Samples and data acquired under this protocol will be tracked using either Microsoft Excel (for 
input into the Biological Specimen Inventory) or REDCap.
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 for research purposes. If the planned research falls within the category of “human 
subjects research,” appropriate NIH IRB and NECHR review and approval will be obtained. This 
includes the NIH or CNM researchers sending out coded and linked samples or data and getting 
results that they can link back to their subjects.  

At the time of protocol termination, samples will be either destroyed or, after NIH IRB and 
NECHR approval, transferred to another existing protocol. Data will be archived by the study 
team in compliance with requirements for retention of research records; alternatively, after NIH 
IRB and NECHR approval, the data may be either destroyed or transferred to another repository.
Loss or Destruction:

Any loss or unanticipated destruction of samples (for example, due to freezer malfunction) or 
data (for example, misplacing a printout of data with identifiers) that meets the definition of a 
reportable event will be reported to the NIH IRB and the NECHR according to NIH Policy 801.

Additionally, subjects may decide at any point not to have their samples stored. In this case, the 
PI will destroy all known remaining samples and report what was done to both the subject and 
to the NIH IRB and NECHR. This decision will not affect the individual’s participation in this 
protocol or any other protocols at NIH.

However, withdrawal of consent with regard to bio-sample storage may not be possible after 
the study is completed. If a subject had previously consented to future use and some of their 
blood has already been used for research purposes, then the information from that research 
may still be used even after consent is revoked.
  
10.1.5 CLINICAL AND SAFETY MONITORING
Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct of 
the trial is in compliance with the currently approved protocol/amendment(s), with ICH GCP, and with 
applicable regulatory requirement(s). 
According to ICH-GCP 5.18 clinical protocols are required to be adequately monitored. This study 
monitoring will be conducted according to the “NIAID Intramural Clinical Monitoring Guidelines.” 
Monitors under contract to NIAID/Office of Clinical Research Policy and Regulatory Operations 
(OCRPRO) will visit the 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 ICFs and documentation of the informed consent process for each monitored 
subject; (2) to verify the prompt and accurate recording of all monitored data points, and prompt 
reporting of all SAEs; (3) to compare abstracted information with individual subjects’ records and source 
documents (subjects’ charts, laboratory analyses and test results, physicians’ progress notes, nurses’ 

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notes, and any other relevant original subject 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 (Office for Human Research Protections), 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.
During monitoring visits, the PI (and/or designee) and other study personnel will be available to discuss 
the study progress and monitoring visit. The PI will be provided copies of monitoring follow-up letters 
within 20 days of a visit.
The investigator (and/or designee) will make study documents (e.g., ICFs) and pertinent hospital or 
clinical records readily available for inspection by the NIH IRB, NECHR, the site monitors, and the NIAID 
staff for confirmation of the study data. 
A specific protocol monitoring plan will be discussed with the PI 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.
The data gathered during this study will be monitored by the PI for safety and compliance with protocol-
specified requirements.
10.1.6 QUALITY ASSURANCE AND QUALITY CONTROL
 
QC procedures will be implemented beginning with the data entry system and data QC checks that will 
be run on the database will be generated. Any missing data or data anomalies will be communicated to 
the PI for clarification/resolution.
REDCap will serve as the core data collection component of the data management system. REDCap, 
which has been deployed in approximately 100 countries in support of 244,000 studies, is already in use 
at the site in an ongoing cohort study. It has an extensive range of features, the most important of which 
are the following:

the ability to operate from a web browser on a server or laptop and from Android and Apple 
tablets in disconnected mode and to synchronize data between any of these devices; 

the ability to interface with and receive data from external data management systems and 
adjudicate data imports prior to incorporation into the database;

a lightweight application that requires minimal processing power or memory;

the ability to define and run data quality rules over the duration of the study that can be run on 
both a real-time and ad hoc basis;

support for updates through multiple mechanisms, including programming directly in the 
system at the site and transmission of updates from the Data Coordinating Center;

support for multiple languages, both in the user interface and data collection instruments; and

real-time reports, data summaries, and data exports, including frequencies for outlier detection.
Precision and accuracy of actual data collected will be checked by internal procedures at random (5%). 
Data editing and error resolution will be performed monthly. 

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Following written SOPs, the monitors will verify that the clinical trial is conducted, data are generated, 
and biological specimens are collected, documented (recorded), and reported in compliance with the 
protocol, ICH GCP, and applicable regulatory requirements 
The study site will provide direct access to source data/documents, reports for the purpose of 
monitoring and auditing, and inspection by local and regulatory authorities.
10.1.7 DATA HANDLING AND RECORD KEEPING 
10.1.7.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES 
Upon enrollment in the protocol, all subjects will be assigned a unique identifying combination code of 
four to seven numbers depending on assigned protocol number (e.g. 001-000) as it is a single-site study. 
The identifier will serve to link this identifying code with subject name. CRFs, databases, and samples 
will be coded with unique identifying numbers and will not contain subject names. Study personnel 
involved in data acquisition, entry, and analysis will therefore not have access to names, assuring the 
privacy and confidentiality of subjects. Standard CRFs containing clinical and laboratory data will be 
completed on tablets using the aforementioned REDCap that will be locked with a code and uploaded to 
password-protected servers. Relevant subject data will be entered into a hybrid system (REDCap plus 
Datafax as needed which is already established in the NIAID sites in Cambodia). Electronically captured 
data will be checked by study investigators and verified by a data manager. All electronic data will be 
stored on a secure server. 
Access to subject charts and databases for data analysis will be restricted to the investigators listed on 
this protocol and those working under their direct supervision. Access to ICFs, which link identifying 
numbers to names, will be restricted to those investigators authorized to obtain consent. The OCRPRO 
and their authorized representatives may also have access to the subjects’ study records, including ICFs 
and source documents. CRFs, ICFs, and source documents will eventually be transported from the study 
site to our offices in Phnom Penh, where they will be retained under lock and key by the investigators 
listed on this protocol and made available for clinical monitoring. 
10.1.7.2 STUDY RECORDS RETENTION 
The PI will retain all study records for at least 7 years in compliance with institutional, IRB and ethics 
committee, state, and federal medical records retention requirements, whichever is longest. The PI will 
keep all stored records confidential to the extent required by federal, state, and local law. Data captured 
electronically via tablets will be backed up nightly to the site’s central server and transmitted on a 
weekly basis. All data will be archived at the end of the study and retained for a period of time 
consistent with IRB and ethics committee requirements.
Should the PI wish to assign the study records to another party and/or move them to another location, 
the PI 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. NIAID will be 
notified in writing and written OCRPRO/NIAID permission shall be obtained by the site prior to 
destruction or relocation of research records.

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10.1.8 PROTOCOL DEVIATIONS 
As a result of a protocol deviation (as defined in Section 8.4.1), corrective actions are to be developed by 
the site and implemented promptly. 
These practices are consistent with ICH GCP: 

4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3. 

5.1 Quality Assurance and Quality Control, section 5.1.1. 

5.20 Noncompliance, sections 5.20.1, and 5.20.2. 
Protocol deviations must be sent to the NIH IRB and NECHR per their policies (Section 8.4.3). The site 
investigator is responsible for knowing and adhering to the reviewing IRB and ethics committee 
requirements. 
10.1.9 PUBLICATION AND DATA SHARING POLICY
This study will be conducted in accordance with the following publication and data sharing policies and 
regulations:
NIH Public Access Policy, which ensures that the public has access to the published results of NIH funded 
research. It requires scientists to submit final peer-reviewed journal manuscripts that arise from NIH 
funds to the digital archive PubMed Central upon acceptance for publication.
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-Funded 
Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. As 
such, this trial will be registered at ClinicalTrials.gov, and results information from this trial will be 
submitted to ClinicalTrials.gov. In addition, every attempt will be made to publish results in 
peer-reviewed journals. Data from this study may be requested from other researchers 5 years after the 
completion of the primary endpoint by contacting Jessica Manning (NIAID).
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.

De-identified data in another public repository.

De-identified data with approved outside collaborators under appropriate agreements.

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10.2 ABBREVIATIONS
 
ACL2
Arthropod Containment Level 2
AE
Adverse Event
cDNA
Complementary Deoxyribonucleic Acid
CFR
Code of Federal Regulations
CHIKV
Chikungunya Virus
CNM
National Malaria Center
CONSORT
Consolidated Standards of Reporting Trials
CRF
Case Report Form
DENV
Dengue Virus
DP
Double-positive (T Cell)
ELISA
Enzyme-linked Immunosorbent Assay
FDR
False Discovery Rate
GCP
Good Clinical Practice
GLM
General Linear Modelling
hu-NSG
Humanized NOD/SCID/IL1-Gamma Chain Null Mice
ICF
Informed Consent Form
ICH
International Council on Harmonisation of Technical Requirements for Pharmaceuticals 
for Human Use
IRB
Institutional Review Board
JEV
Japanese Encephalitis Virus
LMVR
Laboratory of Malaria and Vector Research
MVRL
Malaria and Vector Research Laboratory
NECHR
National Ethics Committee for Health Research
NIAID
National Institute of Allergy and Infectious Diseases
NIH 
National Institutes of Health
OCRPRO
Office of Clinical Research Policy and Regulatory Operations
PBMC
Peripheral Blood Mononuclear Cell
PI
Principal Investigator
QC
Quality Control
REDCap
Research Electronic Data Capture
RNASeq
Ribonucleic Acid Sequencing
SAE
Serious Adverse Event
SoA
Schedule of Activities
SOP
Standard Operating Procedure
SST
Serum-separating Tube
TCR
T-cell Receptor
Th
T Helper
UP
Unanticipated Problem Involving Risks to Subjects or Others
US
United States
ZIKV
Zika Virus

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11
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