Defining Skin Immunity of a Bite of Key Insect Vectors in Humans
Phase: NA
Conditions: Zika, Dengue, Malaria, Chikungunya, Leishmaniasis
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Skin Immunity to Vector Bites 
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Defining Skin Immunity of a Bite of Key Insect Vectors in Humans 
 
 
 
NIAID Protocol Number 
18-I-0139 
 
 
 
Principal Investigator 
Matthew J. Memoli, MD, MS 
Laboratory of Infectious Diseases   
National Institute of Allergy and Infectious Diseases  
National Institutes of Health  
Tel: 301-443-5971 
Email: memolim@mail.nih.gov 
 
 
 
Version Number and Date 
Version 3.0 
February 1, 2019 
 
 
 
Conducted by 
Laboratory of Infectious Diseases  
National Institute of Allergy and Infectious Diseases  
National Institutes of Health  
 

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TEAM ROSTER 
Principal Investigator: 
 
 
 
 
 
 
Associate Investigators: 
 
 
  
Matthew J. Memoli, MD, MS 
Laboratory of Infectious Diseases  
National Institute of Allergy and Infectious Diseases  
National Institutes of Health  
Tel: 301-443-5971 
Email: memolim@mail.nih.gov 
 
Jesus G. Valenzuela, PhD 
Laboratory of Malaria and Vector Research   
Vector Molecular Biology Section  
National Institute of Allergy and Infectious Diseases 
Tel: 301-761-5070 
Email: jvalenzuela@niaid.nih.gov 
 
Shaden Kamhawi, PhD 
Laboratory of Malaria and Vector Research  
Vector Molecular Biology Section  
National Institute of Allergy and Infectious Diseases 
Tel: 301-761-5081 
Email: skamhawi@niaid.nih.gov 
 
Alison Han, MD, MS 
Laboratory of Infectious Diseases  
National Institute of Allergy and Infectious Diseases  
Tel: 301-480-5722 
Email: alison.han@nih.gov 
 
Luiz Fabiano Oliveira, MD, PhD 
Laboratory of Malaria and Vector Research  
Vector Molecular Biology Section  
National Institute of Allergy and Infectious Diseases 
Tel: 301-761-5073 
Email: loliveira@niaid.nih.gov 
 
Iliano V. Coutinho-Abreu, PhD 
Laboratory of Malaria and Vector Research  
Vector Molecular Biology Section  
National Institute of Allergy and Infectious Diseases 
Tel: 301-761-3977 
Email: iliano.vieiracoutinhoabreugomes2@nih.gov 
 
Joshua Lacsina, MD, PhD 
Laboratory of Malaria and Vector Research  
Vector Molecular Biology Section  
National Institute of Allergy and Infectious Diseases 
Tel: 301-275-2045 
Email: joshua.lacsina@nih.gov 

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Luca Giurgea, MD 
Laboratory of Infectious Diseases 
National Institute of Allergy and Infectious Diseases 
Tel: 301.538.5235 
Email: luca.giurgea@nih.gov 
 
 
Study Staff NIH: 
Holly Ann Baus, RN, MSN 
Laboratory of Immunoregulation 
National Institute of Allergy and Infectious Diseases 
Tel: 301-761-6800 
Email: holly.baus@nih.gov 
 
Lindsay M. Czajkowski, MSN, NP-C 
Laboratory of Infectious Diseases  
National Institute of Allergy and Infectious Diseases  
Tel: 301-451-7705 
Email: lindsay.czajkowski@nih.gov 
 
Susan M. Reed, BA, CRC 
Laboratory of Infectious Diseases  
National Institute of Allergy and Infectious Diseases  
Tel: 301-451-3354 
Email: susan.reed@nih.gov 
 
Rani S. Athota, PhD 
Laboratory of Infectious Diseases  
National Institute of Allergy and Infectious Diseases  
Tel: 301-594-0803 
Email: rani.athota@nih.gov 
 
 
 
Institutional Review  
Board (IRB): 
National Institute of Allergy and Infectious Diseases  
 
Institutional Review Board 
 
5601 Fishers Lane  
 
Rockville, MD 20852 
 
 
 
 

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TABLE OF CONTENTS 
TEAM ROSTER .............................................................................................................. 2 
LIST OF TABLES ............................................................................................................ 6 
LIST OF FIGURES .......................................................................................................... 6 
LIST OF APPENDICES ................................................................................................... 6 
LIST OF ABBREVIATIONS ............................................................................................. 7 
PROTOCOL SUMMARY ................................................................................................. 8 
1 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ..................... 12 
1.1 
Background Information ..................................................................................... 12 
1.2 
Scientific Rationale ............................................................................................ 13 
2 
STUDY OBJECTIVES ....................................................................................... 15 
2.1 
Primary Objectives ............................................................................................. 15 
2.2 
Secondary Objectives ........................................................................................ 15 
2.3 
Exploratory Objective: ........................................................................................ 15 
3 
STUDY DESIGN ................................................................................................ 16 
3.1 
Description of the Study Design ......................................................................... 16 
3.2 
Study Endpoints ................................................................................................ 16 
3.2.1 
Primary endpoints .................................................................................. 16 
3.2.2 
Secondary endpoints ............................................................................. 17 
3.2.3 
Exploratory endpoint .............................................................................. 17 
4 
STUDY POPULATION ...................................................................................... 17 
4.1 
Rationale for Participant Selection ..................................................................... 17 
4.2 
Recruitment Plan ............................................................................................... 18 
4.3 
Inclusion Criteria ................................................................................................ 19 
4.4 
Exclusion Criteria ............................................................................................... 19 
4.5 
Justification for Exclusion of Special Populations ............................................... 20 
5 
STUDY INTERVENTION ................................................................................... 21 
5.1 
Arthropod Feeding ............................................................................................. 21 
6 
STUDY SCHEDULE .......................................................................................... 22 
6.1 
Enrollment ......................................................................................................... 22 
6.2 
Cohort A ............................................................................................................ 23 
6.2.1 
Vector feeding and biopsy (Day 0) ......................................................... 23 
6.2.2 
24-Hour Post-Feeding Visit (Day 1 +1 day) ............................................ 24 
6.2.3 
Final study contact (Day 7 ±3 days) ....................................................... 24 
6.3 
Cohort B ............................................................................................................ 24 
6.3.1 
Vector feeding and biopsy (Days 0, 14, 28, and 42, +7 days) ................. 24 
6.3.2 
Follow-up and biopsy (Day 44 +3 days) ................................................. 25 
6.3.3 
Final study contact (Day 51 ±3 days) ..................................................... 25 
6.3.4 
Early termination visit ............................................................................. 26 
7 
STUDY PROCEDURES/EVALUATIONS ........................................................... 26 
7.1 
Study Procedures .............................................................................................. 26 
7.1.1 
Vital signs............................................................................................... 26 
7.1.2 
Skin biopsies .......................................................................................... 26 
7.2 
Laboratory Evaluations ...................................................................................... 27 
7.2.1 
Safety laboratory evaluations ................................................................. 27 
7.2.2 
Laboratory testing of samples for study endpoints .................................. 27 
8 
POTENTIAL RISKS AND BENEFITS ................................................................ 28 
8.1 
Potential Risks ................................................................................................... 28 
8.1.1 
Risks of vector feeding ........................................................................... 28 
8.1.2 
Risks of skin punch biopsy ..................................................................... 28 

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8.1.3 
Risks of blood draw ................................................................................ 29 
8.2 
Potential Benefits ............................................................................................... 29 
9 
RESEARCH USE OF STORED HUMAN SAMPLES, SPECIMENS, AND DATA
 
29 
10 
DATA SHARING PLAN ..................................................................................... 30 
11 
REMUNERATION PLAN ................................................................................... 30 
12 
ASSESSMENT OF SAFETY ............................................................................. 31 
12.1 
Toxicity Scale .................................................................................................... 31 
12.2 
Recording/Documentation ................................................................................. 32 
12.3 
Definitions .......................................................................................................... 33 
12.4 
Reporting Procedures to the NIAID IRB ............................................................. 35 
12.4.1 
Special reporting situations .................................................................... 35 
12.4.2 
Expedited reporting to the NIAID IRB ..................................................... 36 
12.4.3 
Waiver of reporting anticipated protocol deviations, and expected 
UPnonAEs to the NIAID IRB .................................................................. 36 
12.4.4 
Annual reporting to the NIAID IRB .......................................................... 36 
12.5 
Pregnancy ......................................................................................................... 36 
12.6 
Type and Duration of the Follow-up of Participants after AEs ............................ 36 
12.7 
Pausing Rules for an Individual Subject ............................................................. 37 
12.7.1 
Reporting a pause .................................................................................. 37 
12.7.2 
Resumption of a paused study ............................................................... 37 
12.8 
Halting Rules for the Protocol ............................................................................ 37 
12.8.1 
Reporting a study halt ............................................................................ 38 
12.8.2 
Resumption of a halted study ................................................................. 38 
12.9 
Premature Withdrawal of a Participant ............................................................... 38 
12.10 Replacement of Withdrawn Participants or Participants Who Discontinue Study 
Intervention ........................................................................................................ 38 
13 
CLINICAL MONITORING STRUCTURE ............................................................ 39 
13.1 
Quality Management Plan .................................................................................. 39 
13.2 
Safety Monitoring Plan ....................................................................................... 39 
14 
STATISTICAL CONSIDERATIONS ................................................................... 39 
14.1 
Study Hypothesis ............................................................................................... 39 
14.2 
Sample Size Justification and Analysis Plan ...................................................... 40 
15 
ETHICS/PROTECTION OF HUMAN PARTICIPANTS ....................................... 40 
15.1 
Informed Consent Process ................................................................................ 40 
15.1.1 
Non-English–speaking participants ........................................................ 41 
15.2 
Participant Confidentiality .................................................................................. 41 
16 
DATA HANDLING AND RECORD KEEPING .................................................... 42 
16.1 
Data Capture and Management ......................................................................... 42 
16.2 
Record Retention ............................................................................................... 42 
SCIENTIFIC REFERENCES ......................................................................................... 43 
 
 
 

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LIST OF TABLES 
Table 1. Sample size estimate per cohort, scheduling group, and vector. ..................... 16 
Table 2. Estimated participant remuneration. ................................................................ 31 
 
 
LIST OF FIGURES 
Figure 1. Parasite burden in mice after exposure to infected sand flies. ........................ 14 
Figure 2. Histology time course after exposure to infected sand flies. ........................... 14 
 
 
LIST OF APPENDICES 
Appendix A: Schedule of Procedures/Evaluations ......................................................... 45 
Appendix B: Blood Volumes for Specimen Collection .................................................... 46 
 

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LIST OF ABBREVIATIONS 
AE 
 
adverse event 
CC 
 
Clinical Center 
cDNA  
complementary DNA 
CFR 
 
Code of Federal Regulations 
CRIMSON 
Clinical Research Information Management System of the NIAID 
ELISA  
enzyme-linked immunosorbent assay 
FDA 
 
Food and Drug Administration 
FDR 
 
false discovery rate 
GCP 
 
Good Clinical Practice 
GLM 
 
general linear modelling 
HRPP  
Human Research Protection Program 
ICH 
 
International Council on Harmonisation 
INR 
 
international normalized ratio 
IRB 
 
institutional review board 
LMVR  
Laboratory of Malaria and Vector Research 
NIAID  
National Institute of Allergy and Infectious Diseases  
NIH 
 
National Institutes of Health 
PBMC  
peripheral blood mononuclear cell 
PI 
 
principal investigator 
RNA 
 
ribonucleic acid 
RNAseq 
RNA sequencing 
SAE 
 
Serious Adverse Event 
SCSU   
Special Clinical Studies Unit 
SUSAR 
suspected unexpected serious adverse reaction 
UP 
 
unanticipated problem 
UPnonAE 
unanticipated problem that is not an AE 

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PROTOCOL SUMMARY 
Full Title:  
Defining Skin Immunity of a Bite of Key Insect Vectors in 
Humans 
Short Title: 
Skin immunity to vector bites 
Sample Size: 
N=90 participants (Cohort A n=45; Cohort B n=45) 
Accrual Ceiling: 
N=140 participants  
Study Population: 
 
Healthy volunteers 18 to 64 years of age 
Accrual Period: 
 
2 years  
 
Study Duration: 
Start Date: May 2018 
 
End Date: May 2020 
Length of individual subject participation is 7 days for 
Cohort A or 51 days for Cohort B. 
Study Design: 
 
This is a single-center, case-control study characterizing 
the innate immune response of human skin to insect bites. 
Participants will undergo vector feeding and have a skin 
biopsy taken from the site of vector bites (case) and from 
unbitten skin (control). Participants will be assigned to 
groups that differ by type of vector and number of 
feedings. Additionally, blood will be collected after selected 
feedings to evaluate systemic immune response. 
Study Intervention  
Description: 
 
Controlled feeding by 1 of 3 colony-reared vectors (Aedes 
aegypti mosquitoes, Anopheles gambiae mosquitoes, or 
Lutzomyia longipalpis sand flies). Participants will undergo 
either 1 feeding (Cohort A) or 4 feedings, each about 2 
weeks apart (Cohort B), with the same vector type. 
Primary Objectives:  
The primary objectives of the study are: 
1. To compare the early innate immune response in the 
skin of bitten (case) versus unbitten (control) skin for 
each of the three vector groups (Aedes, Anopheles, 
sand flies). 
2. To chracterize the local skin adaptive immune 
response after multiple exposures over time to bites of 
each of the three vector groups (Aedes, Anopheles, 
sand flies). 
Secondary Objectives: 
The secondary objectives of this study are:  
1. To analyze systemic immune response to vector 
salivary proteins in vector-naïve participants in each 
group (Aedes, Anopheles, sand flies). 

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2. To analyze systemic immune response to vector 
salivary proteins in vector-experienced participants in 
each group (Aedes, Anopheles, sand flies). 
Exploratory Objective: 
The exploratory objective of this study is: 
1. To evaluate gene expression after selected vector 
feedings to assess for reliable biomarkers of exposure. 
Primary Endpoints: 
1. Measurement of changes in the early innate immune 
response and cellular recruitment in the skin of bitten 
versus unbitten skin in each vector group in Cohort A 
by: 
a. immunohistochemistry of target proteins in 
vector-naïve participants for each of the three 
vector groups at 30 minutes and 4 hours after 1 
feeding/exposure. 
b. Transcriptomics via deep sequencing and/or gene 
expression array of complementary DNA (cDNA) 
libraries made from skin ribonucleic acid (RNA) of 
vector-naïve participants for each of the three 
vector groups at 30 minutes and 4 hours after 1 
feeding/exposure. 
2. Measurement of changes in the adaptive immune 
response and cellular recruitment in the skin of bitten 
versus unbitten skin after the fourth and final feeding in 
each vector group in Cohort B by: 
a. immunohistochemistry of target proteins in 
vector-experienced participants for each of the 
three vector groups at 4 hours and 48 hours after 4 
feedings/exposures. 
b. Transcriptomics via deep sequencing and/or gene 
expression array of cDNA libraries made from skin 
RNA of vector-experienced participants for each of 
the three vector groups at 4 hours and 48 hours 
after 4 feedings/exposures. 
Secondary Endpoints:  
1. Flow cytometry analysis of peripheral blood 
mononuclear cells (PBMCs) and cytokine analysis of 
plasma collected 24 hours after the one feeding in 
each vector group of vector-naïve participants in 
Cohort A. 

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2. Flow cytometry analysis of PBMCs and cytokine 
analysis of plasma collected 48 hours after the fourth 
and final feeding in each vector group of 
vector-experienced participants in Cohort B. 
Exploratory Endpoint: 
1. Comparison of gene expression of circulating white 
blood cells after the one feeding in each vector group 
of vector-naïve participants in Cohort A. 
2. Comparison of gene expression of circulating white 
blood cells after the fourth and final feeding in each 
vector group of vector-experienced participants in 
Cohort B. 
 
 

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Précis 
Vector-borne diseases continue to cause significant morbidity and mortality worldwide 
despite ongoing control efforts. Vectors like sand flies and mosquitoes deliver the 
pathogen into the skin of humans while taking a blood meal. Most vaccines under 
development ignore the importance of the complex infectious inoculum delivered by the 
vector and the local immune response that occurs at the site of the bite. In addition, 
many preclinical studies are carried out in animal models that do not replicate the natural 
route of infection, transmission by vector bites, and often bypass the skin interface 
altogether. As such, these studies do not evaluate what role the vector plays in the 
initiation of these infections. Further compounding this problem, many clinical studies are 
performed in naïve individuals who have never been exposed to the vector, while those 
living in endemic areas will have had long-term exposure to vectors through uninfected 
bites.  
 
A cumulative body of evidence from animal models demonstrates that a variety of 
vector-derived components are co-delivered with the pathogen and may play an 
important role in the establishment of infection. There is limited knowledge of the effect 
of these vector-derived factors on the immune response in human skin and their 
potential impact on infection establishment.  
 
In this protocol, we will examine the early skin immune response to bites of three 
arthropods: the mosquito Aedes aegypti, the vector of Zika, dengue, and chikungunya 
viruses; the mosquito Anopheles gambiae, the vector of malaria; and the sand fly 
Lutzomyia longipalpis, the vector of leishmaniasis. We will also explore how multiple 
vector bite exposures over time modulate future immune response at the bite site. 
Healthy participants will come to the National Institutes of Health (NIH) and undergo 
feeding by one of the three vectors, then have three skin punch biopsies performed by 
trained medical practitioners to evaluate local immune response. Participants in Cohort 
A will have one feeding; participants in Cohort B will have 4 feedings, each 2 weeks 
apart. Biopsies will be collected after the final feeding. Blood will be collected after the 
one feeding in Cohort A and after the fourth and final feeding in Cohort B to assess 
systemic immune response. 
 
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. 

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1 
Background Information and Scientific Rationale 
1.1 
Background Information 
Arthropod-borne diseases are emerging and re-emerging globally at a rapid pace [1]. 
Malaria, leishmaniasis, and dengue cause significant morbidity and mortality in many 
parts of the world, and the emergence of epidemics of Zika, chikungunya, and West Nile 
viruses show how quickly these diseases can spread [2]–[4]. Studies of pathogenesis 
and vaccine development for these diseases have mostly focused on the pathogens 
themselves, and little attention has been given to the role of their vectors. Many of these 
vectors deliver the pathogens into the skin of humans while taking a blood meal, and a 
cumulative body of evidence from animal models demonstrates that in the process of 
delivering a pathogen, a variety of vector-derived components are co-delivered that may 
play an important role in the establishment of infection [5]–[7].  
 
The majority of vaccines developed against vector-borne diseases have not been 
effective in humans despite often showing some effect in certain animal models. Most 
developed vaccines ignore the importance of the complex infectious inoculum delivered 
by the vector and the local immune response that occurs at the bite site. In addition, 
many preclinical studies are carried out in animal models that do not replicate natural 
infection and do not evaluate what role the vector plays in these infections. As a 
consequence, many vaccine candidates fail during development. For example, 
Leishmania candidate vaccines have demonstrated protection in animals challenged 
artificially by introducing the parasite via a needle but not when challenged naturally by 
bites of infected sand flies [8].  
 
We lack a clear understanding of the immune response in skin after insect bites and how 
that response affects disease initiation and efficacy of vaccines against these important 
pathogens. Furthermore, many clinical studies are performed in naïve individuals who 
have never been exposed to the vector, while people living in endemic areas will have 
long-term exposure to these vectors and different immune response to subsequent bites, 
which could interfere with vaccine effectiveness. These are likely important factors in 
why many of these vaccines fail once they move on to clinical trials. 
 
In this protocol, we propose to perform a careful examination of the early skin immune 
response to bites of three arthropods: the mosquito Aedes aegypti, the vector of Zika, 
dengue, and chikungunya viruses; the mosquito Anopheles gambiae, the vector of 
malaria; and the sand fly Lutzomyia longipalpis, the vector of leishmaniasis. We will also 
explore how multiple vector bite exposures over time modulate future immune response 
at the bite site.  
        
                                                                              

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1.2 
Scientific Rationale 
Vector saliva contains a range of molecules including vasodilators, anticoagulants and 
immunomodulators whose purpose is to prevent the disruption of feeding by the host [9]. 
Vector saliva has been shown to inhibit T- and B-cell proliferation and downregulate the 
expression of interferon gamma (IFN-γ), a soluble T helper cell type 1 (Th1) pro-
inflammatory immune mediator known to have in vivo therapeutic and prophylactic 
effects against other arthropod-transmitted diseases such as leishmaniasis [10]–[12]. 
With time and repeated exposure, individuals become desensitized to mosquito bites 
due to a reduction in T helper cell type 2 (Th2) cytokine mediators, thus allowing for the 
more effective development of pro-inflammatory Th1 responses [10].  
 
When comparing the effect on parasite dissemination of needle infection vs. 
transmission of Leishmania donovani by sand fly bites to mice; mice only injected with 
virulent parasites demonstrated that the parasites did not visceralize to the spleens of 
the animals; in contrast, animals infected by the bite of a sand fly had spleen 
visceralization of the Leishmania parasite (Figure 1). 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 was also different. 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 (Figure 
2). 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 [13]. All these results strongly suggest that a vector bite 
(mosquitoes or sand flies) alters the skin environment, and consequently, the 
establishment of the parasite or virus delivered by the vector. Our objective is to 
translate these findings in animal models to human studies, as well as to gain a better 
understanding of the immune responses generated in the skin of humans and how this 
information can help us to design or improve test vaccines targeting vector-borne 
diseases. 
 

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Figure 1. Parasite burden in mice after exposure to infected sand flies.  
Parasite burden in individual mice spleens determined by serial dilution at 5 and 30 
weeks after exposure to 20 infected sand flies (IS) or intradermal injection of 10^5 
metacyclic Leishmania donovani parasites (LI). 
 
 
Figure 2. Histology time course after exposure to infected sand flies. 
Mice ears 3-18 hours after exposure to 20 infected (IS) or intradermal injection with 10^5 
metacyclic parasites (LI). Mice ear sections were stained with anti-Ly6G antibody. 
Pictures are representative of four samples per condition; scale bars indicate 20um. 
 
Concerning adaptive responses after multiple exposures to vector saliva, various groups 
have shown that saliva of insects or bites of an insect can alter the immune response in 
rodents, dogs, and non-human primates [13]–[15]. In rodent models, Teixeira et al. have 
shown that the cytokine and chemokine environment in the skin changes significantly 
when the rodent was previously exposed to bites of sand flies compared to naïve 
animals [16]. In dogs, Collin et al. have shown that multiple sand fly bites generate an 
adaptive immune response that can be recalled by a sand fly bite [14]. In non-human 
primates and in human subjects living in a sand fly-prevalent area, a skin immune 
response to bites of uninfected sand flies was observed [15], [17]. These data show that 

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bites of vectors can generate a specific systemic immune response to vector-derived 
factors; we aim to investigate the nature of the adaptive immune response to vector bites 
in the skin of humans. This may elucidate how the immune response may affect the 
efficacy of vector-based vaccines currently in clinical trials. 
 
This study will be the first to carefully examine the early immune response to bites of 
different vectors in humans and to explore how multiple exposures over time 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 that modulate the innate immune response in naïve 
individuals not previously exposed to vector bites (Cohort A) and recall a saliva-specific 
adaptive immune response to the bite site in previously exposed individuals (Cohort B), 
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 vector-derived 
factors play in the establishment of vector-borne infections in humans. These findings 
may be useful in determining why many current vaccine candidates fail as they move 
further into clinical development. Additionally, the findings will be essential in informing 
future vaccine design and clinical development for both well-established vector-borne 
diseases like malaria and dengue as well as emerging threats requiring the rapid 
development of countermeasures like Zika and chikungunya viruses. 
2 
Study Objectives 
2.1 
Primary Objectives 
The primary objectives of the study are: 
1. To compare the early innate immune response in the skin of bitten (case) 
versus unbitten (control) skin for each of the three vector groups (Aedes, 
Anopheles, sand flies). 
2. To chracterize the local skin adaptive immune response after multiple 
exposures over time to bites of each of the three vector groups (Aedes, 
Anopheles, sand flies). 
2.2 
Secondary Objectives 
The secondary objectives of this study are:  
1. To analyze systemic immune response to vector salivary proteins in 
vector-naïve participants in each group (Aedes, Anopheles, sand flies). 
2. To analyze systemic immune response to vector salivary proteins in 
vector-experienced participants in each group (Aedes, Anopheles, sand flies). 
2.3 
Exploratory Objective: 
 
The exploratory objective of this study is: 

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1. To evaluate gene expression after selected vector feedings to assess for 
reliable biomarkers of exposure. 
3 
Study Design 
3.1 
Description of the Study Design 
This is a single-center, case-control study of healthy skin immune response to bites of 
three species of colony-reared arthropods not infected with any pathogen. Healthy 
participants will undergo vector feeding with one of the three types of vectors as 
described in Table 1. Participants will be assigned to a vector in a 1:1:1 ratio. 
Additionally, participants will be assigned to a cohort: Cohort A will undergo a single 
feeding and Cohort B will undergo 4 feedings, each about 2 weeks apart.  
 
Table 1. Sample size estimate per cohort and vector group.  
Vector 
Cohort A 
(Vector-Naïve) 
Cohort B 
(Vector-Experienced) 
Total 
Aedes 
aegypti 
15 
15 
30 
Anopheles 
gambiae 
15 
15 
30 
Lutzomyia 
longipalpis 
15 
15 
30 
Total 
45 
45 
90 
 
After the one vector feeding for Cohort A, participants will undergo skin punch biopsy at 
three sites: two at the sites of bites at 2 different time points (case) and one from 
unbitten skin (control). After the fourth and final vector feeding for Cohort B, participants 
will undergo skin punch biopsy at three sites: two at the sites of bites at 2 different time 
points (case), and one from unbitten skin (control). Skin biopsies will be used for 
research tests to evaluate local immune response to vector bites. Additionally, systemic 
immune response will be evaluated on blood collected after the one feeding for Cohort A 
and after the fourth and final feeding for Cohort B.  
 
3.2 
Study Endpoints  
3.2.1 
Primary endpoints  
1. Measurement of changes in the early innate immune response and cellular 
recruitment in the skin of bitten versus unbitten skin in each vector group in 
Cohort A by: 

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a. immunohistochemistry of target proteins in vector-naïve participants 
for each of the three vector groups at 30 minutes and 4 hours after 1 
feeding/exposure. Biopsies from eight participants within a group will 
be assigned for immunohistochemical analysis. 
b. Transcriptomics via deep sequencing and/or gene expression array of 
cDNA libraries made from skin RNA of vector-naïve participants for 
each of the three vector groups at 30 minutes and 4 hours after 1 
feeding/exposure. Biopsies from seven participants within a group will 
be assigned for transcriptomic analysis. 
2. Measurement of changes in the adaptive immune response and cellular 
recruitment in the skin of bitten versus unbitten skin after fourth and final feeding 
in each vector group in Cohort B by: 
a. immunohistochemistry of target proteins in vector-experienced 
participants for each of the three vector groups at 4 hours and 48 
hours after 4 feedings/exposures. Biopsies from eight participants 
within a group will be assigned for immunohistochemical analysis. 
b. Transcriptomics via deep sequencing and/or gene expression array of 
cDNA libraries made from skin RNA of vector-experienced 
participants for each of the three vector groups at 4 hours and 48 
hours after 4 feedings/exposures. Biopsies from seven participants 
within a group will be assigned for transcriptomic analysis. 
3.2.2 
Secondary endpoints  
1. Flow cytometry analysis of PBMCs and cytokine analysis of plasma collected 24 
hours after the one feeding in each vector group of vector-naïve participants in 
Cohort A. 
2. Flow cytometry analysis of PBMCs and cytokine analysis of plasma collected 48 
hours after the fourth and final feeding in each vector group of 
vector-experienced participants in Cohort B. 
3.2.3 
Exploratory endpoint 
1. Comparison of gene expression of circulating white blood cells after the one 
feeding in each vector group of vector-naïve participants in Cohort A. 
2. Comparison of gene expression of circulating white blood cells after the fourth 
and final feeding in each vector group of vector-experienced participants in 
Cohort B. 
4 
Study Population 
4.1 
Rationale for Participant Selection  
Participants will be carefully screened using the inclusion and exclusion criteria 
described here to select the optimum participants for completing the study objectives 

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and minimize the risk of adverse events (AEs). We will enroll up to 140 healthy adults in 
this protocol in order to fill all cohorts/groups. 
 
National Institutes of Health (NIH) employees and members of their immediate families 
may participate in this protocol. We will follow the Guidelines for the Inclusion of 
Employees in NIH Research Studies and will give each employee a copy of the “NIH 
Information Sheet on Employee Research Participation.”  
 
For NIH employees:  
• 
NIH staff may be a vulnerable class of participants. 
• 
Neither participation nor refusal to participate will have an effect, either beneficial 
or adverse, on the participant’s employment or work situation.  
• 
The NIH information sheet regarding NIH employee research participation will be 
distributed to all potential participants who are NIH employees.  
• 
The employee participant’s privacy and confidentiality will be preserved in 
accordance with NIH Clinical Center (CC) and National Institute of Allergy and 
Infectious Diseases (NIAID) policies, which define the scope and limitations of 
the protections.  
• 
For NIH employee participants, consent will be obtained by an individual 
independent of the employee’s team. Those in supervisory position to any 
employee and co-workers of the employee will not obtain consent.  
• 
The importance of maintaining confidentiality when obtaining potentially sensitive 
and private information from co-workers or subordinates will be reviewed with the 
study staff at least annually and more often if warranted.  
 
4.2 
Recruitment Plan 
Participants will be recruited through the screening study #11-I-0183 “Screening for LID 
Clinical Studies Unit Healthy Volunteer Protocols”. Individuals will be carefully screened 
and evaluated, and those who meet the study eligibility criteria will be contacted and 
given the opportunity to be enrolled into the study.  
 
If a potential participant has completed the screening study #11-I-0183 more than 90 
calendar days prior to Day 0 of this study, they will be asked to come to the NIH CC for 
another visit under the screening study #11-I-0183 within 90 days prior to Day 0. Under 
that study, they will repeat HIV testing and complete any laboratory or other testing 
deemed necessary by the investigator to ensure that the individual is still eligible to take 
part in this study. All eligible participants will be consented and enrolled for this study 
only after completion of all necessary screening studies within 90 days prior to the first 
vector feeding (Day 0) unless they have completed participation in Cohort A and are 
participating in Cohort B, in which case rescreening is not required.  
 

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If a participant in Cohort A would like to also participate in Cohort B for the same vector 
group after they have completed Cohort A, they may be allowed to be reenrolled and 
counted as a new participant in Cohort B. The participant will be consented again prior to 
enrollment in Cohort B. In addition, any participant in Cohort A or B can also reenroll to 
participate in Cohort A or B of a different vector if eligible. In that case, they will 
participate in all feeding and other procedures for that cohort/vector group as per 
protocol. The participant will be consented again prior to enrolling in a different cohort.   
4.3 
Inclusion Criteria 
Individuals must meet all of the following criteria to be eligible for study participation: 
1. Healthy women and men who are ≥18 and ≤64 years of age. 
2. Able to provide informed consent. 
3. Willingness to complete all study visits and comply with all study requirements. 
4. Willing to have samples stored for future research. 
5. A female is eligible for this study if she meets 1 of the following: 
• 
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). 
• 
Of childbearing potential but agrees to practice effective contraception or 
abstinence for 4 weeks prior to enrollment through the completion of the 
study. Acceptable methods of contraception include a male partner who is 
sterile and is the sole sexual partner of the female participant or a male 
partner who uses a condom with spermicide plus 1 or more of the 
following that is used by the female: 1) implants of levonorgestrel; 2) 
injectable progestogen; 3) an intrauterine device with a documented 
failure rate of <1%; 4) oral contraceptives; and 5) double barrier method 
including diaphragm. 
6. Agrees to not use scented lotions, deodorants, or topical creams on each feeding 
day. 
7. Agrees to not take aspirin or any other NSAID within 7 days of a biopsy. 
8. Agrees to not use topical steroid creams or ointments throughout the study 
without prior permission of Principal Investigator (PI). 
9. Vector-specific antibody enzyme-linked immunosorbent assay (ELISA) to one of 
the three vectors (the one to which the individual is assigned) is <2.5 standard 
deviations above the negative control for Cohort A only. 
 
4.4 
Exclusion Criteria  
Individuals meeting any of the following criteria will be excluded from study participation: 

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1. Any underlying or current medical condition that, in the opinion of the investigator, 
would interfere with participation in the study.  
2. Any participant that is HIV positive. 
3. A clinically significant (as determined by the PI) baseline Grade 1 or greater 
toxicity by the toxicity table.  
4. History of severe allergic reaction (including to mosquito or other insect bites) with 
generalized urticaria, angioedema, anaphylaxis, or anaphylactoid reaction. 
5. Prone to allergic responses and/or significant history of allergies, including 
seasonal or specific allergies as determined by the PI. 
6. Receipt of any investigational drug that is unlicensed within 3 months or 5.5 half-
lives (whichever is greater) prior to enrollment. 
7. Receipt of any unlicensed vaccine within 6 months prior to enrollment. 
8. Self-reported or known history of alcoholism or drug abuse within 6 months prior 
to enrollment, or positive urine test for drugs of abuse at screening (excluding 
positive test for tetrahydrocannabinol [THC] or its metabolites if usage is less than 
3 times per week).  
9. 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.  
10. Any use of medications that affect blood clotting within 3 months, history of 
abnormal blood clotting, or result outside of the normal laboratory range for 
measurements of prothrombin time (PT), partial thromboplastin time (PTT), or 
international normalized ratio (INR) that may suggest a problem with blood 
clotting. 
11. History of significant scarring 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. 
12. Pregnant or breastfeeding.  
 
Co-enrollment Guidelines: Co-enrollment in other trials is restricted, but may take 
place after consultation with the study staff and approval from the PI. 
4.5 
Justification for Exclusion of Special Populations 
Exclusion of Pregnant Women: In this study, participants will have multiple exposures 
to mosquito or sand fly bites. 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.  
 

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Exclusion of Children: This is a study of the adult immune response to insect bites. 
Therefore, children will be excluded from the study. 
5 
Study Intervention 
5.1 
Arthropod Feeding  
At the Laboratory of Malaria and Vector Research (LMVR), established state of the art 
insectaries for both mosquitoes and sand flies have been inspected and certified by the 
USDA as ACL2 (arthropod containment level 2) insectaries. The sand fly colony of 
Lutzomyia longipalpis originates from Brazil and was initially established at the 
Laboratory of Parasitic Diseases and was brought to LMVR in 2004. The Aedes aegypti 
and Anopheles gambiae mosquitoes were first established in 1972 and 1973, 
respectively. The two colonies were brought to LMVR in 2004. The colonies have been 
raised in our insectaries since. The insectaries include temperature- and humidity-
controlled rooms as well as incubators with temperature, humidity and light cycle 
controls. We also 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 in incubators dedicated to mosquitoes for use in human studies, 
located in a separate room from the rest of the colony. Both sand flies and mosquitoes 
used in human studies never come into contact with human blood prior to their use.  
 
Colony-reared, clean female arthropods from each of three species, Aedes aegypti 
mosquitoes, Anopheles gambiae mosquitoes, or Lutzomyia longipalpis sand flies, will be 
bred in the insectary at the LMVR at the Twinbrook campus. In Cohort A, participants will 
undergo one feeding. In Cohort B, participants will undergo 4 feedings by the same 
vector. Participants will be asked to not use scented lotions, deodorants, or topical 
creams on each feeding day. 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 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. 
 
Each feeding will be conducted as follows: 
i. 
Assessment of the skin will be documented pre-feeding.  
ii. 
Depending on the cohort, starved female Aedes aegypti mosquitoes, 
Anopheles gambiae mosquitoes, or Lutzomyia longipalpis sand flies will 
be selected from a colony approved for human feeding studies in 
LMVR, NIAID.  
a. 5-10 female insects will be aspirated or placed into a secured 
feeding device prior to feeding and brought to the CC from the 
LMVR 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 

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the human participant at the end of exposure, where the abdomen of 
blood fed mosquitoes or sand flies will be distended and bright red, and 
clearly visibile 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 with 5-10 fresh insects once.  
b. If a repeat feed is required, the post-feeding biopsies and blood 
draws 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 or sand flies have fed, they will be brought back to 
the lab for evaluation and then disposal. 
 
After each feeding, participants may be offered standard treatment as needed to control 
reactions to vector bites. 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. Any necessary treatment to control bleeding, discomfort, or reduce scarring 
from the biopsy will also be offered. 
6 
Study Schedule 
This study will take place at the NIH CC. All aspects of the protocol will be carried out in 
accordance with NIH guidelines, and International Council on Harmonisation (ICH) Good 
Clinical Practice (GCP) involving human participant research. The study schedule is 
described below and in Appendix A: Schedule of Procedures/Evaluations. 
6.1 
Enrollment 
At the time of enrollment, participants will be assigned to one of two cohorts and one of 
three vector groups based on their history of exposure and vector-specific ELISA results 
performed during screening (for Cohort A only, see Section 4.3). Vector assignment will 
also consider participant availability, since not all vectors may be available for feeding on 
the same feeding day. Participants may be assigned to any of the vectors or groups until 
a group is full. At that point, participants will only be assigned to vectors and groups that 
have not been completely enrolled. If a participant is only eligible for a vector or group 
that is already completely enrolled, they will not continue with consent and enrollment.  
 
The research team will thoroughly discuss the informed consent form with each eligible 
individual and obtain consent prior to performing any study procedures. This process 
may take place within 90 days of or on Day 0 before any research procedures are 
performed.  

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6.2 
Cohort A 
Cohort A participants will undergo 1 vector feeding and 3 biopsy procedures, all on Day 
0. 
6.2.1 
Vector feeding and biopsy (Day 0) 
The vector feeding will take place in the NIH CC Special Clinical Studies Unit (SCSU) or 
other appropriate unit. The following procedures will be performed:  
• 
Review of medical/medication history. 
• 
Review of inclusion/exclusion criteria. 
• 
Documentation of usual responses to mosquito or other insect bites as none 
(0), mild (1), moderate (2), or severe (3). 
• 
Clinician exam/assessment. 
• 
Vital signs.  
• 
Urine pregnancy test (for women of childbearing potential; confirmed negative 
before feeding). 
• 
Blood draw for research labs (section 7.2, Appendix A). 
 
 
After these procedures, participants will undergo feeding with the assigned vector. 
Feeding and monitoring will be conducted as described in section 5.1.  
 
After the feeding, participants will undergo the following procedures:  
• 
3 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. 
• 
1 biopsy from unbitten skin on the opposite arm collected no 
earlier than 30 minutes and no later than 6 hours after the vector 
feeding is completed. 
• 
Biopsies will be placed immediately and no later than one minute 
after excision into pre-labeled Eppendorf tubes containing either a 
fixative (10% buffered formalin) or RNAlater for 
immunohistochemical or transcriptomic analyses, respectively.  
• 
Any other clinical tests that are medically indicated or appropriate to ensure 
the safety of the individual participant as determined by the PI. 
 
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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6.2.2 
24-Hour Post-Feeding Visit (Day 1 +1 day) 
All Cohort A participants will return for a 24-hour post-feeding visit. At this visit, the 
following procedures will be performed. 
• 
Vital Signs. 
• 
Clinician assessment of bites and biopsy sites. 
• 
Blood draw for research labs (section 7.2, Appendix A). 
 
6.2.3 
Final study contact (Day 7 ±3 days) 
All participants will be contacted by telephone about 1 week after Day 0 to ensure there 
are no issues after the feedings or biopsies or adverse events. 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 this phone call, Cohort A participation will be complete.  
6.3 
Cohort B 
Cohort B participants will undergo 4 vector feedings over about 8 weeks and 3 biopsy 
procedures after the fourth and final feeding.  
6.3.1 
Vector feeding and biopsy (Days 0, 14, 28, and 42, +7 days) 
All Cohort B participants will undergo feeding procedures on Days 0, 14 (+7 days), 28 
(+7 days), and 42 (+7 days) for a total of 4 feedings. Feedings will be approximately 2 
weeks apart with a minimum of 7 days in between feedings. These visits will occur in the 
SCSU, or other appropriate unit, and the following procedures will be performed:  
• 
Review of medical/medication history. 
• 
Review of inclusion/exclusion criteria. 
• 
Documentation of usual responses to mosquito bites as none (0), mild (1), 
moderate (2), or severe (3). (only performed on Day 0). 
• 
Clinician exam/assessment.  
• 
Vital signs.  
• 
Urine pregnancy test (for women of childbearing potential; confirmed negative 
before feeding). 
• 
Blood draw for research labs (section 7.2, Appendix A). 
 
 
After these procedures, participants will undergo feeding with the assigned vector. 
Feeding and monitoring will be conducted as described in section 5.1.  
 
After each feeding on Day 0, 14, and 28, participants will be monitored for a minimum of 
1 hour post feeding and the following procedures will be performed: 
• 
Any clinical tests that are medically indicated or appropriate to ensure the 
safety of the individual participant as deemed by the PI. 
• 
Participants will be asked to not apply any topical steroid treatments. Other 
topical treatments, such as antihistamines, may be applied as needed.  

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After the final feeding on Day 42, the following procedures will be performed: 
• 
2 skin punch biopsy collections as follows: 
• 
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. 
• 
1 biopsy from unbitten skin on the opposite arm collected no 
earlier than 30 minutes and no later than 6 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 deemed by the PI. 
• 
Participants will be asked to not apply any topical treatments after the fourth 
and final feeding. Other topical treatments may be applied after the Day 44 
biopsy is completed. 
 
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. 
6.3.2 
Follow-up and biopsy (Day 44 +3 days) 
About 2 days after the final feeding procedure, all participants in Cohort B will return to 
the NIH CC for a follow-up visit. The following procedures will be performed: 
• 
Vital signs.  
• 
Review of medical/medication history. 
• 
Clinician assessment of bite and biopsy sites. 
• 
1 skin punch biopsy from bitten skin at a minimum 40 hours after vector 
feeding is completed. 
• 
Blood draw for research labs (section 7.2, Appendix A). 
 
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. 
6.3.3 
Final study contact (Day 51 ±3 days) 
All Cohort B participants will be contacted by telephone about 1 week after Day 44 to 
ensure there are no issues after the feedings or biopsies or adverse events. 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 this phone call, Cohort B participation will be 
complete. 

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6.3.4 
Early termination visit 
• 
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 (section 7.2, Appendix A) if participant is 
willing. 
 
6.4. 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. 
 
 
7 
Study Procedures/Evaluations 
See Appendix A for schedule of clinical and study procedures and laboratory 
evaluations. Blood volumes are provided in a tabular form in Appendix B: Blood Volumes 
for Specimen Collection. 
7.1 
Study Procedures  
7.1.1 
Vital signs 
Participants must be sitting for a minimum of 5 minutes prior to these procedures being 
performed. Vital signs include blood pressure, mean arterial pressure, heart rate, 
respiratory rate, temperature, weight, and pulse oximetry. Height will be taken at the 
Day 0 visit only. On feeding days, vital signs will be performed pre-feeding. 
7.1.2 
Arthropod Feeding 
Please see Section 5.1 (Arthropod Feeding) 
7.1.3 
Skin biopsies 
From each participant, three 3-mm skin biopsies will be taken, two at the bite sites and 
one at an unbitten site on the opposite arm (control), using the following procedure: 
1. Participant will be placed in a relaxed position with their arm out to expose 
the insect bites or control site on the ventral side of the appropriate forearm. 
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. 

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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 or without 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 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. Sutures may 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.   
7.2 
Laboratory Evaluations 
  
7.2.1 
Safety laboratory evaluations 
 
For women of childbearing potential, a urine pregnancy test will be performed before 
each vector feeding procedure. 
 
7.2.2 
Laboratory testing of samples for study endpoints 
 
Primary Endpoints:  
• 
Immunohistochemistry of target proteins in vector-naïve participant skin 
biopsy samples for each of the three vector groups at 30 minutes and 4 hours 
after the one feeding. 
• 
Transcriptomics via deep sequencing and/or gene expression array of cDNA 
libraries made from skin RNA of vector-naïve participants for each of the 
three vector groups at 30 minutes and 4 hours after the one feeding. 

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• 
Immunohistochemistry of target proteins in vector-experienced participant 
skin biopsy samples for each of the three vector groups at 4 hours and 48 
hours after the fourth and final feeding. 
• 
Transcriptomics via deep sequencing and/or gene expression array of cDNA 
libraries made from skin RNA of vector-experienced participants for each of 
the three vector groups at 4 hours and 48 hours after the fourth and final 
feeding. 
Secondary Endpoints:  
• 
Flow cytometry analysis of PBMCs and cytokine analysis of plasma collected 
24 hours after the one feeding of in each vector group of vector-naïve 
participants. 
• 
Flow cytometry analysis of PBMCs and cytokine analysis of plasma collected 
48 hours after the fourth and final feeding in each vector group of vector-
experienced participants. 
Exploratory Endpoints: 
• 
Comparison of gene expression of circulating white blood cells after the one 
vector feeding for each vector group of vector-naïve participants. 
• 
Comparison of gene expression of circulating white blood cells after the 
fourth and final feeding in each vector group of vector-experienced 
participants. 
 
8 
Potential Risks and Benefits 
8.1 
Potential Risks 
8.1.1 
Risks of vector feeding  
Vector 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. Vector 
feeding or scratching after vector 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. 
8.1.2 
Risks of skin punch biopsy 
Risks of biopsy include local pain, bleeding, redness, infection, a scar and possible 
keloid formation. Oral or topical antibiotics and oral analgesics will be used to manage 
pain and infection as needed. Any non-routine complications resulting from the 

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procedure will be addressed in consultation with the NIH CC Dermatology service. 
Injection of local anesthetic may cause a minimal burning discomfort or bruising at the 
site of the needle puncture. 
8.1.3 
Risks of blood draw 
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 will be within the limits allowed for adult participants by the NIH CC (Medical 
Administrative Policy 95-9: Guidelines for Limits of Blood Drawn for Research Purposes 
in the Clinical Center:  
http://cc-internal.cc.nih.gov/policies/PDF/M95-9.pdf).  
8.2 
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. 
9 
Research Use of Stored Human Samples, Specimens, and Data 
Intended Use: Samples, specimens and data collected under this protocol may be used 
to study aspects of leishmaniasis, Zika virus infection, and other vector-borne diseases.  
Storage: All of the stored study research samples are labeled by a code that only the 
investigators can link to the participant. Samples are stored in a freezer within a secure 
laboratory with limited access. 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 a 
database located on password-protected computers, which will be maintained by the 
investigators and their designees.  
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. Before any sharing of samples, data, or clinical 
information, either institutional review board (IRB) approval must be obtained 
or the NIH Office of Human Subjects Research Protections (OHSRP) must 
determine that the research is exempt from IRB oversight. OHSRP can make 
this determination for some research where the samples or data have no 
personal identifying information about the study participant and the 
researcher is not able to ascertain it.  
• 
At the time of protocol termination, samples will either be destroyed, or after 
IRB 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 IRB approval, the data may be either destroyed or 
transferred to another repository. 

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Reporting the Loss or Destruction of Samples/Specimens/Data to the IRB: 
• 
Any loss or unanticipated destruction of samples or data (for example, due to 
freezer malfunction) that meets the definition of protocol deviation or 
unanticipated problem (UP), and/or compromises the scientific integrity of the 
data collected for the study will be reported to the NIAID IRB.  
• 
Additionally, participants may decide at any point not to have their samples 
stored. In this case, the PI will destroy all known remaining samples and 
report what was done to both the participant and to the IRB. This decision will 
not affect the individual’s participation in this protocol or any other protocols 
at NIH. 
10 Data Sharing Plan 
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.  
• 
Identified data in the Biomedical Translational Research Information System 
(BTRIS, automatic for activities in the CC). 
• 
De-identified or identified data with approved outside collaborators under 
appropriate agreements. 
• 
Data sharing may be complicated or limited in certain cases by contractual 
obligations or agreements with outside collaborators, such as cooperative 
research and development agreements (CRADA), clinical trial agreements 
(CTA), other restraints, etc.  
 
Data will be shared through: 
• 
BTRIS (automatic for activities in the Clinical Center). 
• 
Approved outside collaborators under appropriate individual agreements. 
• 
Publication and/or public presentations. 
 
Data will be shared at the time of publication or shortly thereafter. 
11 Remuneration Plan  
Participants will be compensated according to Table 2 below. Study visits will be 
compensated according to the number of visits the participant completes. Participants 
will only be compensated for the protocol visits and interim visits requested by the 
investigators if the visits are medically necessary. Remuneration will be provided to the 
participants in Cohort A after the completion of the day 7 phone call. Remuneration will 
be provided to participants in Cohort B two times: after the completion of Day 28 and the 
remainder of the compensation will be provided after the day 51 phone call. 
 
If a subject participates in Cohort A and Cohort B of the same vector, he/she will 
undergo Day 0 Feeding only once as part of Cohort A (which will also count for Day 0 
Feeding for Cohort B). Therefore, these participants will not be compensated for Day 0 
of Cohort B. They will receive compensation for the other protocol visits as described 
above and outlined below.  

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Table 2. Estimated participant remuneration. 
Cohort A 
Compensation 
Day 0: Vector feeding and biopsy 
$825 
Day 1: 24-hour post-feeding blood draw 
$100 
Expected total for completion of Cohort A study 
visit 
$925 
 
 
Cohort B 
 
Day 0: Vector Feeding 1 
$325 
Day 14: Vector Feeding 2 
$425 
Day 28: Vector Feeding 3 
$425 
Day 42: Vector Feeding 4 and biopsy 
$725 
Day 44: 48-hour post-feeding biopsy and blood draw 
$200 
Expected total for completion of ALL Cohort B 
study visits 
$2100 
 
 
Investigator-requested interim visits: 
$75 
12 Assessment of Safety 
12.1 
Toxicity Scale 
The investigator will grade the severity of each AE according to the U.S. Food and Drug 
Administration (FDA) “Toxicity Grading Scale for Healthy Adult and Adolescent 
Volunteers Enrolled in Preventive Vaccine Clinical Trials” September 2007, which can be 
found at: https://www.fda.gov/downloads/BiologicsBloodVaccines/ucm091977. 
 
Severity grading for clinical events that are not found in the FDA Healthy Volunteer 
Toxicity Table will be graded according to the following grading scale: 
• 
Grade 1 (Mild)  
Events causing no or minimal interference with daily activity. 
• 
Grade 2 (Moderate) 
 
Events causing greater than minimal interference with daily activity but not 
requiring medical intervention. 
• 
Grade 3 (Severe) 
 
Events causing inability to perform daily activity and/or requiring medical 
intervention. 
• 
Grade 4 (Potentially Life-Threatening)* 
 
Events causing inability to perform basic self-care functions OR medical or 
operative intervention indicated to prevent permanent impairment, persistent 
disability, or death. 
 
*Note: A severity assessment of “potentially life-threatening” is not necessarily the same 
as life-threatening as a “serious adverse event” (SAE) criterion. The latter means that 
the event is an immediate threat to life as opposed to a potential threat to life. 

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12.2 
Recording/Documentation 
At each contact with the participant, information regarding AEs will be elicited by 
appropriate questioning and examinations from consent to the last date on study. All 
events, both expected/unexpected and related/unrelated will be recorded on a source 
document. Source documents will include: progress notes, laboratory reports, consult 
notes, phone call summaries, survey tools, and data collection tools. Source documents 
will be reviewed in a timely manner by the research team. All reportable AEs that are 
identified will be recorded in the Clinical Research Information Management System of 
the NIAID (CRIMSON). The start date, the stop date, the severity of each reportable 
event, and the PI’s judgment of the AE’s relationship and expectedness to the study 
intervention will also be recorded in CRIMSON. 
 
All vital sign, physical exam, and laboratory abnormalities found prior to the first 
arthropod feeding will be documented as a baseline finding (not an AE) and will be 
assessed for clinical significance. The participant will be reassessed for inclusion or 
exclusion to undergo each feeding. After the first arthropod feeding, all new gradable 
abnormalities not found at baseline will be reported as AEs.  
 
Causality (likelihood that the event is caused by the study intervention) will be assessed 
considering the factors listed under the following categories: 
 
Definitely Related 
• 
Reasonable temporal relationship 
• 
Follows a known response pattern 
• 
Clear evidence to suggest a causal relationship 
• 
There is no alternative etiology 
 
Probably Related 
• 
Reasonable temporal relationship 
• 
Follows a suspected response pattern (based on similar products) 
• 
No evidence of a more likely alternative etiology 
 
Possibly Related 
• 
Reasonable temporal relationship 
• 
Little evidence for a more likely alternative etiology 
 
Unlikely Related 
• 
Does not have a reasonable temporal relationship  
OR 
• 
Good evidence for a more likely alternative etiology 
 
Not Related 
• 
Does not have a temporal relationship  
OR 

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• 
Definitely due to an alternative etiology 
 
Note: Other factors should also be considered for each causality category when 
appropriate. Causality assessment is based on available information at the time of the 
assessment of the AE. The investigator may revise the causality assessment as 
additional information becomes available. 
12.3 
Definitions 
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 be not be recorded as adverse events in 
CRIMSON per protocol, unless deemed by the PI to be abnormal or greater than Grade 
2. 
 
Feeding: 
• 
Local swelling 
• 
Pruritis 
• 
Erythema 
Biopsy: 
• 
Local swelling 
• 
Erythema 
• 
Bleeding at biopsy site 
• 
Scar at biopsy site 
 
Reactions should be graded in the following manner: 
 
Grade 1 
Mild transient reaction; interruption not indicated; intervention not 
indicated. 
Grade 2 
 
Interruption indicated but responds promptly to symptomatic treatment 
(e.g., antihistamines, nonsteroidal anti-inflammatory drugs, narcotics, 
intravenous fluids); prophylactic medications indicated for less than 24 
hours. 
Grade 3 
Prolonged (e.g., not rapidly responsive to symptomatic medication 
and/or brief interruption of infusion); recurrence of symptoms following 
initial improvement; hospitalization indicated for clinical sequelae. 
Grade 4 
Life-threatening consequences; urgent intervention indicated. 
Grade 5 
Death. 

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Adverse Event  
Any untoward medical occurrence in a human subject, including any abnormal sign, 
symptom, or disease, temporally associated with the individual’s participation in 
research, whether or not considered related to the individual’s participation in the 
research.  
 
Serious Adverse Event (SAE) 
An SAE is an AE that results in 1 or more of the following outcomes: 
• 
Death 
• 
A life-threatening event (places the subject at immediate risk of death from 
the event as it occurred) 
• 
An inpatient hospitalization or prolongation of an existing hospitalization 
• 
A persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions  
• 
A congenital anomaly/birth defect 
• 
A medically important event* 
*Medical and scientific judgment should be exercised in deciding events that may not be 
immediately life threatening or result in death or hospitalization but may jeopardize the 
participant or may require intervention to prevent one of the other outcomes listed 
above. 
 
Protocol Deviation  
Any change, divergence, or departure from the IRB-approved study procedures in a 
research protocol. Protocol deviations are designated as serious or non-serious and 
further characterized as: 
• 
Those that occur because a member of the research team deviates from the 
protocol. 
• 
Those that are identified before they occur, but cannot be prevented. 
• 
Those that are discovered after they occur. 
 
Serious Protocol Deviation 
A deviation that meets the definition of an SAE or compromises the safety, welfare, or 
rights of subjects or others. 
 
Non-compliance  
The failure to comply with applicable NIH Human Research Protections Program 
(HRPP) policies, IRB requirements, or regulatory requirements for the protection of 
human subjects. Non-compliance is further characterized as: 
1. Serious: Non-compliance that 
a. Increases risks, or causes harm, to participants. 
b. Decreases potential benefits to participants. 
c. Compromises the integrity of the NIH HRPP. 
d. Invalidates the study data. 

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2. Continuing: Non-compliance that is recurring. 
3. Minor: Non-compliance that is neither serious nor continuing. 
 
Unanticipated Problem (UP) 
Any incident, experience, or outcome that meets all 3 of the following criteria: 
1. Unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research 
protocol and informed consent document, investigator’s brochure, or 
other study documents; and 
b. the characteristics of the participant population being studied;  
2. Related or possibly related to participation in the research;  
3. Suggests that the research places participants or others at a greater risk of 
harm (including physical, psychological, economic, or social harm) than was 
previously known or recognized.  
 
Serious UP 
A UP that meets the definition of a SAE or compromises the safety, welfare, or rights of 
subjects or others. 
 
UP that is not an AE (UPnonAE)  
A UP that does not fit the definition of an AE, but which may, in the opinion of the 
investigator, involve risk to the subject, affect others in the research study, or 
significantly impact the integrity of research data. These events may involve a greater 
risk of social or economic harm to subjects or others rather than physical/psychological 
harm. Such events would be considered non-serious UPs. Examples of a UPnonAE 
include a breach of confidentiality, accidental destruction of study records, or 
unaccounted-for study drug. 
12.4 
Reporting Procedures to the NIAID IRB 
12.4.1 
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 12.4.2 and 12.4.3. 

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12.4.2 
Expedited reporting to the NIAID IRB  
Serious and non-serious UPs, deaths, serious deviations, and serious or continuing 
non-compliance will be reported within 7 calendar days of investigator awareness. SAEs 
that are possibly, probably, or definitely related to the research will be reported to the 
NIAID IRB within one calendar day of site awareness, regardless of expectedness. 
12.4.3 
Waiver of reporting anticipated protocol deviations, and 
expected UPnonAEs to the NIAID IRB  
Anticipated deviations in the conduct of the protocol will not be reported to the IRB 
unless they occur at a rate greater than anticipated by the study team.. 
12.4.4 
Annual reporting to the NIAID IRB 
The following items will be reported to the NIAID IRB in summary at the time of 
continuing review: 
• 
Serious and non-serious UPs  
• 
SAEs that are possibly, probably, or definitely related to the research 
• 
SAEs that are not related to the research 
• 
All AEs, except expected AEs granted a waiver of reporting 
• 
Serious and non-serious protocol deviations  
• 
Serious, continuing, and minor non-compliance 
• 
Any trends or events which in the opinion of the investigator should be 
reported 
• 
A summary of accumulated safety data  
12.5 
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 IRB. 
• 
Advise research participant to notify the obstetrician of study participation and 
study intervention exposure. 
12.6 
Type and Duration of the Follow-up of Participants after AEs 
AEs that occur following enrollment of the participant (by signing the informed consent) 
will be followed until the final outcome is known or until the end of the 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 

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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 CRIMSON. 
 
SAEs that have not resolved by the end of the follow-up period will be followed until final 
outcome is known. If it is not possible to obtain a final outcome for an SAE (e.g., the 
participant is lost to follow-up), the reason a final outcome could not be obtained will be 
recorded by the investigator in CRIMSON.  
12.7 
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. 
12.7.1 
Reporting a pause 
If a pausing criterion is met, a description of the AE(s) or safety issue must be reported 
by the PI within 1 business day to the IRB by fax or email. 
12.7.2 
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 IRB of the decision on resumption of 
the study intervention. 
 
A subject who does not resume study interventions will continue to be followed for 
safety. 
12.8 
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: 

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• 
1 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 AE 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. 
 
12.8.1 
Reporting a study halt 
If a halting rule is met, a description of the AE(s) or safety issue must be reported by the 
PI within 1 business day to the IRB by fax or email. 
12.8.2 
Resumption of a halted study 
The PI will determine if it is safe to resume the study. The PI will notify the IRB of the 
decision on resumption of the study. 
 
Participants who do not resume study intervention will continue to be followed for safety. 
12.9 
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.) 
• 
Non-compliance with study procedures to the extent that it is potentially harmful 
to the participant or to the integrity of the study data (e.g., participant misses a 
feeding visit beyond visit window). 
• 
A change in the participant’s baseline condition after enrollment so that the 
participant no longer meets one or more of the inclusion/exclusion criteria. 
• 
The investigator determines that continued participation in the study would not be 
in the best interest of the participant. 
12.10 
Replacement of Withdrawn Participants or Participants Who 
Discontinue Study Intervention  
Participants who withdraw at any time before all biopsies may be replaced. If a 
participant is removed prior to the first 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.  

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13 Clinical Monitoring Structure 
13.1 
Quality Management Plan 
During the study, the PI and study team will be responsible for implementing a quality 
control plan. Additionally, the study team will be responsible for completing and 
submitting a summary report on the quality plan on a quarterly basis to the NIAID 
Clinical Director or designee. A courtesy copy will also be sent to Clinical Trials 
Management. 
13.2 
Safety Monitoring Plan 
The data generated during participation in this protocol will be monitored by the PI and 
the study team for safety and compliance with protocol-specific requirements. The PI 
and the study team will closely monitor and analyze study data as it becomes available 
and will take any steps necessary to ensure that safety is maximized and risks are 
minimized and that subject protections are appropriate and comply with applicable 
regulations and guidance. 
 
13.1 
Monitoring 
To help ensure that NIH/ ORSC Good Clinical Practices are being carried out, a Clinical 
Trials Management (CTM)/ RCHSPP designee on behalf of  OCRPRO will conduct a 
Study Initiation Visit (SIV) prior to study enrollment beginning. The purpose of this 
meeting is to review with the Principal Investigator (PI) and study team their roles and 
responsibilities, as well as their commitment to adhere to the requirements of the 
protocol, especially in terms of AE, SAE and UP reporting requirements 
(NIH/OHSRP).  In addition, the quality management and data management plans for the 
study will be reviewed.  
14 Statistical Considerations  
This study is highly exploratory as very little is known about skin immunity to bites of these 
vectors.  The simple characterization of this skin immunity will be important, as even with a 
small samples size we expect to be able to identify important aspects of the innate and adaptive 
immune response after a vector bite by examining the transcriptomic responses of differentially 
expressed genes.  Assessing the use of next generation transcriptomic and immunohistochemistry 
techniques, we have made estimates that will allow us to see useful differences between bittena 
dn unbitten skin. 
14.1 
Study Hypothesis 
The study hypothesis is that there will be differential transcriptomic gene expression and 
immunihistochemical differences between the cases (bitten skin) and controls (unbitten 
skin from the opposite arm) with regard to innate immune markers at Day 0 after the first 
bite of an insect in a relatively naïve individual.   We also exepect to see differential gene 
expression and immunohistochemical differences of adaptive immune markers between 
cases (bitten skin) biopsies collected on Day 42 and Day 44 (experienced cohort) as 
compared to controls (unbitten skin). 

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14.2 
Sample Size Justification and Analysis Plan 
Previous studies of the skin transcriptomes have estimated approximately 13,000 genes 
per biopsy of healthy individuals [18]. This large number of genes will be examined to 
find a smaller list of differentially expressed genes within cohorts. To analyze the data, 
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 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. FDR adjustment 
will be used for multiple testing correction. An FDR threshold of 0.1 for statistical 
significance is typically applied. Genes with larger differential expression will be defined 
with a log-fold-change threshold of 0.5 (i.e., 50% change between experimental 
conditions). 
 
Currently it is unknown how uniform skin immune responses to bites of these vector are 
and how much variability will be observed.  Therefore it is very difficult estimate an exact 
samples size.  As this study is very exploratory in nature it is likely with the relative small 
number of individuals in each cohort 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 15 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.   
 
15 Ethics/Protection of Human Participants 
15.1 
Informed Consent Process 
Informed consent is a process where information is presented to enable persons to 
voluntarily decide whether or not to participate as a research participant. It is an ongoing 
conversation between the human research participant and the researchers, which 
begins before consent is given and continues until the end of the participant’s 
involvement in the research. Discussions about the research will provide essential 
information about the study and include: purpose, duration, experimental procedures, 
alternatives, risks, and benefits. Participants will be given the opportunity to ask 
questions and have them answered.  
 

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The participants will sign the informed consent document prior to undergoing any 
research procedures. The participants may withdraw consent at any time throughout the 
course of the trial. A copy of the informed consent document will be given to the 
participants for their records. The researcher will document the signing of the consent 
form in the participant’s medical record. The rights and welfare of the participant will be 
protected by emphasizing to them that the quality of their medical care will not be 
adversely affected if they decline to participate or withdraw from this study.  
15.1.1 
Non-English–speaking participants 
If a non-English–speaking participant is unexpectedly eligible for enrollment, the 
participant will be provided with the CC Short Written Consent Form for Non-English–
Speaking Research Participants in the participant’s native language and a verbal 
explanation of the purpose, procedures, and risks of the study as described in NIH 
HRPP standard operating procedure 12, and 45 Code of Federal Regulations (CFR) 
46.117(b)(2). The IRB-approved English consent form will serve as basis for the verbal 
explanation of the study. The investigator will obtain an interpreter unless the 
investigator is fluent in the prospective participant’s language. Preferably, the interpreter 
will be someone who is independent of the participant (i.e., not a family member). 
Interpreters provided by the CC will be used whenever possible. The interpreters will 
interpret all oral communications (English to target language and conversely) between 
the investigator and a limited English-proficient participant, facilitate discussions, and 
clarify information as necessary. 
 
The IRB-approved English consent form will be signed by the investigator obtaining 
consent and a witness to the oral presentation. The CC Short Written Consent Form will 
be signed by the participant and a witness who observed the presentation of information. 
The interpreter may sign the consent document as the witness and, in this case, will note 
“Interpreter” under the signature line. A copy of both signed forms will be provided to the 
participant to take home. 
 
The investigator obtaining consent will document the consent process in the participant’s 
medical record (CRIMSON), including the name of the interpreter. Further, all instances 
of use of the CC Short Written Consent Form will be reported to the IRB at the time of 
annual review. If the CC Short Written Consent Form is used three times or more for the 
same language, this will be reported to the IRB immediately. 
15.2 
Participant Confidentiality 
All records will be kept confidential to the extent provided by federal, state, and local law. 
The study monitors and other authorized individuals may inspect all documents and 
records required to be maintained by the investigator, including but not limited to medical 
records. Records will be kept locked and all computer entry and networking programs 
will be done with coded numbers only. Clinical information will not be released without 
written permission of the participant, except as necessary for monitoring by the IRB, 
NIAID, and Office for Human Research Protections. 

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16 Data Handling and Record Keeping 
16.1 
Data Capture and Management 
Study data will be collected and maintained in CRIMSON and the Clinical Research 
Information System (CRIS) and collected directly from participants during study visits 
and telephone calls, or will be abstracted from participants’ medical records. Source 
documents include all recordings of observations or notations of clinical activities and all 
reports and records necessary to confirm the data abstracted for this study. Data entry 
into these systems will be performed by authorized individuals. The investigator is 
responsible for assuring that the data collected are complete, accurate, and recorded in 
a timely manner. 
16.2 
Record Retention 
The investigator is responsible for retaining all essential documents listed in the ICH 
GCP guidelines. Study records will be maintained by the PI according to the timelines 
specified in CFR 312.62 or a minimum of 5 to 7 years, and in compliance with 
institutional, IRB, state, and federal medical records retention requirements, whichever is 
longest. All stored records will be kept confidential to the extent required by federal, 
state, and local law.  

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[17] F. Oliveira et al., “Delayed-Type Hypersensitivity to Sand Fly Saliva in Humans 
from a Leishmaniasis-Endemic Area of Mali Is TH1-Mediated and Persists to 
Midlife,” J. Invest. Dermatol., vol. 133, no. 2, pp. 452–459, Feb. 2013. 
[18] P.-H. D. Edqvist et al., “Expression of Human Skin-Specific Genes Defined by 
Transcriptomics and Antibody-Based Profiling,” J. Histochem. Cytochem., vol. 63, 
no. 2, pp. 129–141, Feb. 2015. 
[19] Y. Guo, S. Zhao, C.-I. Li, Q. Sheng, and Y. Shyr, “RNAseqPS: A Web Tool for 
Estimating Sample Size and Power for RNAseq Experiment,” Cancer Inform., vol. 
13, no. Suppl 6, pp. 1–5, 2014. 

Skin immunity to vector bites 
Protocol Version 3.0 
February 1, 2019 
Page 45 of 46 
Appendix A: Schedule of Procedures/Evaluations 
Study Cohort 
Cohort A 
Cohort B 
Procedures/Evaluations 
Study Days (window) 
Day 0 
 
Day 1 
(+1 day) 
Day 7 
(±3 days) 
Day 0 
 
Day 14 
(+7 days) 
Day 28 
(+7 days) 
Day 42 
(+7 days) 
Day 44 
(+3 days) 
Day 51 
(±3 days) 
Informed Consent1 
X 
 
 
X 
 
 
 
 
 
Medical/Medication History 
X 
 
 
X 
X 
X 
X 
X 
 
Review of Eligibility Criteria 
X 
 
 
X 
X 
X 
X 
X 
 
Documentation of Usual Insect Bite Response 
X 
 
 
X 
 
 
 
 
 
Clinician Exam/Assessment 
X 
X 
 
X 
X 
X 
X 
X 
 
Vital Signs2 
X 
X 
 
X 
X 
X 
X 
X 
 
Urine Pregnancy Test3 
X 
 
 
X 
X 
X 
X 
 
 
Blood Draw 
X 
X 
 
X 
 
 
 
X 
 
Vector Feeding 
X 
 
 
X 
X 
X 
X 
 
 
Skin Punch Biopsy 
 X4 
 
 
 
 
 
 X5 
 X6 
 
Telephone Call to Assess Symptoms 
 
 
X 
 
 
 
 
 
X 
Skin Biopsy Laboratory Evaluations 
Skin Immunohistochemistry 
X 
 
 
 
 
 
X 
X 
 
Skin Transcriptomics 
X 
 
 
 
 
 
X 
X 
 
Blood Laboratory Evaluations 
Post-feeding Labs (PBMC flow cytometry, 
plasma cytokine analysis, gene expression of 
circulating white blood cells) 
 
X 
 
 
 
 
 
X 
 
Abbreviations: RNA, Ribonucleic acid; PBMC, peripheral blood mononuclear cell 
1Informed consent will be obtained within 90 days of or at Day 0 prior to any study procedures. Screening is performed under protocol 11-I-0183. 
2Participants must be sitting for a minimum of 5 minutes prior to these procedures being performed; vital signs include blood pressure, mean arterial 
pressure, heart rate, respiratory rate, temperature, weight, and pulse oximetry; height will be taken at the Day 0 visit only. On days with feeding, vital 
signs will be performed pre-feeding. 
3Females of childbearing potential only. Insect feeding will not be initiated until results are available. 
 4One biopsy from bitten skin will be collected no earlier than 30 minutes and no later than 90 minutes after the vector feeding is completed. One biopsy 
from bitten skin will be collected no earlier than 3.5 hours and no later than 4.5 hours after the vector feeding is completed. One biopsy will be collected 
from unbitten skin on the opposite arm no earlier than 30 minutes and no later than 6 hours after the vector feeding is completed. 
5 One biopsy from bitten skin will be collected no earlier than 3.5 hours and no later than 4.5 hours after the vector feeding is completed. One biopsy from 
unbitten skin on the opposite arm will be collected no earlier than 30 minutes and no later than 6 hours after the vector feeding is completed. 
6 One biopsy will be collected from bitten skin at a minimum 40 hours after vector feeding is completed. 

Skin immunity to vector bites 
Protocol Version 3.0 
February 1, 2019 
Page 46 of 46 
Appendix B: Blood Volumes for Specimen Collection 
 
Study Cohort 
Cohort A 
Cohort B 
Evaluations 
Study Days (window) 
Day 0 
 
Day 1 
(+1 day) 
Day 0 
 
Day 14 
(+7 days) 
Day 28 
(+7 days) 
Day 42 
(+7 days) 
Day 44 
(+3 days) 
Blood Volume in mL 
Post-Feeding Labs 
 
 
 
 
 
 
 
Whole blood 
(10 mL X 5) 
(10 mL x 5) 
(10 mL X 5) 
0 
0 
0 
(10 mL x 5) 
PAXGene tube 
(2.5 mL X 1) 
(2.5 mL x 1) 
(2.5 mL X 1) 
0 
0 
0 
(2.5 mL x 1) 
Daily Volume 
52.5mL 
52.5 mL 
52.5mL 
0 
0 
0 
52.5 mL 
Cumulative Volume 
52.5 mL 
105 mL 
52.5 mL 
0 
0 
0 
105 mL