Investigating Vector-Borne Determinants of Aedes Transmitted Arboviral Infections in Cambodia: An Observational Longitudinal Cohort Study in Children

Study Overview
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Brief Summary
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Background: Some mosquitos carry viruses that can cause disease. Some examples are dengue and Zika. The mosquitos spread disease by biting people and infecting them with the virus. Children, elderly people, and people who are already sick are especially likely to get infected. Researchers want to learn more to help make new medicines to treat these viral infections. Objective: To learn more about how mosquitos infect people, and why young children are more likely to get sick than other people. Eligibility: Healthy children 2-9 years old who live near the study site. This is Kampong Speu District Referral Hospital in Chbar Mon, Cambodia. Design: At visit 1, participants will have a physical exam. A small amount of blood will be taken from their arm or finger. Parents will answer questions about the participant s general health and medical history. Participants will come back to the study site every wet season and every dry season for the next 3 years. The visits will be the same as visit 1 and take about 1 hour. If at any time during the study the participant gets a fever and has other symptoms that could be caused by these viral diseases, they should be brought to the study site. These symptoms might include headache, pain behind the eyes, muscle pain, or joint pain. They can also include a rash that lasts longer than 12 hours. Participation ends after the final study visit in late 2021.

Detailed Description
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Mosquito-borne viruses continue to cause significant global morbidity and mortality, particularly in Southeast Asia. When mosquitoes deliver the virus into the skin of humans while probing for a blood meal, they deposit also saliva, which contains a myriad of pharmacologically active compounds that modulate the host immune system. Most vaccines against vector-borne diseases under development ignore the importance of the complex infectious inoculum delivered by the mosquito vector and the subsequent host immune response to mosquito salivary proteins. Many studies of vector-borne disease do not evaluate what role vector-derived factors play in the host immune response of these infections. A cumulative body of evidence from animal models and limited retrospective human data demonstrates that a variety of vector-derived components, including salivary components, are codelivered with the pathogen and may play an important role in the establishment and dissemination of arboviral infection. Knowledge of the effect of these vector-derived factors on the development of arboviruses in the human is limited. Here, we will establish and follow a longitudinal pediatric cohort study to describe the burden of dengue virus and to carefully examine the immune response to exposure of the salivary proteins of Aedes aegypti, the mosquito vector of dengue, Zika and chikungunya viruses. This study will serve as a foundation so that future studies may contribute to further understanding how saliva immunity impacts arboviral disease development i Cambodia, a country endemic to these viruses.

Official Title
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Investigating Vector-borne Determinants of Aedes-transmitted Arboviral Infections in Cambodia: An Observational Longitudinal Cohort Study in Children

Conditions
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Dengue Fever

Participation Criteria
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Eligibility Criteria
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INCLUSION CRITERIA: In order to be eligible to participate in this study, an individual must meet all of the following criteria: Provision of signed and dated informed consent form Stated willingness to comply with all study procedures and availability for the duration of the study Male or female, aged 2-9 years Live within approximately 5.5 km of study site In good general health as evidenced by medical history Willing to allow biological samples to be stored for future research. EXCLUSION CRITERIA: Current or prior use within last 6 months of any immunosuppression (e.g. intravenous immunoglobulin, steroids, interferon therapy) Treatment with another investigational drug, vaccine, or other intervention within six months of screening

Ages Eligible for Study
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Minimum Age: 2 Years
Maximum Age: 9 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| 1<br>Healthy children aged 2 - 9 years | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Prevalence of symptomatic and inapparent dengue infection (serotypes 1-4) as detected semiannually via ELISA assay (binary outcome present/absent) over a three-year period in Kampong Speu in children aged 2-9 years old | Detailed knowledge of dengue seroprevalence and transmission season variability will help establish an epidemiological foundation to prepare for larger future studies such as disease incidence studies or vector interventional trials. | Semi-Annual visits and sick/convalescent visits throughout study enrollment | 
| Prevalence of Aedes aegypti salivary gland homogenate reactivity as detected by ELISA assay (binary outcome present/absent) during wet and dry seasons over a three-year period in Kampong Speu in children aged 2-9 years old | Characterizing the Ae. aegypti salivary protein reactivity profile in Cambodians is the first step prior to assessing how Ae. aegypti saliva exposure modulates disease in humans. | Semi-annual visits and sick/convalescent visits throughout study enrollment | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Western blot analysis of sera from participants with strongest ELISA positivity to Ae. aegypti whole salivary gland homogenate compared to Anopheles and Culex to assess cross-reactive immunogenicity to mosquito saliva versus specific Aedes marke... | This next layer of analysis to identify specific immune dominant molecules, from a whole-protein approach, will provide the highestprobability of identifying novel proteins, irrespective of immune-modulatory function, that can then be characterized. It is essentialto assess reactivity to the saliva of Anopheles and Culex in order to exclude crossreactive antigens and select for Aedes-specific markers. | Semi-annual visits and sick/convalescent visits throughout study enrollment | 
| Positive RT-PCR result for diagnosis of dengue, chikungunya, and Zika viruses (or IgM capture ELISAs for dengue as needed) | Dengue is the predominant flavivirus, but other circulating arbovirusescomplicate the serologic diagnosis of prior immunity and it will be important to characterize this burden given the ease and little costadded to run the multiplex RT-PCR compared to a DENV RT-PCR alone. Depending upon local transmission patterns, this could informwhether additional serological assays (e.g. plaque reduction neutralization assays [PRNTs]) may be needed to assess ZIKV, CHIKV, and Culex-transmitted JEV seroprevalence. The study is not powered to detect incidence of these symptomatic Aedestransmitted disease because it would require thousands and thousands of children. | Semi-annual visits and sick/convalescent visits throughout study enrollment | 
| Geographic information system with all data components (mosquito catch sites, houses, schools) referenced by latitude and longitude in addition to a series of map layers (point maps, smoothed maps) to evaluate relationships between IgG intensity... | These data and analyses will allow for detailed spatiotemporal analysis of the data at individual, house and community levels. Similar published methods (except different entomological indices) were used for assessment of Anopheles salivary protein exposure at the Thai-Burma border for risk of malaria transmission. | Semi-annual visits and sick/convalescent visits throughout study enrollment | 
| Seroconversion to Ae. aegypti salivary homogenate in relationship to season (wet versus dry) and collected time-dependent variables defined as mean and maximum rainfall, temperature and humidity inaddition to monthly fingerpricks to evaluate ... | Assessing the persistence of anti-saliva antibody response in the dry season is critical to a biomonitoring strategy to inform future vector control programs in the area. The natural time course of salivary protein exposure, particularly in confirmed arboviral disease settings,has not been wellcharacterized. Retrospective repeat analyses of nondiseased samples have suggested that salivary IgG antibodies may last 30-40 days. 10 Seasonal variation and climate factors are implicated in both disease transmission and vector abundance. | Semi-annual visits and sick/convalescent visits throughout study enrollment | 
| Capture a minimum of 25 female Aedes aegypti mosquitoes for transcriptional comparison to LMVR-reared Aedes aegypti mosquitoes | Wild-caught Ae. aegypti may harbor considerable difference to inbred mosquito strains maintained in insectaries. Differences in salivary and midgut composition may help explain unique vector competence aspects and immunogenicity of the strains of Ae. aegypti in Cambodia. | Duration of study enrollment | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Mosquito Salivary Protein, Dengue Fever, Chikungunya, Kampong Speu, Natural History, Zika