International Research in Cambodia: Tropical Medicine and Vector Research

National Institute Of Allergy And Infectious Diseases

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Abstract

A DIVERSE VECTOR-BORNE PATHOGEN LANDSCAPE IS RESPONSIBLE FOR MOST FEBRILE DISEASE IN CAMBODIA. In 2019, we implemented an in-country pathogen mNGS pipeline for evaluation of clinical samples from febrile patients aged 6 months to 65 years of age in peri-urban Cambodia (NIH 19-I-N109). mNGS analysis of the first 487 patients identified vector-borne pathogens as the largest clinical category responsible for febrile disease. Dengue virus (DENV) was the most abundant although Plasmodium vivax, undetectable on rapid tests used for elimination efforts, was also often identified. mNGS identification of chikungunya virus presaged a larger national outbreak, leading to vector control interventions and the addition of chikungunya PCR to the national surveillance system. Interestingly, underappreciated vector-borne and zoonotic pathogens such as Plasmodium knowlesi, Rickettsia spp., scrub typhus, leptospirosis, and co-infecting HIV were also detected. We demonstrated that pathogen-agnostic mNGS investigation of febrile persons can better identify and monitor the breadth of pathogens, enabling improved diagnostic algorithms. A NOVEL DENV2 GENOTYPE DROVE THE UNPRECEDENTED 2019 DENGUE EPIDEMIC IN CAMBODIA WITH GEOGRAPHICALLY DISPERSED TRANSMISSION PATTERNS IN COMPARISON TO ENDEMIC DENV1. To better understand the 2019 dengue epidemic, we employed phylogeographic analyses of 106 georeferenced DENV genomes (from above finding). We discovered a novel DENV2 genotype, phylogenetically distant from any available sequence data, as likely responsible for the Cambodian epidemic. Further, DENV2 transmission patterns were more intensely local than DENV-1, suggesting DENV1 as the endemic serotype and DENV-2 as an introduction. Older children infected with DENV2, but with likely pre-existing DENV-1 immunity, were more likely to share a transmission chain at a given spatial distance, suggesting those with secondary infections were sicker and more likely to stay at home (and infect others). This novel DENV2 genotype finding raises important questions about viral evolutionary expansion and associated clinical pathogenesis. HIGH LEVELS OF HUMORAL IMMUNITY TO AE. AEGYPTI SALIVARY PROTEINS ARE ASSOCIATED WITH FUTURE DEVELOPMENT OF DENGUE INFECTION, PRIMARILY INAPPARENT DENGUE, IN DENGUE-NAVE CAMBODIAN CHILDREN. In order to better understand endemic hosts immune response to Ae. aegypti mosquito saliva and its relationship to disease development, we initiated a pediatric dengue cohort of 771 children aged two to nine years old in Cambodia since 2018 (NIH 18-I-N100). We follow the children semi-annually for antibodies to dengue and to proteins in Ae. aegypti salivary gland homogenate using enzyme-linked immunosorbent assays and dengue-specific neutralization titers. At any time, children present with fever to undergo dengue PCR. At baseline, 22% (173/770) of children had neutralizing antibodies to one or more dengue serotypes. By April 2020, 51 children had symptomatic dengue while 148 dengue-nave children had clinically inapparent dengue defined by seroconversion on DENV14 neutralization assays. Children with higher total IgG to Ae. aegypti salivary proteins were 1.5x more likely to have dengue infection (HR 1.47 95% CI 1.052.06; p=0.02), particularly individuals with inapparent dengue (HR 1.64 95% CI 1.122.41; p=0.01). The primary isotype of saliva-mediated immunity here is IgG4, typically produced in response to IL-4, a Th2-predominant cytokine. These findings were further replicated in a geospatial model showing an increased risk of dengue infection by 29% per each increase in saliva ELISA unit in a dose-dependent manner in 100m x 100m grid cells. These data represent the first instance of mosquito salivary antibody intensity associated with a disease endpoint. Given that children with clinically inapparent dengue had higher levels of saliva-specific antibodies, a new question arises: does salivary-mediated immunity play a role in the complex picture of arboviral immunopathology? SKIN IMMUNE SIGNATURES AFTER AEDES AEGYPTI MOSQUITO BITES IN CAMBODIAN VOLUNTEERS ARE CONSISTENT AND APPEAR TO BE DOMINATED BY CD8+ T-CELLS AS OPPOSED TO CD4+ T-CELLS. To understand the immunological impact of saliva from a clinically relevant vector, Aedes aegypti mosquito, in the human skin microenvironment, we enrolled 42 healthy Cambodian participants (NIH-I-053) to undergo controlled Aedes aegypti uninfected mosquito feedings. Punch biopsies of unbitten (control) skin and of bitten (case) skin at 4 hours revealed an increased frequency of Langerhans cells, activated dermal dendritic cells, and activated M2 type macrophages (CD1c+CD69+) compared to normal skin (NSK). Individuals with robust reactions (e.g. erythema, induration) to mosquito saliva had higher frequencies of activated dermal dendritic cells, Langerhans cells, and anti-inflammatory macrophages. At 48 hours post-feeding, frequencies of activated CD8+ T cells were increased compared to NSK, particularly those with robust reactions. The T-cell response, primarily observed at 48h, is Th2-skewed and driven by CD8+ T cells. Identification of key cell populations that mediate the response to mosquito saliva in human skin is a fundamental step towards understanding arboviral infections transmitted by Aedes aegypti mosquitos. IDENTIFICATION OF BIOMARKERS OF EXPOSURE TO AEDES AEGYPTI MOSQUITO After confirming a sufficient burden of disease circulating in our cohort, we investigated semi-annual sero-reactivity as total IgG (isotypes pending) to Ae. aegypti salivary gland homogenate (SGH), which was notably higher in the younger participants and during wet season, which is not unexpected. When we surveyed our cohort for reactivity to Ae. aegypti SGH via ELISA and Western blot, we identified five immunodominant proteins by molecular weight. We produced these selected targets, expressed in HEK293 cells, as recombinant proteins and tested them in sera from our cohort and NIH Clinical Center healthy volunteers. As expected, we see reactivity in likely-exposed Cambodian sera but not NIH Clinical Center nave sera, suggesting that these proteins may serve as markers of differential exposure to Ae. aegypti. These markers are now undergoing further validation in 18-I-N100, 19-I-N109, and soon 20-I-N054 protocols.