International Research in Cambodia: Tropical Medicine and Vector Research

柬埔寨的国际研究：热带医学和媒介研究

基本信息

批准号：
10692122
负责人：
Jessica Manning
金额：
$ 73.56万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10692122
关键词：
9 year old Aedes Age-Years Animal Model Anti-Inflammatory Agents Antibodies Arbovirus Infections Arboviruses Area Arthropod Vectors Awareness Behavior Therapy Biodiversity Biological Assay Biological Markers Bite CD4 Positive T Lymphocytes CD8-Positive T-Lymphocytes Cambodia Cambodian Categories Cells Chikungunya fever Chikungunya virus Child Childhood Clinical Communities Complex Complex Mixtures Country Culicidae Data Dendritic Cells Dengue Dengue Infection Dengue Virus Deposition Dermal Development Diagnosis Diagnostic Disease Disease Outbreaks Distant Dose Endemic Diseases Enrollment Enzyme-Linked Immunosorbent Assay Epidemic Epidemiology Erythema Evaluation Exposure to Fever Frequencies Future Genomics Genotype Geography Goals HIV Home Hot Spot Hour Human Humoral Immunities IgG4 Immune Immune response Immune system Immunity Immunoglobulin G Immunologics Individual Induration Infection Interleukin-4 International Intervention Investigation Knowledge Laboratories Langerhans cell Laos Leptospirosis Malaria Mediating Metagenomics Modeling Molecular Weight Monitor Morbidity - disease rate Outcome Participant Pathogenesis Patients Pattern Persons Phylogenetic Analysis Plasmodium knowlesi Plasmodium vivax Play Population Proteins Protocols documentation Public Health Punch Biopsy Reaction Recombinant Proteins Reporting Research Resources Rickettsia Risk Role Rural Saliva Salivary Salivary Glands Salivary Proteins Sampling Scrub Typhus Seasons Serology Serotyping Skin Surveys System T cell response Testing Therapeutic Intervention Time Translating Tropical Medicine Undifferentiated United States National Institutes of Health Vaccines Validation Vector-transmitted infectious disease Viral Viral Genome Western Blotting Yellow fever virus Zoonoses adaptive immune response aged animal data base biomarker identification burden of illness chikungunya clinical center clinically relevant cohort cytokine detection method diagnostic algorithm disorder prevention epidemiological model feeding healthy volunteer i(19)immunopathology improved innovation macrophage microbial mortality mosquito-borne pathogen national surveillance neutralizing antibody next generation next generation sequencing novel novel marker pathogen peri-urban rapid test research clinical testing response risk stratification saliva mediated secondary infection seroconversion targeted biomarker transmission process urban area vaccine distribution vector vector control vector mosquito vector transmission vector-borne vector-borne pathogen volunteer zika fever

9 year old Aedes Age-Years Animal Model Anti-Inflammatory Agents Antibodies Arbovirus Infections Arboviruses Area Arthropod Vectors Awareness Behavior Therapy Biodiversity Biological Assay Biological Markers Bite CD4 Positive T Lymphocytes CD8-Positive T-Lymphocytes Cambodia Cambodian Categories Cells Chikungunya fever Chikungunya virus Child Childhood Clinical Communities Complex Complex Mixtures Country Culicidae Data Dendritic Cells Dengue Dengue Infection Dengue Virus Deposition Dermal Development Diagnosis Diagnostic Disease Disease Outbreaks Distant Dose Endemic Diseases Enrollment Enzyme-Linked Immunosorbent Assay Epidemic Epidemiology Erythema Evaluation Exposure to Fever Frequencies Future Genomics Genotype Geography Goals HIV Home Hot Spot Hour Human Humoral Immunities IgG4 Immune Immune response Immune system Immunity Immunoglobulin G Immunologics Individual Induration Infection Interleukin-4 International Intervention Investigation Knowledge Laboratories Langerhans cell Laos Leptospirosis Malaria Mediating Metagenomics Modeling Molecular Weight Monitor Morbidity - disease rate Outcome Participant Pathogenesis Patients Pattern Persons Phylogenetic Analysis Plasmodium knowlesi Plasmodium vivax Play Population Proteins Protocols documentation Public Health Punch Biopsy Reaction Recombinant Proteins Reporting Research Resources Rickettsia Risk Role Rural Saliva Salivary Salivary Glands Salivary Proteins Sampling Scrub Typhus Seasons Serology Serotyping Skin Surveys System T cell response Testing Therapeutic Intervention Time Translating Tropical Medicine Undifferentiated United States National Institutes of Health Vaccines Validation Vector-transmitted infectious disease Viral Viral Genome Western Blotting Yellow fever virus Zoonoses adaptive immune response aged animal data base biomarker identification burden of illness chikungunya clinical center clinically relevant cohort cytokine detection method diagnostic algorithm disorder prevention epidemiological model feeding healthy volunteer i(19)immunopathology improved innovation macrophage microbial mortality mosquito-borne pathogen national surveillance neutralizing antibody next generation next generation sequencing novel novel marker pathogen peri-urban rapid test research clinical testing response risk stratification saliva mediated secondary infection seroconversion targeted biomarker transmission process urban area vaccine distribution vector vector control vector mosquito vector transmission vector-borne vector-borne pathogen volunteer zika fever

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项目摘要

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.

多样化的媒介传播病原体景观是柬埔寨大多数发热疾病的原因。 2019年，我们实施了一条乡村病原体MNGS管道，以评估柬埔寨周上6个月至65岁的高热患者的临床样本（NIH 19-I-N109）。对前487例患者的MNGS分析确定载体传播病原体是导致发热疾病的最大临床类别。登革热病毒（DENV）是最丰富的，尽管在用于消除努力的快速测试中无法检测到的疟原虫也经常被发现。 MNGS对Chikungunya病毒的识别预示了更大的全国疫情，从而导致媒介控制干预措施，并将Chikungunya PCR添加到国家监测系统中。有趣的是，还检测到还检测到还没有检测到未充分的载体 - 载体和人畜共患病原体，例如疟原虫诺尔斯西，立克属，scrub typhus，钩端螺旋病和共同感染的HIV。我们证明，病原体无形的MNGS对高热患者的研究可以更好地识别和监测病原体的广度，从而改善了诊断算法。一种新型的DENV2基因型驱动了柬埔寨前所未有的2019年登革热流行，与地方性DENV1相比，地理分散的传播模式。为了更好地了解2019年登革热流行，我们采用了106个地理地理分析（从上面的发现）。我们发现了一种新型的DENV2基因型，在系统发育上与任何可用序列数据相距甚远，这可能是柬埔寨流行病的可能。此外，DENV2传输模式比DENV-1更加局部，这表明DENV1是流行的血清型和DENV-2作为引言。感染DENV2的大儿童，但可能先前存在的DENV-1免疫力，更有可能在给定的空间距离上共享传输链，这表明那些患有继发性感染的人病了，更有可能呆在家里（并感染其他人）。这种新型的DENV2基因型发现提出了有关病毒进化扩张和相关临床发病机理的重要问题。高水平的体液免疫对AE。埃及唾液蛋白与登革热柬埔寨儿童的登革热感染的未来发展有关，主要是登革热。为了更好地理解地方性宿主对AE的免疫反应。自2018年以来，我们开始了埃及蚊子唾液及其与疾病发展的关系，该小儿登革热同龄人在柬埔寨（NIH 18-I-N100）成立了2至9岁。我们半年度关注儿童，以获取抗体的登革热和AE中的蛋白质。使用酶联免疫吸附测定和登革热特异性中和滴度，埃及唾液腺匀浆。在任何时候，儿童出现发烧以接受登革热PCR。在基线时，有22％（173/770）的儿童对一种或多种登革热血清型具有中和抗体。到2020年4月，有51名儿童患有症状性登革热，而148名登革热儿童在DENV14中和测定法上通过血清转化定义了临床上的登革热。总IgG较高的儿童到AE。埃及唾液蛋白患有登革热感染的可能性要高1.5倍（HR 1.47 95％CI 1.052.06; P = 0.02），尤其是患有登革热的个体（HR 1.64 95％CI 1.122.41; P = 0.01）。唾液介导的免疫的主要同种型是IgG4，通常是响应IL-4而产生的，它是Th2-促促的细胞因子。这些发现在地理空间模型中进一步复制，以100m x 100m网格细胞中的剂量依赖性方式，唾液ELISA单元的登革热感染风险增加了29％。这些数据代表了与疾病终点相关的蚊子唾液抗体强度的第一个实例。鉴于患有临床上登革热的儿童具有更高水平的唾液特异性抗体，因此出现了一个新问题：唾液介导的免疫力是否在arboviral免疫病理学的复杂情况中起作用？柬埔寨志愿者的埃及埃及蚊子叮咬后的皮肤免疫特征是一致的，并且似乎由CD8+ T细胞主导，而不是CD4+ T细胞。为了了解唾液对临床相关的载体对人类皮肤微环境的艾德斯埃及埃及蚊的影响，我们招募了42位健康的柬埔寨参与者（NIH-I-053）接受受控的埃及埃及埃及埃及埃及埃及的未感染的蚊子的摄食。与正常皮肤（NSK）相比，在4小时内的不受欢迎的（对照）皮肤和咬伤皮肤的手术活检显示出兰格汉细胞，激活的真皮树突状细胞（CD1C+CD69+）的频率增加，激活了兰格汉细胞的频率增加。具有强大反应的个体（例如红斑，硬化）与蚊子唾液具有较高的活化的皮肤树突状细胞，兰格汉斯细胞和抗炎巨噬细胞的频率。喂养后48小时，与NSK相比，激活的CD8+ T细胞的频率增加了，尤其是那些具有牢固反应的NSK。主要在48h处观察到的T细胞响应是由CD8+ T细胞驱动的，并驱动。鉴定介导人皮肤中对蚊子唾液反应的关键细胞种群是理解艾德斯伊蚊传播的弧菌感染的基本步骤。鉴定暴露于伊蚊的生物标志物在确认我们队列中循环的疾病负担后，我们研究了半年度血清反应性，为AE的总IgG（同种型）。埃及唾液腺匀浆（SGH），在年轻的参与者和潮湿季节，这并不意外。当我们调查我们对AE反应性的队列时。通过ELISA和Western印迹，埃及SGH，我们通过分子量确定了五种免疫主导蛋白。我们以HEK293细胞表示这些选择的靶标，作为重组蛋白，并在我们的同类和NIH临床中心健康志愿者中在血清中进行了测试。正如预期的那样，我们看到可能暴露于柬埔寨血清的反应性，而不是NIH临床中心中心血清中的反应性，这表明这些蛋白质可能是暴露于AE的差异标志物。埃及。这些标记现在正在18-I-N100、19-I-N109和20-I-N054协议中进行进一步验证。