Molecular approaches to understand vector-host and vector-pathogen interactions

了解载体-宿主和载体-病原体相互作用的分子方法

• 批准号: 10014101
• 负责人: Jesus Valenzuela
• 金额: $ 172.96万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 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/10014101
• 关键词: Acute; Adjuvant; Aedes; Age; Amino Acids; Animal Model; Animals; Anti-inflammatory; Antibodies; Antibody Response; Antigens; Arboviruses; Arthropods; Automobile Driving; Bacteria; Basic Science; Binding; Biochemical; Bioinformatics; Biological Assay; Biological Markers; Biological Process; Bite; Blood; C-terminal; C57BL/6 Mouse; Cambodia; Canis familiaris; Cells; Chikungunya virus; Child; Clinical Protocols; Code; Cohort Studies; Communities; Country; Crystallization; Culicidae; Cutaneous Leishmaniasis; Data; Dengue; Dengue Infection; Dengue Virus; Deposition; Detection; Development; Diagnostic tests; Disease; Disease Outcome; Ear; Eastern Africa; Endemic Diseases; Entomology; Enzyme-Linked Immunosorbent Assay; Ethiopia; Event; Exhibits; Exposure to; Foundations; Gel; Hemostatic Agents; Human; Human Volunteers; Immune; Immune response; Immune system; Immunity; Immunize; Immunoblotting; Immunoglobulin G; Immunologic Tests; Immunologics; In Vitro; Inbred BALB C Mice; Infection; Inflammatory Response; Insect Bites; Insect Vectors; Insecta; Institution; Interferons; Knowledge; Laboratories; Leishmania; Leishmania infantum; Leishmania major; Leishmaniasis; Lesion; Leukocytes; Leukotrienes; Ligands; Lutzomyia genus; Midgut; Modeling; Molecular; Molecular Biology; Monitor; Morbidity - disease rate; Mus; N-terminal; Names; Needles; Nodule; Nonprofit Organizations; Outcome; Parasites; Parasitic Diseases; Pathology; Pediatric cohort; Phlebotominae; Phlebotomus; Plasmids; Play; Predictive Value; Production; Property; Protein Family; Proteins; Protocols documentation; Province; Public Health; Recombinant Proteins; Research; Reverse Transcriptase Polymerase Chain Reaction; Rodent Model; Role; Saliva; Salivary; Salivary Glands; Salivary Proteins; Sand Flies; Seasons; Seroprevalences; Site; Skin; Source; Southeastern Asia; Specificity; Specimen; Structure; Sudan; System; T-Cell Activation; Tertiary Protein Structure; Testing; Thromboxanes; Time; Translating; Trypanosoma; Trypanosoma brucei brucei; Vaccinated; Vaccination; Vector-transmitted infectious disease; Virus; Virus Diseases; Visceral Leishmaniasis; Zika Virus; arbovirus disease; base; burden of illness; cDNA Library; cell mediated immune response; chikungunya; chronic infection; co-infection; curative treatments; cysteinyl-leukotriene; disorder control; exosome; exposed human population; follow-up; fundamental research; heme oxygenase-1; human data; immunogenic; in vivo; inflammatory milieu; interest; lymph nodes; macrophage; microbiome; molecular vector; mortality; mosquito-borne; neglect; nonhuman primate; pathogen; peri-urban; protective effect; recruit; response; seroconversion; success; transmission process; vaccine candidate; vector; vector mosquito; vector-borne; vector-borne pathogen

The accomplishments of the section are: 1. We discovered a new biological function of a salivary protein, named D7, from the saliva of sand flies. Despite the the notion that blood feeders from independent evolutionary lineages utilize different protein families for various salivary functions, the saliva of sand flies contains apparent homologs of the two-domain long-form D7 proteins of mosquitoes. The similarity is particularly strong in the predicted N-terminal domain of the protein where key amino acids lining the leukotriene/thromboxane binding pocket that interact with the ligand are conserved. We demonstrated, using binding studies and bioassays, that the sand fly long-form D7s do in fact bind both cysteinyl leukotrienes and thromboxanes, and thereby serve as anti-inflammatory and anti-hemostatic agents in both sandflies and mosquitoes. We also solved the crystal structure of the sand fly protein where we show the conserved N-terminal domain as well as the more divergent C-terminal domain and domain interface region related with the mosquito D7 forms and the evolutionary origins of the group as a whole. 2. We identified two sand fly salivary proteins as biomarkers for human exposure to the sand fly Phlebotomus orientalis, a vector of visceral Leishmaniasis in Eastern Africa. Recombinant proteins derived from sequence data on P. orientalis secreted salivary proteins cDNA library were produced using mammalian expression systems HEK293 cells and tested as antigens applicable for detection of anti-P. orientalis IgG in human sera. Human sera from Sudan and Ethiopia, countries endemic for visceral leishmaniasis, were screened by ELISA and immunoblotting to identify the potential markers of exposure to P. orientalis bites. Two recombinant proteins; mAG5 and mYEL1, were identified as the most promising antigens showing high correlation coefficients as well as good specificity in comparison to the whole sand fly salivary gland homogenate. Combination of both proteins led to a further increase of correlation coefficients as well as both positive and negative predictive values of P. orientalis exposure. 3. We demonstrated that co-infection with the parasite Trypanosoma brucei confers protection against cutanoues leishmaniasis. Infection with certain bacteria, parasites, and viruses alters the host immune system to Leishmania major influencing disease outcome. We determined the outcome of a chronic infection with Trypanosoma brucei brucei on cutaneous leishmaniasis (CL) caused by L. major. C57BL/6 mice infected with T. b. brucei were given a sub-curative treatment with diminazene aceturate then coinfected with L. major by vector bites. Our results revealed that infection with T. b. brucei controls CL pathology. Compared to controls, coinfected mice showed a significant decrease in lesion size up to 6 weeks post-infection and a significant decrease in parasite burden at 3 weeks post-infection. Protection against L. major resulted from a non-specific activation of T cells by trypanosomes. This induced a strong immune response characterized by IFN- production at the site of bites and systemically, creating a hostile inflammatory environment for L. major parasites and conferring protection from CL. 4. We identified a sand fly salivary vaccine candidate from the sand fly Phlebotomus sergenti. We tested the immune response in BALB/c mice to 14 different plasmids coding for the most abundant salivary proteins of Ph. sergenti. The plasmid coding for the salivary protein PsSP9 induced a DTH response in the presence of a significant increase of IFN- expression in draining lymph nodes (dLN) as compared to control plasmid and no detectable PsSP9 antibody response. Animals immunized with whole Ph. sergenti SGH developed only a saliva-specific antibody response and no DTH response. Mice immunized with whole Ph. sergenti saliva and challenged intradermally with L. tropica plus Ph. sergenti SGH in their ears, exhibited no protective effect. In contrast, PsSP9-immunized mice showed protection against L. tropica infection resulting in a reduction in nodule size, disease burden and parasite burden compared to controls. These results suggest that this family of proteins in Ph. sergenti, Ph. duboscqi and Ph. papatasi may have similar immunogenic and protective properties against different Leishmania species. Indeed, this anti-saliva immunity may act as an adjuvant to accelerate the cell-mediated immune response to co-administered Leishmania antigens, or even cause the activation of infected macrophages to remove parasites more efficiently. These findings highlight the idea of applying arthropod saliva components in vaccination approaches for diseases caused by vector-borne pathogens. 5. We demonstrated that saliva of the sand fly Lutzomyia longipalpis induces the expression of Hemooxygenase-1 in the skin of animals. Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24-48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections. 6. We developed a human protocol to study the immune response to mosquito bites and dengue cases in humans in Cambodia. Mosquito-borne arboviruses, like dengue virus, continue to cause significant global morbidity and mortality, particularly in Southeast Asia. Cumulative evidence from animal models and limited data from humans have identified various vector-derived components, including salivary components, that are co-delivered with the pathogen and play an important role in the dissemination of infection. Much about the roles and effects of these vector-derived factors remain to be discovered. We developed a longitudinal, pagoda (community)-based pediatric cohort study to evaluate the burden of dengue virus infection and document the immune responses to salivary proteins of Aedes aegypti, the mosquito vector of dengue, Zika, and chikungunya viruses. The study includes community-based seroprevalence assessments in the peri-urban town of Chbar Mon in Kampong Speu Province, Cambodia. The study aims to recruit 771 children between the ages of 2 and 9 years for a three year period of longitudinal follow-up, including twice per year (rainy and dry season) serosurveillance for dengue seroconversion and Ae. aegypti salivary gland homogenate antibody intensity determinations by ELISA assays. Diagnostic tests for acute dengue, Zika and chikungunya viral infections will be performed by RT-PCR. This study will serve as a foundation for further understanding of mosquito saliva immunity and its impact on Aedes-transmitted arboviral diseases endemic to Cambodia.

本节的成就是： 1。我们从沙蝇的唾液中发现了一种名为D7的唾液蛋白的新生物学功能。 尽管从独立进化谱系的血液饲养者利用不同的蛋白质家族来提高各种唾液功能，但沙蝇的唾液包含蚊子两层长形的D7蛋白的明显同源物。在蛋白质的预测的N末端结构域中，相似性尤其强，其中与配体相互作用的白细胞/血栓烷结合口袋内衬的关键氨基酸是保守的。我们使用结合研究和生物测定表明，沙子长形的D7实际上是结合了白细胞三烯和血栓烷的，因此在砂蝇和蚊子中充当抗炎和抗肿瘤剂。我们还解决了沙蝇蛋白的晶体结构，在该蛋白质中，我们显示了保守的N末端结构域以及与蚊子D7形式相关的更具不同的C末端结构域和域界面区域以及整个组的进化起源。 2。我们确定了两种沙蝇唾液蛋白是人类暴露于砂蝇植物外骨东方的生物标志物，这是东非内脏利什曼病的载体。 使用哺乳动物表达系统HEK293细胞产生了来自Orientalis分泌的唾液蛋白cDNA文库的序列数据的重组蛋白，并作为适用于检测抗P的抗原进行了测试。东方IgG在人类血清中。来自苏丹和埃塞俄比亚的人类血清，被ELISA和免疫印迹筛查了内脏利什曼病的特有国家，以识别暴露于Orientalis叮咬的潜在标志。两个重组蛋白；与整个沙蝇唾液腺匀浆相比，MAG5和Myel1被确定为最有前途的抗原，表现出高相关系数以及良好的特异性。两种蛋白质的组合都导致相关系数的进一步增加以及东方疟原虫暴露的正预测值和阴性预测值。 3。我们证明了与寄生虫锥虫的共同感染赋予了对Cutanoues Leishmanisis的保护。 某些细菌，寄生虫和病毒感染会改变宿主免疫系统，从而影响利什曼原虫的主要影响疾病结果。我们确定了Brucei brucei brucei慢性感染对L. Major引起的皮肤利什曼病（CL）的结果。 C57BL/6小鼠感染了T. b。给Brucei用乙二甲酸次尿酸盐进行了亚垂直治疗，然后通过载体叮咬与L. Major共同感染。我们的结果表明，T. b。感染。布鲁氏控制CL病理学。与对照组相比，共感染的小鼠显示感染后6周的病变大小显着降低，并且在感染后3周时，寄生虫负担显着减少。锥虫对T细胞的非特异性激活产生了对乳杆菌的保护。这引起了强烈的免疫反应，其特征是在叮咬部位和系统地产生IFN，从而为主要寄生虫创造了敌对的炎症环境，并赋予了CL的保护。 4。我们从沙蝇Sergenti鉴定出一种沙蝇唾液疫苗候选者。 我们测试了BALB/C小鼠中的免疫反应，以编码为塞金蒂ph。Sergenti的最丰富的唾液蛋白编码的14种不同的质粒。与对照质粒相比，唾液蛋白PSSP9编码的质粒在排水淋巴结（DLN）中显着增加IFN表达的质粒诱导了DTH反应。用全ph。SergentiSGH免疫的动物仅产生唾液特异性抗体反应，没有DTH反应。用整个Ph。Sergenti唾液免疫的小鼠在耳朵内用L. tropica和Sergenti sgh的皮内挑战，没有任何保护作用。相比之下，与对照组相比，PSSP9免疫的小鼠对防波北感染的保护表现出了防止结节大小，疾病负担和寄生虫负担的降低。这些结果表明，在Sergenti，Ph。Duboscqi和Ph。Papatasi博士学位中的蛋白质家族可能对不同的利什曼原虫物种具有相似的免疫原性和保护特性。实际上，这种抗甲硅烷免疫力可以充当辅助物，以加速细胞介导的免疫反应对共同辅助利什曼原虫抗原，甚至引起感染巨噬细胞的激活以更有效地去除寄生虫。这些发现突出了将节肢动物唾液成分应用于媒介传播病原体引起的疾病的疫苗接种方法的想法。 5。我们证明了沙蝇lutzomyia longipalpis的唾液会诱导动物皮肤中半加氧酶-1的表达。 沙蝇咬人哺乳动物宿主获得血液餐，推动宿主炎症反应的变化，以支持建立利什曼原虫感染。这种效果部分归因于沙蝇唾液的成分，这些唾液能够募集和激活白细胞。我们的小组表明，血红素加氧酶-1（HO-1）通过减少炎症反应而利用感染细胞中利什曼原虫的生存。在这里，我们表明暴露于沙蝇叮咬与HO-1在体内的诱导有关。从实验暴露于沙蝇的人类志愿者的皮肤标本的组织病理学分析表明，HO-1和NRF2是在皮肤的咬伤部位产生的。这些结果被概括为注入唾液腺索的小鼠（SG）或暴露于沙蝇咬伤的小鼠耳朵中，表明载体唾液可能是触发HO-1表达的关键因素。咬伤后24-48小时，居民皮肤巨噬细胞是主要来源HO-1。此外，咬伤后的体内和体外的测定均表明巨噬细胞产生的HO-1是NRF2依赖性的。总的来说，我们的数据表明，载体唾液会在咬合部位诱导早期HO-1产生，这代表了与建立自然传播的利什曼尼亚感染有关的重大事件。 6。我们制定了人类方案，以研究柬埔寨人类对蚊子叮咬和登革热病例的免疫反应。 蚊子 - 传播的arboviruses，例如登革热病毒，继续引起全球的显着发病率和死亡率，尤其是在东南亚。来自动物模型的累积证据和人类数据有限的数据确定了与病原体共同交付的各种载体衍生的成分，包括唾液成分，在感染传播中起着重要作用。关于这些向量衍生的因素的作用和影响的许多内容仍有待发现。我们开发了一项基于纵向的宝塔（社区）的小儿队列研究，以评估登革热病毒感染的负担，并记录对伊蚊伊蚊的免疫反应，埃及伊蚊的唾液蛋白，登革热，Zika和Chikungunnya病毒的蚊子媒介。该研究包括在柬埔寨坎ampong Speu省Chbar Mon的基于社区的血清阳性评估。该研究旨在招募771名2至9岁之间的儿童，进行三年的纵向随访，包括每年两次（雨季和干旱季节）血清监视登革热血清转化和AE。 EEGYPTI唾液腺匀浆抗体强度通过ELISA测定确定。 RT-PCR将对急性登革热，Zika和Chikungunya病毒感染进行诊断测试。这项研究将成为进一步了解蚊子免疫及其对柬埔寨特有的埃德斯转移弧病毒疾病的影响的基础。