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: 10927790
• 负责人: Jesus Valenzuela
• 金额: $ 128.19万
• 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/10927790
• 关键词: Active Sites; Adult; Aedes; Age Years; Animals; Antibodies; Antibody Response; Area; Binding; Biochemical; Bioinformatics; Biological Markers; Biopsy; Bite; Borrelia burgdorferi; C-terminal; CD8-Positive T-Lymphocytes; Cambodia; Cell Count; Cell Degranulation; Clinical Protocols; Coagulation Process; Complement; Complement 3b; Complement Activation; Complement Factor B; Complement Inactivators; Complex; Cryoelectron Microscopy; Culicidae; Cutaneous; Cytoplasmic Granules; DNA; Data; Dendritic Cells; Development; Disease; Dose; Double-Blind Method; Elements; Endemic Diseases; Enrollment; Event; Exposure to; Gene Expression Profiling; Generations; Genes; Goals; Hemolytic-Uremic Syndrome; Histamine; Human; IL4 gene; Immune; Immune response; Immunity; Immunization; Immunoglobulin G; Immunologics; Immunophenotyping; Infection; Inflammatory; Inflammatory Response; Injections; Innate Immune Response; Insect Bites; Insect Vectors; Interferons; Interleukin-10; Interleukin-4; Intervention Studies; Knowledge; Laboratories; Leishmania; Leishmania donovani; Leishmania major; Leishmania mexicana; Leishmaniasis; Lesion; Liver; Lyme Disease; Lymphocyte; Macrophage; Measurement; Mediating; Midgut; Modeling; Molecular; Molecular Biology; Molecular Conformation; Monitor; Mosquito-borne infectious disease; N-terminal; NFKB Signaling Pathway; Needles; Neutrophil Infiltration; Pain; Parasite Control; Parasites; Parasitic Diseases; Participant; Pathogenesis; Pathway interactions; Peptide Vaccines; Peptides; Phlebotominae; Phlebotomus; Phosphorylation; Placebo Control; Placebos; Plasma; Play; Proteins; Public Health; RNA vaccination; ROC Curve; Randomized; Recombinants; Regulatory Pathway; Resistance; Role; Safety; Saliva; Salivary; Salivary Proteins; Sand Flies; Serine Protease; Serious Adverse Event; Shapes; Site; Skin; Specificity; Spleen; Spottings; Stains; Structure; Surface Plasmon Resonance; T-Cell Activation; T-Lymphocyte; TNF gene; Testing; Thrombin; Thrombosis; Tick-Borne Diseases; Ticks; Translating; Trimethoprim-Sulfamethoxazole; Tryptase; Vaccines; Viral; Visceral Leishmaniasis; adaptive immune response; aged; anti-tick vaccine; arboviral disease; arm; cell type; clinical efficacy; cytokine; disease transmission; disorder control; experience; experimental study; feeding; immunogenicity; immunoreaction; immunoregulation; inhibitor; mast cell; molecular vector; monocyte; mortality; neglect; neutrophil; novel; novel marker; novel therapeutics; paroxysmal nocturnal hemoglobinuria; pathogen; phase I trial; primary endpoint; recruit; response; screening; success; tick bite; tick-borne pathogen; tool; transmission process; vaccine candidate; vector; vector control; vector-borne

The accomplishment of the section are: 1. We developed a novel biomarker for sand fly exposure in humans using two recombinant salivary proteins from the sand fly Phlebotomus argentipes. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the Indian subcontinent, as immunodominant proteins in humans. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after visceral leishmaniasis elimination. 2. We demonstrated that Leishmania major-Infected sand fly bites enhance mast cell degranulation. Leishmania parasites infect mammalian hosts through the bites of sand fly vectors. The response by mast cells (MC) to the parasite and vector-derived factors, delivered by sand fly bites, has not been characterized. MC were found at the bite sites of infective and non-infected sand flies throughout 48 h, showing the release of granules with intense TNF-, histamine, and tryptase staining. At 30 min and 48 h, the MC numbers were significantly higher (p < 0.001) in infected as compared to non-infected bites or controls. Our data show that MC orchestrate an early inflammatory response after infected and non-infected sand fly bites, leading to neutrophilic recruitment, which potentially provides a safe passage for the parasite within the mammalian host. 3. We demonstrate that the sand fly salivary protein PpSP32 exerts immunomodulatory effects on human monocytes, macrophages, and lymphocytes. We aimed to study the role of PpSP32 by screening of several immunomodulatory activities either on lymphocytes or on monocytes/macrophages. Our data showed that PpSP32 down-modulated the expression of activation markers in LPS-stimulated monocytes and THP1-derived macrophages. This protein negatively modulated the secretion of Th1 and Th2 cytokines by human lymphocytes as well as pro-inflammatory cytokines by monocytes, and THP1-derived macrophages. PpSP32 treatment led to a dose-dependent reduction of IB phosphorylation. Our data indicates that PpSP32 induces a potent immunomodulatory effect on monocytes and THP-1-derived macrophages. This inhibition could be mediated, among others, by the modulation of the NF-kB signaling pathway. 4. We evaluated the human skin immune response to mosquito bites from subjects living in a disease endemic area. Initial viral inoculation occurs in the skin via the mosquito 'bite', eliciting immune responses that shape the establishment of infection and pathogenesis. Here we assess the cutaneous innate and adaptive immune responses to controlled Aedes aegypti feedings in humans living in Aedes-endemic areas. In this single-arm, cross-sectional interventional study (trial registration #NCT04350905), we enroll 30 healthy adult participants aged 18 to 45 years of age from Cambodia between October 2020 and January 2021. We perform 3-mm skin biopsies at baseline as well as 30 min, 4 h, and 48 h after a controlled feeding by uninfected Aedes aegypti mosquitos. The primary endpoints are measurement of changes in early and late innate responses in bitten vs unbitten skin by gene expression profiling, immunophenotyping, and cytokine profiling. The results reveal induction of neutrophil degranulation and recruitment of skin-resident dendritic cells and M2 macrophages. As the immune reaction progresses T cell priming and regulatory pathways are upregulated along with a shift to Th2-driven responses and CD8+ T cell activation. These results identify key immune genes, cell types, and pathways in the human response to mosquito bites and can be leveraged to inform and develop novel therapeutics and vector-targeted vaccine candidates to interfere with vector-mediated disease. 5. We performed a Safety and immunogenicity of AGS-v PLUS, a mosquito saliva peptide vaccine against arboviral diseases: A randomized, double-blind, placebo-controlled Phase 1 trial. Immunity to mosquito salivary proteins could provide protection against multiple mosquito-borne diseases and significantly impact public health. We evaluated the safety and immunogenicity of AGS-v PLUS, a mosquito salivary peptide vaccine, in healthy adults 18-50 years old. Primary endpoints were safety and antibody and cytokine responses. Participants experienced no treatment-emergent or serious adverse events. Only injection site pain was more common in vaccine groups (15/51 after dose 1 and 11/51 after dose 2) versus placebo. Compared to placebo, all vaccine groups had significantly greater fold change in anti-AGS-v PLUS IgG and IFN- from baseline. AGS-v PLUS had favorable safety profile and induced robust immune responses. Next steps will determine if findings translate into clinical efficacy against mosquito-borne diseases. 6. We demonstrated that Centrin-deficient Leishmania mexicana confers protection against Old World visceral leishmaniasis. In this study, we tested whether LmexCen-/- parasites can protect against visceral leishmaniasis caused by L. donovani. We showed that immunization with LmexCen-/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver as well as reduced mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the disease promoting cytokines IL-10 and IL-4 and increased pro-inflammatory cytokine IFN- resulting in higher IFN-/IL-10 and IFN-/IL4 ratios compared to non-immunized groups. LmexCen-/- immunization also resulted in long-lasting protection against L. donovani infection. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against the Old World visceral leishmaniasis. 7. We identified and characterized a novel bispecific inhibitor of complement and coagulation that blocks activation in complementopathy models. Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all -sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo-electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.

本节的成就是： 1。我们使用两种重组唾液蛋白来自砂蝇阿根廷的重组唾液蛋白开发了一种用于人类沙蝇暴露的新型生物标志物。为了开发此工具，我们从印度次大陆的Leishmania Donovani的唾液中鉴定出PagSP02和PAGSP06，是人类中的免疫主导蛋白。 重要的是，通过结合重组RPAGSP02和RPAGSP06，我们获得了比单唾液蛋白更大的抗体识别和特异性。与阴性对照血清相比，RPAGSP02 + RPAGSP06的接收器操作特性曲线预测，具有90％特异性和87％敏感性的Argentipes叮咬暴露（p> .0001）。总体而言，RPAGSP02 + RPAGSP06提供了一种有效的监视工具，用于监测内脏利什曼病后向量控制工作。 2。我们证明了利什曼原虫主要感染的沙蝇可增强肥大细胞的脱粒。利什曼原虫寄生虫感染了哺乳动物的寄主，寄主是通过沙蝇载体的叮咬。肥料细胞（MC）对寄生虫和载体衍生的因素的反应尚未表征。 MC在整个48小时内在感染性和未感染的沙蝇的咬合部位发现，显示了颗粒的释放，其颗粒具有强烈的TNF-，组胺和胰蛋白酶染色。在30分钟和48 h时，与未感染的叮咬或对照组相比，感染的MC数明显更高（P <0.001）。我们的数据表明，MC在感染和未感染的沙蝇咬伤后策划了早期的炎症反应，从而导致中性粒细胞募集，这有可能为哺乳动物宿主内的寄生虫提供安全的通道。 3。我们证明，沙蝇唾液蛋白ppsp32对人的单核细胞，巨噬细胞和淋巴细胞产生免疫调节作用。 我们的目的是通过筛选在淋巴细胞或单核细胞/巨噬细胞上筛选几种免疫调节活性来研究PPSP32的作用。我们的数据表明，ppsp32在LPS刺激的单核细胞和THP1衍生的巨噬细胞中降低了激活标记的表达。该蛋白质对人淋巴细胞以及单核细胞和THP1衍生的巨噬细胞的促炎细胞因子和促炎性细胞因子的分泌负面调节。 PPSP32治疗导致IB磷酸化的剂量依赖性降低。我们的数据表明，PPSP32诱导了对单核细胞和THP-1衍生的巨噬细胞的有效免疫调节作用。该抑制作用可以通过调节NF-KB信号通路来介导。 4。我们评估了人类皮肤对生活在疾病特有区域的受试者的蚊子叮咬的免疫反应。初始病毒接种发生在皮肤中，通过蚊子“咬合”，引起了塑造感染和发病机理的建立的免疫反应。在这里，我们评估了居住在艾德斯流行地区的人类中对受控伊迪斯伊蚊的皮肤和适应性免疫反应。在这项单臂横断面介入研究（试验登记＃NCT04350905）中，我们在2020年10月至2021年1月之间从柬埔寨招募了30名18至45岁的健康的成年参与者。我们在基线和30小时，4 h，48 h，48 h和48 h的摩擦均进行了互动的效果。主要终点是通过基因表达分析，免疫表型和细胞因子谱分析来测量咬伤与未训练皮肤的早期和晚期反应的变化。结果揭示了中性粒细胞脱粒和皮肤居民树突状细胞和M2巨噬细胞的诱导。随着免疫反应的进展，T细胞启动和调节途径随着TH2驱动的反应和CD8+ T细胞激活而上调。这些结果确定了人类对蚊子叮咬的反应中的关键免疫基因，细胞类型和途径，并可以利用以告知和开发新型的疗法和靶向载体的疫苗候选者，以干扰载体介导的疾病。 5。我们进行了AGS-V Plus的安全性和免疫原性，蚊子唾液肽疫苗针对灰烬病毒疾病：一项随机，双盲，安慰剂对照的1期试验。对蚊子唾液蛋白的免疫力可以为多种蚊子传播疾病提供保护，并显着影响公共卫生。我们评估了18至50岁健康的成年人的AGS-V Plus（蚊子唾液肽疫苗）的安全性和免疫原性。 主要终点是安全性，抗体和细胞因子反应。参与者没有经历过治疗效果或严重的不良事件。在疫苗组中，只有注射部位疼痛（剂量1和剂量2之后的15/51）比安慰剂更为常见。与安慰剂相比，所有疫苗组的抗AGS-V和IgG和IFN-基线的折叠变化明显更大。 AGS-V Plus具有良好的安全性和诱导的可靠免疫反应。下一步将确定发现是否转化为针对蚊子传播疾病的临床功效。 6。我们证明了缺乏中心素的利什曼原虫墨西哥人赋予了对旧世界内脏利什曼病的保护。在这项研究中，我们测试了LMEXCEN - / - 寄生虫是否可以预防由L. Donovani引起的内脏利什曼病。 我们表明，使用LMEXCEN - / - 寄生虫免疫是安全的，不会引起病变。此外，这种免疫赋予了针对针刺发起的多诺瓦乳杆菌挑战引起的内脏利什曼病的保护，这表明脾脏和肝脏的寄生虫负担显着降低以及死亡率降低。还观察到了针对沙蝇介导的多诺瓦乳杆菌挑战的类似控制寄生虫负担。重要的是，通过LMEXCEN - / - 下调该疾病，促进细胞因子IL-10和IL-4的疾病，并增加促炎性细胞因子IFN，从而导致IFN-/IL-10和IFN-/IL4比率较高，与非免疫组相比。 LMEXCEN - / - 免疫也导致对Donovani感染的持久保护。综上所述，我们的研究表明，使用LMExcen - / - 寄生虫免疫对旧世界内脏利什曼病是安全有效的。 7。我们确定并表征了一种新型的补体和凝结抑制剂，该抑制剂阻断了补体模型中的激活。表面等离子体共振实验表明，卢法辛稳定丝氨酸蛋白酶因子B（Fb）与C3B的结合，但不能单独结合C3B或FB。抑制剂的晶体结构揭示了一个包含2个结构域的新颖的所有折叠。通过冷冻电子显微镜（EM）获得的Lufaxin-C3BB复合物的结构表明，卢法辛在包含C3B和FB元素的界面上通过其N末端结构域结合。通过占据该位置，抑制剂将FB锁定成一个封闭的构象，在该构型中，FD无法通过FD进行蛋白水解激活。 C3BB结合的卢法辛在其C末端结构域的单独位点结合FXA。在C3BB-lufaxin-FXA复合物的冷冻EM结构中，抑制剂同时与两个靶标结合，而卢法辛通过活性位点的C末端肽的底物样结合以及该区域的其他相互作用抑制FXA。 Lufaxin在非典型溶血性尿毒综合征（AHUS）和阵发性夜间夜间血红蛋白尿（PNH）以及血浆中产生凝血酶的生成中抑制补体激活，从而为双抑制剂的发展提供了一个基于双抑制剂的基础，以治疗补充的分散性分歧是一个预期的杂物。

项目成果

A sand fly salivary protein acts as a neutrophil chemoattractant.

• DOI: 10.1038/s41467-021-23002-5
• 发表时间: 2021-05-28
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Guimaraes-Costa AB;Shannon JP;Waclawiak I;Oliveira J;Meneses C;de Castro W;Wen X;Brzostowski J;Serafim TD;Andersen JF;Hickman HD;Kamhawi S;Valenzuela JG;Oliveira F
• 通讯作者: Oliveira F

Engineering a vector-based pan-Leishmania vaccine for humans: proof of principle.

• DOI: 10.1038/s41598-020-75410-0
• 发表时间: 2020-10-29
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Cecílio P;Oristian J;Meneses C;Serafim TD;Valenzuela JG;Cordeiro da Silva A;Oliveira F
• 通讯作者: Oliveira F

PpSP32, the Phlebotomus papatasi immunodominant salivary protein, exerts immunomodulatory effects on human monocytes, macrophages, and lymphocytes.

• DOI: 10.1186/s13071-022-05627-7
• 发表时间: 2023-01-02
• 期刊: Parasites & vectors
• 影响因子: 3.2

Antibody targeting of a specific region of Pfs47 blocks Plasmodium falciparum malaria transmission.

• DOI: 10.1038/s41541-018-0065-5
• 发表时间: 2018
• 期刊: NPJ vaccines
• 影响因子: 9.2
• 作者: Canepa GE;Molina-Cruz A;Yenkoidiok-Douti L;Calvo E;Williams AE;Burkhardt M;Peng F;Narum D;Boulanger MJ;Valenzuela JG;Barillas-Mury C
• 通讯作者: Barillas-Mury C

Enhanced protective efficacy of nonpathogenic recombinant leishmania tarentolae expressing cysteine proteinases combined with a sand fly salivary antigen.

• DOI: 10.1371/journal.pntd.0002751
• 发表时间: 2014-03
• 期刊: PLoS neglected tropical diseases
• 影响因子: 3.8
• 作者: Zahedifard F;Gholami E;Taheri T;Taslimi Y;Doustdari F;Seyed N;Torkashvand F;Meneses C;Papadopoulou B;Kamhawi S;Valenzuela JG;Rafati S
• 通讯作者: Rafati S