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.

该部分的成果有： 1. 我们使用来自白蛉白蛉的两种重组唾液蛋白，开发了一种用于人类白蛉暴露的新型生物标志物。为了开发这个工具，我们从印度次大陆杜氏利什曼原虫的载体银白蛉唾液中鉴定出 PagSP02 和 PagSP06 作为人类的免疫显性蛋白。 重要的是，通过组合重组 rPagSP02 和 rPagSP06，我们获得了比单一唾液蛋白更高的抗体识别和特异性。与阴性对照血清相比，rPagSP02 + rPagSP06 的受试者工作特征曲线预测银蝽叮咬暴露的特异性为 90%，敏感性为 87% (P >.0001)。总体而言，rPagSP02 + rPagSP06 提供了一种有效的监测工具，用于监测消除内脏利什曼病后的病媒控制工作。 2. 我们证明，感染重度利什曼原虫的白蛉叮咬可增强肥大细胞脱颗粒。利什曼原虫寄生虫通过白蛉媒介的叮咬感染哺乳动物宿主。肥大细胞 (MC) 对白蛉叮咬传递的寄生虫和载体衍生因子的反应尚未得到表征。 在 48 小时内，在感染性和未感染性白蛉的叮咬部位发现了 MC，显示释放出具有强烈 TNF-、组胺和类胰蛋白酶染色的颗粒。在 30 分钟和 48 小时，与未感染的叮咬或对照相比，感染的 MC 数量显着更高 (p < 0.001)。我们的数据显示，MC 在感染和未感染的白蛉叮咬后精心策划早期炎症反应，导致中性粒细胞募集，这可能为哺乳动物宿主体内的寄生虫提供安全通道。 3. 我们证明白蛉唾液蛋白PpSP32对人类单核细胞、巨噬细胞和淋巴细胞具有免疫调节作用。 我们的目的是通过筛选对淋巴细胞或单核细胞/巨噬细胞的几种免疫调节活性来研究 PpSP32 的作用。我们的数据表明，PpSP32 下调 LPS 刺激的单核细胞和 THP1 衍生的巨噬细胞中激活标记的表达。该蛋白负调节人类淋巴细胞分泌 Th1 和 Th2 细胞因子以及单核细胞和 THP1 衍生巨噬细胞分泌的促炎细胞因子。 PpSP32 治疗导致 IB 磷酸化呈剂量依赖性减少。我们的数据表明 PpSP32 对单核细胞和 THP-1 衍生的巨噬细胞产生有效的免疫调节作用。这种抑制可以通过调节 NF-kB 信号通路来介导。 4. 我们评估了居住在疾病流行地区的受试者对蚊虫叮咬的人体皮肤免疫反应。最初的病毒接种通过蚊子“叮咬”发生在皮肤中，引发免疫反应，从而形成感染和发病机制。在这里，我们评估了生活在伊蚊流行地区的人类对受控埃及伊蚊喂养的皮肤先天和适应性免疫反应。在这项单臂横断面介入研究（试验注册#NCT04350905）中，我们于 2020 年 10 月至 2021 年 1 月期间从柬埔寨招募了 30 名年龄在 18 岁至 45 岁之间的健康成年参与者。我们在基线时进行了 3 毫米皮肤活检，如下所示：以及未感染的埃及伊蚊控制进食后 30 分钟、4 小时和 48 小时。主要终点是通过基因表达谱、免疫表型和细胞因子谱来测量被咬伤与未被咬伤皮肤的早期和晚期先天反应的变化。结果揭示了中性粒细胞脱颗粒的诱导以及皮肤驻留树突细胞和 M2 巨噬细胞的募集。随着免疫反应的进展，T 细胞启动和调节途径上调，同时转向 Th2 驱动的反应和 CD8+ T 细胞激活。这些结果确定了人类对蚊虫叮咬反应的关键免疫基因、细胞类型和途径，可用于告知和开发新的疗法和载体靶向候选疫苗，以干扰载体介导的疾病。 5. 我们进行了 AGS-v PLUS（一种针对虫媒病毒疾病的蚊子唾液肽疫苗）的安全性和免疫原性：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提供针对多种蚊媒疾病的保护，并显着影响公共健康。我们评估了 AGS-v PLUS（一种蚊子唾液肽疫苗）在 18-50 岁健康成年人中的安全性和免疫原性。 主要终点是安全性以及抗体和细胞因子反应。参与者没有经历治疗引起的或严重的不良事件。与安慰剂相比，疫苗组中只有注射部位疼痛更常见（第 1 剂后为 15/51，第 2 剂后为 11/51）。与安慰剂相比，所有疫苗组的抗 AGS-v PLUS IgG 和 IFN- 相对于基线的倍数变化显着更大。 AGS-v PLUS 具有良好的安全性并诱导强大的免疫反应。下一步将确定研究结果是否转化为针对蚊媒疾病的临床疗效。 6. 我们证明缺乏 Centrin 的墨西哥利什曼原虫可以预防旧世界内脏利什曼病。在这项研究中，我们测试了 LmexCen-/- 寄生虫是否可以预防杜诺瓦尼利什曼病引起的内脏利什曼病。 我们证明，用 LmexCen-/- 寄生虫进行免疫是安全的，不会引起病变。此外，这种免疫接种可以预防由针头引发的杜氏利什曼病引起的内脏利什曼病，脾脏和肝脏中的寄生虫负担显着减少以及死亡率降低表明了这一点。在白蛉介导的杜氏乳杆菌攻击中也观察到了类似的寄生虫负荷控制。重要的是，与未免疫接种相比，用 LmexCen-/- 免疫下调了促进疾病的细胞因子 IL-10 和 IL-4，并增加了促炎细胞因子 IFN-，从而导致更高的 IFN-/IL-10 和 IFN-/IL4 比率组。 LmexCen-/- 免疫还可以对杜氏乳杆菌感染产生持久的保护。综上所述，我们的研究表明，用 LmexCen-/- 寄生虫进行免疫接种对旧世界内脏利什曼病是安全有效的。 7. 我们鉴定并表征了一种新型的补体和凝血双特异性抑制剂，可阻断补体病模型中的激活。表面等离子共振实验表明，lufaxin 可稳定丝氨酸蛋白酶因子 B (FB) 与 C3b 的结合，但无法检测到单独结合 C3b 或 FB。该抑制剂的晶体结构揭示了一种包含 2 个结构域的新型全片折叠。通过冷冻电子显微镜 (EM) 获得的 lufaxin-C3bB 复合物的结构表明，lufaxin 通过其 N 端结构域在包含 C3b 和 FB 元件的界面处结合。通过占据该位点，抑制剂将 FB 锁定为闭合构象，其中 FD 对 FB 的蛋白水解激活不会发生。 C3bB 结合的 lufaxin 在其 C 端结构域的单独位点结合 fXa。在 C3bB-lufaxin-fXa 复合物的冷冻电镜结构中，抑制剂同时与两个靶标结合，lufaxin 通过活性位点 C 端肽的底物样结合以及该区域的其他相互作用来抑制 fXa 。 Lufaxin 可抑制非典型溶血性尿毒症综合征 (aHUS) 和阵发性睡眠性血红蛋白尿症 (PNH) 离体模型中的补体激活以及血浆中凝血酶的产生，为开发双特异性抑制剂来治疗补体相关疾病（其中血栓形成）提供了理论基础。是一个突出的体现。

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

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

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

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