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: 8555876
• 负责人: Jesus Valenzuela
• 金额: $ 122.27万
• 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/8555876
• 关键词: Accounting; Actins; Activated Partial Thromboplastin Time measurement; Adjuvant; Animals; Antibodies; Anticoagulants; Antigens; Antithrombin III; Apyrase; Arthritis; Binding; Bite; Blood coagulation; Bone Marrow; CD3 Antigens; CD4 Positive T Lymphocytes; Calorimetry; Carotid Arteries; Cell Line; Cells; Chemotaxis; Clinical Research; Coagulation Process; Cutaneous; Dendritic Cells; Development; Disease; Disease Outcome; Edema; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Equilibrium; Experimental Arthritis; Factor Xa; Flow Cytometry; GRK; Gene Expression Profile; Helper-Inducer T-Lymphocyte; Hyaluronidase; IL8RB gene; Immigration; Immune response; Immunity; Immunization; Immunomodulators; Inbred BALB C Mice; Infection; Inflammation; Inflammatory; Injection of therapeutic agent; Interferon Type II; Interferons; Interleukin-10; Interleukin-17; Investigation; Leishmania; Leishmania infantum; Leishmania major; Leishmania tropica; Leishmaniasis; Lesion; Lutzomyia genus; Mammalian Cell; Measures; Mechanics; Midgut; Modeling; Molecular; Molecular Biology; Mus; Names; Needles; Ovalbumin; PAR-2 Receptor; Pain; Parasite Control; Parasites; Pathology; Phlebotomus; Physiological; Plasmids; Production; Property; Proteins; Proteome; Proteomics; Prothrombin time assay; Recombinant Proteins; Recombinants; Reporting; Research; SELL gene; Saliva; Salivary; Salivary Glands; Salivary Proteins; Sand Flies; Silicon Dioxide; Surface Plasmon Resonance; T memory cell; TNF gene; Testing; Thromboplastin; Thrombosis; Thrombus; Time; Titrations; Ulcer; Vaccinated; Vaccines; Virulence; Visceral Leishmaniasis; Work; arginyl-glycyl-glutamic acid; human disease; in vivo; inhibitor/antagonist; lymph nodes; migration; neglect; neutrophil; novel; pathogen; polymerization; prevent; protective efficacy; research study; response; stoichiometry; transcriptomics; transmission process; vaccine candidate; vector; vector-based vaccine

The accomplishments of the section are: 1) Immunity to Sand Fly Salivary Protein LJM11 Modulates Host Response to Vector-Transmitted Leishmania Conferring pathology-Free Protection. Leishmania vaccines that protect against needle challenge fail against the potency of a Leishmania-infected sand fly transmission. Here, we demonstrate that intradermal immunization of mice with 500ng of the sand fly salivary recombinant protein LJM11 (rLJM11) from Lutzomyia longipalpis, in the absence of adjuvant, induces long-lasting immunity that results in ulcer-free protection against Leishmania major delivered by vector bites. This protection is antibody independent and abrogated by depletion of CD4(+) T cells. Two weeks after challenge, early induction of IFN-gamma; specifically to rLJM11 correlates to diminished parasite replication in protected animals. At this time point, Leishmania-specific induction of IFN-gamma; in these mice is low in comparison with its high level in non-protected controls. We hypothesize that early control of parasites in a T-cell helper type 1 environment induced by immunity to LJM11 permits the slow development of Leishmania-specific immunity in the absence of open ulcers. Leishmania-specific immunity observed 5 weeks after infection in rLJM11-immunized mice shows a twofold increase over controls in the percentage of IFN-gamma;-producing CD4(+) T cells. We propose LJM11 as an immunomodulator that drives an efficient and controlled protective immune response to a sand fly-transmitted Leishmania somewhat mimicking "leishmanization"-induced protective immunity but without its associated lesions 2) KSAC, a defined Leishmania antigen, plus adjuvant protects against the virulence of L. major transmitted by its natural vector Phlebotomus duboscqi. Recombinant KSAC and L110f are promising Leishmania vaccine candidates. Both antigens protected BALB/c mice against L. major infection following needle challenge. Considering the virulence of vector-transmitted Leishmania infections, we vaccinated BALB/c mice with either KSAC+GLA-SE or L110f+GLA-SE to assess protection against L. major transmitted via its vector Phlebotomus duboscqi. Mice receiving the KSAC or L110f vaccines were challenged by needle or L. major-infected sand flies. Following sand fly challenge, KSAC-vaccinated mice were protected while L110f-vaccinated animals showed partial protection. Protection correlated with the ability of SLA to induce IFN-gamma;-producing CD4(+)CD62L(low)CCR7(low) effector memory T cells pre- and post-sand fly challenge. This study demonstrates the protective efficacy of KSAC+GLA-SE against sand fly challenge; the importance of vector-transmitted challenge in evaluating vaccine candidates against Leishmania infection; and the necessity of a rapid potent Th1 response against Leishmania to attain true protection. 3) Salivary gland transcriptomes and proteomes of Phlebotomus tobbi and Phlebotomus sergenti, vectors of leishmaniasis. Phlebotomus tobbi is a vector of Leishmania infantum, and P. sergenti is a vector of Leishmania tropica. To better understand the components and possible implications of sand fly saliva in leishmaniasis, the transcriptomes of the salivary glands (SGs) of these two sand fly species were sequenced, characterized and compared. The most abundant, secreted putative proteins were categorized as antigen 5-related proteins, apyrases, hyaluronidases, D7-related and PpSP15-like proteins, ParSP25-like proteins, PpSP32-like proteins, yellow-related proteins, the 33-kDa salivary proteins, and the 41.9-kDa superfamily of proteins. This analysis of P. sergenti is the first description of the subgenus Paraphlebotomus salivary components. The investigation of the subgenus Larroussius sand fly P. tobbi expands the repertoire of salivary proteins in vectors of Le. infantum. Although P. tobbi transmits a cutaneous form of leishmaniasis, its salivary proteins are most similar to other Larroussius subgenus species transmitting visceral leishmaniasis. These transcriptomic and proteomic analyses provide a better understanding of sand fly salivary proteins across species and subgenera that will be vital in vector-pathogen and vector-host research. 4) Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis, Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo. The aim of the study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Several L. longipalpis salivary proteins were expressed in mammalian cells (293 F) and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant of 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. 5) The protein LJM 111 from Lutzomyia longipalpis salivary gland extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model. 5) The protein LJM 111 from Lutzomyia longipalpis salivary gland extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model. Several studies have pointed out the immunomodulatory properties of the Salivary Gland Extract (SGE) from Lutzomyia longipalpis. We aimed to identify the SGE component (s) responsible for its effect on ovalbumin (OVA)-induced neutrophil migration (NM) and to evaluate the effect of SGE and components in the antigen-induced arthritis (AIA) model. We tested the anti-arthritic activities of SGE and the recombinant LJM111 salivary protein (rLJM111) by measuring the mechanical hypernociception and the NM into synovial cavity. Furthermore, we measured IL-17, TNF- and IFN- released by lymph nodes cells stimulated with mBSA or anti-CD3 using enzyme-linked immunosorbent assay (ELISA). Additionally, we tested the effect of SGE and rLJM111 on co-stimulatory molecules expression (MHC-II and CD-86) by flow cytometry, TNF- and IL-10 production (ELISA) of bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, chemotaxis and actin polymerization from neutrophils. Besides, the effect of SGE on CXCR2 and GRK-2 expression on neutrophils was investigated. We identified one plasmid expressing the protein LJM111 that prevented NM in OVA-challenged immunized mice. Furthermore, both SGE and rLJM111 inhibited NM and pain sensitivity in AIA and reduced IL-17, TNF- and IFN-. SGE and rLJM111 also reduced MHC-II and CD-86 expression and TNF- whereas increased IL-10 release by LPS-stimulated BMDCs. SGE, but not LJM 111, inhibited neutrophils chemotaxis and actin polymerization. Additionally, SGE reduced neutrophil CXCR2 expression and increased GRK-2. Thus, rLJM111 is partially responsible for SGE mechanisms by diminishing DC function and maturation but not chemoattraction of neutrophils.

本节的成就是： 1）免疫力对沙蝇唾液蛋白LJM11调节宿主对载体传播利什曼原虫的反应，赋予无病理保护。 防止针头挑战的利什曼原虫疫苗无法通过利什曼尼亚感染的沙蝇传输的效力。在这里，我们证明，在没有佐剂的情况下，用Lutzomyia longipalpis的500ng Sand Fly唾液重组蛋白LJM11（RLJM11）对小鼠进行了皮内免疫，从而诱导了持久的免疫，从而导致了持久的免疫力，从而无效地保护了Leishmania的无效保护Leishmania的vecter vector vector vector vector Bites。该保护是独立的抗体，并且通过CD4（+）T细胞的耗竭消除。挑战两周后，提前诱导IFN-gamma；特别与RLJM11相关，与受保护动物的寄生虫复制减少相关。此时，利什曼尼亚特定于IFN-gamma的诱导；在这些小鼠中，与其在非保护对照中的高水平相比，它很低。我们假设在没有开放性溃疡的情况下，对LJM11免疫引起的T细胞助手1型环境中寄生虫的早期控制允许Leishmania特异性免疫的缓慢发展。在RLJM11免疫化小鼠感染后5周观察到的利什曼原虫特异性免疫力表明，IFN-gamma百分比的对照增长了双重； - 产生CD4（+）T细胞。我们建议LJM11作为一种免疫调节剂，该免疫调节剂可以驱动对沙蝇传输的利什曼原虫的有效和控制的保护性免疫反应，这有些模仿“利什曼化”诱导的保护性免疫，但没有其相关病变 2）KSAC是一种定义的利什曼原虫抗原，加上佐剂可预防其自然载体荧光省Duboscqi传播的L. major的毒力。 重组KSAC和L110F是有希望的利什曼原虫疫苗候选者。两种抗原都保护了针头挑战后的BALB/C小鼠免受重大感染。考虑到载体传播的利什曼原虫感染的毒力，我们用KSAC+GLA-SE或L110F+GLA-SE接种了BALB/C小鼠，以评估通过其通过其载体Phlebotomus duboscQi传播的L. Major的保护。 接收KSAC或L110F疫苗的小鼠受到针或主要感染的沙蝇的挑战。在沙蝇挑战之后，保护了KSAC接种疫苗的小鼠，而L110F接种疫苗的动物显示出部分保护。保护与SLA诱导IFN-GAMMA的能力相关； - 生产CD4（+）CD62L（Low）CCR7（低）效应效应器记忆T细胞前和后蝇后蝇质挑战。这项研究证明了KSAC+GLA-SE免受沙蝇挑战的保护作用。媒介传播挑战在评估候选利什曼原虫感染的挑战中的重要性；以及对利什曼尼亚（Leishmania）进行快速有效的TH1反应以获得真正的保护的必要性。 3）唾液腺转录组和phlebotomus tobbi和phlebotomus sergenti的蛋白质组，利什曼病的载体。 Phlebotomus Tobbi是Leishmania Infantum的载体，P。Sergenti是Tropica Leishmania的载体。 为了更好地理解沙蝇唾液在利什曼病中的成分和可能的含义，对这两种沙蝇物种的唾液腺（SG）的转录组进行了测序，表征和比较。最丰富的，分泌的推定蛋白被归类为抗原5相关蛋白，apyrass，透明质酸酶，与D7相关的蛋白质和PPSP15样蛋白，PARSP25蛋白，PPSP32-like蛋白，PPSP32-like蛋白，黄色相关的蛋白质，黄色相关蛋白，33- kda saliv saliv proteins and Prote蛋白和41个 - 41个 - 41-- 41-k-ks-ked。对Sergenti P. p. per的分析是属于唾液组成分的亚属植物瘤的第一个描述。对Larroussius sand蝇的调查P. Tobbi扩大了LE中的唾液蛋白的曲目。婴儿。尽管P. tobbi传播了一种皮肤形式的利什曼病，但其唾液蛋白与其他传播内脏利什曼病的Larroussius子属物种最相似。这些转录组和蛋白质组学分析为跨物种和亚属的沙蝇唾液蛋白提供了更好的了解，这对载体 - 病原体和载体 - 宿主研究至关重要。 4）Lufaxin是一种新的因子Xa抑制剂，来自沙蝇lutzomyia longipalpis的唾液腺，阻止了蛋白酶激活的受体2激活2，并抑制体内炎症和血栓形成。 该研究的目的是确定沙子lutzomyia longipalpis的独特唾液抗凝剂，该抗凝剂数十年来一直难以捉摸。在哺乳动物细胞（293 F）中表达了几种longipalpis唾液蛋白，并进行筛选以抑制血液凝结。一种名为卢法辛的新型32.4-kDa分子被鉴定为因子Xa（FXA）的缓慢，紧密，无竞争和可逆抑制剂。值得注意的是，卢法辛的主要序列与迄今为止报道的凝血的任何生理或唾液抑制剂没有相似之处。卢法辛（Lufaxin）是FXA的特异性，并且不与FX，Dansyl-Glu-Gly-Gly-Arg-FXA或15种其他酶相互作用。此外，卢法辛会阻断凝血酶原酶，并增加凝血酶原的时间和激活的部分凝血蛋白时间。表面等离子体共振实验表明，FXA结合卢法辛的平衡常数为3 nm，等温滴定量热法测定了1：1的化学计量。卢法辛还可以防止FXA在MDA-MB-231细胞系中通过FXA激活蛋白酶激活的受体2，并消除通过小鼠爪子中FXA触发的水肿形成。此外，卢法辛可防止FECL（3）诱导的颈动脉血栓形成，并延长激活的部分血栓质蛋白时间，这意味着它在体内起着抗凝剂的作用。 5）在实验性关节炎模型中，lutzomyia longipalpis唾液腺提取物（SGE）的蛋白质LJM 111对SGE抑制性抑制作用产生了抑制作用。 5）在实验性关节炎模型中，lutzomyia longipalpis唾液腺提取物（SGE）的蛋白质LJM 111对SGE抑制性抑制作用产生了抑制作用。 几项研究指出了lutzomyia longipalpis的唾液腺提取物（SGE）的免疫调节特性。我们的目的是确定负责其对卵巢蛋白（OVA）诱导的中性粒细胞迁移（NM）的影响的SGE成分，并评估SGE和成分在抗原诱导的关节炎（AIA）模型中的影响。我们通过测量机械性高点心受到摄影和NM来测试SGE和重组LJM111唾液蛋白（RLJM111）的抗关节炎活性。此外，我们使用酶 - 连接的免疫吸附测定法（ELISA）测量了由MBSA或抗CD3刺激的淋巴结细胞释放的IL-17，TNF-和IFN-。此外，我们通过流式细胞仪，TNF-和IL-10产生（ELISA）对SGE和RLJM111对共刺激分子表达（MHC-II和CD-86）的影响进行了测试。此外，研究了SGE对CXCR2和GRK-2表达对中性粒细胞的影响。我们确定了一种表达蛋白LJM111的质粒，该质粒阻止了OVA挑战的免疫小鼠NM。此外，SGE和RLJM111均抑制了AIA的NM和疼痛敏感性，并降低了IL-17，TNF-和IFN-。 SGE和RLJM111还降低了MHC-II和CD-86的表达，而TNF-降低了LPS刺激的BMDC释放IL-10。 SGE，但不是LJM 111，抑制了嗜中性粒细胞趋化性和肌动蛋白聚合。另外，SGE降低了中性粒细胞CXCR2表达并增加了GRK-2。因此，RLJM111通过降低直流功能和成熟而不是中性粒细胞的趋化性来部分负责SGE机制。