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: 7732588
• 负责人: Jesus Valenzuela
• 金额: $ 94.3万
• 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/7732588
• 关键词: Acceleration; Active Sites; Adjuvant; Animals; Anticoagulants; Antigens; Arthropod Vectors; Automobile Driving; Binding; Biochemical; Biological; Biological Assay; Biological Process; Bite; Blood; Bystander Effect; CD4 Positive T Lymphocytes; Cells; Classical Complement Pathway; Clinical Research; Cloning; Coagulants; Complement; Complement 3b; Complement 3b Inactivators; Control Groups; Cutaneous Leishmaniasis; DNA; DNA Vaccines; Data Reporting; Development; Disease; Disease Outcome; Endotoxins; Environment; Event; Facility Construction Funding Category; Factor Xa; Fatal Outcome; Gene Expression Profile; Hamsters; Hour; Human; Immune; Immune System Diseases; Immune response; Immunity; Immunization; Immunologics; Infection; Insect Vectors; Interferons; Interleukin-4; Leishmania; Leishmania infantum; Leishmaniasis; Lutzomyia genus; Mammalian Cell; Medicine; Methodology; Midgut; Modeling; Molecular; Molecular Biology; Molecular Profiling; Mus; Numbers; Operative Surgical Procedures; Outcome; Parasites; Pharmaceutical Preparations; Plasmids; Predisposition; Proteins; Recombinant Proteins; Recombinants; Relative (related person); Research; Resistance; Role; Salivary; Salivary Glands; Salivary Proteins; Sand Flies; Screening procedure; Skin; Staging; Stress; System; Testing; Thromboplastin; Time; Tissues; Transcript; Vaccines; Vector-transmitted infectious disease; Virulent; Visceral Leishmaniasis; Week; Work; base; cDNA Library; complement pathway; expression vector; functional genomics; human disease; inhibitor/antagonist; killings; neglect; novel; pathogen; plasmid DNA; prevent; protective effect; sugar; transmission process; vector; vector-based vaccine; Acceleration; Active Sites; Adjuvant; Animals; Anticoagulants; Antigens; Arthropod Vectors; Automobile Driving; Binding; Biochemical; Biological; Biological Assay; Biological Process; Bite; Blood; Bystander Effect; CD4 Positive T Lymphocytes; Cells; Classical Complement Pathway; Clinical Research; Cloning; Coagulants; Complement; Complement 3b; Complement 3b Inactivators; Control Groups; Cutaneous Leishmaniasis; DNA; DNA Vaccines; Data Reporting; Development; Disease; Disease Outcome; Endotoxins; Environment; Event; Facility Construction Funding Category; Factor Xa; Fatal Outcome; Gene Expression Profile; Hamsters; Hour; Human; Immune; Immune System Diseases; Immune response; Immunity; Immunization; Immunologics; Infection; Insect Vectors; Interferons; Interleukin-4; Leishmania; Leishmania infantum; Leishmaniasis; Lutzomyia genus; Mammalian Cell; Medicine; Methodology; Midgut; Modeling; Molecular; Molecular Biology; Molecular Profiling; Mus; Numbers; Operative Surgical Procedures; Outcome; Parasites; Pharmaceutical Preparations; Plasmids; Predisposition; Proteins; Recombinant Proteins; Recombinants; Relative (related person); Research; Resistance; Role; Salivary; Salivary Glands; Salivary Proteins; Sand Flies; Screening procedure; Skin; Staging; Stress; System; Testing; Thromboplastin; Time; Tissues; Transcript; Vaccines; Vector-transmitted infectious disease; Virulent; Visceral Leishmaniasis; Week; Work; base; cDNA Library; complement pathway; expression vector; functional genomics; human disease; inhibitor/antagonist; killings; neglect; novel; pathogen; plasmid DNA; prevent; protective effect; sugar; transmission process; vector; vector-based vaccine

The Vector Molecular Biology Unit focuses on the molecular aspects of sand fly salivary and midgut proteins with emphasis on the understanding of vector/host and vector/parasite interactions, specifically sand fly/Leishmania interactions. Unit research combines basic approaches together with veterinary and clinical research broadening our understanding of the relationship between immune responses to vector proteins in animal reservoirs and humans and disease outcome, and between the Leishmania parasite and the sand fly midgut proteins to ultimately develop a vector-based vaccine against the neglected disease leishmaniasis. The two main hypotheses driving the research of this unit are: 1) Cellular immune responses to vector arthropod salivary proteins produce an inhospitable environment in the skin of the host to the co injected pathogen, resulting in indirect killing or acceleration of anti-Leishmania immunity. Identifying the vector salivary proteins and the correlates of protection particularly the initial immunological events will help us to understand the immunologic basis of protection and to select vaccine candidates to prevent pathogen transmission. 2) Specific molecular interactions between the sand fly midgut and the Leishmania parasite are required for Leishmania survival and development to the infective stage in the insect vector. Characterization of these molecular interactions will help in the understanding of the molecular basis of Leishmania sand fly interactions and may identify a suitable target for a transmission blocking vaccine. The accomplishment from the two main projects of the unit are: 1) Development of a robust protein recombinant expression and purification methodology to test salivary proteins for biological activities, and validate vaccine candidates identified using DNA immunization. We maximized expression of a properly folded recombinant salivary protein by cloning sand fly salivary gland transcripts into the mammalian expression vector developed by this unit, VR2001-TOPO, the same vector used for DNA immunization, and used it to transfect 293F mammalian cells. Transfected cells produced soluble recombinant proteins and by following an optimized purification methodology we obtained a number of salivary proteins in large quantities, highly pure and with a minimal amount of endotoxin for testing in vaccine, immunological and biological assays. 2) The demonstration that a recombinant sand fly salivary protein protects against challenge with L. major infected sand flies. We demonstrated that the protection observed by immunization with DNA vaccine is not a bystander effect of the DNA plasmid and it is specific to the protein encoded by the plasmid. We tested the protective effect of recombinant protein LJM11 that is encoded in the selected DNA plasmid against the virulent challenge of L. major infected Lu. longipalpis sand fly bites. Mice immunized with a small amount of recombinant LJM11 in the absence of adjuvants were protected against challenge by bites from ten infected sand flies (Figure 4). This is the first demonstration of the protective role of a recombinant sand fly salivary protein against cutaneous leishmaniasis and validates the use of DNA immunization for protection against Leishmania infections. 3) The finding that immunity to distinct sand fly salivary proteins primes the anti-Leishmania immune response towards protection or exacerbation of disease. Two distinct DTH-inducing salivary proteins from P. papatasi were investigated for their effect on L. major infection. DNA immunization with these molecules resulted in contrasting outcomes of infection upon challenge with L. major parasites. PpSP15-immunized mice showed lasting protection while immunization with PpSP44 aggravated the infection (without an increase in parasite load as compared to the control group). This suggests that immunization with these distinct molecules alters the course of anti-Leishmania immunity. Two weeks post-infection 31.5% of CD4+ T cells produced IFN- in PpSP15-mice compared to 7.1% in PpSP44-mice. IL-4 producing cells were three-fold higher in PpSP44-mice. Two hours after challenge with SGH and L. major, the expression profile of PpSP15-mice showed over three-fold higher IFN- and IL-12-R2 and 20-fold lower IL-4 expression, relative to PpSP44-mice suggesting that salivary proteins differentially prime anti-Leishmania immunity. This demonstrates for the first time that immunity to a salivary protein (PpSP44) results in disease enhancement and stresses the importance of the proper selection of vector-based vaccine candidates. 4) The finding that immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model. Two distinct DTH-inducing salivary proteins from P. papatasi were investigated for their effect on L. major infection. DNA immunization with these molecules resulted in contrasting outcomes of infection upon challenge with L. major parasites. PpSP15-immunized mice showed lasting protection while immunization with PpSP44 aggravated the infection (without an increase in parasite load as compared to the control group). This suggests that immunization with these distinct molecules alters the course of anti-Leishmania immunity. In this work, we demonstrated that not all DTH-inducing P. papatasi sand fly salivary molecules are universally protective against L. major infection and that immunization with two DTH-inducing salivary proteins produced distinct immune profiles that correlated with resistance or susceptibility to Leishmania infection. This demonstrates for the first time that immunity to a salivary protein (PpSP44) results in disease enhancement and stresses the importance of the proper selection of vector-based vaccine candidates. 5) Identification of biological activities from novel salivary molecules of unknown sequence or function. Most sand fly salivary proteins, including the identified protective molecules, are novel and do not have an assigned biological function. Soluble recombinant proteins were produced in a mammalian expression system to test potential biological activities. We identified a potent anticoagulant salivary protein from Lu. longipalpis that binds specifically the active site of factor Xa. We also identified four salivary recombinant proteins that inhibited the alternate pathway of complement by binding specifically to C3b and one protein that inhibits the classical pathway of complement. These molecules have the potential for use in the treatment of a variety of human ailments. This work not only demonstrates that the anticoagulant inhibitor of sand flies is a novel prothrombinase inhibitor, but also makes it a potential active drug to correct pro-coagulant disorders in human medicine. C3b-inhibitors could be potential new drugs that correct human diseases related to complement such as auto-immune diseases and post-surgery complications due to complement. 6) Identification of the most abundant midgut transcripts of Lutzomyia longipalpis, the vector of Leishmania infantum chagasi. We have generated and sequenced five cDNA libraries from the midgut tissue of Lu. longipalpis; including midgut tissue after a sugar meal, a blood meal and a blood meal in the presence of L. infantum chagasi. Combining all cDNA library sequences produced 655 contigs, 2279 singletons and an average of 9.45 sequences per contig with approximately 10,000 sequences in total. This transcriptome analysis represents the largest set of sequence data reported from a specific sand fly tissue and provides further information of the transcripts present in the gut of the sand fly Lu. longipalpis.

载体分子生物学单元着重于沙蝇唾液和中肠蛋白的分子方面，重点是对载体/宿主和载体和载体/寄生虫相互作用，特别是沙蝇/利什曼原虫相互作用的理解。单元研究将基本方法与兽医和临床研究结合在一起，扩大了我们对动物储层中对载体蛋白与人类媒介蛋白的关系的理解，以及人类与疾病的结果，以及利什曼原虫的寄生虫与沙子飞蝇蛋白之间的关系，以最终对基于基于被忽视的疾病的媒介疫苗开发载体的疫苗。 推动该单元研究的两个主要假设是： 1）对载体节肢动物唾液蛋白的细胞免疫反应在宿主皮肤中产生一个不可避免的环境，从而对CO注射的病原体产生了可行的环境，从而导致抗leishmania免疫的间接杀伤或加速。识别载体唾液蛋白以及保护的相关性，特别是最初的免疫事件将有助于我们了解保护的免疫学基础，并选择候选疫苗的候选物，以防止病原体传播。 2）在昆虫载体的感染阶段，砂蝇中肠和利什曼原虫寄生虫之间的特定分子相互作用是必需的。这些分子相互作用的表征将有助于理解利什曼原虫砂蝇相互作用的分子基础，并可能确定适合传输阻断疫苗的靶标。 该单元的两个主要项目的成就是： 1）开发可靠的蛋白质重组表达和纯化方法，以测试唾液蛋白的生物活性，并验证使用DNA免疫鉴定的疫苗候选者。 我们通过将沙蝇唾液腺转录物克隆到该单元开发的哺乳动物表达载体，VR2001-TOPO（用于DNA接种的相同的载体，并将其用于转染293F 293F哺乳动物细胞）中最大化正确折叠的重组唾液蛋白的表达。转染的细胞产生可溶性重组蛋白，并遵循优化的纯化方法，我们获得了许多唾液蛋白，大量含量，高度纯净，并且在疫苗，免疫学和生物学测定中进行测试的内毒素量最小。 2）证明重组沙蝇唾液蛋白可以防止主要感染的沙蝇挑战。 我们证明，用DNA疫苗免疫观察到的保护不是DNA质粒的旁观者效应，它特异于质粒编码的蛋白质。我们测试了重组蛋白LJM11的保护作用，该蛋白LJM11在选定的DNA质粒中针对主要受感染的Lu的胁迫挑战进行了编码。 longipalpis sand fly咬。在没有佐剂的情况下，用少量重组LJM11免疫的小鼠受到十只感染的沙蝇的叮咬保护（图4）。这是重组沙蝇唾液蛋白对皮肤利什曼病的保护作用的首次证明，并验证了使用DNA免疫以保护利什曼原虫感染的使用。 3）对不同的沙蝇唾液蛋白免疫的发现，抗leishmania免疫反应对疾病的保护或加剧。 研究了两种与P. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. to的影响的影响。用这些分子免疫DNA免疫导致与主要寄生虫挑战时感染的对比。 PPSP15免疫的小鼠显示出持久的保护，而PPSP44免疫会加剧感染（与对照组相比，寄生虫负荷没有增加）。这表明使用这些不同分子的免疫会改变抗Leishmania免疫力的进程。 感染后两周的CD4+ T细胞中有31.5％在PPSP15小鼠中产生IFN-，而PPSP4444小鼠为7.1％。 PPSP44小鼠的IL-4产生细胞高三倍。在与SGH和L. Major挑战后的两个小时，相对于PPSP44小鼠，PPSP15小鼠的表达谱显示出高于3倍的IFN和IL-12-R2和20倍下的IL-4表达，这表明唾液蛋白差异化蛋白质差异性抗抗乳球的免疫。这首先表明对唾液蛋白的免疫力（PPSP44）导致疾病增强，并强调适当选择基于载体的疫苗候选物的重要性。 4）发现，对沙蝇载体的唾液蛋白免疫可预防仓鼠模型中内脏利什曼病的致命结果。 研究了两种与P. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. to的影响的影响。用这些分子免疫DNA免疫导致与主要寄生虫挑战时感染的对比。 PPSP15免疫的小鼠显示出持久的保护，而PPSP44免疫会加剧感染（与对照组相比，寄生虫负荷没有增加）。这表明使用这些不同分子的免疫会改变抗Leishmania免疫力的进程。在这项工作中，我们证明了并非所有诱导DTH的P. papatasi sand蝇唾液分子都普遍保护重大感染。这首先表明对唾液蛋白的免疫力（PPSP44）导致疾病增强，并强调适当选择基于载体的疫苗候选物的重要性。 5）从未知序列或功能的新型唾液分子中鉴定生物学活性。 大多数沙蝇唾液蛋白，包括鉴定的保护性分子，都是新颖的，没有指定的生物学功能。在哺乳动物表达系统中生产可溶性重组蛋白，以测试潜在的生物学活性。我们从LU鉴定出有效的抗凝唾液蛋白。 longipalpis特定结合因子Xa的活性位点。我们还确定了四种唾液重组蛋白，它们通过特异性结合与C3B和一种抑制经典补体途径的蛋白质来抑制补体的替代途径。这些分子具有用于治疗各种人类疾病的潜力。这项工作不仅表明沙蝇的抗凝剂抑制剂是一种新型的凝血酶原酶抑制剂，而且还使其成为纠正人类医学中促凝的活性药物的潜在活性药物。 C3B抑制剂可能是潜在的新药物，可以纠正与补体有关的人类疾病，例如自身免疫性疾病和由于补体而引起的手术后并发症。 6）鉴定lutzomyia longipalpis的最丰富的中肠转录本，即chagasi利什曼原虫的载体。 我们已经从LU的中肠组织生成并测序了五个cDNA文库。 longipalpis;在糖餐后，包括摄食乳杆菌的糖粉和血液粉包括中肠组织。 将所有cDNA库序列结合起来，每一个重叠群产生了655个重叠群，2279个单例和9.45个序列，总共约有10,000个序列。 该转录组分析代表了从特定的砂蝇组织报告的最大序列数据集，并提供了在sand蝇lu肠道中存在的转录本的进一步信息。 longipalpis。