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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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专注于白蛉唾液和中肠蛋白的分子方面，重点是了解载体/宿主和载体/寄生虫相互作用，特别是白蛉/利什曼原虫相互作用。单元研究将基本方法与兽医和临床研究相结合，拓宽了我们对动物宿主和人类中载体蛋白的免疫反应与疾病结果之间关系的理解，以及利什曼原虫寄生虫和白蛉中肠蛋白之间的关系，最终开发出基于载体的载体针对被忽视的疾病利什曼病的疫苗。推动本单元研究的两个主要假设是： 1) 对媒介节肢动物唾液蛋白的细胞免疫反应在宿主皮肤中产生了对共同注射的病原体不利的环境，导致间接杀死或加速抗利什曼原虫免疫。识别载体唾液蛋白和保护的相关性，特别是初始免疫事件，将有助于我们了解保护的免疫学基础，并选择候选疫苗来预防病原体传播。 2) 白蛉中肠和利什曼原虫寄生虫之间的特定分子相互作用是利什曼原虫在昆虫媒介中生存和发展到感染阶段所必需的。这些分子相互作用的表征将有助于了解利什曼原虫白蛉相互作用的分子基础，并可能确定传播阻断疫苗的合适靶点。该单位的两个主要项目的成果是： 1) 开发强大的蛋白质重组表达和纯化方法，以测试唾液蛋白质的生物活性，并验证使用 DNA 免疫鉴定的候选疫苗。我们通过将白蛉唾液腺转录物克隆到本单位开发的哺乳动物表达载体VR2001-TOPO（与DNA免疫相同的载体）中，最大限度地表达正确折叠的重组唾液蛋白，并用它转染293F哺乳动物细胞。转染细胞产生可溶性重组蛋白，通过采用优化的纯化方法，我们获得了大量、高纯度且内毒素含量极低的唾液蛋白，用于疫苗、免疫学和生物测定中的测试。 2) 证明重组白蛉唾液蛋白可防止受 L. Major 感染的白蛉的攻击。我们证明，通过 DNA 疫苗免疫所观察到的保护并不是 DNA 质粒的旁观者效应，而是对质粒编码的蛋白质具有特异性。我们测试了所选 DNA 质粒中编码的重组蛋白 LJM11 对受 L. Major 感染的 Lu 的毒力攻击的保护作用。长须沙蝇叮咬。在没有佐剂的情况下用少量重组 LJM11 免疫的小鼠可以免受 10 只受感染白蛉叮咬的攻击（图 4）。这是重组白蛉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 免疫来预防利什曼原虫感染。 3) 研究发现，对不同白蛉唾液蛋白的免疫力会引发抗利什曼原虫免疫反应，从而保护或加剧疾病。研究了来自 P. papatasi 的两种不同的 DTH 诱导唾液蛋白对 L. Major 感染的影响。使用这些分子进行 DNA 免疫，在受到大型利斯特氏菌寄生虫攻击后，会产生截然不同的感染结果。 PpSP15 免疫小鼠显示出持久的保护作用，而 PpSP44 免疫则加剧了感染（与对照组相比，寄生虫负荷没有增加）。这表明用这些不同的分子进行免疫改变了抗利什曼原虫免疫的过程。感染后两周，PpSP15 小鼠中 31.5% 的 CD4+ T 细胞产生 IFN-，而 PpSP44 小鼠中这一比例为 7.1%。 PpSP44 小鼠中产生 IL-4 的细胞数量高出三倍。在用 SGH 和 L. Major 攻击两小时后，与 PpSP44 小鼠相比，PpSP15 小鼠的表达谱显示 IFN- 和 IL-12-R2 高出三倍多，IL-4 表达低 20 倍，这表明唾液中蛋白质差异性地引发抗利什曼原虫免疫力。这首次证明对唾液蛋白（PpSP44）的免疫会导致疾病加重，并强调了正确选择基于载体的候选疫苗的重要性。 4) 研究发现，在仓鼠模型中，对白蛉载体唾液蛋白的免疫力可以防止内脏利什曼病的致命后果。研究了来自 P. papatasi 的两种不同的 DTH 诱导唾液蛋白对 L. Major 感染的影响。使用这些分子进行 DNA 免疫，在受到大型利斯特氏菌寄生虫攻击后，会产生截然不同的感染结果。 PpSP15 免疫小鼠显示出持久的保护作用，而 PpSP44 免疫则加剧了感染（与对照组相比，寄生虫负荷没有增加）。这表明用这些不同分子进行免疫改变了抗利什曼原虫免疫的过程。在这项工作中，我们证明并非所有诱导 DTH 的白蛉唾液分子都能普遍预防利什曼原虫感染，并且用两种诱导 DTH 的唾液蛋白进行免疫产生了与利什曼原虫感染的抵抗力或易感性相关的独特免疫特征。这首次证明对唾液蛋白（PpSP44）的免疫会导致疾病加重，并强调了正确选择基于载体的候选疫苗的重要性。 5) 鉴定序列或功能未知的新型唾液分子的生物活性。大多数白蛉唾液蛋白，包括已识别的保护性分子，都是新颖的，并且不具有指定的生物学功能。在哺乳动物表达系统中产生可溶性重组蛋白以测试潜在的生物活性。我们从 Lu 中鉴定出一种有效的抗凝血唾液蛋白。 longipalpis 特异性结合因子 Xa 的活性位点。我们还鉴定了四种通过特异性结合 C3b 来抑制补体替代途径的唾液重组蛋白和一种抑制补体经典途径的蛋白。这些分子具有用于治疗多种人类疾病的潜力。这项工作不仅证明了白蛉抗凝抑制剂是一种新型凝血酶原酶抑制剂，而且使其成为人类医学中纠正促凝血障碍的潜在活性药物。 C3b抑制剂可能是纠正与补体相关的人类疾病的潜在新药，例如自身免疫性疾病和补体引起的手术后并发症。 6) 鉴定婴儿利什曼原虫恰加斯利什曼原虫的载体 Lutzomyia longipalis 最丰富的中肠转录本。我们从 Lu 的中肠组织中生成了 5 个 cDNA 文库并对其进行了测序。长须;包括糖粉、血粉和存在婴儿乳杆菌的情况下的血粉后的中肠组织。组合所有 cDNA 文库序列产生了 655 个重叠群、2279 个单一重叠群，每个重叠群平均有 9.45 个序列，总共约 10,000 个序列。该转录组分析代表了特定白蛉组织报告的最大序列数据集，并提供了白蛉 Lu 肠道中存在的转录本的进一步信息。长触须。

项目成果

期刊论文数量（20）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies.

DOI：
10.1186/1471-2164-9-15
发表时间：
2008-01-14
期刊：
BMC genomics
影响因子：
4.4
作者：
Jochim RC;Teixeira CR;Laughinghouse A;Mu J;Oliveira F;Gomes RB;Elnaiem DE;Valenzuela JG
通讯作者：
Valenzuela JG

High-throughput approaches to study salivary proteins and genes from vectors of disease.

研究疾病载体唾液蛋白和基因的高通量方法。

DOI：
10.1016/s0965-1748(02)00083-8
发表时间：
2002
期刊：
Insect biochemistry and molecular biology
影响因子：
3.8
作者：
Valenzuela,JesusG
通讯作者：
Valenzuela,JesusG

Exploring the midgut transcriptome of Phlebotomus papatasi: comparative analysis of expression profiles of sugar-fed, blood-fed and Leishmania-major-infected sandflies.

DOI：
10.1186/1471-2164-8-300
发表时间：
2007-08-30
期刊：
BMC GENOMICS
影响因子：
4.4
作者：
Ramalho-Ortigao, Marcelo;Jochim, Ryan C;Anderson, Jennifer M;Lawyer, Phillip G;Pham, Van-My;Kamhawi, Shaden;Valenzuela, Jesus G
通讯作者：
Valenzuela, Jesus G

Exploring tick saliva: from biochemistry to 'sialomes' and functional genomics.

探索蜱唾液：从生物化学到“唾液酸组”和功能基因组学。