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: 10272099
• 负责人: Jesus Valenzuela
• 金额: $ 169.82万
• 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/10272099
• 关键词: Adjuvant; Adult; Aedes; Allergic Reaction; Animals; Anopheles gambiae; Antibiotic Resistance; Antibodies; Antibody Response; Apyrase; Area; Basic Science; Binding; Biochemical; Bioinformatics; Bite; Blood; Canis familiaris; Chromosomes; Clinical Protocols; Clustered Regularly Interspaced Short Palindromic Repeats; Culicidae; Cutaneous Leishmaniasis; Deposition; Development; Disease; Double-Blind Method; Enrollment; Entomology; Erythema; Event; Exanthema; Exhibits; Exposure to; Gel; Gene Expression Profile; Generations; Genes; Genetic Engineering; Genetic Transcription; Human; Immune; Immune response; Immunity; Immunization; Immunize; Immunocompromised Host; Immunoglobulin G; Immunologic Memory; Immunologics; Infection; Injections; Insect Bites; Insect Vectors; Insecta; Institution; Interferons; Knock-out; Knowledge; Laboratories; Leishmania; Leishmania infantum; Leishmaniasis; Lesion; Lutzomyia genus; Masks; Midgut; Modeling; Molecular; Molecular Biology; Monitor; Montanide ISA-51; Mus; Needles; Nonprofit Organizations; Pain; Parasites; Parasitic Diseases; Participant; Peptide Vaccines; Peripheral Blood Mononuclear Cell; Placebos; Principal Component Analysis; Production; Protein Family; Proteins; Pruritus; Public Health; Randomized; Recombinant Proteins; Recording of previous events; Reporting; Research; Risk; Rodent Model; Safety; Saliva; Salivary; Salivary Glands; Salivary Proteins; Sand Flies; Seasons; Site; Sterility; Subcutaneous Injections; Sustainable Development; Swelling; Symptoms; Testing; Time; Transcript; Translating; Trimethoprim-Sulfamethoxazole; United States National Institutes of Health; Vaccinated; Vaccines; Vector-transmitted infectious disease; Visceral Leishmaniasis; Water; aged; base; clinical center; computer generated; differential expression; disorder control; epidemiology study; exosome; experimental study; feeding; fundamental research; genome editing; immunogenic; immunogenicity; interest; microbiome; molecular vector; mutant; neglect; nonhuman primate; off-target mutation; pathogen; peptide based vaccine; phase I trial; placebo group; success; tool; transcriptome sequencing; transmission process; treatment group; vaccine candidate; vaccine trial; vector; vector competence; vector-borne

The accomplishments of the section are: 1. We assessed the safety and immunogenicity of Anopheles gambiae saliva vaccine (AGS-v), a peptide-based vaccine derived from four A gambiae salivary proteins, in humans. In this randomised, placebo-controlled, double-blind, phase 1 trial, participants were enrolled at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Participants were eligible if they were healthy adults, aged 18-50 years with no history of severe allergic reactions to mosquito bites. Participants were randomly assigned (1:1:1), using block randomisation and a computer-generated randomisation sequence, to treatment with either 200 nmol of AGS-v vaccine alone, 200 nmol of AGS-v with adjuvant (Montanide ISA 51), or sterile water as placebo. Participants and clinicians were masked to treatment assignment. Participants were given a subcutaneous injection of their allocated treatment at day 0 and day 21, followed by exposure to feeding by an uninfected Aedes aegypti mosquito at day 42 to assess subsequent risk to mosquito bites in a controlled setting. No systemic safety concerns were identified; however, one participant in the adjuvanted vaccine group developed a grade 3 erythematous rash at the injection site. Pain, swelling, erythema, and itching were the most commonly reported local symptoms and were significantly increased in the adjuvanted vaccine group compared with both other treatment groups (nine 53% of 17 participants in the adjuvanted vaccine group, two 13% of 16 in the vaccine only group, and one 6% of 16 in the placebo group; p=0004). By day 42, participants who were given the adjuvanted vaccine had a significant increase in vaccine-specific total IgG antibodies compared with at baseline than did participants who were give vaccine only (absolute difference of log10-fold change of 064 95% CI 039 to 089; p=00002) and who were given placebo (062 034 to 091; p=00001). We saw a significant increase in IFN- production by peripheral blood mononuclear cells at day 42 in the adjuvanted vaccine group compared with in the placebo group (absolute difference of log10 ratio of vaccine peptide-stimulated vs negative control 017 95% CI 0061 to 027; p=0009) but we saw no difference between the IFN- production in the vaccine only group compared with the placebo group (0022 -0072 to 0116; p=063). AGS-v was well tolerated, and, when adjuvanted, immunogenic. These findings suggest that vector-targeted vaccine administration in humans is safe and could be a viable option for the increasing burden of vector-borne disease. 2. We demonstrated that humans and dogs residing in Tbilisi have little immunological memory to saliva of P. kandelakii, the principal vector of VL. In Tbilisi, Georgia, an endemic area for visceral leishmaniasis (VL), sand flies are abundant for a short period of 3 months. Only 30% of humans and 50% of dogs displayed a weak antibody response to saliva after the end of the sand fly season. Likewise, their peripheral blood mononuclear cells mounted a negligible cellular immune response after stimulation with saliva. RNA seq analysis of wild-caught P. kandelakii salivary glands established the presence of a typical salivary repertoire that included proteins commonly found in other sand fly species such as the yellow, SP15 and apyrase protein families. This indicates that the absence of immunity to P. kandelakii saliva in humans and dogs from Tbilisi is probably caused by insufficient exposure to sand fly bites. This absence of immunity to vector saliva will influence the dynamics of VL transmission in Tbilisi and other endemic areas with brief sand fly seasons. 3. We evaluated the capacity of Lu. longipalpis sand flies to acquire, maintain, and transmit L. major parasites by reproducing the natural Leishmania transmission cycle under laboratory conditions. Lutzomyia longipalpis sand flies are the major natural vector of Leishmania infantum parasites, responsible for transmission of visceral leishmaniasis in the New World. Several experimental studies have demonstrated the ability of Lu. longipalpis to sustain development of different Leishmania species. However, no study had explored in depth the potential vector competence of Lu. longipalpis for Leishmania species other than L. infantum. Here, we show that Lu. longipalpis is a competent vector of L. major parasites, being able to acquire parasites from active cutaneous leishmaniasis lesions, sustain mature infections, and transmit them to naive hosts, causing disease. 4. We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum. Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies. 5. We tested a second generation leishmanization using a CRISPR genome edited L. major strain (LmCen-/-). LmCen-/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen-/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization results in protection and an immune response comparable to leishmanization. LmCen-/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

该科的成果有： 1. 我们评估了冈比亚按蚊唾液疫苗 (AGS-v) 的安全性和免疫原性，这是一种源自四种冈比亚按蚊唾液蛋白的肽疫苗。在这项随机、安慰剂对照、双盲、1 期试验中，参与者在美国马里兰州贝塞斯达的国立卫生研究院临床中心入组。参与者如果是健康成年人，年龄在 18 至 50 岁之间，没有对蚊虫叮咬的严重过敏反应史，则符合资格。使用分组随机化和计算机生成的随机化序列，将参与者随机分配 (1:1:1)，分别接受 200 nmol AGS-v 疫苗单独治疗、200 nmol AGS-v 佐剂 (Montanide ISA 51) 治疗、或无菌水作为安慰剂。参与者和临床医生不知道治疗分配情况。参与者在第 0 天和第 21 天皮下注射分配的治疗药物，然后在第 42 天接受未感染的埃及伊蚊的喂养，以评估随后在受控环境中被蚊子叮咬的风险。未发现系统性安全问题；然而，佐剂疫苗组的一名参与者在注射部位出现了 3 级红斑皮疹。疼痛、肿胀、红斑和瘙痒是最常报告的局部症状，与其他两个治疗组相比，佐剂疫苗组的症状显着增加（佐剂疫苗组 17 名参与者中的 9 名参与者为 53%，而对照组 16 名参与者中的 2 名参与者为 13%）。仅疫苗组，以及安慰剂组 16 人中的 6%；p=0004）。到第 42 天，与仅接种疫苗的参与者相比，接种佐剂疫苗的参与者的疫苗特异性总 IgG 抗体在基线时显着增加（log10 倍变化的绝对差异 064 95% CI 039 至 089 ；p=00002）以及服用安慰剂的人（062 034 至 091；p=00001）。我们发现，与安慰剂组相比，第 42 天时，佐剂疫苗组外周血单核细胞产生的 IFN- 显着增加（疫苗肽刺激与阴性对照的 log10 比率的绝对差异 017 95% CI 0061 至 027； p=0009），但我们发现仅疫苗组与安慰剂组相比，IFN-产生没有差异（0022 -0072 至0116；p=063)。 AGS-v 具有良好的耐受性，并且在添加佐剂后具有免疫原性。这些发现表明，针对人类的载体疫苗接种是安全的，并且可能是解决日益增加的载体传播疾病负担的可行选择。 2. 我们证明，居住在第比利斯的人类和狗对 VL 的主要载体坎德拉基疟原虫的唾液几乎没有免疫记忆。 格鲁吉亚第比利斯是内脏利什曼病（VL）流行区，白蛉在短短三个月内大量繁殖。白蛉季节结束后，只有 30% 的人类和 50% 的狗对唾液表现出微弱的抗体反应。同样，他们的外周血单核细胞在唾液刺激后产生的细胞免疫反应可以忽略不计。对野生捕获的 P. kandelakii 唾液腺的 RNA seq 分析证实了典型唾液库的存在，其中包括其他白蛉物种中常见的蛋白质，如黄色、SP15 和腺苷三磷酸双磷酸酶蛋白家族。这表明第比利斯人和狗对坎德拉基假单胞菌唾液缺乏免疫力可能是由于白蛉叮咬暴露不足造成的。对媒介唾液缺乏免疫力将影响第比利斯和其他白蛉季节短暂的流行地区的病毒传播动态。 3.我们对卢的能力进行了评估。 longipalpis 沙蝇通过在实验室条件下复制自然利什曼原虫传播周期来获取、维持和传播主要寄生虫。 Lutzomyia longipis 沙蝇是婴儿利什曼原虫寄生虫的主要自然媒介，负责新世界内脏利什曼病的传播。多项实验研究证明了卢的能力。 longipalpis 维持不同利什曼原虫物种的发育。然而，还没有研究深入探讨卢的潜在矢量能力。 longipalpis 为除婴儿利什曼原虫以外的利什曼原虫种。在这里，我们展示一下卢。 longipalpis 是主要利什曼原虫寄生虫的有效载体，能够从活跃的皮肤利什曼病病变处获取寄生虫，维持成熟的感染，并将其传播给幼稚宿主，从而引起疾病。 4. 我们对感染婴儿乳杆菌寄生虫后未改变的长须白蛉中肠进行 RNAseq。 RNAseq 在寄生虫发育过程中的不同时间点进行。主成分分析分离了对应于不同利什曼原虫前鞭毛体阶段、原循环、内单胞、细单胞和后循环的转录本。重要的是，当比较白蛉中利什曼原虫各个阶段的顺序发育时，存在大量差异表达基因。前循环体和长内单胞体前鞭毛体之间存在836个差异表达（DE）基因； 113个内单胞菌和细单胞菌前鞭毛体之间的DE基因；和302个细单胞菌和后循环前鞭毛体之间的DE基因。大多数 DE 基因在不同阶段之间不重叠，突出了利什曼原虫每个阶段的独特性。此外，利什曼原虫寄生虫的不同阶段表现出跨染色体的特定转录富集。利用白蛉中肠发育过程中不同利什曼原虫阶段所表现出的转录特征，我们确定了每个阶段主要富集的基因，从而识别了婴儿利什曼原虫的多个潜在阶段特异性标记。 总体而言，这些发现证明了白蛉载体内利什曼原虫寄生虫的转录可塑性，并提供了一系列潜在的阶段特异性标记，可进一步开发为流行病学研究的分子工具。 5. 我们使用 CRISPR 基因组编辑的大型利什曼菌株 (LmCen-/-) 测试了第二代利什曼化。 LmCen-/- 是一种基因工程中心蛋白基因敲除突变株，无抗生素抗性标记，并且不具有可检测的脱靶突变。用 LmCen-/- 免疫的小鼠在受到 L.major 感染的白蛉攻击后没有明显的病变，而未免疫的动物则出现大的进行性病变，寄生虫负荷高出 2 个对数倍。 LmCen-/- 免疫可产生与利什曼原化相当的保护和免疫反应。 LmCen-/- 是安全的，因为它不会在免疫功能低下的小鼠中引起疾病​​，诱导宿主针对媒介白蛉攻击提供强大的保护，并且由于它不含标记，因此可以进行人体疫苗试验。