Immunobiology, molecular virology and countermeasures of highly pathogenic viruses

免疫生物学、分子病毒学及高致病性病毒对策

• 批准号: 10692213
• 负责人: Andrea Marzi
• 金额: $ 817.99万
• 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/10692213
• 关键词: Africa; Angola; Animal Model; Animals; Anti-CD47; Antibodies; Antibody Therapy; Antigen Presentation; Antigens; Attenuated; Australia; B-Cell Activation; B-Lymphocytes; Blood specimen; Body Weight decreased; CD47 gene; CD8-Positive T-Lymphocytes; Cell Culture Techniques; Chimeric Proteins; Chiroptera; Closure by clamp; Collaborations; Collection; Containment; Cryopreservation; Data; Dengue; Development; Disease; Disease Outbreaks; Disease Outcome; Dose; Ebola Vaccines; Ebola virus; Emerging Communicable Diseases; Exanthema; Exhibits; Family; Ferrets; Fever; Filoviridae Infections; Filovirus; GTP-Binding Protein alpha Subunits, Gs; Gene Expression; Genetic; Genetic Transcription; Genotype; Ghana; Glycoproteins; Guinea; Hamsters; Hemorrhage; Human; Hungary; IFNAR1 gene; Immune; Immune response; Immunobiology; Immunoglobulin G; Immunoglobulin M; Immunology; Infection; Inflammatory; Integration Host Factors; Intramuscular; Ivory Coast; Kentucky; Knowledge; Laboratories; Lassa virus; Macaca fascicularis; Marburg Virus Disease; Marburgvirus; Mediating; Middle East Respiratory Syndrome Coronavirus; Modeling; Molecular; Molecular Virology; Mus; National Institute of Allergy and Infectious Disease; Nipah Virus; Pan Genus; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Phagocytosis; Predisposition; Preparation; Public Health; Queensland; RNA Sequences; Readiness; Recording of previous events; Reporting; Research; Route; Sampling; Sequence Analysis; Serum; Severity of illness; Sierra Leone; Signal Transduction; Subunit Vaccines; Switzerland; Symptoms; T cell response; Taxonomy; Technology; Testing; Therapeutic; Thrombocytopenia; Time; Tissue Sample; United States National Institutes of Health; Up-Regulation; Vaccinated; Vaccination; Vaccines; Vesicular stomatitis Indiana virus; Viral; Viral Antigens; Viral Pathogenesis; Viremia; Virulence Factors; Virus; Virus Diseases; Whole Blood; antiviral immunity; base; cytokine; forest; frontier; genetic approach; human disease; immune activation; immunogenicity; immunopathology; improved; innovation; insight; intraperitoneal; longitudinal analysis; macrophage; mouse model; neutralizing antibody; novel; pathogen; pathogenic virus; prevent; response; reverse genetics; transcriptomics; unvaccinated; vaccine candidate; vaccine development; vaccine evaluation; vaccine platform; viral RNA

Filovirus pathogenesis 1. Tai Forest virus (TAFV) causes severe disease and lethality in cynomolgus macaques TAFV is a lesser-known ebolavirus that has caused one reported human case of infection in Ivory Coast in 1994. The patient developed dengue-like symptoms including fever and rash and was evacuated to Basel, Switzerland. There, the disease was treated, and the patient survived without sequelae. TAFV has also caused lethal disease outbreaks in chimpanzees in the Ta Forest in West Africa. Limited research has been performed with this human-pathogenic virus. With the recent emergence of Ebola virus (EBOV) and Marburg virus (MARV) in West Africa, we wanted to fill knowledge gaps regarding this virus to inform outbreak response preparedness. TAFV disease and pathogenesis was assessed after intramuscular (IM) infection with 1,000 PFU, the standard filovirus infection dose and route for NHPs. We had access to two different TAFV stock preparations (distinctive in their cell culture passaging history) which were derived from the Swiss TAFV isolate. One stock (TAFV-RML) had a confirmed 7U genotype whereas the other one (TAFV-UTMB) presented with a G insertion at the 7U editing sit). Both stocks grew to similar titers in cell culture and were used to infect groups of NHPs. Unexpectedly, infection with the TAFV-RML stock caused no disease despite the 7U genotype, yet the NHPs seroconverted to TAFV GP. In contrast, the TAFV-UTMB stock caused severe disease and lethality in NHPs within 8-11 days (Fletcher et al., in preparation). Histopathologic analysis of key tissue samples revealed severe damage in TAFV-UTMB-infected NHPs. Sequence analysis of terminal blood samples from these NHPs revealed a uniform reversion to a 7U genotype. Currently, host transcriptomics and TAFV RNA sequences are being analyzed in longitudinal blood samples in collaboration with Prof. Ilhem Messaoudi (U Kentucky). This data is providing valuable insight into the human pathogenic potential of this virus. 2. Blocking CD47 signaling does not impact Ebola virus infection in mice In collaboration with Dr. Kim Hasenkrug (NIAID) we tested if anti-CD47 antibody treatment results in a benefit against EBOV infection in mice. Upregulation of CD47 is well-established as a mechanism of inhibiting macrophage-mediated phagocytosis and antigen presentation, possibly to prevent immune overactivation. We found that mice treated with an anti-CD47 antibody exhibited increased activation of B cells and increases in recently cytolytic CD8 T cells. However, anti-CD47-treated mice also exhibited significant weight loss, higher virus titers, and increased inflammatory cytokine expression before succumbing more rapidly to disease. Significantly, these results demonstrate that in the context of a rapidly progressing hemorrhagic viral disease, CD47 attenuates immunopathology and disease severity (Rao et al., Antiviral Research 2021). 3. Pathogenesis of novel filoviruses in IFNAR-/- in mice Filovirus sequences have been found in bats in Sierra Leone, Hungary and other places. We received Bombali virus (BOMV; recovered using reverse genetics) and Lloviou virus (LLOV; isolated from bats in Hungary) from collaborators and propagated these viruses at RML. In order to assess the pathogenic potential of these viruses, we started with infection of IFNAR-/- mice, a model in which some filoviruses can cause disease. First infection studies in these mice investigating intraperitoneal and intranasal infection with a high and a low dose of these viruses did not result in disease (Fletcher et al., unpublished data). Because of the limitations of this mouse model, further pathogenesis studies in ferrets and NHPs are planned. Filovirus vaccine development 1. VSV-MARV rapidly protects NHPs from lethal disease MARV is on the priority list of the WHO for countermeasure development. Cases of MARV disease (MVD) identified in Guinea (August 2021) and Ghana (July 2022) highlighted its potential impact on regional public health. We previously developed a VSV-based vaccine expressing the MARV Angola GP (VSV-MARV) and demonstrated uniform protection of NHPs with a single dose within 28 days. In this project, we investigated the fast-acting potential of this vaccine by challenging NHPs by the IM route with MARV 14, 7 or 3 days after a single IM vaccination with 107 PFU VSV-MARV. We found that 100% of the animals survived when vaccinated at 7 or 14 days and 75% of the animal survived when vaccinated 3 days prior to lethal MARV challenge. Transcriptional analysis of longitudinal whole blood samples indicated activation of B cells and antiviral defense pathways after VSV-MARV vaccination. Only the 14-day vaccination group (d-14) had detectable MARV GP-specific IgM at the time of challenge, likely contributing to the limited transcriptional changes after challenge that were detected in these NHPs in comparison to baseline samples. In the 7-day vaccination group (d-7), we detected gene expression profiles indicative of a recall response on day 9 post-challenge but limited transcriptional changes at all other time points analyzed when compared to baseline samples. In the 3-day vaccination group (d-3), transcriptional analysis of samples from surviving NHPs revealed strong innate immune activation. In contrast, the animal that succumbed to disease in this group lacked signatures of antiviral immunity, likely contributing to disease outcome (Marzi et al., Frontiers in Immunology 2021). This data highlights the applicability of the VSV-MARV vaccine in outbreak situations and the development for human use is underway. 2. A single dose of VSV-TAFV uniformly protects NHPs from disease Because ebolaviruses vary in their antigenicity, we wanted to expand our collection to include a VSV-based vaccine for every species with demonstrated potential to cause human disease. We developed a TAFV-specific VSV vaccine (VSV-TAFV) expressing the TAFV GP as viral antigen to investigate protection against TAFV infection. The immunogenicity of a single dose of 107 PFU VSV-TAFV was assessed in cynomolgus macaques vaccinated one month prior to IM TAFV challenge. Vaccinated NHPs developed antigen-specific IgG reaching peak levels 14 days after vaccination and were uniformly protected from disease after challenge. TAFV RNA was not detected within the vaccinated NHPs in any sample collected throughout the study. In contrast, all unvaccinated NHPs succumbed between day 8 and 11 after challenge presenting with fever, increasing viremia and thrombocytopenia starting on day 6 after challenge. Analysis of serum cytokine levels, antigen-specific B and T cell responses in cryopreserved PBMC preparations is ongoing. In addition, host transcriptomics will be investigated in longitudinal blood samples. These data demonstrate that the VSV-TAFV is a viable single-dose vaccine ideal for use in outbreaks and should be developed for human use (Fletcher et al., in preparation). 3. A molecular clamp vaccine against EBOV In a collaboration with Drs. Daniel Watterson and Keith Chappell (U Queensland, Australia) we characterized a subunit-based clamp vaccine against EBOV in the hamster model. This subunit vaccine platform technology, the molecular clamp, was applied to four viruses from divergent taxonomic families in this project: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), EBOV, Lassa virus (LASV) and Nipah virus (NiV). Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge (Young et al., Frontiers in Immunology - accepted

丝状病毒发病机制 1. 泰森林病毒（TAFV）导致食蟹猴严重患病并致死 TAFV 是一种鲜为人知的埃博拉病毒，1994 年在科特迪瓦报告了一起人类感染病例。该患者出现了发烧和皮疹等登革热样症状，并被疏散到瑞士巴塞尔。在那里，疾病得到了治疗，患者幸存下来，没有留下任何后遗症。 TAFV 还导致西非塔森林黑猩猩爆发致命疾病。对这种人类致病病毒的研究有限。随着最近在西非出现埃博拉病毒 (EBOV) 和马尔堡病毒 (MARV)，我们希望填补有关该病毒的知识空白，为疫情应对准备提供信息。 在用 1,000 PFU 肌内 (IM) 感染后评估 TAFV 疾病和发病机制，这是 NHP 的标准丝状病毒感染剂量和途径。我们获得了两种不同的 TAFV 储备制剂（其细胞培养传代历史不同），它们源自瑞士 TAFV 分离株。一种股票（TAFV-RML）具有确认的 7U 基因型，而另一种股票（TAFV-UTMB）则在 7U 编辑位点处出现 G 插入）。两种原种在细胞培养中都生长到相似的滴度，并用于感染 NHP 组。出乎意料的是，尽管基因型为 7U，TAFV-RML 种群的感染并未引起任何疾病，但 NHP 血清转化为 TAFV GP。相比之下，TAFV-UTMB 库存在 8-11 天内引起 NHP 严重疾病和致死（Fletcher 等人，正在准备中）。对关键组织样本的组织病理学分析显示，TAFV-UTMB 感染的 NHP 存在严重损伤。对这些 NHP 的终末血样进行的序列分析显示，其一致回复为 7U 基因型。目前，正在与 Ilhem Messaoudi 教授（肯塔基大学）合作，对纵向血液样本中的宿主转录组学和 TAFV RNA 序列进行分析。这些数据为了解该病毒对人类的致病潜力提供了宝贵的见解。 2. 阻断CD47信号传导不会影响小鼠的埃博拉病毒感染 我们与 Kim Hasenkrug 博士 (NIAID) 合作，测试了抗 CD47 抗体治疗是否能对小鼠对抗 EBOV 感染产生益处。 CD47 的上调已被证实是一种抑制巨噬细胞介导的吞噬作用和抗原呈递的机制，可能是为了防止免疫过度激活。我们发现，用抗 CD47 抗体治疗的小鼠表现出 B 细胞活化增加以及最近溶细胞性 CD8 T 细胞增加。然而，抗CD47治疗的小鼠在更快地死于疾病之前也表现出显着的体重减轻、更高的病毒滴度和增加的炎症细胞因子表达。值得注意的是，这些结果表明，在快速进展的出血性病毒性疾病的背景下，CD47 可减弱免疫病理学和疾病严重程度（Rao 等人，Antiviral Research 2021）。 3. 新型丝状病毒在小鼠 IFNAR-/- 中的发病机制 在塞拉利昂、匈牙利等地的蝙蝠体内发现了丝状病毒序列。我们从合作者那里收到了 Bombali 病毒（BOMV；通过反向遗传学恢复）和 Lloviou 病毒（LLOV；从匈牙利的蝙蝠中分离出来），并在 RML 传播了这些病毒。为了评估这些病毒的致病潜力，我们从感染 IFNAR-/- 小鼠开始，在该模型中，一些丝状病毒可以引起疾病。对这些小鼠进行的首次感染研究调查了高剂量和低剂量这些病毒的腹膜内和鼻内感染，但没有导致疾病（Fletcher 等人，未发表的数据）。由于该小鼠模型的局限性，计划在雪貂和 NHP 中进行进一步的发病机制研究。 丝状病毒疫苗开发 1. VSV-MARV 快速保护 NHP 免受致命疾病的侵害 MARV 已被列入 WHO 制定对策的优先名单。几内亚（2021 年 8 月）和加纳（2022 年 7 月）发现的 MARV 病 (MVD) 病例凸显了其对区域公共卫生的潜在影响。我们之前开发了一种表达 MARV Angola GP (VSV-MARV) 的基于 VSV 的疫苗，并在 28 天内证明了单剂对 NHP 的均匀保护。在该项目中，我们在使用 107 PFU VSV-MARV 进行单次 IM 疫苗接种后 14、7 或 3 天，通过 IM 途径用 MARV 攻击 NHP，从而研究了该疫苗的速效潜力。我们发现，在 7 或 14 天接种疫苗时，100% 的动物存活，在致命 MARV 攻击前 3 天接种疫苗时，75% 的动物存活。纵向全血样本的转录分析表明，VSV-MARV 疫苗接种后 B 细胞和抗病毒防御途径被激活。只有 14 天疫苗接种组 (d-14) 在攻击时具有可检测到的 MARV GP 特异性 IgM，这可能是与基线样本相比，攻击后在这些 NHP 中检测到的有限转录变化的原因。在 7 天疫苗接种组 (d-7) 中，我们在攻击后第 9 天检测到表明记忆反应的基因表达谱，但与基线样本相比，在分析的所有其他时间点的转录变化有限。在 3 天疫苗接种组 (d-3) 中，对幸存 NHP 样本的转录分析显示出强烈的先天免疫激活。相比之下，该组中死于疾病的动物缺乏抗病毒免疫特征，这可能会导致疾病结果（Marzi 等人，免疫学前沿，2021 年）。该数据凸显了 VSV-MARV 疫苗在疫情爆发情况下的适用性，并且人类用途的开发正在进行中。 2. 单剂量 VSV-TAFV 一致保护 NHP 免受疾病侵害 由于埃博拉病毒的抗原性各不相同，因此我们希望扩大我们的收集范围，为每个已证明有可能导致人类疾病的物种提供基于 VSV 的疫苗。我们开发了一种表达 TAFV GP 作为病毒抗原的 TAFV 特异性 VSV 疫苗 (VSV-TAFV)，以研究针对 TAFV 感染的保护作用。在 IM TAFV 攻击前一个月接种的食蟹猴中评估了单剂量 107 PFU VSV-TAFV 的免疫原性。接种疫苗的 NHP 产生的抗原特异性 IgG 在接种后 14 天达到峰值水平，并且在攻击后得到一致的保护，免受疾病影响。在整个研究过程中收集的任何样本中，未在接种疫苗的 NHP 中检测到 TAFV RNA。相比之下，所有未接种疫苗的 NHP 在攻击后第 8 至 11 天之间死亡，从攻击后第 6 天开始出现发烧、病毒血症增加和血小板减少症。对冷冻保存的 PBMC 制剂中的血清细胞因子水平、抗原特异性 B 和 T 细胞反应的分析正在进行中。此外，还将在纵向血液样本中研究宿主转录组学。这些数据表明，VSV-TAFV 是一种可行的单剂量疫苗，非常适合在疫情爆发时使用，并且应该开发供人类使用（Fletcher 等人，正在准备中）。 3. 埃博拉病毒分子钳疫苗 与博士合作。 Daniel Watterson 和 Keith Chappell（澳大利亚昆士兰大学）在仓鼠模型中描述了一种基于亚单位的针对 EBOV 的钳夹疫苗。该项目中，这种亚单位疫苗平台技术（分子钳）应用于来自不同分类科的四种病毒：中东呼吸综合征冠状病毒（MERS-CoV）、埃博拉病毒、拉沙病毒（LASV）和尼帕病毒（NiV）。在测试的四种疫苗中，钳稳定的 MERS-CoV 刺突、埃博拉病毒糖蛋白和 NiV 融合蛋白刺激了中和免疫反应。只有钳稳定的 LASV 糖蛋白前体未能引发病毒中和抗体。 MERS-CoV 和 EBOV 候选疫苗均在动物模型中进行了测试，发现可以提供针对病毒攻击的保护（Young 等人，免疫学前沿 - 已接受）