Viral Hemorrhagic Fevers: Disease Modeling and Transmission

病毒性出血热：疾病建模和传播

• 批准号: 8336299
• 负责人: Heinrich Feldmann
• 金额: $ 334.74万
• 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/8336299
• 关键词: Acute; Acute Disease; Address; Andes Virus; Animal Model; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Antigens; Antiviral Agents; Arenavirus; Arthropods; Attenuated; Basic Science; Biology; Brain; Breeding; CD4 Positive T Lymphocytes; Cathepsin L; Cathepsins; Cathepsins B; Cause of Death; Cavia; Cell Culture Techniques; Cells; Central Africa; Cercopithecus pygerythrus; Chiroptera; Coagulation Process; Collaborations; Congo; Containment; Crimean Hemorrhagic Fever; Cysteine Protease; Cytomegalovirus Vaccines; Deer Mouse; Development; Diagnostic; Diagnostic tests; Disease; Disease Progression; Disease model; Disulfides; Dose; Ebola virus; Ecology; Emergency Situation; Family suidae; Filovirus; Fostering; Fruit; Functional disorder; Future; Gene Expression Profile; Genes; Glycoproteins; Goals; Hamsters; Hantavirus; Hendra Virus; Henipavirus; Hour; Human; Immune Response Genes; Immune response; Immunity; In Vitro; Indigenous; Infection; Infectious Diseases Research; Inflammatory; Interferons; Intravenous; Investigation; Knockout Mice; Laboratories; Lassa virus; Lesion; Liberia; Link; Lung; Macaca; Mali; Mastomys; Mesocricetus auratus; Modeling; Molecular; Molecular Evolution; Molecular and Cellular Biology; Monitor; Monoclonal Antibodies; Mus; Neurologic; Nipah Virus; Nucleocapsid Proteins; Nucleoproteins; Organ; Orthobunyavirus; Paramyxovirus; Pathogenesis; Pathogenicity; Pathology; Peptide Hydrolases; Phase; Philippines; Play; Pongidae; Population; Preparation; Production; Prophylactic treatment; Proteins; Public Health; Quarantine; RNA Editing; Recombinants; Research; Research Activity; Respiratory distress; Reston; Reverse Transcriptase Polymerase Chain Reaction; Ribavirin; Rodent; Role; STAT1 gene; Sampling; Scientist; Signal Transduction; Sin Nombre virus; Site; Staging; Study models; Symptoms; System; T cell response; T-Lymphocyte Epitopes; Technology; Testing; Therapeutic Intervention; Time; Vaccination; Vaccines; Vesicular stomatitis Indiana virus; Viral; Viral Encephalitis; Viral Hemorrhagic Fevers; Viral Pathogenesis; Virulence; Virus; Virus Diseases; Virus Replication; Work; base; dimer; disease transmission; epidemiology study; genetic analysis; glycoprotein G; immunoregulation; in vivo; innovation; interest; mouse model; neutralizing antibody; nonhuman primate; novel vaccines; pathogen; positional cloning; prevent; research study; respiratory; respiratory distress syndrome; response; success; therapeutic vaccine; tool; transmission process; type I interferon receptor; vaccine candidate; vector; vector vaccine; virus host interaction

(I) Disease Models and Pathogenesis Goal: To develop disease/infection models for studying pathogenesis and research on vaccines/therapeutics. Rodents as disease models: The Syrian hamster has been broadly used in infectious disease research but research tools are limited. We have developed quantitative real time RT-PCR for monitoring hamster immune response genes (Zivcec 2011) and sequenced the transcriptome of the Syrian hamster. Annotation is still ongoing in collaboration with RTB (Dr. S. Porcella). The Andes virus hamster model is characterized by a strong suppression of innate immune responses during the early stage of infection and massive activation of pro-inflammatory and Th1/Th2 responses during the symptomatic phase suggesting that infection-derived immune modulation is important to pathogenesis (Safronetz, in revision). The lethal Syrian hamster model for Nipah and Hendra virus infections is characterized by acute severe respiratory distress and severe neurological symptoms. Respiratory symptoms were more prevalent in animals challenged with a high dose, whereas animals challenged with a low dose mainly showed neurological signs of infection (Rockx 2011). We established an acute disease model for Crimean-Congo hemorrhagic fever (CCHF) using mice lacking the type I interferon receptor. This model recapitulates most hallmarks of human CCHF disease including coagulation abnormalities (Zivcec, in preparation). The emergence of Reston ebolavirus (REBOV) in domestic swine in the Philippines has caused a renewed interest in its pathogenicity. We have established a mouse disease models based on STAT1-/-mice, which may allow future pathogenesis studies (deWit 2011). Nonhuman primates as disease models: We have established lethal disease models in African green monkeys for Nipah and Hendra virus infection. Animals showed severe lesions in lungs and brains which are the main target organs of the infection. The primary cause of death is severe respiratory distress (Rockx 2011). The virulence of a new Lassa strain from Mali was assessed in cynomolgus macaques in comparison with well studied isolates from Sierra Leona and Liberia. This virus (Soromba-R) was found to be less pathogenic which might be explained by a stronger early innate immune response (Safronetz, in preparation). (II) Vaccines and Therapeutics Goal: To develop and characterize fast-acting vaccines and targets for therapeutic intervention for emerging/re-emerging viruses. Our main platform is based on attenuated recombinant vesicular stomatitis virus (rVSV) vaccine vectors. For post-exposure treatment of filovirus infections with rVSV vectors we could increase the treatment window to 24 and 48 hours post-infection with 80 and 30% success, respectively. This supports the use of the rVSV in cases of emergencies such as laboratory exposures (Geisbert 2010). Cross-protective vaccines would be very desirable for filoviruses in Central Africa due to overlapping endemicity zones. Recent studies in guinea pigs demonstrated that an rVSV vaccine expressing a single filovirus glycoprotein cannot achieve cross-protection, but the use of a bivalent vector expressing a second filovirus immunogen in a two-dose regime strongly increased cross-protective immunity (Marzi 2011). Multivalent vaccine vectors targeting different viruses with overlapping endemicity zones are of even greater public health interest. We performed a proof-of-concept study using a bivalent rVSV vector expressing glycoproteins of Ebola and Andes viruses. Complete protection was achieved against lethal challenge with both viruses in the common Syrian hamster model (Tsuda 2011). We have generated a new rVSV vector expressing Andes virus glycoproteins that provided complete protection in the Syrian hamster model. High titered neutralizing antibodies following a single vaccination seem to be important for protection in pre-exposure vaccination. The vaccine was protective when given three days prior to challenge and remained 90% effective even when administered 24 hours post-challenge. Here, a strong innate immune response seems to be the mechanism of protection (Brown 2011). Ebola is a serious problem for the endangered great ape population in Central Africa. To address vaccine needs for remote wildlife populations we evaluated in a proof-of-concept study the use of a disseminating cytomegalovirus vaccine vector expressing a single Ebola nucleoprotein T cell epitope. The strategy provided complete protection against lethal challenge in the Ebola mouse model. Future studies will focus on a disseminating vaccine vector for nonhuman primates (Tsuda 2011). The cellular cysteine proteases cathepsin B & L are proposed to play an important role in Ebola virus replication. We tested Ebola virus replication in cathepsin B & L knockout mice and could demonstrate that cathepsin cleavage is not necessary for Ebola virus replication. We conclude that other proteases can substitute for cathepsins making it difficult to use proteases as antiviral targets (Marzi, in preparation). We identified a new soluble Ebola virus glycoprotein (ssGP) generated through RNA editing of the glycoprotein gene. ssGP is a disulfide-linked dimer and exclusively N-glycosylated, but a function could not yet be assigned to this new protein (Mehedi 2011). We are currently studying the mechanism of editing and its importance for pathogenesis. Preliminary studies have identified Ebola virus VP30 as a necessary factor for RNA editing. We confirmed that the glycoproteins of pathogenic New World hantaviruses appear to be the primary antagonist of RIG-I directed IFN production. In addition, we showed that the ANDV nucleocapsid protein (NP) serves as the primary antagonist in JAK-STAT signaling (Levine 2010). We tested the in vivo efficacy of a monoclonal antibody (mAb) directed against the G glycoprotein of henipaviruses. This mAb has potent in vitro neutralizing activity against Nipah and Hendra viruses. Intravenous application of the antibody as late as three days post infection completely protected African green monkeys against lethal Nipah or Hendra virus challenge (Bossart 2011). We tested the in vivo efficacy of ribavirin against Hendra virus infection in African green monkeys. Treatment did prolong and alter disease progression but was largely unsuccessful (Rockx 2011). We also tested the effect of ribavirin on ANDV replication in the established lethal Syrian hamster model. We concluded that ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses (Safronetz 2011). (III) Ecology and Transmission Goal: Understanding the interaction of virus and reservoir species to prevent transmission into end host. We collected Mastomys natalensis in southern Mali and isolated an arenavirus. Genetic analysis confirmed a unique Lassa virus, Soromba R, and demonstrated for the first time the presence of Lassa in southern Mali (Safronetz 2010). We have developed a framework for animal and human filovirus surveillance in the Republic of Congo. In addition, facilities for basic research will be created for holding, quarantine and breeding of indigenous fruit bat species. This will allow pathogenicity and transmission studies in the potential reservoir species of filoviruses. We have already identified long-term sampling sites (Munster, in preparation). We have established an infection model for Sin nombre virus (hantavirus) to study the immune response to infection in the natural reservoir species, the deer mouse. We have found that the deer mouse mounts a strong anti-inflammatory CD4+ T cell response to infection, thereby limiting pathology and allowing virus persistence (Prescott, in preparation)

（i）疾病模型和发病机理 目标：开发用于研究疫苗/治疗剂的发病机理和研究的疾病/感染模型。 啮齿动物作为疾病模型：叙利亚仓鼠已广泛用于传染病研究中，但研究工具有限。我们已经开发了用于监测仓鼠免疫反应基因的定量实时RT-PCR（ZIVCEC 2011），并测序了叙利亚仓鼠的转录组。注释仍在与RTB（S. Porcella博士）合作。 安第斯山脉病毒仓鼠模型的特征是在感染的早期阶段对先天免疫反应的强烈抑制，以及在症状阶段的促炎和Th1/Th2反应的大规模激活，这表明感染衍生的免疫调节对发病机理很重要（Safronetz，safronetz，Revision）。 NIPAH和HENDRA病毒感染的致命叙利亚仓鼠模型的特征是急性严重呼吸窘迫和严重的神经系统症状。在高剂量挑战的动物中，呼吸道症状更为普遍，而低剂量的动物主要显示出感染的神经系统迹象（Rockx 2011）。 我们使用缺乏I型干扰素受体的小鼠建立了一个用于克里米亚 - 隆戈出血热（CCHF）的急性疾病模型。该模型概括了人类CCHF疾病的大多数标志，包括凝血异常（ZIVCEC，准备中）。 菲律宾家用猪雷斯顿埃博拉病毒（Rebov）的出现引起了对其致病性的重新兴趣。我们已经建立了基于STAT1 - / - 小鼠的小鼠疾病模型，该模型可能允许将来的发病机理研究（Dewit 2011）。 非人类灵长类动物作为疾病模型：我们已经在非洲绿猴中建立了致命的疾病模型，用于尼帕和亨德拉病毒感染。动物在肺和大脑中表现出严重的病变，这是感染的主要靶心器官。 死亡的主要原因是严重的呼吸窘迫（Rockx 2011）。 与从塞拉利昂（Sierra Leona）和利比里亚（Sierra Leona）和利比里亚（Sierra Lela）的良好分离株相比，在cynomolgus猕猴中评估了来自马里新拉萨菌株的毒力。发现该病毒（Soromba-R）的病原体较少，可以通过更强的先天免疫反应（Safronetz，在制备中）来解释。 （ii）疫苗和治疗药 目标：开发和表征快速作用的疫苗和靶标，用于用于新兴/重新出现病毒的治疗干预措施。 我们的主要平台基于减毒的重组囊泡病毒病毒（RVSV）疫苗载体。对于使用RVSV载体对丝状病毒感染的暴露后处理，我们可以分别将治疗窗口增加到24和48小时，分别以80％和30％的成功。这支持在实验室暴露等紧急情况下使用RVSV（Geisbert，2010年）。 由于中非地区重叠，跨保护疫苗对于中非的丝病毒是非常可取的。豚鼠的最新研究表明，表达单一丝状病毒糖蛋白的RVSV疫苗无法实现交叉保护，但是在两剂量方案中表达第二丝病毒免疫原的双价载体的使用强烈增加了跨保护性的免疫力（Marzi 2011）。针对不同病毒的多价疫苗向量具有重叠的地方性区域，其公共卫生的利益更大。我们使用表达埃博拉病毒糖蛋白和安第斯山脉病毒的二价RVSV载体进行了概念验证研究。在普通的叙利亚仓鼠模型中，两种病毒都对两种病毒的致命挑战实现了完全的保护（Tsuda 2011）。 我们已经产生了一种新的RVSV载体，表达了Andes病毒糖蛋白，该糖蛋白在叙利亚仓鼠模型中提供了完全保护。一次疫苗接种后，高滴定的中和抗体对于暴露前疫苗接种似乎很重要。疫苗在挑战前三天给予疫苗保护性，即使在挑战后24小时服用时，疫苗也保持90％。在这里，强烈的先天免疫反应似乎是保护的机制（Brown 2011）。 埃博拉病毒是中非濒临灭绝的大猿人口的严重问题。为了满足远程野生动植物种群的疫苗需求，我们在概念验证研究中评估了传播巨细胞病毒疫苗载体的使用，表达了单个埃博拉蛋白核蛋白T细胞效率。该策略为埃博拉小鼠模型中的致命挑战提供了完全保护。 未来的研究将集中于非人类灵长类动物的传播疫苗载体（Tsuda 2011）。 提议细胞半胱氨酸蛋白酶组织蛋白酶B＆L在埃博拉病毒复制中起重要作用。我们在组织蛋白酶B＆L敲除小鼠中测试了埃博拉病毒复制，并且可以证明埃博拉病毒复制不需要组织蛋白酶裂解。我们得出的结论是，其他蛋白酶可以代替组织蛋白酶，使其难以将蛋白酶用作抗病毒药靶标（Marzi，在制备中）。 我们确定了通过糖蛋白基因的RNA编辑产生的一种新的可溶性埃博拉病毒糖蛋白（SSGP）。 SSGP是一种二硫化合物二聚体，仅是N-糖基化的，但是尚无法分配到该新蛋白质的功能（Mehedi 2011）。 我们目前正在研究编辑机制及其对发病机理的重要性。初步研究已将埃博拉病毒VP30确定为RNA编辑的必要因素。 我们证实，致病性新世界汉坦病毒的糖蛋白似乎是RIG-I指向IFN产生的主要拮抗剂。 此外，我们证明了ANDV核素蛋白（NP）是JAK-STAT信号传导中的主要拮抗剂（Levine 2010）。 我们测试了针对Henipavirus的G糖蛋白的单克隆抗体（MAB）的体内功效。该mAb具有对Nipah和Hendra病毒的体外中和活性。感染后三天，抗体的静脉注射应用完全保护了非洲绿猴，以抵抗致命的尼帕或亨德拉病毒挑战（Bossart 2011）。 我们测试了利巴韦林对非洲绿猴中亨德拉病毒感染的体内功效。治疗确实延长和改变了疾病的进展，但在很大程度上没有成功（Rockx 2011）。 我们还测试了利巴韦林在已建立的致命叙利亚仓鼠模型中对ANDV复制的影响。我们得出的结论是，利巴韦林治疗对暴露后预防抗HPS的汉坦病毒是有益的（Safronetz 2011）。 （iii）生态和传播 目标：了解病毒和水库物种的相互作用，以防止传播到最终宿主。 我们在马里南部收集了Mastomys Natalensis，并隔离了体育病毒。遗传分析证实了一种独特的LASSA病毒，Soromba R，并首次证明了Lassa在马里南部的存在（Safronetz 2010）。 我们已经在刚果共和国开发了一个针对动物和人类货物病毒监测的框架。此外，还将创建基础研究的设施，用于持有，隔离和繁殖土著水果蝙蝠物种。这将允许在潜在的铁病毒物种中进行致病性和传播研究。我们已经确定了长期抽样站点（Munster，在准备中）。 我们已经建立了一种罪恶诺姆布雷病毒（汉塔病毒）的感染模型，以研究天然储层物种鹿小鼠对感染的免疫反应。我们发现，鹿小鼠对感染对感染的强烈抗炎CD4+ T细胞反应进行了限制，从而限制了病理和病毒的持久性（Prescott，在准备中）