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)

（一）疾病模型及发病机制 目标：开发用于研究发病机制和疫苗/治疗研究的疾病/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，但研究工具有限。我们开发了用于监测仓鼠免疫反应基因的定量实时 RT-PCR (Zivcec 2011)，并对叙利亚仓鼠的转录组进行了测序。与 RTB（S. Porcella 博士）的合作仍在继续进行注释。 安第斯病毒仓鼠模型的特点是在感染早期阶段强烈抑制先天免疫反应，并在症状阶段大量激活促炎和 Th1/Th2 反应，这表明感染源性免疫调节对于发病机制很重要（Safronetz） ，修订中）。 尼帕病毒和亨德拉病毒感染的致命叙利亚仓鼠模型的特点是急性严重呼吸窘迫和严重的神经系统症状。在接受高剂量攻击的动物中，呼吸道症状更为普遍，而接受低剂量攻击的动物主要表现出神经系统感染体征（Rockx 2011）。 我们使用缺乏 I 型干扰素受体的小鼠建立了克里米亚-刚果出血热 (CCHF) 急性疾病模型。该模型概括了人类 CCHF 疾病的大多数特征，包括凝血异常（Zivcec，正在准备中）。 菲律宾家猪中出现的雷斯顿埃博拉病毒（REBOV）引起了人们对其致病性的新兴趣。我们已经建立了基于 STAT1-/-小鼠的小鼠疾病模型，这可能允许未来的发病机制研究（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尼帕病毒和亨德拉病毒感染的致命疾病模型。动物的肺部和大脑出现严重病变，这是感染的主要靶器官。 死亡的主要原因是严重的呼吸窘迫（Rockx 2011）。 在食蟹猴中评估了来自马里的一种新拉沙病毒株的毒力，并与来自塞拉利昂和利比里亚的经过充分研究的分离株进行了比较。人们发现这种病毒（Soromba-R）的致病性较低，这可能是由于早期先天免疫反应更强（Safronetz，正在准备中）。 （二）疫苗和治疗药物 目标：开发和表征速效疫苗和针对新出现/重新出现病毒的治疗干预的靶点。 我们的主要平台基于减毒重组水泡性口炎病毒（rVSV）疫苗载体。对于使用 rVSV 载体对丝状病毒感染进行暴露后治疗，我们可以将治疗窗口延长至感染后 24 小时和 48 小时，成功率分别为 80% 和 30%。这支持在实验室暴露等紧急情况下使用 rVSV (Geisbert 2010)。 由于流行区重叠，中非的丝状病毒非常需要交叉保护疫苗。最近对豚鼠的研究表明，表达单一丝状病毒糖蛋白的 rVSV 疫苗无法实现交叉保护，但在双剂量方案中使用表达第二种丝状病毒免疫原的二价载体可大大增加交叉保护性免疫（Marzi 2011）。针对具有重叠流行区的不同病毒的多价疫苗载体具有更大的公共卫生利益。我们使用表达埃博拉病毒和安第斯病毒糖蛋白的二价 rVSV 载体进行了概念验证研究。在常见的叙利亚仓鼠模型中，两种病毒都实现了针对致命攻击的完全保护（Tsuda 2011）。 我们生成了一种表达安第斯病毒糖蛋白的新 rVSV 载体，该载体在叙利亚仓鼠模型中提供了完整的保护。单次疫苗接种后产生的高滴度中和抗体似乎对于暴露前疫苗接种的保护很重要。该疫苗在攻击前 3 天接种时具有保护作用，即使在攻击后 24 小时内接种，仍保持 90% 的有效性。在这里，强烈的先天免疫反应似乎是保护机制（Brown 2011）。 埃博拉病毒对中部非洲濒临灭绝的类人猿种群来说是一个严重的问题。为了满足偏远野生动物种群的疫苗需求，我们在概念验证研究中评估了表达单个埃博拉核蛋白 T 细胞表位的传播性巨细胞病毒疫苗载体的使用。该策略为埃博拉小鼠模型提供了针对致命挑战的完整保护。 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）。 细胞半胱氨酸蛋白酶组织蛋白酶 B 和 L 在埃博拉病毒复制中发挥重要作用。我们在组织蛋白酶 B 和 L 敲除小鼠中测试了埃博拉病毒的复制，并证明组织蛋白酶裂解对于埃博拉病毒复制不是必需的。我们的结论是，其他蛋白酶可以替代组织蛋白酶，因此很难使用蛋白酶作为抗病毒靶点（Marzi，准备中）。 我们鉴定了一种新的可溶性埃博拉病毒糖蛋白（ssGP），它是通过糖蛋白基因的 RNA 编辑产生的。 ssGP 是一种二硫键连接的二聚体，并且完全是 N-糖基化的，但尚未为这种新蛋白质指定功能 (Mehedi 2011)。 我们目前正在研究编辑机制及其对发病机制的重要性。初步研究已确定埃博拉病毒VP30是RNA编辑的必要因子。 我们证实，致病性新世界汉坦病毒的糖蛋白似乎是 RIG-I 指导的 IFN 产生的主要拮抗剂。 此外，我们还发现 ANDV 核衣壳蛋白 (NP) 是 JAK-STAT 信号传导的主要拮抗剂 (Levine 2010)。 我们测试了针对亨尼帕病毒 G 糖蛋白的单克隆抗体 (mAb) 的体内功效。该 mAb 对尼帕病毒和亨德拉病毒具有有效的体外中和活性。感染后三天静脉注射抗体完全保护非洲绿猴免受致命的尼帕病毒或亨德拉病毒的攻击（Bossart 2011）。 我们在非洲绿猴体内测试了利巴韦林对抗亨德拉病毒感染的体内功效。治疗确实延长并改变了疾病进展，但基本上不成功（Rockx 2011）。 我们还在已建立的致命叙利亚仓鼠模型中测试了利巴韦林对 ANDV 复制的影响。我们得出的结论是，利巴韦林治疗有利于针对引起 HPS 的汉坦病毒进行暴露后预防（Safronetz 2011）。 （三）生态与传播 目标：了解病毒和储存宿主物种的相互作用，以防止传播到最终宿主。 我们在马里南部采集了 Mastomys natalensis 并分离出了沙粒病毒。基因分析证实了一种独特的拉沙病毒 Soromba R，并首次证明马里南部存在拉沙病毒（Safronetz 2010）。 我们制定了刚果共和国动物和人类丝状病毒监测框架。此外，还将建立基础研究设施，用于本土果蝠物种的保存、检疫和繁殖。这将允许对丝状病毒的潜在储存物种进行致病性和传播研究。我们已经确定了长期采样点（明斯特，正在准备中）。 我们建立了汉坦病毒（hantavirus）的感染模型，以研究天然宿主物种鹿鼠对感染的免疫反应。我们发现鹿鼠对感染产生强烈的抗炎 CD4+ T 细胞反应，从而限制病理并允许病毒持续存在（Prescott，准备中）