Disease Modeling of Influenza and Other Emerging Respiratory Viral Pathogens

流感和其他新出现的呼吸道病毒病原体的疾病模型

• 批准号: 9566702
• 负责人: Heinrich Feldmann
• 金额: $ 15.35万
• 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/9566702
• 关键词: Acute; Animal Diseases; Animals; Anti-Inflammatory Agents; Antibodies; Antibody Response; Antigens; Antiviral Agents; Apodemus; B-Lymphocyte Epitopes; Binding; Blood Vessels; Brain; CD8-Positive T-Lymphocytes; Callithrix; Callithrix jacchus jacchus; Camels; Cell Culture Techniques; Cells; Cellular Immunity; Clinical; Codon Nucleotides; Collaborations; Comparative Pathology; Consensus; Containment; Coronaviridae; Coronavirus; Coronavirus Infections; Cyclosporine; Cytomegalovirus; DNA Vaccines; Deer Mouse; Development; Diagnostic radiologic examination; Disease; Disease model; Domestic Pig; Dose; Emergency Situation; Emerging Communicable Diseases; Epitopes; Event; Excision; Family suidae; Fostering; Functional disorder; G-substrate; GTP-Binding Proteins; Globular Region; Goals; Hamsters; Hantavirus; Hantavirus Pulmonary Syndrome; Hemagglutinin; Hendra Virus; Henipavirus; Immune response; Infection; Inflammatory; Inflammatory Response; Influenza; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza A virus; International; Lesion; Lung; Lung diseases; Macaca; Macaca mulatta; Manuscripts; Middle East Respiratory Syndrome Coronavirus; Minor; Modeling; Molecular Target; Monoclonal Antibodies; Mus; National Institute of Allergy and Infectious Disease; Nebulizer; Nervous system structure; Nipah Virus; Olfactory Epithelium; Organ; Pathogenesis; Pathogenicity; Peromyscus; Phase; Play; Pneumonia; Preparation; Production; Proteins; Public Health; Pulmonary Pathology; Regulatory T-Lymphocyte; Research; Respiratory System; Respiratory tract structure; Ribavirin; Rodent; Role; SARS coronavirus; Schedule; Sepsis; Severities; Sin Nombre virus; T-Cell Depletion; T-Lymphocyte; Therapeutic; Tissues; Treatment Efficacy; United States National Institutes of Health; Vaccinated; Vaccination; Vaccines; Vesicular stomatitis Indiana virus; Viral; Viral Load result; Viral reservoir; Virulence; Virus; Virus Diseases; Virus Replication; Work; analog; base; burden of illness; co-infection; disease transmission; efficacy testing; glycoprotein G; improved outcome; in vivo; influenzavirus; inhibitor/antagonist; innovation; methicillin resistant Staphylococcus aureus; mouse model; neutralizing antibody; nonhuman primate; novel; pandemic disease; pathogen; prevent; programs; respiratory; response; therapeutic development; therapeutic evaluation; vaccination strategy; vaccine development; vaccine efficacy; vector; vector vaccine

(1) To develop animal disease (end host) and persistence (reservoir host) models: Over the past years we have developed and characterized rodent and nonhuman primate disease models for infections with influenza A viruses, the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), henipaviruses (Nipah and Hendra), and hantaviruses causing Hantavirus Pulmonary Syndrome (HPS). The two nonhuman primate MERS models, rhesus macaque and common marmoset, were further refined with a comparative pathology study. The results suggested that increased virus replication and the local immune response to MERS-CoV infection play a role in the severity of pulmonary pathology. We also investigated whether domestic pigs could serve as an amplifying/intermediate species for MERS-CoV or as a disease model. Pigs were inoculated intranasally and intratracheally with a high dose of MERS-CoV but did not develop signs of disease nor lesions in the respiratory tract. They are unlikely to serve as an amplifying/intermediate species for MERS-CoV. We have also further characterized the rhesus macaque HPS model to investigate mechanisms of disease pathogenesis. We are currently studying specific inflammatory events to gain understanding of what contributes to disease as well as to develop a therapeutic. Much of our work on hantaviruses in the past year was aimed at determining how the natural reservoirs can support elevated levels of virus replication without disease. We have previously shown that Sin Nombre virus elicits an initial inflammatory response in deer mice, the natural reservoir, but this response turned into an active anti-inflammatory response, as indicated by the activity of virus-specific T regulatory cells. Currently we are determining the requirement of these T regulatory cells to the suppression of the anti-inflammatory response by using T cell depletion strategies. (studies ongoing) (2) To identify and characterize determinants of viral pathogenicity to develop antivirals: Severe influenza virus infections are often associated with bacterial co-infections. To study a potentiating effect of co-infection we performed a study in cynomolgus macaques using a moderately severe pandemic H1N1 strain (Ca04) and Methicillin-resistant Staphylococcus aureus (MRSA). Animals infected with MRSA only were largely asymptomatic, whereas animals infected with Ca04 only developed moderate pulmonary disease. Interestingly, animals initially infected with MRSA followed by Ca04 showed a dramatic reduction in clinical signs, whereas those initially infected with Ca04 showed enhanced clinical disease. Similar studies were performed with a seasonal H3N2 virus and MRSA, in which we did not see disease reduction or enhancement. Studies to decipher the mechanisms behind these observations were objectives over the past year and are still ongoing. (studies ongoing) We could identify the early target cells of Nipah virus infection in the hamster disease model. Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination. This has important implications for the development of vaccine and therapeutics/antivirals. We could show that for hantaviruses adaptation to cell culture leads to loss of virulence. Therefore, we have established colonies with different mouse species (Peromyscus maniculatus; Apodemus flavicollis) for studying virus-reservoir interaction. These colonies will also be used to produce stock virus for in vivo work. (studies ongoing) (3) To identify and characterize host responses to viral infection to develop therapeutics: In collaboration with the Molecular Targets Program at NCI, griffithsin, a novel viral entry inhibitor, was identified as having potent (EC50 5nM) activity against MERS-CoV. The post-exposure efficacy of nebulized griffithsin in the rhesus macaque model showed moderate reduction of viral load but did not significantly reduce disease signs. We have now shown that pre-exposure treatment reduces clinical signs of disease and viral titers in target organs. (studies ongoing) We have also tested efficacy of three monoclonal antibodies (mAb) as a treatment for MERS-CoV infection in the common marmoset. These mAb had shown efficacy in mouse models of MERS-CoV infection. Unfortunately, none of the mABs showed significant reduction in disease burden and viral lung load in the nonhuman primate model suggesting that treatment with mABs may likely not very efficacious. Confirmatory studies and treatment with mAB cocktails are either ongoing or planned. We have tested the therapeutic efficacy of alisporivir, a non-immunosuppressive cyclosporin A-analog, against MERS-CoV and SARS-CoV. Low-micromolar concentrations of alisporivir inhibit the replication of four different coronaviruses, including MERS- and SARS-coronavirus. Ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of SARS-CoV infection in a mouse model. We have tested the efficacy of the antiviral compound GS-5734 against MERS-CoV in the rhesus macaque model. Pre-exposure treatment resulted in reduction of disease burden and viral lung loads. In contrast, post-exposure treatment with GS-5734 showed only minor effects. Confirmatory studies in the marmoset model are planned. (studies ongoing) (4) To develop protective vaccines: We continued with our efforts to develop a universal vaccine against influenza A viruses. We currently are applying two approaches: i) expression of highly conserved B cell epitopes from two separate helical regions within the hemagglutinin stalk that have shown to afford heterosubtypic binding and protection, and ii) removal of hemagglutinin globular region to increase antibody responses against otherwise poorly antigenic epitopes. We used the Cytomegalovirus (CMV) vector platform for these studies, which can induce long-lasting immune responses (both T cell and antibody). Unfortunately, first attempts using the mouse model of influenza A viruses were rather discouraging. We will continue to optimize the CMV platform but have also started to use the vesicular stomatitis virus (VSV) as an alternative platform. (studies are ongoing) For MERS, we have obtained very promising results with a DNA vaccine platform encoding a codon-optimized consensus spike protein. This vaccine induced potent cellular immunity and antigen specific neutralizing antibodies in three animal species, mice, macaques and camels using a prime/boost/boost approach. Vaccinated macaques were protected against MERS-CoV challenge and did not show any clinical or radiographic signs of pneumonia. Recently, we were successful in shortening the vaccination strategy for potential application of this vaccination approach in emergency situations to prevent MERS-CoV infection. (manuscript in preparation) To generate a vaccine against Nipah virus infection, we used the VSV platform to express single Nipah virus glycoproteins (G or F) as the immunogens. The vaccines elicited strong antibody responses in hamsters and nonhuman primates and protected them from lethal Nipah virus challenge. We could demonstrate that the vaccines elucidated strong neutralizing responses and primed the CD8+ T cell responses. To investigate the limits of the efficacy of this vaccine, we used the hamster model and showed that this vaccine still provided partial protection when administered on the day of Nipah virus challenge. The VSV vaccine vectors expressing the Nipah virus G protein is currently scheduled for GMP production.

（1）发展动物疾病（最终宿主）和持久性（水库宿主）模型： 在过去的几年中。 通过一项比较病理学研究，进一步完善了两个非人类灵长类动物MERS模型，恒河猕猴和常见的Marmoset。结果表明，增加的病毒复制和对MERS-COV感染的局部免疫反应在肺病理的严重程度中起作用。我们还调查了家猪是MERS-COV还是疾病模型的放大/中间物种。用高剂量的MERS-COV接种鼻子和气管内的猪，但没有出现疾病的迹象或呼吸道病变的迹象。它们不太可能作为MERS-COV的放大/中间物种。 我们还进一步表征了恒河猴HPS模型，以研究疾病发病机理的机理。我们目前正在研究特定的炎症事件，以了解有助于疾病以及发展治疗性的原因。过去一年中，我们在汉坦病毒方面的大部分工作旨在确定自然储层如何支持没有疾病的病毒复制水平升高。我们先前已经表明，罪恶的病毒会引起天然储层鹿小鼠的初始炎症反应，但是这种反应变成了活性的抗炎反应，如病毒特异性T调节细胞的活性所表明。目前，我们通过使用T细胞耗尽策略来确定这些T调节细胞对抑制抗炎反应的需求。 （正在进行的研究） （2）识别和表征病毒致病性的决定因素以发展抗病毒药： 严重的流感病毒感染通常与细菌共感染有关。为了研究共感染的增强作用，我们使用中度严重的大流行H1N1菌株（CA04）和耐甲氧西林的金黄色葡萄球菌（MRSA）进行了一项研究在cynomolgus猕猴方面进行的研究。仅感染MRSA的动物在很大程度上是无症状的，而感染CA04的动物仅患有中度肺部疾病。有趣的是，最初感染MRSA的动物随后CA04显示出临床体征的降低，而最初感染CA04的动物显示出临床疾病的增强。使用季节性H3N2病毒和MRSA进行了类似的研究，其中我们没有看到疾病降低或增强。在过去的一年中，对这些观察结果背后的机制的研究是目标，并且仍在进行中。 （正在进行的研究） 我们可以在仓鼠疾病模型中鉴定NIPAH病毒感染的早期靶细胞。 NIPAH病毒最初针对呼吸系统。在感染的早期阶段，不会发生大脑中的病毒复制和非呼吸组织中血管的感染。但是，病毒在嗅觉上皮早期复制，可以作为神经系统传播的第一步。这对疫苗和治疗剂/抗病毒药的发展具有重要意义。 我们可以证明，对于汉坦病毒适应细胞培养会导致毒力丧失。因此，我们已经建立了与不同的小鼠物种（peromyscus maniculatus; apodemus flavicollis）建立菌落，用于研究病毒 - 保存相互作用。这些菌落还将用于生产用于体内工作的库存病毒。 （正在进行的研究） （3）识别和表征宿主对病毒感染的反应以发展治疗剂： 与NCI分子靶计划合作，一种新型病毒入口抑制剂Griffithsin被确定为对MERS-COV具有有效的（EC50 5Nm）活性。在恒河猴模型中，雾化的griffithsin的暴露后功效显示病毒载量的中度降低，但并未显着降低疾病的体征。现在，我们已经表明，暴露前治疗可减少靶心器官中疾病和病毒滴度的临床迹象。 （正在进行的研究） 我们还测试了三种单克隆抗体（MAB）的疗效，作为对公共果棒中MERS-COV感染的一种治疗方法。这些mAB在MERS-COV感染的小鼠模型中显示出功效。不幸的是，在非人类的灵长类动物模型中，没有任何mAB显示疾病负担和病毒肺负荷的显着减轻，这表明用mAb的治疗可能不太有效。正在进行的或计划的MAB鸡尾酒的验证性研究和治疗。 我们已经测试了Alisporivir（一种非免疫抑制环孢菌素A-Analog）对MERS-COV和SARS-COV的治疗功效。低微摩尔浓度的alisporivir抑制了四种不同的冠状病毒的复制，包括MERS和SARS-核纳病毒。发现利巴韦林在这些基于细胞培养的感染模型中进一步增强了Alisporivir的抗病毒作用，但是这种组合治疗无法改善小鼠模型中SARS-COV感染的结果。 我们已经测试了恒河猕猴模型中抗病毒化合物GS-5734对MERS-COV的功效。暴露前治疗导致疾病负担减轻和病毒肺负荷。相反，使用GS-5734的暴露后处理仅显示出较小的影响。计划在Marmoset模型中进行验证性研究。 （正在进行的研究） （4）开发保护性疫苗： 我们继续努力开发针对流感病毒的普遍疫苗。我们目前正在采用两种方法：i）表达高度保守的B细胞表位来自血凝素茎内两个独立的螺旋素区域的表达抗原表位。我们将巨细胞病毒（CMV）矢量平台用于这些研究，这可以诱导长期的免疫反应（T细胞和抗体）。不幸的是，首次尝试使用流感A病毒的小鼠模型令人沮丧。我们将继续优化CMV平台，但也已经开始使用囊泡气孔病毒（VSV）作为替代平台。 （正在进行研究） 对于MERS，我们通过编码密码子优化共识尖峰蛋白的DNA疫苗平台获得了非常有希望的结果。这种疫苗使用主要/增强/增强方法在三种动物，小鼠，猕猴和骆驼中诱导了有效的细胞免疫和抗原特异性中和抗体。疫苗接种的猕猴受到了MERS-COV挑战的保护，并且没有显示出肺炎的任何临床或射线照相迹象。最近，我们成功地缩短了这种疫苗接种方法在紧急情况下的潜在应用，以防止MERS-COV感染。 （准备手稿） 为了生成针对NIPAH病毒感染的疫苗，我们使用VSV平台表达单个NIPAH病毒糖蛋白（G或F）作为免疫原子。该疫苗在仓鼠和非人类灵长类动物中引起了强抗体反应，并保护了它们免受致命的Nipah病毒挑战。我们可以证明疫苗阐明了强中和反应并启动CD8+ T细胞反应。为了研究该疫苗功效的限制，我们使用了仓鼠模型，并表明该疫苗在NIPAH病毒挑战当天进行给药时仍提供了部分保护。目前，表达NIPAH病毒G蛋白的VSV疫苗向量计划用于GMP生产。