Disease Modeling of Influenza and Other Emerging Respiratory Viral Pathogens

(1) 建立动物疾病（最终宿主）和持久性（储存宿主）模型： 在过去的几年里，我们开发并表征了甲型流感病毒、中东呼吸综合征冠状病毒（MERS-CoV）、亨尼帕病毒（尼帕病毒和亨德拉病毒）以及引起汉坦病毒肺综合征（HPS）的汉坦病毒感染的啮齿动物和非人灵长类动物疾病模型。 通过比较病理学研究进一步完善了两种非人类灵长类 MERS 模型：恒河猴和普通狨猴。结果表明，病毒复制的增加和对中东呼吸综合征冠状病毒感染的局部免疫反应在肺部病理的严重程度中发挥着作用。我们还研究了家猪是否可以作为中东呼吸综合征冠状病毒的放大/中间物种或作为疾病模型。猪鼻内和气管内接种了高剂量的中东呼吸综合征冠状病毒，但没有出现疾病迹象，也没有出现呼吸道病变。它们不太可能充当中东呼吸综合征冠状病毒的放大/中间物种。 我们还进一步表征了恒河猴 HPS 模型，以研究疾病发病机制。我们目前正在研究特定的炎症事件，以了解导致疾病的原因并开发治疗方法。去年，我们对汉坦病毒的大部分工作旨在确定自然宿主如何在不患病的情况下支持病毒复制水平的提高。我们之前已经证明，Sin Nombre 病毒会在自然宿主鹿小鼠体内引发最初的炎症反应，但正如病毒特异性 T 调节细胞的活性所表明的那样，这种反应转变为主动的抗炎反应。目前，我们正在通过使用 T 细胞耗竭策略来确定这些 T 调节细胞对抑制抗炎反应的需求。 （研究正在进行中） (2) 识别和表征病毒致病性的决定因素以开发抗病毒药物： 严重的流感病毒感染通常与细菌合并感染有关。为了研究混合感染的增强作用，我们使用中度严重流行性 H1N1 病毒株 (Ca04) 和耐甲氧西林金黄色葡萄球菌 (MRSA) 对食蟹猴进行了一项研究。仅感染 MRSA 的动物大部分无症状，而感染 CaO4 的动物仅出现中度肺部疾病。有趣的是，最初感染MRSA、随后感染CaO4的动物表现出临床症状的显着减轻，而最初感染CaO4的动物则表现出临床疾病的增强。对季节性 H3N2 病毒和 MRSA 进行了类似的研究，我们没有看到疾病减少或增强。破译这些观察结果背后的机制的研究是过去一年的目标，目前仍在进行中。 （研究正在进行中） 我们可以在仓鼠疾病模型中鉴定尼帕病毒感染的早期靶细胞。尼帕病毒最初以呼吸系统为目标。感染早期不会发生脑内病毒复制和非呼吸组织血管感染。然而，病毒在嗅觉上皮细胞中早期复制，可能是神经系统传播的第一步。这对于疫苗和治疗/抗病毒药物的开发具有重要意义。 我们可以证明，汉坦病毒对细胞培养的适应会导致毒力丧失。因此，我们建立了不同小鼠物种（Peromyscus maniculatus；Apododemus flavicollis）的群体来研究病毒-储存库相互作用。这些菌落还将用于生产用于体内工作的原种病毒。 （研究正在进行中） (3) 识别和表征宿主对病毒感染的反应以开发治疗方法： 与 NCI 的分子目标项目合作，griffithsin（一种新型病毒进入抑制剂）被鉴定为具有有效的 (EC50 5nM) 抗 MERS-CoV 活性。在恒河猴模型中，雾化格里菲辛的暴露后功效显示病毒载量适度减少，但并未显着减少疾病体征。我们现在已经证明，暴露前治疗可以减少疾病的临床症状和靶器官中的病毒滴度。 （研究正在进行中） 我们还测试了三种单克隆抗体 (mAb) 治疗普通狨猴 MERS-CoV 感染的功效。这些单克隆抗体在中东呼吸综合征冠状病毒感染的小鼠模型中显示出功效。不幸的是，在非人灵长类动物模型中，没有一种 mAB 能显着降低疾病负担和病毒肺负荷，这表明 mAB 治疗可能不太有效。验证性研究和 mAB 鸡尾酒治疗正在进行或计划中。 我们测试了阿拉泊韦（一种非免疫抑制性环孢菌素 A 类似物）针对 MERS-CoV 和 SARS-CoV 的治疗效果。低微摩尔浓度的阿拉泊韦可抑制四种不同冠状病毒的复制，包括 MERS 和 SARS 冠状病毒。在这些基于细胞培养的感染模型中，利巴韦林被发现可以进一步增强阿拉泊韦的抗病毒作用，但这种联合治疗无法改善小鼠模型中 SARS-CoV 感染的结果。 我们在恒河猴模型中测试了抗病毒化合物 GS-5734 对抗 MERS-CoV 的功效。暴露前治疗减少了疾病负担和病毒肺负荷。相比之下，使用 GS-5734 进行暴露后处理仅显示出轻微的效果。计划对狨猴模型进行验证性研究。 （研究正在进行中） (4) 开发保护性疫苗： 我们继续努力开发针对甲型流感病毒的通用疫苗。我们目前正在应用两种方法：i) 从血凝素柄内两个独立的螺旋区域表达高度保守的 B 细胞表位，这已证明可以提供异亚型结合和保护；ii) 去除血凝素球状区域以增加针对其他抗原性较差的表位的抗体反应。我们在这些研究中使用了巨细胞病毒 (CMV) 载体平台，它可以诱导持久的免疫反应（T 细胞和抗体）。不幸的是，使用甲型流感病毒小鼠模型的首次尝试相当令人沮丧。我们将继续优化 CMV 平台，但也开始使用水泡性口炎病毒（VSV）作为替代平台。 （研究正在进行中） 对于 MERS，我们通过编码密码子优化共有刺突蛋白的 DNA 疫苗平台获得了非常有希望的结果。该疫苗使用初免/加强/加强方法在小鼠、猕猴和骆驼这三种动物中诱导了有效的细胞免疫和抗原特异性中和抗体。接种疫苗的猕猴可以免受中东呼吸综合征冠状病毒的攻击，并且没有表现出任何肺炎的临床或放射学迹象。最近，我们成功地缩短了疫苗接种策略，以便在紧急情况下应用这种疫苗接种方法来预防中东呼吸综合征冠状病毒感染。 （手稿正在准备中） 为了生成针对尼帕病毒感染的疫苗，我们使用 VSV 平台表达单一尼帕病毒糖蛋白（G 或 F）作为免疫原。这些疫苗在仓鼠和非人类灵长类动物中引发了强烈的抗体反应，并保护它们免受致命的尼帕病毒的攻击。我们可以证明这些疫苗能够产生强烈的中和反应并引发 CD8 T 细胞反应。为了研究这种疫苗功效的局限性，我们使用了仓鼠模型，结果表明，在尼帕病毒攻击当天接种该疫苗仍能提供部分保护。表达尼帕病毒G蛋白的VSV疫苗载体目前计划进行GMP生产。

(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.