Pathogenesis and countermeasures of poxviruses and hemorrhagic fever viruses

痘病毒和出血热病毒的发病机制及对策

• 批准号: 8157024
• 负责人: Peter Jahrling
• 金额: $ 201.43万
• 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/8157024
• 关键词: Acute; Affect; Africa; Amino Acids; Animals; Clinical; Collaborations; Cowpox; Cowpox virus; Diagnosis; Disease; Disease Outbreaks; Disease Progression; Dose; Ebola Vaccines; Ebola virus; Evaluation; Fever; Gene Expression; Genetic; Glycoproteins; Goals; HIV; Health Personnel; Hepatitis C virus; Host Defense Mechanism; Human; Immunity; Immunotherapy; In Vitro; Individual; Infection; Inflammation; Laboratories; Lesion; Licensing; Lung; Lymphatic Diseases; Lymphoid; Membrane Glycoproteins; Metabolic Marker; Military Personnel; Monitor; Monkey Hemorrhagic Disease Virus; Monkeypox; Monkeypox virus; Mus; Nose; Orthopoxvirus; Pathogenesis; Peptide Hydrolases; Peptides; Physiological; Plaque Assay; Positron-Emission Tomography; Poxviridae; Recombinants; Retro-Inverso Peptide; Swab; Viral Hemorrhagic Fevers; Viral Pathogenesis; Viral Pneumonia; Virus Shedding; anthrax protective factor; base; biodefense; dosage; hemorrhagic fever virus; improved; in vivo; lymph nodes; molecular imaging; nonhuman primate; prevent; uptake

Findings from FY10 are summarized below. 1. Animal models of orthopoxviruses and countermeasures. Cases of monkeypox virus (MPXV) infection in humans are expanding in Africa and can be fatal in up to 10% of affected individuals. In addition, the virus was imported into the United States in 2003 and caused a localized outbreak in the Midwest. Based on the growing threat of MPXV disease and concerns that variola might be used as an agent of bioterrorism, we have continued to focus on the study of orthopoxvirus pathogenesis in animals and the identification of countermeasures. We are exploring alternate routes of MPXV inoculation in nonhuman primates (NHP) to identify a model of infection that most closely resembles natural monkeypox and variola disease progression in humans. The commonly utilized intravenous (IV) route of infection results in an accelerated fulminant disease course compared to MPXV infection in humans. Our goal was to determine if intrabronchial (IB) exposure of NHPs to MPXV results in a systemic disease that better resembles the progression of human MPXV infection. We have compared the disease course following IV or IB inoculation of NHPs with 10-fold serial doses of MPXV Zaire. Classical pox-like disease was observed in NHPs administered a high virus dose by either route. Several key events were delayed in the highest doses tested of the IB model when compared to the IV model including the onset of fever, lesion appearance, peak viremia, viral shedding in nasal and oral swabs, peak cytokine levels, and time to reach endpoint criteria. Virus distribution across 19 tissues was largely unaffected by inoculation route at the highest doses tested. The NHPs inoculated by the IB route developed a viral pneumonia that likely exacerbated disease progression. Based on the observations of delayed onset of clinical and virological parameters and endpoint criteria that may more closely resemble human MPXV infection, the IB MPXV model should be considered for further investigation of viral pathogenesis and countermeasures. To further our understanding of MPXV pathogenesis, we have expanded our studies to include molecular imaging applications to investigate factors that contribute to the survival of severe orthopoxvirus infection or that might be used for diagnosis or prognosis. One such factor, lymphadenopathy, is a distinctive feature of MPXV infection and was associated with disease severity in the recent United States outbreak. We have used positron emission tomography and computed tomography (PET/CT) with 18FDG as a nonspecific marker of metabolic activity to monitor inflammation and immune activation in NHPs. Groups of two NHPs were inoculated by the IV or IB route, and one animal survived in each group. Lymphadenopathy and increased immune activation represented by 18FDG uptake in the axillary lymph nodes (LNs) was evident in each NHP. Retrospective analysis of the axillary LNs indicated an association between pre-infection immune activation and survival. Both surviving IV- and IB-infected NHPs had significant 18FDG uptake in the LNs at the time of MPXV challenge with no clinical signs of illness, while the NHPs that succumbed did not. Massive inflammation in the lungs of IB-infected NHPs was also observed, and bilobular involvement was associated with moribundity. Molecular imaging was successfully used to monitor disease progression of an acute, severe viral infection and identify two patterns of immune activation/inflammation associated with survival. We have continued our study of cowpox (CPXV) infection as a BSL-2 model of orthopoxvirus infection and a surrogate model of variola virus infection. We have extended our studies in FY10 by demonstrating that IV inoculation with CPXV Brighton resulted in a uniformly lethal disease characterized by hemorrhagic manifestations. Surprisingly, the uniformly lethal dose for CPXV appears to be at least 100-fold lower than that observed for MPXV. Peak viremia levels reached greater than 8.0 log10genome copies/ml for NHPs infected with 5x10e5 PFU to 5x10e7 PFU CPXV. Lesion counts were less numerous than observed for MPXV with typically 50 or fewer lesions present in CPXV-infected NHPs. However, viral load in respiratory, lymphoid, and reticuloendothelial tissues was similar to levels observed in NHPs infected IV with MPXV. Investigations into countermeasures against the orthopoxviruses have also been pursued. We are currently evaluating broad-spectrum antiviral agents in collaboration with Functional Genetics, Inc. for in vitro and in vivo activity against the orthopoxviruses. In addition, a subset of host defense peptides (HDPs) with previously demonstrated immunomodulatory activities was selected for testing of direct or indirect antiviral function. In vitro antiviral activities were assessed using virus plaque assays or virus-GFP constructs through the following: 1) co-incubation of virus with HDPs, 2) pre-incubation of host cells with HDPs prior to infection, or 3) administration of HDPs to host cells post-infection. Two HDPs, as well as their protease-resistant all D-amino acid and retro-inverso analogs, demonstrated enhanced direct or indirect (immunomodulatory) antiviral activities against a broad range of viruses including cowpox, monkeypox, and vaccinia and were selected for testing in an intraperitoneal murine model of cowpox virus infection. Efficacy was demonstrated for both the immunomodulatory HDP and the all-D amino acid direct antiviral HDP with 40% survival in the lethal murine model following a single administration of peptide concurrent with infection. Currently these studies are being repeated in both intranasal and intraperitoneal models of cowpox infection and gene expression analysis will follow using the most efficacious infection model for determination of a putative mechanism of HDP action in vivo. 2. Countermeasures against viral hemorrhagic fevers. A safe and effective vaccine against Zaire ebola virus (ZEBOV) would potentially be used by multiple groups including healthcare providers, laboratory researchers, and military personnel. A vaccine could also be used to mitigate outbreaks in Africa and possibly be used to protect endemic nonhuman primate populations that have been affected by ZEBOV. While multiple strategies for ZEBOV vaccine development have been pursued, no current vaccine is close to being licensed by the Food and Drug Administration. Unresolved issues with current vaccine candidates include safety concerns, pre-existing immunity to vectored vaccines, and manufacturing and dosage obstacles. We are developing an alternative vaccine approach in collaboration with Dr. Matthias Schnell using attenuated rabies virus vaccine based vectors that express the major protective antigen of ZEBOV, the virion surface glycoprotein (GP). Rabies virus based vectors have previously been generated that express protective antigens from human immunodeficiency virus, hepatitis C virus, and anthrax protective antigen and found to be safe, attenuated, and efficacious in mice and/or nonhuman primates. The main objective of the present study is to determine the safety and level of immunogenicity of rabies virus vaccine-based vectors expressing ZEBOV GP as either live or killed vaccines and determine their efficacy in animal models of ZEBOV infection. Currently, we have generated an infectious cDNA clone of rabies vaccine virus expressing ZEBOV GP and recovered it in tissue culture. The recombinant viruses replicate to similar levels as the parental rabies vaccine strain and express ZEBOV GP in tissue culture. Analysis in mice for safety, immunogenicity, and protective efficacy is ongoing.

FY10的发现如下总结。 1。正托病毒和对策的动物模型。 人类中蒙基毒病毒（MPXV）感染的病例在非洲正在扩大，在多达10％的受影响个体中可能致命。 此外，该病毒于2003年进口到美国，并在中西部引起了本地爆发。 基于MPXV疾病的日益严重的威胁，并担心Variola可能被用作生物恐怖主义的毒剂，我们继续专注于动物正oxoxvirus发病机理的研究和对策的鉴定。 我们正在探索非人类灵长类动物（NHP）中MPXV接种的替代途径，以识别一种最类似于人类天然的蒙基托和Variola疾病进展的感染模型。 与人类的MPXV感染相比，通常使用的静脉内（IV）感染途径导致了加速暴发性病程。 我们的目标是确定NHPS内（IB）对MPXV的暴露是否会导致系统性疾病，从而更好地类似于人类MPXV感染的进展。 我们已经比较了IV或IB接种NHP的疾病病程，该NHP具有10倍连续剂量的MPXV Zaire。 在NHP中观察到经典的痘痘样疾病，通过两种途径施用高病毒剂量。 与IV模型相比，在IB模型测试的最高剂量中，几个关键事件被延迟，包括发烧，病变外观，峰值病毒血症，鼻拭子和口腔拭子的病毒脱落，峰值细胞因子水平以及达到端点标准的时间。 在最高剂量测试的最高剂量下，在19个组织中的病毒分布在很大程度上不受接种途径的影响。 IB路线接种的NHP发生了病毒性肺炎，可能加剧了疾病进展。基于可能更类似于人类MPXV感染的临床和病毒学参数延迟发作和终点标准的观察结果，应考虑进一步研究IB MPXV模型。 为了进一步了解MPXV发病机理，我们将研究扩展到包括分子成像应用，以研究有助于严重正托病毒感染存活或可能用于诊断或预后的因素。 这样的因素是淋巴结肿大，是MPXV感染的独特特征，并且与最近的美国爆发有关疾病的严重程度。 我们已经使用具有18FDG的正电子发射断层扫描和计算机断层扫描（PET/CT）作为代谢活性的非特异性标记，以监测NHP中的炎症和免疫激活。 IV或IB途径接种了两个NHP的组，每组中有一组幸存下来。 在每个NHP中，在腋窝淋巴结（LNS）中以18FDG摄取表示的淋巴结肿大和免疫活化增加。 对腋LN的回顾性分析表明，感染前免疫激活与存活之间存在关联。 在MPXV挑战时，LNS的IV和IB感染的NHP均在LNS中均显着18fdg，而没有临床疾病迹象，而屈服的NHP则没有。 还观察到了IB感染的NHP的肺中的严重炎症，并且双眼受累与死亡相关。分子成像成功地用于监测急性严重病毒感染的疾病进展，并确定与生存相关的两种免疫激活/炎症模式。 我们一直在研究牛波克（CPXV）感染，作为正托病毒感染的BSL-2模型和Variola病毒感染的替代模型。 我们通过证明CPXV Brighton接种IV导致了一种以出血性表现为特征的统一致命疾病，从而扩展了FY10的研究。 出乎意料的是，CPXV的均匀致命剂量似乎比MPXV观察到的剂量至少低100倍。 对于5x10E5 PFU感染至5x10E7 PFU CPXV的NHP的峰值病毒血症水平大于8.0 log10 genome拷贝/mL。 在CPXV感染的NHP中通常存在50个或更少病变的MPXV，病变计数的数量少于观察到的MPXV。 然而，呼吸道，淋巴样和网状内皮组织中的病毒负荷与在用MPXV感染的NHPS中观察到的水平相似。 还进行了针对正托病毒的对策的调查。 我们目前正在与Fuctional Genetics，Inc。合作评估广谱抗病毒剂，以实现对正托病毒的体外和体内活性。 此外，选择了先前证明的免疫调节活性的宿主防御肽（HDP）的子集以测试直接或间接抗病毒功能。 通过以下几种病毒斑块测定或病毒-GFP构建体评估体外抗病毒活性：1）病毒与HDPS合并，2）在感染前与HDP一起预孵育宿主细胞，或3）3）在感染后施用HDPS来施用HDPS。 两个HDP及其抗蛋白酶耐药性所有D-氨基酸和恢复类似物的类似物，表现出针对广泛的病毒的直接或间接或间接（免疫调节）抗病毒活性，包括Cowpox，Monkeypox和vercinia，并被选择用于在腹膜内甲米内尔鼠模型的contapox Murine Murine Moline Mode of Cowpox Virus Intection中进行测试。 在单一同时施用与感染的肽同时给药后，在致命的鼠模型中，在致命的鼠模型中，在致命的鼠模型中，免疫调节的HDP和全D氨基酸直接抗病毒hdp都证明了功效。目前，这些研究正在使用最有效的感染模型来确定体内HDP作用的推定机制，在乳晶感染的鼻内和腹膜内模型中都重复进行这些研究。 2。反对病毒出血狂的对策。 针对Zaire Ebola病毒（Zebov）的安全有效疫苗可能会被包括医疗保健提供者，实验室研究人员和军事人员在内的多个群体使用。 疫苗也可用于减轻非洲的爆发，并可能用于保护受Zebov影响的地方性非人类灵长类动物种群。 尽管已经采用了多种开发Zebov疫苗的策略，但目前的疫苗尚未获得食品药品监督管理局的许可。 目前候选疫苗的尚未解决的问题包括安全问题，对媒介疫苗的免疫力以及制造和剂量障碍。 我们正在使用基于减毒的狂犬病病毒疫苗的载体与Matthias Schnell博士合作开发一种替代疫苗方法，该媒介表达了Zebov的主要保护性抗原，Zebov，即Virion表面糖蛋白（GP）。先前已经产生了基于狂犬病病毒的载体，该载体从人类免疫缺陷病毒，丙型肝炎病毒和炭疽保护性抗原中表达保护性抗原，并发现在小鼠和/或非人类灵长类动物中安全，衰减和有效。 本研究的主要目的是确定表达Zebov GP的狂犬病病毒疫苗媒介的免疫原性的安全性和水平，以现场直播或杀死疫苗，并确定其在Zebov感染动物模型中的功效。 目前，我们已经产生了一种表达Zebov GP的狂犬病疫苗病毒的传染性cDNA克隆，并在组织培养中恢复了它。 重组病毒在组织培养中复制到与亲本狂犬病疫苗菌株和表达Zebov GP相似的水平。 针对安全性，免疫原性和保护效力的小鼠分析正在进行中。