Pathogenesis and countermeasures of poxviruses, hemorrhagic fever viruses, MERS

痘病毒、出血热病毒、MERS的发病机制及对策

• 批准号: 8946425
• 负责人: Peter Jahrling
• 金额: $ 105.49万
• 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/8946425
• 关键词: Acute; Aerosols; Animal Model; Antibiotic Therapy; Antibiotics; Antigens; Antiviral Agents; Applied Research; Attenuated; Attenuated Vaccines; Bacterial Infections; Basic Science; Binding; Biological Assay; Bioterrorism; CCL2 gene; Callithrix jacchus jacchus; Cardiac; Cells; Cellular Immunity; Chemistry; Chest; Chiroptera; Clinical; Codon Nucleotides; Collaborations; Communicable Diseases; Complete Blood Count; Coronavirus; Cowpox virus; Data; Democratic Republic of the Congo; Development; Disease; Disease Outcome; Disease Progression; Dose; Ebola virus; Emerging Communicable Diseases; Euthanasia; Evaluation; Event; Filovirus; Genetic Transcription; Genome; Glycoproteins; Goals; Human; Humoral Immunities; Imaging Techniques; Immunization; Immunoprecipitation; Immunotherapy; Inactivated Vaccines; Infection; Infection Control; Inflammation; Interferon Type II; Investigation; Jordan; Knock-out; Knockout Mice; Laboratories; Lethal Dose 50; Life; Lung; Macaca; Macaca mulatta; Medical Imaging; Metabolic; Middle East; Modeling; Molecular Biology; Molecular Virology; Monitor; Monkey Hemorrhagic Disease Virus; Monkeypox virus; Mus; Orthopoxvirus; Outcome; Pathogenesis; Pathogenicity; Phenotype; Phosphorylation; Phosphorylation Site; Physiological; Point Mutation; Positron-Emission Tomography; Poxviridae; Prevention; Process; Proteins; RANTES; RNA; Rabies; Rabies virus; Regulation; Replicon; Reporter; Respiratory Tract Diseases; Reverse Transcriptase Polymerase Chain Reaction; Role; Route; Safety; Sampling; Serum; Signal Transduction; Site; Smallpox; Smallpox Viruses; Structure; Structure-Activity Relationship; Syndrome; System; Testing; Universities; Untranslated Regions; Vaccines; Viral; Viral Proteins; Virus; Virus Diseases; Work; X-Ray Computed Tomography; attenuation; base; biodefense; cytokine; heart function; hemorrhagic fever virus; immunogenicity; improved; interest; longitudinal analysis; macrophage; member; mutant; nonhuman primate; particle; pathogen; polyclonal antibody; positional cloning; prevent; rabies virus glycoprotein G; receptor; recombinant virus; research study; respiratory; sodium-iodide symporter; therapeutic target; vaccine candidate; vector vaccine; virus host interaction

1.Orthopoxvirus pathogenesis A goal of EVPS is to understand virus-host interactions and exploit them for countermeasure development. Previously, we had established that infection of rhesus macaques with cowpox virus (CPXV) resulted in a disease that resembled human hemorrhagic smallpox, a rare and nearly 100% lethal disease associated with secondary bacterial infections. We compared the impact of antibiotic treatment on disease progression. 8 NHPs were IV inoculated with CPXV, 4 were treated with antibiotics, 4 were not. We did not observe a difference in onset of clinical signs or outcome between the groups. We did observe some differences in clinical parameters such as complete blood count and complete metabolic panel serum chemistry values. As an additional step in development of the IV NHP CPXV model we performed a serial euthanasia study with cardiac MR to evaluate cardiac function through the course of disease. We observed acute changes in heart function that coincided with subjects reaching moribund endpoint criteria. We also altered the route of inoculation of CPXV in macaques to determine if small particle aerosol results in disease that more closely resembles human smallpox. We observed an LD100 and LD50 and disease progression was different than IV inoculation and did not result in hemorrhagic signs or secondary bacterial infections. We also performed chest CT to evaluate lung progression for further development of medical imaging techniques for use in infectious disease studies. To further develop our CPXV model as a surrogate for smallpox, large particle aerosol inoculation was performed. In contrast to the small particle aerosol inoculation experiments, large particle aerosol inoculation resulted in reduced inflammation in the lung as evaluated by CT. With large particle aerosol inoculation, we were unable to achieve an LD100, even at 100 fold more virus than small particle aerosol. Our studies indicate the route of inoculation influences disease outcome. Our third project studying orthopox pathogenesis is based on our Backwards Matched Longitudinal Analysis which identified several cytokines that were statistically associated with lethal or non-lethal outcome in NHPs. The duration and intensity of certain cytokines was associated with survival (IFN-gamma and RANTES) or non-survival (MCP-1). The role of these cytokines was investigated using recombinant virus expressing each cytokine and knockout mice of the cytokine or its receptor. MCP-1 expressing virus and MCP-1 and MCP-1 receptor knockout mice had increased pathogenicity when compared to wild type virus. The MCP-1data suggests that macrophages are essential for controlling infection, but excessive MCP-1 alter macrophage function which may exacerbate disease. IFN-gamma expressing virus did not develop disease, and knockout of IFN-gamma or its receptor resulted in increased pathogenicity. Mice inoculated with interferon gamma expressing virus did not develop any signs of disease and survived lethal challenge at the equivalent 100% lethal dose of wild type virus. Expression of interferon gamma at the site of infection via the recombinant virus resulted in protection from challenge with the LD100 of wild type virus. 2. Bivalent vaccines that confer protection against rabies and Ebola virus We have been developing a rabies virus based vaccine that expresses the glycoprotein (GP) from ebolavirus, marburgvirus and sudanvirus. Previously we developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Zaire ebolavirus (EBOV) glycoprotein (GP) using a reverse genetics system based on the SAD B19 RABV wildlife vaccine in collaboration with Matthias Schnell of Thomas Jefferson University. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced cellular and humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. We evaluated our vaccine candidates in a rhesus macaque challenge model. 100% protection was observed with live attenuated RABV-GP. The inactivated and RVdelG-GP viruses provided 50% protection. Strong humoral and cellular immunity was observed. In summary, our findings indicated that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVdelG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use. We also further developed our vaccine constructs to use codon optimized filoviruses and have demonstrated immunogenicity and safety of these constructs in mice. 3.Filovirus Molecular Virology We are also investigating molecular virology of filoviruses. We are studying the role of phosphorylation of the viral proteins in the virus lifecycle. We have chosen to focus on VP35 as it is essential for transcription and replication of the genome. Using a replicon system we have generated point mutations and clustered point mutations of potential phosphorylation sites and evaluate the mutant for replication and transcription. Our data indicates a hierarchical phosphorylation strategy that regulates VP35 function. A second project involves identification of structure and function relationships of the EBOV noncoding regions. We have identified host proteins that interact with these EBOV genome regions. We are working to identify the proteins. We have identified HSC70 as interacting with at least one domain in the EBOV Trailer by pull-down assays and confirmed using immunoprecipitation-RT-PCR. Three HSC70 binding domains have been identified within the EBOV Trailer and mutational analysis of the 5 most region results in a decrease in replicon signal. We have also undertaken SHAPE analysis of the EBOV Trailer to define the secondary structure of the EBOV Trailer. Based on these data we may identify therapeutic targets as well as establish mechanisms of filovirus lifecycle regulation. 4. MERS-CoV We have also been developing NHP models for MERS-CoV. We have established that intratracheal inoculation with MERS-CoV Jordan isolate results in limited respiratory tract disease that peaks between days 4 and 5 post inoculation, resolves by day 9 post inoculation and can be quantified by computed tomography. Inoculation of common marmosets also results in a longer lived respiratory tract disease that peaks between days 3 and 14, but does not completely resolve, and can be monitored and quantified by computed tomography. Infectious virus could not be recovered from serial samples in either rhesus or common marmosets. To further develop the MERS-CoV model we are working with Dr. Ralph Baric to construct and evaluate a MERS-CoV that expresses the human sodium-iodide symporter that can be used as a PET reporter. We expct this recombinant virus to provide enhanced detail on the infectious process of MERS in NHPs and other animal models. We have also entered a collaboration with SAB and NMRC to develop a polyclonal antibody to MERS-CoV as a human therapuetic

1.正托病毒发病机理 EVP的目标是了解病毒宿主相互作用，并利用它们进行对策开发。以前，我们已经确定了与牛肝病毒（CPXV）感染的恒河猴感染导致一种类似于人类出血的天花，一种与继发性细菌感染相关的罕见且接近100％的致死性疾病。我们比较了抗生素治疗对疾病进展的影响。将8个NHP静脉输液使用CPXV接种，4例用抗生素处理，其中4例未进行。我们没有观察到两组之间的临床体征或结果的差异。我们确实观察到了临床参数的一些差异，例如完全的血数和完整的代谢面板血清化学值。作为IV NHP CPXV模型开发的额外步骤，我们通过心脏MR进行了一项序列的安乐死研究，以通过疾病的过程评估心脏功能。 我们观察到心脏功能的急性变化，这与达到垂死终点标准的受试者相吻合。 我们还改变了猕猴中CPXV接种的途径，以确定小颗粒气溶胶是否导致疾病是否与人类天花更相似。我们观察到LD100和LD50，疾病的进展与IV接种不同，并且不会导致出血迹象或继发细菌感染。我们还进行了胸部CT来评估肺的进展，以进一步开发用于传染病研究的医学成像技术。为了进一步开发我们的CPXV模型作为天花的替代物，进行了大型颗粒气雾剂接种。 与小粒子气溶胶接种实验相反，大型颗粒气雾剂接种导致肺部炎症减少，如CT评估。 通过大型颗粒气雾剂接种，我们无法实现LD100，即使比小颗粒气溶胶多100倍。我们的研究表明接种途径会影响疾病结果。 我们研究东毒发病机理的第三个项目基于我们的向后纵向分析，该纵向分析确定了几种与NHP中致死性或非致命结局相关的细胞因子。某些细胞因子的持续时间和强度与生存（IFN-gamma和Rantes）或非生存（MCP-1）有关。这些细胞因子的作用是使用表达细胞因子或其受体的每种细胞因子和敲除小鼠的重组病毒研究的。与野生型病毒相比，表达病毒和MCP-1和MCP-1受体敲除小鼠的MCP-1具有致病性。 MCP-1DATA表明巨噬细胞对于控制感染至关重要，但是过度的MCP-1会改变巨噬细胞功能，这可能加剧疾病。表达病毒的IFN-gamma不会发展疾病，而IFN-gamma或其受体的敲除导致致病性增加。在表达病毒的干扰素伽马接种的小鼠中没有产生任何疾病迹象，并且在同等的100％致命剂量的野生型病毒中幸存下来挑战。通过重组病毒在感染部位的干扰素伽马表达可保护野生型病毒的LD100挑战。 2。赋予狂犬病和埃博拉病毒保护的双价疫苗 我们一直在开发一种基于狂犬病病毒的疫苗，该疫苗表达了来自埃博拉病毒，马堡病毒和苏丹病毒的糖蛋白（GP）。 以前，我们开发了（a）富有复制的，（b）复制缺陷，（c）化学失活的狂犬病病毒（RABV）疫苗（RABV）疫苗，使用基于SAD B19 Rabv Wildlife jeffersors in Matthias Schan的SAD B19 Rabv Wildlife viccine in Consigation for Matthias Schnell in thanel的sad reverse遗传系统，表达Zaire Ebolavirus（EBOV）糖蛋白（GP）糖蛋白（GP）。用活或灭活的RABV疫苗免疫，表达Zebov GP诱导每种病毒的细胞和体液免疫，并赋予小鼠致命的RABV和EBOV挑战的保护。我们在恒河猕猴挑战模型中评估了我们的疫苗候选物。通过现场衰减的RABV-GP观察到100％的保护。灭活和RVDELG-GP病毒提供了50％的保护。 观察到强烈的体液和细胞免疫。总而言之，我们的发现表明，RV-GP保留了活体衰减的RABV疫苗的衰减表型，RVDELG-GP似乎是用于野生动植物或用于人类使用的考虑的更安全的替代品。我们还进一步开发了疫苗构建体以使用密码子优化的丝病毒，并证明了这些构建体在小鼠中的免疫原性和安全性。 3. Filovirus分子病毒学 我们还正在研究丝状病毒的分子病毒学。 我们正在研究病毒蛋白在病毒生命周期中磷酸化的作用。我们选择专注于VP35，因为它对于基因组的转录和复制至关重要。使用复制子系统，我们已经生成了潜在磷酸化位点的点突变和簇点突变，并评估突变体的复制和转录。我们的数据表明一种调节VP35功能的分层磷酸化策略。第二个项目涉及识别EBOV非编码区域的结构和功能关系。 我们已经确定了与这些EBOV基因组区域相互作用的宿主蛋白。 我们正在努力识别蛋白质。我们已经将HSC70确定为与EBOV拖车中至少一个域相互作用，并使用免疫沉淀-RT-PCR确认。在EBOV拖车中已经确定了三个HSC70结合结构域，对5个区域的突变分析导致复制子信号降低。我们还对EBOV预告片进行了形状分析，以定义EBOV预告片的二级结构。基于这些数据，我们可以确定治疗靶标，并建立FILOVIRUS生命周期调节的机制。 4。MERS-COV 我们还一直在为MERS-COV开发NHP模型。 我们已经确定，在接种后第4至5天之间，接种MERS-COV JORDAN分离株的气管内接种导致有限的呼吸道疾病，在接种后第4和第5天达到峰值，在接种后第9天消退，可以通过计算机断层扫描来量化。 接种常见的摩尔果会导致较长的呼吸道疾病，在第3至14天之间达到峰值，但不能完全解决，可以通过计算机断层扫描来监测和量化。 在恒河类或普通果果中，无法从连续样品中回收传染病。 为了进一步开发MERS-COV模型，我们正在与Ralph Baric博士合作，构建和评估表达可以用作宠物记者的人类钠 - 碘分类器的MERS-COV。 我们揭示这种重组病毒，以增强NHP和其他动物模型中MER的传染性过程的细节。 我们还与SAB和NMRC进行了合作，以开发对MERS-COV的多克隆抗体作为人类治疗