Laboratory And Preclinical Studies Of Flaviviruses

黄病毒的实验室和临床前研究

• 批准号: 10692068
• 负责人: Stephen Whitehead
• 金额: $ 104.83万
• 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/10692068
• 关键词: Accounting; Admixture; Antibodies; Asia; Attention; Attenuated; Attenuated Live Virus Vaccine; Attenuated Vaccines; Basic Science; Binding; Biochemical; Biological Assay; Brain; Brazil; Case Study; Cells; Cessation of life; China; Clinical; Clinical Distribution; Clinical Research; Clinical Trials; Collaborations; Consultations; Culicidae; Cyclic GMP; Data; Dengue Fever; Dengue Vaccine; Dengue Virus; Development; E protein; Engineering; Epididymis; Epitopes; Evaluation; Family; Flaviviridae; Flavivirus; Formulation; Generations; Genome; Goals; Harvest; Human; Immune; Immune response; Immunology; Individual; Infection; Innate Immune Response; Institution; Japanese Encephalitis; Japanese encephalitis virus; Laboratories; Laboratory Study; Length; Macaca mulatta; Maintenance; Methods; MicroRNAs; Modeling; Molecular; Monkeys; Mus; Mutation; Neutralizing antibody assay; Pathogenicity; Pathology; Phase I Clinical Trials; Phase II Clinical Trials; Placenta; Preclinical Testing; Preparation; RNA; Recombinants; Research Personnel; Rivers; Saliva; Seeds; Sequence Analysis; Serotyping; Shipping; Southeastern Asia; Sterility; Structure; Syndrome; Techniques; Technology; Time; Tissues; Untranslated Regions; Vaccination; Vaccines; Viral; Viral Encephalitis; Viral Pathogenesis; Virulent; Virus; Virus Replication; Visit; West Nile Fever; Work; Yellow Fever; ZIKA; ZIKV infection; Zika Virus; cancer type; clinical application; clinical lot; human subject; immunogenicity; interest; macrophage; manufacturing process development; member; mosquito-borne; nonhuman primate; phase 3 testing; preclinical study; programs; recombinant virus; research clinical testing; vaccine candidate; vaccine formulation; vaccine platform; virology; Accounting; Admixture; Antibodies; Asia; Attention; Attenuated; Attenuated Live Virus Vaccine; Attenuated Vaccines; Basic Science; Binding; Biochemical; Biological Assay; Brain; Brazil; Case Study; Cells; Cessation of life; China; Clinical; Clinical Distribution; Clinical Research; Clinical Trials; Collaborations; Consultations; Culicidae; Cyclic GMP; Data; Dengue Fever; Dengue Vaccine; Dengue Virus; Development; E protein; Engineering; Epididymis; Epitopes; Evaluation; Family; Flaviviridae; Flavivirus; Formulation; Generations; Genome; Goals; Harvest; Human; Immune; Immune response; Immunology; Individual; Infection; Innate Immune Response; Institution; Japanese Encephalitis; Japanese encephalitis virus; Laboratories; Laboratory Study; Length; Macaca mulatta; Maintenance; Methods; MicroRNAs; Modeling; Molecular; Monkeys; Mus; Mutation; Neutralizing antibody assay; Pathogenicity; Pathology; Phase I Clinical Trials; Phase II Clinical Trials; Placenta; Preclinical Testing; Preparation; RNA; Recombinants; Research Personnel; Rivers; Saliva; Seeds; Sequence Analysis; Serotyping; Shipping; Southeastern Asia; Sterility; Structure; Syndrome; Techniques; Technology; Time; Tissues; Untranslated Regions; Vaccination; Vaccines; Viral; Viral Encephalitis; Viral Pathogenesis; Virulent; Virus; Virus Replication; Visit; West Nile Fever; Work; Yellow Fever; ZIKA; ZIKV infection; Zika Virus; cancer type; clinical application; clinical lot; human subject; immunogenicity; interest; macrophage; manufacturing process development; member; mosquito-borne; nonhuman primate; phase 3 testing; preclinical study; programs; recombinant virus; research clinical testing; vaccine candidate; vaccine formulation; vaccine platform; virology

The mosquito-borne members of the Flaviviridae family, contain a single-stranded positive-sense RNA genome and are the cause of yellow fever, dengue fever, Japanese encephalitis, Zika, and West Nile fever syndromes. In recent years, much of our laboratory effort was focused on the development and preclinical testing of dengue virus vaccine candidates suitable for inclusion in a live attenuated tetravalent vaccine. Clinical lots of each of these vaccine candidates were manufactured in prior years and have been evaluated individually and in combination in numerous Phase I and II clinical trials, Optimal tetravalent admixtures have been selected and have now completed a first Phase III evaluation. Although the dengue virus vaccine program is predominantly in a clinical mode at this time, considerable effort is currently devoted to support a number of important functions, including, 1) manufacture, replacement, maintenance, stability/sterility analysis, and distribution of clinical lots of vaccines suitable for study in human subjects, 2) basic research on virus stabilization,, 3) submission and laboratory support of IND applications for the clinical evaluation of tetravalent dengue vaccine formulations, 4) support of the seven companies/institutions that have licensed our vaccine technology or virus products, which includes consultative visits and clinical trial planning, development of manufacturing processes, preparation and shipping of vaccine seed or clinical lot viruses, assistance with sequence analysis, and sharing of IND/clinical trial data, 5) support of collaborations with investigators interested in basic virology or immunology studies, 6) use of immune cells collected from our clinical studies to investigate the innate immune response to vaccination, 7) characterization of epitopes recognized following dengue or Zika virus infection, and 8) refinement/qualification of laboratory assays such as the antibody neutralization assay, and 9) isolation and recombinant construction of suitable DENV-4 and DENV-1 strains for use as a potential clinical challenge strain. With the emergence of Zika virus in the Western hemisphere ur attention remains focused on the development of live attenuated vaccine candidates that would be compatible for co-formulation with our tetravalent dengue vaccine. Initially, recombinant chimeric viruses expressing the prM and E proteins of Zika virus on either the DENV-2 or DENV-4 background were generated and evaluated successfully in rhesus monkeys and human subjects. Although the chimeric candidate appeared to be safe for use in humans, it was observed to be over-attenuated with low infectivity and subsequently limited immunogenicity, even at an increased inoculation potency. Additional candidates were evaluated in preclinical studies in collaboration with LID and include full-length ZIKV candidates containing 3-UTR deletions and microRNA targets that restrict virus replication in tissues such as brain, placenta, epididymis, and certain macrophage types. Three of these candidates have been manufactured under cGMP by Charles River Laboratories and are IND applications are pending. In addition, collaborative studies continue to look at the pathology and immune responses of ZIKV in monkey models of infection and the use of the virus as a cytolytic viral agent against several cancer types. Mosquito-borne Japanese encephalitis virus (JEV) causes the most important viral encephalitis in the Asia Pacific region, accounting for more than 20,000 reported cases and 6,000 deaths annually. Efforts to develop a JEV vaccine continue in our laboratory and it is envisioned that a suitable live attenuated JEV vaccine could be combined with our live attenuated DEN virus vaccine to create a second-generation pentavalent vaccine for the control of these viruses in Southeast Asia. The laboratory has recovered numerous engineered viruses and has evaluated their pathogenicity in mice. The recombinant rJEV virus remains fully virulent in mice and provides a background for the evaluation of attenuating mutations. Sets of mutations derived from the attenuated SA14-14-2 vaccine virus produced in China have been introduced into the rJEV virus clone in order to evaluate the attenuating potential of mutations found in both the structural and non-structural genes. Following earlier evaluation in mice, a number of these recombinant viruses look suitable for evaluation in non-human primates and consideration as vaccine candidates. We are awaiting the re-commissioning of our BSL3 suite to enable us to move the JEV vaccine candidates forward. We plan to investigate the maturation state of DENV produced by live mosquitoes with a central hypothesis that viral the maturation state contributes to overall viral pathogenesis. To accomplish this we will optimize several techniques and variables: identify a specific combination of DENV and mosquito strains capable of producing sufficient virus titers for analysis, develop an efficient mosquito saliva/virus harvesting technique, and optimize molecular and biochemical methods for quantifying levels of viral maturation.

Flaviviridae家族的蚊子传播成员包含一个单链的阳性RNA基因组，是黄热病，登革热，日本脑炎，Zika和West Nile Fever综合征的原因。近年来，我们的许多实验室努力都集中在登革热病毒疫苗候选物的开发和临床前测试，适合纳入活衰减的四载疫苗。这些候选疫苗的临床批次是在前几年生产的，并且已经在许多I期和II期临床试验中进行了单独评估，并且已经选择了最佳的四位化助剂，现在已经完成了第一阶段的III期评估。尽管目前，登革热病毒疫苗计划主要是在临床模式下，但目前大量精力致力于支持许多重要功能，包括1），包括1），制造，替代，维持，维持，稳定性/不育分析，稳定性/不育分析，临床疫苗的分布大量适用于人类受试者研究的临床疫苗，以进行人类稳定和实验性研究，2）3）3）3）3）3）3）3）3）3）3）3）3）3）3）3） dengue vaccine formulations, 4) support of the seven companies/institutions that have licensed our vaccine technology or virus products, which includes consultative visits and clinical trial planning, development of manufacturing processes, preparation and shipping of vaccine seed or clinical lot viruses, assistance with sequence analysis, and sharing of IND/clinical trial data, 5) support of collaborations with investigators interested in basic virology or immunology studies, 6) use of immune cells collected from我们研究对疫苗接种的先天免疫反应的临床研究，7）表征登革热或Zika病毒感染后识别的表位，以及8）实验室测定的细化/资格，例如抗体中和测定等实验室测定，9）隔离和重组的隔离和重组构建适当的DENV-4和DENV-1菌株，用于使用潜在的临床挑战。 随着西半球的寨卡病毒的出现，您的注意力仍然集中在活衰减的疫苗候选物的发展上，这些疫苗候选物将与我们的四卫登革热疫苗共同成型。最初，在DENV-2或DENV-4背景上表达Zika病毒的PRM和E蛋白的重组嵌合病毒是在恒河猴和人类受试者中成功评估的。 尽管嵌合候选者似乎可以安全地用于人类，但观察到它的感染率低，随后被限制了免疫原性，即使在接种效力上也是如此。在临床前研究中评估了其他候选者，并包括包含3-UTR缺失和microRNA靶标的全长ZIKV候选者，这些候选者限制了脑，胎盘，附子膜和某些巨噬细胞等组织中病毒复制的范围。这些候选人中有三名是由查尔斯河实验室（Charles River Laboratories）在CGMP下制造的，并且正在申请IND。 此外，协作研究继续研究猴子感染模型中ZIKV的病理和免疫反应，并将病毒用作几种癌症类型的细胞溶解病毒剂。 蚊子传播的日本脑炎病毒（JEV）在亚太地区引起最重要的病毒脑炎，每年占20,000多例病例和6,000例死亡。在我们的实验室中，仍在继续开发JEV疫苗的努力，可以预见，可以将合适的活死的JEV疫苗与我们的活衰减的Den病毒疫苗结合使用，以创建第二代五角星疫苗，以控制东南亚这些病毒。该实验室已经回收了许多工程病毒，并评估了其在小鼠中的致病性。重组RJEV病毒在小鼠中仍然充满活力，并提供了评估衰减突变的背景。为了评估在结构和非结构性基因中发现的突变的衰减潜力，已将在中国产生的衰减的SA14-14-2疫苗病毒中得出的突变集已引入中。在较早的小鼠评估之后，许多这些重组病毒看起来适合于非人类灵长类动物的评估，并将其视为候选疫苗。我们正在等待重新委托BSL3套件，以使我们能够将JEV疫苗候选者前进。 我们计划研究活体蚊子产生的DENV的成熟状态，其中一个中心假设，即成熟状态有助于整体病毒发病机理。为此，我们将优化几种技术和变量：确定DENV和蚊子菌株的特定组合，能够产生足够的病毒滴度进行分析，开发有效的唾液/病毒收集技术，并优化分子和生物化学方法，以量化病毒成熟水平。