Optimization of a mucosal RSV vaccine

摘要 人呼吸道合胞病毒(RSV)是副粘病毒家族中具有高度传染性的成员，可引起 上呼吸道和下呼吸道感染。呼吸道合胞病毒是引起低收入人群肺部疾病的主要原因 病毒引起的婴幼儿呼吸道感染(毛细支气管炎、肺炎和呼吸衰竭) 破坏和复杂的炎症过程，每年估计造成16万人死亡 全世界。呼吸道合胞病毒还会导致免疫受损和老年人群的发病率/死亡率。在美国， 治疗呼吸道合胞病毒肺炎和毛细支气管炎每年的医疗费用超过4亿美元。因为无论是因为 免疫原性低和/或出于安全原因，以前试图研制预防RSV的疫苗- 介导性疾病并没有成功。福尔马林灭活疫苗候选疫苗(FI-RSV)诱导 随后自然感染呼吸道合胞病毒后的严重疾病，接种疫苗的儿童被发现患有 来自疾病严重性的增加，甚至死亡。严重的肺部炎症反应，其特征是 扭曲的CD4+T细胞反应(在没有中和抗体的情况下)和嗜酸性粒细胞的涌入 肺组织检测。由于对安全有效的RSV疫苗的需求尚未得到满足，新的方法正在开发中 需求量很大。因为对于RSV疫苗来说，保护上呼吸道和下呼吸道感染 在随后的RSV感染中，理想的RSV候选疫苗应该引起持久的粘膜反应和 保护作为东道主的第一道防线。能够诱导持久的RSV特异性的疫苗 呼吸道中的粘膜免疫球蛋白A反应可能比产生系统性肺炎的粘膜免疫球蛋白A更具保护性 单独的抗体反应。 幸运的是，RSV研究中最令人兴奋的进展之一是由一名合作研究员在这方面取得的 格兰特。具体地说，在稳定融合前F(Pre-F)上发现了一个新发现的抗原位点(？) 蛋白和这个前F蛋白，以及所有四个中和抗原位点(？、I、II和IV)，被证明是一种 免疫原性比融合后的F(后F)具有三个中和抗原位点(I，II和IV)，即 以前曾被用于疫苗接种方法。发展一种安全有效的粘膜RSV 疫苗，在这项提议中，我们计划将这种新抗原的优点与我们的专利粘膜结合起来 疫苗接种平台，优化小鼠和棉大鼠的最佳接种条件。这个平台有 已经显示出强大的免疫反应和对两种不同的粘膜 挑战HSV-2和RSV等病毒。这种强大的疫苗方案可以产生Th1偏向的、广泛的 以及强大的体液、粘膜和T细胞反应，包括大量的粘膜IgA和CTL。这个 我们专利免疫方案的独特之处在于：1)粘膜(特别是粘膜中和) 抗体)、系统免疫反应和完全的粘膜保护，而不使用任何病毒 媒介和/或有毒佐剂；2)未检测到免疫病理学或疫苗增强型疾病 病毒挑战了动物。因此，这个黏膜疫苗平台是开发一种 防止进入粘膜表面的病原体的粘膜疫苗，如呼吸道合胞病毒。 使用这种获得专利的粘膜免疫策略，我们将建立安全有效的粘膜RSV 疫苗是在以前资助的SBIR赠款中开发的，并通过广泛的测试和 比较小鼠和棉花大鼠的F前抗原和F后抗原。一支非常强大的科学团队，包括 四个合作研究所：生物医学研究模型公司、达特茅斯学院、爱荷华大学和 西格莫夫生物系统公司将：1)准备和优化疫苗配方，在两只BALB/c小鼠身上进行测试 和棉花鼠，2)优化最佳免疫剂量和免疫程序，比较F前和F后的诱导效果 小鼠的免疫应答和保护；3)优化棉鼠免疫的最佳条件 使用前F和后F，测试和比较免疫反应的持久性、保护性和安全性 (肺组织病理学)由RSV候选疫苗在棉花大鼠中提供。在完成 格兰特，我们希望：1)完全或显著地保护动物免受肺部和鼻腔病毒复制的影响 通过；2)没有疫苗增强的肺部疾病；3)持久的粘膜免疫和保护性免疫。最好的 将确定抗原、最佳配方/剂量和免疫时间表，以供进一步开发。这 研究是我们可以推进第二阶段研究的关键一步，这将需要cGMP的主要合作伙伴 制造最好的疫苗抗原配方，进行毒理学研究，并测试粘膜 在非洲绿猴模型中使用RSV疫苗，并最终将IND提交临床试验。

Abstract Human respiratory syncytial virus (RSV) is a highly infectious member of the paramyxovirus family causing upper and lower respiratory tract infections. RSV is the leading cause of pulmonary disease of the lower respiratory tract (bronchiolitis, pneumonia and respiratory failure) in infants due to virus-induced airway damage and complex inflammatory processes and responsible for an estimated 160,000 deaths annually worldwide. RSV also causes morbidity/mortality to immunocompromised and elderly populations. In the USA, treatment of RSV pneumonia and bronchiolitis annual health care costs exceed $400 million. Because of either low immunogenicity and/or for safety reasons, previous attempts to formulate a vaccine to prevent RSV- mediated disease have not been successful. The formalin-inactivated vaccine candidate (FI-RSV) induced severe disease upon subsequent natural infection with RSV wherein vaccinated children were found to suffer from enhanced disease severity and even death. Severe lung inflammatory responses characterized by a skewed CD4+ T-cell response (in the absence of neutralizing antibodies) and an influx of eosinophils in the lung were detected. Because of the unmet need of a safe and effective RSV vaccine, novel approaches are in high demand. Since it is important for an RSV vaccine to protect the upper and lower respiratory tracts from subsequent RSV infection, the ideal RSV vaccine candidate should elicit a durable mucosal response and protection as the first line of defense in the host. Vaccines that are able to induce durable RSV-specific mucosal IgA responses in the respiratory tract may be more protective than those that generate a systemic antibody response alone. Fortunately, one of the most exciting advances in RSV research was made by a co-investigator on this grant. Specifically, a newly discovered antigenic site (Ø) was identified on stabilize the pre-fusion F (pre-F) protein and this pre-F, with all four neutralizing antigen sites (Ø, I, II, and IV), was proven to be a far more immunogenic antigen than the post-fusion F (post-F) with three neutralizing antigen sites (I, II, and IV) that have been previously employed in vaccination approaches. To develop a safe and effective mucosal RSV vaccine, in this proposal we plan to combine the merits of this novel antigen and our patented mucosal vaccination platform, and optimize the best vaccination conditions in mice and cotton rats. This platform has already demonstrated potent immune responses and significant protection against two different, mucosally challenged viruses such as HSV-2 and RSV. This powerful vaccine regimen can generate a Th1 biased, broad and potent humoral, mucosal and T cell responses including substantial mucosal IgA and CTL. The uniqueness of our patented immunization regimen are:1) both mucosal (especially mucosal neutralizing antibodies), systemic immune responses and complete mucosal protection were raised without using any virus vectors and/or toxic adjuvants; 2) no immunopathology or vaccine-enhanced diseases have been detected in virus challenged animals. Therefore, this mucosal vaccine platform is an ideal candidate for developing a mucosal vaccine that protects against pathogens which enter at mucosal surfaces, as is the case for RSV. Using this patented mucosal immunization strategy, we will build on a safe and effective mucosal RSV vaccine that was developed in a previously funded SBIR grant and further optimize by extensively testing and comparing the pre-F and the post-F antigens in mice and cotton rats. A very strong scientific team including four collaborating Institutes: Biomedical Research Models, Inc., Dartmouth College, University of Iowa and Sigmovir Biosystem, Inc. will: 1) prepare and optimize vaccine formulations to be tested in both BALB/c mice and cotton rats, 2) optimize the best immunization dose and schedule for comparing pre-F vs. post F in eliciting the immune responses and protection in mice, 3) Optimize the best conditions for immunization of cotton rats with pre-F and post F, test and compare the durability of immune responses, protection, and safety (pulmonary histopathology) provided by the RSV vaccine candidates in cotton rats. At the completion of the grant, we expect: 1) complete or significant protection of animals from virus replication in both lung and nasal passage; 2) no vaccine-enhanced pulmonary diseases; 3) durable mucosal and protective immunity. The best antigen, optimal formulation/dose, and immunization schedule will be identified for further development. This study is a critical step before we can advance with phase II studies, which will require key partners for cGMP manufacturing the best vaccine antigen formulation, performing toxicology studies, and testing of the mucosal RSV vaccine in the African Green Monkey model and eventually filing the IND for clinical trials.