Development of a Recombinant Tetravalent Dengue Vaccine Based on a Subunit Envelo

描述（由申请人提供）：登革热对全球超过25亿人构成重大威胁，目前没有获得许可的疫苗。有效和安全的疫苗需要对所有四种登革热病毒血清型（DENV-1至-4）的平衡免疫应答，因为不同登革热病毒血清型的连续感染可导致危及生命的登革出血热或登革休克综合征。基于活减毒或嵌合病毒的疫苗是有希望的，但它们由于干扰而难以充分减毒或不平衡的免疫应答。虽然可以通过12个月的免疫接种时间表克服干扰，但这种时间表使疫苗不适合需要快速发展免疫力的接种者。此外，与17 D相关的安全性问题可能会限制其在免疫功能低下的个体、儿童或老年人中的使用。我们已经开发了一系列的黄病毒疫苗的基础上，我们的临床证明的疫苗平台在三种形式，不同的抗原附着到鞭毛蛋白（TLR 5配体）的网站。这些候选疫苗 可以有效地产生并引发保护水平的针对两种登革热血清型的中和抗体。 TLR信号传导触发先天免疫级联反应，其增强抗原摄取和呈递，并促进诱导适应性免疫应答，从而消除对亚单位疫苗中通常使用的佐剂的需要。鞭毛蛋白含有四个不同的结构域（D 0、D1、D2和D3），其中D1含有TLR 5结合位点。抗原可以以C端形式融合到C端，代替R3形式中的D3，或以R3.2x形式融合到两个拷贝中的两个位置。在这三种疫苗中，R3.2x形式在流感疫苗的I期试验中被发现是最安全和最具免疫原性的。为了开发黄病毒疫苗，我们最初将包膜的结构域III（EIII）融合到鞭毛蛋白的C-末端。我们已经证明，基于鞭毛蛋白-EIII融合的单价西尼罗和DENV-2疫苗候选物诱导有效的抗体应答，并且在小鼠攻击模型中是有效的。我们随后产生了各种形式的鞭毛蛋白-EIII登革热疫苗，并已将R3.2x鉴定为用于小鼠和非人灵长类动物（NHP）的四价候选疫苗的主要形式。基于人多克隆抗体和单克隆抗体的中和表位的最新发现，我们已经产生了备份亚单位鞭毛蛋白-E疫苗，其包括E蛋白（EIEIII）的结构域III和I中的主要中和表位 作为EI/EII结区。 在此，我们建议在小鼠免疫原性研究中优化先导TDV制剂（剂量水平和组分比例），然后优化这些候选物的发酵条件和纯化工艺。接下来，我们将评价前导TDV在NHP DENV攻毒模型中的免疫原性和有效性，然后使用完善的兔反应原性模型评价剂量范围以及制剂开发。我们将证明四价疫苗诱导持久、平衡的免疫应答，能够中和各种血清型的广谱毒株和基因型，并在小鼠和猴模型中提供针对所有四种DENV血清型的保护。一旦锁定了主要候选产品，我们将开发一种适合4 º C储存至少一年的液体配方。此外，我们将确认TDV候选药物的安全有效窗口，以指导后续GLP家兔毒性研究以及I期试验中剂量递增研究的剂量选择。这项研究的积极结果将是成功开发更安全，更便宜，更有效的疫苗的坚实一步，可用于预防DENV相关疾病。最后，VaxInnate的快速和低成本的细菌为基础的生产可以很容易地转移到流行的国家，证明了最近成功的过程转移的2009年H1N1大流行疫苗。

DESCRIPTION (provided by applicant): Dengue disease poses a significant threat to over 2.5 billion people worldwide and currently, no licensed vaccine is available. An effective and safe vaccine requires a balanced immune response to all four serotypes of dengue viruses (DENV-1 to -4) because sequential infection by different dengue virus serotypes can lead to life-threatening dengue hemorrhagic fever or dengue shock syndrome. Live attenuated or chimeric virus-based vaccines are promising but they struggle with inadequate attenuation or an imbalanced immune response due to interference. While interference can be overcome by a 12-month immunization schedule, this schedule renders the vaccine unsuitable for recipients requiring rapid development of immunity. Additionally, safety concerns associated with 17D may limit its use in immunocompromised individuals, children or elderly. We have developed a series of flavivirus vaccines based on our clinically proven vaccine platform in three formats, which differ in the site of antigen attachment to the flagellin (a TLR5 ligand). These vaccine candidates can be efficiently produced and elicit protective levels of neutralizing antibodies to both dengue serotypes. TLR signaling triggers an innate immune cascade that enhances antigen uptake and presentation, and facilitates induction of adaptive immune response, thereby eliminating the need for adjuvants typically used in subunit vaccines. Flagellin contains of four distinct domains (D0, D1, D2, and D3) where D1 contains the TLR5 binding site. Antigens can be fused to the C-terminus in the C-term format, in place of D3 in the R3 format, or to both positions in two copies in the R3.2x formats. Among the three, the R3.2x format was found to be the safest and the most immunogenic in Phase I trials of influenza vaccine. To develop flavivirus vaccines, we initially fused the domain III of the envelope (EIII) to the C-terminus of the flagellin. We have demonstrated that monovalent West Nile and DENV-2 vaccine candidates based on the flagellin-EIII fusion induce potent antibody responses and are efficacious in mouse challenge models. We have subsequently generated flagellin-EIII dengue vaccines in various formats, and have identified R3.2x as the lead format for use in a tetravalent vaccine candidate in mice and non-human primates (NHP). Based on recent findings of neutralizing epitopes of human polyclonal and monoclonal antibodies, we have generated backup subunit flagellin-E vaccines that include major neutralizing epitopes in the domains III and I of the E protein (EIEIII) as well as the EI/EII junction region. We here propose to optimize the lead TDV formulation (dose level and component ratio) in immunogenicity studies in mice, and then to optimize the fermentation conditions and purification process for those candidates. We will next evaluate the immunogenicity and efficacy of the lead TDV in NHP DENV challenge models, followed by evaluation of dose range using a well-established rabbit reactogenicity model as well as formulation development. We will demonstrate that the tetravalent vaccine induces durable, balanced immune responses that are capable of neutralizing a broad spectrum of strains and genotypes of each serotype, and provides protection against all four DENV serotypes in mouse and monkey models. Once the lead candidates are locked we will develop a liquid formulation suitable for 4 ¿C storage for at least one year. Further, we will confirm a safe and effective window of the TDV candidate to guide the dose selection in the subsequent GLP rabbit toxicity study as well as dose escalating study in phase I trial. The positive outcome of this study will be a solid step towards the successful development of a safer, cheaper, and effective vaccine, which can be used to prevent DENV-associated diseases. Lastly, VaxInnate's rapid and low cost bacteria-based manufacturing can easily be transferred to endemic countries as demonstrated by a recent successful process transfer of the 2009 H1N1 pandemic vaccine.