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

描述(申请人提供)：登革热疾病对全球超过25亿人构成重大威胁，目前还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)做出平衡的免疫反应，因为不同血清型登革热病毒的顺序感染可能导致危及生命的登革热出血热或登革热休克综合征。减毒活疫苗或嵌合病毒疫苗很有希望，但它们存在衰减不足或由于干扰而导致免疫反应不平衡的问题。虽然可以通过12个月的免疫时间表克服干扰，但这一时间表使疫苗不适合需要快速发展免疫力的接受者。此外，与17D相关的安全问题可能会限制免疫功能低下的个人、儿童或老年人使用17D。我们已经开发了一系列基于我们临床证明的疫苗平台的黄病毒疫苗，有三种形式，在抗原与鞭毛蛋白(TLR5配体)的结合位置上有所不同。这些候选疫苗 可以有效地产生并诱导针对两种登革热血清型的中和抗体的保护性水平。 TLR信号触发先天免疫级联反应，增强抗原的摄取和呈递，促进适应性免疫反应的诱导，从而消除对亚单位疫苗中通常使用的佐剂的需求。鞭毛蛋白包含四个不同的结构域(D0、D1、D2和D3)，其中D1包含TLR5结合位点。抗原可以融合到C-Term形式的C-末端，代替R3形式的D3，或者融合到R3.2x形式的两个拷贝中的两个位置。在这三种疫苗中，在流感疫苗的第一阶段试验中，R3.2x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)与鞭毛蛋白的C-末端融合。我们已经证明了基于鞭毛蛋白-EIII融合的单价西尼罗河和DENV-2候选疫苗能够诱导强大的抗体反应，并在小鼠攻击模型中有效。随后，我们产生了各种形式的鞭毛蛋白-EIII登革热疫苗，并确定R3.2x为用于小鼠和非人灵长类(NHP)的四价疫苗候选的主要形式。基于人类多克隆和单抗中和表位的最新发现，我们已经研制出备用的鞭毛蛋白-E亚单位疫苗，该疫苗还包括E蛋白(EIEIII)结构域III和I的主要中和表位 作为EI/EII结合区。 我们建议在小鼠免疫原性研究中优化铅TDV配方(剂量水平和成分比例)，然后优化这些候选TDV的发酵条件和纯化工艺。接下来，我们将评估先导TDV在NHP DENV攻击模型中的免疫原性和有效性，然后使用成熟的兔反应性模型评估剂量范围以及配方开发。我们将证明，四价疫苗诱导持久、平衡的免疫反应，能够中和各种血清型的广泛毒株和基因型，并在小鼠和猴子模型中提供对所有四种DENV血清型的保护。一旦候选铅被锁定，我们将开发一种适合4C储存至少一年的液体配方。此外，我们将确定TDV候选的安全有效的窗口，以指导随后的GLP兔毒性研究以及I期试验的剂量递增研究中的剂量选择。这项研究的积极结果将是朝着成功开发更安全、更便宜和有效的疫苗迈出的坚实一步，该疫苗可用于预防DENV相关疾病。最后，VaxInnate以细菌为基础的快速和低成本制造可以很容易地转移到流行国家，正如最近成功转移的2009年H1N1大流行疫苗所表明的那样。

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.