Generation of a polycistronic universal influenza virus vaccine based on rare species adenoviral vectors

摘要：甲型流感病毒（IAV）在人类中引起严重的呼吸道疾病，约250，000 - 500，000 全球每年死亡人数。除了季节性流行病外，新的、 新出现的、耐药的流感病毒，人类对它们的免疫是幼稚的， 公共卫生问题。目前的流感疫苗受到几个问题的影响。其中包括， 狭隘的，菌株特异性的免疫反应，对鸡蛋为基础的制造方法的过度依赖， 长期生产过程（>6个月），需要提前预测哪些菌株将在 即将到来的季节和对多种流感的细胞和体液免疫应答的最小诱导 抗原（Ags）。近年来季节性流感疫苗的次优性能 加速了对开发通用流感病毒疫苗的兴趣，该疫苗能够提供广泛和长期的 对季节性和流行性亚型的有效保护。实现这一目标的战略包括重新确定重点， 针对流感病毒抗原上的高度保守表位的免疫应答，例如流感病毒的茎， 主要表面糖蛋白、血凝素（HA）、神经氨酸酶（NA）或内部核蛋白（NP）。 我的研究旨在开发一种替代的，优化的，通用的流感疫苗平台， 使用三种方法克服与当前疫苗相关的问题。(1)首先，我们将优化 多顺反子Ag表达盒，其中多个IAV Ag同时表达。这些将 包括双顺反子或三顺反子Ag盒，其特征在于来自第1组或第2组IAV组合无头HA NA和/或NP。我将通过将抗原靶向宿主来增强/扩大无头HA或NA的免疫识别- 衍生的细胞外囊泡（EV），包括体内的外来体。这将通过工程融合Ag 构建体以将Ag拴系到富含外泌体的蛋白质结构域。外泌体是纳米大小的电动汽车， 在免疫应答的调节中起重要作用，这是由于它们除了MHC之外还能够呈递Ag 和共刺激分子，对T细胞和B细胞。(2)其次，我会把这些Ag结构改造成非- 复制的、稀有物种的腺病毒（Ad）载体疫苗，其已经建立了临床应用的方案。 在无冷链条件下， 在临床试验中，已在婴儿、成人和老年人中证明了其安全性和免疫原性。 (3)最后，我将全面评估和表型的大小和概况，这些普遍的 单次注射流感疫苗。这些数据将为设计提供有价值的信息。 随后的预充：加强方案和未来的挑战实验。总之，普遍 本提案所述流感疫苗平台非常适合为大流行病储备疫苗 准备，并可以提供异源保护后，一个单一的镜头，这可能是 足以在新出现的大流行病的早期阶段减轻医疗保健系统的负担。

SUMMARY: Influenza A viruses (IAVs) cause serious respiratory illness in humans, with ~250,000-500,000 deaths per year globally. In addition to seasonal epidemics, the ongoing pandemic threat posed by new, emerging, reassortant influenza viruses, for which humans are immunologically naive, represents a major public health concern. Current influenza vaccines are impacted by several issues. These include, the elicitation of narrow, strain-specific immune responses, an over-reliance on egg-based manufacturing methods, a protracted production process (>6 months), the need to predict in advance which strains will circulate in forthcoming seasons and the minimal induction of cellular and humoral immune responses to multiple influenza antigens (Ags) simultaneously. The sub-optimal performance of seasonal influenza vaccines in recent years has accelerated interest in developing a universal influenza virus vaccine, capable of providing broad and long- lived protection against seasonal and pandemic subtypes. Strategies to achieve this include refocusing immune responses towards highly conserved epitopes on influenza virus antigens such as the stalk of the major surface glycoprotein, hemagglutinin (HA), the neuraminidase (NA) or the internal nucleoprotein (NP). My research aims to develop an alternative, optimized, universal influenza vaccine platform which will overcome issues associated with current vaccines using three approaches. (1) Firstly, I will optimize polycistronic Ag expression cassettes, in which multiple IAV Ags are expressed simultaneously. These will include bi- or tri-cistronic Ag cassettes featuring headless HAs from group 1 or group 2 IAVs in combination with NA and/or NP. I will augment/broaden immune recognition of headless HA or NA by targeting Ags to host- derived extracellular vesicles (EVs) including exosomes in vivo. This will be achieved by engineering fusion-Ag constructs to tether Ag to a protein domain enriched in exosomes. Exosomes are nano-sized EVs which play important roles in the regulation of immune responses, due to their ability to present Ag, in addition to MHC and co-stimulatory molecules, to T- and B-cells. (2) Secondly, I will engineer these Ag constructs into non- replicating, rare species adenoviral (Ad) vectored vaccines, which have established protocols for clinical manufacturing, can be thermostabilized with minimal losses to immunogenicity under cold-chain free conditions and have demonstrated safety and immunogenicity in infants, adults and the elderly in clinical trials. (3) Finally, I will comprehensively evaluate and phenotype the magnitude and profile of these universal influenza vaccines in single-shot regimens. These data will provide valuable information for the design of subsequent prime:boost regimens and for challenge experiments in the future. In summary, the universal influenza vaccine platform described in this proposal would be well-suited to stockpiling for pandemic preparedness, and could provide heterologous protection following a single shot, which may be sufficient to ease the burden on the healthcare system in the early phase of an emerging pandemic.