Universal VLP-Based Flu Vaccine:Broadly protective (universal) virus-like particle (VLP) based influenza vaccine that can neutralize a broad spectrum of influenza A virus subtypes.

1.摘要/摘要 我们建议继续我们的SBIR第一阶段工作，并进一步发展一个广泛的 由病毒样颗粒(VLP)组成的中和、可能通用的流感疫苗 展示重塑的HA分子，这些分子表现出原本神秘的表位。这些 重塑的HAS将以缺少或脱落的形式进行重组表达 显性高变表位，表现为明显保守的亚显性表位 已知的抗原部位可以促进抗体反应，从而中和广泛的 流感病毒的谱系。此外，VLP结构显示了改建的 HA和NA含有更保守的流感抗原，如M1、M2和 核蛋白NP。目前流感疫苗的保护范围仅限于 同源病毒或密切相关的变种和疫苗效力随着 流感病毒的快速抗原性进化。大多数保护性抗体具有很高的靶向性 HA分子球状头部的可变和优势部位，尽管更多 也存在保守的和免疫识别较少的构象抗原位点。 在HA的茎(HA2)和球形头部(HA1)与茎部分之间。 针对这些部位的分离的人类抗体已被发现能中和 广谱的流感病毒。因此，准备和测试似乎是合理的 显示这些高度保守的亚优势抗原位点的疫苗与 其他重要的流感抗原，并确定它们是否会刺激广泛的抗体 在自然流感感染中或接种疫苗后的最小反应 含有完整HA分子的配方。修饰的HA分子的掺入 变成流感病毒样颗粒(VLP)应该提供一个极好的机会 开发一种广泛中和的疫苗。VLP是由五个基因共同表达产生的 流感结构蛋白(M1、M2、HA、NA和NP)，不含传染性病毒 遗传物质，因此不能复制或引起感染。研究 在第一阶段SBIR期间执行的结果显示，使用改装的HA生成的VLP不 不仅能诱导中和抗体，而且还能提供显著的保护，防止致命的 流感单次免疫后挑战。为了继续这项工作，我们建议 进一步评估基于VLP的疫苗，该疫苗包含不同构象的 改型HA与天然或改性NA一起作为M1、M2的表面组分 和NP流感病毒样颗粒集结。这种疫苗是悬浮式生产的。 适合最先进发酵技术的哺乳动物细胞培养和 为连续生产提供稳定的细胞系发展。这种疫苗 其组成和构型可诱导体液中和免疫以及细胞免疫。 对M1和NP抗原的中介免疫(CMI)。免疫原性和 VLP疫苗组合物的效力将在小鼠和雪貂身上进行测试，使用AS 挑战代表1组和2组流感病毒的流感病毒亚型。 将对两种B型流感病毒采取类似的策略。这些 研究将为推进流感疫苗的开发提供基础 不仅拓宽和加强了保护的范围，而且还扩大了 几年的豁免期。流感是一个持续的威胁，目前 预防季节性或潜在大流行爆发的疫苗技术正在开发中 永久追赶模式不仅要按时生产疫苗，而且要正确 与流行的流行病毒的抗原成分相匹配。即使在最好的情况下 在某些情况下，流感疫苗的整体效力可达40%至 70%。为了改变这种模式，我们需要一种疫苗技术来克服和 抵御抗原变异，延长诱导免疫持续时间以及 利用快速、高效和具有成本效益的疫苗制造方法。这项建议 解决这些问题，旨在推动疫苗技术的发展 和生产方法，以克服现有疫苗及其 制作。

1. ABSTRACT / SUMMARY We propose to continue our SBIR phase I work and further develop a broadly neutralizing, possibly universal influenza vaccine composed of virus-like particles (VLPs) displaying remodeled HA molecules which exhibit otherwise cryptic epitopes. These remodeled HAs will be expressed recombinantly in forms lacking or shedding the dominant hypervariable epitopes and instead display distinctly conserved subdominant antigenic sites known to promote an antibody response that will neutralize a broad spectrum of influenza viruses. Additionally, the VLP structure displaying the remodeled HA and NA contains more conserved influenza antigens like matrices M1, M2 and the nucleoprotein NP. The protective scope of current influenza vaccines is restricted to homologous viruses or closely related variants and vaccine efficacy wanes following the fast antigenic evolution of the influenza virus. Most protective antibodies target highly variable and dominant sites on the globular head of the HA molecule, although more conserved and less immune-recognized conformational antigenic sites are also present in the stem (HA2) and between the globular head (HA1) and stem portions of HA. Isolated human antibodies directed toward these sites have been found to neutralize a broad spectrum of influenza viruses. It seems reasonable therefore to prepare and test vaccines that display these highly conserved subdominant antigenic sites together with other important influenza antigens and determine if they stimulate a broad antibody response which is minimal in a natural influenza infection or following vaccination with formulations containing whole HA molecules. Incorporation of remodeled HA molecules into influenza virus-like particles (VLPs) should provide an excellent opportunity to develop a broadly neutralizing vaccine. VLPs are generated by the co-expression of five structural influenza proteins (M1, M2, HA, NA and NP) and do not contain infectious viral genetic material and are therefore unable to replicate or cause infection. Studies performed during the phase I SBIR showed that VLP generated with remodeled HA not only elicit neutralizing antibodies but also afford significant protection against a lethal influenza challenge after a single immunization. To continue this work, we propose to further evaluate the VLP based vaccine containing alternative conformations of the remodeled HA together with native or modified NA as surface component of a M1, M2 and NP influenza virus-like particle assembly. This vaccine is produced in suspension culture of mammalian cells suitable for the most advanced fermentation technology and amenable for stable cells line development for continuous manufacturing. This vaccine composition and configuration will elicit humoral neutralizing immunity as well as cell- mediated immunity (CMI) toward the M1 and NP antigens. The immunogenicity and efficacy of the VLP vaccine compositions will be tested in mice and ferrets using as challenge influenza virus subtypes that represent group 1 and group 2 Influenza A virus. A similar strategy will be pursued with the two lineages of influenza B viruses. These studies will provide the foundation to advance development of an influenza vaccine able not only to broaden and enhanced the spectrum of protection but also extend the duration on immunity for several years. Influenza is a constant threat and the current vaccine technology to prevent seasonal or potential pandemic outbreaks is in a permanent catch up mode not only to produce vaccine on time but also to correctly match the antigenic composition of prevailing circulating virus. Even in the best of circumstances the overall effectiveness of the influenza vaccine may range from 40% to 70%. To change this paradigm, we need a vaccine technology that overcomes and withstands antigenic variation and prolongs the duration of induced immunity as well as utilizes rapid, efficient and cost-effective vaccine manufacturing methods. This proposal addresses these issues aiming to advance the development of the vaccine technology and production methods to overcome the limitation of the current vaccine and its production.