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/总结 我们建议继续进行中小企业改革第一阶段的工作，并进一步制定一个广泛的 由病毒样颗粒（VLP）组成的可能通用的中和流感疫苗 显示出重塑的HA分子，其表现出另外的隐蔽表位。这些 重构的HA将以缺乏或脱落HA的形式重组表达。 显性高变表位，而是显示明显保守的亚显性表位 已知促进抗体应答的抗原位点，所述抗体应答将中和广泛的 流感病毒谱。此外，显示重塑的VLP结构 HA和NA含有更保守的流感抗原，如基质M1、M2和M3。 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体和疫苗效力减弱后， 流感病毒的快速抗原进化。大多数保护性抗体高度靶向 HA分子球状头部的可变和优势位点，尽管更多 也存在保守的和较少免疫识别的构象抗原位点 在HA的柄部（HA 2）中以及球形头部（HA 1）和柄部之间。 已经发现针对这些位点的分离的人抗体中和了 广谱流感病毒。因此，似乎有理由准备和测试 显示这些高度保守的亚显性抗原位点以及 其他重要的流感抗原，并确定它们是否刺激广泛的抗体 在自然流感感染或接种以下疫苗后的最小反应 含有完整HA分子的制剂。重组HA分子的掺入 流感病毒样颗粒（VLP）应该提供一个很好的机会， 研制出广泛中和的疫苗VLP是通过共表达五个 结构流感蛋白（M1、M2、HA、NA和NP），不含感染性病毒 遗传物质，因此无法复制或引起感染。研究 在I期SBIR期间进行的研究表明，用重塑HA产生的VLP 不仅能引发中和抗体，而且还能提供有效的保护， 单次免疫接种后流感攻毒。为了继续这项工作，我们建议 进一步评价含有替代构象的VLP疫苗， 重塑的HA与天然或修饰的NA一起作为M1、M2 和NP流感病毒样颗粒组装体。这种疫苗是悬浮生产的 培养适合最先进发酵技术的哺乳动物细胞， 适合用于连续生产的稳定细胞系开发。这种疫苗 组合物和构型将引发体液中和免疫以及细胞- 介导的免疫（CMI）对M1和NP抗原。的免疫原性和 VLP疫苗组合物的效力将在小鼠和雪貂中测试， 代表1组和2组甲型流感病毒的攻毒流感病毒亚型。 将对两个谱系的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， 不仅要扩大和加强保护范围， 豁免权持续数年。流感是一个持续的威胁，目前 预防季节性或潜在的大流行爆发的疫苗技术正处于 一种既能按时生产疫苗，又能正确 与流行病毒的抗原成分相匹配即使在最好的 在某些情况下，流感疫苗的整体有效性可能介于40%至 百分之七十为了改变这种范式，我们需要一种疫苗技术来克服并 耐受抗原变异并延长诱导免疫的持续时间， 利用快速、有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，旨在促进疫苗技术的发展 和生产方法，以克服现有疫苗的局限性， 生产

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.