Improving mRNA vaccines with extracellular vesicle-associated immunogens

使用细胞外囊泡相关免疫原改进 mRNA 疫苗

• 批准号: 10850617
• 负责人: Michael R. Farzan
• 金额: $ 18.96万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-09 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10850617
• 关键词: 2019-nCoV; Acting Out; Adenovirus Vector; Animals; Antibody Formation; Antibody Response; Antibody titer measurement; Antigen-Presenting Cells; Antigens; Behavior; COVID-19; COVID-19 vaccine; Capsid Proteins; Carrier Proteins; Cell Fraction; Cell Line; Cell membrane; Cells; Characteristics; Communicable Diseases; DNA; Data; Development; Dose; Effectiveness; Eukaryotic Cell; Feline Immunodeficiency Virus; Fluzone; Formulation; Glycoproteins; HIV; HIV-1; Hemagglutinin; Human; Immune; Improve Access; Influenza A virus; Injections; Interferons; Membrane; Messenger RNA; Modification; Molecular Conformation; Mus; Nonstructural Protein; Patients; Performance; Preparation; Production; Proteins; RNA vaccine; Recombinants; Reporting; Resistance; Route; SARS-CoV-2 spike protein; Sampling; Serum; Site; Structure; Subunit Vaccines; Tail; Tertiary Protein Structure; Testing; Transfection; Translating; Vaccinated; Vaccines; Viral; Viral Antigens; Viral Envelope Proteins; Viral Matrix Proteins; Viral Proteins; Viral Vaccines; Virus; Virus Diseases; Work; adaptive immunity; antagonist; antigen binding; antigen test; cellular transduction; chicken egg; design; draining lymph node; efficacy testing; empowerment; env Gene Products; exosome; experimental study; extracellular vesicles; immunogenic; immunogenicity; improved; in vivo; influenza virus vaccine; interest; iterative design; lipid nanoparticle; manufacturing process; microvesicles; panacea; pathogen; prevent; process optimization; resistance mechanism; response; scaffold; success; tissue culture; vaccine delivery; vaccine efficacy; vaccine immunogenicity; vaccine platform; vector vaccine

Abstract The central hypothesis of this proposal is that the efficacy of mRNA vaccines that deliver membrane-anchored immunogens can be improved by localizing the immunogen to extracellular vesicles (EVs, small membrane- limited structures shed by eukaryotic cells). Our rationale is that EVs provide a natural scaffold for immunogen multimerization while also enabling membrane-bound antigens to access antigen presenting cells, both local to the site of injection, and in the draining lymph node. To localize immunogens to EVs and promote EV shedding we propose two complimentary approaches. In Aim 1, we will append a viral “late domain” to the carboxy terminus of our immunogen. Viral late domains are small protein domains, usually associated with a matrix or capsid protein, used by enveloped viruses to facilitate budding and egress. We have found that these domains can act out of context; fusing a late domain from feline immunodeficiency virus Gag to a SARS-CoV-2 spike protein immunogen caused the immunogen to re- localize to EVs and improved its immunogenicity nearly two-fold. We will expand this work by testing late domains from other viruses for their ability to promote EV localization and/or production. We will thoroughly characterize these EVs to determine correlates of vaccine immunogenicity. In Aim 2, we will modify our immunogens to overcome the activity of the host anti-viral restriction factor BST-2 (a.k.a. tetherin). Tetherin inhibits viral egress by “tethering” budding enveloped viruses to the host cell membranes and also inhibits the release of EVs by the same mechanism. Therefore, we will explore strategies for antagonizing tetherin in order to promote release of our immunogen-laden EVs. Enveloped viruses have evolved different strategies for tetherin evasion that we will attempt to incorporate into our immunogen designs. Indeed, we have identified a portion of the SARS-CoV-2 spike protein that we suspect is responsible for tetherin antagonism. Incorporating this S protein domain into our immunogen dramatically increases the amount of immunogen recovered from EV fractions of tissue culture supernatants. We will also explore similar strategies based on tetherin resistance mechanisms from other viruses. Finally, in Aim 3, promising immunogen design strategies in the context of different viral envelope protein immunogens (SARS-CoV-2, influenza A virus, HIV) will be compared in mice. These tests will allow us to establish correlations between the behavior of our vaccine immunogens in tissue culture (quantity and characteristics of the EVs, cytoxicity, etc.) and performance of the vaccine in vivo and determine if our modifications universally improve vaccine efficacy, or if particular immunogen designs are better suited for specific viral antigens.

抽象的 该提案的中心假设是，提供膜锚定的 mRNA 疫苗的功效 通过将免疫原定位于细胞外囊泡（EV、小膜- 真核细胞脱落的有限结构）。我们的理由是 EV 为免疫原提供了天然支架 多聚化，同时还使膜结合抗原能够进入抗原呈递细胞，两者都局部 注射部位和引流淋巴结。 为了将免疫原定位于 EV 并促进 EV 脱落，我们提出了两种互补的方法。瞄准 1，我们将在免疫原的羧基末端附加一个病毒“晚期结构域”。病毒晚期结构域很小 蛋白质结构域，通常与基质或衣壳蛋白相关，被包膜病毒用来促进 发芽和出口。我们发现这些域可能会断章取义。融合来自的后期域 猫免疫缺陷病毒 Gag 对 SARS-CoV-2 刺突蛋白免疫原导致免疫原重新 定位于 EV 并将其免疫原性提高了近两倍。我们将通过后期测试来扩展这项工作 来自其他病毒的域，因为它们能够促进电动汽车本地化和/或生产。我们将彻底 表征这些 EV 以确定疫苗免疫原性的相关性。 在目标 2 中，我们将修改我们的免疫原以克服宿主抗病毒限制因子 BST-2 的活性 （又名系链素）。 Tetherin 通过将出芽的有包膜病毒“拴在”宿主细胞上来抑制病毒流出 膜并通过相同的机制抑制 EV 的释放。因此，我们将探索 拮抗系链蛋白的策略，以促进携带免疫原的 EV 的释放。信封式 病毒已经进化出不同的逃避束缚蛋白的策略，我们将尝试将其纳入我们的研究中 免疫原设计。事实上，我们已经鉴定出 SARS-CoV-2 刺突蛋白的一部分，我们怀疑它是 负责tetherin拮抗作用。将这个 S 蛋白结构域戏剧性地整合到我们的免疫原中 增加从组织培养上清液的 EV 级分中回收的免疫原的量。我们还将 探索基于其他病毒的系链素抗性机制的类似策略。 最后，在目标 3 中，在不同病毒包膜蛋白的背景下有前途的免疫原设计策略 免疫原（SARS-CoV-2、甲型流感病毒、HIV）将在小鼠中进行比较。这些测试将使我们能够 建立我们的疫苗免疫原在组织培养中的行为之间的相关性（数量和 EV 的特性、细胞毒性等）和疫苗的体内性能，并确定我们的 修改普遍提高疫苗功效，或者特定的免疫原设计是否更适合 特异性病毒抗原。