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.

摘要 这一提议的中心假设是，递送膜锚定的mRNA疫苗的效力 通过将免疫原定位于细胞外小泡(EVS，小膜-EV)，可以改进免疫原。 真核细胞脱落的有限结构)。我们的理由是电动汽车为免疫原提供了一个天然的支架。 多聚体，同时也使膜结合的抗原能够访问抗原提呈细胞，既局部到 注射部位和引流淋巴结内。 为了将免疫原定位于电动汽车，并促进电动汽车的脱落，我们提出了两种免费的方法。在AIM 1，我们将把病毒的“晚期结构域”附加到免疫原的羧基末端。病毒的晚期区域很小 蛋白结构域，通常与基质或衣壳蛋白有关，被包膜病毒用来促进 萌芽和出土。我们发现，这些域可以断章取义；将较晚的域从 猫免疫缺陷病毒与SARS-CoV-2刺突蛋白免疫原结合可使该免疫原重获 定位于EVS，并将其免疫原性提高近两倍。我们将通过晚些时候的测试来扩展这项工作 与其他病毒不同的域名，因为它们有能力促进EV的本地化和/或生产。我们会彻底 确定这些EV的特征，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 (也就是系留)。Tetherin通过将被包裹的病毒“拴”在宿主细胞上来抑制病毒的外泄 也通过同样的机制抑制EVS的释放。因此，我们将探索 对抗Tetherin的策略，以促进我们携带免疫原的EVS的释放。封套的 病毒已经进化出不同的Tetherin逃避策略，我们将尝试将这些策略整合到我们的 免疫基因设计。事实上，我们已经确定了SARS-CoV-2尖峰蛋白的一部分，我们怀疑 对系留蛋白的对抗负责。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EV组织培养上清液中回收的免疫原量。我们还将 根据其他病毒的Tetherin抗性机制，探索类似的策略。 最后，在目标3中，针对不同的病毒包膜蛋白，提出了有希望的免疫原设计策略 免疫原(SARS-CoV-2、A型流感病毒、HIV)将在小鼠身上进行比较。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 电动汽车的特性、细胞毒性等)以及疫苗在体内的表现，并确定我们的 修改普遍提高疫苗效力，或者如果特定的免疫原设计更适合于 特定的病毒抗原。