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: 10573644
• 负责人: Michael R. Farzan
• 金额: $ 8.28万
• 依托单位: UNIVERSITY OF FLORIDA
• 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 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10573644
• 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 该提议的中心假设是传递膜锚定的mRNA疫苗的效率 可以通过将免疫原定位到细胞外蔬菜（EV，小膜 - 真核细胞脱离的有限结构）。我们的理由是，电动汽车为免疫机提供了自然的脚手架 多媒体同时还可以使膜结合的抗原进入抗原呈现细胞，均可 注射部位，在图纸淋巴结中。 为了将免疫原位定位到电动汽车并促进电动汽车脱落，我们提出了两种免费的方法。目标 1，我们将在免疫原的羧基末端附加病毒“晚域”。病毒后期域很小 蛋白质结构域通常与基质或衣壳蛋白有关，被包膜病毒用于促进 萌芽和出口。我们发现这些领域可以脱离上下文。融合从 猫免疫缺陷病毒对SARS-COV-2尖峰蛋白免疫原的堵嘴导致免疫原病原 定位于电动汽车并提高其免疫原性几乎两倍。我们将通过迟到来扩展这项工作 来自其他病毒的域，以促进EV定位和/或生产的能力。我们会彻底 这些电动汽车的特征是确定疫苗免疫原性的相关性。 在AIM 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Tetherin）。 Tetherin通过“束缚”萌芽的包裹病毒抑制病毒出口 膜并通过相同的机制抑制电动汽车的释放。因此，我们将探索 拮抗Tetherin的策略，以促进我们的免疫原电动汽车的释放。包裹 病毒已经进化了Tetherin Evolution的不同策略，我们将尝试将其纳入我们的 免疫原设计。确实，我们已经确定了我们怀疑的SARS-COV-2峰值蛋白的一部分 负责系绳的拮抗作用。将此S蛋白结构域纳入我们的免疫原中 增加了从组织培养上清液的EV级分中回收的免疫原的量。我们也会 基于其他病毒的Tetherin耐药机制探索类似的策略。 最后，在AIM 3中，在不同的病毒包膜蛋白的背景下有希望的免疫原设计策略 在小鼠中，将比较免疫原（SARS-COV-2，影响力病毒，HIV）。这些测试将使我们能够 在组织培养中疫苗免疫原子的行为之间建立相关性（数量和 电动汽车的特征，细胞毒性等）和体内疫苗的性能 修改普遍提高疫苗效率，或者是否更适合特定的免疫原设计 特定的病毒抗原。