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 该提议的中心假设是，递送膜锚定的mRNA疫苗的功效是有效的。 免疫原可以通过将免疫原定位于细胞外囊泡（EV，小膜- 由真核细胞脱落的有限结构）。我们的理由是，电动汽车提供了一个天然的支架免疫原 多聚化，同时也使膜结合抗原能够接近抗原呈递细胞，两者都是局部的， 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。在Aim中 1，我们将附加一个病毒的“晚期结构域”到我们的免疫原的羧基末端。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，被包膜病毒用于促进 萌芽和出口。我们已经发现，这些域可以在上下文之外起作用; 猫免疫缺陷病毒Gag对SARS-CoV-2刺突蛋白免疫原的作用导致免疫原重新 定位于EV并将其免疫原性提高近两倍。我们将通过后期测试来扩展这项工作 结构域与其他病毒的区别在于它们促进EV定位和/或产生的能力。深入 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 （又称栓系）。Tetherin通过将出芽的包膜病毒“拴系”到宿主细胞来抑制病毒外出 膜，并通过相同的机制抑制EV的释放。因此，我们将探讨 拮抗tetherin的策略，以促进我们的免疫原负载EV的释放。笼罩 病毒已经进化出不同的策略来逃避tetherin，我们将尝试将其纳入我们的 免疫原设计。事实上，我们已经确定了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蛋白结构域插入我们的免疫原 增加从组织培养上清液的EV级分回收的免疫原的量。我们还将 探索基于来自其他病毒的tetherin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希望的免疫原设计策略 将在小鼠中比较免疫原（SARS-CoV-2、甲型流感病毒、HIV）。这些测试可以让我们 建立我们的疫苗免疫原在组织培养中的行为（数量和 EV的特性、细胞毒性等）和疫苗在体内的性能，并确定我们的 修饰普遍提高疫苗效力，或者如果特定的免疫原设计更适合于 特异性病毒抗原。