Design and validation of VLP/mRNA-based vaccine strategies against SARS-CoV2

该项目取得了以下成果： 1. 合成尾修饰的SARS-CoV 2包膜刺突（S）mRNA以促进VLP与慢病毒核心蛋白的形成。为了促进含有慢病毒（HIV-1或SIV）核心蛋白的VLP的组装，我们设计了通过用HIV-1或SIV gp 41 CT替换天然尾来修饰其胞质尾（CT）的S蛋白。 基于我们对HIV-1疫苗的原始设计，已经使用了截短形式的慢病毒尾，以促进更有效的表面膜表达。 Moderna公司根据我们的设计产生了特定的mRNA。 2. SARS-CoV 2包膜刺突糖蛋白在哺乳动物细胞中的重组表达表达由Moderna Inc.在哺乳动物细胞中进行测试，以验证它们的表达效率和抗原谱，使用市售的抗S蛋白的单克隆抗体（mAb）。 3. 生产VLP。将刺突蛋白mRNA与编码HIV-1或SIV Gag或Gag-Pol的mRNA以所有可能的组合共转染到哺乳动物细胞中。收获细胞外VLP并通过蔗糖垫上的超离心浓缩。 4. VLP的广泛表征。已经使用广泛的物理和免疫学测定，包括通过mAb捕获病毒体，然后进行ELISA用于慢病毒核心抗原定量、Western印迹等，对产生的VLP进行了广泛的定量和定性表征。 初步结果表明，可以有效地产生包含CoV 2刺突蛋白和慢逆转录病毒核心的VLP，并在其表面上展示S蛋白的天然样形式。广泛的表征仍在进行中，随后将在小动物模型中进行免疫原性研究。

The project has yielded the following results: 1. Synthesis of tail-modified SARS-CoV2 envelope spike (S) mRNAs to facilitate VLP formation with lentivirus core proteins. In order to facilitate the assembly of VLPs containing lentivirus (HIV-1 or SIV) core proteins, we have designed S proteins modified in their cytoplasmic tails (CT) by replacement of the natural tail with either the HIV-1 or SIV gp41 CT. A truncated form of the lentivirus tails has been used, based on our original design for the HIV-1 vaccine, to promote a more efficient surface membrane expression. Moderna Inc. has produced the specific mRNAs according to our design. 2. Recombinant expression of the SARS-CoV2 envelope spike (S) glycoprotein in mammalian cells. The mRNA expressing the various CoV2 envelope spike (S) glycoproteins (native, HIV-CT and SIV-CT chimeric) produced by Moderna Inc. were tested in mammalian cells to verify their expression both in terms of efficiency and antigenic profile using commercially available monoclonal antibodies (mAbs) against the S protein. 3. Production of VLPs. The spike protein mRNAs were co-transfected into mammalian cells in all possible combinations with mRNAs encoding HIV-1 or SIV Gag or Gag-Pol. The extracellular VLPs were harvested and concentrated by ultracentrifugation on sucrose cushions. 4. Extensive characterization of VLPs. The produced VLPs have been extensively characterized both quantitatively and qualitatively using a wide array of physical and immunological assays, including virion capture by mAbs followed by ELISA for lentivirus core antigen quantification, Western blot and others. The preliminary results indicate that VLPs comprising the CoV2 Spike protein and lentiretrovirus core can be efficiently produced and display the native-like form of the S-protein on their surface. Extensive characterization is still under way and will be followed by immunogenicity studies in small animal models.