以免疫細胞受體為標靶提升 SARS-CoV-2 RBD mRNA 疫苗免疫原性之探討

Abstract

目前的SARS-CoV-2疫苗若單獨使用棘蛋白受體結合區作為疫苗抗原，便無法誘發良好的免疫反應，過去的研究指出，使用免疫細胞標靶策略即可提升疫苗的免疫原性與產出強大的免疫反應，但如何選擇合適的標靶配體至今仍是未解之謎，因此，我們使用了mRNA的技術來探索多種不同配體的效能，以促進疫苗之發展。在此研究中，我們進行了三種配體的比較，包含補體成分C3b（mC3）、分化群40配體（mCD40L）以及免疫球蛋白G1之結晶區域片段（mFc），並將它們分別結合上SARS-CoV-2 Omicron變異株（B.1.1.529）的棘蛋白受體結合區，接著生產可以轉譯出這些抗原蛋白的mRNA，優化它們並將其包進脂質奈米顆粒中製成疫苗，我們將mRNA轉染至HEK293T細胞株之後，具有完整抗原性的抗原能夠有效地被表達，在免疫染色實驗中也呈現了三種配體（mC3、mCD40L、mFc）能夠分別標定並結合各自的受體（CR1、CD40、Fc𝛄R），展現出強而有力的免疫細胞標靶特性。在小鼠（C57BL/6）實驗中測試了不同的疫苗劑量，由每劑1 微克至每劑40 微克，每隻小鼠共接種三劑，使用肌肉注射方式施打疫苗，我們所生產的疫苗可保存至少三個月且不會在小鼠體內引起不良副作用，尤其，我們的三種標靶樹突細胞mRNA疫苗能夠有效誘發強力的抗體反應，並且這三種疫苗皆達到相當的抗體效價，此外，三種疫苗也展現了各自的獨特性，有利於我們更了解不同標靶配體所引起的免疫反應，並作為未來疫苗發展的應用依據。此篇研究作為第一個探討不同標靶配體的差異的先驅，首先嘗試使用了mRNA技術作為免疫細胞標靶疫苗的疫苗型式，研究中三種標靶配體皆展現了高效的標靶能力，期待在未來，此研究能夠被廣泛應用，並有效地改善抗原傳遞效率、增進疫苗免疫原性與促進體液免疫反應。

Current SARS-CoV-2 vaccines using receptor binding domain (RBD) alone induce weak immune responses. Studies have shown that immune cell-targeting strategies can improve vaccine immunogenicity and generate potent immunity. However, what to select as an appropriate targeting ligand is still a secret. Hence, we applied mRNA technology to explore targeting ligands, including mC3, mCD40L, and mFc, shedding light on the enhancement of vaccines. Ligands were fused to the RBD of SARS-CoV-2 Omicron variant (B.1.1.529). The mRNAs were optimized for promoting expression, produced with high quality, and encapsulated in lipid nanoparticles (LNPs) for effective delivery. HEK293T cell line, transfected by each mRNA, efficiently expressed the vaccine antigens with correct antigenicity. Immunofluorescence staining revealed that the three targeting ligands, mC3, mCD40L, and mFc, were successfully co-expressed with the RBD and were able to target their receptors, CR1, CD40, and Fc𝛄 receptors, respectively, presenting highly correlated interaction and showing convincing targeting properties. In animal studies, C57BL/6 mice were vaccinated intramuscularly with different doses, from 1 µg to 40 µg, and each mouse underwent three doses of vaccination. The mRNA-LNP vaccines displayed great stability for lasting over three months and presented safety without causing adverse effects in mice. Notably, targeting mRNA vaccines elicited a promising elevation of antigen-specific antibody responses compared to the non-targeting vaccine. Furthermore, they achieved a similar antibody level, demonstrating their analogously robust targeting capabilities. Each vaccine has exhibited its own unique characterization and potential future application. This is the first study that uses mRNA vaccine to explore various targeting ligands and proves their promising targeting ability. These innovative vaccine candidates hold tremendous potential for improving antigen delivery, increasing vaccine immunogenicity, and enhancing humoral immunity.