Disease Intervention and Prevention, and Population Health Programs, Texas Biomedical Research Institute, San Antonio, Texas, USA.
Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA.
Microbiol Spectr. 2023 Jun 15;11(3):e0065323. doi: 10.1128/spectrum.00653-23. Epub 2023 May 16.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a worldwide coronavirus disease 2019 (COVID-19) pandemic. Despite the high efficacy of the authorized vaccines, there may be uncertain and unknown side effects or disadvantages associated with current vaccination approaches. Live-attenuated vaccines (LAVs) have been shown to elicit robust and long-term protection by the induction of host innate and adaptive immune responses. In this study, we sought to verify an attenuation strategy by generating 3 double open reading frame (ORF)-deficient recombinant SARS-CoV-2s (rSARS-CoV-2s) simultaneously lacking two accessory ORF proteins (ORF3a/ORF6, ORF3a/ORF7a, and ORF3a/ORF7b). We report that these double ORF-deficient rSARS-CoV-2s have slower replication kinetics and reduced fitness in cultured cells compared with their parental wild-type (WT) counterpart. Importantly, these double ORF-deficient rSARS-CoV-2s showed attenuation in both K18 hACE2 transgenic mice and golden Syrian hamsters. A single intranasal dose vaccination induced high levels of neutralizing antibodies against SARS-CoV-2 and some variants of concern and activated viral component-specific T cell responses. Notably, double ORF-deficient rSARS-CoV-2s were able to protect, as determined by the inhibition of viral replication, shedding, and transmission, against challenge with SARS-CoV-2 in both K18 hACE2 mice and golden Syrian hamsters. Collectively, our results demonstrate the feasibility of implementing the double ORF-deficient strategy to develop safe, immunogenic, and protective LAVs to prevent SARS-CoV-2 infection and associated COVID-19. Live-attenuated vaccines (LAVs) are able to induce robust immune responses, including both humoral and cellular immunity, representing a very promising option to provide broad and long-term immunity. To develop LAVs for SARS-CoV-2, we engineered attenuated recombinant SARS-CoV-2 (rSARS-CoV-2) that simultaneously lacks the viral open reading frame 3a (ORF3a) in combination with either ORF6, ORF7a, or ORF7b (Δ3a/Δ6, Δ3a/Δ7a, and Δ3a/Δ7b, respectively) proteins. Among them, the rSARS-CoV-2 Δ3a/Δ7b was completely attenuated and able to provide 100% protection against an otherwise lethal challenge in K18 hACE2 transgenic mice. Moreover, the rSARS-CoV-2 Δ3a/Δ7b conferred protection against viral transmission between golden Syrian hamsters.
严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）导致了全球 2019 年冠状病毒病（COVID-19）大流行。尽管授权疫苗的功效很高，但目前的疫苗接种方法可能存在不确定和未知的副作用或缺点。减毒活疫苗（LAV）通过诱导宿主先天和适应性免疫反应，已被证明能产生强大和长期的保护作用。在这项研究中，我们试图通过同时缺失两个辅助开放阅读框（ORF）蛋白（ORF3a/ORF6、ORF3a/ORF7a 和 ORF3a/ORF7b）生成 3 种双开放阅读框（ORF）缺失的重组 SARS-CoV-2（rSARS-CoV-2）来验证一种减毒策略。我们报告说，与亲本野生型（WT）相比，这些双 ORF 缺失的 rSARS-CoV-2 在培养细胞中的复制动力学更慢，适应性更低。重要的是，这些双 ORF 缺失的 rSARS-CoV-2 在 K18 hACE2 转基因小鼠和金黄地鼠中均表现出减毒作用。单次鼻腔内接种一剂可诱导针对 SARS-CoV-2 和一些关注变体的高水平中和抗体，并激活病毒成分特异性 T 细胞反应。值得注意的是，双 ORF 缺失的 rSARS-CoV-2 能够通过抑制病毒复制、脱落和传播来保护 K18 hACE2 小鼠和金黄地鼠免受 SARS-CoV-2 的攻击。总的来说，我们的研究结果表明，实施双 ORF 缺失策略来开发安全、免疫原性和保护性的 LAV 以预防 SARS-CoV-2 感染和相关的 COVID-19 是可行的。减毒活疫苗（LAV）能够诱导强大的免疫反应，包括体液免疫和细胞免疫，是提供广泛和长期免疫的非常有前途的选择。为了开发针对 SARS-CoV-2 的 LAV，我们设计了一种减毒重组 SARS-CoV-2（rSARS-CoV-2），该病毒同时缺失病毒开放阅读框 3a（ORF3a），并与 ORF6、ORF7a 或 ORF7b（Δ3a/Δ6、Δ3a/Δ7a 和 Δ3a/Δ7b）蛋白缺失（分别）。其中，rSARS-CoV-2Δ3a/Δ7b 完全减毒，能够在 K18 hACE2 转基因小鼠中提供 100%的致死性攻击保护。此外，rSARS-CoV-2Δ3a/Δ7b 能够防止金黄地鼠之间的病毒传播。
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