表达新冠病毒S基因重组狂犬病病毒的构建及其免疫原性

Construction and immunogenicity of recombinant rabies virus expressing S gene of SARS-CoV-2

摘要:

【背景】新型冠状病毒肺炎(coronavirus disease 2019，COVID-19)在全球流行已近3年，除对人类造成了巨大伤害，也影响了人类的伴侣动物。人的COVID-19疫苗已在全球应用，但动物用的新冠病毒疫苗却鲜有报道。【目的】研制兽用新冠病毒(severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)和狂犬病病毒(rabies virus，RABV)的二联苗。【方法】将合成的SARS-CoV-2 S基因和S1基因分别克隆至RABV弱毒疫苗株rHEP-Flury基因组G与L基因间，并将2个重组质粒分别与辅助质粒共转染至BHK-21细胞中，拯救重组病毒rHEP-nCOV-S和rHEP-nCOV-S1。通过RT-PCR、Western blotting和荧光抗体染色，验证重组病毒、确证S和S1蛋白在RABV中成功表达。再将重组病毒接种NA细胞及成年小白鼠，测定病毒的体外生长特性、重组病毒的致病性及免疫原性。【结果】免疫荧光结果显示，转染7d后细胞上清均出现了绿色免疫荧光，表明已成功拯救嵌合SARS-CoV-2S和S1基因的重组病毒RABV rHEP-nCOV-S和rHEP-nCOV-S1，并且rHEP-nCOV-S1的增殖和扩散能力强于亲本株rHEP-Flury，但rHEP-nCOV-S与亲本株无显著差异。Western blotting结果显示，在目的位置处均出现72kDa和144kDa特异性条带，表明S和S1蛋白在重组RABV中高效表达。重组病毒免疫6周KM小鼠后，小鼠的体重变化与亲本RABV基本一致，重组病毒诱导小鼠产生狂犬中和抗体。【结论】本研究拯救出了嵌合SARS-CoV-2 S/S1基因的重组RABV，为动物COVID-19载体疫苗的研发奠定了基础。

Abstract:

[Background] The coronavirus disease 2019 (COVID-19) pandemic has lasted for nearly three years in the globe, which has not only caused serious harm to humans but also affected companion animals. The COVID-19 vaccines for human have been used globally, while those for animals are rarely reported. [Objective] To develop a bivalent vaccine against both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and rabies virus (RABV) for animal use.[Methods] We cloned the S and S1 genes of SARS-CoV-2 into the region between G and L genes of the attenuated RABV vaccine strain rHEP-Flury to construct the recombinant plasmids pHEP-nCOV-S and pHEP-nCOV-S1, respectively. The two plasmids were respectively co-transfected into BHK-21 cells with the helper plasmids and finally the recombinant viruses rHEP-nCOV-S and rHEP-nCOV-S1 were rescued. The recombinant viruses were confirmed by RT-PCR and direct fluorescent antibody staining against RABV N protein. Western blotting was employed to detect the expression of S and S1 proteins in the cells infected with the recombinant viruses. The growth curves, pathogenicity, and immunogenicity of recombinant viruses were confirmed in NA cells and mice. [Results] The rescued recombinant viruses rHEP-nCOV-S and rHEP-nCOV-S1 respectively carrying the S and S1 genes of SARS-CoV-2 were confirmed by direct fluorescent antibody assay based on the green fluorescence from the supernatants 7 days post infection. rHEP-nCOV-S1 rather than rHEP-nCOV-S showed stronger proliferation and diffusion abilities than the parental virus rHEP-Flury in NA cells. The specific bands at 72 kDa and 144 kDa in the Western blotting confirmed the efficient expression of S and S1 in the recombinant viruses, respectively. The mice vaccinated with the recombinant viruses did not show significant changes in the body weight compared with those vaccinated with rHEP-Flury, and the recombinant viruses induced the production of neutralizing antibody against RABV in mice. [Conclusion] The production of the recombinant RABV carrying the S/S1 gene of SARS-CoV-2 provides a foundation for the development of the bivalent vaccine against both SARS-CoV-2 and rabies virus for animal use.