猪德尔塔冠状病毒双脯氨酸突变S蛋白的表达及免疫原性评价

Expression and immunogenicity of S protein with two proline residue mutations of porcine deltacoronavirus

摘要:

【目的】 猪德尔塔冠状病毒(porcine deltacoronavirus, PDCoV)是一种重要的猪肠道冠状病毒，给全球生猪养殖业带来了巨大的经济损失，截至目前，尚无可用的商品化疫苗。本研究选择宿主免疫反应主要诱因因子棘突(spike, S)蛋白，并将PDCoV S蛋白七肽重复序列1 (heptapeptide repeat-1, HR1)与中心螺旋之间的环中855和856位点均突变为双脯氨酸(E855P和V856P)，然后利用ExpiCHO-S真核表达系统表达、纯化重组S蛋白和双脯氨酸突变S蛋白(S2P)并评价其免疫原性及免疫保护性，以初步研制一种免疫效果较好的PDCoV亚单位疫苗。【方法】 利用间接酶联免疫吸附法检测免疫小鼠血清特异性抗体IgG水平；利用血清中和试验检测免疫小鼠血清中和抗体滴度；通过流式细胞术检测免疫小鼠T淋巴细胞增殖情况；通过细胞因子检测免疫小鼠干扰素(interferon, IFN)-γ、IFN-α、白细胞介素(interleukin, IL)-2和IL-4等细胞因子分泌情况；采用RT-qPCR检测攻毒后小鼠肠道组织PDCoV病毒载量；采用病理组织切片检测小鼠肠道有无病理损伤；利用免疫组织化学检测小鼠肠道组织PDCoV抗原分布情况。【结果】 免疫原性结果显示，小鼠在肌肉注射S组和S2P组亚单位疫苗后能够产生较高水平的抗PDCoV特异性IgG抗体，免疫后42 d的小鼠血清均对PDCoV具有中和作用，其中，S2P组对LLC-PK细胞50%中和保护效价显著高于S组。此外，S组和S2P组显著地诱导小鼠CD₄⁺ T淋巴细胞增殖，并且S2P组高于S组，而S2P组能够诱导小鼠CD₈⁺ T淋巴细胞增殖，但S组与PBS组并无差异。同时，S组和S2P组诱导的IFN-γ、IFN-α、IL-2和IL-4等细胞因子水平均显著高于PBS组，但S组和S2P组并无差异。免疫保护性结果显示，PBS组肠组织中可检测到PDCoV病毒，肠组织出现病理损伤且可见大量PDCoV抗原，而S组和S2P组未检测到PDCoV病毒且未观察到肠组织损伤，二者之间无明显差异。【结论】 S2P组较之S组能够诱导小鼠产生更高水平的抗PDCoV体液免疫应答，两组疫苗对小鼠均有一定的保护效果，本研究为后续PDCoV亚单位疫苗研发奠定坚实的基础。

关键词:猪德尔塔冠状病毒(porcine deltacoronavirus,PDCoV);S蛋白;双脯氨酸突变;免疫原性

Abstract:

[Objective] Porcine deltacoronavirus (PDCoV) is a major porcine enteric coronavirus, causing huge economic losses to the pig breeding industry worldwide. However, there is no commercial vaccine available for this virus. The spike (S) protein is a key factor inducing host immune response. In this study, the two sites 855 and 856 in the loop between the heptapeptide repeat-1 (HR1) and the central helix of PDCoV S protein were mutated to proline (E855P and V856P). Then, the recombinant S protein and mutated S protein (S2P) were expressed and purified by the ExpiCHO-S eukaryotic expression system, and their immunogenicity and immunoprotecive performance were evaluated for developing a PDCoV subunit vaccine with good immune effect. [Methods] The serum level of the specific antibody IgG in immunized mice was measured by indirect enzyme-linked immunosorbent assay. The serum neutralization test was carried out to determine the titer of neutralizing antibodies in the immunized mice. The proliferation of T lymphocytes in immunized mice was detected by flow cytometry. The secretion levels of interferon (IFN)-γ, IFN-α, interleukin (IL)-2, and IL-4 were determined. RT-qPCR was employed to measure the PDCoV load in the intestinal tissue of mice after challenge. Tissue sections were prepared to observe the intestinal lesions of mice. The distribution of PDCoV antigen in the intestinal tissue of mice was detected by immunohistochemistry. [Results] High levels of anti-PDCoV specific IgG antibodies were produced in mice after intramuscular injection of S and S2P subunit vaccines, and the serum of mice 42 days after immunization had a neutralizing effect on PDCoV. The 50% neutralizing protective titer of LLC-PK cells in the S2P group was significantly higher than that in the S group. In addition, the immunization with S and S2P significantly induced the proliferation of CD₄⁺ T lymphocytes in mice, which was higher in the S2P group than in the S group. The immunization with S2P induced the proliferation of CD₈⁺ T lymphocytes in mice, and the level of CD₈⁺ T lymphocytes showed no difference between the S group and the PBS group. The levels of IFN-γ, IFN-α, IL-2, and IL-4 in the S and S2P groups were significantly higher than those in the PBS group and had no difference between the S and S2P groups. The challenge assay results showed that the PBS group presented PDCoV, pathological damage, and a large number of PDCoV antigens in the intestinal tissue, while neither PDCoV nor intestinal tissue damage was detected in the S and S2P groups, which showed no significant difference between the two groups. [Conclusion] S2P induces higher level of the humoral immune response against PDCoV in mice than S. The vaccines prepared with both S2P and S have protective effects on mice. The findings lays a foundation for the follow-up study of PDCoV subunit vaccines.

Key words:porcine deltacoronavirus (PDCoV);S protein;bisproline mutation;immunogenicity