Immunogenicity and Antigenicity of the Ectodomain of Rabies Virus Glycoprotein Stably Expressed in HEK293T Cells.
Immunogenicity and Antigenicity of the Ectodomain of Rabies Virus Glycoprotein Stably Expressed in HEK293T Cells.
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DOI：
10.7150/ijms.87134
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发表时间：
2023
影响因子：
3.6
通讯作者：
Mao Y
中科院分区：
文献类型：
--
作者：
Li Q;Yan R;Bai N;Tan Z;Yu Q;Su H;Wei X;Li A;Chen X;Li Z;He Y;Li H;Li X;Mao Y
狂犬病仍然是对公共卫生的巨大威胁。狂犬病病毒包膜糖蛋白（RABV G）是狂犬病病毒的主要抗原，含有中和表位，是亚单位疫苗和诊断抗原的主要候选抗原。然而，生产和纯化rRABV G，同时保留其抗原性和免疫原性仍然是一个挑战。本研究旨在建立rRABV G的生产和纯化平台，并检测rRABV G的免疫原性和抗原性。合成了编码可溶性RABV G的cDNA片段，并克隆到慢病毒表达载体中。将重组慢病毒载体LV-CMV-RABV G-eGFP包装、滴定，然后转导到HEK 293 T细胞中。使用镍亲和层析纯化细胞培养上清液，随后通过Western印迹分析和间接酶联免疫吸附测定（ELISA）进行确认。ELISA使用了从接种人商业纯化Vero细胞狂犬病疫苗（PVRV）的个体获得的人血清。值得注意的是，我们在免疫的猪而不是小鼠中观察到中和抗体应答。这种差异可能归因于诸如rRABV G蛋白的不稳定性、宿主反应的变化和所用佐剂的变化等因素。考虑到所有这些发现，本研究中产生的rRABV G蛋白有望成为预防狂犬病的潜在候选疫苗。
Rabies continues to be a huge threat to public health. The rabies virus envelope glycoprotein (RABV G) is a major rabies virus antigen and contains neutralizing epitopes, which are primary candidates for subunit vaccines and diagnostic antigens. However, the production and purification of rRABV G while retaining its antigenic and immunogenic remains to be a challenge. Here, we aimed to establish a platform for rRABV G production and purification, and determine the immunogenicity and antigenicity of rRABV G. The cDNA fragment encoding the soluble form of RABV G was synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-RABV G-eGFP was packaged, titered, and then transduced into HEK 293T cells. The cell culture supernatant was purified using nickel affinity chromatography and subsequently confirmed through Western Blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). The ELISA utilized human sera obtained from individuals who had been vaccinated with the human commercial Purified Vero Cells Rabies Vaccine (PVRV). Notably, we observed a neutralizing antibody response in immunized pigs rather than in mice. This discrepancy could potentially be attributed to factors such as the instability of the rRABV G protein, variations in host responses, and variances in the adjuvant used. Taking all these findings into account, the rRABV G protein generated in this study exhibits promise as a potential vaccine candidate for the prevention of rabies.
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