携带HCV核心蛋白原核表达质粒与真核表达质粒的减毒伤寒沙门菌诱导小鼠免疫应答之比较

Comparison of Immune Responses Induced by Recombinant Attenuated Salmonella typhi Carrying Eukaryotic Expression Plasmid or Prokaryotic Expression Plasmid of HCV Core Protein

摘要:

丙型肝炎病毒（HCV）核心蛋白是丙肝疫苗的重要候选抗原，然而，该蛋白因具有免疫调控作用而影响免疫应答的诱导。构建了HCV核心蛋白的两种表达质粒，一种是体内激活型原核表达质粒pZW-C，另一种是真核表达质粒pCI-C。将该两种质粒转化减毒鼠伤寒沙门菌SL7207，得到重组菌SL7207/pZW-C和SL7207/pCI-C，分别将重组菌口服接种小鼠，检测小鼠的免疫应答，结果发现：① SL7207/pCI-C免疫鼠的CD3⁺CD4⁺ T细胞持续降低，而SL7207/pZW-C免疫鼠的CD3⁺CD4⁺ T细胞无明显改变；② SL7207/pCI-C免疫只诱导低水平抗HCV核心蛋白抗体，加强免疫对抗体阳转率及抗体水平无明显影响，而SL7207/pZW-C免疫组所有小鼠均产生较高水平的抗核心蛋白抗体。③ SL7207/pCI-C免疫鼠脾细胞的体外增殖活性、细胞毒性T细胞活性以及加强免疫对细胞免疫应答的增强作用均明显不及SL7207/pZW-C免疫鼠。结果提示：携带真核表达质粒pCI-C的沙门菌因在小鼠细胞内表达天然形式（结构以及磷酸化修饰）的HCV核心蛋白，可能通过对T细胞的免疫抑制作用而弱化免疫应答。而以携带原核表达质粒pZW-C的沙门菌免疫可避免这一问题，并具有接种方便，成本低廉等优点，从而可望作为基于HCV核心蛋白为靶抗原的HCV疫苗的候选免疫方式。

Abstract:

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for development of protective HCV vaccines. However, this protein may attenuate the induction of systemic immune responses due to its immunomodulatory properties. In this study, we constructed a HCV core gene-containing eukaryotic expression plamid pCI-C, and an in vivo-inducible prokaryotic expression plasmid pZW-C, and transformed the recombinant plasmids into an attenuated Salmonella typhimurium aroA strain SL7207. The resulting bacterial strains SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and the immune responses specific to HCV core protein were assessed. Immunization with the recombinant bacteria SL7207/pCI-C led to a persistent drop in percentage of CD3⁺CD4⁺+ T cells, and induced a weak anti-core IgG production. Splenocytes from SL7207/pCI-C immunized mice developed a relatively weak proliferation response and inferior cytotoxic activity compared to those from the mice immunized with bacteria SL7207/pZW-C. Boost immunization with SL7207/pCI-C yielded limited improvement in immune strength, while the boost with bacteria SL7207/pZW-C significantly enhanced the immune response. These results suggest that de novo synthesis of native HCV core protein may blunt the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.