利用毕赤酵母表达的登革 1 型病毒样颗粒诱导病毒中和抗体和 T 细胞应答。

Induction of virus-neutralizing antibodies and T cell responses by dengue virus type 1 virus-like particles prepared from Pichia pastoris.

机构信息

Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, China.

出版信息

Chin Med J (Engl). 2012 Jun;125(11):1986-92.

Abstract

BACKGROUND

Dengue is currently a significant global health problem but no vaccines are available against the four dengue serotypes virus infections. The development of safe and effective vaccines has been hampered by the requirement of conferring complete protection against all four dengue serotypes and the lack of a convenient animal model. Virus-like particles (VLPs) have emerged as a promising subunit vaccine candidate. One strategy of vaccine development is to produce a tetravalent dengue subunit vaccine by mixing recombinant VLPs, corresponding to all four dengue virus serotypes. Towards this end, this study aimed to establish a Pichia pastoris (P. pastoris) expression system for production of dengue virus type 1 (DENV-1) VLPs and evaluate the humoral and cellular immune response of this particle in mice.

METHODS

A recombinant yeast P. pastoris clone containing prM and E genes of DENV-1 was constructed and DENV-1 VLPs expressed by this clone were analyzed by sucrose density gradient centrifugation, Western blotting, and transmission electron microscope. Groups of mice were immunized by these particles plus adjuvant formulations, then mice were tested by ELISA and neutralization assay for humoral immune response, and by lymphocyte proliferation and cytokine production assays for a cellular immune response.

RESULTS

Our data demonstrated that recombinant DENV-1 VLPs consisting of prM and E protein were successfully expressed in the yeast P. pastoris. Sera of VLPs immunized mice were shown to contain a high-titer of antibodies and the neutralization assay suggested that those antibodies neutralized virus infection in vitro. Data from the T lymphocyte proliferation assay showed proliferation of T cell, and ELISA found elevated secretion levels of interferon IFN-γ and IL-4.

CONCLUSIONS

P. pastoris-expressed DENV-1 VLPs can induce virus neutralizing antibodies and T cell responses in immunized mice. Using P. pastoris to produce VLPs offers a promising and economic strategy for dengue virus vaccine development.

摘要

背景

登革热目前是一个重大的全球健康问题，但尚无针对四种登革热血清型病毒感染的疫苗。由于需要针对所有四种登革热血清型提供完全保护，并且缺乏方便的动物模型，因此安全有效的疫苗的开发受到了阻碍。病毒样颗粒（VLPs）已成为有前途的亚单位疫苗候选物。疫苗开发的一种策略是通过混合重组 VLPs 来生产四价登革热亚单位疫苗，这些 VLPs 对应于所有四种登革热病毒血清型。为此，本研究旨在建立毕赤酵母（Pichia pastoris，P. pastoris）表达系统，用于生产登革热病毒 1 型（DENV-1）VLPs，并评估该颗粒在小鼠中的体液和细胞免疫应答。

方法

构建了含有 DENV-1 的 prM 和 E 基因的重组酵母 P. pastoris 克隆，并通过蔗糖密度梯度离心、Western blot 和透射电子显微镜分析该克隆表达的 DENV-1 VLPs。将这些颗粒与佐剂制剂一起免疫小鼠，然后通过 ELISA 和中和试验检测体液免疫应答，通过淋巴细胞增殖和细胞因子产生试验检测细胞免疫应答。

结果

我们的数据表明，在酵母 P. pastoris 中成功表达了由 prM 和 E 蛋白组成的重组 DENV-1 VLPs。VLPs 免疫小鼠的血清中含有高滴度的抗体，中和试验表明这些抗体在体外中和了病毒感染。T 淋巴细胞增殖试验的数据显示 T 细胞增殖，ELISA 发现干扰素 IFN-γ和 IL-4 的分泌水平升高。