Recombinant Gene Products Group, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.
Vaccine. 2013 Jan 30;31(6):873-8. doi: 10.1016/j.vaccine.2012.12.016. Epub 2012 Dec 20.
Currently, dengue represents one of the most significant arboviral disease worldwide, for which a vaccine is not yet available. Persistent challenges in live viral dengue vaccines have sparked a keen interest in exploring non-replicating dengue vaccines. We have examined the feasibility of using the methylotrophic yeast Pichia pastoris to develop a chimeric vaccine candidate displaying the dengue virus type-2 (DENV-2) envelope domain III (EDIII), implicated in host receptor binding and in the induction of virus-neutralizing antibodies, on the surface of non-infectious virus-like particles (VLP)-based on the Hepatitis B virus core antigen (HBcAg).
We designed a fusion antigen by inserting DENV-2 EDIII into c/e1 loop of HBcAg. A codon-optimized gene encoding this fusion antigen was integrated into the genome of P. pastoris, under the control of the Alcohol Oxidase 1 promoter. The antigen was expressed by methanol induction and purified to near homogeneity by Ni(2+) affinity chromatography. The purified antigen was characterized physically and functionally to evaluate its ability to assemble into VLPs, and elicit DENV-2-specific antibodies in mice.
This fusion antigen was expressed successfully to high yields and purified to near homogeneity. Electron microscopy and competitive ELISA analyses showed that it formed VLPs in which the EDIII moiety was accessible to different EDIII-specific antibodies. These VLPs were immunogenic in mice, stimulating the production of antibodies that could specifically recognize DENV-2 and neutralize its infectivity. However, virus-neutralizing antibody titers were modest.
Our data show: (i) insertion of EDIII into the c/e1 loop of HBcAg does not compromise particle assembly; and (ii) the chimeric VLPs elicit a specific humoral response against DENV-2. The strategy of displaying dengue virus EDIII using a VLP platform will need further optimization before it may be developed into a viable alternative option.
目前，登革热是全球最重要的虫媒病毒病之一，尚无可用的疫苗。活病毒登革热疫苗持续面临挑战，这激发了人们对探索非复制性登革热疫苗的浓厚兴趣。我们研究了甲醇营养型酵母毕赤酵母（Pichia pastoris）在开发嵌合疫苗方面的可行性，该疫苗在乙型肝炎病毒核心抗原（HBcAg）的基础上展示了登革病毒 2 型（DENV-2）包膜域 3（EDIII），该结构域与宿主受体结合和诱导病毒中和抗体有关。
我们通过将 DENV-2 EDIII 插入 HBcAg 的 c/e1 环，设计了融合抗原。该融合抗原的密码子优化基因被整合到毕赤酵母基因组中，受醇氧化酶 1 启动子的控制。通过甲醇诱导表达抗原，并通过 Ni（2+）亲和层析进行近均一纯化。对纯化的抗原进行物理和功能特性分析，以评估其组装成病毒样颗粒（VLPs）的能力，并在小鼠中引发 DENV-2 特异性抗体。
该融合抗原成功高表达并进行了近均一纯化。电子显微镜和竞争 ELISA 分析表明，它形成了 VLPs，其中 EDIII 结构域可被不同的 EDIII 特异性抗体识别。这些 VLPs在小鼠中具有免疫原性，可刺激产生能够特异性识别 DENV-2 并中和其感染性的抗体。然而，病毒中和抗体滴度适中。
我们的数据表明：（i）将 EDIII 插入 HBcAg 的 c/e1 环不影响颗粒组装；（ii）嵌合 VLPs 可引发针对 DENV-2 的特异性体液反应。使用 VLP 平台展示登革热病毒 EDIII 的策略需要进一步优化，然后才能开发为可行的替代方案。
