基于多功能T细胞的汉滩病毒新型多表位长肽疫苗的研究

Background: Although millions of doses of inactivated rodent brain- or cell culture-derived HFRS vaccines are annually given, the effectiveness of the vaccine continues to be debated. It has been suggested that virus-specific T cells with a “polyfunctional” profile, defined by the capacity to proliferate, degranulate and secrete cytokines, are most competent in controlling viral replication in virus infection. Our previous study showed that IFN-γ–producing T cells specific for the nucleocapsid protein were strongly associated with reduced risk of progression to acute renal failure in patients with hemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus (HTNV) infection. The majority of the antigenic peptides in nucleocapsid protein were clustered within an amino acid region of N115–429. However, the functional and phenotypic characteristics of the T cells and their correlations with immunoprotection remain unknown. Study design: Mice were injected with 35 amino acid long overlapping peptides covering amino acids 115 to 429 of nucleocapsid protein in combination with various adjuvants including CpG-ODN. Immunomonitoring techniques, such as direct ex vivo ELISPOT, intracellular cytokine staining and tetramer staining, were used to evaluate the production of the cytokines IFN-γ, TNF-α, and IL-2, the ability to proliferate, and surface mobilization of the degranulation marker CD107a on mouse CD4+ and CD8+ T cells induced by the long peptide vaccines. Surface expression of CD127, PD-1 and Tim-3 on responding cells was used to classify the phenotype of CD4+ and CD8+ T cells. Significance: Taken together with the viral clearance and survival of lethally challenged mice immunized with long peptides, this study will provide the new insights into the relationship between the frequencies, functions and phenotypes of CD4+ and CD8+ T cell subsets, and the immunoprotective or immunopathologic effect of vaccination with HTNV long peptides, define the long neucleocapsid protein peptides which can induce protective T cell responses, and provide the basis for vaccinations with the HTNV-derived long peptides containing both CTL and Th epitopes.

汉坦病毒灭活疫苗是迄今唯一获准上市的肾综合征出血热疫苗，但国际上对灭活疫苗的保护作用一直存在着争议。新近研究表明多功能T细胞在病毒的清除中至关重要。我们前期研究发现，汉滩病毒（HTNV）核蛋白特异的分泌IFN-γ的T细胞应答与免疫保护有关，其针对的表位主要位于N115-429，但其中多功能T细胞的作用还有待进一步探讨。本项目拟采用HTNV N115-429部分重叠长肽制备疫苗免疫小鼠，用ELISPOT、胞内细胞因子染色等技术，探讨长肽诱导的特异性T细胞各亚群的频率分布，增殖、杀伤和分泌细胞因子的功能特点，以及CD127、PD-1和Tim-3的表达特征，结合疫苗免疫后HTNV致死性感染小鼠病毒清除的程度及存活的情况，阐明HTNV长肽诱导的T细胞应答（亚群、功能和表型）的规律及其与免疫保护或免疫损伤的关系，明确诱导保护性免疫应答的长肽组合，为新型HTNV长肽疫苗的设计提供重要的理论和实验依据。

本项目瞄准了汉滩病毒在军事医学中的重要地位及现有灭活疫苗的局限性，在我们对肾综合征出血热患者急性期汉滩病毒特异性T细胞免疫应答规律研究的基础上，我们以覆盖汉滩病毒核蛋白N110–429序列的长度为25–35个氨基酸残基的长肽为免疫原，与佐剂混合后制备疫苗免疫小鼠，发现这些长肽可诱导小鼠产生强烈的、多表位的、多功能（增殖、杀伤、分泌IFN-γ、TNF-α和IL-2等细胞因子）的以Th1为主的病毒特异性T细胞应答，突破了传统灭活疫苗不产生T细胞应答或T细胞应答极弱的局限性；鉴定出了15个小鼠HTNV特异的19肽T细胞表位，并筛选得到了17条候选长肽，有望成为新型的预防和治疗性疫苗的抗原组分之一，为进一步研究人用的新型汉坦病毒预防和治疗性疫苗提供了重要的理论和实验依据；发现在同一种病毒结构蛋白上的某些肽段之间，尤其是在一级结构上相距较远的肽段之间存在着“抗原竞争”现象，从理论上解释了灭活病毒或蛋白质疫苗为何不能有效地诱导机体产生细胞免疫应答，为长肽疫苗研究的重要性和可行性提供了理论保证。