复制型重组痘苗病毒TTK-EG特异性T细胞表位研究

目的筛选和确定TTK-EG包含的H-2~d限制的HIV-1特异性T细胞表位。方法 6~8周龄雌性BALB/c小鼠0周时分别经肌肉注射、皮内注射和滴鼻途径免疫5×10~6PFU TTK-EG,间隔4个月进行加强免疫。末次免疫后2周取小鼠脾细胞,采用IFN-γELISPOT方法筛选HIV-1 Env和Gag肽库,反应阳性的肽库进行第二轮单肽筛选。用流式细胞术(胞内因子染色,intracellular cytokine staining,ICS)对筛选出的单肽进行验证(小鼠免疫途径为肌肉注射),确定H-2~d限制的HIV-1特异性T细胞表位。结果经过ELISPOT筛选和ICS验证,确定2条Env多肽(gp140-9,氨基酸位点60~77;gp140-74,氨基酸位点548~565)和3条Gag多肽(Gag-49,氨基酸位点196~210;Gag-64,氨基酸位点256~270;Gag-65,氨基酸位点260~274)包含H-2~d限制的HIV-1特异性T细胞表位,其中gp140-74(IVQQQSNLLRAIEAQQHL)是首次发现的H-2~d限制的HIV-1特异性T细胞表位。Env、Gag优势表位肽与Env、Gag全肽池刺激小鼠T细胞分泌IFN-γ、IL-2、TNF-α三种细胞因子的能力基本相同。结论 Env多肽gp140-9、gp140-74和Gag多肽Gag-49、Gag-64、Gag-65包含H-2~d限制的T细胞表位,可用于重组痘苗病毒TTK-EG的T细胞免疫研究。

Specific T-cell epitope mapping of replication-competent recombinant vaccinia virus TTK-EG

Objective To screen and identify the H-2d restricted HIV-1 specific T-cell epitopes in TTK-EG.Methods 6-8 week-old female BALB/c mice were primed with 5× 106 PFU of TTK-EG at month 0 intramuscularly,intracutaneously and intranasally,respectively,and then boosted at month 4.2 weeks after the final inoculation,the spleen cells were isolated and tested by IFN-γ ELISPOT using the Env and Gag peptide pools.The positive pools were further screened to identify the dominant peptides.ICS was used to confirm that the screened peptides were H-2d restricted HIV-1 specific T-cell epitopes or not(female BALB/c mice were immunized by intramuscular injection).Results Two Env-specific(gp140-9,aa 60-77;gp140-74,aa 548-565) and three Gag-specific(Gag-49,aa 196-210;Gag-64,aa 256-270;Gag-65,aa 260-274) peptides were identified containing HIV-1 specific T-cell epitopes,and gp140-74(IVQQQSNLLRAIEAQQHL) was first discovered containing H-2d restricted T-cell epitopes.The ability to stimulate HIV specific CD8+ T cells secreting IFN-γ,IL-2 and TNF-α cytokines between the immunodominant epitope peptides and all-peptide pools was comparative.Conclusion The Env peptides gp140-9,gp140-74 and Gag peptides Gag-49,Gag-64,Gag-65 contained H-2d restricted T-cell epitopes,and could be used to study the T cell responses in BALB/c mice.