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为了筛选和确定用于检测表达HIV-1 B'/C亚型病毒6种抗原(gp160、gag、polr、evt、at和nef)的艾滋病疫苗免疫小鼠后H-2d限制的特异性T细胞表位,本研究使用表达上述6种抗原的复制型DNA疫苗和非复制型重组痘苗病毒疫苗联合免疫BALB/c小鼠,通过矩阵设计将HIV-1 B(C)亚型6种相应抗原全序列肽库分别混合成肽池,使用肽池对免疫小鼠进行IFN-γELISPOT检测,根据检测结果确定肽库中特异反应的优势表位肽。结果显示:筛选到七条针对Gag的特异表位肽,其中有5条与文献报道相同,另2条为新表位肽;筛选到3条针对Pol蛋白特异表位肽,其中一条为新表位肽;筛选到2条针对gp160特异表位肽,其中一条为新表位肽;在Nef肽库中筛选到一条新的表位肽;从Tat肽库中筛选到3条表位肽,这三条肽在肽库中是连续的序列,都包含(或部分包含)网上公布的表位序列;在Rev肽库中没有筛选到能够产生阳性反应的特异性表位肽。本研究使用IFN-γELISPOT方法筛选和确定了可用于检测表达HIV-1 B'/C亚型病毒6种抗原(gp160、gag、pol、revt、at和nef)的艾滋病疫苗免疫小鼠后H-2d限制的特异性T细胞表位。
Abstract：The purpose is to screen and identify the specific H-2d restricted T-cell epitopes.These epitopes are used to investigate the cellular immune response of BALB/c(H-2d) mice immunized with a HIV-1 vaccine which expresses six antigens of gp160,gag,pol,rev,tat and nef of HIV subtype B'/C.A replicating DNA vaccine and a non-replicating recombinant vaccinia virus vector,both expressing the six antigens mentioned above,were used to immune BALB/c(H-2d) mice in a prime-boost regiment.The six peptide libraries of HIV B'/C corresponding respectively to the six complete antigens were pooled according to a designed matrix format and used to test for IFN-γ production from splenocytes of immunized mice by an enzyme-linked immunospot(IFN-γ ELISPOT) assay.The ELISPOT data indicated that two of seven Gag-specific T-cell epitope peptides were identified to be the novel epitopes.One of three Pol-specific T-cell epitope is unreported.One novel epitope was confirmed in two gp160-specific T-cell epitope peptides.One Nef-specific T-cell epitope was identified.Three Tat-specific T-cell epitope peptides were continuous sequences in Tat peptide library and all contained either complete or partial sequence reported.Rev-specific T-cell epitope was not be found.The specific T-cell epitopes(H-2d restricted) were identified by IFN-γ ELISPOT assay,which could be used to detect the cellular immune response of BALB/c mice immunized with the HIV-1 vaccine expressing six antigens of gp160,gag,pol,rev,tat and nef of HIV subtype B'/C.
[1]Girard M P,Osmanov S K,Kieny M P.A review ofvaccine research and development:The human immuno-deficiency virus(HIV)[J].Vaccine,2006,24(19):4062-4081.
[2]Addo M M,Yu X G,Rathod A,et al.Comprehensiveepitope analysis of human immunodeficiency virus type 1(HIV-1)-specific T-cell responses directed against theentire expressed HIV-1 genome demonstrate broadly di-rected responses,but no correlation to viral load[J].JVirol,2003,77(3):2081-2092.
[3]Kumar S,Seth P.Immunogenicity of recombinant modi-fied vaccinia Ankara viruses(rMVA)expressing HIV-1Indian subtype C gag-protease and env-gp120 genes inmice[J].Viral Immunol,2004,17(4):574-579.
[4]Yang K,Li S,He F,et al.Expression and purificationof soluble HIV-1 envelope glycoprotein gp160 mutantfrom Saccharomyces cerevisiae[J].J Biosci Bioeng,2009,108(1):5-10.
[5]Luo L,Li Y,Dales S,et al.Mapping of functional do-mains for HIV-2 gag assembly into virus-like particles[J].Virology,1994,205(2):496-502.
[6]Griffiths J C,Harris S J,Layton G T,et al.Hybrid hu-man immunodeficiency virus gag particles as an antigencarrier system:lnduction of cytotoxic T-cell and humor-al responses by a gag:V3 fusion[J].J Virol,1993,67(6):3191-3198.
[7]Casimiro D R,Tang A,Perry H C,et al.Vaccine-in-duced immune responses in rodents and nonhuman pri-mates by use of a humanized human immunodeficiencyvirus type 1 pol gene[J].J Virol,2002,76(1):185-1894.
[8]Mabrouk K,Van Rietschoten J,Vives E,et al.Lethalneurotoxicity in mice of the basic domains of HIV andSIV Rev proteins.Study of these regions by circular di-chroism[J].FEBS Lett,1991,289(1):13-17.
[9]ButtòS,Fiorelli V,Tripiciano A,et al.Sequence con-servation and antibody cross-recognition of clade B hu-man immunodeficiency virus(HIV)type 1 Tat proteinin HIV-1-infected Italians,Ugandans,and South Afri-cans[J].J Infect Dis,2003,188(8):1171-1180.
[10]Goldstein G,Tribbick G,Manson K.Two B cellepitopes of HIV-1 Tat protein have limited antigenicpolymorphism in geographically diverse HIV-1 strains[J].Vaccine,2001,19(13-14):1738-1746.
[11]Ranasinghe C,Turner S J,McArthur C,et al.Muco-sal HIV-1 pox virus prime-boost immunization induceshigh-avidity CD8+T cells with regime-dependent cyto-kine/granzyme B profiles[J].J Immunol,2007,178(4):2370-2379.
[12]Malm M,Rollman E,Ustav M,et al.Cross-clade pro-tection induced by human immunodeficiency virus-1DNA immunogens expressing consensus sequences ofmultiple genes and epitopes from subtypes A,B,C,and FGH[J].Viral Immunol,2005,18(4):678-688.
[13]Mata M,Travers P J,Liu Q,et al.The MHC class I-restricted immune response to HIV-gag in BALB/cmice selects a single epitope that does not have a pre-dictable MHC-binding motif and binds to Kd throughinteractions between a glutamine at P3 and pocket D[J].J Immunol,1998,161(6):2985-2993.
[14]Mata M,Paterson Y.Th1 T cell responses to HIV-1Gag protein delivered by a Listeria monocytogenes vac-cine are similar to those induced by endogenous listerialantigens[J].J Immunol,1999,163(3):1449-1456.
[15]Casimiro D R,Tang A,Perry H C,et al.Vaccine-in-duced immune responses in rodents and nonhuman pri-mates by use of a humanized human immunodeficiencyvirus type 1 pol gene[J].J Virol,2002,76(1):185-194.
[16]Xu J,Ren L,Huang X,et al.Sequential priming andboosting with heterologous HIVimmunogens predomi-nantly stimulated T cell immunity against conservedepitopes[J].AIDS,2006,20(18):2293-2303.
[17]MacGregor R R,Ginsberg R,Ugen K E,et al.T-cellresponses induced in normal volunteers immunized witha DNA-based vaccine containing HIV-1 env and rev[J].AIDS,2002,16(16):2137-2143.
[18]Addo M M,Altfeld M,Rosenberg E S,et al.TheHIV-1 regulatory proteins Tat and Rev are frequentlytargeted by cytotoxic T lymphocytes derived fromHIV-1-infected individuals[J].Proc Natl Acad SciUSA,2001,98(4):1781-1786.
[19]Fogg M H,Garry D,Awad A,et al.The BZLF1 hom-olog of an epstein-barr-related-γherpesvirus is a fre-quent target of the CTL response in persistently infec-ted rhesus macaques[J].J Immunol,2006,176(6):3391-3401.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002139
中图分类号:R392
引用信息:
[1]齐香荣,高瑛瑛,陆柔剑,等.应用ELISPOT方法筛选确定HIV-1 B'/C亚型疫苗六种抗原的H-2~d限制的T细胞表位[J].病毒学报,2011,27(01):34-43.DOI:10.13242/j.cnki.bingduxuebao.002139.
基金信息:
CIPRA项目(1U19A15191501); 863项目(2003AA219080); “十一五”重大专项(2008ZX10001-012)
2011-01-15
2011-01-15