Antigen epitopes of animal coronaviruses:a mini-review

Coronaviruses are widespread in nature and can infect mammals and poultry,making them a public health concern.Globally,prevention and control of emerging and re-emerging animal coronaviruses is a great challenge.The mecha-nisms of virus-mediated immune responses have important implications for research on virus prevention and control.The antigenic epitope is a chemical group capable of stimulating the production of antibodies or sensitized lympho-cytes,playing an important role in antiviral immune responses.Thus,it can shed light on the development of diagnos-tic methods and novel vaccines.Here,we have reviewed advances in animal coronavirus antigenic epitope research,aiming to provide a reference for the prevention and control of animal and human coronaviruses.

An immunoglobulin Y that specifically binds to an in silico-predicted unique epitope of Zika virus non-structural 1 antigen

Objective:To identify unique immunogenic epitopes of Zika virus non-structural 1(NS1)antigen and produce immunoglobulin Y(IgY)for potential use in he diagnosis of of Zika virus infection.Methods:Immunogenic epitopes were identified using in silico B-cell epitope prediction.A synthetic peptide analog of the predicted epitope was used to induce antipeptide IgY production in hens which was purified using affinity chromatography.Presence of purified IgY and its binding specificity were performed by gel electrophoresis and ELISA,respectively.Results:Out of the nine continuous epitopes identified,the sequence at position 193-208(LKVREDYSLECDPAVI)was selected and used to produce anti-peptide IgY.The produced IgY was found to bind to the synthetic analog of the Zika virus NS1 immunogenic epitope but not to other flaviviruses and random peptides from other pathogens.Conclusions:In this study,we identified an immunogenic epitope unique to Zika virus that can be used to develop a serodiagnostic tool that specifically detect Zika virus infection.

Prokaryotic Expression and Immunogenicity of Major Antigen Epitope of Rabbit Haemorrhagic Disease Virus VP60

[ Objective] To investigate the immunogenicity of main antigen epitope of RHDV （ Rabbit haemorrhagic disease virus） VP60 expressed in a prokaryotic system. [ Method] The major antigen epitope gene of RHDV VP60 was amplified by RT-PCR. It was cloned into pET-28b （ ＋ ） and expressed in E. coli Rosetta strain. The recombinant protein was detected by Western blot. The pudfied recombinant protein was used to immunize rabbits in order to observe its immunogenicity. [ Result] Western blot analysis revealed a clear band at approximately 24.0 kDa. The purified recom- binant protein reacted with the purified RHDV in ELISA. [ Conclusion] The prokaryotically expressed main antigen epitope of RHDV VP60 shows good immunogenicity.

An Indirect ELISA of Classical Swine Fever Virus Based on Quadruple Antigenic Epitope Peptide Expressed in E.coli

In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.

Hepatocellular carcinoma-specific immunotherapy with synthesized α1,3-galactosyl epitope-pulsed dendritic cells and cytokine-induced killer cells

AIM: To evaluate the safety and clinical efficacy of a new immunotherapy using both α-Gal epitope-pulsed dendritic cells (DCs) and cytokine-induced killer cells.METHODS: Freshly collected hepatocellular carcinoma(HCC) tumor tissues were incubated with a mixture of neuraminidase and recombinant α1,3-galactosyltransferase (α1,3GT) to synthesize α-Gal epitopes on carbohydrate chains of the glycoproteins of tumor membranes. The subsequent incubation of the processed membranes in the presence of human natural anti-Gal IgG resulted in the effective phagocytosis to the tumor membrane by DCs. Eighteen patients aged 38-78 years with stage Ⅲ primary HCC were randomLy chosen for the study; 9 patients served as controls, and 9 patients were enrolled in the study group.RESULTS: The evaluation demonstrated that the procedure was safe; no serious side effects or autoimmune diseases were observed. The therapy significantly prolonged the survival of treated patients as compared with the controls (17.1 ± 2.01 mo vs 10.1 ± 4.5 mo,P = 0.00121). After treatment, all patients in the study group had positive delayed hyper sensitivity and robust systemic cytotoxicity in response to tumor lysate as measured by interferon-γ-expression in peripheral blood mononuclear cells using enzyme-linked immunosorbent spot assay. They also displayed increased numbers of CD8-, CD45RO-and CD56-positive cells in the peripheral blood and decreased α-fetoprotein level in the serum.CONCLUSION: This new tumor-specific immunotherapy is safe, effective and has a great potential for the treatment of tumors.

Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8％ of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1immunogenicity and antigenicity.

乙型肝炎病毒前S2抗原决定簇（HBVPreS2epitope）与乙型肝炎病毒核心抗原（HBcAg）的基因融合与表达

乙肝前Ｓ２（ＨＢＶＰｒｅＳ２）肽段由５５个氨基酸组成，其Ｎ端肽段含Ｔｈ和Ｂ细胞抗原决定簇。我们将化学合成的ＰｒｅＳ２ｅｐｉｔｏｐｅ（１２０－１４５）基因与ＨＢｃＡｇ基因不同位点进行融合，融合基因在大肠杆菌中获得表达，并对融合蛋白进行了纯化。经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验表明，融合蛋白具有ＰｒｅＳ２和ＨＢｃＡｇ两者的抗原性。此外，研究还表明，强启动子能使表达水平有一定提高。

Prediction of promiscuous T-cell epitopes in the Zika virus polyprotein:An in silico approach

Objective:To predict immunogenic promiscuous T-cell epitopes from the polyprotein of the Zika virus using a range of bioinformatics tools.To date,no epitope data are available for the Zika virus in the IEDB database.Methods:We retrieved nearly 54 full length polyprotein sequences of the Zika virus from the NCBI database belonging to different outbreaks.A consensus sequence was then used to predict the promiscuous T cell epitopes that bind MHC 1 and MHC II alleles using Propred1 and Propred immunoinformatic algorithms respectively.The antigencity predicted score was also calculated for each predicted epitope using the Vaxi Jen 2.0 tool.Results:By using Pro Pred1,23 antigenic epitopes for HLA class I and 48 antigenic epitopes for HLA class II were predicted from the consensus polyprotein sequence of Zika virus.The greatest number of MHC class I binding epitopes were projected within the NS5(21%),followed by Envelope(17%).For MHC class II,greatest number of predicted epitopes were in NS5(19%) followed by the Envelope,NS1 and NS2(17% each).A variety of epitopes with good binding affinity,promiscuity and antigenicity were predicted for both the HLA classes.Conclusion:The predicted conserved promiscuous T-cell epitopes examined in this study were reported for the first time and will contribute to the imminent design of Zika virus vaccine candidates,which will be able to induce a broad range of immune responses in a heterogeneous HLA population.However,our results can be verified and employed in future efficacious vaccine formulations only after successful experimental studies.

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM To identify hepatitis C virus (HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL).METHODS Utilizing the method of computer prediction followed by a 4 h 51 Cr-release assay confirmation.RESULTS The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide 'ALAHGVFAL (core TS0-158)'.The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%,respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis.But blocking of CTL response with anti-CD8 mAb could abolish the Iysis.CONCLUSION The peptide (core 150 - 158 ) is the candidate epitope recognized by HLA-A2 restricted CTL.

Hepatitis B virus S gene enhances immune responses of a multiple-epitope DNA construct of hepatitis C virus in mice

The purpose of this study was to construct an eukaryotic DNA vector encoding a multiple-epitope antigen (MFC) of hepatitis C virus (HCV) and a hepatitis B surface antigen (HBsAg) , and explore the effect of HBsAg gene on the immunity of HCV multiple-epitope DNA construct in vitro and in vivo in mice. An HCV DNA vector (pVAX1-HBs-MFC) was constructed by fusing HBsAg gene to the N-terminal of an HCV multiple-epitope antigen gene. The pVAX1-HBs-MFC was transfected into HEK 293T cells and its expression was measured by ELISA and Western blotting. BALB/c mice were intramuscularly immunized with the pVAX1-HBs-MFC, and an ELISA approach was applied to determine the specific antibody titers and subtypes in the mouse serum. The cross-reactivity of the antibodies was also checked with two synthesized HCV hypervariable region 1 (HVR1) peptides. The IFN-γ production and cell proliferation of the mouse spleen cells were evaluated by ELISA and MTS (3-[4, 5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assays, respectively. The expression of pVAX1-HBs-MFC was detectable in the transfected HEK 293T cells. The serum antibody response was effectively elicited in BALB/c mice injected with pVAX1-HBs-MFC. The highest titer of antibody against HCV (MFC) was 1∶1280, and the ratio of IgG2a/IgG1 was 1.50±0.12 at the fifth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the two synthesized HCV HVR1 peptides. The stimulation index of the mouse splenocytes to MFC was 1.79 ±0.07, and the IFN-γ level was 287±6?pg/ml at week 21 after first immunization. The highest titer of the antibody in control BALB/c mice immunized with pVAX1-MFC was 1∶320, and the ratio of IgG2a/IgG1 was 1.33±0.11 at week 5 post-immunization. Furthermore, the stimulation index of the mouse splenocytes cells to MFC was 1.52±0.06, and the IFN-γ level was 225±9.3?pg/ml at week 21 post-immunization. The HBsAg gene can enhance the effects of an HCV multiple-epitope DNA construct on its humoral and cellular immune responses. This HBsAg enhanced HCV multiple-epitope DNA vector may be of potential use in the development of HCV vaccines.

为提升IPTR患者血小板输注后CCI值建立分级规避HLA抗体对应抗原方法及HLAMatchmaker的应用研究

目的:建立分级规避HLA抗体MFI阈值对应抗原方法,联合应用HLAMatchmaker表位计算法,选择供患者表位最小错配评分值,评估两种方法为免疫性血小板输注无效(Immune platelet transfusion refractoriness,IPTR)患者选择HLA相容性血小板供者,在提升血小板输注后校正增加值(CCI)的应用价值。方法:采用SPRCA法完成51例IPTR患者的7807次血小板交叉配型实验,判断其免疫反应阴/阳性结果。采用Luminex单抗原流式微珠法检测患者的HLA-I类抗体,获得不同特异性抗体对应HLA-I类抗原MFI值,并将其分组及分级,强阳性组(MFI>4000,1级)、中阳性组(1000中阳性组>弱阳性组)。强阳性和中阳性组与阴性对照组之间的SPRCA实验免疫反应阳性结果检出数存在统计学差异(P<0.001),弱阳性位组和阴性对照组之间的SPRCA实验免疫反应阳性结果检出数无统计学差异(P>0.05)。设置强阳性组为相应特异性HLA位点对应抗原1级规避阈值,中阳性组为2级规避阈值,弱阳性组为3级规避阈值,在供者血小板紧缺情况下,可以不需要规避弱阳性组。规避1和2级HLA-I类抗体对应供者抗原及选择HLAMatchmaker表位错配评分数≤7血小板供者策略,24 h内CCI值均>4.5×109/L,均可获得临床血小板输注有效。结论:在为IPTR患者选择HLA-I类相容性供者时,分级规避HLA-I类抗体对应供者抗原,综合选择供受者HLAMatchmaker表位错配评分数≤7,经血小板交叉配型实验确认为阴性结果的供者选择策略,对提升IPTR患者血小板计数具有一定实际应用价值。

猪细小病毒病研究进展

猪细小病毒(Porcine parvovirus,PPV)是一种无囊膜DNA病毒,主要引起母猪繁殖障碍。该病毒在全世界广泛流行,我国猪场PPV感染率高达90%以上,给养猪业带来巨大经济损失。PPV经口、鼻传播,主要侵染猪的生殖器官,引起母猪的死胎、木乃伊胎和公猪的精液质量下降。临床采用接种疫苗的方式进行防控,起到了一定的效果。论文对病毒的基因组和蛋白特征、流行病学和疫苗研究进行综述,以期为PPV的基础研究和疫苗开发提供参考。

伪狂犬病病毒VP22蛋白单克隆抗体制备及其抗原表位鉴定

为制备伪狂犬病病毒(PRV)VP22蛋白单克隆抗体,将目的基因克隆至原核表达载体pET-28a(+),构建pET-28a-VP22原核表达质粒,并利用大肠杆菌表达系统,获得重组VP22蛋白。将纯化后的蛋白免疫6~8周龄BALB/c雌鼠,通过杂交瘤细胞融合技术及间接ELISA方法筛选,经3次亚克隆后获得4株PRV VP22蛋白的单克隆抗体1D6、1D9、1B12和2C10。间接ELISA检测4株杂交瘤细胞传至15代均能稳定分泌抗体,且细胞上清液抗体效价均为1∶6400。经亚型鉴定,4株单克隆抗体的重链均是IgG1亚类,轻链皆属于κ型。Western blot和间接免疫荧光试验(IFA)结果表明,制备的单克隆抗体均可与PRV发生特异性反应,并鉴定出2个抗原表位;Western blot和共聚焦试验结果表明,VP22蛋白是病毒早期蛋白,且早期存在于细胞质,晚期移位至细胞核并在核中积累。本研究制备的单克隆抗体将为后续探索PRV VP22蛋白的功能提供材料支撑。

Ⅱ型鹅星状病毒VP27蛋白生物信息学分析

为探究Ⅱ型鹅星状病毒(GAstⅤ-Ⅱ)VP27蛋白的结构和功能,运用生物信息学软件,对具有良好免疫原性的GAstⅤ-ⅡZYL02株进行分析。通过Expasy-ProtParam、PSORTⅡPrediction、ProtScale、SignalP 4.1、TMHMM 2.0和SOPMA等在线软件预测分析VP27蛋白的生物学特性,运用BepiPred-2.0、ABCpred、IEDB、ToxinPred2和VaxiJenv2.0等在线软件预测分析VP27蛋白的B细胞抗原表位,从中筛选出优势抗原表位;使用Phyre2在线软件对VP27蛋白进行建模,运用PyMOL软件准确定位预测的优势表位在模型中的分布。结果显示:VP27蛋白是不稳定的亲水蛋白,其亚细胞定位位于细胞质,无跨膜结构域和信号肽;二级、三级结构中无规卷曲平均占比为41.91%,延伸链平均占比为29.05%,α螺旋平均占比为22.41%,β转角平均占比为6.64%;共预测出26条B细胞抗原表位,筛选出4条B细胞优势抗原表位,分别位于26~41aa(FGIPQADSRSRYNANI)、48~57 aa(RGRTSTSFTL)、111~126 aa(TSTGGQITELRNRLNI)、174~189 aa(PVLINWSVSDSQEQYN);三级结构定位发现,4条优势B细胞表位均位于该蛋白表面。结果表明,GAstⅤ-ⅡVP27蛋白有多处可诱导体液免疫的潜在位点。本研究运用生物信息学分析方法预测了GAstⅤ-ⅡVP27蛋白的理化性质和结构,对进一步深入研究VP27蛋白功能,研发相应疫苗具有重要指导意义。

微小扇头蜱microRNA文库建立及miR-275靶蛋白Vg-2的生物信息学分析

【目的】建立饱血状态雌性微小扇头蜱的microRNA文库,鉴定所含microRNA的种类,分析miR-275靶蛋白Vg-2的二、三级结构,检测其信号肽、跨膜结构域、磷酸化及糖基化位点,预测其优势抗原表位。【方法】以雌性、饱血状态的微小扇头蜱为研究对象,通过高通量测序技术建立其microRNA文库,利用软件miRDP及检索miRBase数据库,鉴定饱血状态雌性微小扇头蜱所含的microRNA种类;运用在线软件EXPASY、PRABI与SWISS-MODEL等分析Vg-2蛋白质理化性质,推断其二、三级结构;运用在线软件SignalP 5.0、TMHMM、NetPhos 3.1及NETCGlyc 1.0检测该蛋白的信号肽、跨膜结构域、磷酸化与糖基化位点;利用在线软件ABCpred Prediction、Scratch、IEDB和NetCTL预测Vg-2蛋白的B、T细胞优势抗原表位。【结果】获得成熟的microRNA 383个,其中67个为已被鉴定和注释的microRNA,316个为新发现的microRNA;微小扇头蜱的Vg-2基因序列全长5040 bp,共编码1679个氨基酸;Vg-2蛋白为亲水性酸蛋白,分子大小为189.89 kDa,理论等电点(pI)为6.08,其二级结构以无规则卷曲为主要结构成分,而其三级结构却以β-折叠为主要结构成分;Vg-2蛋白存在268个磷酸位点,20个糖基化位点,无信号肽和跨膜结构域,拥有58个B细胞优势抗原表位和8个T细胞优势抗原表位。【结论】在饱血状态雌性微小扇头蜱体内含有383个microRNA,受miR-275调控的Vg-2蛋白为结构不稳定的亲水性酸蛋白,具有良好的抗原性与免疫源性。

两步酶解法制备低致敏性乳清蛋白及其致敏性评价

目的探究两步复合酶解工艺处理对牛乳清蛋白(whey protein isolate,WPI)结构和致敏性的影响。方法实验采用邻苯二甲醛法、体积排阻色谱法分析WPI水解物在不同酶解时间下的水解程度,同时采用酶联免疫吸附法(enzyme-linked immunoassay,ELISA)、细胞模型和小鼠模型对水解物进行体外和体内的致敏性评估,并利用超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)分析水解物中的残留过敏原表位。结果WPI经碱性蛋白酶和木瓜蛋白酶顺序酶解3.0 h后水解度达到14.19%,在水解2.0~3.0 h期间,WPI水解物主要由小肽(<5.0 kDa)组成。水解3.0 h后,水解产物与特异性抗体的结合能力降低了97.83%,细胞模型和小鼠模型结果证实,酶解产物的体外、体内致敏性显著降低,与空白对照组无显著性差异。UPLC-MS/MS结果表明WPI水解3.0 h产物中70.59%的肽段不含过敏原表位。结论复合酶解工艺有效降低了WPI致敏性,本研究构建的致敏性综合评价体系可为低敏乳制品水解工艺的开发提供指导。

应用噬菌体展示随机12肽库筛选诺如病毒抗原模拟表位

目的:利用Ph.D.-12噬菌体展示肽库筛选诺如病毒(NoV)的抗原模拟表位。方法:包被与GⅡ.4、GⅡ.6、GⅡ.17型NoV具有高特异性及中和能力较强的单链可变片段抗体(scFv),用Ph.D.-12噬菌体展示肽库进行3轮生物淘选。ELISA鉴定淘选所得噬菌体与scFv的结合活性及其与NoV P蛋白的竞争作用;阳性克隆测序后进行生物信息学分析,合成多肽鉴定其抗原性。结果:发现1段与GⅡ.6 VP1区同源性较高的氨基酸序列“MG-D-W”,综合分析提示其可能为GⅡ.6 NoV的抗原模拟表位,且合成的包含“MG-D-W”的多肽可竞争抑制P蛋白与人类组织血型抗原(HBGAs)受体的结合。结论:“MG-DW”是与NoV单链抗体高亲和力的肽段,可能模拟了GⅡ.6 NoV与scFv结合的抗原表位。

一株高病毒载量PCV2毒株的基因组特征及序列分析

【目的】了解广东省某猪群中猪圆环病毒2型(Porcine circovirus type 2,PCV2)流行毒株的遗传进化情况,丰富PCV2分子流行病学数据,为当地PCV2疫苗候选株的选用和研发提供参考。【方法】使用qPCR方法对疑似PCV2的样品进行检测,发现1株具有高病毒载量的PCV2毒株,命名为GD222858。通过PCR方法进行全基因组分子克隆及遗传进化分析。使用MegAlign软件将该毒株ORF1、ORF2基因编码的氨基酸序列与PCV2同亚型参考毒株进行比对,分析氨基酸序列的相似性;采用DNAStar预测该毒株的Cap蛋白二级结构及B细胞表位,并与4株疫苗株DBN-SX07-2(HM641752)、LG(HM038034)、SH(HM038027)、ZJ(AY686764)的Cap蛋白抗原指数进行比对分析。【结果】GD222858毒株基因组长度为1767 bp。遗传进化分析表明该毒株属于PCV2d亚型。与国内外82株参考毒株的核苷酸相似性为91.4%~99.6%,与越南毒株Han8(GenBank登录号:JQ181600)的亲缘关系最近。在ORF1编码的Rep蛋白处发现多个特异性突变位点F70Y、F77L、W202R、N256S;ORF2编码的Cap蛋白相对保守。Protean预测Cap蛋白的氨基酸第5~18、24~25、39~41、48~49、57~65、99、101、112~114、139~140、145~150、162~165、175~181、188~189、205~211、227~232位置处均可能存在潜在的B细胞表位。GD222858毒株的Cap蛋白抗原指数与4株疫苗株均有差异,在氨基酸45~57、124~132、223~233位置处抗原指数明显高于4株疫苗株,且与疫苗株HM038034差异最大。【结论】GD222858毒株感染猪群的原因可能是Rep蛋白多个位点发生特异性突变及疫苗株选用不当所致。

Key role of human leukocyte antigen in modulating human immunodeficiency virus progression: An overview of the possible applications

Host and viral factors deeply influence the human immunodeficiency virus(HIV) disease progression. Among them human leukocyte antigen(HLA) locus plays a key role at different levels. In fact, genes of the HLA locus have shown the peculiar capability to modulate both innate and adaptive immune responses. In particular, HLA class Ⅰmolecules are recognized by CD8+ T-cells and natural killers(NK) cells towards the interaction with T cell receptor(TCR) and Killer Immunoglobulin Receptor(KIR) 3DL1 respectively. Polymorphisms within the different HLA alleles generate structural changes in HLA classⅠpeptide-binding pockets. Amino acid changes in the peptide-binding pocket lead to the presentation of a different set of peptides to T and NK cells. This review summarizes the role of HLA in HIV progression toward acquired immunodeficiency disease syndrome and its receptors. Recently, many studies have been focused on determining the HLA binding-peptides. The novel use of immune-informatics tools, from the prediction of the HLA-bound peptides to the modification of the HLAreceptor complexes, is considered. A better knowledge of HLA peptide presentation and recognition are allowing new strategies for immune response manipulation to be applied against HIV virus.

Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the “a” region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the “a” determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.