Three Candidate Peptide-Vaccines in Combination To Induce High Levels of Multiantibodies Against HIV-1

IntroductionIt has been demonstrated that humanimmunodeficiency virus type1 ( HIV- 1 ) gp41 isinvolved in HIV- 1 entry into the cell. Recentcrystal structure analysis of gp41 revealed that thebinding of gp1 2 0 to CD4and a co- receptor inducesthe conformational changes of gp41 ,resulting inthe exposure of the N- and C- domains and creationof a fusion intermediate and then the fusionprocess[1,2 ] .Itis generally agreed thatthe V3loopof gp1 2 0 plays a major role in the binding of gp1 2 0wit…     

中国流行株HIV—1gp120及其与IFNα基因共表达核酸疫苗质粒的构建及表达

目的 研究pGP和pGPIFNα在真核细胞中的表达。方法 以表达中国流行株HIV－1gp120基因的核酸疫苗质粒pGP及其表达中国流行株HIV－1gp120基因与IFNα基因与IFNα基因的核酸疫苗质粒pGPIFNα。转染BHK－21细胞，以间接免疫荧光鉴定其表达产物。结果 荧光显微镜下，pGP和pGPIFNα转染细胞可见绿色荧光，而HK－21细胞和空质粒则不见绿色荧光。结论 pGP和pGPIFNα可在真核细胞中表达目的蛋白gp120。     

Immune responses of a designed HIV-1 DNA vaccine on rhesus monkeys

According to the UNAIDS/WHO report (http:// www.unaids.org/Epi2005/doc/report.html), there are about 40.3 million HIV/AIDS survivors, and the new HIV infection number amounts to about 4.9 million, while the death number is some 3.1 million in the world in…     

Repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response

Based on the hypothesis suggested by us that epitope-vaccine may be a new strategy against HIV mutation, we have studied several neutralizing epitopes on HIV envelope proteins. However we do not know whether a repeated epitope in a recombinant epitope-peptide can enhance epitope-specific antibody response or not. ELDKWA-epitope (aa669-674) on the C-domain of HIV-1 gp41 is a neutralizing epitope defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. In this study, we designed and prepared a series of the recombinant epitope-peptides bearing 1, 4 and 8 copies of ELDKWA-epitope respectively. In the comparison of the antisera induced by the three recombinant antigens, an obviously increased titre of ELDKWA-epitope-specific antibody was observed in the case of four and eight repeated epitopes. In flow cytometry analysis, the epitope-specific antibodies in both antisera showed stronger activity to bind the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response, which could contribute to designing an effective recombinant epitope-vaccine.     

HIV-1C亚型DNA疫苗诱导小鼠免疫反应的研究

 构建含中国流行株HIV-1 C亚型核心蛋白gag基因的重组质粒pVAX-gag,并在体外进行了表达与鉴定.同时构建了含此gag基因的原核表达质粒pGEX-gag,表达纯化并鉴定重组蛋白Gag.以质粒pVAX-gag免疫Balb/C小鼠后,用ELISpot和流式细胞仪检测其细胞免疫反应.再以纯化后的重组蛋白Gag作为包被抗原,用ELISA检测其体液免疫反应.结果显示重组质粒pVAX-gag免疫小鼠后可有效地诱导机体产生细胞免疫和体液免疫反应,且免疫剂量和免疫效果存在一定的正相关性.重组原核表达质粒pGEX-gag的表达产物能与抗p24单克隆抗体发生特异性反应,可用于抗HIV抗体检测.     

Induction of multi-epitopespecific antibodies against HIV-1 by multi-epitopevaccines

Some neutralizing antibodies against HIV-1 envelope proteins were highly effective to inhibit the infection of different strainsin vitro, and existed in the infected individuals with very low levels. We suggested multi-epitope-vaccine as a new strategy to increase levels of neutralizing antibodies and the abilities against HIV mutationin vivo. Two candidate multi-epitope-vaccines induced antibodies with predefined multi-epitope-specificity in rhesus macaque. These antibodies recognized corresponding neutralizing epitopes on epitope-peptides, gp41 peptides, V3 loop peptide, rsgp41 and rgp120. Besides, three candidate epitope-vaccines in combination (another kind of multi-epitopevaccines) showed similar potency to induce predefined multiple immune responses in rabbits. These results suggest that multi-epitope-vaccines may be a new strategy to induce multi-antiviral activities against HIV-1 infection and mutations.     

应用梭状芽孢杆菌口服疫苗载体表达HIV p24蛋白的研究

应用一种不产毒的梭状芽孢杆菌（Clostridium perfringens,C.P.）作为口服疫苗载体表达HIVp24蛋白。用PCR扩增HIVp24基因,酶切后插入到含有C.P.序列的pJRC200质粒的CPEORF中,将重组的cp24质粒转化无致病作用的ATCC3624菌株。使HIVp24基因在C.P.形成芽孢时大量表达。结果应用SDS_PAGE和West Blot分析HIVp24在重组C.P.繁殖体和芽孢中的表达情况,发现HIVp24蛋白在C.P.芽孢体中大量表达,而在C.P.繁殖体中却没有表达。证明在体外构建了含有HIVp24基因的重组pJRC200质粒,成功地表达了HIVp24蛋白并得到了HIVp24的高表达ATCC3624菌株。为成功研究C.P.作为一种口服HIV疫苗载体奠定了基础。     

小麦GLB1蛋白单克隆抗体的制备及鉴定

以小麦主要过敏原Glb-1蛋白为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。采用细胞融合和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞株,用杂交瘤细胞株诱生小鼠腹水并用蛋白A亲和层析法纯化抗体后检测。采用间接ELISA法鉴定该单克隆抗体的IgG亚型;通过间接ELISA鉴定该单克隆抗体的特性和交叉性。利用双单抗夹心ELISA法检测单抗的抗原表位特异性。结果表明:共获得4株可稳定分泌小鼠抗小麦主要过敏原Glb-1蛋白的单克隆抗体,分别命名为1C4、4H5、1A9、4F5,经检测其Ig亚型均为IgG1,且4株单抗效价均在10-9以上。ELISA结果分析表明该4株单抗均能特异性识别小麦主要过敏原Glb-1蛋白且和其他常见食物无交叉反应性。将4株单抗两两配对进行ELISA实验,结果发现1C4与4H5可能有不同的抗原表位,以此建立的双抗夹心ELISA系统可以检测小麦Glb1-G3蛋白。实验成功制备了鼠抗小麦主要过敏原Glb-1蛋白抗原的单克隆抗体,并且建立了双单抗夹心ELISA检测系统,为小麦主要过敏原蛋白的检测奠定了基础。     

Long-Term Exposure to High Corticosterone Levels Inducing a Decrease of Adenylate Kinase 1 Activity

Corticosterone, a principal glucocorticoid synthesized in the rodent adrenal cortex, can be cumulatively toxic to hippocampal neurons, the cause of which is not known. The present study determined whether the cytosol adenylate kinase (AK) system was involved in the neuronal damage induced by long-term exposure to high corticosterone levels. We investigated the effects of long-term exposure to high corticosterone levels on AK1 activity, AK1 mRNA expression, and energy levels in cultured hippocampal neurons. The results show that long-term exposure to high corticosterone levels induces a reduction of the cultured hippocampal neuron viability, significantly reduces energy levels, and causes a time-dependant reduction of the AK1 activity. These findings indicate that changes in the AK system might be the mechanism underlying neuronal damage induced by long-term exposure to high corticosterone levels.     

Preliminary Evaluation of a Candidate Multi-Epitope-Vaccine Against the Classical Swine Fever Virus

A multi-epitope-vaccine MEVABc consisting of two linear neutralizing determinants （BCI： aa693-716; A6： aa844-865） located on antigenic unit B/C and unit A of glycoprotein E2 was prepared to evaluate whether a combination strategy is effective in the design of peptide vaccines. After immunization, pig sera collected every one to two weeks were evaluated by enzyme linked immunosorbent assay. C-straininduced anti-sera and hyper-immune sera cannot recognize overlapping peptides that cover the E2 N-terminus, while MEVAgC is able to elicit high levels of peptide-specific antibody response. When compared with previously studied peptide vaccines PV-BC1 and PV-A6, the same dose of either component in the MEMABc increases the BC1- or A6-specific antibodies （to 1/3-1/2 of the levels of the separate vaccines）. However, the synergy between the antibodies may make MEVAgc much more potent. Moreover, anti-C-strain immunity pre-existing in pigs does not disturb the sequent MEVABc vaccination. Thus, MEVABc can be administrated to pigs which already possess anti-classical swine fever virus immunity. MEVAgC is a promising candidate marker vaccine.     

LAGE-1DNA肿瘤疫苗的抗肿瘤治疗效果研究

以LAGE-1转染EMT6构建小鼠乳腺癌肿瘤模型;将BalB/c小鼠随机分为4组,以pcDNA3.1/LAGE-1及对照pcDNA3.1分别接种实验组和对照组各三次.于末次免疫后10 d在小鼠左背部、右背部皮下分别种植EMT6肿瘤细胞和EMT6/LAGE1肿瘤细胞．荷瘤后观察成瘤时间、肿瘤大小,并对小鼠脾细胞进行CTL细胞杀伤活性实验来观察DNA疫苗引起的免疫反应.结果显示LAGE-1DNA疫苗在体内能诱导有效的特异性肿瘤免疫应答,该疫苗简便有效.