Demonstration of neutralizing mucosal IgA response to intranasal HIV-1 env DNA vaccines with or without the V3 glycosylation site

HIV-1 env based DNA vaccines are generally found to be poor B-cell immunogens. We examined the role of an N-glycan located in the V3 loop of HIV-1 (N306) that is known to modulate the immunogenicity of gp120. Here we describe intranasal immunizations with env (HIV-1 BRU) based genetic immunogens in combination with subcutaneous boosts of recombinant gp160 (rgp160) in mice. Immunization with DNA alone resulted in detectable IgA responses to rgp160 in both faeces and bronchoalveolar lavage (BAL) fluid, but the additional boosting increased the faecal IgA titres only. Protein boosting was required for induction of faecal IgA antibodies capable of neutralizing a homologous laboratory strain and a subtype B primary isolate. The B-cell response towards V3 loop peptides was not only directed against the homologous subtype B but also against the subtype F. In contrast to our previous observations on IgG, there were no differences in anti-gp160 IgA titres elicited by the N-glycan mutant and the wild-type immunogen. These results indicate that intranasal administration of plasmids containing env in combination with a subcutaneous boost proved to be an effective way of eliciting neutralizing mucosal IgA against HIV-1.     

狂犬病病毒CTN-1株在鸡胚细胞驯化过程中G基因和滴度分析

目的 研究狂犬病病毒CTN-1株在鸡胚细胞驯化过程中的特性。方法 利用RT-PCR反应扩增CTN-1株19个代次的G蛋白基因,获得cDNA进行序列测定;并以免疫荧光法测定各代次毒株在细胞中的滴度。结果 序列测定显示:CTN-1株在鸡胚细胞驯化过程中G蛋白基因序列发生不同程度变异且逐步积累,从第30代开始,G蛋白基因序列趋于稳定。与CTN-1株相比,CTN-1鸡胚细胞驯化株在G蛋白的第147、333、389、421和485位氨基酸发生了突变。与此相对应,CTN-1株鸡胚细胞驯化株滴度逐渐趋于稳定,并于第33-58代滴度维持在10~10^{1 g}细胞荧光转化灶单位/mL左右。G蛋白同源性分析表明,CTN-1株鸡胚细胞驯化株与我国近期不同区域分离得到的街毒株具有较高同源性,其G蛋白氨基酸同源性为89.3%~99.0%。结论 CTN-1株在鸡胚细胞驯化过程中G蛋白基因发生稳定变异,滴度在传代后33-58代达到较高水平,稳定性较好,适于我国狂犬病防治的实际应用。     

Immunogenicity of a novel DNA vaccine cassette expressing multiple human immunodeficiency virus (HIV-1) accessory genes

OBJECTIVE: To develop an HIV-1 accessory gene immunogen using a DNA vaccine approach. METHODS: HIV-1 accessory genes vif, vpu and nef were modified to express under the control of a single promoter with cellular proteolytic cleavage sites between the coding sequences (VVN-P). Immune responses induced by these constructs were evaluated in mice. RESULTS: DNA vaccine construct (pVVN-P) expressing Vif, Vpu and Nef was processed and the fusion protein was cleaved appropriately. Vif, Vpu and Nef as a fusion protein with proteolytic cleavage sites (VVN-P) is able to induce a significant level of cellular immune responses. We also observed that accessory genes Vif, Vpu and Nef (VVN-P) induced an effective T helper 1 proliferative response measured by cytokine production. Furthermore, expression cassette pVVN-P was able to induce cytotoxic T lymphocyte (CTL) responses against diverse HIV-1 viruses in infected target cells. CONCLUSION: We conclude that cell-mediated immune responses induced by accessory gene constructs from clade B may have a broader recognition of divergent HIV-1 viruses and should be further examined for both prophylactic and therapeutic vaccination schemes against HIV-1.     

A canarypox vaccine expressing multiple human immunodeficiency virus type 1 genes given alone or with rgp120 elicits broad and durable CD8+ cytotoxic T lymphocyte responses in seronegative volunteers.

Induction of CD8+ cytotoxic T cells is considered one of the important correlates for the protective efficacy of candidate human immunodeficiency virus type 1 (HIV-1) vaccines. To induce CD8+ cytotoxic T lymphocytes (CTLs) along with neutralizing antibody and CD4+ T cell help, a live canarypox virus construct expressing gp120, transmembrane gp41, the gag and protease genes, and sequences containing CTL epitopes in nef and pol was given simultaneously with, or followed by, rgp120 SF2. CD8+ CTLs were detected in 61% of volunteers at some time during the trial. Three to 6 months after the last immunization, the gene-specific responses were gag, 26/81; env, 17/77; nef, 12/77; and pol, 3/16. Simultaneous immunization with the canarypox vector and the subunit, beginning with the initial immunization, resulted in earlier antibody responses. In summary, a strategy of immunization with a canarypox vector expressing multiple genes of HIV-1 given with gp120 results in durable CD8+ CTL responses to a broad range of epitopes.     

Chimeric influenza virus hemagglutinin containing either the NH2 terminus or the COOH terminus of G protein of vesicular stomatitis virus is defective in transport to the cell surface.

Chimeric cDNA clones of influenza virus hemagglutinin (HA) were constructed in which the DNA encoding either the NH2 terminus or the COOH terminus of HA was replaced with that of a vesicular stomatitis virus G protein. The chimeric cDNAs (GHA or HAG) were expressed in CV1 cells using the simian virus 40 late replacement promoter. Both chimeric proteins are synthesized, glycosylated, and transported to the rough endoplasmic reticulum. These results show that the NH2-terminal sequences of vesicular stomatitis virus G protein can provide a signal function for translocation and the COOH-terminal sequences can provide the anchor function for the influenza virus HA, when substituted for similar sequences. However, the chimeric glycoproteins were not transported to the Golgi complex or the plasma membrane. The implication of these results in translocation, sorting, and transport processes is discussed.     

Safety of 2 recombinant human immunodeficiency virus type 1 (HIV-1) envelope vaccines in neonates born to HIV-1-infected women.

To determine the safety of 2 candidate vaccines against human immunodeficiency virus type 1 (HIV-1), a randomized, placebo-controlled, multicenter trial compared low, medium, and high doses of the vaccines or an adjuvant among infants born to HIV-infected women. No local or systemic reactions of grade 2 or greater were reported 48 h after the subjects underwent immunization. Grade 3 or 4 chemistry toxicities occurred in 5 (3%) and grade 3 or 4 hematologic toxicities in 17 (11%) of 154 vaccinated subjects (not significantly different from 29 adjuvant recipients). CD4(+) cell percentages of < or = 20% occurred at least once in 9 vaccinated subjects and 1 control subject. Sustained CD4(+) cell percentages of < or = 20% occurred in 4 HIV-infected children. Fourteen infants (8%) were confirmed to be HIV-infected; median CD4(+) cell counts among these children were 2074, 1674, 1584, and 821 cells/mm(3) at birth and weeks 24, 52, and 104, respectively. Thus, both vaccines were safe and well tolerated in neonates, and there was no evidence of accelerated immunologic decline in HIV-infected infants.     

Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein.

A recombinant rabies virus (RV) mutant deficient for the surface spike glycoprotein (G) gene was used to study the incorporation of envelope proteins from HIV-1 expressed from transfected plasmids. A hybrid HIV-1 protein in which the cytoplasmic domain was replaced with that of RV G was incorporated into the virus envelope and rescued the infectivity of the RV mutant. The RV(HIV-1) pseudotype viruses could infect only CD4+ cells, and their infectivity was neutralized specifically by anti-HIV-1 sera. In contrast to the chimeric protein, wild-type HIV-1 envelope protein or mutants with truncated cytoplasmic domains failed to produce pseudotyped particles. This indicates the presence of a specific signal in the RV G cytoplasmic domain, allowing correct incorporation of a spike protein into the envelope of rhabdovirus particles. The possibility of directing the cell tropism of RV by replacement of the RV G with proteins of defined receptor specificity should prove useful for future development of targetable gene delivery vectors.     

狂犬病病毒G基因的克隆表达及间接ELISA检测方法的初步建立与应用

目的以重组G蛋白为检测抗原,应用间接ELISA方法检测狂犬病病毒抗体。方法根据GenBank 公布的狂犬病病毒LEP Flury株基因序列设计引物,PCR扩增出目的基因,连接pGM T载体,重组质粒经BamH I和XhoⅠ双酶切,连接到表达载体pGEX 6P 1,构建重组表达载体pGEX G。将重组表达载体转化大肠杆菌BL21(DE3)感受态细胞中,在IPTG诱导下表达目的蛋白,表达蛋白纯化后SDS PAGE电泳分析并经Western blotting鉴定。纯化的G蛋白作为包被抗原,建立间接ELISA方法检测93份犬血清抗体。结果成功构建了克隆载体pGM G和表达载体pGEX G,高效表达了狂犬病病毒G蛋白,融合蛋白分子量大小为79kDa,表达蛋白可与狂犬病病毒抗血清发生特异性反应。建立的检测方法灵敏度达到1∶1 600,与商品化的试剂盒相比符合率为96.8%。结论成功表达狂犬病病毒G蛋白,表达产物可作为检测抗原用于间接ELISA方法中狂犬病病毒抗体的检测。     

云南省2006-2010年狂犬病病毒N和G基因分子结构特征分析

目的了解云南省狂犬病毒的流行情况以及街毒株N、G基因结构特征,为有效控制狂犬病疫情提供初步科学依据。方法对云南省2006-2010年10株狂犬病街毒株N、G基因进行全基因克隆、测序,并与已知国内外代表毒株核苷酸及推导氨基酸进行序列比对及系统发育分析。结果序列分析表明所研究的10个云南毒株与Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ型代表毒株N、G基因核苷酸序列同源性分别为72.1%～78.5%、58.4%～71.0%,与基因Ⅰ型毒株序列同源性为85.2%～90.1%、82.7～99.6%,云南毒株之间核苷酸序列同源性为89.3%～99.2%、86.0～99.6%。云南10个毒株均为基因Ⅰ型和血清1型毒株,分为2个进化分支,除YNTC06与GXN119和HM88单独属于分支Ⅲ外,其余云南毒株均分布在分支Ⅰ上,且进一步分为3个小亚分支。遗传进化分析表明,各毒株氨基酸位点均存在不同程度的变异,其中YNTC06在360、369、375等多个NP抗原位点存在变异;云南毒株在GP330、333位毒力决定位点未发生变异,均为强毒株。结论云南狂犬病毒属于基因Ⅰ型,存在2个分支;云南狂犬病毒NP和GP存在特异性氨基酸变异位点,其基因结构及变异、亚组群划分与地理位置无相关性。     

Breakthrough infections during phase 1 and 2 prime-boost HIV-1 vaccine trials with canarypox vectors (ALVAC) and booster dose of recombinant gp120 or gp160

Candidate human immunodeficiency virus (HIV)-1 vaccines that elicit cytotoxic T lymphocytes may modulate HIV infection, requiring a prototype evaluation to assess participants who become infected with HIV. Of 1497 participants in canarypox HIV-1 vaccine prime-boost trials, 28 (1.9%) acquired HIV-1 infection after vaccination. Median plasma HIV-1 RNA levels (vaccinees, 4.78 log10 copies/mL; placebo recipients, 4.27 log10 copies/mL) and CD4 cell counts (vaccinees, 552 cells/mm3; placebo recipients, 657 cells/mm3) before administration of antiretroviral therapy (ART) and time to a composite end point (plasma HIV-1 RNA level >55,000 copies/mL, CD4 cell count <350 cells/mm3, or initiation of ART) did not differ significantly between vaccinees and placebo recipients (P =.4, P =.1, and P =.7, respectively). Persons who acquire HIV-1 infection while enrolled in HIV-1 vaccine trials can be successfully followed after infection, to determine whether vaccines alter the course of HIV-1 infection.     

狂犬病毒核蛋白基因的克隆及表达

目的获得狂犬病毒CVS株核蛋白(N)基因并在大肠杆菌中表达,为进一步研发狂犬血清学检测试剂盒提供抗原。方法RT-PCR扩增狂犬病毒CVS株核蛋白测序、并定向克隆入原核表达载体pET-28a,构建原核重组表达质粒pET-28a-N,转化大肠杆菌E.coliBL21,并经IPTG诱导在大肠杆菌中表达CVS株核蛋白。结果与结论扩增的CVS株N基因与发表的CVS株N基因序列完全一致,并在大肠杆菌中得到了高效表达,且表达产物具有免疫反应性,为进一步研发狂犬血清学检测试剂盒提供了抗原。     

Efficacy of the anti-Candida rAls3p-N or rAls1p-N vaccines against disseminated and mucosal candidiasis

We have shown that vaccination with the recombinant N terminus of Als1p (rAls1p-N) protects mice against disseminated and oropharyngeal candidiasis. We now report that vaccination of mice with a related candidate, rAls3p-N, induces a broader antibody response than rAls1p-N and a similar cell-mediated immune response. The rAls3p-N vaccine was equally as effective as rAls1p-N against disseminated candidiasis but was more effective than rAls1p-N against oropharyngeal or vaginal candidiasis. Antibody titers did not correlate with protection against disseminated candidiasis, but delayed-type hypersensitivity did. The rAls3p-N vaccine is a promising new vaccine candidate for further exploration to prevent systemic and mucosal candidal infections.     

Systemic and mucosal immunity is elicited after both intramuscular and intravaginal delivery of human immunodeficiency virus type 1 DNA plasmid vaccines to pregnant chimpanzees.

DNA vaccines encoding human immunodeficiency virus type 1 (HIV-1) env/rev and gag/pol were delivered intravaginally (IVAG) and intramuscularly (IM) to 2 pregnant chimpanzees. Vaccination was well tolerated and each chimpanzee developed antibodies (up to 1 year later) to both vaccines. Placental transfer of anti-Env and anti-Gag IgG was demonstrated in both maternal/infant pairs. Specific IgG was also demonstrated in saliva, vaginal, and rectal washes after IVAG immunization. Predominantly anti-HIV-1 IgA was detected in the milk of both mothers after both IM and IVAG immunization. Cellular responses included Gag-specific proliferation of lymphocytes and cytotoxic T lymphocytes against both antigens. These data suggest a strategy for induction of mucosal and systemic responses after both IM and IVAG delivery of DNA vaccines in a primate model and could ultimately be useful in lowering maternal-to-fetal transmission of HIV-1, perinatally and through breastfeeding.     

An ELISA suitable for the detection of rabies virus antibodies in serum samples from human vaccinated with either cell-culture vaccine or suckling-mouse-brain vaccine.

An indirect ELISA for determination of post-vaccination rabies antibody was applied. Purified rabies virus was used as antigen to coat plates, and staphylococcal protein A linked with horseradish peroxidase was used for detecting IgG antibody in human sera. Sera from humans, vaccinated with cell-culture vaccine or suckling-mouse-brain vaccine, were examined. ELISA results were compared to those obtained from the virus neutralization test. The mean and standard deviation of OD were determined for 126 negative sera (pre-vaccination) and for 73 sera from vaccinated persons showing antibody titers lower than 0.5 IU/ml. Results were defined as ELISA -positive, -negative or -doubtful. Establishment of a doubtful region reduced the number of sera otherwise classified as positive (false-positive sera). In this way, the sensitivity, specificity and agreement values were respectively 87.5%, 92.4% and 88.5%. No significant differences were observed in these values when the group vaccinated with cell-culture vaccine and the group vaccinated with suckling-mouse-brain vaccine were compared. It was shown that much of the disagreement between the values obtained by neutralization test and ELISA occurred in sera obtained at the beginning of the immunization process, and was probably due to the presence of IgM in the serum samples, detected only by the former test. This ELISA method can be used as a screening test in rabies laboratories regardless of the kind of vaccine used for immunization.     

Neutralization of pseudotyped vesicular stomatitis virus expressing hepatitis C virus envelope glycoprotein 1 or 2 by serum from patients

Infection with hepatitis C virus (HCV) generally progresses to chronic disease, although a minority of patients appear to clear viremia spontaneously. In this investigation, serum samples were analyzed for virological parameters, serum alanine aminotransferase (ALT) levels, and neutralizing antibody response against pseudotyped vesicular stomatitis virus (VSV) generated using chimeric envelope glycoprotein 1 (E1) or 2 (E2) of HCV. Testing of sequential serum samples that were collected beginning at the onset of acute-phase disease demonstrated intermittent viremia, elevated ALT levels, and detectable neutralization activity against VSV in 9 of 10 patients. Serum neutralization activity did not exhibit a correlation with the genotype of the infecting HCV or with virus load. On the other hand, patients with chronic HCV infection consistently had detectable amounts of virus present but no significant variation in ALT levels, and serum samples from a majority (>90%) of patients failed to show detectable neutralization activity.     

丙型肝炎病毒E2结合蛋白1结合蛋白的酵母双杂交筛选研究

目的 应用酵母技术筛选丙型肝炎病毒(HCV)7包膜蛋白E2结合蛋白1(E2BPl)的结合蛋白。方法 应用酵母双杂交系统3，构建E2BP1诱饵质粒，转化酵母AH109，与含人肝细胞cDNA文库质粒的酵母Y187进行配合，于涂有x-α-gal营养缺陷型培养基(SD／-Trp-Leu-His-Ade)上筛选生长，对于阳性克隆的插入基因片段序列进行测定，并进行生物信息学分析。结果 53个阳性克隆测序，结果与GenBank数据库进行初步比较。其中6个克隆为未知功能基因，其余47个均与己知基因的序列高度同源(90％～100％)。这47个己知基因包括人类酶原颗粒蛋白16、人类染色体序列、人类乙酰辅酶A转乙酰酶1(ACATl)、人类4．羟基苯丙酮酸二加氧酶、人类硒蛋白P1(SEPP1)、人类组织蛋白酶F(CTSF)、人类prosaposin(PSAP)、人类金属硫蛋白2A等16种。结论 HCVE2BP结合蛋白的酵母双杂交技术筛选为进一步研究E2BP1蛋白相互作用的分子生物学机制和探索新型治疗技术奠定了基础。