Genotypes and S-gene variability of Mexican hepatitis B virus strains

The genotypes and subtypes of 15 Mexican hepatitis B virus strains were determined by sequencing and phylogenetic analysis of the small S-gene. The most predominant strains were found to be divergent genotype/subtype F/adw4 strains (66.6%), followed by A/adw2 (20.0%), D/ayw3 (6.7%), and G/adw2 (6.7%). The S-genes of the Mexican genotype F strains and two Nicaraguan strains described previously formed a subcluster with more than 4% divergence from the other strains within this genotype. The Mexican strains within genotypes A and D showed the highest homology with strains from Europe and the United States. Ten amino acid substitutions not described previously were found in the S-genes of strains from nine chronic carriers, whereas the S gene in strains from six acute hepatitis B patients were highly conserved as compared to their respective genotypes. One genotype F strain from an HBsAg positive chronic carrier had a T to A mutation at position 647, forming a translational stop at codon 216. Two genotype F strains from HBsAg negative chronic carriers had a Val180 instead of an Ala found in the other genotype F strains. This study shows that a divergent genotype F predominates in Mexican strains analyzed, which presented amino acid substitutions not reported previously outside the a determinant.     

Clinical significance of a highly sensitive enzyme immunoassay of hepatitis B surface antigen using a novel electron spin resonance technique.

We developed a highly sensitive enzyme immunoassay (EIA), the p-AP/HHTIO method, that detects serum hepatitis B surface antigen (HBsAg) by measuring stabilized nitroxide radicals using a novel electron spin resonance technique [Matsuo et al. (1998) Free Radic Biol Med 25:929-935]. To demonstrate the clinical significance of this method and to reveal occult hepatitis B virus (HBV) infection in patients, we used the method to analyze serum samples of 30 patients with acute or fulminant hepatitis who were negative for HBsAg by standard EIA, and those of seven chronic HBV carriers who became negative for HBsAg during a follow-up period by standard EIA. We also examined serum HBV DNA by amplification of the HBV S gene, using the polymerase chain reaction (PCR) technique. The p-AP/HHTIO method showed that 9 of 20 (45%) patients with acute hepatitis and 2 of 10 (20%) with fulminant hepatitis were positive for HBsAg; PCR detected HBV DNA in these HBsAg-positive patients. Antibody against hepatitis B core antigen was detected in one patient with fulminant hepatitis. The p-AP/HHTIO method demonstrated prolonged seropositivity of HBsAg even after standard EIA showed a loss of HBsAg in all seven HBV carriers. Our p-AP/HHTIO method is useful for screening and diagnosing HBV infection in patients with liver diseases who are negative for conventional HBV-related serological markers.     

Immunization with Combined HSV-2 Glycoproteins B2:D2 Gene DNAs: Protection against Lethal Intravaginal Challenges in Mice

The immunity of a combined DNA vaccine of HSV-2 glycoproteins B2 (gB2) and D2 (gD2) genes in comparison to individual vaccines was studied with regard to protecting against the HSV infection. Two recombinant DNA vaccines of the pHS2-gB2 or pHS2-gD2 were constructed and formulated. The neutralizing antibody titers appeared higher in the B2:D2 gene cocktail-vaccinated mice than that of the individual B2 or D2 gene-vaccinated group alone, and the positive KOS control induced higher titer of the neutralizing antibody than combined or individual gene vaccines. The mock-immunized mice failed to induce enough. The ranks for the CTL activity and the protection rates against the lethal intravaginal challenge were shown as KOS>B2:D2 cocktail>D2>B2 gene vaccines. The vaginal external diseases in the B2:D2 or D-vaccinated mice were significantly reduced against the challenging dosages. The virus titers in the vaginal secretions of the vaccinated mice significantly reduced with time, and the B2:D2 gene vaccine decreased more than each individual vaccine alone. It can be concluded that the cocktailed vaccines are more effective in the humoral and cellular immune responses in the mice, and in the protection of the mice against the intravaginal challenging dosages when compared with individual gene vaccines. All the DNA vaccines failed to block the latent infection in sensory nerves.     

Isolation of vaccine-derived type 1 polioviruses displaying similar properties to virulent wild strain Mahoney from sewage in Japan

Type 1, 2, and 3 vaccine-derived polioviruses were isolated from a sewage disposal plant located downstream of the Oyabe River in Toyama Prefecture, Japan, between October 1993 and September 1995. Neurovirulence was analyzed in 13 type 1 vaccine-derived strains, using mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC). Nine strains (69%) were estimated to have marked neurovirulence. Some of the neutralizing antigenic sites, temperature sensitivity, and plaque-forming ability of two virulent vaccine-derived poliovirus strains were similar to Mahoney strain. The neutralizing activity of human sera obtained after oral poliomyelitis vaccine (OPV) administration against one of the virulent vaccine-derived polioviruses was examined. Although all human sera showed sufficient neutralizing activity for the prevention of poliomyelitis by vaccine-derived poliovirus strains, a lower titer than that against Sabin type 1 strain was observed. Vaccination against virulent vaccine-derived poliovirus will be effective. However, the environmental presence of viruses that have properties similar to those Mahoney strain is a threat. The introduction of inactivated poliovirus vaccine (IPV), and well-maintained herd immunity, together with reinforced environmental surveillance is important for the final phase of the polio eradication program by the World Health Organization (WHO).     

大鼠抗—HBs的单克隆抗独特型抗体杂交瘤细胞系的建立

用纯化的小鼠单克隆抗—HBs和纯化的大鼠多克隆抗—HBs免疫LOU/C大鼠,取免疫大鼠脾细胞与IR983F骨髓瘤细胞融合,获得两株(TD_1、TD_2)持续分泌抗—HBs的单克隆抗独特型抗体(抗—Id,Ab_2)杂交瘤细胞系。对TD_2进行了较全面的鉴定,TD_2的Ig类型为IgG,亚类为IgG_(2?),核型分析结果染色体数为65条。通过ELISA竞争抑制和中和抑制试验表明,TD_2所分泌的Ab_2既能被不同来源的抗—HBs所中和。又能被HBsAg.所竞争,且都呈现剂量依赖关系。将纯化的TD_2IgG免疫同系大鼠,诱导出了具有抗—HBs活性的抗—抗—独特型抗体(Ab_3)。上述结果证实,TD_2所分泌的单克隆抗体(McAb)是带有HBsAg表位内影像,具有HBsAg免疫源性的抗—HBs的单克隆抗—Id。     

CVB3-VP1基因免疫诱导特异性抗病毒免疫应答及保护作用

目的 :构建表达柯萨奇病毒B3(CVB3)主要包膜蛋白VP1的基因疫苗 ,并研究该疫苗诱导CVB3特异性免疫应答及免疫保护的作用。方法 :抽提CVB3RNA ,以RT PCR扩增VP1基因 ,克隆于真核表达载体 pcDNA3中 ,构建质粒pcDNA3 VP1。将该质粒转染Hela细胞 ,观察其表达情况 ;以 5 0 μgpcDNA3 VP1质粒DNA肌注免疫BALB/c小鼠 3次 ,检测CVB3特异性体液和细胞免疫应答。间隔 4wk以 5×LD50 的CVB3攻击小鼠 ,观察攻击后小鼠的存活情况。结果 :构建了重组质粒 pcDNA3 VP1,并在体外获得有效表达。以该质粒肌肉免疫BALB/c小鼠 ,可诱生高水平的IgM和IgG ,VP1多肽特异性淋巴细胞增殖反应及CTL活性均显著高于 pcDNA3免疫的对照组。病毒攻击试验表明 ,pcDNA3 VP1免疫组33.3%小鼠可长期存活 ,其心肌组织未见明显的病理学改变 ;而对照小鼠平均仅存活 6 .7d ,心肌显示大量的局灶性坏死和炎性细胞浸润。结论 :pcDNA3 VP1免疫可诱生CVB3特异性体液及细胞免疫应答 ,保护免疫小鼠抵抗CVB3的致死性攻击     

Histopathological studies on the local reactions induced by complete Freund's adjuvant (CFA), bacterial lipopolysaccharide (LPS), and synthetic lipopeptide (P3C) conjugates

The inflammatory reactions following subcutaneous application of adjuvants revealed characteristic pathological patterns. The injection of complete Freund's adjuvant (CFA) resulted in the formation of large lipid deposits encircled by an inflammatory reaction and concentrically arranged collagen bundles. Bacterial lipopolysaccharide (LPS) caused granulomatous aggregations of mononuclear cells with thrombotic vessel occlusions. Inoculation of the lipopeptide adjuvants induced accumulation of mononuclear cells with only minimal fibrotic changes which were resolved after day 28. Lipopeptide conjugates based on the head group tripalmitoyl-S-glyceryl-cysteinyl-serin (P3CS) can thus be used as effective immunogens and adjuvants without long-term tissue damage.     

巨噬细胞肿瘤疫苗抗瘤作用的实验研究

目的：探究巨噬细胞肿瘤疫苗的细胞形态与表型特征,并观察其CTL反应的诱导效果。方法：分别采用透射与扫描电子显微镜、免疫荧光染色及流式细胞术等技术对巨噬细胞肿瘤疫苗的超微结构及CD14、CD68、CD80、CD86、MHCⅡ等分子进行测定。采用生长状态良好的H22肿瘤细胞移植于接种不同肿瘤疫苗的实验小鼠,分别采用直接测量法、MTT法及比色法测定瘤重量、瘤体积、肿瘤细胞杀伤率和培养上清液LDH活性。结果：巨噬细胞肿瘤疫苗细胞表面有许多的伪足皱褶、囊泡,胞浆内有大量大小不一、形态不规则的吞噬体；CD14、CD68、CD80、CD86及MHCⅡ的阳性细胞率分别为53.90%、98.60%、26.50%、90.20%和25.40%。小鼠体内实验结果显示,巨噬细胞肿瘤疫苗接种组的肿瘤形成率、瘤体积与瘤重量明显低于对照组和石蜡诱生的巨噬细胞接种组（P均〈0.05）；巨噬细胞肿瘤疫苗接种组的成瘤率与灭活肿瘤细胞接种组无明显差异（P〉0.05）,但瘤体积与瘤重量明显低于灭活肿瘤细胞接种组（P均〈0.05）。另外,巨噬细胞肿瘤疫苗接种组的淋巴细胞自体肿瘤细胞杀伤率和培养上清液LDH活性分别高于对照组、灭活肿瘤细胞接种组和石蜡诱生的巨噬细胞接种组。结论：巨噬细胞肿瘤疫苗具备巨噬细胞的典型特征,该种细胞接种后可诱导机体产生特异性抗肿瘤免疫反应。     

表达鼠精子Cyritestin蛋白的减毒沙门氏菌疫苗株的构建及其鉴定

目的 构建表达鼠精子eyritestin蛋白的可口服减毒沙让氏菌活疫苗株。方法 应用质粒TAZ83／13，将之进行双酶切获得含有成熟cyritestin蛋白cDNA序列的酶切片段。将其插入表达载体yA3149中，构建重组质粒pCFL。将该重组质粒转入鼠沙门氏伤寒杆菌疫苗株X4632和X4550中，以获得重组菌株X4632（pCFL)和X4550（pCFL)。结果 该两种无抗药性的重组菌株在体外营养选择压力下，可较稳定地携带重组质粒传代繁殖。X4550(pCFL)在体内可较稳定地害居于Peyer‘s结，肠系膜淋巴结和脾脏；X4632（pCFL)可较稳定地定居于Peyer‘s结。二者均可表达被抗cyritestin单抗（mAb)识别的重组cyritestin蛋白。口服该疫苗菌株无明显的毒性作用。结论 X4632（pCFL),X4550（pCFL)疫苗株的构建，为研究以cyritestin为靶抗原的精子抗原避孕疫苗打下了基础。     

IL-15真核表达质粒的构建及对乙肝疫苗诱导的体液免疫应答的影响

目的 研究两种不同的IL-15真核表达质粒对乙肝蛋白疫苗诱导的免疫应答的影响。方法：构建IL-15真核表达质粒(简称pIL-15)和含有IL-12信号肽的IL-15真核表达质粒(简称pIL-2s-15)，CTLL-2细胞增殖实验验证两种质粒真核表达产物的生物学活性。将这两种质粒分别与HBsAg共免疫BALB／C小鼠，用ELISA法检测小鼠血清抗-HBs IgG及IgGl、IgG2a亚类的效价。结果：与HBsAg蛋白疫苗共免疫时，pIL-15可使HBsAg诱导的抗-HBsIgG效价升高，显著高于载体pcDNA3．1与HB—sAg共免疫对照组，pIL-2s-15对HBsAg诱导抗-HBsIgC效价没有明显影响。与HBsAg pcDNA3．1组相比，HBsAg pIL-2s-15组和HBsAg pIL-15组诱生的抗HBsIgG2a亚类均升高，但前者IgG2a／IgG1比值最高，与HBsAg pcDNA3．1组相比差别有显著性；HBsAg pIL-15组IgG2a／IgG1比值与HBsAg pcDNA3．1组相比差别无显著性。结论 pIL-15真核表达质粒可增强蛋白疫苗诱导的体液免疫应答，pIL-2s-15真核表达质粒则主要使免疫应答趋向Th1型。