人源抗狂犬病毒G蛋白单链抗体的生物学鉴定及其小鼠体内中和活性测定

目的对从噬菌体抗体库中筛选的人源抗狂犬病毒单链抗体A12进行生物学活性鉴定及小鼠体内中和活性检测。方法用免疫荧光法检测其结合感染狂犬病毒CVS的鼠脑组织的能力,用小鼠中和实验测定A12表达产物的体内中和活性。结果免疫荧光试验显示A12表达产物与感染CVS的鼠脑细胞有强的荧光反应。小鼠中和试验结果表明,A12ScFv样品组小鼠有9只存活,而对照组小鼠全部死亡。A12在729倍稀释时能100%保护小鼠抵抗致死量狂犬病毒的脑内攻击。结论A12对狂犬病毒具有一定的中和活性,有可能被用于暴露后狂犬病的预防。     

河南省南阳地区黄牛狂犬病病毒的分离和鉴定

应用鼠脑传代,从具有神经症状的病牛脑组织中分离获得5株病毒,经电子显微镜观察呈典型弹状病毒样粒子。用抗狂犬病毒CVS(狂大病毒1型代表株)免疫血清作ELISA检测及小鼠中和试验,证明分离株病毒为狂大病毒。用狂犬病相关病毒Lagos bat(狂犬病毒2型)、Mokola(狂犬病毒3型)和Duvenhage(狂犬病毒4型)免疫血清对分离株病毒作交叉中和试验,结果发现分离株病毒与Duvenhage有较密切的抗原联系,而与Lagos bat及Mokola病毒没有抗原联系。用自制狂犬病毒“核蛋白”单克隆抗体作夹心间接ELISA检测分离株病毒的感染鼠脑均呈阳性反应,5株病毒与CVS及其互相之间没有明显差异。结论认为,由河南省黄牛分离的病毒为狂犬病毒。     

ELISA用于人血清抗狂犬病毒抗体的检测

为了检测狂犬疫苗免疫后的人血清中和抗体,国内外先后建立了很多种方法。七十年代以来,酶联免疫吸附试验(ELISA)由于具有快速、敏感、特异,重复性好等优点而得到较快发展。本文报告利用ELISA间接法检测狂犬疫苗免疫后人血清抗体的实验结果,并证明了ELISA效价与传统的小鼠中和试验保护指数有良好的一致性。 材料和方法 抗原:狂犬固定毒CVS毒株,经兔脑传代,取发病濒死兔脑经低速及超速离心,再经蔗糖密度梯度离心制备纯的狂犬病毒抗原。用于ELISA微孔板包被;抗原最适浓度经棋盘格式测定10μg/ml。     

狂犬病毒抗原和抗体快速检测法的应用

关于狂犬病毒抗原和抗体的检测法,常规使用的以小鼠脑内法和免疫荧光法居多,此两法虽被公认具有良好的实用价值,但也有费时,繁锁或需要一定设备等不足之处。ELISA法用于病毒病的诊断检测已被提出多年,并被证明方法简便,重复性强,用其取代小鼠法进行科研或流行病学调查是可行的。本文采用不同的ELISA系统检测人、马抗狂犬血清及研究狂犬病毒在Vero细胞上的生长繁殖动态,同时与经典的小鼠脑内法进行对比试验,获得一定结果。现将结果简述如下。     

Hca-F抗原-HSP70BCG疫苗免疫对小鼠肝癌Hca-F的免疫保护效应

目的探讨化学交联法构建的Hca-F抗原-卡介苗热休克蛋白70(HSP70BCG)复合物对Hca-F肝癌的主动免疫保护效应及其机制。方法采用ADP亲和层析方法提取HSP70BCG,超速离心法提取粗制肿瘤抗原,采用化学连接法或加入ADP构建Hca-F抗原-HSP70BCG复合物,免疫正常小鼠,3次后检测对Hca-F攻击的主动免疫保化效应,用四甲基偶氮唑蓝(MTT)法检测Hca-F刺激下的脾细胞增殖反应,用流式细胞仪检测脾细胞表型的变化。结果化学交联法和加入ADP构建的Hca-F抗原-HSP70BCG复合物免疫可延长小鼠的存活时间,增强脾细胞对抗原刺激的应答能力和细胞介导的细胞毒活性、提高γδT细胞的百分率。结论 Hca-F抗原-HSP70BCG复合物免疫可诱导抗瘤免疫保护效应。     

紫外线减毒弓形虫ZS1株滋养体在小鼠体内的细胞免疫反应

目的： 探讨紫外线减毒弓形虫ＺＳ１ 株在小鼠体内的免疫保护性和细胞免疫反应。方法： 用波长为２５３７°Ａ 的紫外线照射弓形虫ＺＳ１株滋养体, 照射高度为５ ｃｍ , 照射时间６０ ｍ ｉｎ。小鼠于免疫后４５ ｄ 用同株滋养体攻击感染, 攻击后４ ｄ 剖杀, 与单免疫组、单感染组及正常对照组小鼠比较其脾Ｔ淋巴细胞增殖反应及其亚群的变化。结果： 小鼠接种紫外线减毒弓形虫ＺＳ１ 株滋养体后能正常存活, 于接种后４９ ｄ 各组织未查见滋养体、包囊或假包囊; 免疫组攻击感染后存活时间较单感染组延长; 体外特异抗原刺激后, 可诱导免疫组及免疫攻击组强的脾Ｔ淋巴细胞增殖反应; 免疫攻击组ＣＤ４＋ Ｔ细胞明显下降, ＣＤ４＋ ／ＣＤ８＋ 比率倒置; 免疫组、免疫攻击组及感染组的ＮＫ细胞活性均明显增强。结论： 紫外线减毒弓形虫ＺＳ１株滋养体疫苗能够诱导免疫小鼠产生一定的抗攻击感染保护力, 其中ＣＤ８＋ Ｔ细胞和ＮＫ细胞可能发挥着重要作用。     

紫外线减毒弓形虫ZS1株滋养体在小鼠体内的细胞免疫反应

目的： 探讨紫外线减毒弓形虫ＺＳ１ 株在小鼠体内的免疫保护性和细胞免疫反应。方法： 用波长为２５３７°Ａ 的紫外线照射弓形虫ＺＳ１株滋养体, 照射高度为５ ｃｍ , 照射时间６０ ｍ ｉｎ。小鼠于免疫后４５ ｄ 用同株滋养体攻击感染, 攻击后４ ｄ 剖杀, 与单免疫组、单感染组及正常对照组小鼠比较其脾Ｔ淋巴细胞增殖反应及其亚群的变化。结果： 小鼠接种紫外线减毒弓形虫ＺＳ１ 株滋养体后能正常存活, 于接种后４９ ｄ 各组织未查见滋养体、包囊或假包囊; 免疫组攻击感染后存活时间较单感染组延长; 体外特异抗原刺激后, 可诱导免疫组及免疫攻击组强的脾Ｔ淋巴细胞增殖反应; 免疫攻击组ＣＤ４＋ Ｔ细胞明显下降, ＣＤ４＋ ／ＣＤ８＋ 比率倒置; 免疫组、免疫攻击组及感染组的ＮＫ细胞活性均明显增强。结论： 紫外线减毒弓形虫ＺＳ１株滋养体疫苗能够诱导免疫小鼠产生一定的抗攻击感染保护力, 其中ＣＤ８＋ Ｔ细胞和ＮＫ细胞可能发挥着重要作用。     

鼻腔接种伤寒杆菌Fe-SOD的抗体应答及对鼠伤寒杆菌攻击的免疫保护作用

目的　探讨鼻腔接种伤寒杆菌Fe SOD后血液和粘膜系统的抗体应答及免疫保护作用。方法　用IL - 1作为佐剂 ,将伤寒杆菌Fe SOD经鼻腔接种小鼠 ,检测小鼠血液及肠液中的抗体应答 ,并用鼠伤寒杆菌攻击小鼠 ,观察小鼠的存活情况 ,以确定免疫保护作用。结果　当用IL - 1作为佐剂时 ,经鼻腔接种伤寒杆菌Fe SOD的小鼠血液和肠液中可产生高水平特异性IgG和IgA ;而腹腔注射仅在血液中产生高水平特异性IgG。另外发现经鼻腔接种伤寒杆菌Fe SOD的小鼠在 2LD50 鼠伤寒杆菌攻击后 ,3d和 7d的存活率均明显高于对照组 (P <0 0 5 ) ,且以 5LD50 鼠伤寒杆菌攻击时 ,小鼠 7d的存活率明显高于对照组 (P <0 0 1)。结论　结果说明伤寒杆菌Fe SOD经鼻腔接种后可引起小鼠粘膜系统和血液中的抗体应答 ;对鼠伤寒杆菌的攻击可产生一定的免疫保护作用 ,也进一步说明了Fe SOD是沙门菌的共同保护性抗原。     

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。     

免疫酶组织化学染色法检测脑组织中的狂犬病毒抗原

荧光抗体试验(FA)直接检测脑组织、唾液腺或皮肤组织中的狂犬病毒抗原,可以确诊狂犬病。但FA法需要昂贵的荧光显微镜和一定的观察经验,难于普遍摧广应用。本文参照WHO推荐的实验程序,建立了检测狂犬病毒抗原的免疫酶组织化学染色法,检查了104例脑组织标本,并同FA法讲行了比较,取得了满意的结果,现报告如下。 材料和方法 一、标本 狂犬病毒CVS-7株(卫生部成都生物制品研究所提供)感染发病死亡的小鼠41只,狂犬     

Immunization against rabies with plant-derived antigen

We previously demonstrated that recombinant plant virus particles containing a chimeric peptide representing two rabies virus epitopes stimulate virus neutralizing antibody synthesis in immunized mice. We show here that mice immunized intraperitoneally or orally (by gastric intubation or by feeding on virus-infected spinach leaves) with engineered plant virus particles containing rabies antigen mount a local and systemic immune response. After the third dose of antigen, given intraperitoneally, 40% of the mice were protected against challenge infection with a lethal dose of rabies virus. Oral administration of the antigen stimulated serum IgG and IgA synthesis and ameliorated the clinical signs caused by intranasal infection with an attenuated rabies virus strain.     

A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection

To investigate the importance of the rabies virus (RV) glycoprotein (G) in protection against rabies, we constructed a recombinant RV (rRV) in which the RV G ecto- and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein (rRV-VSV-G). We were able to recover rRV-VSV-G and found that particle production was equal to rRV. However, the budding of the chimeric virus was delayed and infectious titers were reduced 10-fold compared with the parental rRV strain containing RV G. Biochemical analysis showed equal replication rates of both viruses, and similar amounts of wild-type and chimeric G were present in the respective viral particles. Additional studies were performed to determine whether the immune response against rRV-VSV-G was sufficient to protect against rabies. Mice were primed with rRV or rRV-VSV-G and challenged with a pathogenic strain of RV 12 days later. Similar immune responses against the internal viral proteins of both viruses indicated successful infection. All mice receiving the rRV vaccine survived the challenge, whereas immunization with rRV-VSV-G did not induce protection. The results confirm the crucial role of RV G in an RV vaccine.     

Interleukin 2 acts as an adjuvant to increase the potency of inactivated rabies virus vaccine.

Interleukin 2 (IL-2) occupies a central position in the cascade of events involved in the immune response. We were interested in determining whether IL-2 could function as an adjuvant to vaccination, to increase the immune response to vaccine immunogens. Using the National Institutes of Health test for rabies vaccine potency, we found that daily systemic administration of IL-2 in conjunction with inactivated rabies virus can increase the potency of vaccination in outbred mice at least 25-fold, as measured by survival following challenge with virulent rabies virus. Enhanced protection is not correlated with an increase in virus-neutralizing antibody titers, and we suggest that IL-2 acts to increase the cellular immune response to vaccination.     

Cellular and humoral immune responses to heat shock protein 65 are both involved in promoting fatty-streak formation in LDL-receptor deficient mice

OBJECTIVES: This study was designed to determine the role of cellular and humoral immune responses to heat shock protein 65 (HSP65) in murine atherosclerosis. BACKGROUND: Inflammatory processes appear to influence the progression of atherosclerosis. Immunization with HSP65 was previously shown to induce arteriosclerosis in rabbits and to enhance fatty-streak formation in mice. However, it has not been demonstrated directly whether HSP65-reactive antibodies and lymphocytes are separately capable of influencing lesion formation. METHODS: Low density lipoprotein-receptor deficient (LDL-RD) mice were immunized with HSP65 or control bovine serum albumin (BSA). Lymph-node cells, splenocytes and immunoglobulin G (IgG) were obtained from the immunized mice and transferred separately to six groups of syngenic LDL-RD mice. RESULTS: Adoptive transfer of HSP65-reactive lymph node cells increased fatty-streak formation in comparison with mice treated with BSA-primed cells. Similarly, transfer of splenocytes reactive with HSP65 led to enhanced fatty-streak generation compared with mice injected with BSA-sensitized splenocytes. Repeated intraperitoneal administration of IgG from serum of HSP65-immunized mice (every 10 days) enhanced fatty-streak formation in mice in comparison with their anti-BSA-IgG injected littermates. CONCLUSIONS: Antibodies and lymphocytes reactive to HSP65 promote fatty-streak formation in mice, providing direct evidence for the proatherogenic properties of cellular and humoral immunity to HSP65.     

Induction of protective immunity against rabies by immunization with rabies virus ribonucleoprotein.

We have studied the ability of rabies virus ribonucleoprotein (RNP) to induce a protective immune response in animals against lethal challenge with rabies and rabies-related lyssa viruses. Liposomes containing either RNP or the glycoprotein (G protein) of a variant virus with multiple alterations in the G antigenic structure conferred no or poor protection, respectively, against lethal intracerebral challenge with rabies virus. By contrast, liposomes containing RNP and the variant G protein induced a good protective response, comparable to that achieved with inactivated virus vaccine against intracerebral challenge. Moreover, mice or raccoons immunized with RNP alone resisted lethal peripheral challenge with homologous or heterologous virus strains. These results indicate that the RNP of rabies virus plays a crucial role in induction of protective immunity.     

Successful prophylaxis against rabies in mice and Rhesus monkeys: the interferon system and vaccine.

Addition of interferon to ineffective rabies virus vaccines by the local injection of either exogenous interferon or a potent interferon inducer (a complex of polyriboinosinic-polyribocytidylic acid containing poly-L-lysine and carboxymethylcellulose) into the footpads of mice previously challenged with rabies virus dramatically reduced the mortality rate. A significant reduction in mortality rate was also noted when the interferon system was administered to rhesus monkeys, but only when treatment was given 6 hr after challenge with rabies virus. Since the monkeys were given an overwhelming challenge of virus, the treatment had to be given quickly to obtain results comparable to those in mice.     

Protection against hepatitis B virus infection by immunization with hepatitis B core antigen

Although antibody to the hepatitis B surface antigen usually provides protection against hepatitis B virus (HBV) infection, recent reports indicate that this is not always the case. To study the possible role of immune responses to hepatitis B core antigen in immunity to HBV infection, chimpanzees were immunized with chimpanzee liver-derived or genetically cloned hepatitis B core antigen and later challenged with known infectious HBV. Two chimpanzees, which received liver-derived or cloned hepatitis B core antigen in Freund's adjuvant and developed hepatitis B core antibody and low-titer hepatitis B e antibody, were completely protected against HBV infection following challenge. In contrast, another chimpanzee, which received liver-derived hepatitis B core antigen without adjuvant, developed hepatitis B core antibody only in serum and had a subclinical HBV infection when challenged. These findings demonstrate that protection against HBV infection can be induced by immunization with hepatitis B core antigen in adjuvant and that protection, in this case, is not solely dependent on hepatitis B surface antibody. This fact has important implications in our understanding of the biology of HBV infection and in the design of future hepatitis B vaccines.     

Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene.

Inoculation of rabbits and mice with a vaccinia-rabies glycoprotein recombinant (V-RG) virus resulted in rapid induction of high concentrations of rabies virus-neutralizing antibodies and protection from severe intracerebral challenge with several strains of rabies virus. Protection from virus challenge also was achieved against the rabies-related Duvenhage virus but not against the Mokola virus. Effective immunization by V-RG depended on the expression of a rabies glycoprotein that registered proline rather than leucine as the eighth amino acid from its NH2 terminus (V-RGpro8). A minimum dose required for effective immunization of mice was 10(4) plaque-forming units of V-RGpro8 virus. beta-propiolactone-inactivated preparations of V-RGpro8 virus also induced high levels of rabies virus-neutralizing antibody and protected mice against intracerebral challenge with street rabies virus. V-RGpro8 virus was highly effective in priming mice to generate a secondary rabies virus-specific cytotoxic T-lymphocyte response following culture of lymphocytes with either ERA or PM strains of rabies virus.     

Highly attenuated rabies virus-based vaccine vectors expressing simian-human immunodeficiency virus89.6P Env and simian immunodeficiency virusmac239 Gag are safe in rhesus macaques and protect from an AIDS-like disease

We analyzed the safety and immunogenicity of attenuated rabies virus vectors expressing simian-human immunodeficiency virus (SHIV)-1(89.6P) Env or simian immunodeficiency virus (SIV)(mac239) Gag in rhesus macaques. Four test macaques were immunized with both vaccine constructs, and 2 control macaques received an empty rabies vector. Seroconversion against rabies virus glycoprotein (G) and SHIV(89.6P) Env was detected after the initial immunization, but no cellular responses against SHIV antigens were observed. HIV/SIV-specific immune responses were not enhanced by boosts with the same vectors. Therefore, we constructed vectors expressing SHIV(89.6P) Env and SIV(mac239) Gag in which the rabies G was replaced with the G protein of vesicular stomatitis virus (VSV). Two years after initial immunization, a boost with the rabies-VSV G vectors resulted in SIV/HIV-specific immune responses. Upon challenge with SHIV(89.6P) test macaques controlled the infection, whereas control macaques had high levels of viremia and a profound loss of CD4(+) T cells, with 1 control macaque dying of an AIDS-like disease.     

Immunization with a DNA plasmid encoding the SAG1 (P30) protein of Toxoplasma gondii is immunogenic and protective in rodents

Immunization with DNA can induce humoral and cell-mediated immune responses, both of which are important in conferring immunity to Toxoplasma gondii. The efficacy of genetic vaccination with a cDNA encoding the T. gondii SAG1 (P30) surface antigen was evaluated. Sera of immunized mice showed recognition of T. gondii tachyzoites by immunofluorescence and exhibited high titers of antibody to SAG1 by ELISA. SAG1-stimulated splenocytes from vaccinated mice produced primarily interferon-gamma and interleukin-2. Vaccinated mice survived challenge with 80 tissue cysts of ME49 strain, whereas all control mice died; challenge with 20 tissue cysts resulted in fewer brain cysts, compared with controls. Challenge of vaccinated rats with VEG strain oocysts resulted in a reduction in brain cysts. No protection was observed when mice were challenged with the highly virulent RH strain tachyzoites. These results suggest that nucleic acid vaccination can provide protection against T. gondii infection in mice.