Cell-mediated and humoral immune response to non-viable Mycoplasma pulmonis in mice enhanced by cross-linked ricin

The enhancing effect of cross-linked ricin (CL-ricin) on the cell-mediated and humoral immune response of mice to non-viable Mycoplasma pulmonis was studied. The cell-mediated immune response was evaluated by means of the delayed-type footpad swelling, and the humoral immune response by means of the indirect hemagglutination test. Mice pre-treated subcutaneously with non-viable M. pulmonis and CL-ricin showed significantly increased delayed-type footpad swelling when they were injected in the footpad with the same antigen 7 days later. Delayed-type footpad swelling was not detected in mice pre-treated only with non-viable M. pulmonis or CL-ricin followed by footpad injection with non-viable M. pulmonis. Injection of non-viable M. pulmonis in the footpad on Days 3, 7, 14, 21, 28, 35 and 42 after pre-treatment with non-viable M. pulmonis and CL-ricin resulted in significant footpad swelling. Delayed-type footpad swelling was transferred by intravenous injection of spleen cells from mice which had been pre-treated 7 days previously with non-viable M. pulmonis and CL-ricin into non-treated recipient mice. Intravenous injection of anti-mouse thymus cell serum into mice previously pre-treated with non-viable M. pulmonis and CL-ricin reduced the delayed-type footpad swelling significantly. Mice pre-treated subcutaneously with non-viable M. pulmonis and CL-ricin showed a marked increase in serum antibody titers compared with those that received non-viable M. pulmonis alone.     

Immunoreactive protein antigens of Mycoplasma pulmonis in experimentally infected rats

Mycoplasma pulmonis was cultured in modified Hayflick's medium, washed in 0.25 M NaCl, and solubilized by 2.5% sodium dodecyl sulfate. Protein antigens of M pulmonis separated by polyacrylamide-gel electrophoresis were blotted onto nitrocellulose strips. Specific-pathogen-free rats were inoculated intranasally with M pulmonis. The serum samples of these rats were obtained periodically and used to react with fractionated M pulmonis antigens which were fixed on the nitrocellulose strips. The antigen-antibody reactions were further recognized by 125I-labeled antiglobulin. Detection of immunoreactive antigens was obtained by autoradiography. Antibody response was not detected in serum obtained 7 days after rats were inoculated, and by 14 days, a slight response to several proteins was found. At 28 days after rats were inoculated, many immunoreactive antigens were detected. Generally, antibodies against antigens of moderate to low molecular weight appeared early in the infection, and antibodies against antigens of high molecular weight appeared late. Important immunoreactive antigens thus identified can readily be distinguished from more than 58 different M pulmonis antigens detectable by sodium dodecyl sulfate polyacrylamide-gel electrophoresis. The humoral antibody response was measured by an enzyme-linked immunosorbent assay. The immunoglobulin G antibodies were initially detected at low level at 7 days after rats were inoculated. These humoral antibody responses reached maximum by 28 days. The increase in serum antibody titer corresponded with numbers of immunoreactive antigens detected by immunoradio-binding assay. The information gained by this investigation may improve our understanding of the antigenicity of M pulmonis and the immune response of rats exposed to M pulmonis.     

Cell-mediated and humoral immune response to Mycoplasma hyopneumoniae in pigs enhanced by dextran sulfate

The effect of dextran sulfate (DS), known to be cytotoxic to macrophages, on the cell-mediated and humoral immune response to nonviable Mycoplasma hyopneumoniae in pigs was investigated. The cell-mediated immune response was determined by means of lymphocyte transformation a test, using uptake of [3H]thymidine in a microculture system and the humoral immune response by means of a microplate complement-fixation test. Peripheral blood lymphocytes from pigs vaccinated with nonviable M hyopneumoniae and DS incorporated substantially more [3H]thymidine than did those from pigs given Mycoplasma or DS alone. The transformation of lymphocytes from M hyopneumoniae-DS vaccinated pigs was enhanced when M hyopneumoniae cells used in the assay system were heated at 60 C for 30 minutes. Similarly prepared M flocculare and M hyorhinis cells also stimulated lymphocytes from M hyopneumoniae-DS vaccinated pigs, but not nearly as great as when M hyopneumoniae cells were used. The humoral antibody response and the cell-mediated immune response to nonviable M hyopneumoniae was markedly enhanced by DS. Pigs were vaccinated with nonviable M hyopneumoniae and/or DS 4 times and challenge exposed intratracheally with viable M hyopneumoniae. Pigs vaccinated with M hyopneumoniae and DS had less severe pneumonia than did nonvaccinated pigs.     

Testing the immunity to orthopoxviruses in the white mouse with vaccinia virus

An infection model was developed, which allows the study of humoral and cellular immune response mechanisms induced by Orthopox viruses in mice. The optimal infection route for neurovirulent vaccinia virus strains was investigated and resulted in well-defined clinical symptoms in non-immunized susceptible mice. Signs of disease were taken as a basis for comparison. Challenge infections by intracutaneous (i.c.) and intraperitoneal (i.p.) application of vaccinia strain Munich 1 (M1) appeared to be most suited to testing immunities of different status in vivo. Mice passively immunized with an anti-vaccinia immune serum survived intraperitoneal challenge infection with 4LD50/mouse. After an intracutaneous challenge infection with 10(4) TCID50/animal, however, they were fully susceptible. Mice immunized with live vaccinia virus showed a solid immunity to both intracutaneous and intraperitoneal challenge.     

猪囊虫病基因工程疫苗的体液与细胞免疫反应

为了探讨以 Ｉ Ｓ Ｃ Ｏ Ｍ 作佐剂的猪囊虫病基因工程疫苗的免疫机理,对其诱导的体液免疫与细胞免疫反应进行了测定。用上述疫苗免疫 ９ 头试验猪,采用间接 Ｅ Ｌ Ｉ Ｓ Ａ 检测体液免疫反应及通过淋巴细胞转化试验、 Ａ Ｎ Ａ Ｅ染色试验、 Ｅ玫瑰花环形成试验等检测细胞免疫反应;用该疫苗和铝胶苗分别免疫昆明小鼠各 ２０ 只,分别检测体液免疫反应和 Ｔ 淋巴细胞抑制／杀伤亚群的动态变化。体液免疫的检测结果显示,免疫后 ７ 天即出现抗体,２１ 天后抗体全部转阳,持续的时间不少于 １９３ 天,效价明显高于铝胶苗;细胞免疫检测结果显示,免疫猪外周血 Ｔ淋巴细胞转化率、 Ａ Ｎ Ａ Ｅ＋ 细胞和粗粒型 Ａ Ｎ Ａ Ｅ＋ 细胞、 Ｅ Ｒ Ｆ Ｃ和 Ｅａ Ｒ Ｆ Ｃ细胞显著升高,免疫小鼠 Ｔ淋巴细胞抑制／杀伤亚群显著升高;与铝胶苗及对照组比较,差异极显著。以上结果表明猪囊虫病基因工程疫苗可同时激发动物的体液免疫和细胞免疫反应,增强了机体的免疫调节功能及杀伤性 Ｔ 淋巴细胞功能。     

Induction of immune response in mice with a DNA vaccine encoding outer membrane protein (omp31) of Brucella melitensis 16M

Brucellosis causes serious economic losses to goat farmers by way of reproductive losses in the form of abortions and stillbirths. Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system. In this study, we evaluated the ability of DNA vaccine encoding the omp31 protein of Brucella melitensis 16M to induce cellular and humoral immune responses in mice. We constructed eukaryotic expression vectors called pTargeTomp31, encoding outer membrane protein (omp31) of B. melitensis 16M. pTargeTomp31 was injected intramuscularly three times, at 3-week intervals in groups of mice 6 weeks of age. pTargeTomp31 induced good antibody response in ELISA . pTargeTomp31 elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the omp31 antigen or B. melitensis 16M extract. We also demonstrate that animals immunized with this plasmid elicited a strong and long-lived memory immune response. Furthermore, pTargeTomp31 elicited a typical T-helper 1-dominated immune response in mice, as determined by immunoglobulin G isotype analysis. This vaccine also provided the moderate degree of protection to the mice. This study for the first time focuses on DNA immunization of a gene from B. melitensis. These results may lead to the development of a DNA-based vaccine for the control of brucellosis in goats.     

Ginsenoside Rg1 enhances immune response induced by recombinant Toxoplasma gondii SAG1 antigen

Ginsenoside, the most important component isolated from Panax ginseng, exhibits a variety of biological activities. Particularly, ginsenoside Rg1 is known to have immune-modulating activities such as increase of immune activity of T helper (Th) cells. In the present study, we evaluated the immunomodulatory potentials of the Rg1 at three dose levels on the cellular and humoral immune responses of ICR mice against T. gondii recombinant surface antigen 1 (rSAG1). ICR mice were immunized subcutaneously with 50 μg Rg1 alone, 100 μg rSAG1 alone or with 100 μg rSAG1 dissolved in saline containing ginsenoside Rg1 (10 μg, 50 μg or 100 μg). After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that the groups immunized with rSAG1 and Rg1 (50 μg, 100 μg) developed a high level of specific antibody responses against T. gondii rSAG1, a strong lymphoproliferative response, and significant levels of cytokine production, compared with the other groups. After lethal challenge, the mice immunized with the rSAG1 and Rg1 (50 μg, 100 μg) showed a significantly increased survival time compared with control mice which died within 6 days of challenge. Our data demonstrate that by addition of ginsenoside Rg1, the rSAG1 triggered a stronger humoral and cellular response against T. gondii, and that Rg1 is a promising vaccine adjuvant against toxoplasmosis, worth further development.     

Frequent exposure of mice to crude Brucella abortus proteins down-regulates immune response

Mice repeatedly immunized via the intraperitoneal route with a Brucella abortus antigen lost their ability to develop a strong in vitro lymphoproliferative response. This result correlates with a decreased tendency of the lymphoid population to produce interferon-gamma when stimulated in culture with the immunizing antigen. With respect to the humoral response, as the number of immunizations increased, the animals produced more specific immunoglobulin M and immunoglobulin G1 antibodies. It is postulated that the long-term exposure of an animal to Brucella antigen changes the nature of the immune response from a T-cell-mediated response to a humoral response favouring the establishment of the disease.     

牛源金黄色葡萄球菌黏附素纤连结合蛋白A分泌型DNA疫苗诱导小鼠免疫效应的研究

采用PCR方法对编码金黄色葡萄球菌黏附素纤连结合蛋白A(FnbpA)的A区基因片段进行了特异性扩增,构建了真核表达载体pVAX-SFn,转染BHK-21细胞后经ELISA可检测出分泌表达的FnbpA蛋白。将真核重组表达质粒肌肉注射C57BL/6小鼠,免疫后检测小鼠血清抗体效价、淋巴细胞增殖及对试验小鼠攻毒试验。结果表明,该重组表达载体诱导细胞和体液免疫应答的强度均明显超过对照组。对小鼠的攻毒试验结果提示该重组DNA经肌肉注射途径接种可对小鼠产生免疫保护。本试验的结果对该DNA疫苗今后在实际中的应用奠定了良好的试验基础。     

猪传染性胃肠炎病毒S基因核酸疫苗免疫小鼠体液免疫功能变化的研究

本实验对TGEV S基因核酸疫苗免疫后体液免疫功能变化进行了研究,同时比较了两种核酸疫苗单独免疫小鼠后体液免疫应答的变化,证明重组核酸疫苗诱导小鼠产生体液免疫应答。     

BALB/c鼠口服免疫猪水肿病大肠杆菌基因突变菌株的免疫效果

为了研究猪水肿病(ED)大肠杆菌SLT-Ⅱe基因突变菌株作为口服疫苗的免疫效果,本实验用已构建的猪ED大肠杆菌SLT-Ⅱe基因突变菌株口服免疫BALB/c小鼠,检测其血清中的IgG抗体及粪便和肠黏液中的slgA抗体水平,并进行淋巴细胞增殖检测及攻毒保护实验.结果表明该基因突变菌株具有良好的免疫性,能诱导小鼠体内产生IgG和sIgA抗体,并且能引起T淋巴细胞增殖反应.攻毒保护实验结果显示,口服免疫突变菌株能对小鼠提供良好的保护,保护率为75%(15/20).本研究结果证明,该大肠杆菌基因突变菌株在小鼠体内能激发体液免疫和细胞免疫反应,可作为猪ED口服疫苗的候选菌株.     

中国流行株HIV及其与细胞因子基因共表达核酸疫苗质粒的构建及表达

以表达中国流行株HIV-1gp120基因的核酸疫苗质粒pGP及共表达中国流行株HIV-1gp120基因与IFN基因的核酸疫苗质粒pG-PIFN转染BHK-21细胞,以间接免疫荧光鉴定其表达产物。将上述两种质粒经胫前肌注射BALB/c鼠,检测gp120抗体的产生情况及ConA和Lps诱导的T细胞增殖能力。结果显示,荧光显微镜下pGPIFN转染细胞可见绿色荧光而pGP转染细胞未见绿色荧光。pGPIFN与pGP均可刺激小鼠产生抗gp120抗体,协同注射gp120和IFN基因的小鼠产生的抗体滴度明显高于单独注射gp120的小鼠。pGPIFN免疫鼠对ConA和Lps诱导的T细胞增殖能力明显高于单独注射gp120的小鼠。实验结果表明,协同应用IFN基因可明显加强HIV-1gp120基收稿日期:2002-07-19基金项目:国家自然科学基金项目(3977066);国家杰出青年基金项目(39825119)作者简介:郭　焱(1963-),女,吉林省长春市人,长春中医学院基础医学部副教授,硕士,主要从事分子病毒学研究工作。因的免疫反应,为中国流行株HIV-1核酸疫苗的可行性提供了重要实验依据。     

Immune response in mice and cattle after immunization with a Boophilus microplus DNA vaccine containing bm86 gene

Plasmid pBMC2 encoding antigen Bm86 from a Colombian strain of cattle tick Boophilus microplus, was used for DNA-mediated immunization of BALB/c mice, employing doses of 10 and 50microg, delivered by intradermic and intramuscular routes. Anti-Bm86 antibody levels were significantly higher compared to control mice treated with PBS. In the evaluation of immunoglobulin isotypes, significant levels of IgG2a and IgG2b were observed in mice immunized with 50microg of pBMC2. Measurement of interleukine (IL) levels (IL-4, IL-5, IL-12(p40)) and interferon-gamma (IFN-gamma) in the sera of mice immunized with pBMC2 indicated high levels of IL-4 and IL-5, although there were also significant levels of IFN-gamma. Mice immunized with pBMC2 showed antigen-specific stimulation of splenocytes according to the incorporation of bromodeoxyuridine and IFN-gamma secretion. In all trials, mice injected intramuscularly with 50microg of pBMC2 presented the highest immune response. Moreover, cattle immunized with this DNA vaccine showed antibody production significantly different to the negative control. In conclusion, these results suggest the potential of DNA immunization with pBMC2 to induce humoral and cellular immune responses against B. microplus.     

Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas

Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freund's complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a detectable immune response in green turtles.     

Frequent Exposure of Mice to Crude Brucella abortus Proteins Down‐regulates Immune Response

Mice repeatedly immunized via the intraperitoneal route with a Brucella abortus antigen lost their ability to develop a strong in vitro lymphoproliferative response. This result correlates with a decreased tendency of the lymphoid population to produce interferon‐γ when stimulated in culture with the immunizing antigen. With respect to the humoral response, as the number of immunizations increased, the animals produced more specific immunoglobulin M and immunoglobulin G1 antibodies. It is postulated that the long‐term exposure of an animal to Brucella antigen changes the nature of the immune response from a T‐cell‐mediated response to a humoral response favouring the establishment of the disease.     

Fusion of C3d molecule with bovine rotavirus VP7 or bovine herpesvirus type 1 glycoprotein D inhibits immune responses following DNA immunization

The binding of the complement C3d molecule with receptors on B cells and/or follicular dendritic cells (FDCs) influences the induction of humoral immune responses. For example, C3d fused to an antigen has been shown to have a strong adjuvant effect on antibody production. We investigated the possibility that co-expression of antigen and C3d as a fusion protein could enhance antigen-specific immune responses, following plasmid immunization. One or two copies of murine C3d-cDNA, C3d or (C3d)(2), respectively, were cloned together with bovine rotavirus (BRV) VP7 or bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) genes. All constructs contained a signal peptide that resulted in the secretion of the expressed proteins. In vitro, the characterization of the chimeric proteins indicated that both VP7 and gD retained their antigenicity and the C3d remained biologically active. However, immunization with plasmids encoding VP7-C3d chimeras did not enhance rotavirus-specific antibody responses and the frequency of BRV-specific IFN-gamma secreting cells in the spleens were significantly lower in mice immunized with pVP7-(C3d)(2) when compared with mice immunized with plasmid encoding VP7. The same pattern of immune responses was observed for plasmids encoding gD-C3d. Both gD-specific antibody responses and the frequency of gD-specific IFN-gamma secreting cells were significantly lower in mice immunized with plasmid expressing gD-C3d chimeras when compared with mice immunized with plasmid encoding gD alone. These results indicate that co-expression of C3d with an antigen actually inhibit both humoral and cell-mediated antigen-specific immune responses.     

Immune response to Rhodococcus equi ATCC 33701-secreted proteins in mice and identification of immunogenic recombinant proteins by dot-blotting

Rhodococcus equi remains a significant pathogen, causing severe pneumonia in foals. The development of vaccines and serologic diagnosis could be greatly facilitated by studying the humoral immune response to this equine pathogen. In this study, a crude extract of R. equi ATCC 33701-secreted proteins combined with the Montanide® ISA70 adjuvant was found to be highly immunogenic in mice with the highest titer of 99,000 on day 42 after the first subcutaneous immunization. This immune response was dependent on the quantity of proteins injected and the presence of adjuvant. By dot-blotting, eight recombinant secreted proteins were identified to react strongly with sera from immunized mice. Of these eight proteins, four were detected as immunogenic only when administered in conjunction with adjuvant. This screening strategy led to the identification of promising new candidates for vaccine development.     

Partial inhibition of the humoral immune response of pigs after early postnatal immunization

The ability of young pigs to be immunized during the postnatal period was studied. Eight groups of pigs born on the same day from 3 sows were injected with hen egg-white lysozyme in Freund's incomplete adjuvant at days 0, 1, 2, 3, 4, 5, 7, and 10 after birth. The serum antibody titers were determined each week. Results indicated that pigs injected within the first 3 days of life exhibited a delay of 10 days in the appearance of the humoral antibodies, compared with the antibody response observed in pigs injected later. Serum antibody titers were markedly lower in the early immunized pigs. The secondary immune response was similar in all pigs. This partial inhibition is not directly linked to the corticoids present in the serum at the immunization day. Possible reasons for this impairment of the humoral immune response were reviewed.     

小鼠对猪囊虫抗原基因TS76的免疫应答

将猪囊虫抗原基因 TS76的真核表达型质粒 VTS76单独或与 p UC18联合肌肉免疫注射于 BAL B/ c小鼠 ,以MTT比色法检测小鼠脾淋巴细胞 Con A刺激的增殖反应及 IL - 2的诱生活性 ,EL ISA方法检测免疫小鼠 Ig G总量和特异性抗体水平 ,常规法检测外周血免疫细胞数量的动态变化。结果发现 ,VTS76免疫小鼠各项细胞和体液免疫应答反应指数均比空白对照组小鼠显著提高 ;联合免疫组小鼠的细胞免疫应答和体液免疫应答反应指数均比 VTS76小鼠提高。由此可见 ,以 VTS76免疫小鼠可诱导其特异性细胞和体液免疫反应 ,而 p UC18又明显提高了 VTS76免疫小鼠的免疫应答水平 ,具有显著的免疫增强作用