The effect of anti-IFN-gamma antibody on the protective effect of Lyt-2+ immune T cells against toxoplasmosis in mice

The effect of an IFN-gamma mAb on the protective activity of immune T cells against Toxoplasma infection was examined in a murine model of toxoplasmosis. Mice that received anti-IFN-gamma antibody and immune spleen cells all died of toxoplasmosis after challenge with Toxoplasma tachyzoites. In contrast, mice that received normal IgG and immune spleen cells all survived the infection. The protective activity of Lyt-2+ immune T cells, previously shown to be the principal mediators of resistance against Toxoplasma in mice was completely ablated by the anti-IFN-gamma mAb. These results suggest that IFN-gamma is the major mediator of the resistance against Toxoplasma infection in mice which is conferred by immune T cells.     

Monoclonal IgM antibody protection in mice against infection with an encapsulated strain of Staphylococcus epidermidis.

Passive protective activities of three different classes of monoclonal antibodies in mice against challenge with strain ATCC 31432 (capsular type I) of Staphylococcus epidermidis were examined. Monoclonal IgM antibody passively protected mice against challenge with the homologous strain, whereas monoclonal IgG1 and IgG2b antibodies did not. The protective activity of IgM was absorbed by the cell surface antigen extracted from the homologous strain but not by the antigen from heterologous strains. Rapid reduction of viable cells took place in the peritoneal cavity of mice immunized with monoclonal IgM as early as 6 h after the challenge with the homologous strain. An enzyme-linked immunosorbent inhibition assay showed there was remarkable inhibition with the homologous cell surface antigen but not with heterologous preparations from other strains. Results suggest that in the mouse the major passive protection against the S. epidermidis strain is provided by the IgM antibody to the cell surface antigen.     

Specific cross-protective antigonococcal immunity in the murine genital tract

Specific acquired immunity to gonococci was studied in systemically immunized mice, challenged with 10(7) gonococci by intrauterine inoculation. Protection after intraperitoneal immunization was monitored by vaginal cultures taken 24 h post-challenge, since events during the first 24 h postexposure to gonococci are crucial in determining the outcome of infection. Mice were protected against gonococcal challenge by two inoculations with either live or boiled gonococci given 4 weeks apart, whereas immunization with one inoculation did not protect against challenge 1 week later. Protection was correlated with high titers of IgG antibody in serum after two immunizations, but not with the high titers of serum IgM antibody found after the one immunization. IgG antibodies, but not IgM antibodies, were shown to pass into genital secretions. Protection could be passively transferred by serum with high titers of antibody. Of most practical importance was the finding that not only were heat-stable antigens protective, but also heterologous protection resulted after immunization with three strains differing in source (disseminated gonococcal infection versus gonorrhea), opacity-transparency characteristics, and serum sensitivity. The data indicate that IgG antibodies resulting from systemic immunization with heat-stable antigens may be able to provide cross-protection immunity against gonorrhea.     

Reinvestigating the Role of IgM in Rabies Virus Postexposure Vaccination

B cells secreting IgG antibodies, but not IgM, are thought to be solely responsible for vaccine-induced protection against rabies virus (RABV) infections in postexposure settings. In this report, we reinvestigated the potential for IgM to mediate protection in a mouse model of RABV vaccination. Immunocompetent mice immunized with an experimental live replication-deficient RABV-based vaccine produced virus neutralizing antibodies (VNAs) within 3 days of vaccination. However, mice unable to produce soluble IgM (sIgM^−/−) did not produce VNAs until 7 days postimmunization. Furthermore, sIgM^−/− mice were not protected against RABV infection when challenged 3 days postimmunization, while all wild-type mice survived challenge. Consistent with the lack of protection against pathogenic RABV challenge, approximately 50- to 100-fold higher viral loads of challenge virus were detected in the muscle, spinal cord, and brain of immunized sIgM^−/− mice compared to control mice. In addition, IgG antibody titers in vaccinated wild-type and sIgM^−/− mice were similar at all time points postimmunization, suggesting that protection against RABV challenge is due to the direct effects of IgM and not the influence of IgM on the development of effective IgG antibody titers. In all, early vaccine-induced IgM can limit dissemination of pathogenic RABV to the central nervous system and mediate protection against pathogenic RABV challenge. Considering the importance for the rapid induction of VNAs to protect against RABV infections in postexposure prophylaxis settings, these findings may help guide the development of a single-dose human rabies vaccine.     

Intranasal vaccination with a double mutant of staphylococcal enterotoxin C provides protection against Staphylococcus aureus infection

Staphylococcus aureus expresses a repertoire of factors including staphylococcal exotoxins (SEs), exoenzymes, and numerous cell-associated components that contribute to the pathogenesis of disease. We constructed and expressed a nontoxic double mutant SEC (dmSEC), devoid of superantigenic activity, and investigated the ability of intranasal vaccination with dmSEC plus cholera toxin (CT) adjuvant to protect mice against S. aureus infection. Mice were vaccinated with dmSEC and inoculated with a viable S. aureus clinical isolate strain. The survival rate in the immunized mice was higher, and bacterial counts in the organs were significantly lower than those in the control group. Intranasal vaccination with dmSEC induced the production of SEC-specific antibodies such as IgG1, IgG2b and IgA. dmSEC-vaccinated mice elicited significantly higher titers of interleukin-4 (IL-4) and IL-10, and lower levels of interferon-gamma (IFN-gamma) after challenge with S. aureus compared with the control group. Furthermore, the sera from dmSEC-immunized mice significantly inhibited IFN-gamma and tumor necrosis factor-alpha production in vitro. These results indicate that intranasal vaccination with dmSEC devoid of superantigenic properties induces systemic immune responses and provides protection against S. aureus infection.     

Monoclonal antibodies to Salmonella lipopolysaccharide: anti-O-polysaccharide antibodies protect C3H mice against challenge with virulent Salmonella typhimurium

The present investigation reports the production of monoclonal antibodies to antigenic determinants of the O-polysaccharide of Salmonella typhimurium lipopolysaccharide (LPS), and assesses the effectiveness of these antibodies in protecting C3H mice against the lethal effects of Salmonella infection. Hybridomas were generated by fusing spleen cells from (BALB/c X A/J)F1 (CAF1) mice hyperimmunized by i.v. injection with acetone-killed S. typhimurium SR-11 with X63-Ag8.653 murine myeloma cells. Hybridomas producing antibodies reactive with S. typhimurium SR-11 whole cells were subcloned, and seven of the resulting clones as well as one previously described clone were selected for use in the studies reported here. Monoclonal antibodies from these eight clones were of the IgG1 (1), IgG3 (6), or IgM (1) isotype and were specific for the O-polysaccharide region of Salmonella LPS, reacting with LPS from smooth S. typhimurium SR-11 and LT-2, but not with LPS from rough S. minnesota R60 (Ra), R345 (Rb), or R595 (Re). The effectiveness of each monoclonal antibody in protecting C3H/HeN and C3H/HeJ mice against the lethal effects of Salmonella infection was evaluated by comparing the median length of survival of groups of mice given antibody by i.p. injection before i.p. challenge with virulent S. typhimurium SR-11 to that of animals that received no antibody. Three out of eight monoclonal anti-O-polysaccharide antibodies, ST-1 (IgM), 10-5-47 (IgG3), and 10-5-6 (IgG3), provided significant (p less than 0.01) protection to C3H/HeN mice challenged with approximately 10(4) LD100 of Salmonella. Only antibodies ST-1 and 10-5-6, however, extended the median length of survival of C3H/HeJ mice beyond that of infected controls. Mouse antiserum prepared against S. typhimurium SR-11 was equally protective in C3H/HeJ mice. In an attempt to understand the contribution of antibody specificity to the relative differences in the protective capacities of the monoclonal antibodies, their reactivities with several Salmonella reference strains of different classical serotypes were examined. Although some differences in reactivity against the different strains were apparent, this approach was not adequate for defining the fine specificity of these monoclonal antibodies. The results of this study provide evidence that monoclonal antibodies with specificity to the O-polysaccharide region of Salmonella LPS can protect C3H mice against challenge with the homologous bacterial strain.     

Vaccination with Staphylococcus aureus fibrinogen binding proteins (FgBPs) reduces colonisation of S. aureus in a mouse mastitis model

Abstract A mouse mastitis model was used to study the effect of vaccination with fibrinogen binding proteins and collagen binding protein from Staphylococcus aureus against challenge infection with S. aureus . The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibronogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. Histopathological examination of tissue sections from challenged glands showed that most glands from mice vaccinated with fibrinogen binding protein developed disseminated necrosis or had no pathological changes. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. In a similar study, immunization of mice with collagen binding protein did not induce protection against challenge infection with S. aureus .     

金黄色葡萄球菌isdb基因的克隆表达及其小鼠免疫试验

为了研究金黄色葡萄球菌表面Isdb蛋白的免疫原性,应用PCR方法扩增出金黄色葡萄球菌Wood46株的isdb 基因并进行序列分析,再将isdb 基因插入到pET32-a(+) 载体上,构建了pET32-a(+)-isdb重组质粒,将重组质粒转化到宿主菌大肠埃希菌BL21中并诱导表达和纯化Isdb蛋白。用纯化的Isdb蛋白免疫小鼠,检测小鼠血清中抗体水平;在二次免疫之后的第2周,用金黄色葡萄球菌Wood46、HLJ23-1株对小鼠攻毒,每组8只小鼠。研究结果发现：isdb基因在不同菌株中高度保守;Isdb蛋     

Role of endogenous tumor necrosis factor alpha and gamma interferon in resistance to Corynebacterium pseudotuberculosis infection in mice

The production and roles of endogenous tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) in the infection of Corynebacterium (C.) pseudotuberculosis were investigated in mice. The maximum levels of TNF-alpha and IFN-gamma were detected on day 4 after infection. The administration of anti-TNF-alpha monoclonal antibody (mAb) as well as anti-IFN-gamma mAb increased bacterial proliferation in the organs, leading to the death of infected mice, but anti-IFN-gamma mAb showed a less marked effect than anti-TNF-alpha mAb. The suppressive effect of anti-TNF-alpha and anti-IFN-gamma mAbs on anticorynebacterial resistance was augmented by the simultaneous administration of these antibodies. Anti-TNF-alpha mAb was found to be highly effective when administered on day 0 and day 4, suggesting that TNF-alpha produced during the early stage of infection is critical for the generation of resistance. Histologically, many microabscesses, severe follicular swelling and lymphocyte destruction were observed in mice treated with anti-TNF-alpha or anti-IFN-gamma mAb. Injection of anti-CD4 or anti-CD8 mAb also resulted in significantly increased mortality and a marked suppression of IFN-gamma production, but had no effect on TNF-alpha production. Carrageenan also showed a marked effect on the exacerbation of infection. Taken together, these results suggest that endogenously produced TNF-alpha and IFN-gamma are both essential to the host defense against C. pseudotuberculosis infection and that these cytokines may have an additive effect.     

Antibodies to immune response gene products inhibit the IgM and IgG antibody response but not the development of resistance to Toxoplasma gondii

We have examined the effects of a monoclonal antibody directed against immune response gene products on the appearance of antibodies and development of resistance to Toxoplasma gondii. In vivo administration of a single dose of anti-I-Ak antibody to C3H/He (H-2k) and not BALB/c (H-2d) mice suppressed both the IgM and IgG response to two different strains of Toxoplasma. Administration of anti-I-Ak antibody to mice 5 days before and 10 days after infection resulted in complete inhibition of IgM and a more pronounced inhibition of IgG response to Toxoplasma. Under these experimental conditions, development of resistance against a subsequent challenge with a virulent strain of Toxoplasma was not affected. The microbicidal and tumoricidal activities of macrophages obtained from anti-I-Ak-treated, Toxoplasma-infected mice and mice infected with Toxoplasma alone were equivalent. Mice treated with anti-I-Ak antibody demonstrated a decreased proliferative response to lipopolysaccharide, a B-cell mitogen. Enumeration of B-cell numbers in anti-I-Ak-treated mice revealed a pronounced decrease in B-cell counts.     

The in vivo antiviral activity of interleukin-12 is mediated by gamma interferon.

The injection of 20 ng of mouse interleukin-12 (IL-12) protects mice from a lethal infection with encephalomyocarditis virus. In vitro, an anti-gamma interferon (anti-IFN-gamma) monoclonal antibody but not an anti-IL-12 monoclonal antibody neutralizes the antiviral activity present in the supernatants of splenocytes stimulated with IL-12. Finally, IL-12 fails to protect 129 Sv/Ev IFN-gamma R0/0 mice against encephalomyocarditis virus infection. These results demonstrate that IL-12 exerts its antiviral activity through the induction of endogenous IFN-gamma.     

Role of L3T4+ and Lyt-2+ T cell subsets in protective immune responses of mice against infection with a low or high virulent strain of Toxoplasma gondii

In order to elucidate the role of T cell subsets in protective immunity against infection with high virulent and low virulent strains of Toxoplasma gondii, monoclonal antibodies specific for T cell subsets were injected into mice before immunization or challenge infection. Treatment of mice with monoclonal antibody to either L3T4+ or Lyt-2+ T cells before they were immunized with Toxoplasma cell homogenate prepared from high virulent RH strain tachyzoites markedly reduced survival after mice were challenged with low virulent bradyzoites of the Beverley strain. Thus, induction of protective immunity against bradyzoites of the Beverley strain requires the presence of both L3T4+ and Lyt-2+ T cells. In contrast, mice injected with living bradyzoites of the low virulent Beverley strain after immunization with Toxoplasma cell homogenate acquired protective immunity against high virulent tachyzoites of the RH strain. Lyt-2+ T cells alone appear to be final effector cells for protection against the challenge with high virulent RH strain tachyzoites, since treatment of the bradyzoite-immune mice with anti-Lyt-2 antibody, but not anti-L3T4 antibody, before challenge significantly increased mortality.     

Resistance to Streptococcus pneumoniae is induced by a phosphocholine-protein conjugate

Previous studies demonstrated that naturally occurring antibodies to the pneumococcal cell wall hapten phosphocholine (PC) are important for the survival of mice against infection with Streptococcus pneumoniae, and that passively administered hybridoma antibody to PC results in added resistance. To determine if a PC-protein conjugate could elicit protective levels of anti-PC antibody, mice were immunized with PC-keyhole limpet hemocyanin (KLH) and tested for their ability to resist challenge with virulent S. pneumoniae. PC-KLH-immunized mice were observed to be resistant to 10- to 1000-fold more organisms than unimmunized control animals. The levels of protection were comparable to those induced with capsular polysaccharide antigens, but had the advantage of not being type-specific; immunization with PC-KLH protected mice against both type 1 and type 3 organisms. The induced immunity appeared to be antibody-mediated; it could be passively transferred with immune serum, and absorption of the immune serum with PC-Sepharose removed its protective capacity. Anti-PC antibodies in the serum of immunized mice were primarily IgM and IgG3 and possessed predominantly the T15 idiotype. Antibodies with these particular isotypes and this idiotype also arise after immunization with heat-killed rough pneumococci and recently were shown to be important in the resistance of mice to pneumococcal infection.     

Endogenous IFN-gamma production is induced and required for protective immunity against pulmonary chlamydial infection in neonatal mice

Chlamydia trachomatis infection in neonates, not adults, has been associated with the development of chronic respiratory sequelae. Adult chlamydial infections induce Th1-type responses that subsequently clear the infection, whereas the neonatal immune milieu in general has been reported to be biased toward Th2-type responses. We examined the protective immune responses against intranasal Chlamydia muridarum challenge in 1-day-old C57BL/6 and BALB/c mice. Infected C57BL/6 pups displayed earlier chlamydial clearance (day 14) compared with BALB/c pups (day 21). However, challenged C57BL/6 pups exhibited prolonged deficits in body weight gain (days 12-30) compared with BALB/c pups (days 9-12), which correlated with continual pulmonary cellular infiltration. Both strains exhibited a robust Th1-type response, including elevated titers of serum antichlamydial IgG2a and IgG2b, not IgG1, and elevated levels of splenic C. muridarum-specific IFN-gamma, not IL-4, production. Additionally, elevated IFN-gamma, not IL-4 expression, was observed locally in the infected lungs of both mouse strains. The immune responses in C57BL/6 pups were significantly greater compared with BALB/c pups after chlamydial challenge. Importantly, infected mice deficient in IFN-gamma or IFN-gamma receptor demonstrated enhanced chlamydial dissemination, and 100% of animals died by 2 wk postchallenge. Collectively, these results indicate that neonatal pulmonary chlamydial infection induces a robust Th1-type response, with elevated pulmonary IFN-gamma production, and that endogenous IFN-gamma is important in protection against this infection. The enhanced IFN-gamma induction in the immature neonatal lung also may be relevant to the development of respiratory sequelae in adult life.     

Defective vaccine-induced immunity to Schistosoma mansoni in P strain mice. I. Analysis of antibody responses

Inbred P4 strain mice have previously been shown to be uniquely defective in their resistance to challenge infection induced by irradiated cercariae of Schistosoma mansoni. To assess whether the low levels of resistance developed by vaccinated P mice could be due to a defective antibody response, we compared the anti-schistosomulum antibody responses in vaccinated P animals with those occurring in vaccinated C57BL/6J (B6) mice, a strain that consistently develops high levels of resistance to challenge infection. Our results indicate that vaccinated P mice develop levels of total anti-schistosomulum antibodies that are significantly lower than those occurring in B6 mice for at least 15 wk after immunization, with the exception of the fifth week, at which time the responses are indistinguishable. Further analysis revealed that the defect in P strain antibody response occurs specifically in the IgM isotype and that specific IgM levels in P mice are less than one-half the levels in B6 mice at every time point examined. In contrast, no differences in total IgM immunoglobulins were evident when sera from normal (nonvaccinated) P and B6 mice were compared. P mouse anti-schistosomulum IgG antibody responses reached the same levels as those observed in B6 mice by 5 wk after vaccination. However, a much faster decay in IgG antibody levels occurred after this time point in P animals. No differences were observed when the levels of anti-schistosomulum antibodies occurring in each of the major IgG isotypes (IgG1, IgG2a, IgG2b, IgG3) were compared in sera from P and B6 mice vaccinated 4 wk previously. Similarly, vaccinated P and B6 mice were found to mount indistinguishable IgG anamnestic responses after challenge infection. Finally, no differences between vaccinated P and B6 mice were observed when immediate (30 min) skin test and mast cell degranulation responses to a soluble schistosome antigenic preparation were compared. The above findings suggest that P strain mice have a specific defect in their ability to mount IgM antibody responses after immunization with irradiated cercariae. The possible contribution of this defect in IgM response to the decreased resistance of vaccinated P mice to challenge infection is discussed.     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance.     

Involvement of endogenously synthesized interleukin (IL)-18 in the protective effects of IL-12 against pulmonary infection with Cryptococcus neoformans in mice

We previously demonstrated that interleukin (IL)-12 protected mice against fatal pulmonary infection with a highly virulent strain of Cryptococcus neoformans, which correlated well with the production of interferon (IFN)-gamma as well as IL-18 in the primary infected site. In the present study, we examined the role of endogenously synthesized IL-18 in IL-12-induced host resistance to this pathogen. There was little or no production of IFN-gamma and IL-18 both at mRNA and protein levels in lungs of mice infected with C. neoformans, while treatment with IL-12 induced a marked production of these cytokines. Caspase-1 mRNA was expressed in infected mice even without IL-12 treatment. Administration of neutralizing anti-IFN-gamma monoclonal antibody (mAb) clearly inhibited production of IFN-gamma and IL-18 induced by IL-12, while control IgG did not show such an effect. However, administration of IFN-gamma did not induce the production of both cytokines in infected mice, although tumor necrosis factor (TNF)-alpha and IFN-gamma-inducible protein (IP)-10 were synthesized by the same treatment. Finally, neutralizing anti-IL-18 antibody (Ab) significantly interfered with the production of IFN-gamma and elimination of the microorganism from the lung induced by IL-12 treatment. Furthermore, both IFN-gamma synthesis and host protection caused by IL-12 were profoundly diminished in IL-18 gene-disrupted mice. Considered collectively, our results indicated that host protection against C. neoformans induced by IL-12 involved endogenously synthesized IL-18 and that the production of IL-18 was mediated at least in part by endogenous IFN-gamma.     

Gamma 3 gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides. II. Increased susceptibility to fatal pneumococcal sepsis due to absence of anti-polysaccharide IgG3 is corrected by induction of anti-polysaccharide IgG1

Bacterial polysaccharides (PS) are type 2 T-independent Ags that elicit Abs restricted in isotype to IgM and predominantly IgG2 in humans and IgM, and IgG3 in mice. Humans with IgG2 subclass deficiency are susceptible to sinus and pulmonary infections with PS-encapsulated bacteria. We previously developed an IgG3-deficient mouse by disrupting the gamma3 H chain constant region gene via targeted mutagenesis. Mutant mice lacking IgG3 were backcrossed for 10 generations to wild-type (WT) BALB/c mice to generate BALB/c mice that have complete absence of IgG3. WT mice immunized with type 3 Streptococcus pneumoniae capsular PS made anti-PS IgM, IgG3, and small quantities of IgG1, which opsonized S. pneumoniae for killing by polymorphonuclear leukocytes. These mice were protected against death from lethal doses of type 3 S. pneumoniae. In contrast, IgG3(-/-) mice made similar titers of anti-PS IgM and IgG1 as WT mice but no IgG3, and had poorly opsonic sera with significantly increased mortality after S. pneumoniae challenge. Immunization of IgG3(-/-) mice with type 3 S. pneumoniae PS conjugated to carrier protein CRM(197)-elicited IgM and high-titer IgG1 Abs, restored serum opsonization, and gave protection from mortality after S. pneumoniae, challenge comparable to WT mice. We conclude that mice lacking the dominant IgG3 subclass made to bacterial PS are more susceptible to fatal S. pneumoniae sepsis than WT mice, but that IgG1 induced by a S. pneumoniae glycoconjugate can adequately protect against S. pneumoniae sepsis. This model suggests that IgG subclass of anti-PS Ab is an important component of immunity to encapsulated bacteria.     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。