Comparative immune response to PE and PE_PGRS antigens of Mycobacterium tuberculosis

Sequencing of the entire genome of Mycobacterium tuberculosis identified a novel multigene family composed of two closely related subfamilies designated PE and PE_PGRS. The major difference between these two families is the presence of a domain containing numerous Gly-Ala repeats extending to the C terminus of the PE_PGRS genes. We have used a representative PE_PGRS gene from M. tuberculosis, Rv1818c (1818PE_PGRS), and its amino-terminal PE region (1818PE), to investigate the immunological response to these proteins during experimental tuberculosis and following immunization with DNA constructs. During infection of mice with M. tuberculosis, a significant humoral immune response was observed against recombinant 1818PE_PGRS but not toward the 1818PE protein. Similarly, immunization with a 1818PE_PGRS DNA construct induced antibodies directed against 1818PE_PGRS but not against 1818PE proteins, and no humoral response was induced by 1818PE DNA. These results suggest that certain PE_PGRS genes are expressed during infection of the host with M. tuberculosis and that an antibody response is directed solely against the Gly-Ala-rich PGRS domain. Conversely, splenocytes from 1818PE-vaccinated mice but not mice immunized with 1818PE_PGRS secreted gamma interferon following in vitro restimulation and demonstrated protection in the mouse tuberculosis challenge model. These results suggest that the PE vaccine can elicit an effective cellular immune response and that immune recognition of the PE antigen is influenced by the Gly-Ala-rich PGRS domain.     

Immunological memory after somatic transgene immunization is positively affected by priming with GM-CSF and does not require bone marrow- derived dendritic cells

Inoculation of plasmid DNA is a promising vaccination approach but optimal regimes and ways to enhance immunogenicity remain to be established. Among natural immunological adjuvants, granulocyte-macrophage colony-stimulating factor (GM-CSF) was shown to increase the potency of immunization against tumor cells and protein antigens. Here we studied the effect of GM-CSF on memory responses against a 12-mer B cell epitope in mice primed with a single DNA inoculation. The results show that GM-CSF given at priming as a DNA/GM-CSF chimeric vaccine enhances the magnitude of the anamnestic response irrespective of the form of antigen used subsequently in the booster immunization. Using mice lacking bone marrow-derived dendritic cells we also determined that the enhancing effect is not strictly dependent on these cells. These results expand our understanding of the activity of GM-CSF in vivo as a modulator of the immune response including immunological memory.     

CD8(+) T cells participate in the memory immune response to Mycobacterium tuberculosis

The contribution of CD8(+) T cells to the control of tuberculosis has been studied primarily during acute infection in mouse models. Memory or recall responses in tuberculosis are less well characterized, particularly with respect to the CD8 T-cell subset. In fact, there are published reports that CD8(+) T cells do not participate in the memory immune response to Mycobacterium tuberculosis. We examined the CD8(+) T-cell memory and local recall response to M. tuberculosis. To establish a memory immunity model, C57BL/6 mice were infected with M. tuberculosis, followed by treatment with anti-mycobacterial drugs and prolonged rest. The lungs of memory immune mice contained CD4(+) and CD8(+) T cells with the cell surface phenotype characteristic of memory cells (CD69(low) CD25(low) CD44(high)). At 1 week postchallenge with M. tuberculosis via aerosol, > or =30% of both CD4(+) and CD8(+) T cells in the lungs of immune mice expressed the activation marker CD69 and could be restimulated to produce gamma interferon (IFN-gamma). In contrast, <6% of T cells in the lungs of naive challenged mice were CD69(+) at 1 week postchallenge, and IFN-gamma production was not observed at this time point. CD8(+) T cells from the lungs of both naive and memory mice after challenge were cytotoxic toward M. tuberculosis-infected macrophages. Our data indicate that memory and recall immunity to M. tuberculosis is comprised of both CD4(+) and CD8(+) T lymphocytes and that there is a rapid response of both subsets in the lungs following challenge.     

Gamma interferon-producing CD4+ T lymphocytes in the lung correlate with resistance to infection with Mycobacterium tuberculosis

The human immune system efficiently limits the replication of Mycobacterium tuberculosis in most infected individuals. Only 5 to 10% of infected people develop clinical tuberculosis, a sign of the inability of the immune system to control the infection. We have studied the C3H/HeJ (C3H) and C57BL/6 (B6) inbred mouse strains, which differ in their susceptibility to tuberculosis, in order to ascertain the immunological determinants of a successful immune response against M. tuberculosis and to establish a system to identify genes that influence susceptibility to tuberculosis. We found that the resistant B6 mice were able to control infection in both the lung and spleen, while susceptible C3H mice were incapable of limiting bacteria growth, especially in the lung, and succumbed to infection within 4 weeks. We determined that the susceptibility of C3H mice was independent of the Toll-like receptor 4 (tlr4) genetic locus and allelic major histocompatibility complex differences. Although the splenic immune responses were similar in the two mouse strains, the local immune responses in the lungs of the infected mice differed greatly. The pulmonary immune response in resistant B6 mice was characterized by an early influx of both CD4+ and CD8+ lymphocytes that produced gamma interferon (IFN-gamma). In contrast, the immune response of C3H mice in the lung was characterized by a delayed and decreased influx of lymphocytes, which produced little IFN-gamma. These results suggest an important role for the early appearance of IFN-gamma-producing lymphocytes in the lung in resistance to infection with M. tuberculosis.     

Effect of Mycobacterium tuberculosis chaperonins on bronchial eosinophilia and hyper-responsiveness in a murine model of allergic inflammation

BACKGROUND: Epidemiological evidence suggests that infection with Mycobacterium tuberculosis protects children against asthma. Several laboratories have shown that, in mouse models of allergic inflammation, administration of the whole live tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guerin (BCG), prevents ovalbumin (OVA)-induced pulmonary eosinophilia. OBJECTIVE: The aim of this study was to characterize specific M. tuberculosis molecules that are known to modulate immune responses to see if they affected pulmonary eosinophilia and bronchial hyper-responsiveness. METHODS: C57Bl/6 mice were sensitized to OVA on days 0 and 7 and subsequently challenged with OVA on day 14 over a 3-day period. Pulmonary eosinophilia and bronchial hyper-responsiveness were measured 24 h following the last antigen challenge. In some groups, mice were pre-treated with M. tuberculosis or M. tuberculosis chaperonins (Cpns)60.1, 60.2 and 10, and the effect of this treatment on the allergic inflammatory response to aerosolized OVA was established. RESULTS: We show that M. tuberculosis Cpns inhibit allergen-induced pulmonary eosinophilia in the mouse. Of the three Cpns produced by M. tuberculosis, Cpn60.1, Cpn10 and Cpn60.2, the first two are effective in preventing eosinophilia when administered by the intra-tracheal route. Furthermore, the increase in airways sensitivity to inhaled methacholine following OVA challenge of immunized mice was suppressed following treatment with Cpn60.1. The allergic inflammatory response was also characterized by an increase in Th2 cytokines IL-4 and IL-5 in bronchoalveolar lavage fluid, which was also suppressed following treatment with Cpn60.1. CONCLUSION: These data show that bacterial Cpns can suppress eosinophil recruitment and bronchial hyper-responsiveness in a murine model of allergic inflammation.     

Dissemination of Mycobacterium tuberculosis is influenced by host factors and precedes the initiation of T-cell immunity

We report that dissemination of Mycobacterium tuberculosis in the mouse is under host control and precedes the initiation of T-cell immunity. Nine to eleven days after aerosol inoculation, M. tuberculosis disseminates to the pulmonary lymph nodes (LN), where M. tuberculosis-specific T cells are detected 2 to 3 days thereafter. This indicates that the initial spread of bacteria occurs via lymphatic drainage and that the acquired T-cell immune response is generated in the draining LN. Dissemination to peripheral sites, such as the spleen and the liver, occurs 11 to 14 days postinfection and is followed by the appearance of M. tuberculosis-specific T cells in the lung and the spleen. In all cases studied, dissemination to the LN or the spleen preceded activation of M. tuberculosis-specific T cells in that organ. Interestingly, bacteria disseminate earlier from the lungs of resistant C57BL/6 mice than from the lungs of susceptible C3H mice, and consequently, C57BL/6 mice generate an immune response to M. tuberculosis sooner than C3H mice generate an immune response. Thus, instead of spreading infection, early dissemination of M. tuberculosis may aid in the initiation of an appropriate and timely immune response. We hypothesize that this early initiation of immunity following inoculation with M. tuberculosis may contribute to the superior resistance of C57BL/6 mice.     

The mycobacterial heparin-binding hemagglutinin is a protective antigen in the mouse aerosol challenge model of tuberculosis

The heparin-binding hemagglutinin (HBHA) of Mycobacterium tuberculosis is a surface-expressed adhesin that can affect binding to host cells via a unique, methylated, carboxyl-terminal, lysine-, alanine-, and proline-rich repeat region. It has been implicated in extrapulmonary dissemination of M. tuberculosis from the lung following the initial infection of the host. To assess the vaccine potential of this protein, purified preparations of HBHA were emulsified in a dimethyldioctadecylammonium bromide-monophosphoryl lipid A adjuvant and tested for the ability to reduce M. tuberculosis infection in the mouse aerosol challenge model for tuberculosis. The HBHA-containing vaccine gave a approximately 0.7-log reduction in CFU in both mouse lungs and spleens compared to adjuvant controls 28 days following challenge. Although a notable level of serum antibody to HBHA was elicited after three immunizations and the antibodies were able to bind to the surface of M. tuberculosis, passive immunization with monoclonal antibodies directed against HBHA did not protect in the challenge model. Compared to adjuvant controls, an elevated gamma interferon response was generated by splenic and lymph node-derived T cells from immunized mice in the presence of macrophages pulsed with purified HBHA or infected with live M. tuberculosis, suggesting that the effective immunity may be cell mediated. Efforts to construct effective recombinant HBHA vaccines in fast-growing Mycobacterium smegmatis have been unsuccessful so far, which indicates that distinctive posttranslational modifications present in the HBHA protein expressed by M. tuberculosis are critical for generating effective host immune responses. The vaccine studies described here demonstrate that HBHA is a promising new vaccine candidate for tuberculosis.     

Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 T-cell epitope mapping and role of gamma interferon

There is an urgent need for an efficacious vaccine against tuberculosis (TB). Cellular immune responses are key to an effective protective response against TB. Recombinant adenovirus (rAd) vectors are especially suited to the induction of strong T-cell immunity and thus represent promising vaccine vehicles for the prevention of TB. We have previously reported on rAd vector serotype 35, the serotype of choice due to low preexisting immunity worldwide, which expresses a unique fusion protein of Mycobacterium tuberculosis antigens Ag85A, Ag85B, and TB10.4 (Ad35-TBS). Here, we demonstrate that Ad35-TBS confers protection against M. tuberculosis when administered to mice through either an intranasal or an intramuscular route. Histological evaluation of lung tissue corroborated the protection and, in addition, demonstrated differences between two mouse strains, with diffuse inflammation in BALB/c mice and distinct granuloma formation in C57BL/6 mice. Epitope mapping analysis in these mouse strains showed that the major T-cell epitopes are conserved in the artificial fusion protein, while three novel CD8 peptides were discovered. Using a defined set of T-cell epitopes, we reveal differences between the two mouse strains in the type of protective immune response, demonstrating that different antigen-specific gamma interferon (IFN-gamma)-producing T cells can provide protection against M. tuberculosis challenge. While in BALB/c (H-2(d)) mice, a dominant CD8 T-cell response was detected, in C57BL/6 (H-2(b)) mice, more balanced CD4/CD8 T-cell responses were observed, with a more pronounced CD4 response in the lungs. These results unify conflicting reports on the relative importance of CD4 versus CD8 T-cell responses in protection and emphasize the key role of IFN-gamma.     

A DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis

The Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine has variable efficacy for both human and bovine tuberculosis. There is a need for improved vaccines or vaccine strategies for control of these diseases. A recently developed prime-boost strategy was investigated for vaccination against M. bovis infection in mice. BALB/c and C57BL/6 mice were primed with a DNA vaccine, expressing two mycobacterial antigens, ESAT-6 and antigen 85 A and boosted with attenuated M. bovis strains, BCG or WAg520, a newly attenuated strain, prior to aerosol challenge. Before challenge, the antigen-specific production of interferon-gamma (IFN-gamma) was evaluated by ELISPOT and antibody responses were measured. The prime-boost stimulated an increase in the numbers of IFN-gamma producing cells compared with DNA or live vaccination alone, but this varied according to the attenuated vaccine strain, time of challenge and the strain of mouse used. Animals vaccinated with DNA alone generated the strongest antibody response to mycobacterial antigens, which was predominantly IgG1. BCG and WAg520 alone generally gave a 1-2 log10 reduction in bacterial load in lungs or spleen, compared to non-vaccinated or plasmid DNA only control groups. The prime-boost regimen was not more effective than BCG or WAg520 alone. These observations demonstrate the comparable efficacy of BCG and WAg520 in a mouse model of bovine tuberculosis. However, priming with the DNA vaccine and boosting with an attenuated M. bovis vaccine enhanced IFN-gamma immune responses compared to vaccinating with an attenuated M. bovis vaccine alone, but did not increase protection against a virulent M. bovis infection.     

Humanized NOG mice as a model for tuberculosis vaccine‐induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4⁺ and CD8⁺ T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG‐C, a liposome‐based formulation containing the M. tuberculosis antigen ESAT‐6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T‐cell response. Humanized mice provide a crucial pre‐clinical platform for evaluating human T‐cell immune responses in vaccine development against M. tuberculosis.     

Immunological responses and protective immunity against tuberculosis conferred by vaccination of Balb/C mice with the attenuated Mycobacterium tuberculosis (phoP) SO2 strain

The Mycobacterium tuberculosis phoP mutant strain SO2 has been shown previously to be more attenuated than Mycobacterium bovis bacillus Calmette-Guérin (BCG) and confers protective immunity against tuberculosis in mice and guinea pig models. In this study we have investigated the survival and immunological responses of Balb/c mice infected with the M. tuberculosis SO2 strain. All Balb/C mice survived intratracheal infection with M. tuberculosis SO2 strain under conditions where all the mice infected with the parental M. tuberculosis MT103 had died after 9 weeks. Infection of Balb/c mice with M. tuberculosis SO2 was associated with comparatively lower levels of interferon (IFN)-gamma, interleukin (IL)-4 and tumour necrosis factor (TNF)-alpha and higher levels of inducible nitric oxide synthase (iNOS) during the late stage of infection, when compared with M. tuberculosis MT103 infection. The delayed-type hypersensitivity (DTH) response against M. tuberculosis culture filtrates was similar in mice infected with either the M. tuberculosis phoP SO2 strain or M. tuberculosis MT103. The protective efficacy of M. tuberculosis SO2 was compared with M. bovis BCG when delivered subcutaneously to groups of Balb/C mice. Following intratracheal challenge with M. tuberculosis H37Rv, protection was generated by 60 days post-challenge in mice vaccinated with either vaccine. At day 120 post-challenge the levels of protection were still significantly greater when compared with the non-vaccinated control group. The levels of protection conferred by vaccination with M. tuberculosis SO2 or with M. bovis BCG were similar, as measured by granuloma coalescence and pneumonia in addition to growth reduction of M. tuberculosis H37Rv.     

Toll-like receptor 4-defective C3H/HeJ mice are not more susceptible than other C3H substrains to infection with Mycobacterium tuberculosis

Mycobacterium tuberculosis produces a variety of molecules capable of activating Toll-like receptors, a family of pattern recognition receptors expressed by macrophages and a variety of other cells. To determine whether Toll-like receptor 4 (TLR4) was critical in resistance to M. tuberculosis infection, we compared the morbidity and mortality of TLR4-defective C3H/HeJ mice to those of TLR4-sufficient C3H mouse substrains. TLR4-defective C3H/HeJ mice and TLR4-sufficient C3H/HeSnJ, C3HeB/FeJ, and C3H/HeOuJ mice were infected by the aerosol route with M. tuberculosis. TLR4-defective C3H/HeJ mice had levels of cytokines in their bronchoalveolar lavage fluids and in vitro mycobacterial antigen-specific recall responses similar to those of other C3H mouse substrains. In addition, bacterial replication and long-term survival of mice following infection appeared to be independent of TLR4. Interestingly, C3HeB/FeJ mice were significantly more susceptible to M. tuberculosis infection, indicating that genetic heterogeneity among inbred C3H mouse substrains modifies resistance to infection. Therefore, cautious interpretation is required when the C3H/HeJ strain is used as a model of a TLR4-defective mouse strain, as there are significant allelic differences between C3H/HeJ and other C3H mouse substrains in response to M. tuberculosis infection. With this caveat, our data indicate that TLR4 may not be required for optimal immunity of mice to M. tuberculosis.     

Immunologic Memory Induced by a Glycoconjugate Vaccine in a Murine Adoptive Lymphocyte Transfer Model

We have developed an adoptive cell transfer model in mice to study the ability of a glycoprotein conjugate vaccine to induce immunologic memory for the polysaccharide moiety. We used type III capsular polysaccharide from the clinically relevant pathogen group B streptococci conjugated to tetanus toxoid (GBSIII-TT) as our model vaccine. GBS are a major cause of neonatal infections in humans, and type-specific antibodies to the capsular polysaccharide protect against invasive disease. Adoptive transfer of splenocytes from mice immunized with the GBSIII-TT conjugate vaccine conferred anti-polysaccharide immunologic memory to naive recipient mice. The transfer of memory occurred in a dose-dependent manner. The observed anamnestic immune response was characterized by (i) more rapid kinetics, (ii) isotype switching from immunoglobulin M (IgM) to IgG, and (iii) 10-fold-higher levels of type III-specific IgG antibody than for the primary response in animals with cells transferred from placebo-immunized mice. The adoptive cell transfer model described in this paper can be used for at least two purposes: (i) to evaluate conjugate vaccines with different physicochemical properties for their ability to induce immunologic memory and (ii) to study the cellular interactions required for an immune response to these molecules.     

Protection and Long-Lived Immunity Induced by the ID93/GLA-SE Vaccine Candidate against a Clinical Mycobacterium tuberculosis Isolate

Mycobacterium tuberculosis HN878 represents a virulent clinical strain from the W-Beijing family, which has been tested in small animal models in order to study its virulence and its induction of host immune responses following infection. This isolate causes death and extensive lung pathology in infected C57BL/6 mice, whereas lab-adapted strains, such as M. tuberculosis H37Rv, do not. The use of this clinically relevant isolate of M. tuberculosis increases the possibilities of assessing the long-lived efficacy of tuberculosis vaccines in a relatively inexpensive small animal model. This model will also allow for the use of knockout mouse strains to critically examine key immunological factors responsible for long-lived, vaccine-induced immunity in addition to vaccine-mediated prevention of pulmonary immunopathology. In this study, we show that the ID93/glucopyranosyl lipid adjuvant (GLA)-stable emulsion (SE) tuberculosis vaccine candidate, currently in human clinical trials, is able to elicit protection against M. tuberculosis HN878 by reducing the bacterial burden in the lung and spleen and by preventing the extensive lung pathology induced by this pathogen in C57BL/6 mice.     

CD4(+) T-cell subsets that mediate immunological memory to Mycobacterium tuberculosis infection in mice

We have studied CD4(+) T cells that mediate immunological memory to an intravenous infection with Mycobacterium tuberculosis. The studies were conducted with a mouse model of memory immunity in which mice are rendered immune by a primary infection followed by antibiotic treatment and rest. Shortly after reinfection, tuberculosis-specific memory cells were recruited from the recirculating pool, leading to rapidly increasing precursor frequencies in the liver and a simultaneous decrease in the blood. A small subset of the infiltrating T cells was rapidly activated (<20 h) and expressed high levels of intracellular gamma interferon and the T-cell activation markers CD69 and CD25. These memory effector T cells expressed intermediate levels of CD45RB and were heterogeneous with regard to the L-selectin and CD44 markers. By adoptive transfer into nude mice, the highest level of resistance to a challenge with M. tuberculosis was mediated by CD45RB(high), L-selectin(high), CD44(low) cells. Taken together, these two lines of evidence support an important role for memory cells which have reverted to a naive phenotype in the long-term protection against M. tuberculosis.     

Iccosomes and the secondary antibody response.

Iccosomes derived from follicular dendritic cells (FDC) are believed to play an important role in dispersion of antigen necessary for induction of anamnestic responses. Because FDC are aberrant and iccosome release has not been observed in aged mice, we hypothesized that these animals would be impaired in their ability to mount anamnestic responses. To test this, anamnestic responses were compared in aged and control mice. To ensure the presence of functional lymphocytes, some aged mice were reconstituted with T- and B-memory cells obtained from control mice. Anamnestic responses in aged mice were markedly depressed even when given functional memory cells. To more directly relate the impaired antibody response of aged mice to FDC function, antigen-bearing FDC from either aged or control mice were incubated with T- and B-memory cells from control mice to induce an anamnestic response. Antigen retained by FDC from aged mice was much less immunogenic than antigen retained by FDC from control mice. Since iccosome formation does not appear in aged mice, iccosome-like fragments were generated by sonicating FDC from aged mice and tested for their ability to induce an anamnestic response. This procedure restored the ability of antigen retained on FDC from aged mice to induce a normal anamnestic response. These data support the concept that the inability to form and disperse iccosomes contributes to the impaired ability of aged mice to mount anamnestic antibody responses and provides further support for the role of iccosomes in anamnestic responses.     

Interleukin-6 regulates the phenotype of the immune response to a tuberculosis subunit vaccine

We investigated the role of interleukin-6 (IL-6) in the development of the immune response to a subunit vaccine against tuberculosis consisting of the culture filtrate proteins of Mycobacterium tuberculosis emulsified in the adjuvant dimethyldioctadecylammonium bromide (DDA). C57Bl/6 mice immunized with this vaccine developed a strong T helper 1 (Th1) response characterized by an increased production of interferon-gamma (IFN-gamma) secreted by CD4+ T cells. Neutralization of IL-6 during in vivo priming resulted in marked reduction in the ability of T cells to secrete IFN-gamma and IL-2 and to proliferate. IL-6 gene-disrupted mice primed with the vaccine showed a decrease in the number of IFN-gamma-producing cells and an increase in IL-4-secreting cells as compared to control mice. In contrast, neutralization of IL-6 during a boost of the vaccine in previously primed mice did not affect the development of IFN-gamma-producing cells but still increased the number of IL-4-producing cells. Our work shows that IL-6 plays a major role in the priming but not in the later expression of a Th1 response to a tuberculosis vaccine.     

结核分枝杆菌Ag85B基因疫苗免疫保护作用的初步研究

目的:研究编码结核分枝杆菌分泌蛋白Ag85B的基因疫苗pTB30m和pTB30s对免疫动物的保护作用。方法:以基因疫苗pTB30m和pTB30s肌注免疫BALB/c小鼠。免疫完成6 wk后,用5×105 CFU的MTB H37Rv毒株经小鼠尾静脉攻击感染。同时用尼龙毛柱分离基因免疫BALB/c小鼠的T细胞,并以5×106 T细胞/只小鼠过继免疫正常BALB/c小鼠,立即用105 CFU的MTB毒株经小鼠尾静脉攻击感染。4 wk后分别计数脾脏中的细菌负荷。结果:与生理盐水对照组相比较,pTB30m及pTB30s质粒免疫组BALB/c小鼠脾脏中的细菌负荷均减少,分别为0.645(log10 CFU,P<0.01)和0.839(log10CFU,P<0.001);而空质粒对照组小鼠脾脏中的细菌负荷减少较少。经质粒pTB30m和pTB30s免疫的BALB/c小鼠的T细胞,过继免疫的正常BALB/c小鼠,对攻击感染的MTB H37Rv毒株在脾脏中的增殖具有部分抑制作用。结论:pTB30s免疫的BALB/c小鼠,对MTB H37 Rv毒株攻击的保护作用优于pTB30m质粒免疫,有望进一步用于结核病的防治研究。     

The recall response induced by genital challenge with Chlamydia muridarum protects the oviduct from pathology but not from reinfection

The significant morbidities of ectopic pregnancy and infertility observed in women after Chlamydia trachomatis genital infection result from ascension of the bacteria from the endocervix to the oviduct, where an overly aggressive inflammatory response leads to chronic scarring and Fallopian tube obstruction. A vaccine to prevent chlamydia-induced disease is urgently needed. An important question for vaccine development is whether sterilizing immunity at the level of the oviduct is essential for protection because of the possibility that a chlamydial component drives a deleterious anamnestic T cell response upon oviduct reinfection. We show that mice inoculated with attenuated plasmid-cured strains of Chlamydia muridarum are protected from oviduct pathology upon challenge with wild-type C. muridarum Nigg despite induction of a response that did not prevent reinfection of the oviduct. Interestingly, repeated abbreviated infections with Nigg also elicited recall responses that protected the oviduct from pathology despite low-level reinfection of this vulnerable tissue site. Challenged mice displayed significant decreases in tissue infiltration of inflammatory leukocytes with marked reductions in frequencies of neutrophils but significant increases in frequencies of CD4 Th1 and CD8 T cells. An anamnestic antibody response was also detected. These data indicate that exposure to a live attenuated chlamydial vaccine or repeated abbreviated genital infection with virulent chlamydiae promotes anamnestic antibody and T cell responses that protect the oviduct from pathology despite a lack of sterilizing immunity at the site.     

Cloning of the gene encoding a 22-kilodalton cell surface antigen of Mycobacterium bovis BCG and analysis of its potential for DNA vaccination against tuberculosis

Using spleen cells from mice vaccinated with live Mycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687-2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage lambdagt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test.