Enhanced effect of BCG vaccine against pulmonary Mycobacterium tuberculosis infection in mice with lung Th17 response to mycobacterial heparin‐binding hemagglutinin adhesin antigen

Although the BCG vaccine can prevent tuberculosis (TB) in infants, its ability to prevent adult pulmonary TB is reportedly limited. Therefore, development of a novel effective vaccine against pulmonary TB has become an international research priority. We have previously reported that intranasal vaccination of mice with a mycobacterial heparin‐binding hemagglutinin adhesin (HBHA) plus mucosal adjuvant cholera toxin (CT) enhances production of IFN‐γ and anti‐HBHA antibody and suppresses extrapulmonary bacterial dissemination after intranasal infection with BCG. In the present study, the effects of intranasal HBHA + CT vaccine on murine pulmonary Mycobacterium tuberculosis (Mtb) infection were examined. Intranasal HBHA + CT vaccination alone failed to reduce the bacterial burden in the infected lung. However, a combination vaccine consisting of s.c. BCG priming and an intranasal HBHA + CT booster significantly enhanced protective immunity against pulmonary Mtb infection on day 14 compared with BCG vaccine alone. Further, it was found that intranasal HBHA + CT vaccine enhanced not only IFN‐γ but also IL‐17A production by HBHA‐specific T cells in the lung after pulmonary Mtb infection. Therefore, this combination vaccine may be a good candidate for a new vaccine strategy against pulmonary TB.     

O-mannosylation of the Mycobacterium tuberculosis Adhesin Apa Is Crucial for T Cell Antigenicity during Infection but Is Expendable for Protection

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.     

Protective and therapeutic efficacy of Mycobacterium smegmatis expressing HBHA-hIL12 fusion protein against Mycobacterium tuberculosis in mice

Tuberculosis (TB) remains a major worldwide health problem. The only vaccine against TB, Mycobacterium bovis Bacille Calmette-Guerin (BCG), has demonstrated relatively low efficacy and does not provide satisfactory protection against the disease. More efficient vaccines and improved therapies are urgently needed to decrease the worldwide spread and burden of TB, and use of a viable, metabolizing mycobacteria vaccine may be a promising strategy against the disease. Here, we constructed a recombinant Mycobacterium smegmatis (rMS) strain expressing a fusion protein of heparin-binding hemagglutinin (HBHA) and human interleukin 12 (hIL-12). Immune responses induced by the rMS in mice and protection against Mycobacterium tuberculosis (MTB) were investigated. Administration of this novel rMS enhanced Th1-type cellular responses (IFN-γ and IL-2) in mice and reduced bacterial burden in lungs as well as that achieved by BCG vaccination. Meanwhile, the bacteria load in M. tuberculosis infected mice treated with the rMS vaccine also was significantly reduced. In conclusion, the rMS strain expressing the HBHA and human IL-12 fusion protein enhanced immunogencity by improving the Th1-type response against TB, and the protective effect was equivalent to that of the conventional BCG vaccine in mice. Furthermore, it could decrease bacterial load and alleviate histopathological damage in lungs of M. tuberculosis infected mice.     

Targeting of Mycobacterium tuberculosis heparin-binding hemagglutinin to mitochondria in macrophages

Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA), a virulence factor involved in extrapulmonary dissemination and a strong diagnostic antigen against tuberculosis, is both surface-associated and secreted. The role of HBHA in macrophages during M. tuberculosis infection, however, is less well known. Here, we show that recombinant HBHA produced by Mycobacterium smegmatis effectively induces apoptosis in murine macrophages. DNA fragmentation, nuclear condensation, caspase activation, and poly (ADP-ribose) polymerase cleavage were observed in apoptotic macrophages treated with HBHA. Enhanced reactive oxygen species (ROS) production and Bax activation were essential for HBHA-induced apoptosis, as evidenced by a restoration of the viability of macrophages pretreated with N-acetylcysteine, a potent ROS scavenger, or transfected with Bax siRNA. HBHA is targeted to the mitochondrial compartment of HBHA-treated and M. tuberculosis-infected macrophages. Dissipation of the mitochondrial transmembrane potential (ΔΨ(m)) and depletion of cytochrome c also occurred in both macrophages and isolated mitochondria treated with HBHA. Disruption of HBHA gene led to the restoration of ΔΨ(m) impairment in infected macrophages, resulting in reduced apoptosis. Taken together, our data suggest that HBHA may act as a strong pathogenic factor to cause apoptosis of professional phagocytes infected with M. tuberculosis.     

Vaccination with the T cell antigen Mtb 8.4 protects against challenge with Mycobacterium tuberculosis

The development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest. Mounting evidence suggests that protective immunity to M. tuberculosis relies on both MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells. By purifying polypeptides present in the culture filtrate of M. tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w. immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4. Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guérin as well as in mice infected i.v. with M. tuberculosis. More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4(+) T cell and CD8(+) CTL responses and induced protection on challenge with virulent M. tuberculosis. Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.     

In vivo depletion of CD11c+ cells delays the CD4+ T cell response to Mycobacterium tuberculosis and exacerbates the outcome of infection

Although dendritic cells (DC) are potent APC that prime T cells against many pathogens, there is no direct evidence that DC are required for immunity to Mycobacterium tuberculosis (Mtb) infection. The requirement for DC to prime the CD4+ T cell response following Mtb infection was investigated using pCD11c-diptheria toxin receptor/GFP transgenic mice, in which DC can be transiently ablated in vivo. We show a critical role for DC in initiation of the CD4+ T cell response to the mycobacterial Ag early secretory Ag of tuberculosis 6. The delay in initiating the Ag-specific T cell response led to impaired control of Mtb replication. Interestingly, DC were not required for the secondary CD4+ T cell response following Mtb infection in peptide-vaccinated mice. Thus, this study shows that DC are essential for the initiation of the adaptive T cell response to the human pathogen Mtb.     

Enzymatic methylation of the Mycobacterium tuberculosis heparin-binding haemagglutinin

Heparin-binding haemagglutinin (HBHA) is an important Mycobacterium tuberculosis virulence factor. It displays a complex methylation pattern in its C-terminal, functional domain, which protects this domain against proteolysis. Here, it is shown that HBHA methylation is catalysed by mycobacterial enzymes and a radio-enzymatic and a nonradioactive enzyme assay are described, based on the recognition of methylated HBHA by monoclonal antibodies. MS analysis of in vitro methylated HBHA shows a complex methylation pattern similar to that of naturally methylated HBHA. Using recombinant hybrid molecules as acceptor substrates, it was found that the N-terminal domain of HBHA is not required for recognition by the HBHA-methyltransferase(s), although it is required for in vivo methylation.     

Subcutaneous boosting with heparin binding haemagglutinin increases BCG-induced protection against tuberculosis

Pulmonary tuberculosis remains a major health problem. Effective vaccination strategies are urgently needed. It was previously demonstrated that purified Mycobacterium bovis BCG Heparin Binding Haemagglutinin (HBHA) is able to induce in BALB/c mice protection levels against a Mycobacterium tuberculosis infection that are similar to those offered by BCG. Here we developed a heterologous prime/boost immunisation approach using a combination of BCG and HBHA in order to increase the protective immune response. We show that the time period between BCG priming and HBHA boosting strongly influences the efficacy of the boost. The optimized vaccine protocol consisting of a BCG administration followed 8 months later by boosting with HBHA resulted in an increase in the level of protection of up to 0.7 log when compared to BCG alone. These results suggest an immunisation strategy where BCG is administered to neonates and is followed by subcutaneous HBHA boosting in young adults.     

Interleukin-23 restores immunity to Mycobacterium tuberculosis infection in IL-12p40-deficient mice and is not required for the development of IL-17-secreting T cell responses

Host control of Mycobacterium tuberculosis is dependent on the activation of CD4+ T cells secreting IFN-gamma and their recruitment to the site of infection. The development of more efficient vaccines against tuberculosis requires detailed understanding of the induction and maintenance of T cell immunity. Cytokines important for the development of cell-mediated immunity include IL-12 and IL-23, which share the p40 subunit and the IL-12Rbeta1 signaling chain. To explore the differential effect of IL-12 and IL-23 during M. tuberculosis infection, we used plasmids expressing IL-23 (p2AIL-23) or IL-12 (p2AIL-12) alone in dendritic cells or macrophages from IL-12p40(-/-) mice. In the absence of the IL-12/IL-23 axis, immunization with a DNA vaccine expressing the M. tuberculosis Ag85B induced a limited Ag-specific T cell response and no control of M. tuberculosis infection. Co-delivery of p2AIL-23 or p2AIL-12 with DNA85B induced strong proliferative and IFN-gamma-secreting T cell responses equivalent to those observed in wild-type mice immunized with DNA85B. This response resulted in partial protection against aerosol M. tuberculosis; however, the protective effect was less than in wild-type mice owing to the requirement for IL-12 or IL-23 for the optimal expansion of IFN-gamma-secreting T cells. Interestingly, bacillus Calmette-Guérin immune T cells generated in the absence of IL-12 or IL-23 were deficient in IFN-gamma production, but exhibited a robust IL-17 secretion associated with a degree of protection against pulmonary infection. Therefore, exogenous IL-23 can complement IL-12 deficiency for the initial expansion of Ag-specific T cells and is not essential for the development of potentially protective IL-17-secreting T cells.     

CD4+ T cells mediate IFN-gamma-independent control of Mycobacterium tuberculosis infection both in vitro and in vivo

Although IFN-gamma is necessary for survival of Mycobacterium tuberculosis infection in people and animal models, it may not be sufficient to clear the infection, and IFN-gamma is not a reliable correlate of protection. To determine whether IFN-gamma-independent mechanisms of immunity exist, we developed a murine ex vivo culture system that directly evaluates the ability of splenic or lung lymphocytes to control the growth of M. tuberculosis within infected macrophages, and that models in vivo immunity to tuberculosis. Surprisingly, CD4(+) T cells controlled >90% of intracellular M. tuberculosis growth in the complete absence of IFN-gamma stimulation of macrophages, via a NO-dependent mechanism. Furthermore, bacillus Calmette-Guerin-vaccinated IFN-gamma-deficient mice exhibited significant protection against M. tuberculosis challenge that was lost upon depletion of CD4(+) T cells. These findings demonstrate that CD4(+) T cells possess IFN-gamma-independent mechanisms that can limit the growth of an intracellular pathogen and are dominant in secondary responses to M. tuberculosis.     

Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4(+) and CD8(+) T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8(+) T cell responses against even low doses of protein Ags, and this effect was independent of CD4(+) T cell help. Ag-specific CD8(+) T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.     

Characterization of T cell antigens associated with the cell wall protein-peptidoglycan complex of Mycobacterium tuberculosis

Mycobacterium tuberculosis cell walls are likely to contain critical T cell Ag capable of inducing protective immunity against the development of tuberculosis in animal models. Therefore, we characterized cell wall-associated Ag that stimulate T lymphocytes in tuberculosis patients and clinically well tuberculin-positive individuals. A protein-peptidoglycan complex isolated from the M. tuberculosis cell wall had potent immunologic activity, evoking PBMC proliferative responses similar to those induced by sonicated whole M. tuberculosis. In order to characterize the immunoreactive protein determinants associated with the protein-peptidoglycan complex, T cell lines were established to cell wall Ag and used to probe M. tuberculosis proteins separated by SDS-PAGE. These T cell lines proliferated primarily to protein Ag of 10, 19, 23, 28, 30, 40 to 50, and 65 kDa. Cell wall-reactive T cell clones that recognized the 10-, 23-, 28-, and 30-kDa proteins as single bands on SDS-PAGE did so under reducing and nonreducing conditions, suggesting that these are not proteolytic fragments or subunits of larger protein aggregates. We propose that these protein monomers, when post-translationally complexed with peptidoglycan, are the key ingredients of the immunogenic protein-peptidoglycan complex. In order to assess the relationship of the cell wall-associated Ag to those secreted proteins from "early culture filtrates" of actively growing M. tuberculosis recently implicated in eliciting protective immunity, cell wall-reactive T cell clones were tested for their ability to recognize early culture filtrates. Results revealed that at least three proteins shared with the cell wall complex are contained within early culture filtrates. Our data indicate that antigenic determinants associated with the protein-peptidoglycan complex of the M. tuberculosis cell wall may be involved in protective immunity and hence are potential candidates for inclusion in an effective antituberculosis vaccine.     

The LFA-1 adhesion molecule is required for protective immunity during pulmonary Mycobacterium tuberculosis infection

Host immunity to Mycobacterium tuberculosis is mediated by T cells that recognize and activate infected macrophages to control intracellular bacterial replication. The early appearance of T cells in the lungs of infected mice correlates with greater resistance to infection. However, it is unknown whether the trafficking of T cells to the lung following infection is dependent upon the expression of certain adhesion molecules. To address this question, we infected knockout (KO) mice that have defective expression of CD11a, CD11b, CD18, CD62, CD103, or beta7. We found that the integrins CD11a and CD18 are absolutely required for host resistance following infection with aerosolized M. tuberculosis. Although Ag-specific T cells are generated following infection of CD11a KO mice, T cell priming is delayed, T cell trafficking to the lung is impaired, and fewer ESAT6-specific CD4+ T cells are found in the lungs of CD11a KO mice compared with control mice. Thus, LFA-1 (CD11a/CD18) plays an essential role in immunity to M. tuberculosis infection.     

TIM3 Mediates T Cell Exhaustion during Mycobacterium tuberculosis Infection

While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain–containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis.     

Transmembrane TNF is sufficient to initiate cell migration and granuloma formation and provide acute, but not long-term, control of Mycobacterium tuberculosis infection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF-/-, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF-/- mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF-/- mice, into either RAG-/- or TNF-/- mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.     

B cells moderate inflammatory progression and enhance bacterial containment upon pulmonary challenge with Mycobacterium tuberculosis

Though much is known about the function of T lymphocytes in the adaptive immune response against Mycobacterium tuberculosis, comparably little is understood regarding the corresponding role of B lymphocytes. Indicating B cells as components of lymphoid neogenesis during pulmonary tuberculosis, we have identified ectopic germinal centers (GCs) in the lungs of infected mice. B cells in these pulmonary lymphoid aggregates express peanut agglutinin and GL7, two markers of GC B cells, as well as CXCR5, and migrate in response to the lymphoid-associated chemokine CXCL13 ex vivo. CXCL13 is negatively regulated by the presence of B cells, as its production is elevated in lungs of B cell-deficient (B cell(-/-)) mice. Upon aerosol with 100 CFU of M. tuberculosis Erdman, B cell(-/-) mice have exacerbated immunopathology corresponding with elevated pulmonary recruitment of neutrophils. Infected B cell(-/-) mice show increased production of IL-10 in the lungs, whereas IFN-gamma, TNF-alpha, and IL-10R remain unchanged from wild type. B cell(-/-) mice have enhanced susceptibility to infection when aerogenically challenged with 300 CFU of M. tuberculosis corresponding with elevated bacterial burden in the lungs but not in the spleen or liver. Adoptive transfer of B cells complements the phenotypes of B cell(-/-) mice, confirming a role for B cells in both modulation of the host response and optimal containment of the tubercle bacillus. As components of ectopic GCs, moderators of inflammatory progression, and enhancers of local immunity against bacterial challenge, B cells may have a greater role in the host defense against M. tuberculosis than previously thought.     

Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

表达HBHA和hIL-12融合蛋白的重组耻垢分枝杆菌在小鼠体内诱导的免疫应答及保护力

目的:测定表达肝素结合血凝素(HBHA)和人白细胞介素12(hIL-12)融合蛋白的重组耻垢分枝杆菌在小鼠体内诱导产生的免疫应答及对结核分枝杆菌感染的保护作用。方法:将表达HBHA和hIL-12融合蛋白的重组耻垢分枝杆菌采用同源加强免疫的方法免疫小鼠,检测小鼠外周血中IFN-γ、IL-2和IL-12的表达水平;用结核分枝杆菌感染免疫小鼠,检测小鼠肺部荷菌量和组织病理变化。结果:表达HBHA和hIL-12融合蛋白的重组耻垢分枝杆菌诱导小鼠产生以IFN-γ、IL-2分泌量增加为主的Th1型免疫应答,并能有效减少感染小鼠肺部结核分枝杆菌的荷菌量和病理损伤。结论:表达HBHA和hIL-12融合蛋白的重组耻垢分枝杆菌免疫小鼠可诱导产生与卡介苗相当的保护作用,可能成为控制结核病的有效疫苗。     

Bacillus Calmette Guerin vaccination of human newborns induces a specific, functional CD8+ T cell response

Mounting evidence points to CD8+ T cells playing an important role in protective immunity against Mycobacterium tuberculosis. The only available vaccine against tuberculosis, bacillus Calmette Guérin (BCG), has traditionally been viewed not to induce these cells optimally. In this study, we show that vaccination of human newborns with BCG does indeed induce a specific CD8+ T cell response. These cells degranulated or secreted IFN-gamma, but not both, when infant blood was incubated with BCG. This stimulation also resulted in proliferation and up-regulation of cytotoxic molecules. Overall, the specific CD8+ T cell response was quantitatively smaller than the BCG-induced CD4+ T cell response. Incubation of whole blood with M. tuberculosis also caused CD8+ T cell IFN-gamma expression. We conclude that BCG induces a robust CD8+ T cell response, which may contribute to vaccination-induced protection against tuberculosis.