Epitope‐specific CD4+, but not CD8+, T‐cell responses induced by recombinant influenza A viruses protect against Mycobacterium tuberculosis infection

Tuberculosis remains a global health problem, in part due to failure of the currently available vaccine, BCG, to protect adults against pulmonary forms of the disease. We explored the impact of pulmonary delivery of recombinant influenza A viruses (rIAVs) on the induction of Mycobacterium tuberculosis (M. tuberculosis)‐specific CD4⁺ and CD8⁺ T‐cell responses and the resultant protection against M. tuberculosis infection in C57BL/6 mice. Intranasal infection with rIAVs expressing a CD4⁺ T‐cell epitope from the Ag85B protein (PR8.p25) or CD8⁺ T‐cell epitope from the TB10.4 protein (PR8.TB10.4) generated strong T‐cell responses to the M. tuberculosis‐specific epitopes in the lung that persisted long after the rIAVs were cleared. Infection with PR8.p25 conferred protection against subsequent M. tuberculosis challenge in the lung, and this was associated with increased levels of poly‐functional CD4⁺ T cells at the time of challenge. By contrast, infection with PR8.TB10.4 did not induce protection despite the presence of IFN‐γ‐producing M. tuberculosis‐specific CD8⁺ T cells in the lung at the time of challenge and during infection. Therefore, the induction of pulmonary M. tuberculosis epitope‐specific CD4⁺, but not CD8⁺ T cells, is essential for protection against acute M. tuberculosis infection in the lung.     

Exosomes carrying mycobacterial antigens can protect mice against Mycobacterium tuberculosis infection

Approximately 2 billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), and an estimated 1.5 million individuals die annually from TB. Presently, Mycobacterium bovis BCG remains the only licensed TB vaccine; however, previous studies suggest its protective efficacy wanes over time and fails in preventing pulmonary TB. Therefore, a safe and effective vaccine is urgently required to replace BCG or boost BCG immunizations. Our previous studies revealed that mycobacterial proteins are released via exosomes from macrophages infected with M. tuberculosis or pulsed with M. tuberculosis culture filtrate proteins (CFP). In the present study, exosomes purified from macrophages treated with M. tuberculosis CFP were found to induce antigen‐specific IFN‐γ and IL‐2‐expressing CD4⁺ and CD8⁺ T cells. In exosome‐vaccinated mice, there was a similar T_H1 immune response but a more limited T_H2 response compared to BCG‐vaccinated mice. Using a low‐dose M. tuberculosis mouse aerosol infection model, exosomes from CFP‐treated macrophages were found to both prime a protective immune response as well as boost prior BCG immunization. The protection was equal to or superior to BCG. In conclusion, our findings suggest that exosomes might serve as a novel cell‐free vaccine against an M. tuberculosis infection.     

Heat‐Shock Protein gp96 Enhances T Cell Responses and Protective Potential to Bacillus Calmette‐Guérin Vaccine

The commonly used Bacillus Calmette‐Guérin (BCG) vaccine only induces moderate T cell responses and is less effective in protecting against pulmonary tuberculosis (TB) in adults and ageing populations. Thus, developing new TB vaccine candidates is an important strategy against the spread of Mycobacterium tuberculosis. Here, we demonstrated that immunization with heat‐shock protein gp96 as an adjuvant led to a significantly increased CD4⁺ and CD8⁺ T cell response to a BCG vaccine. Secretion of the Th1‐type cytokines was increased by splenocytes from gp96‐immunized mice. In addition, adding gp96 as an adjuvant effectively improved the protection against intravenous challenge with Mycobacterium bovis BCG in mice. Our study reveals the novel property of gp96 in boosting the vaccine‐specific T cell response and its potential use as an adjuvant for BCG vaccines against mycobacterial infection.     

Immunogenicity and Protective Efficacy Conferred by a Novel Recombinant Mycobacterium bovis Bacillus Calmette‐Guérin Strain Expressing Interleukin‐12p70 of Human Cytokine and Ag85A of Mycobacterium tuberculosis Fusion Protein

Mycobacterium bovis bacillus Calmette‐Guérin (BCG) immunization provides protection against tuberculosis (TB) in infants, but the antituberculosis protective immunity wanes gradually after initial immunization and lasts less than 15 years. Therefore, more efficacious vaccines are urgently needed. In this study, we constructed a new tuberculosis vaccine of recombinant BCG strain (rBCG‐IA), which could express IL‐12p70 of human cytokine and Ag85A of M. tuberculosis fusion protein, and investigated its immunogenicity in BALB/c mice by measuring antibody titres, proliferation rate of splenocytes, ratios of CD4⁺ T and CD8⁺ T cells stimulated by specific antigens and levels of IFN‐γ production in antigen‐stimulated splenocyte cultures. Meanwhile, we evaluated its protective efficacy against M. tuberculosis H37Rv infection through detecting lung histopathology, organ bacterial loads and lung acid‐fast stain. Immunogenicity experiments illustrated that from 2nd to 8th week after immunization, the rBCG‐IA vaccine was able to induce the highest level of antibody titres, proliferation rate of splenocytes and IFN‐γ production among groups and gained improved ratio of CD4⁺ T and CD8⁺ T cells from 6th to 8th week after vaccination. And from 2nd to 8th week after M. tuberculosis H37Rv infection, the score of pathology and bacterial loads in the rBCG‐IA group were obviously lower than that in rBCG‐I group, rBCG‐A group or control group (PBST group), but similar to that in BCG group. This study suggested that rBCG‐IA was able to elicit stronger humoral and cellular immune responses, but could only confer similar protective efficacy compared with its parental BCG vaccine.     

Mycobacterium chelonae sensitisation induces CD4+‐mediated cytotoxicity against BCG

Significant variability in efficacy of live Mycobacterium bovis BCG as a tuberculosis vaccine is observed globally. Effects of pre‐vaccination sensitisation to non‐tuberculous environmental mycobacteria (Env) are suspected to underlie this phenomenon, but the mechanisms remain unclear. We postulated that it could be due to Env‐specific T cells exerting cytotoxicity against BCG‐infected host cells. After murine sensitisation with heat‐killed antigens of different Env species, splenocytes from M. chelonae (CHE)‐sensitised mice exerted the strongest cytotoxicity against autologous BCG‐infected macrophages. This cytotoxicity was correlated with reduced BCG viability. The cytotoxicity was reduced by the depletion of CD4⁺, but not CD8⁺ or CD56⁺ cells, and CD4⁺ cells showed higher percentage of cytotoxicity than CD4^? cells, supporting a role for CD4⁺ cells in CHE‐induced, BCG‐specific cytotoxicity. Additionally, this cytotoxicity was IFN‐γ, perforin and FasL dependent. After CHE‐sensitisation and subsequent BCG intranasal infection, there was significant expansion of lung CD4⁺ cells, the main cell type producing IFN‐γ. This was associated with 2‐ and 6‐fold reductions in lung BCG counts 1 and 3 wk, respectively post‐ infection, relative to non‐sensitised mice. This is the first report describing cytotoxicity against BCG‐infected cells as a mechanism underlying the influence of Env sensitisation on subsequent BCG responses.     

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.     

Exposure to human alveolar lining fluid enhances Mycobacterium bovis BCG vaccine efficacy against Mycobacterium tuberculosis infection in a CD8+ T-cell-dependent manner

Current tuberculosis (TB) treatments include chemotherapy and preventative vaccination with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In humans, however, BCG vaccination fails to fully protect against pulmonary TB. Few studies have considered the impact of the human lung mucosa (alveolar lining fluid (ALF)), which modifies the Mycobacterium tuberculosis (M.tb) cell wall, revealing alternate antigenic epitopes on the bacterium surface that alter its pathogenicity. We hypothesized that ALF-induced modification of BCG would induce better protection against aerosol infection with M.tb. Here we vaccinated mice with ALF-exposed BCG, mimicking the mycobacterial cell surface properties that would be present in the lung during M.tb infection. ALF-exposed BCG-vaccinated mice were more effective at reducing M.tb bacterial burden in the lung and spleen, and had reduced lung inflammation at late stages of M.tb infection. Improved BCG efficacy was associated with increased numbers of memory CD8⁺ T cells, and CD8⁺ T cells with the potential to produce interferon-γ in the lung in response to M.tb challenge. Depletion studies confirmed an essential role for CD8⁺ T cells in controlling M.tb bacterial burden. We conclude that ALF modifications to the M.tb cell wall in vivo are relevant in the context of vaccine design.     

Prime–boost bacillus Calmette–Guérin vaccination with lentivirus‐vectored and DNA‐based vaccines expressing antigens Ag85B and Rv3425 improves protective efficacy against Mycobacterium tuberculosis in mice

To prevent the global spread of tuberculosis (TB), more effective vaccines and vaccination strategies are urgently needed. As a result of the success of bacillus Calmette–Guérin (BCG) in protecting children against miliary and meningeal TB, the majority of individuals will have been vaccinated with BCG; hence, boosting BCG‐primed immunity will probably be a key component of future vaccine strategies. In this study, we compared the ability of DNA‐, protein‐ and lentiviral vector‐based vaccines that express the antigens Ag85B and Rv3425 to boost the effects of BCG in the context of immunity and protection against Mycobacterium tuberculosis in C57BL/6 mice. Our results demonstrated that prime–boost BCG vaccination with a lentiviral vector expressing the antigens Ag85B and Rv3425 significantly enhanced immune responses, including T helper type 1 and CD8⁺ cytotoxic T lymphocyte responses, compared with DNA‐ and protein‐based vaccines. However, lentivirus‐vectored and DNA‐based vaccines greatly improved the protective efficacy of BCG against M. tuberculosis, as indicated by a lack of weight loss and significantly reduced bacterial loads and histological damage in the lung. Our study suggests that the use of lentiviral or DNA vaccines containing the antigens Ag85B and Rv3425 to boost BCG is a good choice for the rational design of an efficient vaccination strategy against TB.     

Enhanced protective efficacy against Mycobacterium tuberculosis afforded by BCG prime‐DNA boost regimen in an early challenge mouse model is associated with increased splenic interleukin‐2‐producing CD4 T‐cell frequency post‐vaccination

The development of improved vaccines and vaccination strategies against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti‐tuberculosis protective immunity. Simple measurement of interferon‐γ frequency or production per se does not provide adequate prediction of immune protection. In this study, we examined the relationship between T‐cell immune responses and protective efficacy conferred by the heterologous vaccination strategy, bacillus Calmette–Guérin (BCG) prime‐Ag85A DNA boost (B/D), in an early challenge mouse model of pulmonary tuberculosis. The results demonstrated that mice vaccinated with the B/D regimen had a significantly reduced bacillary load compared with BCG‐vaccinated mice, and the reduction in colony‐forming units was associated with decreased pathology and lower levels of inflammatory cytokines in the infected lungs. Further analysis of immunogenicity showed that the superior protection afforded by the B/D regimen was associated with significantly increased frequency of splenic interleukin‐2 (IL‐2) ‐producing CD4 T cells and increased IL‐2 production when measured as integrated mean fluorescence intensity post‐vaccination as well. These data suggest that measurement of elevated frequency of IL‐2‐producing CD4 T cells or IL‐2 production in the spleens of vaccinated mice can predict vaccine efficacy, at least in the B/D strategy, and add to the accumulating body of evidence suggesting that BCG prime‐boost strategies may be a useful approach to the control of M. tuberclosis infection.     

Immunotherapeutic effects of recombinant adenovirus encoding granulocyte–macrophage colony‐stimulating factor in experimental pulmonary tuberculosis

BALB/c mice with pulmonary tuberculosis (TB) develop a T helper cell type 1 that temporarily controls bacterial growth. Bacterial proliferation increases, accompanied by decreasing expression of interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and inducible nitric oxide synthase (iNOS). Activation of dendritic cells (DCs) is delayed. Intratracheal administration of only one dose of recombinant adenoviruses encoding granulocyte–macrophage colony‐stimulating factor (AdGM‐CSF) 1 day before Mycobacterium tuberculosis (Mtb) infection produced a significant decrease of pulmonary bacterial loads, higher activated DCs and increased expression of TNF‐α, IFN‐γ and iNOS. When AdGM‐CSF was given in female mice B6D2F1 (C57BL/6J X DBA/2J) infected with a low Mtb dose to induce chronic infection similar to latent infection and corticosterone was used to induce reactivation, a very low bacilli burden in lungs was detected, and the same effect was observed in healthy mice co‐housed with mice infected with mild and highly virulent bacteria in a model of transmissibility. Thus, GM‐CSF is a significant cytokine in the immune protection against Mtb and gene therapy with AdGM‐CSF increased protective immunity when administered in a single dose 1 day before Mtb infection in a model of progressive disease, and when used to prevent reactivation of latent infection or transmission.     

Accelerated induction of mycobacterial antigen-specific CD8+ T cells in the Mycobacterium tuberculosis-infected lung by subcutaneous vaccination with Mycobacterium bovis bacille Calmette–Guérin

Both CD4⁺ and CD8⁺ T cells are important in protection against Mycobacterium tuberculosis infection. To evaluate the effect of vaccination with Mycobacterium bovis bacille Calmette–Guérin (BCG) on the CD8⁺ T-cell response to pulmonary M. tuberculosis infection, we analyzed the kinetics of CD8⁺ T cells specific to the mycobacterial Mtb32a_309–318 epitope, which is shared by M. tuberculosis and M. bovis BCG, in the lung of mice infected with M. tuberculosis. The CD8⁺ T cells were detected by staining lymphocytes with pentameric major histocompatibility complex (MHC) class I H-2D^b–Mtb32a_209–318 peptide complex and were analysed by flow cytometry. Mtb32a-specific CD8⁺ T cells became detectable on day 14, and reached a plateau on day 21, in the lung of M. tuberculosis-infected unvaccinated mice. Subcutaneous vaccination with M. bovis BCG in the footpads induced Mtb32a-specific CD8⁺ T cells in the draining lymph nodes (LNs) on day 7 and their numbers further increased on day 14. When M. bovis BCG-vaccinated mice were exposed to pulmonaryinfection with M. tuberculosis 4 weeks after vaccination, the Mtb32a-specific CD8⁺ T cells in the infected lung became detectable on day 7 and reached a plateau on day 14, which was 1 week earlier than in the unvaccinated mice. The pulmonary CD8⁺ T cells from the BCG-vaccinated M. tuberculosis-infected mice produced interferon-γ in response to Mtb32a_209–318 peptide on day 7 of the infection, whereas those of unvaccinated mice did not. The results demonstrate that induction of mycobacterial antigen-specific protective CD8⁺ T cells in the M. tuberculosis-infected lung is accelerated by subcutaneous vaccination with M. bovis BCG.     

Immunogenicity and protective efficacy of a DNA vaccine encoding the fusion protein of mycobacterium heat shock protein 65(Hsp65) with human interleukin‐2 against Mycobacterium tuberculosis in BALB/c mice

Developing a new generation of vaccines is important for preventing tuberculosis (TB). DNA vaccine is one promising candidate. In this study we evaluated the immunogenicity and protective efficacy of the DNA vaccine encoding the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) with human interleukin‐2 (hIL‐2) in BALB/c mice. We showed that the DNA vaccine pcDNA‐Hsp65‐hIL‐2 could induce high levels of antigen‐specific antibody, IFN‐γ, CD4⁺ and CD8⁺ T cell production. When the immunized mice were infected with M. tuberculosis H37Rv, the organ bacterial loads in the DNA immunized group were significantly reduced compared to those of the saline control group, but the ability to reduce bacteria was not better than for BCG. The histopathology in lungs of the DNA vaccine immunized mice was similar to that of BCG immunized mice, which was obviously ameliorated compared to that of the saline control group. Overall, the DNA vaccine could afford protection against M. tuberculosis infection, though the protection efficacy was not as great as that of conventional BCG.     

MHC‐restricted Ag85B‐specific CD8+ T cells are enhanced by recombinant BCG prime and DNA boost immunization in mice

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8⁺ T cell response to Bacillus Calmette‐Guérin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8⁺ T cells with an rBCG‐based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG‐Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA‐Mkan85B). We identified two MHC‐I (H2‐K^d)‐restricted epitopes that induce cross‐reactive responses to Mtb and other related mycobacteria in both BALB/c (H2^d) and CB6F1 (H2^b/d) mice. The H2‐K^d‐restricted peptide epitopes elicited polyfunctional CD8⁺ T cell responses that were also highly cross‐reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2‐K^d‐restricted epitopes elicit distinct CD8⁺ T cell populations, a result explained by the X‐ray structure of the two peptide/H2‐K^d complexes. These results suggest that rBCG‐Mkan85B vector‐based immunization and DNA‐Mkan85B boost may enhance CD8⁺ T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.     

Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4+/CD4+ Foxp3+ cells

CD4⁺ Foxp3⁺ regulatory T cells inhibit the production of interferon‐γ, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat‐shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4⁺ Foxp3⁺ cells compared with non‐immunized mice. Heterologous immunization using bacillus Calmette–Guérin (BCG) to prime and DNA‐hsp 65 to boost (BCG/DNA‐hsp 65) or BCG to prime and culture filtrate proteins (CFP)‐CpG to boost (BCG/CFP‐CpG) induced a significantly higher ratio of spleen CD4⁺/CD4⁺ Foxp3⁺ cells compared with non‐immunized mice. In addition, the protection conferred by either the BCG/DNA‐hsp 65 or the BCG/CFP‐CpG vaccines was significant compared with the DNA‐hsp 65 vaccine. Despite the higher ratio of spleen CD4⁺/CD4⁺ Foxp3⁺ cells found in BCG/DNA‐hsp 65‐immunized or BCG/CFP‐CpG‐immunized mice, the lungs of both groups of mice were better preserved than those of DNA‐hsp 65‐immunized mice. These results confirm the protective efficacy of BCG/DNA‐hsp 65 and BCG/CFP‐CpG heterologous prime‐boost vaccines and the DNA‐hsp 65 homologous vaccine. Additionally, the prime‐boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4⁺ and regulatory (CD4⁺ Foxp3⁺) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.     

Sca‐1+ cardiac fibroblasts promote development of heart failure

The causative effect of GM‐CSF produced by cardiac fibroblasts to development of heart failure has not been shown. We identified the pathological GM‐CSF‐producing cardiac fibroblast subset and the specific deletion of IL‐17A signaling to these cells attenuated cardiac inflammation and heart failure. We describe here the CD45^?CD31^?CD29⁺mEF‐SK4⁺PDGFRα⁺Sca‐1⁺periostin⁺ (Sca‐1⁺) cardiac fibroblast subset as the main GM‐CSF producer in both experimental autoimmune myocarditis and myocardial infarction mouse models. Specific ablation of IL‐17A signaling to Sca‐1⁺periostin⁺ cardiac fibroblasts (Postn^CreIl17ra^fl/fl) protected mice from post‐infarct heart failure and death. Moreover, Postn^CreIl17ra^fl/fl mice had significantly fewer GM‐CSF‐producing Sca‐1⁺ cardiac fibroblasts and inflammatory Ly6C^hi monocytes in the heart. Sca‐1⁺ cardiac fibroblasts were not only potent GM‐CSF producers, but also exhibited plasticity and switched their cytokine production profiles depending on local microenvironments. Moreover, we also found GM‐CSF‐positive cardiac fibroblasts in cardiac biopsy samples from heart failure patients of myocarditis or ischemic origin. Thus, this is the first identification of a pathological GM‐CSF‐producing cardiac fibroblast subset in human and mice hearts with myocarditis and ischemic cardiomyopathy. Sca‐1⁺ cardiac fibroblasts direct the type of immune cells infiltrating the heart during cardiac inflammation and drive the development of heart failure.     

Using a prime and pull approach,lentivector vaccines expressing Ag85A induce immunogenicity but fail to induce protection against Mycobacterium bovis bacillus Calmette–Guérin challenge in mice

Although bacillus Calmette–Guérin (BCG) is an established vaccine with excellent efficacy against disseminated Mycobacterium tuberculosis infection in young children, efficacy in adults suffering from respiratory tuberculosis (TB) is suboptimal. Prime‐boost viral vectored vaccines have been shown to induce effective immune responses and lentivectors (LV) have been shown to improve mucosal immunity in the lung. A mucosal boost to induce local immunogenicity is also referred to as a ‘pull’ in a prime and pull approach, which has been found to be a promising vaccine strategy. The majority of infants worldwide receive BCG immunization through current vaccine protocols. We therefore aimed to investigate the role of a boost (or pull) immunization with an LV vaccine expressing the promising TB antigen (Ag85A). We immunized BALB/c mice subcutaneously with BCG or an LV vaccine expressing a nuclear factor‐κB activator vFLIP together with Ag85A (LV vF/85A), then boosted with intranasal LV vF/85A. Prime and pull immunization with LV85A induced significantly enhanced CD8⁺ and CD4⁺ T‐cell responses in the lung, but did not protect against intranasal BCG challenge. In contrast, little T‐cell response in the lung was seen when the prime vaccine was BCG, and intranasal vF/85A provided no additional protection against mucosal BCG infection. Our study demonstrates that not all LV prime and pull approaches may be successful against TB in man and careful antigen and immune activator selection is therefore required.     

Novel Recombinant BCG Coexpressing Ag85B, ESAT-6 and Rv2608 Elicits Significantly Enhanced Cellular Immune and Antibody Responses in C57BL/6 Mice

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate vaccine, the Bacille Calmette‐Guérin (BCG), is urgently needed. BCG has proven to be an effective recombinant delivery vehicle for foreign antigens because of its ability to induce long‐lived specific humoral and cellular immunity. Experimental evidences have revealed that Ag85B, ESAT‐6 and Rv2608 are important immunodominant antigens of Mycobacterium tuberculosis and are all promising vaccine candidate molecules. In this study, we have constructed a novel recombinant BCG (rBCG) expressing fusion protein Ag85B‐ESAT6‐Rv2608 and evaluated the immunogenicity of rBCG in C57BL/6 mice. Results show there is strong TB‐specific CD4⁺ and CD8⁺ T lymphocytes proliferative response in mice immunized with rBCG vaccine, especially the cytotoxic CD8⁺ T cells playing an important role in protection against TB. And rBCG immunization has induced a significantly strong Th1 immune response, characterized by the increased ratio of IgG2b/IgG1. Results also show that rBCG immunization could increase the secretion of Th1 cytokines such as TNF‐α and IL‐2 and could decrease the secretion of Th2 cytokine IL‐10. Moreover, it was shown that rBCG immunization induced a strong humoral response in mice, characterized by the elevated IgG titre. Therefore, we conclude that this rBCG immunization could increase both cellular immune response and antigen‐specific humoral response significantly as compared to BCG immunization in mice. The above results illustrated that rBCG::Ag85B‐ESAT6‐Rv2608 is a potential candidate against M. tuberculosis for further study.     

Modified vaccinia Ankara‐expressing Ag85A,a novel tuberculosis vaccine,is safe in adolescents and children,and induces polyfunctional CD4+ T cells

Modified vaccinia Ankara‐expressing Ag85A (MVA85A) is a new tuberculosis (TB) vaccine aimed at enhancing immunity induced by BCG. We investigated the safety and immunogenicity of MVA85A in healthy adolescents and children from a TB endemic region, who received BCG at birth. Twelve adolescents and 24 children were vaccinated and followed up for 12 or 6 months, respectively. Adverse events were documented and vaccine‐induced immune responses assessed by IFN‐γ ELISpot and intracellular cytokine staining. The vaccine was well tolerated and there were no vaccine‐related serious adverse events. MVA85A induced potent and durable T‐cell responses. Multiple CD4⁺ T‐cell subsets, based on expression of IFN‐γ, TNF‐α, IL‐2, IL‐17 and GM‐CSF, were induced. Polyfunctional CD4⁺ T cells co‐expressing IFN‐γ, TNF‐α and IL‐2 dominated the response in both age groups. A novel CD4⁺ cell subset co‐expressing these three Th1 cytokines and IL‐17 was induced in adolescents, while a novel CD4⁺ T‐cell subset co‐expressing Th1 cytokines and GM‐CSF was induced in children. Ag‐specific CD8⁺ T cells were not detected. We conclude that in adolescents and children MVA85A safely induces the type of immunity thought to be important in protection against TB. This includes induction of novel Th1‐cell populations that have not been previously described in humans.     

Mycobacterium bovis BCG immunization induces protective immunity against nine different Mycobacterium tuberculosis strains in mice

Recent preclinical and epidemiologic studies have suggested that certain Mycobacterium tuberculosis genotypes (in particular, Beijing lineage strains) may be resistant to Mycobacterium bovis BCG vaccine-induced antituberculosis protective immunity. To investigate the strain specificity of BCG-induced protective responses in a murine model of pulmonary tuberculosis, C57BL/6 mice were vaccinated with BCG vaccine and then challenged 2 months later with one of nine M. tuberculosis isolates. Four of these strains were from the W-Beijing lineage (HN878, N4, NHN5, and ChS) while four were non-Beijing-type isolates (C913, CDC1551, NY669, and NY920). As a control, the WHO standard M. tuberculosis Erdman strain was evaluated in these vaccination/challenge experiments. To assess the protective responses evoked by BCG immunization, organ bacterial burdens and lung pathology were assessed in vaccinated and naïve mice at 4, 12, and 20 weeks postchallenge as well as during the day of infection. At 4 weeks after the aerosol challenge with each of these strains, significantly reduced bacterial growth in the lungs and spleens and significantly improved lung pathology were seen in all vaccinated animals compared to naïve controls. After 12 weeks, reduced organ bacterial burdens were detected in vaccinated animals infected with six of nine challenge strains. Although lung CFU values were lower in vaccinated mice for only three of nine groups at 20 weeks postchallenge, significantly decreased lung inflammation was seen in all immunized animals relative to controls at 20 weeks postchallenge. Taken together, these data demonstrate that BCG vaccination protects against infection with diverse M. tuberculosis strains in the mouse model of pulmonary tuberculosis and suggest that strain-specific resistance to BCG-induced protective immunity may be uncommon.