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.     

A DNA vaccine expressing CFP21 and MPT64 fusion protein enhances BCG-induced protective immunity against Mycobacterium tuberculosis infection in mice

The efficacy of Bacillus Calmette–Guérin (BCG) vaccine in preventing adult tuberculosis (TB) is highly variable. Genetic differences between BCG vaccine substrains, which can be divided into early strains and late strains based on the loss of region of difference two (RD2), may result in the variability and BCG substrains. The effect of lack of RD2 on the protective efficacy of BCG substrains against TB remains unknown. In this study, we demonstrated that CFP21 and MPT64(rCM) fusion protein, encoded by RD2 of Mycobacterium tuberculosis, could stimulate higher level of interferon (IFN)-γ in tuberculin skin test (TST)-positive healthy population than in TST-negative healthy population. Compared with naive mice challenged with virulent M. tuberculosis H37Rv, C57BL/6 mice vaccinated with pcD2164 DNA expressing rCM protein resulted in a greater decrease in the bacterial load of lung. Moreover, pcD2164 could boost the protective immunity in mice primed by BCG than BCG alone or DNA vaccination alone, as evidenced by lower bacterial load in the lung tissue and reduced lung pathology. The protection induced by BCG prime-DNA vaccine boost strategy was associated with significant increases in rCM protein-specific IFN-γ. Therefore, our results clearly indicate that the loss of RD2 has an important influence on the protective efficacy of different BCG substrains. These findings will benefit the optimal choice of BCG substrain for neonatal immunization and rational design of new vaccines for the prevention of TB.     

Modulation of pulmonary DC function by vaccine‐encoded GM‐CSF enhances protective immunity against Mycobacterium tuberculosis infection

The rational design of new vaccines engineered to target key components of the host immune response is crucial to aid control of important infectious diseases such as tuberculosis. In this report, we determined whether modifying the function of pulmonary APC could improve protection against infection with Mycobacterium tuberculosis. Targeted delivery to the lung of the cytokine GM‐CSF, expressed by the Mycobacterium bovis BCG vaccine strain, increased pulmonary DC numbers and secretion of the immunoregulatory cytokine IL‐12, compared with parental BCG immunization. This impact on APC number by BCG:GM‐CSF resulted in accelerated priming of antigen‐specific CD4⁺ T cells in the mediastinal lymph nodes and increased migration of activated CD4⁺ T cells into the lung. i.n. administration of BCG:GM‐CSF resulted in significantly increased protection against M. tuberculosis infection compared with mice vaccinated with BCG alone. BCG:GM‐CSF exhibited an improved safety profile, as immunodeficient RAG1^?/? mice vaccinated i.n. with BCG:GM‐CSF survived significantly longer than control BCG‐vaccinated mice. These data demonstrate that manipulating immune cells in the lung by BCG‐based delivery of GM‐CSF can assist the development of protective mucosal immunity against pulmonary bacterial infection.     

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.     

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.     

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.     

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.     

Robust immune response elicited by a novel and unique Mycobacterium tuberculosis protein using an optimized DNA/protein heterologous prime/boost protocol

An efficacious tuberculosis (TB) vaccine will probably need to induce both CD4 and CD8 T‐cell responses specific to a protective Mycobacterium tuberculosis antigen(s). To achieve this broad cellular immune response we tested a heterologous DNA/protein combination vaccine strategy. We used a purified recombinant protein preparation of a unique M. tuberculosis antigen (rMT1721) found in the urine of TB patients, an optimized plasmid DNA expressing this protein (DNA‐MT1721), and a Toll‐like receptor 4 agonist adjuvant. We found that priming mice with DNA‐MT1721 and subsequently boosting with rMT1721 elicited high titres of specific IgG1 and IgG2a antibodies as well as high magnitude and polyfunctional CD4⁺ T‐cell responses. However, no detectable CD8⁺ T‐cell response was observed using this regimen of immunization. In contrast, both CD4⁺ and CD8⁺ T‐cell responses were detected after a prime/boost vaccination regimen using rMT1721 as the priming antigen and DNA‐MT1721 as the boosting immunogen. These findings support the exploration of heterologous DNA/protein immunization strategies in vaccine development against TB and possibly other infectious diseases.     

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.     

Difference in TB10.4 T‐cell epitope recognition following immunization with recombinant TB10.4, BCG or infection with Mycobacterium tuberculosis

Most novel vaccines against infectious diseases are based on recombinant Ag; however, only few studies have compared Ag‐specific immune responses induced by natural infection with that induced by the same Ag in a recombinant form. Here, we studied the epitope recognition pattern of the tuberculosis vaccine Ag, TB10.4, in a recombinant form, or when expressed by the pathogen Mycobacterium tuberculosis (M.tb), or by the current anti‐tuberculosis vaccine, Mycobacterium bovis BCG. We showed that BCG and M.tb induced a similar CD4⁺ T‐cell specific TB10.4 epitope‐pattern, which differed completely from that induced by recombinant TB10.4. This difference was not due to post‐translational modifications of TB10.4 or because TB10.4 is secreted from BCG and M.tb as a complex with Rv0287. In addition, BCG and TB10.4/CAF01 were both taken up by DC and macrophages in vivo, and in vitro uptake experiments revealed that both TB10.4 and BCG were transported to Lamp⁺‐compartments. BCG and TB10.4 however, were directed to different types of Lamp⁺‐compartments in the same APC, which may lead to different epitope recognition patterns. In conclusion, we show that different vectors can induce completely different recognition of the same protein.     

Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by “dormant” M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ⁺/TNF⁺) and IFN-γ⁺ CD4⁺ T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.     

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.     

Immunogenicity and Protective Efficacy of Prime-Boost Regimens with Recombinant ΔureC hly+ Mycobacterium bovis BCG and Modified Vaccinia Virus Ankara Expressing M. tuberculosis Antigen 85A against Murine Tuberculosis

In the light of the recent emergence of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, the epidemic of tuberculosis (TB) in populations coinfected with human immunodeficiency virus, and the failure of Mycobacterium bovis bacillus Calmette-Guerin (BCG) to protect against disease, new vaccines against TB are urgently needed. Two promising new vaccine candidates are the recombinant ΔureC hly⁺ BCG (recBCG), which has been developed to replace the current BCG vaccine strain, and modified vaccinia virus Ankara (MVA) expressing M. tuberculosis antigen 85A (MVA85A), which is a leading candidate vaccine designed to boost the protective efficacy of BCG. In the present study, we examined the effect of MVA85A boosting on the protection afforded at 12 weeks postchallenge by BCG and recBCG by using bacterial CFU as an efficacy readout. recBCG-immunized mice were significantly better protected against aerosol challenge with M. tuberculosis than mice immunized with the parental strain of BCG. Intradermal boosting with MVA85A did not reduce the bacterial burden any further. In order to identify a marker for the development of a protective immune response against M. tuberculosis challenge, we analyzed splenocytes after priming or prime-boosting by using intracytoplasmic cytokine staining and assays for cytokine secretion. Boosting with MVA85A, but not priming with BCG or recBCG, greatly increased the antigen 85A-specific T-cell response, suggesting that the mechanism of protection may differ from that against BCG or recBCG. We show that the numbers of systemic multifunctional cytokine-producing cells did not correlate with protection against aerosol challenge in BALB/c mice. This emphasizes the need for new biomarkers for the evaluation of TB vaccine efficacy.     

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.     

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.     

Recombinant adenovirus delivery of calreticulin-ESAT-6 produces an antigen-specific immune response but no protection against a Mycobacterium tuberculosis challenge

Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT‐6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell‐mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT‐6 to calreticulin and constructed a recombinant replication‐deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT‐6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT‐6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon‐γ and tumour necrosis factor‐α in response to ESAT‐6. This immune response was not improved by the addition of CFP‐10 to the CRT‐ESAT‐6 fusion protein (AdCRT–ESAT‐6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low‐dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis.     

Pattern and diversity of cytokine production differentiates between Mycobacterium tuberculosis infection and disease

Tuberculosis (TB) remains a global health problem. The solution involves development of an effective vaccine, but has been limited by incomplete understanding of what constitutes protective immunity during natural infection with Mycobacterium tuberculosis. In this study, M. tuberculosis‐specific responses following an overnight whole‐blood assay were assessed by intracellular cytokine staining and luminex, and compared between TB cases and exposed household contacts. TB cases had significantly higher levels of IFN‐γ⁺TNF‐α⁺IL‐2⁺CD4⁺T cells compared with contacts. TB cases also had a significantly higher proportion of cells single‐positive for TNF‐α, but lower proportion of cells producing IL‐2 alone and these differences were seen for both CD4⁺and CD8⁺ T cells. Cytokine profiles from culture supernatants were significantly biased toward a Th1 phenotype (IFN‐γ and IL‐12(p40)) together with a complete abrogation of IL‐17 secretion in TB cases. Our data indicate that despite a robust response to TB antigens in active TB disease, changes in the pattern of cytokine production between TB infection and disease clearly contribute to disease progression.     

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.     

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.     

CD4+CD25highforkhead box protein 3+ regulatory T lymphocytes suppress interferon‐γ and CD107 expression in CD4+ and CD8+ T cells from tuberculous pleural effusions

Tuberculous pleural effusion is characterized by a T helper type 1 (Th1) profile, but an excessive Th1 response may also cause tissue damage that might be controlled by regulatory mechanisms. In the current study we investigated the role of regulatory T cells (T_reg) in the modulation of Th1 responses in patients with tuberculous (TB) pleurisy. Using flow cytometry we evaluated the proportion of T_reg (CD4⁺CD25^highforkhead box protein 3⁺), interferon (IFN)‐γ and interleukin (IL)‐10 expression and CD107 degranulation in peripheral blood (PB) and pleural fluid (PF) from patients with TB pleurisy. We demonstrated that the proportion of CD4⁺CD25⁺, CD4⁺CD25^highFoxP3⁺ and CD8⁺CD25⁺ cells were increased in PF compared to PB samples. Mycobacterium tuberculosis stimulation increased the proportion of CD4⁺CD25^low/negIL‐10⁺ in PB and CD4⁺CD25^low/negIFN‐γ⁺ in PF; meanwhile, CD25^high mainly expressed IL‐10 in both compartments. A high proportion of CD4⁺CD107⁺ and CD8⁺CD107⁺ cells was observed in PF. T_reg depletion enhanced the in‐vitro M. tuberculosis‐induced IFN‐γ and CD4⁺ and CD8⁺ degranulation responses and decreased CD4⁺IL‐10⁺ cells in PF. Our results demonstrated that in TB pleurisy T_reg cells effectively inhibit not only IFN‐γ expression but also the ability of CD4⁺ and CD8⁺ cells to degranulate in response to M. tuberculosis.