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.     

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.     

High‐frequency vaccine‐induced CD8+ T cells specific for an epitope naturally processed during infection with Mycobacterium tuberculosis do not confer protection

Relatively few MHC class I epitopes have been identified from Mycobacterium tuberculosis, but during the late stage of infection, CD8⁺ T‐cell responses to these epitopes are often primed at an extraordinary high frequency. Although clearly available for recognition during infection, their role in resistance to mycobacterial infections still remain unclear. As an alternative to DNA and viral vaccination platforms, we have exploited a novel CD8⁺ T‐cell‐inducing adjuvant, cationic adjuvant formulation 05 (dimethyldioctadecylammonium/trehalose dibehenate/poly (inositic:cytidylic) acid), to prime high‐frequency CD8 responses to the immunodominant H2‐K^b‐restricted IMYNYPAM epitope contained in the vaccine Ag tuberculosis (TB)10.4/Rv0288/ESX‐H (where ESX is mycobacterial type VII secretion system). We report that the amino acid C‐terminal to this minimal epitope plays a decisive role in proteasomal cleavage and epitope priming. The primary structure of TB10.4 is suboptimal for proteasomal processing of the epitope and amino acid substitutions in the flanking region markedly increased epitope‐specific CD8⁺ T‐cell responses. One of the optimized sequences was contained in the closely related TB10.3/Rv3019c/ESX‐R Ag and when recombinantly expressed and administered in the cationic adjuvant formulation 05 adjuvant, this Ag promoted very high CD8⁺ T‐cell responses. This abundant T‐cell response was functionally active but provided no protection against challenge, suggesting that CD8⁺ T cells play a limited role in protection against M. tuberculosis in the mouse model.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Crosstalk between human DC subsets promotes antibacterial activity and CD8+ T‐cell stimulation in response to bacille Calmette‐Guérin

To date, little is known about the unique contributions of specialized human DC subsets to protection against tuberculosis (TB). Here, we focus on the role of human plasmacytoid (p)DCs and myeloid (m)DCs in the immune response to the TB vaccine bacille Calmette‐Guérin (BCG). Ex vivo DC subsets from human peripheral blood were purified and infected with BCG expressing GFP to distinguish between infected and noninfected cells. BDCA‐1⁺ myeloid DCs were more susceptible than BDCA‐3⁺ mDCs to BCG infection. Plasmacytoid DCs have poor phagocytic activity but are equipped with endocytic receptors and can be activated by bystander stimulation. Consequently, the mutual interaction of the two DC subsets in response to BCG was analyzed. We found that pDCs were activated by BCG‐infected BDCA‐1⁺ mDCs to upregulate maturation markers and to produce granzyme B, but not IFN‐α. Reciprocally, the presence of activated pDCs enhanced mycobacterial growth control by infected mDCs and increased IL‐1β availability. The synergy between the two DC subsets promoted BCG‐specific CD8⁺ T‐cell stimulation and the role of BCG‐infected BDCA‐1⁺ mDCs could not be efficiently replaced by infected BDCA‐3⁺ mDCs in the crosstalk with pDCs. We conclude that mDC–pDC crosstalk should be exploited for rational design of next‐generation TB vaccines.     

MyD88 and TRIF synergistic interaction is required for TH1‐cell polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant‐stable emulsion (GLA‐SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly‐functional T_H1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR‐domain‐containing adapter inducing IFN‐beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal T_H1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA‐SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild‐type mice. We find that both MyD88 and TRIF are necessary for GLA‐SE to induce a poly‐functional T_H1 immune response characterized by CD4⁺ T cells producing IFN‐γ, TNF, and IL‐2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA‐SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo T_H1‐skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.     

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.     

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.     

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.     

Mucosal delivery of antigen‐coated nanoparticles to lungs confers protective immunity against tuberculosis infection in mice

Mucosal boosting of BCG‐immunised individuals with a subunit tuberculosis (TB) vaccine would be highly desirable, considering that the lungs are the principal port of entry for Mycobacterium tuberculosis (MTB) and the site of the primary infection and reactivation. However, the main roadblock for subunit TB vaccine development is the lack of suitable adjuvants that could induce robust local and systemic immune responses. Here, we describe a novel vaccine delivery system that was designed to mimic, in part, the MTB pathogen itself. The surface of yellow carnauba wax nanoparticles was coated with the highly immunogenic Ag85B Ag of MTB and they were directed to the alveolar epithelial surfaces by the incorporation of the heparin‐binding hemagglutinin adhesion (HBHA) protein. Our results showed that the i.n. immunisation of BCG‐primed BALB/c mice with nanoparticles adsorbed with Ag85B‐HBHA (Nano‐AH vaccine) induced robust humoral and cellular immune responses and IFN‐γ production, and multifunctional CD4⁺ T cells expressing IFN‐γ, IL‐2 and TNF‐α. Mice challenged with H37Rv MTB had a significantly reduced bacterial load in their lungs when compared with controls immunised with BCG alone. We therefore conclude that this immunisation approach is an effective means of boosting the BCG‐induced anti‐TB 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.