Evaluation of specific humoral immune response and cross reactivity against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> antigens induced in mice immunized with liposomes composed of total lipids extracted from <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

The development of a new tuberculosis (TB) vaccine has become one of the main objectives of the scientific community. Protein antigens have been widely explored as subunit TB vaccines, however lipid antigens could be equally important to be used or included in such a vaccine. The aim of this study was to demonstrate the potential of a liposome formulation composed of an extract of lipids from Mycobacterium smegmatis (Ms) as a TB vaccine candidate. We evaluated the immunogenicity of this formulation as well as the cross reactive response against antigens from Mycobacterium tuberculosis (MTb) in BALB/c mice. We determined the anti-liposome IgG response in sera from TB patients and from healthy subjects who displayed a positive (PPD+) or negative (PPD-) tuberculin skin test. A significant increase in anti-liposome IgG (p<0.05) was detected in animals immunized with Bacille Calmette-Guérin (BCG) compared with all groups, and in the group immunized with liposomes from Ms (LMs) compared to animals immunized with either LMs adjuvanted with aluminium (LMs-A) or the negative control group (phosphate buffered saline, PBS) respectively. With respect to the cross reactive response against a cocktail of cell wall antigens (CWA) from MTb, significantly higher IgG levels were observed in animals immunized with BCG and LMs compared to negative controls and either, aluminium-adjuvanted liposomes (LMs-A) or montanide (LMs-M) (p<0.05). Furthermore, the anti-liposome IgG response was significantly superior in sera from pulmonary TB patients compared to PPD+ and PPD- healthy subjects (p<0.001) suggesting the expression of these antigens in vivo during active MTb infection. The results obtained provide some evidence for the potential use of liposomes containing total lipid extracts of Ms as a TB vaccine candidate.     

Intradermal and oral immunization with recombinant Mycobacterium bovis BCG expressing the simian immunodeficiency virus Gag protein induces long-lasting, antigen-specific immune responses in guinea pigs

To develop a new recombinant BCG (rBCG) vaccine, we constructed rBCG that expresses the full-length Gag protein of simian immunodeficiency virus (rBCG-SIVGag) at a level of 0.5 ng/mg after 3 weeks of bacterial cell culture. Intradermal (i.d.) inoculation of guinea pigs with 0.1 mg of rBCG-SIVGag resulted in the induction of delayed-type hypersensitivity (DTH) responses to both purified protein derivative (PPD) of tuberculin and SIV Gag p27 protein; responses that were maintained for the duration of the 50-week study. In contrast, guinea pigs orally vaccinated with 160 mg of the same antigen exhibited a long-lasting DTH response to the SIV Gag p27 protein, but mounted no response to PPD. Proliferative responses to SIV Gag p27 and PPD antigens were detected in both i.d. and orally immunized animals; however, the levels of PPD-specific responses were significantly higher in guinea pigs immunized by the i.d. than the oral route. A significant increase in the level of PPD- and SIV Gag p27-specific IFNgamma mRNA expression was also detected in both immunization groups receiving rBCG-SIVGag. In addition, both i.d. and oral immunization with rBCG-SIVGag induced PPD- and SIV Gag p27-specific serum IgG responses. Insertion of the SIV gag gene into BCG did not appear to change the ability of rBCG-immunized animals to elicit PPD-specific immune responses. These results indicate that rBCG-SIVGag has the ability to effectively induce long-lasting, cell-mediated and humoral immunity against both viral and bacterial antigens in guinea pigs, suggesting that rBCG-Gag has the potential to elicit immunities specific not only for tuberculosis but also for HIV at human doses.     

Immunogenicity and cross-reactivity against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> of proteoliposomes derived from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG

The only currently available vaccine against tuberculosis (TB) is Mycobacterium bovis Bacille Calmette-Guerin (BCG), which has inconsistent efficacy to protect against the disease in adults. M. tuberculosis (MTB) cell wall components have been implicated in the pathogenicity of TB and therefore have been a prime target for the identification and characterization of cell wall proteins with potential application in vaccine development. In this regard, proteoliposomes (PLs) derived from mycobacteria containing lipids and cell wall proteins could be potential vaccine candidates against TB. In the present study PLs derived from BCG were prepared. These homogeneous population of spherical microparticles was then immunized into Balb/c mice. Sera of immunized animals showed high IgG response and strong cross-reactivity against different MTB antigens.These results showed that BCG PLs could be potential vaccine candidates against TB.     

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.     

The ID93 Tuberculosis Vaccine Candidate Does Not Induce Sensitivity to Purified Protein Derivative

The tuberculin skin test (TST) is a simple and inexpensive test to determine whether individuals have been exposed to Mycobacterium tuberculosis. This test is not always reliable, however, in people previously immunized with BCG and/or who have been exposed to environmental mycobacterial species due to a reaction to purified protein derivative (PPD) used in the skin test. An issue with BCG, therefore, is that the resulting sensitization to PPD in some individuals compromises the diagnostic use of the skin test. The ability to induce protective immune responses without sensitizing to the tuberculin skin test will be important properties of next-generation tuberculosis (TB) vaccine candidates. We show here that guinea pigs immunized with the candidate TB vaccine ID93/GLA-SE, currently in clinical trials, do not react to intradermal PPD administration. In contrast, positive DTH responses to both ID93 and components thereof were induced in ID93/GLA-SE-immunized animals, indicating robust but specific cellular responses were present in the immunized animals. Noninterference with the TST is an important factor for consideration in the development of a vaccine against M. tuberculosis.     

Immune Responses in Mice After Gastric and Subcutaneous Immunization with BCG

Mucosal immunization can induce immune responses different from those induced by systemic immunization. In this study, murine immune responses were analysed after immunization through gastric and subcutaneous routes with Mycobacterium bovis BCG. The number of circulating cells secreting antigen-specific immunoglobulins (Ig), and the number of gamma interferon (IFN-γ) secreting cells in spleen-cell cultures after in vitro stimulation with mycobacterial antigens were analysed at the single cell level by the ELISPOT method. Levels of antigen-specific antibodies in sera were determined by ELISA. In the subcutaneously immunized mice the authors found approximately 100 times more splenic cells secreting antigen-specific IgG than in gastrically immunized mice or control mice. Their levels of antigen-specific IgG in sera were 66–6700 times higher than in sera from gastrically immunized mice or control mice. In contrast, the numbers of IFN-γ producing cells in spleen-cell cultures after in vitro activation with BCG were equally high in the immunized groups of mice, and for both groups higher than in non-immunized controls. Furthermore, IFN-γ producing cells could be demonstrated in gastrically immunized animals even without in vitro activation. The results demonstrate that gastric immunization with the BCG vaccine can induce a systemic T cell-mediated immune response against mycobacterial antigens.     

Development of new vaccines and diagnostic reagents against tuberculosis

Tuberculosis (TB) is a major infectious disease problem with one-third of the world population infected, 8 million people developing the active disease and 2 million dying of TB each year. The attenuated Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only available vaccine against TB. However, the trials conducted in different parts of the world have shown that this vaccine doe not provide consistent protection against TB. The purified protein derivative (PPD) of Mycobacterium tuberculosis is the commonly used reagent for the diagnosis of TB. However, PPD lacks specificity because of the presence of antigens crossreactive with M. bovis BCG and other mycobacteria. The studies to identify M. tuberculosis antigens and epitopes as candidates for new protective vaccines and specific diagnostic reagents against TB have led to the identification and characterization of several major antigens of M. tuberculosis including heat shock proteins (hsp) and secreted antigens present in the culture filtrate (CF) of M. tuberculosis. Some of these antigens have shown promise as new candidate vaccines (hsp60, Ag85 and ESAT-6, etc.) and specific diagnostic reagents (ESAT-6 and CFP10, etc.) for TB. Moreover, in the mouse model of TB, vaccination with DNA-hsp60 has immunotheraputic effects and helps in eradication of persisters. In addition, identification of proper adjuvant and delivery systems has shown the promise to overcome the problem of poor immunogenicity associated with subunit and peptide based vaccines. More recently, the comparison of the genome sequence of M. tuberculosis with M. bovis BCG and other mycobacteria has led to the identification of M. tuberculosis-specific genomic regions. Evaluation of these regions for encoding proteins with immunological reactivity can lead to the identification of additional antigens of M. tuberculosis useful as new vaccines and reagents for specific diagnosis of TB.     

A new vaccine against tuberculosis affords greater survival after challenge than the current vaccine in the guinea pig model of pulmonary tuberculosis

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate vaccine, Mycobacterium bovis BCG, is urgently needed. We describe a recombinant BCG vaccine (rBCG30) expressing and secreting the 30-kDa major secretory protein of Mycobacterium tuberculosis, the primary causative agent of TB, that affords greater survival after challenge than parental BCG in the highly demanding guinea pig model of pulmonary TB. Animals immunized with rBCG30 and then challenged by aerosol with a highly virulent strain of M. tuberculosis survived significantly longer than animals immunized with conventional BCG. The parental and recombinant vaccine strains are comparably avirulent in guinea pigs, as they display a similar pattern of growth and clearance in the lung, spleen, and regional lymph nodes. The pMTB30 plasmid encoding the 30-kDa protein is neither self-transmissible nor mobilizable to other bacteria, including mycobacteria. The pMTB30 plasmid can be stably maintained in Escherichia coli but is expressed only in mycobacteria. The recombinant and parental strains are sensitive to the same antimycobacterial antibiotics. rBCG30, the first vaccine against TB more potent than nearly century-old BCG, is being readied for human clinical trials.     

Evaluation of the humoral immune response and cross reactivity against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> of mice immunized with liposomes containing glycolipids of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Mycobacterium smegmati s (Ms) is a nonpathogenic mycobacteria of rapid growth, which shares many characteristics with Mycobacterium tuberculosis (MTB), the major causative agent of tuberculosis. MTB has several cell wall glycolipids in common with Ms, which play an important role in the pathogenesis of tuberculosis and the induction of a protective immune response against MTB infection in some animal models. In this study, the humoral immune response and cross reactivity against MTB, of liposomes containing a mixture of cell wall glycolipids of Ms and commercial lipids was evaluated, in order to study its possible use as a component of a vaccine candidate against tuberculosis. Liposomes containing total lipids extracted from Ms, distearoyl phosphatidyl choline and cholesterol were prepared by the dehydration-rehydration technique. Balb/c mice were immunized with the liposomes obtained and the antibody response and cross reactivity against MTB were tested by ELISA. Total lipids extract from Ms showed the presence of several polar glycolipids in common with MTB, such as phosphatidylinositol mannosides. Liposomes that contained glycolipids of Ms were capable of inducing a specific IgG antibody response that allowed the recognition of surface antigens of MTB. The results of this study demonstrated the presence of immunogenic glycolipids in Ms, which could be included to enhance the protective effects of subunit vaccine formulations against tuberculosis.     

Recombinant BCG coexpressing Ag85B,ESAT-6 and Rv3620c elicits specific Th1 immune responses in C57BL/6 mice

Tuberculosis (TB) remains to be an enormous global health problem. The inconsistent protection efficacy of Bacille Calmette-Guérin (BCG) calls for new vaccines for TB. One choice to improve the efficacy of BCG vaccine is recombinant BCG (rBCG). Experimental evidences have revealed that Ag85B, ESAT-6 and Rv3620c are important immunodominant antigens of Mycobacterium tuberculosis. In this study, we have constructed a novel rBCG expressing fusion protein Ag85B-ESAT6-Rv3620c and evaluated the immunogenicity of this rBCG in C57BL/6 mice. Results show that there is a strong TB-specific CD4⁺ and CD8⁺ T lymphocytes proliferation in mice immunized with this rBCG vaccine. A single dose immunization of rBCG could induce a significantly strong Th1 immune response characterized by an increasing ratio of antigen-specific IgG2b/IgG1 as well as a high expression level of Th1 cytokines such as IFN-γ, TNF-α and IL-2. This conclusion was confirmed by a decreased secretion of Th2 cytokine IL-10. Moreover, this rBCG induced a strong humoral response in mice with an increasing antigen-specific IgG titer. Therefore, we concluded that this rBCG could significantly increase both Th1 type cellular immune response and antigen-specific humoral response compared with BCG. The above observations demonstrated that rBCG::Ag85B-ESAT6-Rv3620c is a potential candidate vaccine against M. tuberculosis for further study.     

Supplementation with RD antigens enhances the protective efficacy of BCG in tuberculous mice

Different combinations of ESAT-6, CFP-10, CFP-21, MPT-64, encoded by RD1 and RD2 of Mycobacterium tuberculosis were evaluated on the basis of antigenicity in PPD positive TB contacts and immunogenicity in C57BL/6J mice immunized with the combination of all four RD antigens. The peripheral blood mononuclear cells of TB contacts showed maximum recognition in response to the combination of ESAT-6+MPT-64 in terms of predominant lymphoproliferation, IFN-gamma levels and the number of responders. On the contrary, the combination of ESAT-6+CFP-21+MPT-64 was found to be most immunogenic based on both T-cell and antibody responses in immunized mice. Prophylactic potential of the selected combinations was assessed as supplementation vaccines to BCG against intravenous challenge with M. tuberculosis in mice. BCG supplementation with the selected combinations resulted in significantly greater protection as compared to BCG alone against experimental tuberculosis and thus appears to be a promising approach to enhance the protective efficacy of the existing vaccine.     

Inhibition of Mycobacterial Growth In Vitro following Primary but Not Secondary Vaccination with Mycobacterium bovis BCG

Despite the widespread use of the Mycobacterium bovis BCG vaccine, there are more than 9 million new cases of tuberculosis (TB) every year, and there is an urgent need for better TB vaccines. TB vaccine candidates are selected for evaluation based in part on the detection of an antigen-specific gamma interferon (IFN-γ) response. The measurement of mycobacterial growth in blood specimens obtained from subjects immunized with investigational TB vaccines may be a better in vitro correlate of in vivo vaccine efficacy. We performed a clinical study with 30 United Kingdom adults who were followed for 6 months to evaluate the abilities of both a whole-blood- and a novel peripheral blood mononuclear cell (PBMC)-based mycobacterial growth inhibition assay to measure a response to primary vaccination and revaccination with BCG. Using cryopreserved PBMCs, we observed a significant improvement in mycobacterial growth inhibition following primary vaccination but no improvement in growth inhibition following revaccination with BCG (P < 0.05). Mycobacterial growth inhibition following primary BCG vaccination was not correlated with purified protein derivative (PPD) antigen-specific IFN-γ enzyme-linked immunospot (ELISPOT) responses. We demonstrate that a mycobacterial growth inhibition assay can detect improved capacity to control growth following primary immunization, but not revaccination, with BCG. This is the first study to demonstrate that an in vitro growth inhibition assay can identify a difference in vaccine responses by comparing both primary and secondary BCG vaccinations, suggesting that in vitro growth inhibition assays may serve as better surrogates of clinical efficacy than the assays currently used for the assessment of candidate TB vaccines.     

Antigen-binding small lymphocytes in the guinea-pig: II. The immunological response to purified protein derivative of mammalian tuberculin

A mean of 6.9 per cent of small lymphocytes in peripheral blood preparations and between 1.8 and 2.4 per cent of small lymphocytes in lymph node, spleen, bone marrow and thymus preparations from unimmunized guinea-pigs bound ¹²⁵I-labelled purified protein derivative of mammalian tuberculin (mammalian PPD). The percentage of these cells fluctuated but did not alter substantially after immunization with BCG or with BCG emulsified with human thyroglobulin (HTg) in Freund's incomplete adjuvant (FIA). Blocking experiments indicated that the binding of ¹²⁵I-labelled mammalian PPD was specific and there was tentative evidence that the lymphocyte receptors may be IgG. A comparison is drawn between the observed time course of ¹²⁵I-labelled mammalian PPD-binding small lymphocytes and the response of lymphocytes sensitive to strong histocompatibility antigens, and it is proposed that the propensity of certain antigens to induce a delayed hypersensitivity-type response is related to the presence of substantial numbers of antigen-binding cells in unimmunized animals.

A noteworthy incidental finding was an unexplained depression in the cellular and humoral responses to mammalian PPD in guinea-pigs that had been immunized with HTg—BCG—FIA emulsion.

     

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.     

Skewing of the Th1/Th2 responses in mice due to variation in the level of expression of an antigen in a recombinant BCG system

In spite of rapid developments in the study of mycobacteria during the last two decades, tuberculosis (TB) has maintained its status as the leading killer among all infectious diseases. Extensive evidence exists to support a central role for a T-helper type 1 (Th1) immune response for protection against TB in mice and humans. Bacille Calmette-Guerin (BCG), the only vaccine against TB, although not perfect in its ability to protect against the adult form of TB, is a strong inducer of Th1 responses and is being increasingly used as a delivery vehicle for the presentation of foreign antigens to the immune system. It has been proposed that expression of immunodominant antigens or cytokine genes in BCG can enhance the ability of BCG to induce a Th1 immune response. Since dose of the antigen is considered as one of the parameters that influence the Th cell responses, the level of expression of the candidate antigen should influence the final Th response against the recombinant BCG (rBCG). In the present study, the effect of over-expression of a candidate antigen Antigen 85B (Ag 85B) in a rBCG system, on the Th-priming ability of BCG has been investigated in the murine model. BALB/c mice were immunized with three different rBCG constructs expressing Ag 85B to various levels. Induction of Th1/Th2 responses was analyzed by measuring levels of interferon-gamma (Th1) and interleukin-10 (Th2) in antigen-stimulated splenocyte cultures and by quantifying the antigen-specific IgG2a (Th1) and IgG1 (Th2) antibody responses. By varying the level of expression of Ag 85B, specific immune responses against Ag 85B were observed to range from mixed Th1/Th2 to Th1. However, the BCG-specific immune responses in case of all rBCG-immunized animals remained predominantly Th1.     

Manipulation of BCG vaccine: a double-edged sword

Mycobacterium bovis Bacillus Calmette–Guérin (BCG), an attenuated vaccine derived from M. bovis, is the only licensed vaccine against tuberculosis (TB). Despite its protection against TB in children, the protective efficacy in pulmonary TB is variable in adolescents and adults. In spite of the current knowledge of molecular biology, immunology and cell biology, infectious diseases such as TB and HIV/AIDS are still challenges for the scientific community. Genetic manipulation facilitates the construction of recombinant BCG (rBCG) vaccine that can be used as a highly immunogenic vaccine against TB with an improved safety profile, but, still, the manipulation of BCG vaccine to improve efficacy should be carefully considered, as it can bring in both favourable and unfavourable effects. The purpose of this review is not to comprehensively review the interaction between microorganisms and host cells in order to use rBCG expressing M. tuberculosis (Mtb) immunodominant antigens that are available in the public domain, but, rather, to also discuss the limitations of rBCG vaccine, expressing heterologous antigens, during manipulation that pave the way for a promising new vaccine approach.     

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.     

Correlation of PPD and BCG-induced leukocyte migration inhibition,delayed cutaneous hypersensitivity,lymphocyte transformation in vitro and humoral antibodies to PPD in man

In a study of healthy human individuals a complete lack of correlation between the results of the agarose leukocyte migration inhibition (LMI) test, using purified protein derivative (of tuberculin) (PPD) and Bacillus Calmette Guérin (BCG) as antigens, and delayed cutaneous hypersensitivity and lymphocyte transformation in vitro to PPD was found. There was a reasonable correlation between PPD- and BCG-induced LMI. Antibodies to PPD proved to have no influence on PPD-induced LMI. Purified polymorphonuclear leukocytes, whether derived from donors sensitive to PPD in the agarose LMI test or from nonsensitive donors, did not show migration inhibition to PPD. It was concluded that polymorphonuclear leukocytes and lymphocytes need to be present simultaneously for migration inhibition of peripheral blood leukocytes by PPD. Furthermore, because a consistent relation with conventional parameters of cell-mediated immunity was lacking, it is doubtful whether the agarose LM1 test can be considered as an alternative parameter of this kind of immunity.     

Failure of the Mycobacterium bovis BCG Vaccine: Some Species of Environmental Mycobacteria Block Multiplication of BCG and Induction of Protective Immunity to Tuberculosis

The efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine against pulmonary tuberculosis (TB) varies enormously in different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment, but the precise mechanism has so far not been clarified. Our study demonstrates that prior exposure to live environmental mycobacteria can result in a broad immune response that is recalled rapidly after BCG vaccination and controls the multiplication of the vaccine. In these sensitized mice, BCG elicits only a transient immune response with a low frequency of mycobacterium-specific cells and no protective immunity against TB. In contrast, the efficacy of TB subunit vaccines was unaffected by prior exposure to environmental mycobacteria. Six different isolates from soil and sputum samples from Karonga district in Northern Malawi (a region in which BCG vaccination has no effect against pulmonary TB) were investigated in the mouse model, and two strains of the Mycobacterium avium complex were found to block BCG activity completely.     

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.