Intranasal boosting with MVA encoding secreted mycobacterial proteins Ag85A and ESAT-6 generates strong pulmonary immune responses and protection against M. tuberculosis in mice given BCG as neonates

Bacille-Calmette-Guerin (BCG) has variable efficacy as an adult tuberculosis (TB) vaccine but can reduce the incidence and severity of TB infection in humans. We have engineered modified vaccinia Ankara (MVA) strain vaccine constructs to express the secreted mycobacterial proteins Ag85A and ESAT-6 (MVA-AE) and evaluated their immunogenicity and protective efficacy as mucosal booster vaccines for BCG given subcutaneously in early life. Intranasal delivery of MVA-AE to young adult mice induced CD4⁺ and CD8⁺ T cell responses to both Ag85A and ESAT-6 in lung mucosae. These responses were markedly enhanced in mice that had been primed neonatally with BCG prior to intranasal MVA-AE immunization (BCG/MVA-AE), as evidenced by numbers of pulmonary Ag85A-, ESAT-6-, and PPD-specific CD4⁺ and CD8⁺ T cells and by their capacity to secrete multiple antimicrobial factors, including IFNγ, IL-2 and IL-17. Moreover, MVA-AE boosting generated multifunctional lung CD4⁺ T cells responding to ESAT-6, which were not, as expected, detected in control mice given BCG, and elevated Ag85A-specific circulating antibody responses. After aerosol challenge with M. tuberculosis H37Rv (Mtb), the BCG/MVA-AE group had significantly reduced mycobacterial burden in the lungs, compared with either BCG primed mice boosted with control MVA or mice given only BCG. These data indicate that intranasal delivery of MVA-AE can boost BCG-induced Th1 and Th17-based immunity locally in the lungs and improve the protective efficacy of neonatally-administered BCG against M. tuberculosis infection.     

Using an effective TB vaccination regimen to identify immune responses associated with protection in the murine model

A vaccine against tuberculosis (TB), a disease resulting from infection with Mycobacterium tuberculosis (M.tb), is urgently needed to prevent more than a million deaths per year. Bacillus Calmette–Guérin (BCG) is the only available vaccine against TB but its efficacy varies throughout the world. Subunit vaccine candidates, based on recombinant viral vectors expressing mycobacterial antigens, are one of the strategies being developed to boost BCG-primed host immune responses and efficacy. A promising vaccination regimen composed of intradermal (i.d.) BCG prime, followed by intranasally (i.n.) administered chimpanzee adenoviral vector (ChAdOx1) and i.n. or i.d. modified vaccinia Ankara virus (MVA), both expressing Ag85A, has been previously reported to significantly improve BCG efficacy in mice. Effector and memory immune responses induced by BCG-ChAdOx1.85A-MVA85A (B-C-M), were evaluated to identify immune correlates of protection in mice. This protective regime induced strong Ag85A-specific cytokine responses in CD4⁺ and CD8⁺ T cells, both in the systemic and pulmonary compartments. Lung parenchymal CXCR3⁺ KLRG1^- Ag85A-specific memory CD4⁺ T cells were significantly increased in B-C-M compared to BCG immunised mice at 4, 8 and 20 weeks post vaccination, but the number of these cells decreased at the latter time point. This cell population was associated with the protective efficacy of this regime and may have an important protective role against M.tb infection.     

An attenuated Salmonella-vectored vaccine elicits protective immunity against Mycobacterium tuberculosis

There is a need to develop protective vaccines against tuberculosis (TB) that elicit full immune responses including mucosal immunity. Here, a live attenuated Salmonellatyphimurium aroA SL7207 vector TB vaccine, namely SL(E6-85B), harboring the Mycobacterium tuberculosis (M. tb) H37Rv ESAT6-Ag85B fusion gene was developed. The experimental data demonstrated that this SL(E6-85B) vaccine, or when it is combined with BCG vaccination, induced the strongest TB Ag-specific mucosal, humoral, and cellular immune responses comprised of increased proliferation of T cells, IFN-gamma expression, granzyme B production, as well as the greatest IFN-gamma production of effector-memory T (T_EM) or effector CD8⁺ T cell responses and exerted high protective efficacy in mice against virulent M. tb H37Rv challenge compared to the other vaccinated groups (mice immunized with SL(Ag85B), a DNA vaccine or BCG only). This strategy may represent a novel promising mucosal vaccine candidate for the prevention of TB which are inexpensive to produce, efficacious, and able to be given orally rather than by injection.     

A recombinant adenovirus expressing immunodominant TB antigens can significantly enhance BCG-induced human immunity

Background

Despite the availability of Bacille Calmette Guérin (BCG) vaccines, Mycobacterium tuberculosis currently infects billions of people and millions die annually from tuberculosis (TB) disease. New TB vaccines are urgently needed.

Methods

We studied the ability of AERAS-402, a recombinant, replication-deficient adenovirus type 35 expressing the protective M. tuberculosis antigens Ag85A, Ag85B, and TB10.4, to boost BCG immunity in an area of low TB endemicity.

Results

In volunteers primed with BCG 3 or 6 months prior to AERAS-402 boosting, significant CD4⁺ and CD8⁺ T cell responses were induced. Ag85-specific responses were more strongly boosted than TB10.4-specific responses. Frequencies of TB-specific CD8⁺ T cells reached > 50 fold higher than pre-AERAS boosting levels, remarkably higher than reported in any previous human TB vaccine trial. Multiparameter flow cytometric assays demonstrated that AERAS-402-boosted CD4⁺ and CD8⁺ T cells were multifunctional, producing multiple cytokines and other immune effector molecules. Furthermore, boosted T cells displayed lymphoproliferative capacity, and tetramer analyses confirmed that antigen-specific CD8⁺ T cells were induced. BCG and AERAS-402 vaccinations given 3 and 6 months apart appeared equivalent.

Conclusions

Our results indicate that AERAS-402 is a promising TB vaccine candidate that can significantly enhance both CD4⁺ and CD8⁺ TB-specific T cell responses after BCG priming.ClinicalTrials.gov Identifier: NCT01378312.     

Enhanced protection against tuberculosis by vaccination with recombinant BCG over-expressing HspX protein

Immunization with Mycobacterium bovis Bacille Calmette–Guerin (BCG) did not induce adequate Th1 responses to the latency antigen, HspX of M. tuberculosis. To increase the immunogenicity and protective efficacy of BCG, a recombinant BCG strain over-expressing antigen HspX (rBCG::X) was constructed. The recombinant strain rBCG::X expressed high levels of both HspX protein in the cytosol and Ag85B protein in the cytosol and supernatant. Mice vaccinated with rBCG::X produced a more consistent and enduring protective effect against infection with M. tuberculosis, showing lower bacterial load in lung and less severe lung pathology, than the control mice vaccinated with BCG strain containing the vector pMV261. The long-term protection induced by rBCG::X was associated with significant increases in antigen-specific IFN-γ to both HspX and Ag85B proteins, while PPD-specific IFN-γ responses declined. Our results suggest that latency antigens of M. tuberculosis may be promising targets for developing more effective recombinant BCG strains to protect against TB.     

Fusion protein Ag85B-MPT64190–198-Mtb8.4 has higher immunogenicity than Ag85B with capacity to boost BCG-primed immunity against Mycobacterium tuberculosis in mice

Tuberculosis (TB) remains a major infectious disease worldwide despite chemotherapy and BCG vaccine. The efficacy of the current TB vaccine BCG varies from 0 to 80%. New vaccines that have better protection than BCG or have the capability to boost BCG-primed immunity are urgently needed. We have previously constructed a fusion protein Ag85B-MPT64_190–198-Mtb8.4 (AMM). In this study, we investigated the immunogenicity of the fusion protein AMM in a novel adjuvant of dimethyl-dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA–BCG PSN), and its capacity to boost BCG-primed immunity. The anti-Ag85B antibodies IgG1 and IgG2a were determined using ELISA and the number of spleen cells secreting IFN-γ was determined by ELISPOT. In addition, the ability of the subunit vaccine AMM to boost BCG-primed immunity against Mycobacterium tuberculosis was analyzed. The fusion protein AMM induced more effective humoral and cell-mediated immune responses in mice than Ag85B alone. Mice primed with BCG vaccination followed by boosting with AMM produced a stronger immune response and afforded a better protection against M. tuberculosis infection than mice immunized with BCG alone or BCG priming followed by boosting with Ag85B. These findings suggest that AMM is a promising candidate subunit vaccine to enhance the protective efficiency of BCG.     

Protective efficacy of recombinant BCG over-expressing protective,stage-specific antigens of Mycobacterium tuberculosis

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, yet current control strategies, including the existing BCG vaccine, have had little impact on disease control. CysVac2, a fusion protein comprising stage-specific Mycobacterium tuberculosis antigens, provided superior protective efficacy against chronic M. tuberculosis infection in mice, compared to BCG. To determine if the delivery of CysVac2 in the context of BCG could improve BCG-induced immunity and protection, we generated a recombinant strain of BCG overexpressing CysVac2 (rBCG:CysVac2). Expression of CysVac2 in BCG was facilitated by the M. tuberculosis hspX promoter, which is highly induced inside phagocytic cells and induces strong cellular immune responses to antigens expressed under its regulation. Intradermal vaccination with rBCG:CysVac2 resulted in increased monocyte/macrophage recruitment and enhanced antigen-specific CD4⁺ T cell priming compared to parental BCG, indicating CysVac2 overexpression had a marked effect on rBCG induced-immunity. Further, rBCG:CysVac2 was a more potent inducer of antigen-specific multifunctional CD4⁺ T cells (CD4⁺IFN-γ⁺TNF⁺IL-2⁺) than BCG after vaccination of mice. This improved immunogenicity however did not influence protective efficacy, with both BCG and rBCG:CysVac2 affording comparable level of protection aerosol infection with M. tuberculosis. Boosting either BCG or rBCG:CysVac2 with the CysVac2 fusion protein resulted in a similar improvement in protective efficacy. These results demonstrate that the expression of protective antigens in BCG can augment antigen-specific immunity after vaccination but does not alter protection against infection, further highlighting the challenge of developing effective vaccines to control TB.     

A multi-valent vaccinia virus-based tuberculosis vaccine molecularly adjuvanted with interleukin-15 induces robust immune responses in mice

Tuberculosis caused by Mycobacterium tuberculosis is responsible for nearly two million deaths every year globally. A single licensed vaccine derived from Mycobacterium bovis, bacille Calmette-Guerin (BCG) administered perinatally as a prophylactic vaccine has been in use for over 80 years and confers substantial protection against childhood tuberculous meningitis and miliary tuberculosis. However, the BCG vaccine is virtually ineffective against the adult pulmonary form of tuberculosis that is pivotal in the transmission of tuberculosis that has infected almost 33% of the global population. Thus, an effective vaccine to both prevent tuberculosis and reduce its transmission is urgently needed. We have generated a multi-valent, vectored vaccine candidate utilizing the modified virus Ankara (MVA) strain of vaccinia virus to tandemly express five antigens, ESAT6, Ag85A, Ag85B, HSP65 and Mtb39A of M. tuberculosis that have been reported to be protective individually in certain animal models together with an immunostimulatory cytokine interleukin-15 (MVA/IL-15/5Mtb). Although, immunological correlates of protection against tuberculosis in humans remain to be established, we demonstrate that our vaccine induced comparable CD4⁺ T cell and greater CD8⁺ T cell and antibody responses against M. tuberculosis in vaccinated mice in a direct comparison with the BCG vaccine and conferred protection against an aerogenic challenge of M. tuberculosis, thus warranting its further preclinical development.     

Novel recombinant BCG expressing perfringolysin O and the over-expression of key immunodominant antigens; pre-clinical characterization,safety and protection against challenge with Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected approximately two billion individuals worldwide with approximately 9.2 million new cases and 1.6 million deaths annually. Current efforts are focused on making better BCG priming vaccines designed to induce a comprehensive and balanced immunity followed by booster(s) targeting a specific set of relevant antigens in common with the BCG prime. We describe the generation and immunological characterization of recombinant BCG strains with properties associated with lysis of the endosome compartment and over-expression of key Mtb antigens. The endosome lysis strain, a derivative of BCG SSI-1331 (BCG₁₃₃₁) expresses a mutant form of perfringolysin O (PfoA_G137Q), a cytolysin normally secreted by Clostridium perfringens. Integration of the PfoA_G137Q gene into the BCG genome was accomplished using an allelic exchange plasmid to replace ureC with pfoA_G137Q under the control of the Ag85B promoter. The resultant BCG construct, designated AERAS-401 (BCG₁₃₃₁ ΔureC::ΩpfoA_G137Q) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice. A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401. Compared to the parental BCG strain, vaccination of mice and guinea pigs with AFRO-1 resulted in enhanced immune responses. Mice vaccinated with AFRO-1 and challenged with the hypervirulent Mtb strain HN878 also survived longer than mice vaccinated with the parental BCG. Thus, we have generated improved rBCG vaccine candidates that address many of the shortcomings of the currently licensed BCG vaccine strains.     

Sequential pulmonary immunization with heterologous recombinant influenza A virus tuberculosis vaccines protects against murine M. tuberculosis infection

Tuberculosis (TB) infection affects a quarter of the global population resulting in a large burden of TB disease and mortality. The long-term control of TB requires vaccines with greater efficacy and durability than the current Mycobacterium bovis Bacille Calmette-Guérin (BCG). Pulmonary immunization may increase and prolong immunity at the site of Mycobacterium tuberculosis infection. We have investigated recombinant influenza A viruses (rIAVs) expressing the p25 CD4⁺ T cell epitope of M. tuberculosis Ag85B_240–254 for single and sequential immunization against M. tuberculosis infection. Intranasal immunization with single dose of rIAV X31 (H3N2 strain) expressing the p25 epitope (X31-p25), induced p25-specific CD4⁺ T cells and conferred protection against aerosol delivery of M. tuberculosis infection in the lungs. To enhance this effect, prime-boost immunization with hetero-subtypic rIAVs was examined. Sequential immunization with X31-p25 and a second rIAV, PR8 (H1N1 strain) expressing the same epitope (PR8-p25), increased the frequency of p25-specific IFN-γ T cell responses and polyfunctional CD4⁺ T cells producing IFN-γ, IL-2, and TNF, compared to immunization with each rIAV alone. This combination resulted in protection against M. tuberculosis in both the lungs and spleen. Therefore, our study revealed that rIAV is not only an efficient vector to induce protective immunity in the lungs, but also has a potential use for sequential immunization with heterologous rIAV to boost the immunogenicity and improve the protection against M. tuberculosis.     

The BCGΔBCG1419c strain,which produces more pellicle in vitro,improves control of chronic tuberculosis in vivo

Mycobacterium tuberculosis (Mtb) has been a threat to humans since ancient times, and it is the main causative agent of tuberculosis (TB). Until today, the only licensed vaccine against Mtb is the live attenuated M. bovis Bacillus Calmette-Guérin (BCG), which has variable levels of protection against the pulmonary form of infection. The quest for a new vaccine is a priority given the rise of multidrug-resistant Mtb around the world, as well as the tremendous burden imposed by latent TB. The objective of this study was to evaluate the immunogenicity and capacity of protection of a modified BCG strain (BCGΔBCG1419c) lacking the c-di-GMP phosphodiesterase gene BCG1419c, in diverse mice models. In a previous report, we have shown that BCGΔBCG1419c was capable of increasing biofilm production and after intravenous infection of immunocompetent mice; this strain persisted longer in lungs than parental BCG Pasteur. This led us to hypothesize that BCGΔBCG1419c might therefore possess some advantage as vaccine candidate. Our results in this report indicate that compared to conventional BCG, vaccination with BCGΔBCG1419c induced a better activation of specific T-lymphocytes population, was equally effective in preventing weight loss despite being used at lower dose, reduced tissue damage (pneumonic scores), increased local IFNγ⁺ T cells, and diminished bacterial burden in lungs of BALB/c mice infected intratracheally with high dose Mtb H37Rv to induce progressive TB. Moreover, vaccination with BCGΔBCG1419c improved resistance to reactivation after immunosuppression induced by corticosterone in a murine model of chronic infection similar to latent TB. Furthermore, despite showing increased persistence in immunocompetent mice, BCGΔBCG1419c was as attenuated as parental BCG in nude mice. To our knowledge, this is the first demonstration that a modified BCG vaccine candidate with increased pellicle/biofilm production has the capacity to protect against Mtb challenge in chronic and reactivation models of infection.     

Weaker protection against tuberculosis in BCG-vaccinated male 129 S2 mice compared to females

BCG – the only available vaccine against tuberculosis (TB) - was first given to babies 100 years ago in 1921. While it is effective against TB meningitis and disseminated TB, its efficacy against pulmonary TB is variable, notably in adults and adolescents. TB remains one of the world’s leading health problems, with a higher prevalence among men. Male sex is associated with increased susceptibility to Mycobacterium tuberculosis in mice, but sex-specific responses to BCG vaccination have not been examined. In this study we vaccinated TB-susceptible 129 S2 mice with BCG and challenged with low-dose M. tuberculosis H37Rv by aerosol infection. BCG was protective against TB in both sexes, as unvaccinated mice lost weight more rapidly than vaccinated ones and suffered from worse lung pathology. However, female mice were better protected than males, showing lower lung bacterial burdens and less weight loss. Overall, vaccinated female mice had increased numbers of T cells and less myeloid cells in the lungs compared to vaccinated males. Principal component analysis of measured features revealed that mice grouped according to timepoint, sex and vaccination status. The features that had the biggest impact on grouping overall included numbers of CD8 T cells, CD8 central memory T cells and CD4 T effector cells, with neutrophil and CD11b⁺GR-1⁻ cell numbers having a big impact at day 29. Hierarchical clustering confirmed that the main difference in global immune response was due to mouse sex, with only a few misgrouped mice. In conclusion, we found sex-specific differences in response to M. tuberculosis H37Rv -challenge in BCG-vaccinated 129 S2 mice. This highlights the need to include both male and female mice in preclinical testing of vaccine candidates.     

Targeted induction of antigen expression within dendritic cells modulates antigen-specific immunity afforded by recombinant BCG

The targeted modulation of antigen expression by recombinant vaccine vehicles would significantly aid development of effective immunotherapeutic strategies. In this report we demonstrate that the Mycobacterium tuberculosis hspX promoter can be used to regulate in vivo induction of antigens expressed by recombinant Bacille Calmette Guérin (rBCG). HspX promoter induction occurred rapidly upon entry of rBCG into cultured dendritic cells (DCs), as evidenced by GFP levels in DCs when infected with BCG:P_hspX-GFP, in which P_hspX controlled GFP expression. Vaccination of mice with BCG:P_hspX-GFP led to rapid in vivo induction of GFP associated with an influx of GFP⁺ DCs at the infection site. P_hspX-driven antigen expression resulted in an improved capacity of DCs to prime antigen-specific T cells, as infection of DCs with BCG:P_hspX-85B, where the hspX promoter controls expression of M. tuberculosis Ag85B, led to enhanced proliferation of Ag85B-reactive CD4⁺ T cells compared to BCG overexpressing Ag85B using the strong Mycobacterium bovis hsp60 promoter. This enhancement of rBCG-induced immunity was also evident in vivo; mice vaccinated with BCG:P_hspX-85B displayed markedly improved generation of Ag85B-reactive IFN-γ-secreting T cells compared to control BCG-vaccinated mice, which was most pronounced at extended times points post-vaccination. These data reveal a novel strategy to enhance the development and maintenance of vaccine-specific T cell responses.     

Delivery of a multivalent scrambled antigen vaccine induces broad spectrum immunity and protection against tuberculosis

The development of effective anti-Tuberculosis (TB) vaccines is an important step towards improved control of TB in high burden countries. Subunit vaccines are advantageous in terms of safety, particularly in the context of high rates of HIV co-infection, but they must contain sufficient Mycobacterium tuberculosis antigens to stimulate immunity in genetically diverse human populations. We have used a novel approach to develop a synthetic scrambled antigen vaccine (TB-SAVINE), comprised of overlapping, recombined peptides from four M. tuberculosis proteins, Ag85B, ESAT-6, PstS3 and Mpt83, each of which is immunogenic and protective against experimental TB. This polyvalent TB-SAVINE construct stimulated CD4 and CD8T cell responses against the individual proteins and M. tuberculosis in C57BL/6 and Balb/c mice, when delivered as DNA, Fowl Pox Virus or Vaccinia Virus vaccines. In addition, the DNA-TBS vaccine induced protective immunity against pulmonary M. tuberculosis infection in C57BL/6 mice. Co-immunization of Balb/c mice with virally expressed TBS and HIV1-SAVINE vaccine stimulated strong T cell responses to both the M. tuberculosis and HIV proteins, indicating no effects of antigenic competition. Further development of this TB-SAVINE vaccine expressing components from multiple M. tuberculosis proteins may prove an effective vaccine candidate against TB, which could potentially form part of a safe, combined preventative strategy together with HIV immunisations.     

ESX-5-targeted export of ESAT-6 in BCG combines enhanced immunogenicity & efficacy against murine tuberculosis with low virulence and reduced persistence

Tuberculosis (TB) is the leading infectious cause of death globally. The only licensed TB vaccine, Bacille Calmette–Guérin (BCG), has low efficacy against TB in adults and is not recommended in people with impaired immunity. The incorporation of the Mycobacterium tuberculosis (Mtb) secretion system ESX-1 into BCG improves immunogenicity and protection against TB in animal models, which is associated with the secretion of the ESX-1-dependent protein ESAT-6. However, the resulting strain, BCG::ESX1^Mtb, has been deemed unsafe as a human vaccine, due to prolonged persistence and increased virulence in immunocompromised mice. In this study, we describe a new recombinant BCG strain that uncouples the beneficial aspects of ESAT-6 secretion from the detrimental ESX-1effects on virulence and persistence. The strain was constructed by fusing the ESAT-6-encoding gene esxA to the general secretion signal for the mycobacterial type VII secretion pathway protein PE25. This new strain, BCG::ESAT6-PE25SS, secretes full-length ESAT-6 via the ESX-5 secretion system, which in contrast to ESX-1 is also present in BCG. In vivo testing revealed that ESX-5-targeted ESAT-6 export, induces cytosolic contact, generates ESAT-6-specific T cells and enhances the protective efficacy against TB disease, but is associated with low virulence and reduced persistence in immunocompetent and immunocompromised mice. Additionally, compared to BCG::ESX1^Mtb and parental BCG, mucosal administration of BCG::ESAT6-PE25SS is associated with more rapid clearance from the lung. These results warrant further studies to evaluate BCG::ESAT6-PE25SS as a potential live attenuated vaccine candidate for TB.     

Increased protective efficacy of recombinant BCG strains expressing virulence-neutral proteins of the ESX-1 secretion system

BackgroundMycobacterium bovis BCG is presently the only available anti-tuberculosis vaccine used, worldwide. While BCG protects against miliary tuberculosis (TB) and tuberculoid meningitis in children, it often fails to protect against adult pulmonary TB. It is thus imperative that new improved anti-TB vaccines are developed. The integration of the ESX-1 secretion system, absent from BCG due to the deletion of region of difference 1 (RD1), into the genome of BCG has been shown to confer to BCG::ESX-1 enhanced protection against TB as compared to BCG.MethodsIn the present study, to counterbalance the increase in virulence resulting from the integration of the RD1 region into BCG, we have constructed and evaluated several BCG::ESX-1 variants that carry selected amino-acid changes in the ESX-1-secreted antigen ESAT-6. In order to find the candidate that combines low virulence with high protective efficacy, these novel recombinant BCG::ESX-1 strains were tested for their virulence properties and their protective efficacy against Mycobacterium tuberculosis in two different animal models (mouse and guinea-pig).ResultsAmong several candidates tested, the BCG::ESAT-L28A/L29S strain, carrying modifications at residues Leu²⁸-Leu²⁹ of the ESAT molecule, showed strong attenuation in mice and high protective efficiency both in mouse and guinea-pig vaccination-infection models.ConclusionThis strain thus represents a promising candidate that merits further investigations and development. Our research also provides the proof of concept that selected ESX-1-complemented BCG strains may show low virulence and increased protective potential over parental strains.     

A practical in vitro growth inhibition assay for the evaluation of TB vaccines

New vaccines and novel immunization strategies are needed to improve the control of the global tuberculosis epidemic. To facilitate vaccine development, we have been creating in vitro mycobacterial intra-macrophage growth inhibition assays. Here we describe the development of an in vitro assay designed for BSL-2 laboratories which measures the capacity of vaccine-induced immune splenocytes to control the growth of isoniazid-resistant Mycobacterium bovis BCG (INH^r BCG). The use of the INH^r BCG as the infecting organism allows the discrimination of BCG bacilli used in murine vaccinations from BCG used in the in vitro assay. In this study, we showed that protective immune responses evoked by four different types of Mycobacterium tuberculosis vaccines [BCG, an ESAT6/Antigen 85B fusion protein formulated in DDA/MPL adjuvant, a DNA vaccine expressing the same fusion protein, and a TB Modified Vaccinia Ankara construct expressing four TB antigens (MVA-4TB)] were detected. Importantly, the levels of vaccine-induced protective immunity seen in the in vitro assay correlated with the results from in vivo protection studies in the mouse model of pulmonary tuberculosis. Furthermore, the growth inhibition data for the INH^r BCG assay was similar to the previously reported results for a M. tuberculosis infection assay. The cytokine expression profiles at day 7 of the INH^r BCG growth inhibition studies were also similar but not identical to the cytokine patterns detected in earlier M. tuberculosis co-culture assays. Overall, we have shown that a BSL-2 compatible in vitro growth inhibition assay using INH^r BCG as the intra-macrophage target organism should be useful in developing and evaluating new TB immunization strategies.     

Lipoprotein LpqS deficient M. tuberculosis mutant is attenuated for virulence in vivo and shows protective efficacy better than BCG in guinea pigs

Bacterial lipoproteins are a functionally diverse class of membrane anchored proteins. Lipoproteins constitute nearly 2.5% of the Mycobacterium tuberculosis proteome. Inactivation of genes coding for individual lipoproteins results in attenuated phenotype of the mutants. LpqS is a lipoprotein highly conserved among slow growing pathogenic mycobacteria. Our previous study has shown that the lpqS gene deletion mutant of M. tuberculosis (MtbΔlpqS) poorly replicates in THP1-(human acute monocytic leukemia cell line) derived macrophagic cell line. In addition, guinea pigs, when infected with the mutant strain exhibited significantly reduced bacterial burden and pathological damage in the infected tissues in comparison with the parental strain infected group. Subsequently, we evaluated the protective efficacy of the mutant by immunization of guinea pigs through aerosol and subcutaneous routes. We observed that immunization of guinea pigs with MtbΔlpqS offered superior protection in lungs as compared to BCG. In addition, MtbΔlpqS also prevented the haematogenous spread of the disease which was evident from the significantly reduced splenic bacillary load compared to saline vaccinated animals. The gross pathological observations and the histopathological observations well corroborated the bacterial findings. We also observed that aerogenic route of immunization imparts superior protection compared to subcutaneous route of immunization. These findings well establishes the efficacy of M. tuberculosis mutant in imparting protection against pulmonary TB.