Recombinant BCG approach for development of vaccines: cloning and expression of immunodominant antigens of M. tuberculosis

In spite of major advances in our understanding of the biology and immunology of tuberculosis, the incidence of the disease has not reduced in most parts of the world. In an attempt to improve the protective efficacy of Mycobacterium bovis bacille Calmette-Guérin (BCG), we have developed a generic vector system, pSD5, for expression of genes at varying levels in mycobacteria. In this study, we have cloned and overexpressed three immunodominant secretory antigens of M. tuberculosis, 85A, 85B and 85C, belonging to the antigen 85 complex. All the genes were cloned under the control of a battery of mycobacterial promoters of varying strength. The expression was analysed in the fast-growing strain M. smegmatis and the slow-growing vaccine strain M. bovis BCG. The recombinant BCG constructs were able to express the antigens at high levels and the majority of the expressed antigens was secreted into the medium. These results show that by using this strategy the recombinant BCG approach can be successfully used for the development of candidate vaccines against infections associated with mycobacteria as well as other pathogens.     

Cytolytic T-cell responses to human dendritic cells and macrophages infected with Mycobacterium bovis BCG and recombinant BCG secreting listeriolysin

Cytolytic T-cell responses from 63 normal blood donors were monitored in a Mycobacterium bovis BCG infection system in vitro. We wanted to know whether cultured dendritic cells were capable of potentiating the cytolytic T-cell responses to M. bovis BCG. Infected cultured dendritic cells were up to ten times more effective antigen-presenting cells than macrophages in proliferative assays, while cytolytic T-cell induction did not differ significantly between dendritic cells and macrophages. Separated CD4+ and CD8+ T-cell subsets contributed equally to lysis of infected targets. Experiments comparing wild-type M. bovis BCG strain with two new recombinant M. bovis BCG strains secreting listeriolysin revealed statistically significant higher maximal lysis values for recombinant M. bovis BCG. We conclude from our in vitro infection system with mycobacteria that dendritic cells are superior to macrophages in proliferative assays but equal to macrophages in their ability to induce cytolytic T-cell responses. Moreover, our data suggest that recombinant M. bovis BCG vaccine strains secreting listeriolysin improve cytolytic T-cell responses.     

Development of BCG as a live recombinant vector system: potential use as an HIV vaccine.

Bacille Calmette-Guèrin (BCG), a live attenuated tubercle bacillus, is currently the most widely used vaccine in the world. Because of its unique characteristics, including low toxicity, adjuvant potential, and long-lasting immunity, BCG represents a novel vaccine vehicle with which to deliver protective antigens of multiple pathogens. We have developed episomal and integrative expression vectors employing regulatory sequences of major BCG heat shock proteins for stable maintenance and expression of foreign antigens in BCG vaccine strains (22). Shuttle plasmids capable of autonomous replication in Escherichia coli and BCG were constructed with a DNA cassette containing a minimal replicon derived from the Mycobacterium fortuitum plasmid pAL5000. Efficient and stable chromosomal integration of recombinant plasmids into BCG was achieved using a DNA segment containing the mycobacteriophage L5 attachment site and integrase coding sequence. Using the BCG hsp60 and hsp70 stress gene promoters, we were able to express Escherchia coli beta-galactosidase to levels in excess of 10% of total cell protein. The major antigens of HIV-1 gag, pol, and env were also stably expressed using our vector systems. The recombinant BCG elicited long-lasting humoral and cellular immune responses to these antigens in mice. Antibody responses to beta-galactosidase using as few as 200 colony-forming units were detected 6 weeks after immunization, and titers (1:30,000) were sustained for more than 10 weeks. Cellular immune responses, of both cytotoxic T cell (CTL) and helper T lymphocytes, were detected to beta-galactosidase. CTL responses were also induced to the HIV-1 envelope protein. Thus, we have demonstrated stable recombinant antigen expression, processing, and presentation using our recombinant BCG vector system. This live recombinant vector system shows promise as a universally applicable and safe vaccine vehicle for protection against various infectious diseases.     

Molecular cloning and sequencing of the merozoite surface antigen 2 gene from Plasmodium falciparum strain FCC-1/HN and expression of the gene in mycobacteria

Strain bacillus Calmette-Guerin (BCG) of Mycobacterium bovis has been used as a live bacterial vaccine to immunize more than 3 billion people against tuberculosis. In an attempt to use this vaccine strain as a vehicle for protective antigens, the gene encoding merozoite surface antigen 2 (MSA2) was amplified from strain FCC-1/HN Plasmodium falciparum genome, sequenced, and expressed in M. bovis BCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from Mycobacterium tuberculosis. The recombinant shuttle plasmid pBCG/MSA2 was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/MSA2 could be induced by heating to express MSA2; the molecular mass of recombinant MSA2 was about 31 kDa. This first report of expression of the full-length P. falciparum MSA2 gene in BCG provides evidence for use of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria.     

Induction of humoral immunity in response to immunization with recombinant Mycobacterium bovis BCG expressing the S1 subunit of Bordetella pertussis toxin

Two recombinant Mycobacterium bovis BCG (rBCG) vaccine strains were developed for the expression of cytoplasmically located S1 subunit of pertussis toxin, with expression driven by the hsp60 promoter of M. bovis (rBCG/pPB10) or the pAN promoter of Mycobacterium paratuberculosis (rBCG/pPB12). Both strains showed stable expression of equivalent levels of recombinant S1 in vitro and induced long-term (up to 8 months) humoral immune responses in BALB/c mice, although these responses differed quantitatively and qualitatively. Specifically, rBCG/pPB12 induced markedly higher levels of IgG1 than did rBCG/pPB10, and mice immunized with the former strain developed specific long-term memory to S1, as indicated by the production of high levels of S1-specific IgG in response to a sublethal challenge with pertussis toxin 15 months after initial immunization. When considered in combination with previous studies, our data encourage further evaluation of rBCG as a potential means of developing a low-cost whooping cough vaccine based on defined antigens.     

Expression of Escherichia coliβ-galactosidase in Mycobacterium bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular immune responses

A promoter sequence, PAN, was isolated from Mycobacterium paratuberculosis and characterized. This promoter lies adjacent to, and outside, the 3' end of an IS900 insertion element. IS900 contains an open reading frame, ORF2, on the complementary strand which codes for the putative transposase of this insertion sequence. A DNA fragment containing PAN and part of ORF2 was fused to the lacZ gene and inserted into the replicative shuttle vector pRR3. Mycobacterium smegmatis and Mycobacterium bovis BCG (BCG) transformed with this plasmid exhibited beta-galactosidase activity. However, lacZ was only expressed in Escherichia coli under the control of PAN, when ORF2 was deleted. Immunization of mice with the recombinant M. bovis BCG expressing lacZ resulted in the induction of a high humoral and cellular response directed against beta-galactosidase. The PAN-ORF2 expression system may prove to be particularly useful for cloning and expression of heterologous genes in the BCG vaccine strain.     

Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.     

A stable Escherichia coli-mycobacteria shuttle vector 'pSO246' in Mycobacterium bovis BCG

Abstract The most widely used plasmid vector system in mycobacteria is based on pAL5000 from Mycobacterium fortuitum . The derivatives of the pAL5000-based shuttle vectors between Escherichia coli and mycobacteria, which we have utilized to secrete recombinant antigens, were generated. The stability of the vectors was assessed in Mycobacterium bovis BCG (BCG). The plasmid vector pSO246 was stable in BCG for at least 50 generations.     

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.     

Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG

A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed. In contrast to r-S. typhimurium control, oral vaccination of mice with the r-S. typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M. bovis BCG strain. The immune response induced by r-S. typhimurium p30 was accompanied by augmented interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels produced by restimulated splenocytes. These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r-S. typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens.     

Genome sequence of Mycobacterium bovis BCG Moreau, the Brazilian vaccine strain against tuberculosis

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis, and the strains used worldwide represent a family of daughter strains with distinct genotypic characteristics. Here we report the complete genome sequence of M. bovis BCG Moreau, the strain in continuous use in Brazil for vaccine production since the 1920s.     

Generation of CD8+ T-cell responses by a recombinant nonpathogenic Mycobacterium smegmatis vaccine vector expressing human immunodeficiency virus type 1 Env

Because the vaccine vectors currently being evaluated in human populations all have significant limitations in their immunogenicity, novel vaccine strategies are needed for the elicitation of cell-mediated immunity. The nonpathogenic, rapidly growing mycobacterium Mycobacterium smegmatis was engineered as a vector expressing full-length human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope protein. Immunization of mice with recombinant M. smegmatis led to the expansion of major histocompatibility complex class I-restricted HIV-1 epitope-specific CD8(+) T cells that were cytolytic and secreted gamma interferon. Effector and memory T lymphocytes were elicited, and repeated immunization generated a stable central memory pool of virus-specific cells. Importantly, preexisting immunity to Mycobacterium bovis BCG had only a marginal effect on the immunogenicity of recombinant M. smegmatis. This mycobacterium may therefore be a useful vaccine vector.     

A novel differential expression system for gene modulation in Mycobacteria

Tuberculosis (TB) remains a major global health problem, and successful genetic manipulation of mycobacteria is crucial for developing new approaches to study the mechanism of pathogenesis of Mycobacterium tuberculosis (M.tb) and to combat TB. In this study, a series of M.tb furA gene operator/promoter (pfurA) mutants were generated aiming at optimization of the promoter activities in mycobacterial strains. Measured by the lacZ gene-fusion reporter system, change of the initial codon GTG to the preferred ATG resulted in a double increase of beta-galactosidase activity, while a 6-bp substitution in the conserved FurA binding AT-rich region upstream of furA gene led to 4-6 folds increase of the activity. It is significant that combination of both mutations showed about 10 folds of beta-galactosidase activity higher than that of the prototype pfurA. Furthermore, all of the furA promoters were expressed continuously in vivo during intracellular growth of Mycobacterium bovis BCG, and were induced early upon infection in macrophages. Employing the series of pfurA-based differential expression vectors, M.tb chimeric antigen Ag856A2 known for its excellent immunogenicity, was shown to be expressed at different levels in the recombinant Mycobacterium smegmatis and BCG strains. These results indicated that this differential expression system is feasible to express any target antigen of interest in a modular fashion for the study of gene regulation in mycobacterial strains, and also for the development of different recombinant BCG vaccine candidates against TB or other infectious diseases, which would be beneficial for elicitation of optimal immune response.     

Cultivation of Mycobacterium bovis BCG in bioreactors

The Mycobacterium bovis BCG vaccine for commercial use is classically produced as surface pellicles by culture on synthetic medium. Under these conditions, reproducibility of the cultures and quality assessment are hampered by slow growth of the bacilli, the formation of bacterial aggregates and a high proportion of dead bacilli after processing and final formulation of the vaccine. Here, we established dispersed cultures of M. bovis BCG in synthetic media in small-scale bioreactors. These cultures allow recording and adjusting of culture parameters and give rise to single bacilli with a high degree of live bacteria. In the murine model, bioreactor-grown M. bovis BCG exhibited slightly stronger replication and persistence than the vaccine produced under the classical conditions. The protective efficacy against challenge with M. tuberculosis was identical for both vaccine preparations.     

Recombinant Mycobacterium bovis BCG producing the circumsporozoite protein of Plasmodium falciparum FCC-1/HN strain induces strong immune responses in BALB/c mice

The current vaccine against tuberculosis, Mycobacterium bovis strain bacillus Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for expression and delivery of protective recombinant antigens. Malaria is one of the severest parasitic diseases in humans especially in the developing world. No efficacious vaccine is currently available. However, circumsporozoite protein (CSP) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the immune response to recombinant BCG (rBCG) vaccine expressing Plasmodium falciparum CSP (BCG-CSP) under the control of heat shock protein 70 promoter in BALB/c mice. The lymphocytes proliferative response to P. falciparum soluble antigen was significantly higher than those in the groups of BCG and normal saline, and the production of cytokines (IFN-gamma and IL-2) in response to malaria antigen was significantly higher in rBCG and BCG groups than control group of normal saline. A specific IgG antibody response against P. falciparum antigen of CSP was also characterized. The booster injection could enhance the production of cytokine, proliferation responses of spleen lymphocytes and the antibodies titer of BCG-CSP. The results in the study demonstrated that rBCG vaccine producing CSP is an appropriate vaccine for further evaluation in non-human primates.     

Live attenuated vaccines for human use.

Live attenuated vaccines have been successfully used for the prevention of a number of viral and bacterial diseases. Several vaccine strains have been utilized recently as expression vectors for cloned heterologous antigens. Through the use of recombinant DNA technology, candidate vaccine strains and vector systems have been developed and are undergoing clinical evaluation.     

Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti

Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock-in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1-encoded proteins were localized in the cell wall, and two of them, the immunodominant T-cell antigens ESAT-6 and CFP-10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock-ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock-in, whereas BCG controls were cleared. Knocking-in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.     

Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.     

Priming with a recombinant pantothenate auxotroph of Mycobacterium bovis BCG and boosting with MVA elicits HIV-1 Gag specific CD8+ T cells

A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔpanCD) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔpanCD[pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8(+) T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4(+) T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge.