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Mycobacterium tuberculosis remains a threat to global health, and a more efficacious vaccine is needed to prevent disease caused by M. tuberculosis We previously reported that the mycobacterial ribosome is a major target of CD4+ T cells in mice immunized with a genetically modified Mycobacterium smegmatis strain (IKEPLUS) but not in mice immunized with Mycobacterium bovis BCG. Two specific ribosomal proteins, RplJ and RpsA, were identified as cross-reactive targets of M. tuberculosis, but the breadth of the CD4+ T cell response to M. tuberculosis ribosomes was not determined. In the present study, a library of M. tuberculosis ribosomal proteins and in silico-predicted peptide libraries were used to screen CD4+ T cell responses in IKEPLUS-immunized mice. This identified 24 out of 57 M. tuberculosis ribosomal proteins distributed over both large and small ribosome subunits as specific CD4+ T cell targets. Although BCG did not induce detectable responses against ribosomal proteins or peptide epitopes, the M. tuberculosis ribosomal protein RplJ produced a robust and multifunctional Th1-like CD4+ T cell population when administered as a booster vaccine to previously BCG-primed mice. Boosting of BCG-primed immunity with the M. tuberculosis RplJ protein led to significantly reduced lung pathology compared to that in BCG-immunized animals and reductions in the bacterial burdens in the mediastinal lymph node compared to those in naive and standard BCG-vaccinated mice. These results identify the mycobacterial ribosome as a potential source of cryptic or subdominant antigenic targets of protective CD4+ T cell responses and suggest that supplementing BCG with ribosomal antigens may enhance protective vaccination against M. tuberculosis.

Mycobacterium tuberculosis remains a threat to global health, and a more efficacious vaccine is needed to prevent disease caused by M. tuberculosis. We previously reported that the mycobacterial ribosome is a major target of CD4(+) T cells in mice immunized with a genetically modified Mycobacterium smegmatis strain (IKEPLUS) but not in mice immunized with Mycobacterium bovis BCG. Two specific ribosomal proteins, RplJ and RpsA, were identified as cross-reactive targets of M. tuberculosis, but the breadth of the CD4(+) T cell response to M. tuberculosis ribosomes was not determined. In the present study, a library of M. tuberculosis ribosomal proteins and in silico-predicted peptide libraries were used to screen CD4(+) T cell responses in IKEPLUS-immunized mice. This identified 24 out of 57 M. tuberculosis ribosomal proteins distributed over both large and small ribosome subunits as specific CD4(+) T cell targets. Although BCG did not induce detectable responses against ribosomal proteins or peptide epitopes, the M. tuberculosis ribosomal protein RplJ produced a robust and multifunctional Th1-like CD4(+) T cell population when administered as a booster vaccine to previously BCG-primed mice. Boosting of BCG-primed immunity with the M. tuberculosis RplJ protein led to significantly reduced lung pathology compared to that in BCG-immunized animals and reductions in the bacterial burdens in the mediastinal lymph node compared to those in naive and standard BCG-vaccinated mice. These results identify the mycobacterial ribosome as a potential source of cryptic or subdominant antigenic targets of protective CD4(+) T cell responses and suggest that supplementing BCG with ribosomal antigens may enhance protective vaccination against M. tuberculosis.

Persistence ofMycobacterium tuberculosisin humans represents a major roadblock to elimination of tuberculosis. We describe identification of a locus inM. tuberculosis, mel2, that displays similarity to bacterial bioluminescent loci and plays an important role during persistence in mice. We constructed a deletion of themel2locus and found that the mutant displays increased susceptibility to reactive oxygen species (ROS). Upon infection of mice by aerosol the mutant grows normally until the persistent stage, where it does not persist as well as wild type. Histopathological analyses show that infection with themel2mutant results in reduced pathology and both CFU and histopathology indicate that dissemination of themel2mutant to the spleen is delayed. These data along with growth in activated macrophages and infection of Phox−/−and iNOS−/−mice and bone marrow-derived macrophages suggest that the primary mechanism by whichmel2affects pathogenesis is through its ability to confer resistance to ROS. These studies provide the first insight into the mechanism of action for this novel class of genes that are related to bioluminescence genes. The role ofmel2in resistance to ROS is important for persistence and dissemination ofM. tuberculosisand suggests that homologues in other bacterial species are likely to play a role in pathogenesis.

Little is known about the immune distribution and localization of antigen-specific T cells in mucosal interfaces of tissues/organs during infection of humans. In this study, we made use of a macaque model ofMycobacterium tuberculosisinfection to assess phosphoantigen-specific Vγ2Vδ2 T cells regarding their tissue distribution, anatomical localization, and correlation with the presence or absence of tuberculosis (TB) lesions in lymphoid and nonlymphoid organs/tissues in the progression of severe pulmonary TB. Progression of pulmonaryM. tuberculosisinfection generated diverse distribution patterns of Vγ2Vδ2 T cells, with remarkable accumulation of these cells in lungs, bronchial lymph nodes, spleens, and remote nonlymphoid organs but not in blood. Increased numbers of Vγ2Vδ2 T cells in tissues were associated withM. tuberculosisinfection but were independent of the severity of TB lesions. In lungs with apparent TB lesions, Vγ2Vδ2 T cells were present within TB granulomas. In extrathoracic organs, Vγ2Vδ2 T cells were localized in the interstitial compartment of nonlymphoid tissues, and the interstitial localization was present despite the absence of detectable TB lesions. Finally, Vγ2Vδ2 T cells accumulated in tissues appeared to possess cytokine production function, since granzyme B was detectable in the γδ T cells present within granulomas. Thus, clonally expanded Vγ2Vδ2 T cells appeared to undergotrans-endothelial migration, interstitial localization, and granuloma infiltration as immune responses toM. tuberculosisinfection.

Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines byMycobacterium smegmatisorM. bovisBCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer usingM. smegmatisor BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected withM. smegmatishyperconjugation mutants.M. smegmatisstrains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation inoriMresulted in a threefold increase in gene transfer to J774 cells. Moreover,M. smegmatisstrains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120hE) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P< 0.05) and threefold (P< 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120hE.M. smegmatisand other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

We generated four individual glutamine synthetase (GS) mutants (Δ glnA1 , Δ glnA2 , Δ glnA3 , and Δ glnA4 ) and one triple mutant (Δ glnA1EA2 ) of Mycobacterium tuberculosis to investigate the roles of GS enzymes. Subcutaneous immunization with the Δ glnA1EA2 and Δ glnA1 glutamine auxotrophic mutants conferred protection on C57BL/6 mice against an aerosol challenge with virulent M. tuberculosis , which was comparable to that provided by Mycobacterium bovis BCG vaccination.

Approximately one-third of the human population is latently infected withMycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintainingM. tuberculosisin the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five relatedM. tuberculosisproteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role ofM. tuberculosis rpfgenes in an established mouse model ofM. tuberculosispersistence and reactivation. After an aerosol infection with theM. tuberculosisErdman wild type (Erdman) or single-deletionrpfmutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the fiverpfdeletion mutants tested, one (ΔRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and ΔRv1009-infected mice at advanced stages of reactivation. Analysis ofrpfgene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the fiverpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting thatrpfgene expression may be regulated in a growth phase-dependent manner. To our knowledge, ΔRv1009 is the firstM. tuberculosismutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.

We developed a live, fully attenuated Mycobacterium tuberculosis vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. The Δ leuD Δ panCD double auxotroph is fully attenuated in the SCID mouse model and highly immunogenic and protective in the extremely sensitive guinea pig tuberculosis model, reducing both bacterial burden and disease pathology.

Mycobacterium tuberculosispossesses five genes with significant homology to the resuscitation-promoting factor (Rpf) ofMicrococcus luteus. TheM. luteusRpf is a secreted ∼16-kDa protein which restores active growth to cultures ofM. luteusrendered dormant by prolonged incubation in stationary phase. More recently, the Rpf-like proteins ofM. tuberculosishave been shown to stimulate the growth of extended-stationary-phase cultures ofMycobacterium bovisBCG. These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function ofM. tuberculosisRpf homologues in vivo. To address these questions, we have disrupted each of the fiverpf-like genes inM. tuberculosisErdman, and analyzed the mutants for their growth in vitro and in vivo. In contrast toM. luteus, for whichrpfis an essential gene, we find that all of theM. tuberculosis rpfdeletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection. Analysis ofrpfexpression inM. tuberculosiscultures from early log phase through late stationary phase indicates that expression of therpf-like genes is growth phase-dependent, and that the expression patterns of the fiveM. tuberculosis rpfgenes, while overlapping to various degrees, are not uniform. We also provide evidence that mycobacterialrpfgenes are expressed in vivo in the lungs of mice acutely infected with virulentM. tuberculosis.

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8 + T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8 + T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8 + T cell responses in vivo . This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8 + T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors.

Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosis relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovis and protection of the spleens of guinea pigs infected with M. tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.

Attenuated mutants ofMycobacterium tuberculosisrepresent potential vaccine candidates for the prevention of tuberculosis. It is known that auxotrophs of a variety of bacteria are attenuated in vivo and yet provide protection against challenge with wild-type organisms. A leucine auxotroph ofM. tuberculosiswas created by allelic exchange, replacing wild-typeleuD(Rv2987c), encoding isopropyl malate isomerase, with a mutant copy of the gene in which 359 bp had been deleted, creating a strain requiring exogenous leucine supplementation for growth in vitro. The frequency of reversion to prototrophy was −11. In contrast to wild-typeM. tuberculosis, the ΔleuDmutant was unable to replicate in macrophages in vitro. Its attenuation in vivo and safety as a vaccine were established by the fact that it caused no deaths in immunodeficient SCID mice. Complementation of the mutant with wild-typeleuDabolished the requirement for leucine supplementation and restored the ability of the strain to grow both in macrophages and in SCID mice, thus confirming that the attenuated phenotype was due to the ΔleuDmutation. As a test of the vaccine potential of the leucine auxotroph, immunocompetent BALB/c mice, susceptible to fatal infection with wild-typeM. tuberculosis, were immunized with the ΔleuDmutant and subsequently challenged with virulentM. tuberculosisby both the intravenous and aerosol routes. A comparison group of mice was immunized with conventionalMycobacterium bovisBCG vaccine. Whereas all unvaccinated mice succumbed to intravenous infection within 15 weeks, mice immunized with either BCG or the ΔleuDmutant ofM. tuberculosisexhibited enhanced and statistically equivalent survival curves. However, theleuDauxotroph was less effective than live BCG in reducing organ burdens and tissue pathology of mice challenged by either route. We conclude that attenuation and protection againstM. tuberculosischallenge can be achieved with a leucine auxotroph and suggest that to induce optimal protection, attenuated strains ofM. tuberculosisshould persist long enough and be sufficiently metabolically active to synthesize relevant antigens for an extended period of time.

We report the safety and immunogenicity of a double lysine and pantothenate auxotroph ofMycobacterium tuberculosisin mice. The ΔlysAΔpanCDmutant is completely attenuated in immunocompromised SCID and gamma interferon knockout mice yet induces short-term and long-term protection in immunocompetent and CD4-deficient mice following single-dose subcutaneous vaccination.

Tuberculosis (TB) is characterized by lifetime persistence ofMycobacterium tuberculosis. Despite the induction of a vigorous host immune response that curtails disease progression in the majority of cases, the organism is not eliminated. Subsequent immunosuppression can lead to reactivation after a prolonged period of clinical latency. Thus, while it is clear that protective immune mechanisms are engaged duringM. tuberculosisinfection, it also appears that the pathogen has evolved effective countermechanisms. Genetic studies with animal infection models and with patients have revealed a key role for the cytokine gamma interferon (IFN-γ) in resistance to TB. IFN-γ activates a large number of antimicrobial pathways. Three of these IFN-γ-dependent mechanisms have been implicated in defense againstM. tuberculosis: inducible nitric oxide synthase (iNOS), phagosome oxidase (phox), and the phagosome-associated GTPase LRG-47. In order to identify bacterial genes that provide protection against specific host immune pathways, we have developed the strategy of differential signature-tagged transposon mutagenesis. Using this approach we have identified threeM. tuberculosisgenes that are essential for progressiveM. tuberculosisgrowth and rapid lethality in iNOS-deficient mice but not in IFN-γ-deficient mice. We propose that these genes are involved in pathways that allowM. tuberculosisto counter IFN-γ-dependent immune mechanisms other than iNOS.

Strains of Escherichia coli K-12 were constructed that permitted the amplification of in vitro-packaged recombinant cosmid-transducing particles by in vivo repackaging of recombinant cosmid molecules. Thermal induction of these thermoinducible, excision-defective lysogens containing recombinant cosmid molecules yielded high titers of packaged recombinant cosmids and low levels of PFU. These strains were used to amplify packaged recombinant cosmid libraries of Mycobacterium leprae, Mycobacterium vaccae, Salmonella typhimurium, and Streptococcus mutans DNA. Contiguous and noncontiguous libraries were compared for the successful identification of cloned genes. Construction of noncontiguous libraries allowed the dissociation of desired genes from genes that were deleterious to the survival of a cosmid recombinant and permitted selection for unlinked traits that resulted in a selected phenotype. In vivo repackaging of recombinant cosmids permitted amplification of the original in vitro-packaged collection of transducing particles, storage of cosmid libraries as phage lysates, facilitation of complementation screening, expression analysis of repackaged recombinant cosmids after UV-irradiated cells were infected, in situ enzyme or immunological screening, and facilitation of recovery of recombinant cosmid molecules containing transposon inserts.