INTRODUCTION {#h0.0}
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Vaccination is the most cost-efficient measure for controlling infectious diseases ([@B1]), and yet, we lack efficient vaccines for the major infectious diseases AIDS, malaria, and tuberculosis (TB).  The only TB vaccine in use today, bacillus Calmette-Guérin (BCG), was introduced into clinical practice more than 90 years ago ([@B2]).  It resulted from repeated passage of virulent *Mycobacterium bovis*.  Although the protection provided by BCG against pulmonary TB is incomplete, it significantly reduces TB-related childhood morbidity and mortality and is relatively safe in immunocompetent individuals ([@B3]).  The global AIDS pandemic has raised new challenges: human immunodeficiency virus (HIV)-*Mycobacterium tuberculosis* coinfection accounts for one-fourth of the 1.3 million deaths due to TB annually ([@B4]), and BCG vaccination of HIV-infected newborns can result in a disseminated disease with TB-like symptoms named BCGosis ([@B3]).  Therefore, a major focus of live TB vaccine research and development is improving vaccine safety in immunocompromised individuals. 

Novel genetic tools for effective mutagenesis of the mycobacterial genome have paved the way for the development of recombinant live TB vaccines based on attenuated mycobacteria ([@B5], [@B6]).  Auxotrophic mutants have been explored as potential vaccine candidates for more than a decade ([@B7], [@B8]).  Although the auxotroph *M. tuberculosis* Δ*panCD*, which fails to synthesize vitamin B~5~ (pantothenic acid), was markedly attenuated, subcutaneous (s.c.)  vaccination of mice provided short-term (28 days) protection against *M. tuberculosis* challenge comparable to that afforded by BCG ([@B9]).  To address safety concerns, notably reversion of a single mutation to full virulence, a second, independent attenuating mutation (*leuD* gene) was included ([@B10]).  The protection induced by the double auxotroph *M. tuberculosis* Δ*leuD* Δ*panCD* was comparable to that of BCG in guinea pigs, but booster immunization with the same construct did not improve efficacy in this model ([@B10]).  Several other auxotrophic mutants have been tested as TB vaccines either alone ([@B11], [@B12]) or in combination ([@B13], [@B14]).  Although auxotrophic constructs have shown improved safety in preclinical models, they collectively failed to provide better protection than canonical BCG. 

Vitamin B~6~ is a water-soluble essential cofactor for humans, which in humans must be supplied by dietary intake.  In contrast, plants, fungi, and bacteria, including *M. tuberculosis*, synthesize pyridoxal-5′-phosphate (PLP), the bioactive form of vitamin B~6~, from glutamine and derivatives of the carbohydrate metabolism in a two-step reaction catalyzed by Pdx1 (*Rv2606c*) and Pdx2 (*Rv2604c*) ([@B15]).  Both proteins form a functional class I glutamine amidotransferase ([@B16], [@B17]).  The protein sequences and crystal structures of Pdx1 orthologs from *Bacillus subtilis*, *Plasmodium* species, *Saccharomyces cerevisiae*, and *M. tuberculosis* are highly similar, suggesting evolutionary conservation ([@B18][@B19][@B21]).  Deletion of the *pdx1* gene renders *M. tuberculosis* auxotrophic for vitamin B~6~ and markedly compromises persistence in mice ([@B15]). 

We previously engineered BCG to secrete pore-forming listeriolysin O (Hly) of *L. monocytogenes* ([@B22]).  BCG Δ*ureC*::*hly* (VPM1002) showed superior protection and safety compared to the results for BCG in preclinical models and proved to be safe and immunogenic in humans ([@B22], [@B23]).  VPM1002 has successfully completed phase IIa clinical assessment in newborns (<http://ClinicalTrials.gov> identifier NCT01479972).  Here, we deleted *pdx1* in the genetic background of BCG Δ*ureC*::*hly* to further improve its safety.  The immunogenicity, protective capacity, and safety of the new construct, BCG Δ*ureC*::*hly* Δ*pdx1*, were evaluated in mice that received either a normal or vitamin B~6~-enriched diet.  Our studies provide an innovative basis for novel vaccination strategies in immunocompromised individuals. 

RESULTS {#h1}
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Disruption of *pdx1* renders BCG auxotrophic for vitamin B~6~. {#h1.1}
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The *pdx1* loss-of-function mutant of *M. tuberculosis* was shown to be fully auxotrophic for vitamin B~6~ ([@B15]).  The operon consisting of *pdx1* (also called *snzP*, *Rv2606c*, and *BCG2631c*), *tesB2* (also called *Rv2605c* and *BCG2630c*), and *pdx2* (also called *snoP*, *Rv2604c*, and *BCG2629c*) encodes the functional vitamin B~6~ synthase complex composed of Pdx1 and Pdx2 and is identical in *M. tuberculosis* and BCG (see [Fig.  S1](#figS1){ref-type="supplementary-material"} in the supplemental material) ([@B24], [@B25]).  Thus, we expected a phenotype for a *pdx1* knockout in BCG similar to that described for *M. tuberculosis* ([@B15]).  Indeed, disruption of *pdx1* by allelic exchange prevented the growth of BCG Δ*ureC*::*hly* in vitamin B~6~-free culture medium ([Fig.  1A](#fig1){ref-type="fig"}).  Replication of bacilli was fully restored when cultures were supplemented with 5 µM B~6~ vitamer pyridoxine.  Lower pyridoxine concentrations resulted in suboptimal growth rates of the *pdx1* mutant, demonstrating concentration-dependent complementation.  Moreover, genetic complementation of the mutant with a functional copy of *pdx1* under the control of its native promoter completely reversed the observed growth defect in minimal medium ([Fig.  1B](#fig1){ref-type="fig"}).  Altogether, deletion of *pdx1* rendered BCG auxotrophic for vitamin B~6~, and this phenotype could be reverted by chemical and genetic means. 

![ Vitamin B~6~ controls growth *in vitro* and partially controls survival *in vivo* of the BCG Δ*ureC*::*hly* Δ*pdx1* auxotroph.  Genetic complementation with *pdx1* (A) and chemical complementation by supplementation of Sauton's minimal medium with vitamin B~6~ (VB6) (B) restored the growth defect of BCG Δ*ureC*::*hly* Δ*pdx1 in vitro*. ( C) Dissemination and clearance of BCG, BCG Δ*ureC*::*hly*, and BCG Δ*ureC*::*hly* Δ*pdx1* in mice on a normal diet or vitamin B~6~-enriched nutrition.  Vaccines (10^6^ CFU) were administered s.c.  at the tail base.  Independent from vitamin B~6~ administration, BCG Δ*ureC*::*hly* and BCG Δ*ureC*::*hly* Δ*pdx1* were not detected in lungs.  Shown are means ± standard deviations (SD) (*n* = 5) analyzed using two-way analysis of variance (ANOVA) and Tukey's *post hoc* test.  Significant statistical differences between BCG Δ*ureC*::*hly* and BCG Δ*ureC*::*hly* Δ*pdx1* groups are indicated as follows: \*, *P* \< 0.05; \*\*, *P* \< 0.01; \*\*\*, *P* \< 0.001; \*\*\*\*, *P* \< 0.0001.  Experiments were performed twice.](mbo0031418610001){#fig1}

Dissemination and survival of BCG Δ*ureC*::*hly* Δ*pdx1* in mice is profoundly compromised. {# h1.2}
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The spread and survival of live vaccines in mice provide key information for safety assessment.  We examined the bacterial loads at the site of vaccination and in draining lymph nodes (dLNs), spleen, and lungs over 90 days in s.c.-vaccinated mice that received either a standard or vitamin B~6~-enriched diet.  BCG disseminated from the injection site to dLNs and spleen, where the bacterial counts remained relatively stable until the end of the experiment, in contrast to BCG Δ*ureC*::*hly*, which was cleared from these organs after 45 or 28 days, respectively ([Fig.  1C](#fig1){ref-type="fig"}).  These observations are in line with previous experiments where BCG persisted for up to 120 days in dLNs and 90 days in the spleen, while at 60 days postvaccination, BCG Δ*ureC*::*hly* was undetectable ([@B26]).  BCG was the only vaccine tested that disseminated to lungs ([Fig.  1C](#fig1){ref-type="fig"}).  The survival of BCG and BCG Δ*ureC*::*hly* in mice remained independent of dietary vitamin B~6~ levels.  Intriguingly, BCG Δ*ureC*::*hly* Δ*pdx1* did not persist beyond 45 days (vaccination site) or 14 days (dLNs and spleen) ([Fig.  1C](#fig1){ref-type="fig"}), suggesting that its attenuation is determined by the *pdx1* deletion rather than *ureC* disruption or Hly expression.  Vitamin B~6~ supplementation restored the persistence of the auxotrophic construct to that of the parental BCG Δ*ureC*::*hly* in dLNs but not at the site of vaccination or in the spleen ([Fig.  1C](#fig1){ref-type="fig"}).  We conclude that knockout of *pdx1* profoundly attenuated BCG constructs in mice.  Elevated vitamin B~6~ administration could partially compensate for reduced persistence but did not achieve complete chemical complementation *in vivo*. 

BCG Δ*ureC*::*hly* Δ*pdx1* is safer than parental BCG Δ*ureC*::*hly* in immunodeficient mice. {# h1.3}
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Having demonstrated that the deletion of *pdx1* profoundly attenuated BCG in immunocompetent mice ([Fig.  1](#fig1){ref-type="fig"}), we went on to determine the safety of BCG Δ*ureC*::*hly* Δ*pdx1* in mice with severe combined immunodeficiency (SCID).  Groups of animals were s.c.  vaccinated with 10^6^ CFU of the constructs under investigation.  The SCID mice receiving the parental BCG Δ*ureC*::*hly* survived significantly longer than the BCG-vaccinated group, confirming the superior safety of the recombinant construct ([Fig.  2](#fig2){ref-type="fig"}) ([@B22]).  When the experiment was terminated 500 days postvaccination, the survival of groups that received BCG Δ*ureC*::*hly* Δ*pdx1* (with or without vitamin B~6~ supplementation) or phosphate-buffered saline (PBS) was significantly improved over that of mice receiving BCG Δ*ureC*::*hly*, demonstrating profound attenuation of the auxotrophic construct in immunodeficient mice ([Fig.  2](#fig2){ref-type="fig"}). 

![ Deletion of *pdx1* improved the superior safety profile of parental BCG Δ*ureC*::*hly* over that of canonical BCG in SCID mice.  Survival of SCID mice s.c.  vaccinated with 10^6^ CFU of indicated strains was monitored over time.  Selected groups received vitamin B~6~ (VB6)-enriched diet from 2 weeks prior to vaccination to the end of the experiment.  Median times of survival were 162.5 days (BCG) and 430 days (BCG Δ*ureC*::*hly*).  Data shown were analyzed using the Mantel-Cox log-rank test. \*, * P* \< 0.05; \*\*, *P* \< 0.01; \*\*\*, *P* \< 0.001; \*\*\*\*, *P* \< 0.0001.  Experiment was performed twice.](mbo0031418610002){#fig2}

Vitamin B~6~ controls acquired but not innate immunity induced by BCG Δ*ureC*::*hly* Δ*pdx1*. {# h1.4}
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The systemic innate immune responses measured by cytokine secretion were independent of dietary vitamin B~6~ levels ([Fig.  3A](#fig3){ref-type="fig"}).  However, 24 h following inoculation, BCG Δ*ureC*::*hly* Δ*pdx1* showed a more pronounced stimulation of the inflammatory mediators interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), keratinocyte cytokine (KC), and macrophage inflammatory protein-1α (MIP-1α) than BCG SSI 1331 and BCG Δ*ureC*::*hly*, which depended on *pdx1* deletion (G-CSF and MIP-1α) or vitamin B~6~ supplementation (IL-6 and KC) ([Fig.  3A](#fig3){ref-type="fig"}).  In order to detect mycobacterium-specific T cells, we stimulated splenocytes with antigen *ex vivo*, in the presence of a CD40-blocking monoclonal antibody.  Antigen-specific CD4 T cells, which consequently upregulated CD154 under these conditions, were then enumerated by flow cytometry and compared to those from naive controls ([@B27]).  Twenty-eight days postvaccination, the absolute numbers and proportions of antigen-specific CD4 T cells were increased in mice vaccinated with BCG Δ*ureC*::*hly*, as well as in mice vaccinated with BCG Δ*ureC*::*hly* Δ*pdx1* under vitamin B~6~ supplementation ([Fig.  3B and C](#fig3){ref-type="fig"}).  The proportion of CD4 T cells secreting gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) was also increased in mice vaccinated with BCG Δ*ureC*::*hly* and in the supplemented auxotrophic-strain-vaccinated groups compared to the proportion in naive mice ([Fig.  3B](#fig3){ref-type="fig"}).  This immune response persisted for 3 months postvaccination.  We conclude that the improved survival of the auxotrophic BCG Δ*ureC*::*hly* Δ*pdx1* strain due to the administration of vitamin B~6~ supported the generation of memory T cells, which persist after clearance of the vaccine strain ([Fig.  1C](#fig1){ref-type="fig"} and [3D](#fig3){ref-type="fig"}).  Vaccination also generated long-lived, IgG-secreting plasma cells, which homed to bone marrow.  These cells were detected 3 months postvaccination with either BCG Δ*ureC*::*hly* or vitamin B~6~-supplemented BCG Δ*ureC*::*hly* Δ*pdx1* ([Fig.  3E](#fig3){ref-type="fig"}).  Thus, adaptive immunity, particularly the generation of antigen-specific CD4 T cells and B cells in response to mycobacterial antigens, depended on the persistence of the vaccine strains, and the persistence of the auxotrophic strain could be regulated by exogenous administration of vitamin B~6~.

![ Dietary vitamin B~6~ supplementation restores adaptive immune responses in BCG Δ*ureC*::*hly* Δ*pdx1*-vaccinated mice to levels in parental BCG Δ*ureC*::*hly*-vaccinated mice. ( A) Influence of vitamin B~6~ (VB6) on serum cytokine responses following immunization with indicated constructs.  Significant differences were only observed 1 day postvaccination.  Shown are means ± standard errors of the means (SEM) (*n* = 5) analyzed using two-way ANOVA and Tukey's posttest. ( B) Representative flow cytometry results showing percentages of antigen-specific CD154^+^ T cells among CD3^+^ CD4^+^ T cells and levels of intracellular IFN-γ and TNF-α in spleen after *in vitro* culture with *M. tuberculosis* lysate 28 days postvaccination. ( C and D) Graphs showing proportions of antigen-specific CD154^+^ T cells among total splenic CD3^+^ CD4^+^ cultured as in B, at 28 or 90 days postvaccination. ( E) The proportion of IgG-secreting cells in bone marrow measured by ELISPOT 3 months postvaccination.  One-way ANOVA followed by Bonferroni's posttest was used for statistical analysis. \*, * P* \< 0.05; \*\*, *P* \< 0.01; \*\*\*, *P* \< 0.001; \*\*\*\*, *P* \< 0.0001; *n* = 3 or 4.  Representative data from two independent experiments are shown.](mbo0031418610003){#fig3}

Vitamin B~6~ controls early protection against *M. tuberculosis* in BCG Δ*ureC*::*hly* Δ*pdx1*-vaccinated mice. {# h1.5}
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In order to assess the protective capacity of BCG Δ*ureC*::*hly* Δ*pdx1*, we aerosol challenged mice with the *M. tuberculosis* laboratory strain H37Rv 90 days postvaccination.  The bacterial burdens in lungs and spleens were determined at 30 and 180 days postinfection ([Fig.  4A](#fig4){ref-type="fig"}).  As in previous experiments ([@B23], [@B26]), BCG Δ*ureC*::*hly* consistently induced better protection than BCG ([Fig.  4B](#fig4){ref-type="fig"}).  The early protection provided by BCG Δ*ureC*::*hly* Δ*pdx1* depended on vitamin B~6~ supplementation and was comparable to that induced by BCG but was completely lost at 180 days postinfection ([Fig.  4B](#fig4){ref-type="fig"}).  Presumably, the vitamin B~6~-dependent persistence of the construct in dLNs and spleen ([Fig.  1C](#fig1){ref-type="fig"}) was not sufficient to induce long-lasting protection.  We conclude that a single immunization with BCG Δ*ureC*::*hly* Δ*pdx1* does not suffice for long-term protection against aerogenic *M. tuberculosis* infection in mice. 

![ Early protection induced by primary BCG Δ*ureC*::*hly* Δ*pdx1* vaccination depends on vitamin B~6~ supplementation of mice. ( A) Schematic design of protection studies. ( B) Impact of vitamin B~6~ on bacterial burdens in lungs and spleen after *M. tuberculosis* aerosol infection of mice that received primary vaccination only.  Groups of mice received vitamin B~6~ (VB6)-enriched diet as indicated starting 2 weeks prior to vaccination until *M. tuberculosis* challenge.  Shown are means ± SEM (*n* = 5 or 6) analyzed using one-way ANOVA and Tukey's posttest. \*, * P* \< 0.05; \*\*, *P* \< 0.01; \*\*\*, *P* \< 0.001; \*\*\*\*, *P* \< 0.0001.  Data from one experiment representative of two independent biological replicates are shown.](mbo0031418610004){#fig4}

Prime-boost vaccination improves the efficacy of BCG Δ*ureC*::*hly* Δ*pdx1*. {# h1.6}
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The fast clearance of the auxotrophic vaccine construct from mouse organs ([Fig.  1C](#fig1){ref-type="fig"}) led us to hypothesize that the protective capacity of BCG Δ*ureC*::*hly* Δ*pdx1* could be improved by homologous prime-boost immunization.  After vaccination, mice were aerosol challenged with the *M. tuberculosis* laboratory strain H37Rv or bacilli of the clinically relevant Beijing/W lineage ([@B28]).  Mice vaccinated with BCG Δ*ureC*::*hly* Δ*pdx1* and fed a normal diet showed bacterial burdens in lungs equal to those of animals vaccinated with BCG ([Fig.  5A and B](#fig5){ref-type="fig"}).  The early protection (day 30) in lungs provided by the auxotrophic strain consistently depended on vitamin B~6~-enriched nutrition and, for Beijing/W-infected groups, was equal to the protection provided by the parental BCG Δ*ureC*::*hly* and significantly better than that of BCG ([Fig.  5A](#fig5){ref-type="fig"}).  Homologous prime-boost vaccination with BCG Δ*ureC*::*hly* Δ*pdx1* improved the long-term efficacy against *M. tuberculosis* H37Rv to the level afforded by BCG and performed significantly better than the mock-vaccinated control group against Beijing/W infection ([Fig.  5A and B](#fig5){ref-type="fig"}).  The protection of the lung at late time points (180 days for *M. tuberculosis* H37Rv and 160 days for *M. tuberculosis* Beijing/W) remained independent from vitamin B~6~ supplementation ([Fig.  5A and B](#fig5){ref-type="fig"}).  The results obtained from the spleen were less pronounced and often lacked statistical significance.  Taken together, homologous prime-boost immunization improved the efficacy afforded by BCG Δ*ureC*::*hly* Δ*pdx1* compared to that of single prime vaccination and consistently depended on a dietary vitamin B~6~ supply at early time points following *M. tuberculosis* challenge. 

![ Homologous prime-boost vaccination with BCG Δ*ureC*::*hly* Δ*pdx1* improves protection and maintains early vitamin B~6~ dependence.  Mice vaccinated with the auxotrophic construct received a homologous booster immunization after 30 days ([Fig.  4A](#fig4){ref-type="fig"}).  Animals were aerosol infected with the *M. tuberculosis* laboratory strain H37Rv (A) or a clinical isolate of the Beijing/W lineage (B).  Groups of mice received vitamin B~6~ (VB6)-enriched diet as indicated starting 2 weeks prior to vaccination until *M. tuberculosis* challenge.  Bacterial burdens of lungs and spleen were assessed 30 and 180 (or 160) days postchallenge.  Shown are means ± SEM (*n* = 6-8) analyzed using one-way ANOVA and Tukey's posttest. \*, * P* \< 0.05; \*\*, *P* \< 0.01; \*\*\*, *P* \< 0.001; \*\*\*\*, *P* \< 0.0001.  Data from one experiment representative of two independent biological replicates are shown.](mbo0031418610005){#fig5}

DISCUSSION {#h2}
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The increasing realization that the current BCG vaccine provides insufficient protection against TB, even in newborns, prompted us to develop a recombinant BCG, BCG Δ*ureC*::*hly*, which had improved efficacy and safety in preclinical models and is currently undergoing phase II clinical assessment ([@B29]).  The increasing proportion of immunocompromised vaccinees has now led us to further modify this construct toward a superior safety profile by rendering it auxotrophic for vitamin B~6~. Several auxotrophic mycobacterial mutants have been described ([@B15], [@B30]).  Due to their inability to synthesize certain amino acids or cofactors, such mutants rely on uptake of the missing factor in order to survive.  During infection, mycobacteria reside in the phagosome of antigen-presenting cells, notably, macrophages ([@B31]).  The phagosome is an organelle specialized in the eradication of microbial intruders and, therefore, poor in nutrients but rich in reactive radicals and hydrogen ions ([@B31]).  Pathogenic mycobacteria can survive and replicate in this hostile environment and eventually egress to the host cell cytosol ([@B32], [@B33]).  In contrast, auxotrophic mutants of *M. tuberculosis* are severely attenuated *in vivo*, demonstrating that infected host cells do not provide sufficient concentrations of the respective factors to overcome deficient persistence ([@B8], [@B9], [@B11], [@B15]). 

The cytosolic concentration of PLP, the bioactive form of vitamin B~6~, in eukaryotic cells is regulated by "metabolic trapping," a process in which the uncharged vitamers become phosphorylated upon entering cells by passive diffusion ([@B34]).  As a result, leakage of the negatively charged B~6~ vitamer phosphoesters through biological membranes is prevented.  Indeed, PLP could not restore the growth of *M. tuberculosis* Δ*pdx1* in cultures, confirming the absence of a dedicated uptake system ([@B15]).  As a corollary, mycobacterial *pdx1* mutants are attenuated in mice ([Fig.  1C](#fig1){ref-type="fig"}) ([@B15]).  Intriguingly, when we fed mice an \~10-fold daily dose of vitamin B~6~---more than 10,000-fold below the 50% lethal dose reported for mice ([@B35])---the persistence of BCG Δ*ureC*::*hly* Δ*pdx1* in dLNs improved and the vaccine candidate was as immunogenic as its parental BCG Δ*ureC*::*hly* and superior to BCG ([Fig.  1C](#fig1){ref-type="fig"}).  Clearance of the auxotrophic strain at the site of vaccination and in the spleen was independent of vitamin B~6~ supplementation, suggesting that the availability of the cofactor could not be raised by elevated uptake into these organs.  Intriguingly, the superiority of the safety profile of BCG Δ*ureC*::*hly* over that of canonical BCG in SCID mice that serve as an immunodeficiency model was further improved by *pdx1* deletion and remained independent of vitamin B~6~ supplementation ([Fig.  2](#fig2){ref-type="fig"}). 

The assessment of blood serum cytokine concentrations as indicators of innate immunity suggests a proinflammatory phenotype for BCG Δ*ureC*::*hly* Δ*pdx1* compared to that of its parental strain ([Fig.  3A](#fig3){ref-type="fig"}).  While the IL-6 and KC release induced by the auxotrophic strain depended on a vitamin B~6~-enriched diet, the increased levels of G-CSF and MIP-1α were related to the *pdx1* mutation.  Vitamin B~6~ supplementation left these innate immune parameters in response to BCG or BCG Δ*ureC*::*hly* unaffected ([Fig.  3A](#fig3){ref-type="fig"}), thus linking our observations to the persistence of the auxotrophic construct.  While inflammatory mediators of innate immunity were stimulated by the attenuated strain, long-lasting antigen-specific T cell responses were only induced when bacterial persistence had been re-established by vitamin B~6~ supplementation.  This is particularly relevant as CD4 T cell responses are thought to be an important component of protection against pulmonary TB ([@B36]) and are frequently harnessed as biomarkers of vaccine efficacy ([@B37], [@B38]).  Although the role of antibodies in protection remains controversial, the ability of vitamin B~6~ supplementation to induce long-lived plasma cells supports the importance of vaccine persistence for effective stimulation of adaptive immune responses. 

The blood serum concentrations of B6 vitamers (pyridoxal 5-phosphate, 5 to 111 nM; 4 to pyridoxic acid, 6 to 93 nM; and pyridoxal, 3 nM) vary considerably in humans on a normal diet but stabilize (pyridoxal 5-phosphate, \~350 nM; 4-pyridoxic acid, \~1,000 nM; and pyridoxal, \~700 nM) 3 days after daily intake of 40 mg of vitamin B~6~ ([@B39]).  Approximately 70 to 80% of overall vitamin B~6~ is found bound to glycogen phosphorylase in muscles ([@B40]).  The upper tolerated dose of the vitamin in adults is 100 mg/day ([@B41]).  Vitamin B~6~ deficiency affects innate and acquired immune responses, but physiological functions can be restored by standard supplementation with the cofactor, while larger doses were not more beneficial ([@B42]).  Accordingly, increasing dietary vitamin B~6~ supplementation beyond sufficient doses did not influence either the protective efficacy of BCG and BCG Δ*ureC*::*hly* in a murine challenge model or the course of *M. tuberculosis* infection *per se* in naive mice (see [Fig.  S2](#figS2){ref-type="supplementary-material"} and S3 in the supplemental material).  Therefore, the early protection induced by BCG Δ*ureC*::*hly* Δ*pdx1* consistently depended on the persistence of the construct under the control of vitamin B~6~ supplementation ([Fig.  4B](#fig4){ref-type="fig"}).  Mice immunized with the auxotrophic strain in a homologous prime-boost vaccination scheme ([Fig.  4A](#fig4){ref-type="fig"}) maintained the vitamin B~6~-related differences but revealed improved protection, to a degree similar to that of parental BCG Δ*ureC*::*hly* 30 days postchallenge with *M. tuberculosis* Beijing/W ([Fig.  5A and B](#fig5){ref-type="fig"}).  Whether protection could be further improved by a second booster vaccination remains the topic of future studies. 

Altogether, we demonstrate for the first time that a nutritional supplement, namely, the small-molecule vitamin B~6~, controls the persistence of a recombinant BCG auxotroph *in vivo*.  Most importantly, this translated into vitamin B~6~ dependency of acquired early immune protection in response to the auxotrophic construct.  This principle will enable the development of safer BCG vaccines that are urgently needed for the growing proportion of immunocompromised individuals among vaccinees. 

MATERIALS AND METHODS {#h3}
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Bacterial strains and growth conditions. {# h3.1}
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BCG (BCG SSI 1331; ATCC 35733; American Type Culture Collection), BCG Δ*ureC*::*hly* (BCG Prague background) ([@B22], [@B23]), *M. tuberculosis* H37Rv (ATCC 27294), and Beijing/W (RIVM 17919, isolated in Mongolia) were maintained in Middlebrook 7H9 medium (Becton, Dickinson) supplemented with 0.2% glycerol, 0.05% Tween 80, 10% albumin-dextrose-catalase supplement (Becton, Dickinson), in Sauton's minimal medium (0.5 g/liter KH~2~PO~4~, 0.5 MgSO~4~, 2 g/liter citric acid, 0.05 g/liter ferric ammonium citrate, 4 g/liter asparagine, 6% glycerol, 0.05% Tween 80, pH 6.8), or on Middlebrook 7H11 agar (Becton, Dickinson) containing 10% (vol/vol) oleic acid-albumin-dextrose-catalase enrichment (Becton, Dickinson) and 0.2% glycerol.  Mycobacterial cultures were grown to mid-log phase in 1-liter roller bottles (450 cm^2^) at 37°C and 2 rpm.  For *in vitro* growth studies, mycobacteria were grown in Sauton's minimal medium supplemented with 5 µM pyridoxine.  Cultures were pelleted at 3,200 rpm and washed three times prior to resuspension in fresh Sauton's medium with or without pyridoxine.  The optical density at 600 nm (OD~600~) was determined daily.  For vaccine stock preparations, bacilli were collected by centrifugation at 3,200 rpm, washed with phosphate-buffered saline (PBS), and stored at −80°C as a suspension in PBS\--10% glycerol.  Prior to vaccination, vials were thawed and cells harvested and resuspended in an appropriate volume of PBS.  For CFU enumeration, serial dilutions were performed in PBS\--0.05% Tween 80 (PBST) and plated on Middlebrook 7H11 agar.  The plates were incubated at 37°C for 3 to 4 weeks prior to CFU counting. 

Generation of BCG Δ*ureC*::*hly* Δ*pdx1* and genetic complementation. {# h3.2}
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The *pdx1* gene of BCG Δ*ureC*::*hly* was disrupted as described earlier for *M. tuberculosis* ([@B15]).  Briefly, 1-kb fragments flanking *pdx1* were amplified by PCR using the specific oligonucleotides ko5′pdx1.fwd (5′ TA[CTTAAG]{.ul}CGGGTCAGCGGGCATTCC 3′)/ko5′pdx1.rev (5′ AT[TCTAGA]{.ul}CCGGGGTGACAACGTCCATGAT 3′) and ko3′pdx1.fwd (5′ TA[AAGCTT]{.ul}TGTGCTGGCCAAGGTGTCG 3′)/ko3′pdx1.rev (5′ AT[ACTAGT]{.ul}CGACCCGTGGAACGCTCACAG 3′) (restriction sites are underlined) and inserted into pYUB854 (43).  The knockout plasmid was then electroporated into BCG Δ*ureC*::*hly*, and transformants were selected on Middlebrook 7H11 agar (contains vitamin B~6~) supplemented with 80 µg/ml hygromycin B (Roche).  The hygromycin resistance cassette was subsequently removed by standard methods described previously (44).  Site-directed mutagenesis and selection marker removal were confirmed by automated sequencing of the *pdx1* region.  For genetic complementation, *pdx1*, including its putative promoter, was amplified by PCR (comppdx1.fwd \[5′ GCT[GGTACC]{.ul}AGGGAAAGGTTGCCGATG 3′\] and comppdx1.rev \[5′ GCT[GGTACC]{.ul}AGGGAAAGGTTGCCGATG 3′\]) and inserted into the integrative vector pMV306 (45).  BCG Δ*ureC*::*hly* Δ*pdx1* was then electroporated with the complementing vector, and transformants were selected on Middlebrook 7H11 agar containing 25 µg/ml kanamycin (Sigma-Aldrich). 

Multiplex cytokine assays. {# h3.3}
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The Bio-Rad mouse cytokine 23-plex panel was used for analysis of cytokines in sera of vaccinated mice.  In all multiplex assays, the volume of the coupled beads, detection antibodies, and streptavidin-phycoerythrin (PE) conjugate was halved and topped up with the appropriate buffer.  The assays were otherwise performed according to the manufacturer's instructions.  The assay plates were read using a Bio-Plex 200 instrument (Bio-Rad). 

Flow cytometry. {# h3.4}
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Cytokine-secreting antigen-specific T cells were enumerated as described previously ([@B27]).  In brief, single-cell suspensions of splenocytes were cultured in complete RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco), 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM [l]{.smallcaps}-glutamine, and 50 µM 2-mercaptoethanol (Sigma) for 14 h along with 10 µg/ml *M. tuberculosis* H37Rv whole-cell lysate in the presence of 2 µg/ml anti-CD40 blocking antibody (HM40-3; BioLegend) and 0.5 µg/ml PE-conjugated anti-CD154 (clone MR1; BioLegend).  Ten micrograms/milliliter Brefeldin A (eBioscience) was added for the final 3 h of culture.  After stimulation, cells were washed and stained on ice in PBS\--2% FCS with fluorescent antibodies against cell surface markers CD4, CD3, CD8 (BD Biosciences), and CD62L (BioLegend), followed by fixation and permeabilization with the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's instructions.  Intracellular cytokines were detected with antibodies against IFN-γ and TNF-α (BD Biosciences).  Cells were analyzed on an LSRII cytometer using Diva (Becton, Dickinson) and FlowJo software (Tree Star). 

Enzyme-linked immunosorbent spot assay (ELISPOT). {# h3.5}
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Ninety-six-well multiscreen filtration plates (Millipore) were soaked with 30% ethanol and then rinsed and coated overnight at 4°C with 5 µg/ml polyclonal goat anti-mouse IgM+IgG+IgA (Millipore).  The following day, the plates were washed and blocked with PBS\--3% bovine serum albumin for 2 h at 37°C.  Serial dilutions of single-cell suspensions of bone marrow were cultured on plates for 3 h at 37°C in medium prepared as described above.  Cells were washed off, and the plates incubated with anti-mouse IgG-alkaline phosphatase (SouthernBiotech) for 1 h at 37°C.  Finally, plates were washed with water before detection of IgG^+^ spots with 1-Step NBT-BCIP (nitro-blue tetrazolium chloride\--5-bromo-4-chloro-3′-indolyphosphate *p*-toluidine salt) substrate (Thermo Scientific). 

Mouse strains and housing conditions. {# h3.6}
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Mice were 9 to 10 weeks old at the beginning of experiments.  Female BALB/c (Janvier) were housed in groups of 5 or 6 in individually ventilated cages.  Drinking water and food pellets (Ssniff R/M-H autoclavable, 0.032 mg pyridoxine per g) were offered *ad libitum*.  Experimental groups that were maintained with vitamin B~6~-enriched nutrition received drinking water with 0.1 mg/ml pyridoxine hydrochloride (Sigma-Aldrich).  Female SCID mice (CB-17/Icr-*Prkdc*^SCID^/Rj; Janvier) were kept under sterile conditions.  All experimental procedures involving mice were approved by the State Office for Health and Social Services, Berlin, Germany (Landesamt für Gesundheit und Soziales Berlin, LAGeSo).  Mice were sacrificed by cervical dislocation, and all efforts were made to minimize suffering and pain. 

Dissemination, protective efficacy, and safety of BCG and derivatives in mice. {# h3.7}
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Mice were s.c.  immunized in the tail base with 10^6^ CFU BCG or recombinant derivative strains.  At designated time points postvaccination, mice were sacrificed, and then a 1-cm^2^ skin portion at the side of injection and the dLNs, spleen, and lungs of each animal were aseptically removed and homogenized in PBS\--0.05% Tween 80 prior to CFU enumeration.  For protective efficacy studies, mice were aerosol challenged 90 days postvaccination with 100 to 200 CFU of *M. tuberculosis*.  At designated time points, lungs and spleens were aseptically removed, homogenized in PBS\--0.05% Tween 80, and plated in serial dilutions onto Middlebrook 7H11 agar for CFU enumeration.  After vaccination, SCID mice did not receive further experimental manipulations.  Weight was monitored weekly, and mice were euthanized when weight loss exceeded 20%. 

SUPPLEMENTAL MATERIAL {#h4}
=====================

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Vitamin B~6~ biosynthesis operons of *M. tuberculosis* and BCG are identical.  The genes *pdx1* and *pdx2* encode subunits of functional vitamin B~6~ synthase in mycobacteria.  In the presence of free ammonia, Pdx1 alone has limited capacity to synthesize vitamin B~6~. The acyl-coenzyme A (CoA) thioesterase II (TesB2) has no direct association with vitamin B~6~ biosynthesis.  Download

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Figure S1, EPS file, 0.3 MB

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Vitamin B~6~ supplementation does not influence vaccine efficacy of BCG and BCG Δ*ureC*::*hly* in mice.  Effects of vitamin B~6~ on bacterial burdens in lungs (A) and spleen (B) after *M. tuberculosis* aerosol infection of mice that received only primary vaccination are shown.  Experimental design is shown in Fig.  4A. Groups of mice received vitamin B~6~ (VB6)-enriched diet as indicated starting 2 weeks before vaccination until *M. tuberculosis* challenge.  Shown are means ± SEM (*n* = 5 or 6) analyzed using one-way ANOVA and Tukey's posttest. \*, * P* \< 0.05; \*\*, *P* \< 0.01; \*\*\*, *P* \< 0.001; \**\*\**\*, *P* \< 0.0001.  The experiment was performed twice.  Download

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Figure S2, EPS file, 0.7 MB

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Vitamin B~6~ supplementation does not influence *M. tuberculosis* infection in immunocompetent mice.  Indicated groups received vitamin B~6~-enriched diet from 2 weeks prior to aerosol challenge of mice with 200 CFU *M. tuberculosis* H37Rv until the end of the experiment.  Bacterial burdens of lungs and spleen were assessed over the course of TB infection.  Differences between experimental groups were analyzed by Student's *t* test (*n* = 5).  A *P* value of \>0.05 was considered statistically insignificant.  Download

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Figure S3, EPS file, 0.3 MB

**Citation** Gengenbacher M, Vogelzang A, Schuerer S, Lazar D, Kaiser P, Kaufmann SHE.  2014.  Dietary pyridoxine controls efficacy of vitamin B~6~-auxotrophic tuberculosis vaccine bacillus Calmette-Guérin Δ*ureC*::*hly* Δ*pdx1* in mice.  mBio 5(3):e01262-14.  doi:10.1128/mBio.01262-14. 

We thank Mary Louise Grossman for excellent editorial support and Ulrike Zedler for technical assistance. 

This work was funded by the European Research Council's 7th Framework Program (FP7), NEWTBVAC (grant no.  HEALTH-F3-2009-241745). 

[^1]: Present address: Martin Gengenbacher, National University of Singapore, Yong Loo Lin School of Medicine, Department of Microbiology, Singapore. 

[^2]: **Editor** Rino Rappuoli, Novartis Vaccines

[^3]: This article is a direct contribution from a member of the American Academy of Microbiology.