Genomic dissection of antibiotic resistance in bacterial pathogens has largely focused on genetic changes conferring growth above a single critical concentration of drug. However, reduced susceptibility to antibiotics—even below this breakpoint—is associated with poor treatment outcomes in the clinic, including in tuberculosis. Clinical strains of Mycobacterium tuberculosis exhibit extensive quantitative variation in antibiotic susceptibility but the genetic basis behind this spectrum of drug susceptibility remains ill-defined. Through a genome wide association study, we show that non-synonymous mutations in dnaA, which encodes an essential and highly conserved regulator of DNA replication, are associated with drug resistance in clinical M. tuberculosis strains. We demonstrate that these dnaA mutations specifically enhance M. tuberculosis survival during isoniazid treatment via reduced expression of katG, the activator of isoniazid. To identify DnaA interactors relevant to this phenotype, we perform the first genome-wide biochemical mapping of DnaA binding sites in mycobacteria which reveals a DnaA interaction site that is the target of recurrent mutation in clinical strains. Reconstructing clinically prevalent mutations in this DnaA interaction site reproduces the phenotypes of dnaA mutants, suggesting that clinical strains of M. tuberculosis have evolved mutations in a previously uncharacterized DnaA pathway that quantitatively increases resistance to the key first-line antibiotic isoniazid. Discovering genetic mechanisms that reduce drug susceptibility and support the evolution of high-level drug resistance will guide development of biomarkers capable of prospectively identifying patients at risk of treatment failure in the clinic.

Drug-resistant TB remains a public health challenge. Rifamycins are among the most potent anti-TB drugs. They are known to target the RpoB subunit of RNA polymerase; however, our understanding of how rifamycin resistance is genetically encoded remains incomplete. Here we investigated rpoB genetic diversity and cross-resistance between the two rifamycin drugs rifampicin and rifabutin. Methods: We performed WGS of 1003 Mycobacterium tuberculosis clinical isolates and determined MICs of both rifamycin agents on 7H10 agar using the indirect proportion method. We generated rpoB mutants in a laboratory strain and measured their antibiotic susceptibility using the alamarBlue reduction assay. Results: Of the 1003 isolates, 766 were rifampicin resistant and 210 (27%) of these were rifabutin susceptible; 102/210 isolates had the rpoB mutation D435V (Escherichia coli D516V). Isolates with discordant resistance were 17.2 times more likely to harbour a D435V mutation than those resistant to both agents (OR 17.2, 95% CI 10.5-27.9, P value ,10 #40). Compared with WT, the D435V in vitro mutant had an increased IC 50 of both rifamycins; however, in both cases to a lesser degree than the S450L (E. coli S531L) mutation. Conclusions: The observation that the rpoB D435V mutation produces an increase in the IC 50 of both drugs contrasts with findings from previous smaller studies that suggested that isolates with the D435V mutation remain rifabutin susceptible despite being rifampicin resistant. Our finding thus suggests that the recommended critical testing concentration for rifabutin should be revised.

Despite the existence of well-characterized, canonical mutations that confer high-level drug resistance to Mycobacterium tuberculosis (Mtb), there is evidence that drug resistance mechanisms are more complex than simple acquisition of such mutations. Recent studies have shown that Mtb can acquire non-canonical resistance-associated mutations that confer survival advantages in the presence of certain drugs, likely acting as stepping-stones for acquisition of high-level resistance. Rv2752c/rnj, encoding RNase J, is disproportionately mutated in drug-resistant clinical Mtb isolates. Here we show that deletion of rnj confers increased tolerance to lethal concentrations of several drugs. RNAseq revealed that RNase J affects expression of a subset of genes enriched for PE/PPE genes and stable RNAs and is key for proper 23S rRNA maturation. Gene expression differences implicated two sRNAs and ppe50-ppe51 as important contributors to the drug tolerance phenotype. In addition, we found that in the absence of RNase J, many short RNA fragments accumulate because they are degraded at slower rates. We show that the accumulated transcript fragments are targets of RNase J and are characterized by strong secondary structure and high G+C content, indicating that RNase J has a rate-limiting role in degradation of highly structured RNAs. Taken together, our results demonstrate that RNase J indirectly affects drug tolerance, as well as reveal the endogenous roles of RNase J in mycobacterial RNA metabolism.

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ⌬leuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freezethaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ⌬leuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10 24 -fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10 68 -fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ⌬leuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. The immense global burden of human immunodeficiency virus (HIV) infection necessitates the development of an efficacious vaccine. There is increasing interest in the use of live recombinant bacterial vectors as HIV vaccines due to the inherent advantages of utilizing a replicating antigen delivery system that is itself an effective adjuvant (1, 2). Previous studies have examined the use of live Gram-positive and Gram-negative bacterial vectors, including recombinant Salmonella, Listeria, Streptococcus, and Escherichia coli, for heterologous expression of HIV antigens, with varying success (3-8).Mycobacterium bovis BCG is the most widely administered vaccine in the world (9). Its extensively documented safety in immunocompetent individuals, relatively low production cost, and well-established infrastructure for vaccine administration make it an ideal candidate for use as an anti-HIV vaccine vehicle (10-12). In addition to the logistical advantages of using BCG, mycobacterial antigen delivery systems possess inherent adjuvant properties which activate innate immunity (13,14). Mycobacteria such as BCG ...

cLive attenuated nonpathogenic Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediates long-lasting immune responses, has been safely administered as a tuberculosis vaccine to billions of humans, and is affordable to produce as a vaccine vector. These characteristics make it very attractive as a human immunodeficiency virus (HIV) vaccine vector candidate. Here, we assessed the immunogenicity of recombinant BCG (rBCG) constructs with different simian immunodeficiency virus (SIV)gag expression cassettes as priming agents followed by a recombinant replication-incompetent New York vaccinia virus (NYVAC) boost in rhesus macaques. Unmutated rBCG constructs were used in comparison to mutants with gene deletions identified in an in vitro screen for augmented immunogenicity. We demonstrated that BCG-SIVgag is able to elicit robust transgene-specific priming responses, resulting in strong SIV epitope-specific cellular immune responses. While enhanced immunogenicity was sustained at moderate levels for >1 year following the heterologous boost vaccination, we were unable to demonstrate a protective effect after repeated rectal mucosal challenges with pathogenic SIVmac251. Our findings highlight the potential for rBCG vaccines to stimulate effective cross-priming and enhanced major histocompatibility complex class I presentation, suggesting that combining this approach with other immunogens may contribute to the development of effective vaccine regimens against HIV.

c 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.

Background A recombinant Mycobacterium bovis BCG (rBCG) vector expressing HIV transgenes is an attractive candidate as a dual vaccine against HIV and TB. However, pre-existing immune responses to mycobacteria may influence immune responses to rBCG. We analyzed data from a rhesus rBCG trial to determine the effect of pre-existing mycobacterial immune responses on the vaccine-induced responses to the vector and expressed transgene. Methods Indian-origin rhesus macaques were primed with rBCG expressing simian immunodeficiency virus (SIV) Gag and boosted with attenuated vaccinia NYVAC gag-pol. Mycobacteria responses were measured by M. tuberculosis (Mtb) purified protein derivative (PPD) interferon-γ ELISpot and Mtb whole cell lysate (WCL) ELISA. SIV Gag responses were measured by SIV Gag ELISpot and by p11C tetramer binding. Results Baseline Mtb PPD ELISpot responses and Mtb WCL antibody responses in rhesus macaques overlapped those in human populations. Cellular and antibody responses boosted sharply 4 weeks after rBCG vaccination. Mtb WCL antibody titers at 4 weeks correlated with baseline titers. Primates vaccinated with rBCG developed strong SIV Gag ELISpot and p11C tetramer responses after rBCG prime and NYVAC boost. There were no correlations between the pre-existing mycobacterial immune responses and the SIV Gag T cell responses after vaccination. Conclusions Rhesus immune responses to SIV Gag expressed by rBCG vectors were independent from pre-existing anti-mycobacterial immunity. Rhesus macaques may serve as a surrogate for investigations of pre-existing anti-mycobacterial immunity in humans.