Killer whale respiration rates.

Measuring breathing rates is a means by which oxygen intake and metabolic rates can be estimated to determine food requirements and energy expenditure of killer whales (Orcinus orca) and other cetaceans. This relatively simple measure also allows the energetic consequences of environmental stressors to cetaceans to be understood but requires knowing respiration rates while they are engaged in different behaviours such as resting, travelling and foraging. We calculated respiration rates for different behavioural states of southern and northern resident killer whales using video from UAV drones and concurrent biologging data from animal-borne tags. Behavioural states of dive tracks were predicted using hierarchical hidden Markov models (HHMM) parameterized with time-depth data and with labeled tracks of drone-identified behavioural states (from drone footage that overlapped with the time-depth data). Dive tracks were sequences of dives and surface intervals lasting ≥ 10 minutes cumulative duration. We calculated respiration rates and estimated oxygen consumption rates for the predicted behavioural states of the tracks. We found that juvenile killer whales breathed at a higher rate when travelling (1.6 breaths min-1) compared to resting (1.2) and foraging (1.5)-and that adult males breathed at a higher rate when travelling (1.8) compared to both foraging (1.7) and resting (1.3). The juveniles in our study were estimated to consume 2.5-18.3 L O2 min-1 compared with 14.3-59.8 L O2 min-1 for adult males across all behaviours based on estimates of mass-specific tidal volume and oxygen extraction. Our findings confirm that killer whales take single breaths between dives and indicate that energy expenditure derived from respirations requires using sex, age, and behavioural-specific respiration rates. These findings can be applied to bioenergetics models on a behavioural-specific basis, and contribute towards obtaining better predictions of dive behaviours, energy expenditure and the food requirements of apex predators.

Identification of bacterial determinants of tuberculosis infection and treatment outcomes: a phenogenomic analysis of clinical strains.

BACKGROUND: Bacterial diversity could contribute to the diversity of tuberculosis infection and treatment outcomes observed clinically, but the biological basis of this association is poorly understood. The aim of this study was to identify associations between phenogenomic variation in Mycobacterium tuberculosis and tuberculosis clinical features. METHODS: We developed a high-throughput platform to define phenotype-genotype relationships in M tuberculosis clinical isolates, which we tested on a set of 158 drug-sensitive M tuberculosis strains sampled from a large tuberculosis clinical study in Ho Chi Minh City, Viet Nam. We tagged the strains with unique genetic barcodes in multiplicate, allowing us to pool the strains for in-vitro competitive fitness assays across 16 host-relevant antibiotic and metabolic conditions. Relative fitness was quantified by deep sequencing, enumerating output barcode read counts relative to input normalised values. We performed a genome-wide association study to identify phylogenetically linked and monogenic mutations associated with the in-vitro fitness phenotypes. These genetic determinants were further associated with relevant clinical outcomes (cavitary disease and treatment failure) by calculating odds ratios (ORs) with binomial logistic regressions. We also assessed the population-level transmission of strains associated with cavitary disease and treatment failure using terminal branch length analysis of the phylogenetic data. FINDINGS: M tuberculosis clinical strains had diverse growth characteristics in host-like metabolic and drug conditions. These fitness phenotypes were highly heritable, and we identified monogenic and phylogenetically linked variants associated with the fitness phenotypes. These data enabled us to define two genetic features that were associated with clinical outcomes. First, mutations in Rv1339, a phosphodiesterase, which were associated with slow growth in glycerol, were further associated with treatment failure (OR 5·34, 95% CI 1·21-23·58, p=0·027). Second, we identified a phenotypically distinct slow-growing subclade of lineage 1 strains (L1.1.1.1) that was associated with cavitary disease (OR 2·49, 1·11-5·59, p=0·027) and treatment failure (OR 4·76, 1·53-14·78, p=0·0069), and which had shorter terminal branch lengths on the phylogenetic tree, suggesting increased transmission. INTERPRETATION: Slow growth under various antibiotic and metabolic conditions served as in-vitro intermediate phenotypes underlying the association between M tuberculosis monogenic and phylogenetically linked mutations and outcomes such as cavitary disease, treatment failure, and transmission potential. These data suggest that M tuberculosis growth regulation is an adaptive advantage for bacterial success in human populations, at least in some circumstances. These data further suggest markers for the underlying bacterial processes that contribute to these clinical outcomes. FUNDING: National Health and Medical Research Council/A∗STAR, National Institutes of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the Wellcome Trust Fellowship in Public Health and Tropical Medicine.

Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis.

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) - the variability of gene expression in response to environmental changes - remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also find that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.

Age and sex influence antibody profiles associated with tuberculosis progression.

Antibody features vary with tuberculosis (TB) disease state. Whether clinical variables, such as age or sex, influence associations between Mycobacterium tuberculosis-specific antibody responses and disease state is not well explored. Here we profiled Mycobacterium tuberculosis-specific antibody responses in 140 TB-exposed South African individuals from the Adolescent Cohort Study. We identified distinct response features in individuals progressing to active TB from non-progressing, matched controls. A multivariate antibody score differentially associated with progression (SeroScore) identified progressors up to 2 years before TB diagnosis, earlier than that achieved with the RISK6 transcriptional signature of progression. We validated these antibody response features in the Grand Challenges 6-74 cohort. Both the SeroScore and RISK6 correlated better with risk of TB progression in adolescents compared with adults, and in males compared with females. This suggests that age and sex are important, underappreciated modifiers of antibody responses associated with TB progression.

Antibiotic treatment modestly reduces protection against Mycobacterium tuberculosis reinfection in macaques.

Concomitant immunity is generally defined as an ongoing infection providing protection against reinfection . Its role in prevention of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is supported by epidemiological evidence in humans as well as experimental evidence in mice and non-human primates (NHPs). Whether the presence of live Mtb, rather than simply persistent antigen, is necessary for concomitant immunity in TB is still unclear. Here, we investigated whether live Mtb plays a measurable role in control of secondary Mtb infection. Using cynomolgus macaques, molecularly barcoded Mtb libraries, positron emission tomography-computed tomography (PET CT) imaging, flow cytometry, and cytokine profiling, we evaluated the effect of antibiotic treatment after primary infection on immunological response and bacterial establishment, dissemination, and burden post-secondary infection. Our data provide evidence that, in this experimental model, treatment with antibiotics after primary infection reduced inflammation in the lung but was not associated with a significant change in bacterial establishment, dissemination, or burden in the lung or lymph nodes. Nonetheless, treatment of the prior infection with antibiotics did result in a modest reduction in protection against reinfection: none of the seven antibiotic-treated animals demonstrated sterilizing immunity against reinfection, while four of the seven non-treated macaques were completely protected against reinfection. These findings support that antibiotic-treated animals were still able to restrict bacterial establishment and dissemination after rechallenge compared to naïve macaques, but not to the full extent of non-antibiotic-treated macaques.

Ongoing evolution of the Mycobacterium tuberculosis lactate dehydrogenase reveals the pleiotropic effects of bacterial adaption to host pressure.

The bacterial determinants that facilitate Mycobacterium tuberculosis (Mtb) adaptation to the human host environment are poorly characterized. We have sought to decipher the pressures facing the bacterium in vivo by assessing Mtb genes that are under positive selection in clinical isolates. One of the strongest targets of selection in the Mtb genome is lldD2, which encodes a quinone-dependent L-lactate dehydrogenase (LldD2) that catalyzes the oxidation of lactate to pyruvate. Lactate accumulation is a salient feature of the intracellular environment during infection and lldD2 is essential for Mtb growth in macrophages. We determined the extent of lldD2 variation across a set of global clinical isolates and defined how prevalent mutations modulate Mtb fitness. We show the stepwise nature of lldD2 evolution that occurs as a result of ongoing lldD2 selection in the background of ancestral lineage-defining mutations and demonstrate that the genetic evolution of lldD2 additively augments Mtb growth in lactate. Using quinone-dependent antibiotic susceptibility as a functional reporter, we also find that the evolved lldD2 mutations functionally increase the quinone-dependent activity of LldD2. Using 13C-lactate metabolic flux tracing, we find that lldD2 is necessary for robust incorporation of lactate into central carbon metabolism. In the absence of lldD2, label preferentially accumulates in dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) and is associated with a discernible growth defect, providing experimental evidence for accrued lactate toxicity via the deleterious buildup of sugar phosphates. The evolved lldD2 variants increase lactate incorporation to pyruvate while altering triose phosphate flux, suggesting both an anaplerotic and detoxification benefit to lldD2 evolution. We further show that the mycobacterial cell is transcriptionally sensitive to the changes associated with altered lldD2 activity which affect the expression of genes involved in cell wall lipid metabolism and the ESX- 1 virulence system. Together, these data illustrate a multifunctional role of LldD2 that provides context for the selective advantage of lldD2 mutations in adapting to host stress.

Antibiotic treatment modestly reduces protection against Mycobacterium tuberculosis reinfection in macaques.

Concomitant immunity is generally defined as an ongoing infection providing protection against reinfection1. Its role in prevention of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is supported by epidemiological evidence in humans as well as experimental evidence in mice and non-human primates (NHPs). Whether the presence of live Mtb, rather than simply persistent antigen, is necessary for concomitant immunity in TB is still unclear. Here, we investigated whether live Mtb plays a measurable role in control of secondary Mtb infection. Using cynomolgus macaques, molecularly barcoded Mtb libraries, PET-CT imaging, flow cytometry and cytokine profiling we evaluated the effect of antibiotic treatment after primary infection on immunological response and bacterial establishment, dissemination, and burden post-secondary infection. Our data provide evidence that, in this experimental model, treatment with antibiotics after primary infection reduced inflammation in the lung but was not associated with a significant change in bacterial establishment, dissemination or burden in the lung or lymph nodes. Nonetheless, treatment of the prior infection with antibiotics did result in a modest reduction in protection against reinfection: none of the 7 antibiotic treated animals demonstrated sterilizing immunity against reinfection while 4 of the 7 non-treated macaques were completely protected against reinfection. These findings support that antibiotic-treated animals were still able to restrict bacterial establishment and dissemination after rechallenge compared to naïve macaques, but not to the full extent of non-antibiotic treated macaques.

Mycobacterial DnaQ is an Alternative Proofreader Ensuring DNA Replication Fidelity.

Remove of mis-incorporated nucleotides ensures replicative fidelity. Although the ε-exonuclease DnaQ is a well-established proofreader in the model organism Escherichia coli, proofreading in mycobacteria relies on the polymerase and histidinol phosphatase (PHP) domain of replicative polymerase despite the presence of an alternative DnaQ homolog. Here, we show that depletion of DnaQ in Mycolicibacterium smegmatis results in increased mutation rate, leading to AT-biased mutagenesis and elevated insertions/deletions in homopolymer tract. We demonstrated that mycobacterial DnaQ binds to the b-clamp and functions synergistically with the PHP domain to correct replication errors. Further, we found that the mycobacterial DnaQ sustains replicative fidelity upon chromosome topological stress. Intriguingly, we showed that a naturally evolved DnaQ variant prevalent in clinical Mycobacterium tuberculosis isolates enables hypermutability and is associated with extensive drug resistance. These results collectively establish that the alternative DnaQ functions in proofreading, and thus reveal that mycobacteria deploy two proofreaders to maintain replicative fidelity.

Development of an Engineered Mycobacterium tuberculosis Strain for a Safe and Effective Tuberculosis Human Challenge Model.

Human challenge experiments could greatly accelerate the development of a tuberculosis (TB) vaccine. Human challenge for tuberculosis requires a strain that can both replicate in the host and be reliably cleared. To accomplish this, we designed Mycobacterium tuberculosis (Mtb) strains featuring up to three orthogonal kill switches, tightly regulated by exogenous tetracyclines and trimethoprim. The resultant strains displayed immunogenicity and antibiotic susceptibility similar to wild-type Mtb under permissive conditions. In the absence of supplementary exogenous compounds, the strains were rapidly killed in axenic culture, mice and nonhuman primates. Notably, the strain that contained three kill switches had an escape rate of less than 10 -10 per genome per generation and displayed no relapse in a SCID mouse model. Collectively, these findings suggest that this engineered Mtb strain could be a safe and effective candidate for a human challenge model.

Ongoing evolution of the Mycobacterium tuberculosis lactate dehydrogenase reveals the pleiotropic effects of bacterial adaption to host pressure.

The bacterial determinants that facilitate Mycobacterium tuberculosis (Mtb) adaptation to the human host environment are poorly characterized. We have sought to decipher the pressures facing the bacterium in vivo by assessing Mtb genes that are under positive selection in clinical isolates. One of the strongest targets of selection in the Mtb genome is lldD2 , which encodes a quinone-dependent L-lactate dehydrogenase (LldD2) that catalyzes the oxidation of lactate to pyruvate. Lactate accumulation is a salient feature of the intracellular environment during infection and lldD2 is essential for Mtb growth in macrophages. We determined the extent of lldD2 variation across a set of global clinical isolates and defined how prevalent mutations modulates Mtb fitness. We show the stepwise nature of lldD2 evolution that occurs as a result of ongoing lldD2 selection in the background of ancestral lineage defining mutations and demonstrate that the genetic evolution of lldD2 additively augments Mtb growth in lactate. Using quinone-dependent antibiotic susceptibility as a functional reporter, we also find that the evolved lldD2 mutations functionally increase the quinone-dependent activity of LldD2. Using 13 C-lactate metabolic flux tracing, we find that lldD2 is necessary for robust incorporation of lactate into central carbon metabolism. In the absence of lldD2 , label preferentially accumulates in methylglyoxal precursors dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) and is associated with a discernible growth defect, providing experimental evidence for accumulated lactate toxicity via a methylglyoxal pathway that has been proposed previously. The evolved lldD2 variants increase lactate incorporation to pyruvate but also alter flux in the methylglyoxal pathway, suggesting both an anaplerotic and detoxification benefit to lldD2 evolution. We further show that the mycobacterial cell is transcriptionally sensitive to the changes associated with altered lldD2 activity which affect the expression of genes involved in cell wall lipid metabolism and the ESX-1 virulence system. Together, these data illustrate a multifunctional role of LldD2 that provide context for the selective advantage of lldD2 mutations in adapting to host stress.

Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis.

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also found that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.

Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs.

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is hypothesized to play a major role in mRNA degradation because of its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the nonpathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in degradation of the transcripts encoded by at least 89% of protein-coding genes, with leadered transcripts often being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in M. smegmatis and Mycobacterium tuberculosis and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negative bacteria. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming that RNase E has a broad impact on the M. tuberculosis transcriptome.

CD8+ lymphocytes are critical for early control of tuberculosis in macaques.

The functional role of CD8+ lymphocytes in tuberculosis remains poorly understood. We depleted innate and/or adaptive CD8+ lymphocytes in macaques and showed that loss of all CD8α+ cells (using anti-CD8α antibody) significantly impaired early control of Mycobacterium tuberculosis (Mtb) infection, leading to increased granulomas, lung inflammation, and bacterial burden. Analysis of barcoded Mtb from infected macaques demonstrated that depletion of all CD8+ lymphocytes allowed increased establishment of Mtb in lungs and dissemination within lungs and to lymph nodes, while depletion of only adaptive CD8+ T cells (with anti-CD8ß antibody) worsened bacterial control in lymph nodes. Flow cytometry and single-cell RNA sequencing revealed polyfunctional cytotoxic CD8+ lymphocytes in control granulomas, while CD8-depleted animals were unexpectedly enriched in CD4 and γδ T cells adopting incomplete cytotoxic signatures. Ligand-receptor analyses identified IL-15 signaling in granulomas as a driver of cytotoxic T cells. These data support that CD8+ lymphocytes are required for early protection against Mtb and suggest polyfunctional cytotoxic responses as a vaccine target.

Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis.

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also found that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.

Humoral correlates of protection against Mycobacterium tuberculosis following intravenous Bacille Calmette-Guérin vaccination in rhesus macaques.

Altering the route of Bacille Calmette-Guérin (BCG) immunization from low-dose intradermal vaccination to high-dose intravenous (IV) vaccination resulted in a high level of protection against Mycobacterium tuberculosis ( Mtb ) infection, providing an opportunity to uncover immune correlates and mechanisms of protection. In addition to strong T cell immunity, IV BCG vaccination was associated with a robust expansion of humoral immune responses that tracked with bacterial control. However, given the near complete protection afforded by high-dose IV BCG immunization, a precise correlate of immune protection was difficult to define. Here we leveraged plasma and bronchoalveolar lavage fluid (BAL) from a cohort of rhesus macaques that received decreasing doses of IV BCG and aimed to define the correlates of immunity across macaques that experienced immune protection or breakthrough infection following Mtb challenge. We show an IV BCG dose-dependent induction of mycobacterial-specific humoral immune responses, both in the plasma and in the airways. Moreover, antibody responses at peak immunogenicity significantly predicted bacterial control following challenge. Multivariate analyses revealed antibody-mediated complement and NK cell activating humoral networks as key functional signatures associated with protective immunity. Collectively, this work extends our understanding of humoral biomarkers and potential mechanisms of IV BCG mediated protection against Mtb .

Protective intravenous BCG vaccination induces enhanced immune signaling in the airways.

Intradermal (ID) Bacillus Calmette-Guérin (BCG) is the most widely administered vaccine in the world. However, ID-BCG fails to achieve the level of protection needed in adults to alter the course of the tuberculosis epidemic. Recent studies in non-human primates have demonstrated high levels of protection against Mycobacterium tuberculosis ( Mtb ) following intravenous (IV) administration of BCG. However, the protective immune features that emerge following IV BCG vaccination remain incompletely defined. Here we used single-cell RNA-sequencing (scRNAseq) to transcriptionally profile 157,114 unstimulated and purified protein derivative (PPD)-stimulated bronchoalveolar lavage (BAL) cells from 29 rhesus macaques immunized with BCG across routes of administration and doses to uncover cell composition-, gene expression-, and biological network-level signatures associated with IV BCG-mediated protection. Our analyses revealed that high-dose IV BCG drove an influx of polyfunctional T cells and macrophages into the airways. These macrophages exhibited a basal activation phenotype even in the absence of PPD-stimulation, defined in part by IFN and TNF-α signaling up to 6 months following BCG immunization. Furthermore, intercellular immune signaling pathways between key myeloid and T cell subsets were enhanced following PPD-stimulation in high-dose IV BCG-vaccinated macaques. High-dose IV BCG also engendered quantitatively and qualitatively stronger transcriptional responses to PPD-stimulation, with a robust Th1-Th17 transcriptional phenotype in T cells, and augmented transcriptional signatures of reactive oxygen species production, hypoxia, and IFN-γ response within alveolar macrophages. Collectively, this work supports that IV BCG immunization creates a unique cellular ecosystem in the airways, which primes and enables local myeloid cells to effectively clear Mtb upon challenge.

IFN-γ independent markers of Mycobacterium tuberculosis exposure among male South African gold miners.

BACKGROUND: The prevalence of tuberculosis among men who work in the gold mines of South Africa is among the highest in the world, but a fraction of miners demonstrate consistently negative results upon tuberculin skin test (TST) and IFN-γ release assay (IGRA). We hypothesized that these "resisters" (RSTRs) may display unconventional immune signatures of exposure to M. tuberculosis (M.tb). METHODS: In a cohort of RSTRs and matched controls with latent TB infection (LTBI), we profiled the functional breadth of M.tb antigen-specific T cell and antibody responses using multi-parameter flow cytometry and systems serology, respectively. FINDINGS: RSTRs and LTBI controls both exhibited IFN-γ independent T-cell and IgG antibody responses to M.tb-specific antigens ESAT-6 and CFP-10. Antigen-specific antibody Fc galactosylation and sialylation were higher among RSTRs. In a combined T-cell and antibody analysis, M.tb lysate-stimulated TNF secretion by T cells correlated positively with levels of purified protein derivative-specific IgG. A multivariate model of the combined data was able to differentiate RSTR and LTBI subjects. INTERPRETATION: IFN-γ independent immune signatures of exposure to M.tb, which are not detected by approved clinical diagnostics, are readily detectable in an occupational cohort uniquely characterized by intense and long-term infection pressure. Further, TNF may mediate a coordinated response between M.tb-specific T-cells and B-cells. FUNDING: This work was supported by the US National Institutes of Health (R01-AI124348 to Boom, Stein, and Hawn; R01-AI125189 and R01-AI146072 to Seshadri; and 75N93019C00071 to Fortune, Alter, Seshadri, and Boom), the Doris Duke Charitable Foundation (Davies), the Bill & Melinda Gates Foundation (OPP1151836 and OPP1109001 to Hawn; and OPP1151840 to Alter), Mass Life Science Foundation (Fortune), and Good Ventures Fund (Fortune).

Airway T cells are a correlate of i.v. Bacille Calmette-Guerin-mediated protection against tuberculosis in rhesus macaques.

Bacille Calmette-Guerin (BCG), the only approved Mycobacterium tuberculosis (Mtb) vaccine, provides limited durable protection when administered intradermally. However, recent work revealed that intravenous (i.v.) BCG administration yielded greater protection in macaques. Here, we perform a dose-ranging study of i.v. BCG vaccination in macaques to generate a range of immune responses and define correlates of protection. Seventeen of 34 macaques had no detectable infection after Mtb challenge. Multivariate analysis incorporating longitudinal cellular and humoral immune parameters uncovered an extensive and highly coordinated immune response from the bronchoalveolar lavage (BAL). A minimal signature predicting protection contained four BAL immune features, of which three remained significant after dose correction: frequency of CD4 T cells producing TNF with interferon γ (IFNγ), frequency of those producing TNF with IL-17, and the number of NK cells. Blood immune features were less predictive of protection. We conclude that CD4 T cell immunity and NK cells in the airway correlate with protection following i.v. BCG.

Maternal HIV infection drives altered placental Mtb-specific antibody transfer.

Introduction: Placental transfer of maternal antibodies is essential for neonatal immunity over the first months of life. In the setting of maternal HIV infection, HIV-exposed uninfected (HEU) infants are at higher risk of developing severe infections, including active tuberculosis (TB). Given our emerging appreciation for the potential role of antibodies in the control of Mycobacterium tuberculosis (Mtb), the bacteria that causes TB, here we aimed to determine whether maternal HIV status altered the quality of Mtb-specific placental antibody transfer. Methods: Antigen-specific antibody systems serology was performed to comprehensively characterize the Mtb-specific humoral immune response in maternal and umbilical cord blood from HIV infected and uninfected pregnant people in Uganda. Results: Significant differences were noted in overall antibody profiles in HIV positive and negative maternal plasma, resulting in heterogeneous transfer of Mtb-specific antibodies. Altered antibody transfer in HIV infected dyads was associated with impaired binding to IgG Fc-receptors, which was directly linked to HIV viral loads and CD4 counts. Conclusions: These results highlight the importance of maternal HIV status on antibody transfer, providing clues related to alterations in transferred maternal immunity that may render HEU infants more vulnerable to TB than their HIV-unexposed peers.