Single-Cell Transcriptomics of Mtb/HIV Co-Infection.

Tuberculosis (TB) and Human Immunodeficiency Virus (HIV) co-infection continues to pose a significant healthcare burden. HIV co-infection during TB predisposes the host to the reactivation of latent TB infection (LTBI), worsening disease conditions and mortality. There is a lack of biomarkers of LTBI reactivation and/or immune-related transcriptional signatures to distinguish active TB from LTBI and predict TB reactivation upon HIV co-infection. Characterizing individual cells using next-generation sequencing-based technologies has facilitated novel biological discoveries about infectious diseases, including TB and HIV pathogenesis. Compared to the more conventional sequencing techniques that provide a bulk assessment, single-cell RNA sequencing (scRNA-seq) can reveal complex and new cell types and identify more high-resolution cellular heterogeneity. This review will summarize the progress made in defining the immune atlas of TB and HIV infections using scRNA-seq, including host-pathogen interactions, heterogeneity in HIV pathogenesis, and the animal models employed to model disease. This review will also address the tools needed to bridge the gap between disease outcomes in single infection vs. co-infection. Finally, it will elaborate on the translational benefits of single-cell sequencing in TB/HIV diagnosis in humans.

Inhibition of indoleamine dioxygenase leads to better control of tuberculosis adjunctive to chemotherapy.

The expression of indoleamine 2,3-dioxygenase (IDO), a robust immunosuppressant, is significantly induced in macaque tuberculosis (TB) granulomas, where it is expressed on IFN-responsive macrophages and myeloid-derived suppressor cells. IDO expression is also highly induced in human TB granulomas, and products of its activity are detected in patients with TB. In vivo blockade of IDO activity resulted in the reorganization of the granuloma with substantially greater T cells being recruited to the core of the lesions. This correlated with better immune control of TB and reduced lung M. tuberculosis burdens. To study if the IDO blockade strategy can be translated to a bona fide host-directed therapy in the clinical setting of TB, we studied the effect of IDO inhibitor 1-methyl-d-tryptophan adjunctive to suboptimal anti-TB chemotherapy. While two-thirds of controls and one-third of chemotherapy-treated animals progressed to active TB, inhibition of IDO adjunctive to the same therapy protected macaques from TB, as measured by clinical, radiological, and microbiological attributes. Although chemotherapy improved proliferative T cell responses, adjunctive inhibition of IDO further enhanced the recruitment of effector T cells to the lung. These results strongly suggest the possibility that IDO inhibition can be attempted adjunctive to anti-TB chemotherapy in clinical trials.

Isoniazid and rifapentine treatment effectively reduces persistent M. tuberculosis infection in macaque lungs.

A once-weekly oral dose of isoniazid and rifapentine for 3 months (3HP) is recommended by the CDC for treatment of latent tuberculosis infection (LTBI). The aim of this study is to assess 3HP-mediated clearance of M. tuberculosis bacteria in macaques with asymptomatic LTBI. Twelve Indian-origin rhesus macaques were infected with a low dose (~10 CFU) of M. tuberculosis CDC1551 via aerosol. Six animals were treated with 3HP and 6 were left untreated. The animals were imaged via PET/CT at frequent intervals. Upon treatment completion, all animals except 1 were coinfected with SIV to assess reactivation of LTBI to active tuberculosis (ATB). Four of 6 treated macaques showed no evidence of persistent bacilli or extrapulmonary spread until the study end point. PET/CT demonstrated the presence of significantly more granulomas in untreated animals relative to the treated group. The untreated animals harbored persistent bacilli and demonstrated tuberculosis (TB) reactivation following SIV coinfection, while none of the treated animals reactivated to ATB. 3HP treatment effectively reduced persistent infection with M. tuberculosis and prevented reactivation of TB in latently infected macaques.

Antiretroviral therapy timing impacts latent tuberculosis infection reactivation in a Mycobacterium tuberculosis/SIV coinfection model.

Studies using the nonhuman primate model of Mycobacterium tuberculosis/simian immunodeficiency virus coinfection have revealed protective CD4+ T cell-independent immune responses that suppress latent tuberculosis infection (LTBI) reactivation. In particular, chronic immune activation rather than the mere depletion of CD4+ T cells correlates with reactivation due to SIV coinfection. Here, we administered combinatorial antiretroviral therapy (cART) 2 weeks after SIV coinfection to study whether restoration of CD4+ T cell immunity occurred more broadly, and whether this prevented reactivation of LTBI compared to cART initiated 4 weeks after SIV. Earlier initiation of cART enhanced survival, led to better control of viral replication, and reduced immune activation in the periphery and lung vasculature, thereby reducing the rate of SIV-induced reactivation. We observed robust CD8+ T effector memory responses and significantly reduced macrophage turnover in the lung tissue. However, skewed CD4+ T effector memory responses persisted and new TB lesions formed after SIV coinfection. Thus, reactivation of LTBI is governed by very early events of SIV infection. Timing of cART is critical in mitigating chronic immune activation. The potential novelty of these findings mainly relates to the development of a robust animal model of human M. tuberculosis/HIV coinfection that allows the testing of underlying mechanisms.

Characterizing Early T Cell Responses in Nonhuman Primate Model of Tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading infectious disease killer worldwide with 1.4 million TB deaths in 2019. While the majority of infected population maintain an active control of the bacteria, a subset develops active disease leading to mortality. Effective T cell responses are critical to TB immunity with CD4+ and CD8+ T cells being key players of defense. These early cellular responses to TB infection have not yet been studied in-depth in either humans or preclinical animal models. Characterizing early T cell responses in a physiologically relevant preclinical model can provide valuable understanding of the factors that control disease development. We studied Mtb-specific T cell responses in the lung compartment of rhesus macaques infected with either a low- or a high-dose of Mtb CDC1551 via aerosol. Relative to baseline, significantly higher Mtb-specific CD4+IFN-γ+ and TNF-α+ T cell responses were observed in the BAL of low dose infected macaques as early as week 1 post TB infection. The IFN-γ and TNF-a response was delayed to week 3 post infection in Mtb-specific CD4+ and CD8+T cells in the high dose group. The manifestation of earlier T cell responses in the group exposed to the lower Mtb dose suggested a critical role of these cytokines in the antimycobacterial immune cascade, and specifically in the granuloma formation to contain the bacteria. However, a similar increase was not reflected in the CD4+ and CD8+IL-17+ T cells at week 1 post infection in the low dose group. This could be attributed to either a suppression of the IL-17 response or a lack of induction at this early stage of infection. On the contrary, there was a significantly higher IL-17+ response in Mtb-specific CD4+ and CD8+T cells at week 3 in the high dose group. The results clearly demonstrate an early differentiation in the immunity following low dose and high dose infection, largely represented by differences in the IFN-γ and TNF-α response by Mtb-specific T cells in the BAL. This early response to antigen expression by the bacteria could be critical for both bacterial growth control and bacterial containment.

Phage-encoded cationic antimicrobial peptide required for lysis.

Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp28 in OM disruption. Additionally, gp28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp28 protein prior to lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.

Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies.

Vaccine strategies for the Mtb/HIV copandemic.

One-third of world's population is predicted to be infected with tuberculosis (TB). The resurgence of this deadly disease has been inflamed by comorbidity with human immunodeficiency virus (HIV). The risk of TB in people living with HIV (PLWH) is 15-22 times higher than people without HIV. Development of a single vaccine to combat both diseases is an ardent but tenable ambition. Studies have focused on the induction of specific humoral and cellular immune responses against HIV-1 following recombinant BCG (rBCG) expressing HIV-1 antigens. Recent advances in the TB vaccines led to the development of promising candidates such as MTBVAC, the BCG revaccination approach, H4:IC31, H56:IC31, M72/AS01 and more recently, intravenous (IV) BCG. Modification of these vaccine candidates against TB/HIV coinfection could reveal key correlates of protection in a representative animal model. This review discusses the (i) potential TB vaccine candidates that can be exploited for use as a dual vaccine against TB/HIV copandemic (ii) progress made in the realm of TB/HIV dual vaccine candidates in small animal model, NHP model, and human clinical trials (iii) the failures and promising targets for a successful vaccine strategy while delineating the correlates of vaccine-induced protection.

Antiretroviral therapy does not reduce tuberculosis reactivation in a tuberculosis-HIV coinfection model.

While the advent of combination antiretroviral therapy (ART) has significantly improved survival, tuberculosis (TB) remains the leading cause of death in the HIV-infected population. We used Mycobacterium tuberculosis/simian immunodeficiency virus-coinfected (M. tuberculosis/SIV-coinfected) macaques to model M. tuberculosis/HIV coinfection and study the impact of ART on TB reactivation due to HIV infection. Although ART significantly reduced viral loads and increased CD4+ T cell counts in blood and bronchoalveolar lavage (BAL) samples, it did not reduce the relative risk of SIV-induced TB reactivation in ART-treated macaques in the early phase of treatment. CD4+ T cells were poorly restored specifically in the lung interstitium, despite their significant restoration in the alveolar compartment of the lung as well as in the periphery. IDO1 induction in myeloid cells in the inducible bronchus-associated lymphoid tissue (iBALT) likely contributed to dysregulated T cell homing and impaired lung immunity. Thus, although ART was indispensable for controlling viral replication, restoring CD4+ T cells, and preventing opportunistic infection, it appeared inadequate in reversing the clinical signs of TB reactivation during the relatively short duration of ART administered in this study. This finding warrants the modeling of concurrent treatment of TB and HIV to potentially reduce the risk of reactivation of TB due to HIV to inform treatment strategies in patients with M. tuberculosis/HIV coinfection.

Chronic Immune Activation in TB/HIV Co-infection.

HIV co-infection is the most critical risk factor for the reactivation of latent tuberculosis (TB) infection (LTBI). While CD4+ T cell depletion has been considered the major cause of HIV-induced reactivation of LTBI, recent work in macaques co-infected with Mycobacterium tuberculosis (Mtb)/simian immunodeficiency virus (SIV) suggests that cytopathic effects of SIV resulting in chronic immune activation and dysregulation of T cell homeostasis correlate with reactivation of LTBI. This review builds on compelling data that the reactivation of LTBI during HIV co-infection is likely to be driven by the events of HIV replication and therefore highlights the need to have optimum translational interventions directed at reactivation due to co-infection.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

IL-22 Defect During Streptococcus pneumoniae Infection Triggers Exacerbation of Chronic Obstructive Pulmonary Disease.

Progression of chronic obstructive pulmonary disease (COPD) is linked to episodes of exacerbations caused by bacterial infections due to Streptococcus pneumoniae. Our objective was to identify during COPD, factors of susceptibility to bacterial infections among cytokine network and their role in COPD exacerbations. S. pneumoniae was used to sub-lethally challenge mice chronically exposed to air or cigarette smoke (CS) and to stimulate peripheral blood mononuclear cells (PBMC) from non-smokers, smokers and COPD patients. The immune response and the cytokine production were evaluated. Delayed clearance of the bacteria and stronger lung inflammation observed in infected CS-exposed mice were associated with an altered production of IL-17 and IL-22 by innate immune cells. This defect was related to a reduced production of IL-1ß and IL-23 by antigen presenting cells. Importantly, supplementation with recombinant IL-22 restored bacterial clearance in CS-exposed mice and limited lung alteration. In contrast with non-smokers, blood NK and NKT cells from COPD patients failed to increase IL-17 and IL-22 levels in response to S. pneumoniae, in association with a defect in IL-1ß and IL-23 secretion. This study identified IL-17 and IL-22 as susceptibility factors in COPD exacerbation. Therefore targeting such cytokines could represent a potent strategy to control COPD exacerbation.

•Increased bacterial susceptibility during COPD is related to a defect in Th17 cytokines.•Cigarette smoke alters the production of immunoregulatory cytokines by lung APC.•Immunotherapy restoring the defective IL-22 response could represent an ideal therapy to prevent exacerbation in COPD patients.The progression of chronic obstructive pulmonary disease (COPD) is linked to episodes of exacerbations mostly due to bacterial infections. It is not well understood why COPD patients are more susceptible to infections. In our experimental model of COPD as well as in COPD patients, we identified a defect in the IL-17/IL-22 response to S. pneumoniae, leading to the bacterial outgrowth. This was mainly due to the alteration of lung antigen-presenting cells by cigarette smoke. Restoring the defective IL-22 response represents a promising therapeutic approach for the treatment and/or the prevention of COPD exacerbations.

Traditional copper water storage vessels and sub-lethal injury of Salmonella enterica serovar Typhi and Vibrio cholerae.

Recent studies on Escherichia coli have demonstrated sub-lethal injury-sensitivity to oxygen and selective agents prior to irreversible inactivation when kept in water in a brass vessel. The present study was carried out to investigate whether equivalent responses occur in copper vessels using the pathogens Salmonella enterica serovar Typhi and Vibrio cholerae. Bacterial suspensions were stored in water in a traditional copper vessel for up to 24 h at 30 °C. Samples were withdrawn and plated on selective and non-selective media, then incubated under (a) aerobic conditions and (b) conditions where reactive oxygen species were neutralized to enumerate injured bacteria. Short-term incubation in water kept in a copper vessel caused a greater decrease in counts for both pathogens on selective media, compared to non-selective media with greater differences between aerobic and reactive oxygen species-neutralized counts using selective media compared to non-selective nutrient agar. These findings have practical implications for the short-term storage of water samples in copper storage vessel as the possibility of bacterial injury is high, hence enumeration under conventional aerobic conditions may not be sufficient to give a count of all viable bacteria.

Inactivation and sub-lethal injury of salmonella typhi, salmonella typhimurium and vibrio cholerae in copper water storage vessels.

BACKGROUND: This study provides information on the antibacterial effect of copper against the water-borne pathogens Salmonella Typhi, Salmonella Typhimurium and Vibrio cholerae. METHODS: Suspensions of each pathogen were kept in water within a traditional copper vessel at 30°C for 24 h. Samples were withdrawn, diluted and plated onto suitable growth media. Conventional enumeration of healthy (uninjured) bacteria was carried out using standard aerobic incubation conditions. Additionally, reactive oxygen species-neutralised (ROS-n) conditions were achieved by adding the peroxide scavenger sodium pyruvate to the medium with anaerobic incubation, to enumerate uninjured (ROS-insensitive) and injured (ROS-sensitive) bacteria. Differences between log-transformed means of conventional (aerobic) and ROS-n counts were statistically evaluated using t tests. RESULTS: Overall, all three pathogens were inactivated by storage in copper vessels for 24 h. However, for shorter-term incubation (4-12 h), higher counts were observed under ROS-n conditions than under aerobic conditions, which demonstrate the presence of substantial numbers of sub-lethally injured cells prior to their complete inactivation. CONCLUSIONS: The present study has for the first time confirmed that these bacterial pathogens are inactivated by storage in a copper vessel within 24 h. However, it has also demonstrated that it is necessary to account for short-term sub-lethal injury, manifest as ROS-sensitivity, in order to more fully understand the process. This has important practical implications in terms of the time required to store water within a copper vessel to completely inactivate these bacteria and thereby remove the risk of water-borne disease transmission by this route.

A murine model to study the antibacterial effect of copper on infectivity of Salmonella enterica serovar Typhimurium.

This study investigated the effect of copper as an antibacterial agent on the infectivity of Salmonella enterica serovar Typhimurium. Mice were infected orally with a standardized dose of unstressed Salmonella Typhimurium and copper-stressed cells of Salmonella Typhimurium. Bacterial counts in ileum, blood, liver and spleen were observed up to 168 h under normal aerobic conditions. Serum sensitivity, phagocytosis, malondialdehyde levels and histopathology were studied for both set of animals. A decreased bacterial count in the organs with mild symptoms of infection and a complete recovery by 48 h was observed in mice infected with copper-stressed bacteria. Histopathological examination of ileum tissue demonstrated regeneration of damaged tissue post-infection with copper-stressed bacteria and no malondialdehyde levels were detected after 24 h in ileum, spleen and liver. Exposure to copper sensitized Salmonella Typhimurium to the lytic action of serum and intracellular killing by peritoneal macrophages. It can be concluded that copper stress confers a decrease in the infectivity of healthy Salmonella Typhimurium in normal mice. This study highlights the significance of use of copper as an antibacterial agent against Salmonella Typhimurium in reducing the risk of incidence of Salmonella infections from contaminated water.

Inactivation and sub-lethal injury of Escherichia coli in a copper water storage vessel: effect of inorganic and organic constituents.

This study provides information on the effects of inorganic and organic constituents on inactivation and sub-lethal injury of Escherichia coli in water stored in a copper vessel. E. coli suspensions were stored for up to 24 h in copper vessels containing one of the following dissolved constituents at 1 g/l: salts of inorganic ions, carbohydrates, proteins and complex natural organic mixtures. Samples were surface plated onto (i) nutrient agar, incubated under standard aerobic conditions to provide conventional counts for uninjured bacteria count and onto (ii) nutrient agar plates with 0.5 g/l sodium pyruvate incubated under anaerobic conditions to enumerate sub-lethally injured (oxygen-sensitive) bacteria alongside their healthy counterparts. The concentration of dissolved copper in the stored water was determined using atomic absorption spectrophotometry. The addition of chloride salts resulted in a faster inactivation of E. coli compared to pure water with no dissolved additives, irrespective of the counterion. In contrast, a slower inactivation was observed in the presence of Na(2)SO(4), NaNO(3) and NaNO(2) when compared to NaCl. Addition of the carbohydrates glucose, lactose and starch gave broadly similar results to those obtained using unsupplemented water. However, the addition of amino acids, proteins, humic acid or complex organic mixtures caused a dramatic decrease in inactivation of E. coli, with evidence of a greater number of sub-lethally injured bacteria than was seen with other added constituents. The amount of copper was highest in stored water containing amino acids and complex organic constituents, with the slow inactivation most likely to be due to complex formation between leached copper and these organic constituents. The present study clearly demonstrates that water composition, particularly natural organic constituents, has a substantial impact on the antibacterial effectiveness and dissolved copper concentration of water stored in copper vessels and that both aspects will need to be considered in terms of their impact on the practical use of copper-based systems for small-scale water treatment.

Inactivation and injury of Escherichia coli in a copper water storage vessel: effects of temperature and pH.

Copper has been used as a disinfectant since ancient times and recent research has demonstrated that antimicrobial copper surfaces may have practical applications in healthcare and related areas. The present study was carried out to establish the effects of temperature and pH on inactivation and sub-lethal injury of Escherichia coli in water stored in a copper vessel, to determine the operational limits of the process in terms of these variables. To investigate the effects of temperature, a bacterial suspension at pH 7.0 was stored for up to 48 h in copper vessels at 5, 15, 25 and 35 degrees C. For pH, a bacterial suspension was stored at 30 degrees C for up to 48 h in copper vessels at pH 6.0, 7.0, 8.0 and 9.0. Both temperature and pH had substantial effects on inactivation and injury, with the fastest inactivation observed at elevated temperature and at pH values furthest from neutrality, while the greatest amount of sub-lethal injury, manifest as sensitivity to conventional aerobic enumeration, was observed at a temperature of 35 degrees C. These findings have important implications for the practical application of copper-based water disinfection methods, in terms of their likely efficacy under environmental conditions.