Optimizing tuberculosis treatment efficacy: Comparing the standard regimen with Moxifloxacin-containing regimens.

Tuberculosis (TB) continues to be one of the deadliest infectious diseases in the world, causing ~1.5 million deaths every year. The World Health Organization initiated an End TB Strategy that aims to reduce TB-related deaths in 2035 by 95%. Recent research goals have focused on discovering more effective and more patient-friendly antibiotic drug regimens to increase patient compliance and decrease emergence of resistant TB. Moxifloxacin is one promising antibiotic that may improve the current standard regimen by shortening treatment time. Clinical trials and in vivo mouse studies suggest that regimens containing moxifloxacin have better bactericidal activity. However, testing every possible combination regimen with moxifloxacin either in vivo or clinically is not feasible due to experimental and clinical limitations. To identify better regimens more systematically, we simulated pharmacokinetics/pharmacodynamics of various regimens (with and without moxifloxacin) to evaluate efficacies, and then compared our predictions to both clinical trials and nonhuman primate studies performed herein. We used GranSim, our well-established hybrid agent-based model that simulates granuloma formation and antibiotic treatment, for this task. In addition, we established a multiple-objective optimization pipeline using GranSim to discover optimized regimens based on treatment objectives of interest, i.e., minimizing total drug dosage and lowering time needed to sterilize granulomas. Our approach can efficiently test many regimens and successfully identify optimal regimens to inform pre-clinical studies or clinical trials and ultimately accelerate the TB regimen discovery process.

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.

CD4+ T cells are homeostatic regulators during Mtb reinfection.

Immunological priming - either in the context of prior infection or vaccination - elicits protective responses against subsequent Mycobacterium tuberculosis (Mtb) infection. However, the changes that occur in the lung cellular milieu post-primary Mtb infection and their contributions to protection upon reinfection remain poorly understood. Here, using clinical and microbiological endpoints in a non-human primate reinfection model, we demonstrate that prior Mtb infection elicits a long-lasting protective response against subsequent Mtb exposure and that the depletion of CD4+ T cells prior to Mtb rechallenge significantly abrogates this protection. Leveraging microbiologic, PET-CT, flow cytometric, and single-cell RNA-seq data from primary infection, reinfection, and reinfection-CD4+ T cell depleted granulomas, we identify differential cellular and microbial features of control. The data collectively demonstrate that the presence of CD4+ T cells in the setting of reinfection results in a reduced inflammatory lung milieu characterized by reprogrammed CD8+ T cell activity, reduced neutrophilia, and blunted type-1 immune signaling among myeloid cells, mitigating Mtb disease severity. These results open avenues for developing vaccines and therapeutics that not only target CD4+ and CD8+ T cells, but also modulate innate immune cells to limit Mtb disease.

Vaccination with intravenous BCG protects macaques with pre-existing SIV infection from tuberculosis.

Tuberculosis (TB) is the most common cause of death in people living with HIV. BCG delivered intradermally (ID) is the only licensed vaccine to prevent TB. However, it offers little protection from pulmonary TB in adults. Intravenous (IV) BCG, but not ID BCG, confers striking protection against Mycobacterium tuberculosis (Mtb) infection and disease in rhesus macaques. We investigated whether IV BCG could protect against TB in macaques with a pre-existing SIV infection. There was a robust influx of airway T cells following IV BCG in both SIV-infected and SIV-naïve animals, with elevated antibody titers in plasma and airways. Following Mtb challenge, all 7 SIV-naïve and 9 out of 12 SIV-infected vaccinated animals were completely protected, without any culturable bacilli in their tissues. PBMC responses post-challenge indicated early clearance of Mtb in vaccinated animals regardless of SIV infection. These data support that IV BCG is immunogenic and efficacious in SIV-infected animals.

Intravenous Bacille Calmette-Guérin vaccination protects simian immunodeficiency virus-infected macaques from tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is the most common cause of death in people living with human immunodeficiency virus (HIV). Intra-dermal Bacille Calmette-Guérin (BCG) delivery is the only licensed vaccine against tuberculosis; however, it offers little protection from pulmonary tuberculosis in adults and is contraindicated in people living with HIV. Intravenous BCG confers protection against Mtb infection in rhesus macaques; we hypothesized that it might prevent tuberculosis in simian immunodeficiency virus (SIV)-infected macaques, a model for HIV infection. Here intravenous BCG-elicited robust airway T cell influx and elevated plasma and airway antibody titres in both SIV-infected and naive animals. Following Mtb challenge, all 7 vaccinated SIV-naive and 9 out of 12 vaccinated SIV-infected animals were protected, without any culturable bacteria detected from tissues. Peripheral blood mononuclear cell responses post-challenge indicated early clearance of Mtb in vaccinated animals, regardless of SIV infection. These data support that intravenous BCG is immunogenic and efficacious in SIV-infected animals.

Murine models of Pneumocystis infection recapitulate human primary immune disorders.

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

Comparison of Atipamezole with Yohimbine for Antagonism of Xylazine in Mice Anesthetized with Ketamine and Xylazine.

The α2 adrenergic agonist xylazine produces a sedative effect and is typically combined with ketamine and used for anesthesia or chemical restraint of laboratory mice. Xylazine's sedative effect-and its undesirable side effects of bradycardia, hypotension, and poor tissue perfusion-can be reversed by administration of α2 antagonists, such as atipamezole or yohimbine. Although atipamezole and yohimbine dosing guidelines are available for mice, no controlled comparison has been performed to guide the lab animal community in the selection of one over the other. This study is a single-dose crossover comparison of these 2 antagonist drugs, given intraperitoneally at clinically recommended doses, to determine which results in more rapid recovery of mice from xylazine-ketamine anesthesia. Time to return of righting reflex was used as the primary outcome measure. Mice were anesthetized with xylazine (10 mg/kg IP) and ketamine (80 mg/kg IP), followed 15 min later by injection of an α2 antagonist or saline (control). Time to return of righting reflex differed significantly among groups, with mice recovering in an average of 10.3 min after administration of atipamezole (1 mg/kg IP) as compared with 21.3 min after yohimbine (1.5 mg/kg IP) and 38.2 min after saline. When rapid recovery of mice after xylazine-ketamine anesthesia is desirable, administration of an antagonist to reverse the effects of the xylazine is indicated. When injection of the antagonist by the technically simple intraperitoneal route is desirable, our data indicate that (at the doses evaluated) atipamezole is more effective than yohimbine.