Mycobacterium tuberculosis (M.tb) is the second leading infectious cause of death worldwide and the primary cause of death in people living with HIV/AIDS. There are several excellent animal models employed to study tuberculosis (TB), but many have limitations for reproducing human pathology and none are amenable to the direct study of HIV/M.tb co-infection. The humanized mouse has been increasingly employed to explore HIV infection and other pathogens where animal models are limiting. Our goal was to develop a small animal model of M.tb infection using the bone marrow, liver, thymus (BLT) humanized mouse. NOD-SCID/γc null mice were engrafted with human fetal liver and thymus tissue, and supplemented with CD34+ fetal liver cells. Excellent reconstitution, as measured by expression of the human CD45 pan leukocyte marker by peripheral blood populations, was observed at 12 weeks after engraftment. Human T cells (CD3, CD4, CD8), as well as natural killer cells and monocyte/macrophages were all observed within the human leukocyte (CD45+) population. Importantly, human T cells were functionally competent as determined by proliferative capacity and effector molecule (e.g. IFN-γ, granulysin, perforin) expression in response to positive stimuli. Animals infected intranasally with M.tb had progressive bacterial infection in the lung and dissemination to spleen and liver from 2–8 weeks post infection. Sites of infection in the lung were characterized by the formation of organized granulomatous lesions, caseous necrosis, bronchial obstruction, and crystallization of cholesterol deposits. Human T cells were distributed throughout the lung, liver, and spleen at sites of inflammation and bacterial growth and were organized to the periphery of granulomas. These preliminary results demonstrate the potential to use the humanized mouse as a model of experimental TB.

The emergence of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an urgent public health crisis. Without available targeted therapies, treatment options remain limited for COVID-19 patients. Using medicinal chemistry and rational drug design strategies, we identify a 2-phenyl-1,2-benzoselenazol-3-one class of compounds targeting the SARS-CoV-2 main protease (M pro ). FRET-based screening against recombinant SARS-CoV-2 M pro identified six compounds that inhibit proteolysis with nanomolar IC 50 values. Preincubation dilution experiments and molecular docking determined that the inhibition of SARS-CoV-2 M pro can occur by either covalent or noncovalent mechanisms, and lead E04 was determined to inhibit M pro competitively. Lead E24 inhibited viral replication with a nanomolar EC 50 value (844 nM) in SARS-CoV-2-infected Vero E6 cells and was further confirmed to impair SARS-CoV-2 replication in human lung epithelial cells and human-induced pluripotent stem cell-derived 3D lung organoids. Altogether, these studies provide a structural framework and mechanism of M pro inhibition that should facilitate the design of future COVID-19 treatments.

Co-infection with HIV increases the morbidity and mortality associated with tuberculosis due to multiple factors including a poorly understood microbial synergy. We developed a novel small animal model of co-infection in the humanized mouse to investigate how HIV infection disrupts pulmonary containment of Mtb. Following dual infection, HIV-infected cells were localized to sites of Mtb-driven inflammation and mycobacterial replication in the lung. Consistent with disease in human subjects, we observed increased mycobacterial burden, loss of granuloma structure, and increased progression of TB disease, due to HIV co-infection. Importantly, we observed an HIV-dependent pro-inflammatory cytokine signature (IL-1β, IL-6, TNFα, and IL-8), neutrophil accumulation, and greater lung pathology in the Mtb-co-infected lung. These results suggest that in the early stages of acute co-infection in the humanized mouse, infection with HIV exacerbates the pro-inflammatory response to pulmonary Mtb, leading to poorly formed granulomas, more severe lung pathology, and increased mycobacterial burden and dissemination.

We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (⌬caf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the ⌬caf mutant was devoid of a capsule. The growth rate of the ⌬caf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37°C, although the mutant's growth dropped slightly during the late phase at 37°C. The ⌬caf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 g/ml of purified F1 antigen or 1 ؋ 10 5 to 5 ؋ 10 5 CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 ؋ 10 8 CFU/ml of the ⌬caf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.

Bacillus Calmette-Guerin (BCG) is the only vaccine against TB and has limited protection efficacy, which wanes past adolescence. Multifunctional CD8+ T cells (IFN-γ+/TNF-α+/IL-2+) are associated with lower reactivation risk and enhanced control of active Mtb infection. Since boosting with BCG is contraindicated, booster vaccines that augment T cell immunity in the lungs of BCG-vaccinated individuals are urgently needed. We developed a vaccination strategy based on self-assembling peptide nanofibers presenting Mtb-specific CD8+ or CD4+ T cell epitopes that induce high frequency and antigen-specific effector memory T cells producing IFN-γ and IL-2. Intranasal immunization with peptide nanofibers was well tolerated in mice leading to increased antigen-specific CD8+ T cell population in the lungs. Co-assembled nanofibers of CD8+ T cell epitopes and toll-like receptor 2 (TLR2) agonists induced a 8-fold expansion in multifunctional CD8+ T cell populations in the lungs of vaccinated mice. Aerosol challenge with Mtb in BCG-primed and nanofiber-boosted mice provided an additional 0.5-log CFU reduction in lung bacterial load and indicating enhanced protection compared to BCG alone. Together, these data suggest that heterologous prime-boost with BCG and peptide nanofiber vaccines induces cell mediated immunity in the lung, reduces bacterial burden, and is a potentially safer alternative for boosting BCG-primed immunity.