KEYWORDS coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), nucleocapsid protein, vaccine D uring the current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there has been an unprecedented level of global collaboration that has led to a rapid characterization of SARS-CoV-2 (1). Its sequence shares 79.6% identity to SARS-CoV (1, 2), the infectious virus that caused an epidemic in 2003 (2, 3). SARS-CoV-2 has a single-stranded, plus-sense, RNA genome of approximately 30 kb, which includes five major open reading frames encoding nonstructural replicase polyproteins and structural proteins (1), namely, spike (S) (4-6), envelope (E), membrane (M), and nucleocapsid (N) (7), and they are in the same order and of approximately the same sizes as those in SARS-CoV. The SARS-CoV-2 S protein is being used as the leading target antigen in vaccine development (8, 9). However, the complex molecular details of viral entry may lead to complications with the vaccine response, similar to those seen with HIV type 1 (HIV-1) Env protein vaccine efforts (10). The SARS-CoV-2 S gene has 76% amino acid similarity to the SARS-CoV S gene (11), and nonsynonymous mutations developed in the S protein as the SARS-CoV epidemic progressed (12, 13). In contrast, the N gene is more conserved and stable, with 90% amino acid homology and fewer mutations over time (2, 3, 11, 14-16). N proteins of many coronaviruses are highly immunogenic and are expressed abundantly during infection (17). High levels of IgG antibodies against N have been detected in sera from SARS patients (18), and the N protein is a representative antigen for the T-cell response in a vaccine setting, inducing SARS-specific T-cell proliferation and cytotoxic activity (19, 20). We have already shown that the middle or C-terminal region of the SARS-CoV N protein is important for eliciting antibodies against SARS-CoV during the immune response (21-23). New reports have additionally shown that the crystal structure of the SARS-CoV-2 nucleocapsid protein is similar to those of previously described coronavirus N proteins, but their surface electrostatic potential characteristics are distinct (7). Sheikh et al. studied the factors influencing N gene variations among 13 coronaviruses and how these affect virus-host relationships, reporting a high AT% and low GC% in the nucleotide contents of SARS coronavirus (24). In this issue, Cong et al. (17) used a mouse hepatitis virus (MHV) model to show that the viral nucleocapsid (N) protein contributes to forming helical ribonucleoproteins during the packaging of the RNA genome, regulating viral RNA synthesis during replication and transcription and modulating metabolism in infected subjects. This study complements others that have shown N to have multiple functions (25). It is becoming more evident just how critical this protein is for multiple steps of the viral life cycle. These reports offer important and timely insights relevant ...

SummaryWidespread resistance to first-line TB drugs is a major problem that will likely only be resolved through the development of new drugs with novel mechanisms of action. We have used structure-guided methods to develop a lead molecule that targets the thioesterase activity of polyketide synthase Pks13, an essential enzyme that forms mycolic acids, required for the cell wall of Mycobacterium tuberculosis. Our lead, TAM16, is a benzofuran class inhibitor of Pks13 with highly potent in vitro bactericidal activity against drug-susceptible and drug-resistant clinical isolates of M. tuberculosis. In multiple mouse models of TB infection, TAM16 showed in vivo efficacy equal to the first-line TB drug isoniazid, both as a monotherapy and in combination therapy with rifampicin. TAM16 has excellent pharmacological and safety profiles, and the frequency of resistance for TAM16 is ∼100-fold lower than INH, suggesting that it can be developed as a new antitubercular aimed at the acute infection.PaperClip

Background Tuberculosis (TB) and AIDS together present a devastating public health challenge. Over three million deaths every year are attributed to these twin epidemics. Annually, ~ 11 million people are co-infected with HIV and Mycobacterium tuberculosis (Mtb). AIDS is thought to alter the spontaneous rate of latent TB reactivation. Methodology Macaques are excellent models of both TB and AIDS. Therefore, it is conceivable that they can also be used to model co-infection. Using clinical, pathological and microbiological data we addressed if latent TB infection in rhesus macaques can be reactivated by infection with Simian Immunodeficiency Virus (SIV). Results A low-dose aerosol infection of rhesus macaques with Mtb caused latent, asymptomatic TB infection. Infection of macaques exhibiting latent TB with a rhesus-specific strain of SIV significantly reactivated TB. Conclusions Rhesus macaques are excellent model of TB/AIDS co-infection and can be used to study the phenomena of TB latency and reactivation.

SUMMARY The aim of this review is to present the current state of knowledge on human latent tuberculosis infection (LTBI) based on clinical studies and observations, as well as experimental in vitro and animal models. Several key terms are defined, including “latency,” “persistence,” “dormancy,” and “antibiotic tolerance.” Dogmas prevalent in the field are critically examined based on available clinical and experimental data, including the long-held beliefs that infection is either latent or active, that LTBI represents a small population of nonreplicating, “dormant” bacilli, and that caseous granulomas are the haven for LTBI. The role of host factors, such as CD4 + and CD8 + T cells, T regulatory cells, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ), in controlling TB infection is discussed. We also highlight microbial regulatory and metabolic pathways implicated in bacillary growth restriction and antibiotic tolerance under various physiologically relevant conditions. Finally, we pose several clinically important questions, which remain unanswered and will serve to stimulate future research on LTBI.

Background TB leads to the annual death of 1.7 million people. The failure of the BCG vaccine, synergy between AIDS and TB, and the emergence of drug-resistance have worsened this situation. It is imperative to delineate the mechanisms employed by Mtb to successfully infect and persist in mammalian lungs. Methods NHPs are arguably the best animal system to model critical aspects of human TB. We studied genes essential for growth/survival of Mtb in the lungs of NHPs experimentally exposed to aerosols of an Mtb transposon mutant library. Results Mutants in 108 Mtb genes (33.13% of all tested) were attenuated for in-vivo growth. Comparable studies have reported the attenuation of only ~6% of mutants in mice. The Mtb mutants attenuated for in-vivo survival in primates were involved in the transport of various biomolecules including lipid virulence factors; biosynthesis of cell-wall arabinan and peptidoglycan; DNA repair; sterol metabolism and mammalian cell-entry. Conclusions Our study highlights the various virulence-mechanisms employed by Mtb to overcome the hostile environment encountered during infection of primates. Prophylactic approaches aimed against bacterial factors which respond to such in-vivo stressors, have the potential to prevent infection at an early stage, thus likely reducing the extent of transmission of Mtb.

BackgroundIn response to Mtb infection, the host remodels the infection foci into a dense mass of cells known as the granuloma. The key objective of the granuloma is to contain the spread of Mtb into uninfected regions of the lung. However, it appears that Mtb has evolved mechanisms to resist killing in the granuloma. Profiling granuloma transcriptome will identify key immune signaling pathways active during TB infection. Such studies are not possible in human granulomas, due to various confounding factors. Nonhuman Primates (NHPs) infected with Mtb accurately reflect human TB in clinical and pathological contexts.Methodology/Principal FindingsWe studied transcriptomics of granuloma lesions in the lungs of NHPs exhibiting active TB, during early and late stages of infection. Early TB lesions were characterized by a highly pro-inflammatory environment, expressing high levels of immune signaling pathways involving IFNγ, TNFα, JAK, STAT and C-C/C-X-C chemokines. Late TB lesions, while morphologically similar to the early ones, exhibited an overwhelming silencing of the inflammatory response. Reprogramming of the granuloma transcriptome was highly significant. The expression of ∼ two-thirds of all genes induced in early lesions was later repressed.Conclusions/SignificanceThe transcriptional characteristics of TB granulomas undergo drastic changes during the course of infection. The overwhelming reprogramming of the initial pro-inflammatory surge in late lesions may be a host strategy to limit immunopathology. We propose that these host profiles can predict changes in bacterial replication and physiology, perhaps serving as markers for latency and reactivation.

The stringent response enables Mycobacterium tuberculosis (Mtb) to shut down its replication and metabolism under various stresses. Here we show that Mtb lacking the stringent response enzyme RelMtb was unable to slow its replication rate during nutrient starvation. Metabolomics analysis revealed that the nutrient-starved relMtb-deficient strain had increased metabolism similar to that of exponentially growing wild-type bacteria in nutrient-rich broth, consistent with an inability to enter quiescence. Deficiency of relMtb increased the susceptibility of mutant bacteria to killing by isoniazid during nutrient starvation and in the lungs of chronically infected mice. We screened a pharmaceutical library of over 2 million compounds for inhibitors of RelMtb and showed that the lead compound X9 was able to directly kill nutrient-starved M. tuberculosis and enhanced the killing activity of isoniazid. Inhibition of RelMtb is a promising approach to target M. tuberculosis persisters, with the potential to shorten the duration of TB treatment.

Pyrazinamide has played a critical role in shortening therapy against drug-sensitive, drug-resistant, active, and latent tuberculosis (TB). Despite widespread recognition of its therapeutic importance, the sterilizing properties of this 60-year-old drug remain an enigma given its rather poor activity in vitro. Here we revisit longstanding paradigms and offer pharmacokinetic explanations for the apparent disconnect between in vitro activity and clinical impact. We show substantial host-mediated conversion of prodrug pyrazinamide (PZA) to the active form, pyrazinoic acid (POA), in TB patients and in animal models. We demonstrate favorable penetration of this pool of circulating POA from plasma into lung tissue and granulomas, where the pathogen resides. In standardized growth inhibition experiments, we show that POA exhibits superior in vitro potency compared to PZA, indicating that the vascular supply of host-derived POA may contribute to the in vivo efficacy of PZA, thereby reducing the apparent discrepancy between in vitro and in vivo activity. However, the results also raise the possibility that subinhibitory concentrations of POA generated by the host could fuel the emergence of resistance to both PZA and POA. In contrast to widespread expectations, we demonstrate good oral bioavailability and exposure in preclinical species in pharmacokinetic studies of oral POA. Baseline exposure of oral POA can be further increased by the xanthine oxidase inhibitor and approved gout drug allopurinol. These promising results pave the way for clinical investigations of oral POA as a therapeutic alternative or an add-on to overcome PZA resistance and salvage this essential TB drug.