Antiviral Effect of Antimicrobial Peptoid TM9 and Murine Model of Respiratory Coronavirus Infection.

New antiviral agents are essential to improving treatment and control of SARS-CoV-2 infections that can lead to the disease COVID-19. Antimicrobial peptoids are sequence-specific oligo-N-substituted glycine peptidomimetics that emulate the structure and function of natural antimicrobial peptides but are resistant to proteases. We demonstrate antiviral activity of a new peptoid (TM9) against the coronavirus, murine hepatitis virus (MHV), as a closely related model for the structure and antiviral susceptibility profile of SARS-CoV-2. This peptoid mimics the human cathelicidin LL-37, which has also been shown to have antimicrobial and antiviral activity. In this study, TM9 was effective against three murine coronavirus strains, demonstrating that the therapeutic window is large enough to allow the use of TM9 for treatment. All three isolates of MHV generated infection in mice after 15 min of exposure by aerosol using the Madison aerosol chamber, and all three viral strains could be isolated from the lungs throughout the 5-day observation period post-infection, with the peak titers on day 2. MHV-A59 and MHV-A59-GFP were also isolated from the liver, heart, spleen, olfactory bulbs, and brain. These data demonstrate that MHV serves as a valuable natural murine model of coronavirus pathogenesis in multiple organs, including the brain.

Peptide-mimetic treatment of Pseudomonas aeruginosa in a mouse model of respiratory infection.

The rise of drug resistance has become a global crisis, with >1 million deaths due to resistant bacterial infections each year. Pseudomonas aeruginosa, in particular, remains a serious problem with limited solutions due to complex resistance mechanisms that now lead to more than 32,000 multidrug-resistant (MDR) infections and over 2,000 deaths annually. While the emergence of resistant bacteria has become concerningly common, identification of useful new drug classes has been limited over the past 40+ years. We found that a potential novel therapeutic, the peptide-mimetic TM5, is effective at killing P. aeruginosa and displays sufficiently low toxicity for mammalian cells to allow for use in treatment of infections. Interestingly, TM5 kills P. aeruginosa more rapidly than traditional antibiotics, within 30-60 minutes in vitro , and is effective against a range of clinical isolates. In vivo , TM5 significantly reduced bacterial load in the lungs within 24 hours compared to untreated mice and demonstrated few adverse effects. Taken together, these observations suggest that TM5 shows promise as an alternative therapy for MDR P. aeruginosa respiratory infections.

Bacillus Calmette-Guérin vaccination as defense against SARS-CoV-2 (BADAS): a randomized controlled trial to protect healthcare workers in the USA by enhanced trained immune responses.

BACKGROUND: A large epidemic, such as that observed with SARS-CoV-2, seriously challenges available hospital capacity, and this would be augmented by infection of healthcare workers (HCW). Bacillus Calmette-Guérin (BCG) is a vaccine against tuberculosis, with protective non-specific effects against other respiratory tract infections in vitro and in vivo. Preliminary analyses suggest that regions of the world with existing BCG vaccination programs have lower incidence and mortality from COVID-19. We hypothesize that BCG vaccination can reduce SARS-CoV-2 infection and disease severity. METHODS: This will be a placebo-controlled adaptive multi-center randomized controlled trial. A total of 1800 individuals considered to be at high risk, including those with comorbidities (hypertension, diabetes, obesity, reactive airway disease, smokers), racial and ethnic minorities, elderly, teachers, police, restaurant wait-staff, delivery personnel, health care workers who are defined as personnel working in a healthcare setting, at a hospital, medical center or clinic (veterinary, dental, ophthalmology), and first responders (paramedics, firefighters, or law enforcement), will be randomly assigned to two treatment groups. The treatment groups will receive intradermal administration of BCG vaccine or placebo (saline) with groups at a 1:1 ratio. Individuals will be tracked for evidence of SARS-CoV-2 infection and severity as well as obtaining whole blood to track immunological markers, and a sub-study will include cognitive function and brain imaging. The majority of individuals will be followed for 6 months, with an option to extend for another 6 months, and the cognitive sub-study duration is 2 years. We will plot Kaplan-Meier curves that will be plotted comparing groups and hazard ratios and p-values reported using Cox proportional hazard models. DISCUSSION: It is expected this trial will allow evaluation of the effects of BCG vaccination at a population level in high-risk healthcare individuals through a mitigated clinical course of SARS-CoV-2 infection and inform policy making during the ongoing epidemic. TRIAL REGISTRATION: ClinicalTrials.gov NCT04348370. Registered on April 16, 2020.

Stabilization of Tuberculosis Reporter Enzyme Fluorescence (REFtb) Diagnostic Reagents for Use at the Point of Care.

Tuberculosis is one of the most frequent causes of death in humans worldwide. One of the primary reasons tuberculosis remains a public health threat is that diagnosis can take weeks to months, is often not very sensitive and cannot be accomplished in many remote environments. A rapid, sensitive and inexpensive point-of-care (POC) diagnostic would have a major impact on tuberculosis eradication efforts. The tuberculosis diagnostic system REFtb is based on specific detection of the constitutively expressed ß-lactamase (BlaC) in Mycobacterium tuberculosis using a custom fluorogenic substrate designated as CDG-3. REFtb has potential as a diagnostic for tuberculosis that could be very inexpensive (

Increased DNA methylation, cellular senescence and premature epigenetic aging in guinea pigs and humans with tuberculosis.

BACKGROUND: Tuberculosis (TB) is the archetypical chronic infection, with patients having months of symptoms before diagnosis. In the two years after successful therapy, survivors of TB have a three-fold increased risk of death. METHODS: Guinea pigs were infected with Mycobacterium tuberculosis (Mtb) for 45 days, followed by RRBS DNA methylation analysis. In humans, network analysis of differentially expressed genes across three TB cohorts were visualized at the pathway-level. Serum levels of inflammation were measured by ELISA. Horvath (DNA methylation) and RNA-seq biological clocks were used to investigate shifts in chronological age among humans with TB. RESULTS: Guinea pigs with TB demonstrated DNA hypermethylation and showed system-level similarity to humans with TB (p-value = 0.002). The transcriptome in TB in multiple cohorts was enriched for DNA methylation and cellular senescence. Senescence associated proteins CXCL9, CXCL10, and TNF were elevated in TB patients compared to healthy controls. Humans with TB demonstrate 12.7 years (95% CI: 7.5, 21.9) and 14.38 years (95% CI: 10.23-18.53) of cellular aging as measured by epigenetic and gene expression based cellular clocks, respectively. CONCLUSIONS: In both guinea pigs and humans, TB perturbs epigenetic processes, promoting premature cellular aging and inflammation, a plausible means to explain the long-term detrimental health outcomes after TB.

COVID-19 and Beyond: Exploring Public Health Benefits from Non-Specific Effects of BCG Vaccination.

Bacille Calmette-Guérin (BCG) vaccination, widely used throughout the world to protect against infant tuberculous meningitis and miliary tuberculosis (TB), can provide broad non-specific protection against infectious respiratory diseases in certain groups. Interest in BCG has seen a resurgence within the scientific community as the mechanisms for non-specific protection have begun to be elucidated. The impact of the COVID-19 pandemic on nearly every aspect of society has profoundly illustrated the pressure that respiratory infections can place on a national healthcare system, further renewing interest in BCG vaccination as a public health policy to reduce the burden of those illnesses. However, the United States does not recommend BCG vaccination due to its variable effectiveness against adult TB, the relatively low risk of Mycobacterium tuberculosis infection in most of the United States, and the vaccine's interference with tuberculin skin test reactivity that complicates TB screening. In this review, we explore the broad immune training effects of BCG vaccination and literature on the effects of BCG vaccination on COVID-19 spread, disease severity, and mortality. We further discuss barriers to scheduled BCG vaccination in the United States and how those barriers could potentially be overcome.

Genomic sequencing is required for identification of tuberculosis transmission in Hawaii.

BACKGROUND: Tuberculosis (TB) caused an estimated 1.4 million deaths and 10.4 million new cases globally in 2015. TB rates in the United States continue to steadily decline, yet rates in the State of Hawaii are perennially among the highest in the nation due to a continuous influx of immigrants from the Western Pacific and Asia. TB in Hawaii is composed of a unique distribution of genetic lineages, with the Beijing and Manila families of Mycobacterium tuberculosis (Mtb) comprising over two-thirds of TB cases. Standard fingerprinting methods (spoligotyping plus 24-loci Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats [MIRU-VNTR] fingerprinting) perform poorly when used to identify actual transmission clusters composed of isolates from these two families. Those typing methods typically group isolates from these families into large clusters of non-linked isolates with identical fingerprints. Next-generation whole-genome sequencing (WGS) provides a new tool for molecular epidemiology that can resolve clusters of isolates with identical spoligotyping and MIRU-VNTR fingerprints. METHODS: We performed WGS and SNP analysis and evaluated epidemiological data to investigate 19 apparent TB transmission clusters in Hawaii from 2003 to 2017 in order to assess WGS' ability to resolve putative Mtb clusters from the Beijing and Manila families. This project additionally investigated MIRU-VNTR allele prevalence to determine why standard Mtb fingerprinting fails to usefully distinguish actual transmission clusters from these two Mtb families. RESULTS: WGS excluded transmission events in seven of these putative clusters, confirmed transmission in eight, and identified both transmission-linked and non-linked isolates in four. For epidemiologically identified clusters, while the sensitivity of MIRU-VNTR fingerprinting for identifying actual transmission clusters was found to be 100%, its specificity was only 28.6% relative to WGS. We identified that the Beijing and Manila families' significantly lower Shannon evenness of MIRU-VNTR allele distributions than lineage 4 was the cause of standard fingerprinting's poor performance when identifying transmission in Beijing and Manila family clusters. CONCLUSIONS: This study demonstrated that WGS is necessary for epidemiological investigation of TB in Hawaii and the Pacific.

Whole genome SNP analysis suggests unique virulence factor differences of the Beijing and Manila families of Mycobacterium tuberculosis found in Hawaii.

While tuberculosis (TB) remains a global disease, the WHO estimates that 62% of the incident TB cases in 2016 occurred in the WHO South-East Asia and Western Pacific regions. TB in the Pacific is composed predominantly of two genetic families of Mycobacterium tuberculosis (Mtb): Beijing and Manila. The Manila family is historically under-studied relative to the families that comprise the majority of TB in Europe and North America (e.g. lineage 4), and it remains unclear why this lineage has persisted in Filipino populations despite the predominance of more globally successful Mtb lineages in most of the world. The Beijing family is of particular interest as it is increasingly associated with drug resistance throughout the world. Both of these lineages are important to the State of Hawaii, where they comprise over two-thirds of TB cases. Here, we performed whole genome sequencing on 82 Beijing family, Manila family, and outgroup clinical Mtb isolates from Hawaii to identify lineage-specific SNPs (SNPs found in all isolates from their respective families, and exclusively in those families) in established virulence factor genes. Six non-silent lineage-specific virulence factor SNPs were found in the Beijing family, including mutations in alternative sigma factor sigG and polyketide synthases pks5 and pks7. The Manila family displayed more than eleven non-silent lineage-specific and characteristic virulence factor mutations, including in genes coding for MCE-family protein Mce1B, two mutations in fatty-acid-AMP ligase FadD26, and virulence-regulating transcriptional regulator VirS. This study further identified an ancient clade that shared some virulence factor mutations with the Manila family, and investigated the relationship of those and other "Manila-like" spoligotypes to the Manila family with this SNP dataset. This work identified a set of virulence genes that are worth pursuing to determine potential differences in transmission or virulence displayed by these two Mtb families.

Genomic analyses of the ancestral Manila family of Mycobacterium tuberculosis.

With its airborne transmission and prolonged latency period, Mycobacterium tuberculosis spreads worldwide as one of the most successful bacterial pathogens and continues to kill millions of people every year. M. tuberculosis lineage 1 is inferred to originate ancestrally based on the presence of the 52-bp TbD1 sequence and analysis of single nucleotide polymorphisms. Previously, we briefly reported the complete genome sequencing of M. tuberculosis strains 96121 and 96075, which belong to the ancient Manila family and modern Beijing family respectively. Here we present the comprehensive genomic analyses of the Manila family in lineage 1 compared to complete genomes in lineages 2-4. Principal component analysis of the presence and absence of CRISPR spacers suggests that Manila isolate 96121 is distinctly distant from lineages 2-4. We further identify a truncated whiB5 gene and a putative operon consisting of genes encoding a putative serine/threonine kinase PknH and a putative ABC transporter, which are only found in the genomes of Manila family isolates. Six single nucleotide polymorphisms are uniquely conserved in 38 Manila strains. Moreover, when compared to M. tuberculosis H37Rv, 59 proteins are under positive selection in Manila family isolate 96121 but not in Beijing family isolate 96075. The unique features further serve as biomarkers for Manila strains and may shed light on the limited transmission of this ancestral lineage outside of its Filipino host population.