Background:The BCG vaccine induces trained immunity, an epigenetic-mediated increase in innate immune responsiveness. Therefore, this clinical trial evaluated if BCG-induced trained immunity could decrease coronavirus disease 2019 (COVID-19)-related frequency or severity. Methods:A double-blind, placebo-controlled clinical trial of healthcare workers randomized participants to vaccination with BCG TICE or placebo (saline). Enrollment included 529 healthcare workers randomized to receive BCG or placebo. Primary analysis evaluated COVID-19 disease frequency, while secondary analysis evaluated coronavirus immunity in a subset of participants. Study enrollment ceased early in December 2020 following introduction of COVID-19-specific vaccines. Results:Study enrollment was halted early, prior to reaching the targeted recruitment, and was not powered to detect a decrease in COVID-19 frequency. Symptomatic COVID-19 occurred in 21 of 263 and 10 of 266 participants in the BCG and placebo arms, respectively (P = .50, Fisher exact test). Participants vaccinated with BCG, but uninfected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demonstrated increased coronavirus vaccine immunity (increase spike-inducible levels of tumor necrosis factor, interleukin 6, and interleukin 1β) 12 months after BCG vaccination compared to participants receiving placebo. Immune responsiveness to SARS-CoV-2 antigens correlated with BCG-induced DNA methylation changes. Conclusions:Due to early study closure, the study was not powered to evaluate COVID-19 frequency. Secondary analysis demonstrated that 12 months following vaccination, BCG increased coronavirus vaccine immunity compared to those who did not receive BCG. This increase in COVID-19 vaccine immunity correlated with BCG-induced DNA methylation changes.

Abstract Background The Bacillus Calmette-Guérin (BCG) vaccine induces trained immunity, an epigenetic-mediated increase in innate immune responsiveness. Therefore, this clinical trial evaluated if BCG-induced trained immunity could decrease COVID-19-related frequency or severity. Methods A double-blind, placebo-controlled clinical trial of healthcare workers randomized participants to vaccination with BCG TICE® or placebo (saline). Enrollment included 529 healthcare workers randomized to receive BCG or placebo. Primary analysis evaluated COVID-19 disease frequency, while secondary analysis evaluated coronavirus immunity in a subset of participants. Study enrollment ceased early in December 2020 following introduction of COVID-19-specific vaccines. Results Study enrollment was halted early, prior to reaching the targeted recruitment and was not powered to detect a decrease in COVID-19 frequency. Symptomatic COVID-19 occurred in 21 of 263 and 10 of 266 participants in the BCG and placebo arms, respectively (p= 0.50, Fisher’s exact test). Participants vaccinated with BCG, but uninfected with COVID-19, demonstrated increased coronavirus vaccine immunity (increase spike-inducible levels of TNF, IL6, and IL-1β) twelve months after BCG vaccination compared to participants receiving placebo. Immune responsiveness to COVID-19 antigens correlated with BCG-induced DNA methylation changes. Conclusion Due to early study closure, the study was not powered to evaluate COVID-19 frequency. Secondary analysis demonstrated that twelve months following vaccination, BCG increased coronavirus vaccine immunity compared to those who did not receive BCG. This increase in COVID-19 vaccine immunity correlated with BCG-induced DNA methylation changes.

Soon after commencement of the SARS-CoV-2 disease outbreak of 2019 (COVID-19), it became evident that the receptor-binding domain of the viral spike protein is the target of neutralizing antibodies that comprise a critical element of protective immunity to the virus. This study addresses the relative lack of information regarding actual antibody concentrations and binding affinities in convalescent plasma (CP) samples from COVID-19 patients and extends these analyses to post-vaccination (PV) samples to estimate protective IgG antibody (Ab) levels. A direct enzyme-linked immunosorbent assay (ELISA) was used to measure IgG anti-spike protein (SP) antibodies (Abs) relative to human chimeric spike S1 Ab standards. Microplate wells were coated with recombinant SP. Affinities of Ab binding to SP were determined by previously described methods. Binding affinities were also determined in an RBD-specific sandwich ELISA. Two indices of protective immunity were determined as permutations of Ab molar concentration divided by affinity as dissociation constant (KD). The range and geometric means of Ab concentrations in 21 CP and 21 PV samples were similar and a protective Ab level of 7.5 μg/ml was determined for the latter population, based on 95% of the normal distribution of the PV population. A population (n = 21) of plasma samples from individuals receiving only one vaccination with the BNT162b2 or mRNA-1273 vaccines (PtV) exhibited a geometric mean Ab concentration significantly (p < 0.03) lower than the PV population. The results of this study have implications for future vaccine development, projection of protective efficacy duration, and understanding of the immune response to SARS-CoV-2 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.

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.

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.

AbstractThe 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 killingP. aeruginosaand displays sufficiently low toxicity for mammalian cells to allow for use in treatment of infections. Interestingly, TM5 killsP. aeruginosamore rapidly than traditional antibiotics, within 30-60 minutesin 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 MDRP. aeruginosarespiratory infections.

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.

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 β-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 (<USD 2.00/test), used at the POC and could provide definitive diagnosis within 10 min. However, the reagents for REFtb are currently in liquid form, making them more susceptible to degradation and difficult to transport. We evaluated the improvement in the stability of REFtb reagents by lyophilization under a variety of conditions through their effects on the performance of REFtb. We found that lyophilization of REFtb components produces an easily reconstituted powder that displays similar performance to the liquid system and that lactose represents one of the most promising excipients for use in a final POC REFtb diagnostic system. These studies provide the foundation for the production of a stable POC REFtb system that could be easily distributed worldwide with minimal or no requirement for refrigeration.

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.

AbstractSoon after commencement of the SARS-CoV-2 disease outbreak of 2019 (COVID-19), it became evident that the receptor-binding domain of the viral spike protein is the target of neutralizing antibodies that comprise a critical element of protective immunity to the virus. This study addresses the relative lack of information regarding actual antibody concentrations in convalescent plasma samples from COVID-19 patients and extends these analyses to post-vaccination samples to estimate protective IgG antibody (Ab) levels. Both sample populations were similar and a protective Ab level of 7.5 µg/ml was determined, based on 95% of the normal distribution of the post-vaccination population. The results of this study have implications for future vaccine development, projection of protective efficacy duration, and understanding of the immune response to SARS-CoV-2 infection.One-Sentence SummaryUsing two quantitative immunoassays, we have found similar IgG antibody responses to the SARS-CoV-2 spike protein in populations of COVID-19 survivors and vaccine recipients that indicate a protective antibody concentration.