Trained Immunity: a Tool for Reducing Susceptibility to and the Severity of SARS-CoV-2 Infection.

SARS-CoV-2 infection is mild in the majority of individuals but progresses into severe pneumonia in a small proportion of patients. The increased susceptibility to severe disease in the elderly and individuals with co-morbidities argues for an initial defect in anti-viral host defense mechanisms. Long-term boosting of innate immune responses, also termed "trained immunity," by certain live vaccines (BCG, oral polio vaccine, measles) induces heterologous protection against infections through epigenetic, transcriptional, and functional reprogramming of innate immune cells. We propose that induction of trained immunity by whole-microorganism vaccines may represent an important tool for reducing susceptibility to and severity of SARS-CoV-2.

Activate: Randomized Clinical Trial of BCG Vaccination against Infection in the Elderly.

BCG vaccination in children protects against heterologous infections and improves survival independently of tuberculosis prevention. The phase III ACTIVATE trial assessed whether BCG has similar effects in the elderly. In this double-blind, randomized trial, elderly patients (n = 198) received BCG or placebo vaccine at hospital discharge and were followed for 12 months for new infections. At interim analysis, BCG vaccination significantly increased the time to first infection (median 16 weeks compared to 11 weeks after placebo). The incidence of new infections was 42.3% (95% CIs 31.9%-53.4%) after placebo vaccination and 25.0% (95% CIs 16.4%-36.1%) after BCG vaccination; most of the protection was against respiratory tract infections of probable viral origin (hazard ratio 0.21, p = 0.013). No difference in the frequency of adverse effects was found. Data show that BCG vaccination is safe and can protect the elderly against infections. Larger studies are needed to assess protection against respiratory infections, including COVID-19 (ClinicalTrials.gov NCT03296423).

Multi-omics analysis of innate and adaptive responses to BCG vaccination reveals epigenetic cell states that predict trained immunity.

Immune responses are tightly regulated yet highly variable between individuals. To investigate human population variation of trained immunity, we immunized healthy individuals with Bacillus Calmette-Guérin (BCG). This live-attenuated vaccine induces not only an adaptive immune response against tuberculosis but also triggers innate immune activation and memory that are indicative of trained immunity. We established personal immune profiles and chromatin accessibility maps over a 90-day time course of BCG vaccination in 323 individuals. Our analysis uncovered genetic and epigenetic predictors of baseline immunity and immune response. BCG vaccination enhanced the innate immune response specifically in individuals with a dormant immune state at baseline, rather than providing a general boost of innate immunity. This study advances our understanding of BCG's heterologous immune-stimulatory effects and trained immunity in humans. Furthermore, it highlights the value of epigenetic cell states for connecting immune function with genotype and the environment.

TCA metabolism regulates DNA hypermethylation in LPS and Mycobacterium tuberculosis-induced immune tolerance.

Severe and chronic infections, including pneumonia, sepsis, and tuberculosis (TB), induce long-lasting epigenetic changes that are associated with an increase in all-cause postinfectious morbidity and mortality. Oncology studies identified metabolic drivers of the epigenetic landscape, with the tricarboxylic acid (TCA) cycle acting as a central hub. It is unknown if the TCA cycle also regulates epigenetics, specifically DNA methylation, after infection-induced immune tolerance. The following studies demonstrate that lipopolysaccharide and Mycobacterium tuberculosis induce changes in DNA methylation that are mediated by the TCA cycle. Infection-induced DNA hypermethylation is mitigated by inhibitors of cellular metabolism (rapamycin, everolimus, metformin) and the TCA cycle (isocitrate dehydrogenase inhibitors). Conversely, exogenous supplementation with TCA metabolites (succinate and itaconate) induces DNA hypermethylation and immune tolerance. Finally, TB patients who received everolimus have less DNA hypermethylation demonstrating proof of concept that metabolic manipulation can mitigate epigenetic scars.

Natural resistance against infections: focus on COVID-19.

Not all individuals exposed to a pathogen develop illness: some are naturally resistant whereas others develop an asymptomatic infection. Epidemiological studies suggest that there is similar variability in susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. We propose that natural resistance is part of the disease history in some individuals exposed to this new coronavirus. Epidemiological arguments for natural resistance to SARS-CoV-2 are the lower seropositivity of children compared to adults, studies on closed environments of ships with outbreaks, and prevalence studies in some developing countries. Potential mechanisms of natural resistance include host genetic variants, viral interference, cross-protective natural antibodies, T cell immunity, and highly effective innate immune responses. Better understanding of natural resistance can help to advance preventive and therapeutic measures against infections for improved preparedness against potential future pandemics.

Plasma metabolome predicts trained immunity responses after antituberculosis BCG vaccination.

The antituberculosis vaccine Bacillus Calmette-Guérin (BCG) induces nonspecific protection against heterologous infections, at least partly through induction of innate immune memory (trained immunity). The amplitude of the response to BCG is variable, but the factors that influence this response are poorly understood. Metabolites, either released by cells or absorbed from the gut, are known to influence immune responses, but whether they impact BCG responses is not known. We vaccinated 325 healthy individuals with BCG, and collected blood before, 2 weeks and 3 months after vaccination, to assess the influence of circulating metabolites on the immune responses induced by BCG. Circulating metabolite concentrations after BCG vaccination were found to have a more pronounced impact on trained immunity responses, such as the increase in IL-1ß and TNF-α production upon Staphylococcus aureus stimulation, than on specific adaptive immune memory, assessed as IFN-γ production in response to Mycobacterium tuberculosis. Circulating metabolites at baseline were able to predict trained immunity responses at 3 months after vaccination and enrichment analysis based on the metabolites positively associated with trained immunity revealed enrichment of the tricarboxylic acid (TCA) cycle and glutamine metabolism, both of which were previously found to be important for trained immunity. Several new metabolic pathways that influence trained immunity were identified, among which taurine metabolism associated with BCG-induced trained immunity, a finding validated in functional experiments. In conclusion, circulating metabolites are important factors influencing BCG-induced trained immunity in humans. Modulation of metabolic pathways may be a novel strategy to improve vaccine and trained immunity responses.

BCG-induced protection against Mycobacterium tuberculosis infection: Evidence, mechanisms, and implications for next-generation vaccines.

The tuberculosis (TB) vaccine Bacillus Calmette-Guérin (BCG) was introduced 100 years ago, but as it provides insufficient protection against TB disease, especially in adults, new vaccines are being developed and evaluated. The discovery that BCG protects humans from becoming infected with Mycobacterium tuberculosis (Mtb) and not just from progressing to TB disease provides justification for considering Mtb infection as an endpoint in vaccine trials. Such trials would require fewer participants than those with disease as an endpoint. In this review, we first define Mtb infection and disease phenotypes that can be used for mechanistic studies and/or endpoints for vaccine trials. Secondly, we review the evidence for BCG-induced protection against Mtb infection from observational and BCG re-vaccination studies, and discuss limitations and variation of this protection. Thirdly, we review possible underlying mechanisms for BCG efficacy against Mtb infection, including alternative T cell responses, antibody-mediated protection, and innate immune mechanisms, with a specific focus on BCG-induced trained immunity, which involves epigenetic and metabolic reprogramming of innate immune cells. Finally, we discuss the implications for further studies of BCG efficacy against Mtb infection, including for mechanistic research, and their relevance to the design and evaluation of new TB vaccines.

A loading dose of clofazimine to rapidly achieve steady-state-like concentrations in patients with nontuberculous mycobacterial disease.

OBJECTIVES: Clofazimine is a promising drug for the treatment of nontuberculous mycobacterial (NTM) diseases. Accumulation of clofazimine to reach steady-state plasma concentrations takes months. A loading dose may reduce the time to steady-state-like concentrations. We evaluated the pharmacokinetics (PK), safety and tolerability of a loading dose regimen in patients with NTM disease. METHODS: Adult participants received a 4-week loading dose regimen of 300 mg clofazimine once daily, followed by a maintenance dose of 100 mg once daily (combined with other antimycobacterial drugs). Blood samples for PK analysis were collected on three occasions. A population PK model for clofazimine was developed and simulations were performed to assess the time to reach steady-state-like (target) concentrations for different dosing regimens. RESULTS: Twelve participants were included. The geometric mean peak and trough clofazimine concentrations after the 4-week loading phase were 0.87 and 0.50 mg/L, respectively. Adverse events were common, but mostly mild and none led to discontinuation of clofazimine. Our loading dose regimen reduced the predicted median time to target concentrations by 1.5 months compared to no loading dose (3.8 versus 5.3 months). Further time benefit was predicted with a 6-week loading dose regimen (1.4 versus 5.3 months). CONCLUSION: A 4-week loading dose regimen of 300 mg once daily reduced the time to target clofazimine concentrations and was safe and well-tolerated. Extending the loading phase to 6 weeks could further decrease the time to target concentrations. Using a loading dose of clofazimine is a feasible strategy to optimize treatment of NTM disease. CLINICAL TRIALS REGISTRATION: NCT05294146.

Clinical presentation, management, and outcome of suspected central nervous system infections in Indonesia: a prospective cohort study.

BACKGROUND: Little is known about the etiology, clinical presentation, management, and outcome of central nervous system (CNS) infections in Indonesia, a country with a high burden of infectious diseases and a rising prevalence of HIV. METHODS: We included adult patients with suspected CNS infections at two referral hospitals in a prospective cohort between April 2019 and December 2021. Clinical, laboratory, and radiological assessments were standardized. We recorded initial and final diagnoses, treatments, and outcomes during 6 months of follow-up. RESULTS: Of 1051 patients screened, 793 were diagnosed with a CNS infection. Patients (median age 33 years, 62% male, 38% HIV-infected) presented a median of 14 days (IQR 7-30) after symptom onset, often with altered consciousness (63%), motor deficits (73%), and seizures (21%). Among HIV-uninfected patients, CNS tuberculosis (TB) was most common (60%), while viral (8%) and bacterial (4%) disease were uncommon. Among HIV-infected patients, cerebral toxoplasmosis (41%) was most common, followed by CNS TB (19%), neurosyphilis (15%), and cryptococcal meningitis (10%). A microbiologically confirmed diagnosis was achieved in 25% of cases, and initial diagnoses were revised in 46% of cases. In-hospital mortality was 30%, and at six months, 45% of patients had died, and 12% suffered from severe disability. Six-month mortality was associated with older age, HIV, and severe clinical, radiological and CSF markers at presentation. CONCLUSION: CNS infections in Indonesia are characterized by late presentation, severe disease, frequent HIV coinfection, low microbiological confirmation and high mortality. These findings highlight the need for earlier disease recognition, faster and more accurate diagnosis, and optimized treatment, coupled with wider efforts to improve the uptake of HIV services.

MUC5AC Genetic Variation Is Associated With Tuberculous Meningitis Cerebral Spinal Fluid Cytokine Responses and Mortality.

BACKGROUND: The purpose of this study was to assess if single nucleotide polymorphisms (SNPs) in lung mucins MUC5B and MUC5AC are associated with Mycobacterium tuberculosis outcomes. METHODS: Independent SNPs in MUC5B and MUC5AC (genotyped by Illumina HumanOmniExpress array) were assessed for associations with tumor necrosis factor (TNF) concentrations (measured by immunoassay) in cerebral spinal fluid (CSF) from tuberculous meningitis (TBM) patients. SNPs associated with CSF TNF concentrations were carried forward for analyses of pulmonary and meningeal tuberculosis susceptibility and TBM mortality. RESULTS: MUC5AC SNP rs28737416 T allele was associated with lower CSF concentrations of TNF (P = 1.8 × 10-8) and IFN-γ (P = 2.3 × 10-6). In an additive genetic model, rs28737416 T/T genotype was associated with higher susceptibility to TBM (odds ratio [OR], 1.24; 95% confidence interval [CI], 1.03-1.49; P = .02), but not pulmonary tuberculosis (OR, 1.11, 95% CI, .98-1.25; P = .10). TBM mortality was higher among participants with the rs28737416 T/T and T/C genotypes (35/119, 30.4%) versus the C/C genotype (11/89, 12.4%; log-rank P = .005) in a Vietnam discovery cohort (n = 210), an independent Vietnam validation cohort (n = 87; 9/87, 19.1% vs 1/20, 2.5%; log-rank P = .02), and an Indonesia validation cohort (n = 468, 127/287, 44.3% vs 65/181, 35.9%; log-rank P = .06). CONCLUSIONS: MUC5AC variants may contribute to immune changes that influence TBM outcomes.

The impact of BCG dose and revaccination on trained immunity.

The innate immune system can display heterologous memory-like responses termed trained immunity after stimulation by certain vaccinations or infections. In this randomized, placebo-controlled trial, we investigated the modulation of Bacille Calmette-Guérin (BCG)-induced trained immunity by BCG revaccination or high-dose BCG administration, in comparison to a standard dose. We show that monocytes from all groups of BCG-vaccinated individuals exerted increased TNFα production after ex-vivo stimulation with various unrelated pathogens. Similarly, we observed increased amounts of T-cell-derived IFNγ after M. tuberculosis exposure, regardless of the BCG intervention. NK cell cytokine production, especially after heterologous stimulation with the fungal pathogen Candida albicans, was predominantly boosted after high dose BCG administration. Cytokine production capacity before vaccination was inversely correlated with trained immunity. While the induction of a trained immunity profile is largely dose- or frequency independent, baseline cytokine production capacity is associated with the magnitude of the innate immune memory response after BCG vaccination.

The impact of BNT162b2 mRNA vaccine on adaptive and innate immune responses.

The mRNA-based BNT162b2 protects against severe disease and mortality caused by SARS-CoV-2 via induction of specific antibody and T-cell responses. Much less is known about its broad effects on immune responses against other pathogens. Here, we investigated the adaptive immune responses induced by BNT162b2 vaccination against various SARS-CoV-2 variants and its effects on the responsiveness of immune cells upon stimulation with heterologous stimuli. BNT162b2 vaccination induced effective humoral and cellular immunity against SARS-CoV-2 that started to wane after six months. We also observed long-term transcriptional changes in immune cells after vaccination. Additionally, vaccination with BNT162b2 modulated innate immune responses as measured by inflammatory cytokine production after stimulation - higher IL-1/IL-6 release and decreased IFN-α production. Altogether, these data expand our knowledge regarding the overall immunological effects of this new class of vaccines and underline the need for additional studies to elucidate their effects on both innate and adaptive immune responses.

Successful addition of topical antibiotic treatment after surgery in treatment-refractory nontuberculous mycobacterial skin and soft tissue infections.

Treatment of skin and soft tissue infections with nontuberculous mycobacteria sometimes fails despite repeated debridements and long-term systemic antibiotic therapy. These treatment-refractory infections can cause significant morbidity and pose a treatment challenge. Following surgery, we treated three patients with negative pressure wound therapy with the instillation and dwell time of topical antibiotics, in addition to systemic antibiotic treatment. Treatment was successful and well tolerated, except for some local irritation.

An integrative genomics approach identifies KDM4 as a modulator of trained immunity.

Innate immune cells are able to build memory characteristics via a process termed "trained immunity." Host factors that influence the magnitude of the individual trained immunity response remain largely unknown. Using an integrative genomics approach, our study aimed to prioritize and understand the role of specific genes in trained immunity responses. In vitro-induced trained immunity responses were assessed in two independent population-based cohorts of healthy individuals, the 300 Bacillus Calmette-Guérin (300BCG; n = 267) and 200 Functional Genomics (200FG; n = 110) cohorts from the Human Functional Genomics Project. Genetic loci that influence cytokine responses upon trained immunity were identified by conducting a meta-analysis of QTLs identified in the 300BCG and 200FG cohorts. From the identified QTL loci, we functionally validated the role of PI3K-Akt signaling pathway and two genes that belong to the family of Siglec receptors (Siglec-5 and Siglec-14). Furthermore, we identified the H3K9 histone demethylases of the KDM4 family as major regulators of trained immunity responses. These data pinpoint an important role of metabolic and epigenetic processes in the regulation of trained immunity responses, and these findings may open new avenues for vaccine design and therapeutic interventions.

Whole sporozoite immunization with Plasmodium falciparum strain NF135 in a randomized trial.

BACKGROUND: Whole sporozoite immunization under chemoprophylaxis (CPS regime) induces long-lasting sterile homologous protection in the controlled human malaria infection model using Plasmodium falciparum strain NF54. The relative proficiency of liver-stage parasite development may be an important factor determining immunization efficacy. Previous studies show that Plasmodium falciparum strain NF135 produces relatively high numbers of large liver-stage schizonts in vitro. Here, we evaluate this strain for use in CPS immunization regimes. METHODS: In a partially randomized, open-label study conducted at the Radboudumc, Nijmegen, the Netherlands, healthy, malaria-naïve adults were immunized by three rounds of fifteen or five NF135-infected mosquito bites under mefloquine prophylaxis (cohort A) or fifteen NF135-infected mosquito bites and presumptive treatment with artemether/lumefantrine (cohort B). Cohort A participants were exposed to a homologous challenge 19 weeks after immunization. The primary objective of the study was to evaluate the safety and tolerability of CPS immunizations with NF135. RESULTS: Relatively high liver-to-blood inocula were observed during immunization with NF135 in both cohorts. Eighteen of 30 (60%) high-dose participants and 3/10 (30%) low-dose participants experienced grade 3 adverse events 7 to 21 days following their first immunization. All cohort A participants and two participants in cohort B developed breakthrough blood-stage malaria infections during immunizations requiring rescue treatment. The resulting compromised immunizations induced modest sterile protection against homologous challenge in cohort A (5/17; 29%). CONCLUSIONS: These CPS regimes using NF135 were relatively poorly tolerated and frequently required rescue treatment, thereby compromising immunization efficiency and protective efficacy. Consequently, the full potential of NF135 sporozoites for induction of immune protection remains inconclusive. Nonetheless, the high liver-stage burden achieved by this strain highlights it as an interesting potential candidate for novel whole sporozoite immunization approaches. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov under identifier NCT03813108.

Induction of trained immunity by influenza vaccination - impact on COVID-19.

Non-specific protective effects of certain vaccines have been reported, and long-term boosting of innate immunity, termed trained immunity, has been proposed as one of the mechanisms mediating these effects. Several epidemiological studies suggested cross-protection between influenza vaccination and COVID-19. In a large academic Dutch hospital, we found that SARS-CoV-2 infection was less common among employees who had received a previous influenza vaccination: relative risk reductions of 37% and 49% were observed following influenza vaccination during the first and second COVID-19 waves, respectively. The quadrivalent inactivated influenza vaccine induced a trained immunity program that boosted innate immune responses against various viral stimuli and fine-tuned the anti-SARS-CoV-2 response, which may result in better protection against COVID-19. Influenza vaccination led to transcriptional reprogramming of monocytes and reduced systemic inflammation. These epidemiological and immunological data argue for potential benefits of influenza vaccination against COVID-19, and future randomized trials are warranted to test this possibility.

Potential drug-drug interactions in males living with HIV who use drugs to treat lower urinary tract symptoms.

OBJECTIVE: Lower urinary tract symptoms (LUTS) are becoming more prevalent in the ageing population of males living with HIV. Drugs to treat LUTS are known for both their potential role as victims in drug-drug interactions (DDIs) and their side effects. We aimed to evaluate the current use of drugs to treat LUTS and to assess potential DDIs in our cohort of adult males living with HIV. DESIGN: This was a retrospective review of pharmacy records. METHODS: We recorded the combination antiretroviral therapy (cART) regimen and any use of drugs to treat LUTS (anatomical therapeutic chemical codes G04CA/CB/CX and G04BD). Potential DDIs were assessed using the interaction checker developed by the University of Liverpool (https://www.hiv-druginteractions.org/checker). RESULTS: A total of 411 adult males living with HIV were included in this analysis. The median (interquartile range [IQR]) age was 53 (41-62) years. Nineteen (4.6%) patients used one or more drugs to treat LUTS. As expected, older patients were more likely to be receiving treatment for LUTS: Q1 (20-40 years) = 0%; Q2 (41-52 years) = 2%; Q3 (53-61 years) = 7%; Q4 (62-79 years) = 10%. Seven potential DDIs between cART and LUTS treatment were noted in six of the 19 (32%) patients. Following medication reviews of these six patients, the following interventions were proposed: evaluate safe use of alpha-blocker (n = 4), change in cART (n = 2), and dose reduction of the anticholinergic agent (n = 1). CONCLUSION: Treatment for LUTS coincided with cART in 7%-10% of patients aged above the median age of 53 years in our cohort. Improvements in DDI management appeared to be possible in this growing cohort of males living with HIV and with LUTS.

Infection incidence, timing, and dose dependency in rheumatoid arthritis patients treated with rituximab: a retrospective cohort study.

OBJECTIVES: Rituximab (RTX) is a safe and effective treatment for rheumatoid arthritis (RA). However, there are some concerns about infection risk and preliminary data suggest dose and time dependency. This study aims to determine the infection incidence in a large real-life population of RA patients using RTX, with special focus on (ultra-)low dosing and time since last infusion. METHODS: RA patients treated with 1000, 500 or 200 mg RTX per cycle between 2012 and 2021 at the Sint Maartenskliniek were included in a retrospective cohort study. Patient-, disease-, treatment- and infection characteristics were retrieved from electronic health records. Infection incidence rates, dose and time relations with RTX infusion were analysed using mixed-effects Poisson regression. RESULTS: Among 490 patients, we identified 819 infections in 1254 patient years. Most infections were mild and respiratory tract infections were most common. Infection incidence rates were 41, 54 and 71 per 100 patient years for doses of 200, 500 and 1000 mg. Incidence rate ratio (IRR) was significantly lower for 200 mg compared to 1000 mg (adjusted IRR 0.35, 95% CI 0.17-0.72, p = 0.004). In patients receiving 1000 or 500 mg RTX, infections seemed to occur more frequently within the first two months after infusion compared to later on in the treatment cycle, suggesting an association with peak concentration. CONCLUSION: Ultra-low dosing (200 mg) of RTX is associated with a lower risk of infections in RA. Future interventions focusing on ultra-low dosing and slow release of RTX (e.g. by subcutaneous administration) may lower infection risk.