Interacting, Nonspecific, Immunological Effects of Bacille Calmette-Guérin and Tetanus-diphtheria-pertussis Inactivated Polio Vaccinations: An Explorative, Randomized Trial.

BACKGROUND: Certain vaccines, such as Bacille Calmette-Guérin (BCG), have nonspecific effects, which modulate innate immune responses and lead to protection against mortality from unrelated infections (trained immunity). In contrast, in spite of the disease-specific effects, an enhanced overall mortality has been described after diphtheria-tetanus-pertussis (DTP) vaccination in females. This randomized trial aimed to investigate the nonspecific immunological effects of BCG and DTP-containing vaccines on the immune response to unrelated pathogens. METHODS: We randomized 75 healthy, female, adult volunteers to receive either BCG, followed by a booster dose of tetanus-diphtheria-pertussis inactivated polio vaccine (Tdap) 3 months later; BCG and Tdap combined; or Tdap followed by BCG 3 months later. Blood was collected before vaccination, as well as at 1 day, 4 days, 2 weeks, and 3 months after the first vaccination(s), plus 2 weeks after the second vaccination. Ex vivo leukocyte responses to unrelated stimuli and pathogens were assessed. RESULTS: Tdap vaccination led to short-term potentiation and long-term repression of monocyte-derived cytokine responses, and short-term as well as long-term repression of T-cell reactivity to unrelated pathogens. BCG led to short-term and long-term potentiation of monocyte-derived cytokine responses. When given together with Tdap or after Tdap, BCG abrogated the immunosuppressive effects of Tdap vaccination. CONCLUSIONS: Tdap induces immunotolerance to unrelated antigens, which is partially restored by concurrent or subsequent BCG vaccination. These data indicate that the modulation of heterologous immune responses is induced by vaccination with Tdap and BCG, and more studies are warranted to investigate whether this is involved in the nonspecific effects of vaccines on mortality. CLINICAL TRIALS REGISTRATION: NCT02771782.

Differential effects of BCG vaccine on immune responses induced by vi polysaccharide typhoid fever vaccination: an explorative randomized trial.

The Vi polysaccharide typhoid fever vaccine (TFV) provides incomplete protection against typhoid fever. BCG, the vaccine against tuberculosis, can potentiate immune responses to other vaccines through induction of trained innate immunity and heterologous adaptive immunity. We performed an explorative, randomized, noncontrolled open trial to investigate whether BCG vaccination increases humoral and cellular response to TFV and whether BCG and TFV modulate nonspecific immune responses. Thirty volunteers were randomized to receive either TFV alone or BCG followed by TFV after 2 weeks. Ex vivo leukocyte responses and anti-Vi IgG antibody titers were measured 2 weeks and 3 months after TFV. BCG administration prior to TFV vaccination did not increase specific humoral or cellular immune responses to Salmonella typhi. TFV vaccination decreased pro-inflammatory responses to non-related stimuli. This effect was counteracted by prior BCG administration, which also led to decreased IL-10 and increased IL-22 responses to non-related stimuli. In an in vitro model of trained immunity TFV led to immunotolerance, which was partially reversed by BCG-induced trained immunity. BCG does not modulate adaptive immune responses to TFV but partially prevents inhibition of innate immune responses induced by TFV. Nonspecific effects of vaccines to unrelated microbial stimuli must be considered in the evaluation of their biological effects (ClinicalTrials.gov NCT02175420).

Opposite effects of Vaccinia and modified Vaccinia Ankara on trained immunity.

Vaccines such as Vaccinia or BCG have non-specific effects conferring protection against other diseases than their target infection, which are likely partly mediated through induction of innate immune memory (trained immunity). MVA85A, a recombinant strain of modified Vaccinia Ankara (MVA), has been suggested as an alternative vaccine against tuberculosis, but its capacity to induce positive or negative non-specific immune effects has not been studied. This study assesses whether Vaccinia and MVA are able to induce trained innate immunity in monocytes. Human primary monocytes were primed in an in vitro model with Vaccinia or MVA for 1 day, after which the stimulus was washed off and the cells were rechallenged with unrelated microbial ligands after 1 week. Heterologous cytokine responses were assessed and the capacity of MVA to induce epigenetic changes at the level of cytokine genes was investigated using chromatin immunoprecipitation and pharmacological inhibitors. Monocytes trained with Vaccinia showed significantly increased IL-6 and TNF-α production to stimulation with non-related stimuli, compared to non-trained monocytes. In contrast, monocytes primed with MVA showed significant decreased heterologous IL-6 and TNF-α responses, an effect which was abrogated by the addition of a histone methyltransferase inhibitor. No effects on H3K4me3 were observed after priming with MVA. It can be thus concluded that Vaccinia induces trained immunity in vitro, whereas MVA induces innate immune tolerance. This suggests the induction of trained immunity as an immunological mechanism involved in the non-specific effects of Vaccinia vaccination and points to a possible explanation for the lack of effect of MVA85A against tuberculosis.

Fungal Chitin Induces Trained Immunity in Human Monocytes during Cross-talk of the Host with Saccharomyces cerevisiae.

The immune system is essential to maintain the mutualistic homeostatic interaction between the host and its micro- and mycobiota. Living as a commensal,Saccharomyces cerevisiaecould potentially shape the immune response in a significant way. We observed thatS. cerevisiaecells induce trained immunity in monocytes in a strain-dependent manner through enhanced TNFα and IL-6 production upon secondary stimulation with TLR ligands, as well as bacterial and fungal commensals. Differential chitin content accounts for the differences in training properties observed among strains, driving induction of trained immunity by increasing cytokine production and direct antimicrobial activity bothin vitroandin vivo These chitin-induced protective properties are intimately associated with its internalization, identifying a critical role of phagosome acidification to facilitate microbial digestion. This study reveals how commensal and passenger microorganisms could be important in promoting health and preventing mucosal diseases by modulating host defense toward pathogens and thus influencing the host microbiota-immune system interactions.

[Glomerular hematuria: an atypical presentation of anti-glomerular basement membrane nephritis]. / Glomerulaire hematurie.

BACKGROUND: Urologic diseases can cause hematuria, but dysmorphic erythrocytes directs to a glomerular disease. The latter might occur isolated or in the presence of systemic complaints, proteinuria or kidney failure. These factors determine the differential diagnosis that ranges from an innocent IgA nephropathy to a fatal anti-glomerular basement membrane (GBM) nephritis. CASE: A 30-year old patient attended the outpatient clinic because of glomerular hematuria and normal kidney function with working diagnosis IgA nephropathy. Three months later he presented to the emergency department with a severe acute kidney injury duo to an anti-GBM nephritis. In retrospect, the anti-GBM titer was already high during the outpatient clinic phase, but due to the preserved kidney function, anti-GBM nephritis was not added to the differential diagnosis. CONCLUSION: Glomerular hematuria with a preserved kidney function could in a rare instance be caused by a subclinical anti-GBM nephritis. Follow-up of the kidney function and comprehensive laboratory testing - or even a kidney biopsy - could potentially lead to an early diagnosis of anti-GBM nephritis.

Association of genetic variations in ACE2, TIRAP and factor X with outcomes in COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can manifest with varying disease severity and mortality. Genetic predisposition influences the clinical course of infectious diseases. We investigated whether genetic polymorphisms in candidate genes ACE2, TIRAP, and factor X are associated with clinical outcomes in COVID-19. METHODS: We conducted a single-centre retrospective cohort study. All patients who visited the emergency department with SARS-CoV-2 infection proven by polymerase chain reaction were included. Single nucleotide polymorphisms in ACE2 (rs2285666), TIRAP (rs8177374) and factor X (rs3211783) were assessed. The outcomes were mortality, respiratory failure and venous thromboembolism. Respiratory failure was defined as the necessity of >5 litres/minute oxygen, high flow nasal oxygen suppletion or mechanical ventilation. RESULTS: Between March and April 2020, 116 patients (35% female, median age 65 [inter quartile range 55-75] years) were included and treated according to the then applicable guidelines. Sixteen patients (14%) died, 44 patients (38%) had respiratory failure of whom 23 required endotracheal intubation for mechanical ventilation, and 20 patients (17%) developed venous thromboembolism. The percentage of TIRAP polymorphism carriers in the survivor group was 28% as compared to 0% in the non-survivor group (p = 0.01, Bonferroni corrected p = 0.02). Genotype distribution of ACE2 and factor X did not differ between survivors and non-survivors. CONCLUSION: This study shows that carriage of TIRAP polymorphism rs8177374 could be associated with a significantly lower mortality in COVID-19. This TIRAP polymorphism may be an important predictor in the outcome of COVID-19.

BCG-induced trained immunity enhances acellular pertussis vaccination responses in an explorative randomized clinical trial.

Acellular pertussis (aP) booster vaccines are central to pertussis immunization programs, although their effectiveness varies. The Bacille Calmette-Guérin (BCG) vaccine is a prototype inducer of trained immunity, which enhances immune responses to subsequent infections or vaccinations. While previous clinical studies have demonstrated that trained immunity can protect against heterologous infections, its effect on aP vaccines in humans is unknown. We conducted a clinical study in order to determine the immunological effects of trained immunity on pertussis vaccination. Healthy female volunteers were randomly assigned to either receive BCG followed by a booster dose of tetanus-diphteria-pertussis inactivated polio vaccine (Tdap-IPV) 3 months later (BCG-trained), BCG + Tdap-IPV concurrently, or Tdap-IPV followed by BCG 3 months later. Primary outcomes were pertussis-specific humoral, T- and B-cell responses and were quantified at baseline of Tdap-IPV vaccination and 2 weeks thereafter. As a secondary outcome in the BCG-trained cohort, ex vivo leukocyte responses were measured in response to unrelated stimuli before and after BCG vaccination. BCG vaccination 3 months prior to, but not concurrent with, Tdap-IPV improves pertussis-specific Th1-cell and humoral responses, and also increases total memory B cell responses. These responses were correlated with enhanced IL-6 and IL-1ß production at the baseline of Tdap-IPV vaccination in the BCG-trained cohort. Our study demonstrates that prior BCG vaccination potentiates immune responses to pertussis vaccines and that biomarkers of trained immunity are the most reliable correlates of those responses.

Human Newborn Monocytes Demonstrate Distinct BCG-Induced Primary and Trained Innate Cytokine Production and Metabolic Activation In Vitro.

Background: Newborns exhibit distinct immune responses and are at high risk of infection. Neonatal immunization with BCG, the live attenuated vaccine against tuberculosis (TB), is associated with broad protection against a range of unrelated pathogens, possibly reflecting vaccine-induced training of innate immune cells ("innate memory"). However, little is known regarding the impact of age on BCG-induced innate responses. Objective: Establish an age-specific human monocyte in vitro training platform to characterize and compare BCG-induced primary and memory cytokine responses and immunometabolic shifts. Design/Methods: Human neonatal and adult CD33-selected monocytes were stimulated for 24h with RPMI (control) or BCG (Danish strain) in 10% autologous serum, washed and cultured for 5 additional days, prior to re-stimulation with the TLR4 agonist LPS for another 24h. Supernatants were collected at Day 1 (D1) to measure primary innate responses and at Day 7 (D7) to assess memory innate responses by ELISA and multiplex cytokine and chemokine assays. Lactate, a signature metabolite increased during trained immunity, was measured by colorimetric assay. Results: Cytokine production by human monocytes differed significantly by age at D1 (primary, BCG 1:750 and 1:100 vol/vol, p<0.0001) and D7 (innate memory response, BCG 1:100 vol/vol, p<0.05). Compared to RPMI control, newborn monocytes demonstrated greater TNF (1:100, 1:10 vol/vol, p<0.01) and IL-12p40 (1:100 vol/vol, p<0.05) production than adult monocytes (1:100, p<0.05). At D7, while BCG-trained adult monocytes, as previously reported, demonstrated enhanced LPS-induced TNF production, BCG-trained newborn monocytes demonstrated tolerization, as evidenced by significantly diminished subsequent LPS-induced TNF (RPMI vs. BCG 1:10, p <0.01), IL-10 and CCL5 production (p<0.05). With the exception of IL-1RA production by newborn monocytes, BCG-induced monocyte production of D1 cytokines/chemokines was inversely correlated with D7 LPS-induced TNF in both age groups (p<0.0001). Compared to BCG-trained adult monocytes, newborn monocytes demonstrated markedly impaired BCG-induced production of lactate, a metabolite implicated in immune training in adults. Conclusions: BCG-induced human monocyte primary- and memory-innate cytokine responses were age-dependent and accompanied by distinct immunometabolic shifts that impact both glycolysis and training. Our results suggest that immune ontogeny may shape innate responses to live attenuated vaccines, suggesting age-specific approaches to leverage innate training for broad protection against infection.

In vitro induction of trained immunity in adherent human monocytes.

A growing number of studies show that innate immune cells can undergo functional reprogramming, facilitating a faster and enhanced response to heterologous secondary stimuli. This concept has been termed "trained immunity." We outline here a protocol to recapitulate this in vitro using adherent monocytes from consecutive isolation of peripheral blood mononuclear cells. The induction of trained immunity and the associated functional reprogramming of monocytes is described in detail using ß-glucan (from Candida albicans) and Bacillus Calmette-Guérin as examples. For complete details on the use and execution of this protocol, please refer to Repnik et al. (2003) and Bekkering et al. (2016).

In Vitro Experimental Model of Trained Innate Immunity in Human Primary Monocytes.

Innate immune memory, or trained immunity, has recently been described to be an important property of cells of the innate immune system. Due to the increased interest in this important new field of immunological investigation, we sought to determine the optimal conditions for an in vitro experimental protocol of monocyte training using three of the most commonly used training stimuli from the literature: ß-glucan, the bacillus Calmette-Guérin (BCG) vaccine, and oxidized low-density lipoprotein (oxLDL). We investigated and optimized a protocol of monocyte trained immunity induced by an initial training period with ß-glucan, BCG, or oxLDL, followed by washing and resting of the cells and, thereafter, restimulation with secondary bacterial stimuli. The training and resting time intervals were varied to identify the optimal setting for the long-term induction of trained immunity. Trained immunity was assessed in terms of the secondary cytokine response, the production of reactive oxygen species, cell morphology, and induction of glycolysis. Monocytes primed with ß-glucan, BCG, and oxLDL showed increased pro- and anti-inflammatory cytokine responses upon restimulation with nonrelated stimuli. Also, all three stimuli induced a switch to glycolysis (the Warburg effect). These effects were most pronounced when the training interval was 24 h and the resting time interval was 6 days. Training with BCG and oxLDL also led to the increased production of reactive oxygen species, whereas training with ß-glucan led to the decreased production of reactive oxygen species. We describe the optimal conditions for an in vitro experimental model with human primary monocytes for study of the induction of trained innate immunity by microbial and metabolic stimuli.

Trained innate immunity as underlying mechanism for the long-term, nonspecific effects of vaccines.

An increasing body of evidence shows that the innate immune system has adaptive characteristics that involve a heterologous memory of past insults. Both experimental models and proof-of-principle clinical trials show that innate immune cells, such as monocytes, macrophages, and NK cells, can provide protection against certain infections in vaccination models independently of lymphocytes. This process is regulated through epigenetic reprogramming of innate immune cells and has been termed "trained immunity." It has been hypothesized that induction of trained immunity is responsible for the protective, nonspecific effects induced by vaccines, such as BCG, measles vaccination, and other whole-microorganism vaccines. In this review, we will present the mechanisms of trained immunity responsible for the long-lasting effects of vaccines on the innate immune system.

Vitamin A induces inhibitory histone methylation modifications and down-regulates trained immunity in human monocytes.

Epidemiologic studies suggest that VAS has long-lasting immunomodulatory effects. We hypothesized that ATRA inhibits inflammatory cytokines in a model of trained immunity in monocytes by inducing epigenetic reprogramming through histone modifications. We used an previously described in vitro model of trained immunity, in which adherent monocytes of healthy volunteers were incubated for 24 h with BCG in the presence or absence of ATRA. After washing the cells, they were incubated for an additional 6 d in culture medium and restimulated with microbial ligands, and cytokine production was assessed. ATRA inhibited cytokine responses upon restimulation of monocytes, and this effect was exerted through increased expression of SUV39H2, a histone methyltransferase that induces the inhibitory mark H3K9me3. H3K9me3 at promoter sites of several cytokines was up-regulated by ATRA, and inhibition of SUV39H2 restored cytokine production. In addition to H3K9me3, the stimulatory histone mark H3K4me3 was down-regulated by ATRA at several promoter locations of cytokine genes. Therefore, we can conclude that ATRA inhibits cytokine production in models of direct stimulation or BCG-induced trained immunity and that these effects are mediated by histone modifications.

Long-term in vitro and in vivo effects of γ-irradiated BCG on innate and adaptive immunity.

BCG vaccination is associated with a reduced mortality from nonmycobacterial infections. This is likely to be mediated by a combination of innate-immune memory ("trained immunity") and heterologous effects on adaptive immunity. As such, BCG could be used to boost host immunity but not in immunocompromised hosts, as it is a live, attenuated vaccine. Therefore, we assessed whether killed γBCG has similar potentiating effects. In an in vitro model of trained immunity, human monocytes were incubated with γBCG for 24 h and restimulated after 6 d. Cytokine production and the role of pattern recognition receptors and histone methylation markers were assessed. The in vivo effects of γBCG vaccination were studied in a proof-of-principle trial in 15 healthy volunteers. γBCG induced trained immunity in vitro via the NOD2 receptor pathway and up-regulation of H3K4me3 histone methylation. However, these effects were less strong than those induced by live BCG. γBCG vaccination in volunteers had only minimal effects on innate immunity, whereas a significant increase in heterologous Th1/Th17 immunity was observed. Our results indicate that γBCG induces long-term training of innate immunity in vitro. In vivo, γBCG induces mainly heterologous effects on the adaptive-immune system, whereas effects on innate cytokine production are limited.