Driving consistency: CEPI-Centralized Laboratory Network's conversion factor initiative for SARS-CoV-2 clinical assays used for efficacy assessment of COVID vaccines.

To date, thousands of SARS-CoV-2 samples from many vaccine developers have been tested within the CEPI-Centralized Laboratory Network. To convert data from each clinical assay to international standard units, the WHO international standard and the CEPI standard generated by the Medicines and Healthcare products Regulatory Agency were run in multiple facilities to determine the conversion factor for each assay. Reporting results in international units advances global understanding of SARS-CoV-2 immunity and vaccine efficacy, enhancing the quality, reliability, and utility of clinical assay data.

Standardised quantitative assays for anti-SARS-CoV-2 immune response used in vaccine clinical trials by the CEPI Centralized Laboratory Network: a qualification analysis.

BACKGROUND: Accurate quantitation of immune markers is crucial for ensuring reliable assessment of vaccine efficacy against infectious diseases. This study was designed to confirm standardised performance of SARS-CoV-2 assays used to evaluate COVID-19 vaccine candidates at the initial seven laboratories (in North America, Europe, and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN). METHODS: Three ELISAs (pre-spike protein, receptor binding domain, and nucleocapsid), a microneutralisation assay (MNA), a pseudotyped virus-based neutralisation assay (PNA), and an IFN-γ T-cell ELISpot assay were developed, validated or qualified, and transferred to participating laboratories. Immune responses were measured in ELISA laboratory units (ELU) for ELISA, 50% neuralisation dilution (ND50) for MNA, 50% neutralisation titre (NT50) for PNA, and spot-forming units for the ELISpot assay. Replicate assay results of well characterised panels and controls of blood samples from individuals with or without SARS-CoV-2 infection were evaluated by geometric mean ratios, standard deviation, linear regression, and Spearman correlation analysis for consistency, accuracy, and linearity of quantitative measurements across all laboratories. FINDINGS: High reproducibility of results across all laboratories was demonstrated, with interlaboratory precision of 4·1-7·7% coefficient of variation for all three ELISAs, 3·8-19·5% for PNA, and 17·1-24·1% for MNA, over a linear range of 11-30 760 ELU per mL for the three ELISAs, 14-7876 NT50 per mL for PNA, and 21-25 587 ND50 per mL for MNA. The MNA was also adapted for detection of neutralising antibodies against the major SARS-CoV-2 variants of concern. The results of PNA and MNA (r=0·864) and of ELISA and PNA (r=0·928) were highly correlated. The IFN-γ ELISpot interlaboratory variability was 15·9-49·9% coefficient of variation. Sensitivity and specificity were close to 100% for all assays. INTERPRETATION: The CEPI CLN provides accurate quantitation of anti-SARS-CoV-2 immune response across laboratories to allow direct comparisons of different vaccine formulations in different geographical areas. Lessons learned from this programme will serve as a model for faster responses to future pandemic threats and roll-out of effective vaccines. FUNDING: CEPI.

Impacts of Eccentric Resistance Exercise on DNA Methylation of Candidate Genes for Inflammatory Cytokines in Skeletal Muscle and Leukocytes of Healthy Males.

Physical inactivity and a poor diet increase systemic inflammation, while chronic inflammation can be reduced through exercise and nutritional interventions. The mechanisms underlying the impacts of lifestyle interventions on inflammation remain to be fully explained; however, epigenetic modifications may be critical. The purpose of our study was to investigate the impacts of eccentric resistance exercise and fatty acid supplementation on DNA methylation and mRNA expression of TNF and IL6 in skeletal muscle and leukocytes. Eight non-resistance exercise-trained males completed three bouts of isokinetic eccentric contractions of the knee extensors. The first bout occurred at baseline, the second occurred following a three-week supplementation of either omega-3 polyunsaturated fatty acid or extra virgin olive oil and the final bout occurred after eight-weeks of eccentric resistance training and supplementation. Acute exercise decreased skeletal muscle TNF DNA methylation by 5% (p = 0.031), whereas IL6 DNA methylation increased by 3% (p = 0.01). Leukocyte DNA methylation was unchanged following exercise (p > 0.05); however, three hours post-exercise the TNF DNA methylation decreased by 2% (p = 0.004). In skeletal muscle, increased TNF and IL6 mRNA expression levels were identified immediately post-exercise (p < 0.027); however, the leukocyte mRNA expression was unchanged. Associations between DNA methylation and markers of exercise performance, inflammation and muscle damage were identified (p < 0.05). Acute eccentric resistance exercise is sufficient to induce tissue-specific DNA methylation modifications to TNF and IL6; however, neither eccentric training nor supplementation was sufficient to further modify the DNA methylation.

DNA methylation of tumour necrosis factor (TNF) alpha gene is associated with specific blood fatty acid levels in a gender-specific manner.

BACKGROUND: Fatty acids, specifically polyunsaturated fatty acids (PUFAs) play an important role in inflammation and its resolution, however, their interaction with the epigenome is relatively unexplored. Here we investigate the relationship between circulating blood fatty acids and the DNA methylation of the cytokine encoding gene tumour necrosis factor (TNF, OMIM 191160). METHODS: Using a cross-sectional study approach, we collected blood samples from adults (N=88 (30 males, 58 females); 18-74 years old) for DNA methylation pyrosequencing analysis at four sites in TNF exon 1 and gas-chromatography mass-spectrometry analysis of the fatty acid profile of dried blood spots (DBS). RESULTS: Methylation levels of TNF exon 1 are significantly correlated with specific fatty acids in a gender-specific manner. In the males the PUFAs Docosahexaenoic Acid (DHA) and Arachidonic Acid (AA) were positively associated with TNF methylation, as was the saturated fatty acid (SFA) Stearic Acid; in contrast, mono-unsaturated fatty acids (MUFAs) had a negative association. In the females, omega-6 PUFA γ-Linolenic acid (GLA) was negatively correlated with TNF methylation; Adrenic acid and Eicosadienoic Acid were positively correlated with TNF methylation. CONCLUSION: These results suggest that one way that fatty acids interact with the inflammation is through altered methylation profiles of cytokine genes; thus, providing potential therapeutic targets for nutritional and health interventions.

Health-related motivational and behavioral processes associated with DNA methylation of the TNF gene.

OBJECTIVE: Epigenetics has been described as one of the most exciting areas of contemporary biology, and research has begun to explore whether epigenetic modifications are influenced by psychological processes. The present research explored the associations of health-related motivation and behavior with the DNA methylation of tumor necrosis factor (TNF) gene. METHOD: Participants (N = 88) completed questionnaires examining engagement with health-related behavior (i.e., physical activity, diet, and smoking) and health-related motivation from the perspective of self-determination theory. They also provided a capillary blood sample for DNA extraction and analysis of four CpG sites via bisulfite conversion within Exon 1 of TNF. RESULTS: Health-related autonomous motivation was weakly but positively associated with TNF methylation (ß = .18, p = .08). Indirect effects were identified in a subsequent step; autonomous motivation was positively associated with fruit consumption (ß = .29, p = .004), negatively associated with smoking (ß = -.22, p = .03), but not associated with physical activity (ß = .10, p = .34). Moreover, TNF methylation was positively associated with lifetime physical activity (ß = .18, p = .08) and negatively associated with smoking (ß = -.23, p = .03). Direct effects of autonomous motivation on DNA methylation did not persist when these indirect effects were included (ß = .09, p = .43). CONCLUSIONS: Results support the idea that autonomous motivation is associated with DNA methylation, albeit indirectly through tobacco consumption. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Characterization of extracellular redox enzyme concentrations in response to exercise in humans.

Redox enzymes modulate intracellular redox balance and are secreted in response to cellular oxidative stress, potentially modulating systemic inflammation. Both aerobic and resistance exercise are known to cause acute systemic oxidative stress and inflammation; however, how redox enzyme concentrations alter in extracellular fluids following bouts of either type of exercise is unknown. Recreationally active men (n = 26, mean ± SD: age 28 ± 8 yr) took part in either: 1) two separate energy-matched cycling bouts: one of moderate intensity (MOD) and a bout of high intensity interval exercise (HIIE) or 2) an eccentric-based resistance exercise protocol (RES). Alterations in plasma (study 1) and serum (study 2) peroxiredoxin (PRDX)-2, PRDX-4, superoxide dismutase-3 (SOD3), thioredoxin (TRX-1), TRX-reductase and interleukin (IL)-6 were assessed before and at various timepoints after exercise. There was a significant increase in SOD3 (+1.5 ng/mL) and PRDX-4 (+5.9 ng/mL) concentration following HIIE only, peaking at 30- and 60-min post-exercise respectively. TRX-R decreased immediately and 60 min following HIIE (-7.3 ng/mL) and MOD (-8.6 ng/mL), respectively. In non-resistance trained men, no significant changes in redox enzyme concentrations were observed up to 48 h following RES, despite significant muscle damage. IL-6 concentration increased in response to all trials, however there was no significant relationship between absolute or exercise-induced changes in redox enzyme concentrations. These results collectively suggest that HIIE, but not MOD or RES increase the extracellular concentration of PRDX-4 and SOD3. Exercise-induced changes in redox enzyme concentrations do not appear to directly relate to systemic changes in IL-6 concentration.NEW & NOTEWORTHY Two studies were conducted to characterize changes in redox enzyme concentrations after single bouts of exercise to investigate the emerging association between extracellular redox enzymes and inflammation. We provide evidence that SOD3 and PRDX-4 concentration increased following high-intensity aerobic but not eccentric-based resistance exercise. Changes were not associated with IL-6. The results provide a platform to investigate the utility of SOD3 and PRDX-4 as biomarkers of oxidative stress following exercise.

Impact of aerobic exercise and fatty acid supplementation on global and gene-specific DNA methylation.

Lifestyle interventions, including exercise and dietary supplementation, can modify DNA methylation and exert health benefits; however, the underlying mechanisms are poorly understood. Here we investigated the impact of acute aerobic exercise and the supplementation of omega-3 polyunsaturated fatty acids (n-3 PUFA) and extra virgin olive oil (EVOO) on global and gene-specific (PPARGC1A, IL6 and TNF) DNA methylation, and DNMT mRNA expression in leukocytes of disease-free individuals. Eight trained male cyclists completed an exercise test before and after a four-week supplementation of n-3 PUFA and EVOO in a double-blind, randomised, repeated measures design. Exercise triggered global hypomethylation (Pre 79.2%; Post 78.7%; p = 0.008), alongside, hypomethylation (Pre 6.9%; Post 6.3%; p < 0.001) and increased mRNA expression of PPARGC1A (p < 0.001). Associations between PPARGC1A methylation and exercise performance were also detected. An interaction between supplement and trial was detected for a single CpG of IL6 indicating increased DNA methylation following n-3 PUFA and decreased methylation following EVOO (p = 0.038). Global and gene-specific DNA methylation associated with markers of inflammation and oxidative stress. The supplementation of EVOO reduced DNMT1 mRNA expression compared to n-3 PUFA supplementation (p = 0.048), whereas, DNMT3a (p = 0.018) and DNMT3b (p = 0.046) mRNA expression were decreased following exercise. In conclusion, we demonstrate that acute exercise and dietary supplementation of n-3 PUFAs and EVOO induce DNA methylation changes in leukocytes, potentially via the modulation of DNMT mRNA expression. Future studies are required to further elucidate the impact of lifestyle interventions on DNA methylation.