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In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4+ T cells from the blood of a healthy donor. CD4+ T cells strongly recognized MAGE-3281–295 and, to a lesser extent, MAGE-3141–155 and MAGE-3146–160. Moreover, CD4+ T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4+ T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3–positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4+ T cells recognized MAGE-3281–295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4+ T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4+ T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4+ T cell epitopes.

This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the selected CYP isoforms.

Background and ObjectiveActivity of human cytochrome P450 enzymes (CYPs) shows high inter-and intra-individual variability, which is determined by genetic and non-genetic factors. Using a combination of CYP-specific probe drugs, phenotyping cocktails allow simultaneous assessment of the activity of different CYP isoforms. The objective of this study was to characterize the phenotyping metrics of the Basel cocktail in healthy male subjects with induced and inhibited CYP activity.MethodsIn a randomized crossover study, the probe drugs for simultaneous phenotyping of CYP1A2 (caffeine), CYP2B6 (efavirenz), CYP2C9 (losartan), 2C19 (omeprazole), CYP2D6 (metoprolol), and CYP3A4 (midazolam) were administered to 16 subjects without pretreatment (baseline), after pretreatment with a combination of CYP inhibitors (ciprofloxacin, ketoconazole, and paroxetine), and after CYP induction with rifampicin. All subjects were genotyped. Pharmacokinetic profiles of the probe drugs and their main metabolites and metabolic ratios 2, 4, 6, and 8 h after probe drug application were determined in plasma and compared with the corresponding area under the plasma concentration-time curve (AUC) ratios.ResultsThe Basel phenotyping cocktail was well tolerated by all subjects independent of pretreatment. Good correlations of metabolic ratios with AUC ratios of the corresponding probe drugs and their metabolites for all three conditions (baseline, CYP inhibition, and CYP induction) were found at 2 h after probe drug administration for CYP3A4, at 4 h for CYP1A2 and CYP2C19, and at 6 h for CYP2B6 and CYP2D6. While CYP inhibition significantly changed AUC ratios and metabolic ratios at these time points for all six CYP isoforms, CYP induction did not significantly change AUC ratios for CYP2C9. For CYP3A4, total 1′-hydroxymidazolam concentrations after pretreatment of samples with β-glucuronidase were needed to obtain adequate reflection of CYP induction by the metabolic ratio.ConclusionsInhibition of CYP activity can be detected with the Basel phenotyping cocktail for all six tested CYP isoforms at the proposed time points. The AUC ratio of losartan:losartan carboxylic acid in plasma does not seem suitable to detect induction of CYP2C9. The observed metabolic ratios for inhibited and induced CYP activity need to be confirmed for extensive metabolizers, and typical ratios for subjects with genetically altered CYP activity will need to be established in subsequent studies.ClinicalTrials.gov-ID: NCT01386593.Electronic supplementary materialThe online version of this article (doi:10.1007/s40262-015-0294-y) contains supplementary material, which is available to authorized users.

Genomic sequencing is essential to track the evolution and spread of SARS-CoV-2, optimize molecular tests, treatments, vaccines, and guide public health responses. To investigate the global SARS-CoV-2 genomic surveillance, we used sequences shared via GISAID to estimate the impact of sequencing intensity and turnaround times on variant detection in 189 countries. In the first two years of the pandemic, 78% of high-income countries sequenced >0.5% of their COVID-19 cases, while 42% of low- and middle-income countries reached that mark. Around 25% of the genomes from high income countries were submitted within 21 days, a pattern observed in 5% of the genomes from low- and middle-income countries. We found that sequencing around 0.5% of the cases, with a turnaround time <21 days, could provide a benchmark for SARS-CoV-2 genomic surveillance. Socioeconomic inequalities undermine the global pandemic preparedness, and efforts must be made to support low- and middle-income countries improve their local sequencing capacity.