Impact of peptide:HLA complex stability for the identification of SARS-CoV-2-specific CD8⁺T cells.

Induction of a lasting protective immune response is dependent on presentation of epitopes to patrolling T cells through the HLA complex. While peptide:HLA (pHLA) complex affinity alone is widely exploited for epitope selection, we demonstrate that including the pHLA complex stability as a selection parameter can significantly reduce the high false discovery rate observed with predicted affinity. In this study, pHLA complex stability was measured on three common class I alleles and 1286 overlapping 9-mer peptides derived from the SARS-CoV-2 Spike protein. Peptides were pooled based on measured stability and predicted affinity. Strikingly, stability of the pHLA complex was shown to strongly select for immunogenic epitopes able to activate functional CD8+T cells. This result was observed across the three studied alleles and in both vaccinated and convalescent COVID-19 donors. Deconvolution of peptide pools showed that specific CD8+T cells recognized one or two dominant epitopes. Moreover, SARS-CoV-2 specific CD8+T cells were detected by tetramer-staining across multiple donors. In conclusion, we show that stability analysis of pHLA is a key factor for identifying immunogenic epitopes

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Identification of Viral and Cancer epitopes using peptide:MHC Stability measurements

The socio-economic threat from pandemics, the most recent example being SARS-CoV-2, as well as the high incidence of cancer in developed countries emphasizes the necessity of having effective anti-viral and anti-cancer therapies. Immune-targeting therapeutics, such as anti-viral vaccines and cancer immunotherapies, are important tools in this aspect. A commonly used way of identifying targets for vaccines or immunotherapy is to assess the affinity of the epitope:HLA complex. The premise being that epitopes with a high binding affinity for HLA molecules are immunogenic. Studies have however showed that the stability of the epitope:HLA complex is a stronger indicator of immunogenicity than affinity alone. The aim of this thesis was to 1) investigate the impact of epitope:HLA complex stability on the immunogenicity of SARS-CoV-2 epitopes and 2) utilize this knowledge on stability and immunogenicity to select an epitope for TCR-like antibodies as a next generation cancer immunotherapy.Here we show that accounting for the stability of epitope:HLA complexes enriches the identification of immunogenic epitopes. This was evident by the induction of strong CD8+ T cell activation in response to stimulation with high stability peptides. These responses were observed for four different HLA-alleles in both SARS-CoV-2 vaccinated and convalescent donors. Deconvolution of the response revealed that the peptides with the highest stability and affinity to A*0101, A*0201 and A*0301 were inducing the response. This was confirmed by tetramer staining of antigen specific CD8+ T cells.The impact of stability in the process of epitope discovery was further elucidated by identifying a novel epitope derived from the cancer testis antigen PRAME. This HLA-A*0201-binding PRAME epitope was discovered using complex-stability assay and the immunogenicity of the epitope was demonstrated by expansion of epitope-specific CD8+ T cells. TCR-LAs were identified using yeast and phage display and generated as scFv-Fc fusion IgGs. The TCR-LAs displayed high affinity and binding specificity to epitope:HLA-A*0201 complex and were shown to induce NK cell-mediated cytotoxicity of HLA-A2+ tumor cells presenting the target epitope.Together, the data presented in this thesis demonstrates that the combination of HLA- affinity and HLA-stability measurements is an effective way of identifying immunogenic epitopes. This strategy can be employed both to viral and cancer antigens and represents a robust tool to identify novel epitopes for immune-targeting therapeutics

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field