MhcVizPipe: A Quality Control Software for Rapid Assessment of Small- to Large-Scale Immunopeptidome Datasets.

MS-based immunopeptidomics is maturing into an automatized and high-throughput technology, producing small- to large-scale datasets of clinically relevant major histocompatibility complex (MHC) class I-associated and class II-associated peptides. Consequently, the development of quality control (QC) and quality assurance systems capable of detecting sample and/or measurement issues is important for instrument operators and scientists in charge of downstream data interpretation. Here, we created MhcVizPipe (MVP), a semiautomated QC software tool that enables rapid and simultaneous assessment of multiple MHC class I and II immunopeptidomic datasets generated by MS, including datasets generated from large sample cohorts. In essence, MVP provides a rapid and consolidated view of sample quality, composition, and MHC specificity to greatly accelerate the "pass-fail" QC decision-making process toward data interpretation. MVP parallelizes the use of well-established immunopeptidomic algorithms (NetMHCpan, NetMHCIIpan, and GibbsCluster) and rapidly generates organized and easy-to-understand reports in HTML format. The reports are fully portable and can be viewed on any computer with a modern web browser. MVP is intuitive to use and will find utility in any specialized immunopeptidomic laboratory and proteomics core facility that provides immunopeptidomic services to the community.

Molecular Characterization of Uropathogenic Escherichia coli Reveals Emergence of Drug Resistant O15, O22 and O25 Serogroups.

Background and Objectives: Uropathogenic Escherichia coli (UPEC) are common pathogens causing urinary tract infections (UTIs). We aimed to investigate the relationship among clinical manifestation, serogroups, phylogenetic groups, and antimicrobial resistance among UPEC. Materials and Methods: One-hundred Escherichia coli isolates recovered from urine and ureteral scrapings were used for the study. The prevalence of antimicrobial resistance was determined by using European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. E. coli serogroups associated with UTI, as well as phylogenetic diversity were analyzed using multiplex PCR reactions. Results: Eighty-seven strains (87%) were isolated from females, while 13 (13%) from males. A high frequency of resistance to cephalosporins (43%) and fluoroquinolones (31%) was observed. Among UTI-associated serogroups O15 (32.8%), O22 (23.4%), and O25 (15.6%) were dominant and demonstrated elevated resistance rates. The E. coli phylogenetic group B2 was most common. These observations extended to pregnant patients with asymptomatic bacteriuria. Conclusions: Due to high rates of resistance, strategies using empirical therapy of second-generation cephalosporins and fluoroquinolones should be reconsidered in this population.

The mutational landscape of SARS-CoV-2 variants diversifies T cell targets in an HLA-supertype-dependent manner.

The rapid, global dispersion of SARS-CoV-2 has led to the emergence of a diverse range of variants. Here, we describe how the mutational landscape of SARS-CoV-2 has shaped HLA-restricted T cell immunity at the population level during the first year of the pandemic. We analyzed a total of 330,246 high-quality SARS-CoV-2 genome assemblies, sampled across 143 countries and all major continents from December 2019 to December 2020 before mass vaccination or the rise of the Delta variant. We observed that proline residues are preferentially removed from the proteome of prevalent mutants, leading to a predicted global loss of SARS-CoV-2 T cell epitopes in individuals expressing HLA-B alleles of the B7 supertype family; this is largely driven by a dominant C-to-U mutation type at the RNA level. These results indicate that B7-supertype-associated epitopes, including the most immunodominant ones, were more likely to escape CD8+ T cell immunosurveillance during the first year of the pandemic.

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis.

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

RHybridFinder: An R package to process immunopeptidomic data for putative hybrid peptide discovery.

Identification of proteasomal spliced peptides (PSPs) by mass spectrometry (MS) is not possible with traditional search engines. Here, we provide a protocol for running RHybridFinder (RHF), an R package for the computational inference of putative PSPs detected by MS. RHF extracts high confidence scored de novo sequenced peptides identified by PEAKS software. Those peptides are then matched to protein databases to infer cis- or trans-spliced major histocompatibility complex (MHC)-associated peptides. RHF is relatively fast and straightforward. PSPs have to be validated experimentally. For complete details on the use and execution of the original protocol, please refer to Faridi et al. (2018).