Human Leukocyte Antigen Molecules Research Articles

The immunogenetics of COVID-19.

The worldwide coronavirus disease 2019 pandemic was sparked by the severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2) that first surfaced in December 2019 (COVID-19). The effects of COVID-19 differ substantially not just between patients individually but also between populations with different ancestries. In humans, the human leukocyte antigen (HLA) system coordinates immune regulation. Since HLA molecules are a major component of antigen-presenting pathway, they play an important role in determining susceptibility to infectious disease. It is likely that differential susceptibility to SARS-CoV-2 infection and/or disease course in COVID-19 in different individuals could be influenced by the variations in the HLA genes which are associated with various immune responses to SARS-CoV-2. A growing number of studies have identified a connection between HLA variation and diverse COVID-19 outcomes. Here, we review research investigating the impact of HLA on individual responses to SARS-CoV-2 infection and/or progression, also discussing the significance of MHC-related immunological patterns and its use in vaccine design.

Rank Wise Effect of HLA-DQ5 Explains Risk for the Development of Anti-IgLON5 Disease

Objective To better characterize the genetic association between human leukocyte antigen (HLA) and anti-IgLON5 disease and to explore auto-antigen binding to associated HLA molecules and their functional involvement in pathophysiology. Background Anti-IgLON5 disease is a rare, but likely underdiagnosed type of autoantibody encephalitis with a heterogeneous clinical phenotype, including sleep, movement and brainstem dysfunction. Its pathophysiology remains elusive, although dominant association with HLA-DRB1*10:01-DQB1*05:01 strongly supports an autoimmune basis. Design/Methods A multicentric cohort of 62 patients and 433 controls matched by principal component analysis was included. Genome-wide association analysis was performed with 4-digit resolution HLA imputation and selected 8-digit resolution validation typing. A generalized logistic model was used to determine the association with individual alleles and haplotype counts to establish haplotype associations. Furthermore, we computationally predicted binding of IgLON5-derived peptides to risk-associated HLA-molecules. Results Our results indicate a rank wise effect of HLA-DQA1*01:05∼DQB1*05:01 (heterozygotes: OR 46.6), HLA-DQA1*01:01∼DQB1*05:01 (homozygotes: OR 26.9; heterozygotes: OR 2.5) and HLA-DQA1*01:04-∼DQB1*05:03 (homozygotes: OR 30.9; heterozygotes: OR 5.6), in order of descending relative risk predisposition. Differences between encoded heterodimers are minimal (a few amino acids outside the main HLA sequence binding region), suggesting a common function. Computational binding predictions support similar, high binding affinity for IgLON5275-283 in a post-translationally modified (N-deglycosylated) form by all three of these HLA-DQ molecules and other common binders. In contrast, association analysis suggests that effects of HLA-DR are likely explained by linkage disequilibrium. Conclusions This study is the, so far, largest genetic study on anti-IgLON5 disease. Our results strongly suggest HLA-DQ over HLA-DR association, with higher reactivity against post-translationally modified versus physiological peptides, in line with reduced T cell priming against these epitopes. Further studies should address the functional implications of these HLA associations.

Association Between Human Leukocyte Antigens and Graft-Versus-Host Disease Occurrence in Allogeneic Hematopoietic Stem Cell Transplantation - A 10-Year Experience on Iranian Patients.

Human leukocyte antigen (HLA) molecules mediate critical roles in determining responsiveness or non-responsiveness of the immune system, especially in transplantation. Some studies have shown a possible association between certain HLA alleles and some allogeneic hematopoietic stem cell transplantation (allo-HSCT) outcomes such as acute/chronic graft-versus-host disease (aGVHD/cGVHD) and overall survival (OS). In the current study, we investigated any possible association of HLA subclasses and acute/chronic GVHD occurrence as well as OS in patients receiving HLA-matched sibling allo-HSCT. We retrospectively evaluated the association of various HLA alleles with the incidence of aGVHD, cGVHD, and OS of 162 patients who received allo-HSCT from HLA-matched sibling between 2009-2018 at Taleghani hospital in Tehran. We found that the incidence of aGVHD grades II-IV was higher among patients who had HLA-B*07 (P=0.031) and HLA-DRB1*07 (P=0.052). The presence of HLA-A*01 was associated with 4.5-fold greater odds of incidence in the extensive-type of cGVHD (P=0.009). Furthermore, HLA-A*03 (P=0.089), HLA-B*13(P=0.013), HLA-B*40 (P=0.042), HLA-DRB1*02 (P=0.074), and HLA-DRB1*04 (P=0.039) were associated with a lower rate of OS. This study suggests that certain HLA alleles might influence the incidence and severity of acute or chronic GVHD in the context of HLA-matched sibling allo-HSCT. In addition, some specific HLA alleles help predict OS in allo-HSCT recipients. These results might be helpful in estimating the incidence of aGVHD, cGVHD, and OS as well as designing personalized therapy.

Human T cells recognize HLA-DP–bound peptides in two orientations

Human leukocyte antigen (HLA) molecules present small peptide antigens to T cells, thereby allowing them to recognize pathogen-infected and cancer cells. A central dogma over the last 50+ y is that peptide binding to HLA molecules is mediated by the docking of side chains of particular amino acids in the peptide into pockets in the HLA molecules in a conserved N- to C-terminal orientation. Whether peptides can be presented in a reversed C- to N-terminal orientation remains unclear. Here, we performed large-scale identification of peptides bound to HLA-DP molecules and observed that in addition to peptide binding in an N- to C-terminal orientation, in 9 out of 14 HLA-DP allotypes, reverse motifs are found, compatible with C- to N-terminal peptide binding. Moreover, we isolated high-avidity human cytomegalovirus (CMV)-specific HLA-DP-restricted CD4+ T cells from the memory repertoire of healthy donors and demonstrate that such T cells recognized CMV-derived peptides bound to HLA-DPB1*01:01 or *05:01 in a reverse C- to N-terminal manner. Finally, we obtained a high-resolution HLA-DPB1*01:01-CMVpp65(142-158) peptide crystal structure, which is the molecular basis for C- to N-terminal peptide binding to HLA-DP. Our results point to unique features of HLA-DP molecules that substantially broaden the HLA class II bound peptide repertoire to combat pathogens and eliminate cancer cells.

IMMU-16. SOMATIC MUTATIONS IN HUMAN LEUKOCYTE ANTIGEN GENES AND ANTIGEN PRESENTATION PATHWAY GENES IN MALIGNANT GLIOMA

Abstract Immunotherapeutic approaches in cancer aim to booster T cell mediated immune responses. Tumor-specific T cells recognize target cells via human leukocyte antigen (HLA) molecules before cell specific lysis is initiated. Malignant gliomas are known for their immunoevasive and immunosuppressive properties which impede effective tumor-specific immune responses, including clinical responses to immune checkpoint inhibition. Our aim was to investigate whether somatic mutation in HLA genes and antigen presentation pathway genes are present in malignant gliomas. METHODS We performed next generation sequencing in 138 patients, including matched tumor and blood samples from 100 IDHwt glioblastomas, 25 IDHmut astrocytomas and 13 IDHmut oligodendrogliomas. Sequencing was performed on tumor and peripheral blood for HLA class I (HLA-A, -B, -C), HLA class II (HLA-DR, -DP, -DQ), non-classical HLA molecules (HLA-E, -F, -G, -H), MICA/B, TAP1/2 and beta2 microglobulin (B2M). Identified calls were validated using targeted nanopore sequencing. RESULTS In total, we identified 28 mutations based on Illumina sequencing. Mutations were located in HLA-B (2 mutations), HLA-C (1), B2M (2), HLA-DQA1 (4), HLA-DQB1 (4), HLA-DPB1 (1), HLA-E (1), HLA-F (2), HLA-G (3), HLA-H (1), MICA (3), MICB (2), TAP1 (1) and TAP2 (1). No mutations were found in HLA-A and HLA-DRB1. A frequency cutoff of 5% was applied. Additional mutations (n = 46) below this cutoff were identified. The mutations were found in 30 patients (21.7%). Some patients harbored up to 7-8 mutations in multiple HLA and antigen presenting pathway genes. Most mutations were found in the MICA (MHC class I polypeptide–related sequence A) gene locus, which is engaged by the NKG2D ligand which is broadly expressed on NK cells, gd and ab T cells. CONCLUSION Our study demonstrates that somatic mutations in HLA and antigen presentation pathway genes are frequently present in malignant gliomas.

Restriction of interleukin-6 alters endothelial cell immunogenicity in an allogenic environment.

The microvascular endothelium of the renal transplant is the first site of graft interaction with the host immune system and is often injured in chronic Antibody Mediated Rejection (AMR). Microvascular inflammation is an independent determinant of AMR and heightens endothelial expression of human leukocyte antigen (HLA) molecules thereby increasing the possibility of Donor Specific Antibody (DSA) binding. Endothelial cells (ECs) produce IL-6 in the steady-state that is increased by inflammation or by HLA-DR antibody binding in an allogeneic setting. Because IL-6 has been implicated in AMR, IL-6 blockade is currently under investigation as a therapeutic target. To further understand the role of IL-6 in EC immunogenicity, we have examined whether humanized antibody blockade of IL-6 altered EC interactions with allogeneic PBMC and after anti-HLA or DSA binding to ECs in an in vitro human experimental model. Soluble factors, endothelial phenotype, Stat-3 activation, CD4+ -T differentiation and C4d deposition were examined. Blockade of IL-6 reduced EC secretion of IL-6 and of the monocyte chemoattractant MCP-1. Pre-activation of ECs by anti-HLA or DSA binding increased IL-6 secretion, that was further increased by concurrent binding of both antibodies and this was inhibited by IL-6 blockade. Activation of Stat-3 in CD4+ -T mediated by soluble factors produced in endothelial-PBMC interactions, and endothelial differentiation of CD4+ -T cell subsets (Th1, Treg), were impaired whereas activation of Complement by anti-HLA antibody binding remained unchanged by IL-6 blockade. Together, these data identify EC-mediated pro-inflammatory responses (T cell expansion, EC auto-activation, chemokine secretion) targeted by IL-6 blockade.

Benchmarking freely available HLA typing algorithms across varying genes, coverages and typing resolutions.

Identifying the specific human leukocyte antigen (HLA) allele combination of an individual is crucial in organ donation, risk assessment of autoimmune and infectious diseases and cancer immunotherapy. However, due to the high genetic polymorphism in this region, HLA typing requires specialized methods. We investigated the performance of five next-generation sequencing (NGS) based HLA typing tools with a non-restricted license namely HLA*LA, Optitype, HISAT-genotype, Kourami and STC-Seq. This evaluation was done for the five HLA loci, HLA-A, -B, -C, -DRB1 and -DQB1 using whole-exome sequencing (WES) samples from 829 individuals. The robustness of the tools to lower depth of coverage (DOC) was evaluated by subsampling and HLA typing 230 WES samples at DOC ranging from 1X to 100X. The HLA typing accuracy was measured across four typing resolutions. Among these, we present two clinically-relevant typing resolutions (P group and pseudo-sequence), which specifically focus on the peptide binding region. On average, across the five HLA loci examined, HLA*LA was found to have the highest typing accuracy. For the individual loci, HLA-A, -B and -C, Optitype's typing accuracy was the highest and HLA*LA had the highest typing accuracy for HLA-DRB1 and -DQB1. The tools' robustness to lower DOC data varied widely and further depended on the specific HLA locus. For all Class I loci, Optitype had a typing accuracy above 95% (according to the modification of the amino acids in the functionally relevant portion of the HLA molecule) at 50X, but increasing the DOC beyond even 100X could still improve the typing accuracy of HISAT-genotype, Kourami, and STC-seq across all five HLA loci as well as HLA*LA's typing accuracy for HLA-DQB1. HLA typing is also used in studies of ancient DNA (aDNA), which is often based on sequencing data with lower quality and DOC. Interestingly, we found that Optitype's typing accuracy is not notably impaired by short read length or by DNA damage, which is typical of aDNA, as long as the DOC is sufficiently high.

Identification of neoantigens in oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) has a relatively poor long-term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumour mutations, presented to cytotoxic T-cells by human leucocyte antigen (HLA) molecules. Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumours from a cohort of seven patients with OAC. We directly identified one HLA-I presented neoantigen from one patient, and report functional T-cell responses from a predicted HLA-II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T-cell responses, emphasizing the need for improved strategies for neoantigen identification.

Occupational health effect of TCE exposure: Experiment evidence of gene-environment interaction in hypersensitivity reaction

BackgroundRecently, Trichloroethylene (TCE) induced TCE hypersensitivity syndrome (THS) has attracted the attention of many researchers in the field of environmental and occupational health. Studies have revealed that Human leukocyte antigen (HLA) polymorphisms were the important genetic determinants of the diseases, but the potential molecular mechanism remains unclear. ObjectiveThis study aimed to investigate the association between THS and HLA at the molecular level. MethodWe chose the human B-lymphoblastoid cell line Hmy2.C1R transfected with cDNA of HLA-B*13:01 and HLA-B*13:02 to analyze the characteristics of HLA-B-binding peptides and investigate the effect of TCE on the binding affinity of peptides to the HLA-B molecules. Further, the mathematical model was used to identify the possible interaction between TCE and HLA-B*13:01 or HLA-B*13:02 molecule. Results54 HLA-B*13:01-binding peptides and 85 HLA-B*13:02-binding peptides were identified. Comparing the protein sequences of HLA-B*13:01 and HLA-B*13:02, amino acids were different at positions 94, 95 and 97. The results of the binding affinity of self-peptides to HLA molecules in the presence of TCE showed that TCE significantly decreased the binding affinity of peptides to HLA-B*13:01 only, but did not affect that of HLA-B*13:02. Molecular docking model showed that there was a unique high-affinity binding mode between TCE and HLA-B*13:01 (but not HLA-B*13:02), and the binding site located in the region of F pocket, suggesting that the unique structure of the F pocket of HLA-B*13:01 might provide the possibility of binding TCE. The pathogenesis of interaction between HLA-B*13:01 and TCE might belong to the model of the alteration of the HLA-presented self-peptide repertoire. DiscussionThis study explored the molecular mechanism of the association between THS and HLA-B*13:01, and had important implications for understanding the role of gene-environment interaction in the development of complex environment-related diseases.

In silico Immunogenicity Assessment for Sequences Containing Unnatural Amino Acids: A Method Using Existing in silico Algorithm Infrastructure and a Vision for Future Enhancements.

The in silico prediction of T cell epitopes within any peptide or biologic drug candidate serves as an important first step for assessing immunogenicity. T cell epitopes bind human leukocyte antigen (HLA) by a well-characterized interaction of amino acid side chains and pockets in the HLA molecule binding groove. Immunoinformatics tools, such as the EpiMatrix algorithm, have been developed to screen natural amino acid sequences for peptides that will bind HLA. In addition to commonly occurring in synthetic peptide impurities, unnatural amino acids (UAA) are also often incorporated into novel peptide therapeutics to improve properties of the drug product. To date, the HLA binding properties of peptides containing UAA are not accurately estimated by most algorithms. Both scenarios warrant the need for enhanced predictive tools. The authors developed an in silico method for modeling the impact of a given UAA on a peptide's likelihood of binding to HLA and, by extension, its immunogenic potential. In silico assessment of immunogenic potential allows for risk-based selection of best candidate peptides in further confirmatory in vitro, ex vivo and in vivo assays, thereby reducing the overall cost of immunogenicity evaluation. Examples demonstrating in silico immunogenicity prediction for product impurities that are commonly found in formulations of the generic peptides teriparatide and semaglutide are provided. Next, this article discusses how HLA binding studies can be used to estimate the binding potentials of commonly encountered UAA and "correct" in silico estimates of binding based on their naturally occurring counterparts. As demonstrated here, these in vitro binding studies are usually performed with known ligands which have been modified to contain UAA in HLA anchor positions. An example using D-amino acids in relative binding position 1 (P1) of the PADRE peptide is presented. As more HLA binding data become available, new predictive models allowing for the direct estimation of HLA binding for peptides containing UAA can be established.

Rare variants and HLA haplotypes associated in patients with neuromyelitis optica spectrum disorders.

Neuromyelitis optica spectrum disorders (NMOSD) are rare, debilitating autoimmune diseases of the central nervous system. Many NMOSD patients have antibodies to Aquaporin-4 (AQP4). Prior studies show associations of NMOSD with individual Human Leukocyte Antigen (HLA) alleles and with mutations in the complement pathway and potassium channels. HLA allele associations with NMOSD are inconsistent between populations, suggesting complex relationships between the identified alleles and risk of disease. We used a retrospective case-control approach to identify contributing genetic variants in patients who met the diagnostic criteria for NMOSD and their unaffected family members. Potentially deleterious variants identified in NMOSD patients were compared to members of their families who do not have the disease and to existing databases of human genetic variation. HLA sequences from patients from Belgrade, Serbia, were compared to the frequency of HLA haplotypes in the general population in Belgrade. We analyzed exome sequencing on 40 NMOSD patients and identified rare inherited variants in the complement pathway and potassium channel genes. Haplotype analysis further detected two haplotypes, HLA-A*01, B*08, DRB1*03 and HLA-A*01, B*08, C*07, DRB1*03, DQB1*02, which were more prevalent in NMOSD patients than in unaffected individuals. In silico modeling indicates that HLA molecules within these haplotypes are predicted to bind AQP4 at several sites, potentially contributing to the development of autoimmunity. Our results point to possible autoimmune and neurodegenerative mechanisms that cause NMOSD, and can be used to investigate potential NMOSD drug targets.

Transplant immunology I: mechanisms of rejection in solid organ transplants

Non-identical transplants (allogeneic) have been a modern medicine milestone; however, the major pitfall for completed succeed is the recognition of foreign organ by the immune system. The myriad variations in sequence of the molecules of the major histocompatibility complex (MHC) or human leukocyte antigen (HLA) between individuals are the major cause of allograft rejection. Thus, the recognition by the B or T lymphocytes of the receptor occurs in the hypervariable regions of the HLA molecules that triggers to the graft 3 types of rejection: hyperacute, acute, and chronic. In addition, the rejection could be cause antibody-mediated (ABMR), T-cell-mediated (TCMR), or both, which depends on the main branch is cause of rejection. This review will explore the mechanisms mentioned above and give an introductory insight into transplant immunology; thus, preparer the reader to delve into further reviews in a Histocompatibility or Immunogenetics laboratory purpose.

Soluble T cell receptor-based immunotherapies empowered by large scale HLA ligandomics

Immunocore focuses on the development of T cell receptor (TCR)-based bispecifics for immunotherapy. Soluble, affinity-enhanced, TCR-based molecules are engineered to target peptides presented on the cell surface in complex with Human Leucocyte Antigen (HLA) molecules (peptide:HLA). Essential to our platform is the identification and validation of peptide:HLA targets, achieved by mass spectrometry (MS). We have particularly focussed on characterizing the immunopeptidome of the most frequent allele, HLA-A*02:01. HLA complexes are isolated from immortalized cancer cells and tumour tissues by affinity purification using various HLA-restriction specific antibodies. HLA peptides, eluted under acidic conditions, are fractionated by reverse phase liquid chromatography and analysed by LC-MS/MS. Mass spectra, searched using PEAKS, are integrated with gene expression and HLA typing data in an in-house database. Fully validated target peptides are used to clonally expand HLA-peptide specific T cells. Isolated TCRs are engineered into soluble molecules (mTCRs) whose affinity is enhanced using phage display technology. Our HLA peptidomic workflow combines novel biochemical techniques with high resolution mass spectrometry and extensive data integration to maximise the depth of the HLA ligandome. Our combined findings across hundreds of cancer cell lines provides peptide information on all protein coding genes, with the very low novelty rate observed in tissue material suggesting that the processing machinery of in vitro model systems accurately represent the tissue environment. Our Immune mobilising monoclonal T cell receptors Against Cancer molecules (ImmTAC®) are generated by coupling high-affinity mTCRs with an anti-CD3 scFv domain to redirect polyclonal T cell responses toward cancer cells. Immunocore has three ImmTAC molecules in clinical trials with our lead candidate, IMCgp100, entered in pivotal trials as a monotherapy for the treatment of patients with metastatic uveal melanoma (NCT03070392, clinicaltrials.gov). Our cell line-based HLA-A*02:01peptide atlas represents a comprehensive resource for effective design of TCR-based immunotherapeutics for cancer treatment.

779TiP Commencement of first-in-human phase I/II TCR-T clinical trial targeting shared tumor-specific hotspot mutations in solid tumors

Adoptive T-cell therapy is an emerging strategy for solid tumors. Cancer cells frequently harbor driver-mutations in KRAS, TP53, and EGFR genes that can be targeted by T-cell receptors (TCRs). These neoepitopes are presented on the tumor cell surface by human leukocyte antigen (HLA) molecules to TCRs. We have developed, using non-viral Sleeping Beauty transposition, a library of TCRs able to target KRAS, TP53 and EGFR mutations for the treatment of solid tumors.

Human Leucocyte Antigens as Prognostic Markers in Head and Neck Squamous Cell Carcinoma.

Simple SummaryHuman leucocyte antigens (HLAs) play a crucial role in the immune defense by presenting antigens to T lymphocytes. The particular combination of HLA alleles determines a patient-specific presentation of cell-derived peptide fragments to the patrolling immune cells. Since deregulated protein expression and mutations are hallmarks of cancer, such abnormal peptides can be presented by tumor cells and eventually recognized, and the presenting tumor cells are then killed by T cells. Therefore, the HLA composition might influence the formation and fate of cancer and might differ between tumor patients and healthy individuals. We performed HLA typing of 84 patients with head and neck cancer, and the results were compared to survival. Two of fifty HLA alleles analyzed showed a significantly different frequency compared with the normal population (HLA-A*25 and HLA-C*06). Patients with HLA-C*04 had poorer oncological outcomes, while patients with HLA-A*02 had significantly better survival.Background: The induction and regulation of immune responses depend on human leucocyte antigen (HLA) molecules that present peptides derived from mutated neoantigens or tumor-associated antigens to cytotoxic T cells. The natural variation of HLA molecules might differ between tumor patients and the normal population. Thus, there might be associations between the frequencies of HLA alleles and the survival of tumor patients. Methods: This issue was studied in a cohort of 84 patients with head and neck squamous cell carcinomas (HNSCCs) of different localizations. The cohort was followed up for more than 10 years. HLA-A/B/C CTS-PCR-SSP typing at 1 field level from blood samples was performed, and the results were correlated with survival. Results: HLA-A*02 was the most prevalent allele in our cohort and was present in 51.1% of patients. The HLA-A*25 and HLA-C*06 alleles exhibited a significantly higher frequency in cancer patients than in the normal population of 174 blood and kidney donors (p = 0.02 and p = 0.01, respectively, Fisher’s exact test). For HLA-C*04, a negative impact on overall survival in univariate analysis (p = 0.045) and a negative, but statistically insignificant effect on survival toward poorer survival in multivariate analysis (HR: 1.82; 95% CI: 0.99–3.34, p = 0.053) were observed. In addition, HLA-A*02 was also beneficial for overall survival and progression-free survival in multivariate analysis (HR 0.54; 95% CI: 0.31–0.92; p = 0.023). Conclusion: HLA-A*02 allele expression might not only predict better survival but might also indicate superior tumor antigen presentation and, thus, help to select patients who could benefit from T-cell-dependent immunotherapies.

New insights and approaches for analyses of immunopeptidomes.

Human leucocyte antigen (HLA) molecules play a key role in health and disease by presenting antigen to T-lymphocytes for immunosurveillance. Immunopeptidomics involves the study of the collection of peptides presented within the antigen-binding groove of HLA molecules. Identifying their nature and diversity is crucial to understanding immunosurveillance especially during infection or for the recognition and potential eradication of tumours. This review discusses recent advances in the isolation, identification, and quantitation of these peptide antigens. New informatics approaches and databases have shed light on the extent of peptide antigens derived from unconventional sources including peptides derived from transcripts associated with frame shifts, long noncoding RNA, incorrectly annotated untranslated regions, post-translational modifications, and proteasomal splicing. Several challenges remain in successful analysis of immunopeptides, yet recent developments point to unexplored biology waiting to be unravelled.

SARS-Arena: Sequence and Structure-Guided Selection of Conserved Peptides from SARS-related Coronaviruses for Novel Vaccine Development.

The pandemic caused by the SARS-CoV-2 virus, the agent responsible for the COVID-19 disease, has affected millions of people worldwide. There is constant search for new therapies to either prevent or mitigate the disease. Fortunately, we have observed the successful development of multiple vaccines. Most of them are focused on one viral envelope protein, the spike protein. However, such focused approaches may contribute for the rise of new variants, fueled by the constant selection pressure on envelope proteins, and the widespread dispersion of coronaviruses in nature. Therefore, it is important to examine other proteins, preferentially those that are less susceptible to selection pressure, such as the nucleocapsid (N) protein. Even though the N protein is less accessible to humoral response, peptides from its conserved regions can be presented by class I Human Leukocyte Antigen (HLA) molecules, eliciting an immune response mediated by T-cells. Given the increased number of protein sequences deposited in biological databases daily and the N protein conservation among viral strains, computational methods can be leveraged to discover potential new targets for SARS-CoV-2 and SARS-CoV-related viruses. Here we developed SARS-Arena, a user-friendly computational pipeline that can be used by practitioners of different levels of expertise for novel vaccine development. SARS-Arena combines sequence-based methods and structure-based analyses to (i) perform multiple sequence alignment (MSA) of SARS-CoV-related N protein sequences, (ii) recover candidate peptides of different lengths from conserved protein regions, and (iii) model the 3D structure of the conserved peptides in the context of different HLAs. We present two main Jupyter Notebook workflows that can help in the identification of new T-cell targets against SARS-CoV viruses. In fact, in a cross-reactive case study, our workflows identified a conserved N protein peptide (SPRWYFYYL) recognized by CD8+ T-cells in the context of HLA-B7+. SARS-Arena is available at https://github.com/KavrakiLab/SARS-Arena.

No replication of previously reported association with genetic variants in the T cell receptor alpha (TRA) locus for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disease with a variety of symptoms such as post-exertional malaise, fatigue, and pain, but where aetiology and pathogenesis are unknown. An increasing number of studies have implicated the involvement of the immune system in ME/CFS. Furthermore, a hereditary component is suggested by the reported increased risk for disease in relatives, and genetic association studies are being performed to identify potential risk variants. We recently reported an association with the immunologically important human leucocyte antigen (HLA) genes HLA-C and HLA-DQB1 in ME/CFS. Furthermore, a genome-wide genetic association study in 42 ME/CFS patients reported significant association signals with two variants in the T cell receptor alpha (TRA) locus (P value <5 × 10−8). As the T cell receptors interact with the HLA molecules, we aimed to replicate the previously reported findings in the TRA locus using a large Norwegian ME/CFS cohort (409 cases and 810 controls) and data from the UK biobank (2105 cases and 4786 controls). We investigated numerous SNPs in the TRA locus, including the two previously ME/CFS-associated variants, rs11157573 and rs17255510. No associations were observed in the Norwegian cohort, and there was no significant association with the two previously reported SNPs in any of the cohorts. However, other SNPs showed signs of association (P value <0.05) in the UK Biobank cohort and meta-analyses of Norwegian and UK biobank cohorts, but none survived correction for multiple testing. Hence, our research did not identify any reliable associations with variants in the TRA locus.

Susceptibility and Severity of COVID-19 Are Both Associated With Lower Overall Viral-Peptide Binding Repertoire of HLA Class I Molecules, Especially in Younger People.

An important number of studies have been conducted on the potential association between human leukocyte antigen (HLA) genes and COVID-19 susceptibility and severity since the beginning of the pandemic. However, case–control and peptide-binding prediction methods tended to provide inconsistent conclusions on risk and protective HLA alleles, whereas some researchers suggested the importance of considering the overall capacity of an individual’s HLA Class I molecules to present SARS-CoV-2-derived peptides. To close the gap between these approaches, we explored the distributions of HLA-A, -B, -C, and -DRB1 1st-field alleles in 142 Iranian patients with COVID-19 and 143 ethnically matched healthy controls, and applied in silico predictions of bound viral peptides for each individual’s HLA molecules. Frequency comparison revealed the possible predisposing roles of HLA-A*03, B*35, and DRB1*16 alleles and the protective effect of HLA-A*32, B*58, B*55, and DRB1*14 alleles in the viral infection. None of these results remained significant after multiple testing corrections, except HLA-A*03, and no allele was associated with severity, either. Compared to peptide repertoires of individual HLA molecules that are more likely population-specific, the overall coverage of virus-derived peptides by one’s HLA Class I molecules seemed to be a more prominent factor associated with both COVID-19 susceptibility and severity, which was independent of affinity index and threshold chosen, especially for people under 60 years old. Our results highlight the effect of the binding capacity of different HLA Class I molecules as a whole, and the more essential role of HLA-A compared to HLA-B and -C genes in immune responses against SARS-CoV-2 infection.

PB1752: ANALYSIS OF HLA-PEPTIDE BINDING POCKETS IN PATIENTS WITH ACUTE LEUKEMIA UNDERGOING UMBILICAL CORD BLOOD TRANSPLANTATION

Background: Despite a better understanding of the genetic heterogeneity of both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), relapse rates remain high and long-term survival is suboptimal. In this context, the association between these malignancies and the human leukocyte antigens (HLA) has been widely studied. The highly polymorphic HLA loci have significance and relevance for disease susceptibility thanks to their peptide-binding grooves-dependent antigen presentation to effector cells. Such processes trigger an algorithm of immune responses based on multiple fine-tuned combinations of amino acid sequence interactions. Thus, the HLA genetic diversity has been a focus of interest in acute leukemia settings as exemplified by studies showing the implication of both HLA class I and II polymorphisms. However, there is scarce data regarding the diversity of peptide-binding pockets per se especially in the context of umbilical cord blood transplantation (UCBT). Aims: To analyze whether variants of HLA-DQB1 and -DRB1 peptide-binding groove pockets 4 and 9 and/or their amino acid positions are associated with the diagnosis of ALL or AML among patients undergoing UCBT. Methods: A retrospective analysis was performed including 849 patients with ALL and AML undergoing an UCBT. Demographics and genotyping data were obtained from the Eurocord database. Pockets and amino acids were imputed from the four-digit HLA genotypes of patients. Easy-HLA was used for haplotype inference when needed. Descriptive statistics were applied. Risk ratios (RRs) and 95% confidence intervals (CI) were estimated by applying log-binomial regression to compare the allelic frequencies between both types of leukemia. Results: ALL and AML were diagnosed in 50.2% and 49.8%, respectively. The median age at UCBT was 8.7 and 17.9 years. Overall, they were mostly pediatric patients (61.8%), males (52%), and in first complete remission (58.7%). From the ten variants analyzed in pocket 9 of the HLA-DQB1 allele, only the variant YYVSY, corresponding to the allele HLA-DQB1*05:02/04, was more frequent in ALL when compared to AML (RR=2.12, 95% CI: 1.30-3.44; p=0.001). Also, within the 21 analyzed pocket 4 variants of the HLA-DRB1 allele, RFDRAY, corresponding to DRB1*16:01/02, was the only variant with a significant higher frequency among ALL patients versus AML patients (RR=3.26, 95% CI: 1.81-5.99; p=0.001). No differences were observed when comparing ALL and AML at either pocket 4 of HLA-DQB1 allele or pocket 9 of HLA-DRB1 allele. Further amino acid analysis revealed that the frequency of serine 57 of the HLA-DQB1 pocket 9 was higher in ALL (RR=2.03, 95% CI: 1.26-3.27; p=0.037). None of the analyzed amino acid positions in HLA-DRB1 pockets was statistically enriched neither in ALL or AML. Summary/Conclusion: Our analyses suggest that specific variants in terms of HLA pockets and amino acids might be unique to ALL. As a result of expressing HLA molecules on their surface, leukemic cells may participate to the disease development and/or to immune evasion. Thus, the immune and genetic expression profile of the leukemic microenvironment has been described as a potential useful biomarker in guiding therapeutic approaches. Interestingly, peptide binding to HLA class II molecules is frequently viewed as a merger of detached anchor residue preferences for pockets 4 and 9. In our study, these associations, at a genotypic level, might be a stepping stone towards analyzing results in acute leukemia after UCBT as there is robust evidence supporting the impact of HLA on important post-transplant outcomes.