Biology subjects

Buus, S.

Publications and source records attributed to Buus, S..

12 recordsLinked to original sources

bioRxiv2024

Profiling of linear B-cell epitopes against human coronaviruses in pooled sera sampled early in the COVID-19 pandemic

BackgroundAntibodies play a key role in the immune defence against infectious pathogens. Understanding the underlying process of B cell recognition is not only of fundamental interest; it supports important applications within diagnostics and therapeutics. Whereas the nature of conformational B cell epitope recognition is inherently complicated, linear B cell epitopes offer a straightforward approach that potentially can be reduced to one of peptide recognition. MethodsUsing an overlapping peptide approach representing the entire proteomes of the seven main coronaviruses known to infect humans, we analysed sera pooled from eight PCR-confirmed COVID-19 convalescents and eight pre-pandemic controls. Using a high-density peptide microarray platform, 13-mer peptides overlapping by 11 amino acids were in situ synthesised and incubated with the pooled primary serum samples, followed by development with secondary fluorochrome-labelled anti-IgG and -IgA antibodies. Interactions were detected by fluorescence detection. Strong Ig interactions encompassing consecutive peptides were considered to represent "high-fidelity regions" (HFRs). These were mapped to the coronavirus proteomes using a 60% homology threshold for clustering. ResultsWe identified 333 human coronavirus derived HFRs. Among these, 98 (29%) mapped to SARS-CoV-2, 144 (44%) mapped to one or more of the four circulating common cold coronaviruses (CCC), and 54 (16%) cross-mapped to both SARS-CoV-2 and CCCs. The remaining 37 (11%) mapped to either SARS-CoV or MERS-CoV. Notably, the COVID-19 serum was skewed towards recognising SARS-CoV-2-mapped HFRs, whereas the pre-pandemic was skewed towards recognising CCC-mapped HFRs. In terms of absolute numbers of linear B cell epitopes, the primary targets are the ORF1ab protein (60%), the spike protein (21%), and the nucleoprotein (15%) in that order; however, in terms of epitope density the order would be reversed. ConclusionWe identified linear B cell epitopes across coronaviruses, highlighting pan-, alpha-, beta-, or SARS-CoV-2-corona-specific B cell recognition patterns. These findings could be pivotal in deciphering past and current exposures to epidemic and endemic coronavirus. Moreover, our results suggest that pre-pandemic anti-CCC antibodies may cross-react against SARS-CoV-2, which could explain the highly variable outcome of COVID-19. Finally, the methodology used here offers a rapid and comprehensive approach to high-resolution linear B-cell epitope mapping, which could be vital for future studies of emerging infectious diseases.

immunology↗

bioRxiv2023

Coxsackievirus infection induces direct pancreatic β-cell killing but poor anti-viral CD8+ T-cell responses

Coxsackievirus B (CVB) infection of pancreatic {beta} cells is associated with {beta}-cell autoimmunity. We investigated how CVB impacts human {beta} cells and anti-CVB T-cell responses. {beta} cells were efficiently infected by CVB in vitro, downregulated HLA Class I and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized only a fraction of these peptides, and only another sub-fraction was targeted by effector/memory T cells that expressed the exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with the {beta}-cell antigen GAD. Infected {beta} cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Thus, our in-vitro and ex-vivo data highlight limited T-cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and non-structural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

immunology↗

bioRxiv2023

Extraordinary peptide-binding mode of a songbird MHC class-I molecule suggests mechanism to counter pathogen immune evasion

Long-distance migratory animals such as birds and bats have evolved to withstand selection imposed by pathogens across the globe, and pathogen richness is known to be particularly high in tropical regions. Immune genes, so-called Major Histocompatibility Complex (MHC) genes, are highly duplicated in songbirds compared to other vertebrates, and this high MHC diversity has been hypothesised to result in a unique adaptive immunity. To understand the rationale behind the evolution of the high MHC genetic diversity in songbirds, we determined the structural properties of an MHC class I protein, Acar3, from a long-distance migratory songbird, the great reed warbler Acrocephalus arundinaceus (in short: Acar). The structure of Acar3 was studied in complex with pathogen-derived antigens and shows an overall antigen presentation similar to human MHC class I. However, the peptides bound to Acar3 display an unusual conformation: Whereas the N-terminal ends of the peptides display enhanced flexibility, the conformation of their C-terminal halves is rather static. This uncommon peptide-binding mode in Acar3 is facilitated by a central Arg residue within the peptide-binding groove that fixes the backbone of the peptide at its central position, and potentially permits successful interactions between MHC class I and innate immune receptors. Our study highlights the importance of investigating the immune system of wild animals, such as birds and bats, to uncover unique immune mechanisms which may neither exist in humans nor in model organisms.

ecology↗

bioRxiv2022

Antibodies to native gluten arise from cross-reactive B cells with implications for epitope spreading in celiac disease

Antibodies to deamidated gluten peptides are accurate diagnostic markers of celiac disease (CeD). However, antibody binding to all possible gluten epitopes has not previously been investigated. To map antibody reactivity in detail and to understand the connection between disease-relevant B-cell and T-cell epitopes, we took advantage of a high-density peptide array for assessment of serum antibody specificity in CeD across the wheat gluten proteome. We confirm the importance of peptide deamidation for antibody binding, and we show that the response is remarkably focused on the known epitope QPEQPFP (where E results from deamidation of Q). In addition, we describe a new epitope in native (non-deamidated) gluten, QQPEQII (where E is gene encoded), which was associated with both B-cell and T-cell reactivity. By generating monoclonal antibodies from peptide-binding gut plasma cells of CeD patients, we show that antibodies to this native gluten epitope are cross-reactive with the major deamidated epitope due to recognition of the shared PEQ motif. Hence, antibodies to native gluten appear to arise from cross-reactive B cells that are generated as a side effect of the immune response to deamidated gluten. Since cross-reactive B cells could present peptides to different gluten-specific T cells, we suspect that such B cells can play a role in epitope spreading by engaging T cells with multiple specificities.

immunology↗

bioRxiv2021

Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

BackgroundHow immune-tolerance is lost to pancreatic {beta}-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin ER chaperone glucose-regulated protein (GRP) 94 leads to mishandling of proinsulin, ER stress and activation of the inducible proteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered {beta}-cell MHC-I bound peptidome and inflammasome activation, sensitizing {beta}-cells to immune attack. MethodsWe used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20{micro}M), or exposed to proinflammatory cytokines interleukin (IL)-1{beta} or IFN{gamma} (15 pg/ml and 10 ng/ml, respectively) for 24 hours. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse phase high performance liquid chromatography (RP-HPLC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). NALP1, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (I{kappa}B), and (pro) IL-1{beta} expression and secretion were investigated by Western blotting. ResultsGRP94 KO increased RT1.A expression in {beta}-cells as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptides diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NALP1 expression was upregulated in GRP94 deficient cells along with decreased I{kappa}B content while proIL-1{beta} cellular levels declined, coupled with an increased secretion of mature IL-1{beta}. Our results suggest that limiting {beta}-cell proinsulin chaperoning enhances RT1.A expression, alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress impeding proinsulin handling may sensitize {beta}-cells to immune-attack.

molecular biology↗

bioRxiv2021

HIV-specific CD8+ T-cells in tonsils express exhaustive TRM-like signatures

Lymphoid tissues are an important HIV reservoir site that persists in the face of antiretroviral therapy and natural immunity. Targeting these reservoirs by harnessing the antiviral activity of local tissue resident memory ( TRM) CD8+ T-cells is of great interest, but limited data exist on TRMs within lymph nodes of people living with HIV (PLWH). Here, we studied tonsil CD8+ T-cells obtained from PLWH and uninfected controls from South Africa. We show that these cells are preferentially located outside the germinal centers (GCs), the main reservoir site for HIV, and display a low cytolytic and transcriptionally TRM-like profile that is distinct from blood. In PLWH, CD8+ TRM-like cells are highly expanded and adopt a more cytolytic, activated and exhausted phenotype characterized by increased expression of CD69, PD-1 and perforin, but reduced CD127. This phenotype was enhanced in HIV-specific CD8+ T-cells from tonsils compared to matched blood. Single-cell profiling of these cells revealed a clear transcriptional signature of T-cell activation, clonal expansion and exhaustion ex-vivo. In contrast, this signature was absent from HIV-specific CD8+ T-cells in tonsils isolated from a natural HIV controller, who expressed lower levels of cell surface PD-1 and CXCR5, and reduced transcriptional evidence of T-cell activation, exhaustion and cytolytic activity. Thus, we show that HIV-specific TRM-like CD8+ T-cells in tonsils from non-HIV controllers are enriched for activation and exhaustion profiles compared to those in blood, suggesting that lymphoid HIV-specific CD8+ TRM cells are potentially ideal candidates for immunotherapy to modulate their ability to targeting the HIV reservoirs.

immunology↗

bioRxiv2020

Integral use of immunopeptidomics and immunoinformatics for the characterization of antigen presentation and rational identification of BoLA-DR-presented peptides and epitopes

Major histocompatibility complex (MHC) peptide binding and presentation is the most selective event defining the landscape of T cell epitopes. Consequently, understanding the diversity of MHC alleles in a given population and the parameters that define the set of ligands that can be bound and presented by each of these alleles (the immunopeptidome) has an enormous impact on our capacity to predict and manipulate the potential of protein antigens to elicit functional T cell responses. Liquid chromatography-mass spectrometry (LC-MS) analysis of MHC eluted ligands (EL data) has proven to be a powerful technique for identifying such peptidomes, and methods integrating such data for prediction of antigen presentation have reached a high level of accuracy for both MHC class I and class II. Here, we demonstrate how these techniques and prediction methods can be readily extended to the bovine leukocyte antigen class II DR locus (BoLA-DR). BoLA-DR binding motifs were characterized by EL data derived from cell lines expressing a range of DRB3 alleles prevalent in Holstein-Friesian populations. The model generated (NetBoLAIIpan - available as a web-server at www.cbs.dtu.dk/services/NetBoLAIIpan) was shown to have unprecedented predictive power to identify known BoLA-DR restricted CD4 epitopes. In summary, the results demonstrate the power of an integrated approach combining advanced MS peptidomics with immunoinformatics for characterization of the BoLA-DR antigen presentation system and provide a novel tool that can be utilised to assist in rational evaluation and selection of bovine CD4 T cell epitopes.

bioinformatics↗

bioRxiv2020

A systematic, unbiased mapping of CD8+ and CD4+ T cell epitopes in Yellow Fever vaccinees

1.Examining CD8+ and CD4+ T cell responses after primary Yellow Fever vaccination in a cohort of 210 volunteers, we have identified and tetramer-validated 92 CD8+ and 50 CD4+ T cell epitopes, many inducing strong and prevalent (i.e. immunodominant) T cell responses. Restricted by 40 and 14 HLA-class I and II allotypes, respectively, these responses have wide population coverage and might be of considerable academic, diagnostic and therapeutic interest. The broad coverage of epitopes and HLA overcame the otherwise confounding effects of HLA diversity and non-HLA background providing the first evidence of T cell immunodomination in humans. Also, double-staining of CD4+ T cells with tetramers representing the same HLA-binding core, albeit with different flanking regions, demonstrated an extensive diversification of the specificities of many CD4+ T cell responses. We suggest that this could reduce the risk of pathogen escape, and that multi-tetramer staining is required to reveal the true magnitude and diversity of CD4+ T cell responses. Our T cell epitope discovery approach uses a combination of 1) overlapping peptides representing the entire Yellow Fever virus proteome to search for peptides containing CD4+ and/or CD8+ T cell epitopes, 2) predictors of peptide-HLA binding to suggest epitopes and their restricting HLA allotypes, 3) generation of peptide-HLA tetramers to identify T cell epitopes, and 4) analysis of ex vivo T cell responses to validate the same. This approach is systematic, exhaustive, and can be done in any individual of any HLA haplotype. It is all-inclusive in the sense that it includes all protein antigens and peptide epitopes, and encompasses both CD4+ and CD8+ T cell epitopes. It is efficient and, importantly, reduces the false discovery rate. The unbiased nature of the T cell epitope discovery approach presented here should support the refinement of future peptide-HLA class I and II predictors and tetramer technologies, which eventually should cover all HLA class I and II isotypes. We believe that future investigations of emerging pathogens (e.g. SARS-CoV-2) should include population-wide T cell epitope discovery using blood samples from patients, convalescents and/or long-term survivors, who might all hold important information on T cell epitopes and responses.

immunology↗

bioRxiv2020

HLA-class II specificity assessed by high-density peptide microarray interactions

The ability to predict and/or identify MHC binding peptides is an essential component of T cell epitope discovery; something that ultimately should benefit the development of vaccines and immunotherapies. In particular, MHC class I (MHC-I) prediction tools have matured to a point where accurate selection of optimal peptide epitopes is possible for virtually all MHC-I allotypes; in comparison, current MHC class II (MHC-II) predictors are less mature. Since MHC-II restricted CD4+ T cells control and orchestrate most immune responses, this shortcoming severely hampers the development of effective immunotherapies. The ability to generate large panels of peptides and subsequently large bodies of peptide-MHC-II interaction data is key to the solution of this problem; a solution that also will support the improvement of bioinformatics predictors, which critically relies on the availability of large amounts of accurate, diverse and representative data. Here, we have used recombinant HLA-DRB1*01:01 and HLA-DRB1*03:01 molecules to interrogate high-density peptide arrays, in casu containing 70,000 random peptides in triplicates. We demonstrate that the binding data acquired contains systematic and interpretable information reflecting the specificity of the HLA-DR molecules investigated. Collectively, with a cost per peptide reduced to a few cents combined with the flexibility of recombinant HLA technology, this poses an attractive strategy to generate vast bodies of MHC-II binding data at an unprecedented speed and for the benefit of generating peptide-MHC-II binding data as well as improving MHC-II prediction tools.

immunology↗

bioRxiv2020

T cell responses induced by attenuated flavivirus vaccination are specific and show limited cross-reactivity with other flavivirus species.

Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. Here, we tested pools of epitopes derived from dengue (DENV), zika (ZIKV), Japanese Encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by Intracellular Cytokine Staining (ICS) using PBMCs of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccines. CD8 T cell responses recognized epitopes from multiple flaviviruses, however, the magnitude of cross-reactive responses was consistently several-fold lower than those to the autologous epitope pools, and associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing twenty-nine different individual epitope/donor combinations. TCL derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted within the DENV serocomplex but were consistently associated with more than 100-fold lower antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV derived peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-reactivity with any other flaviviruses, and in five out of eight cases more than 1000-fold lower antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T cell responses between flaviviruses and has implications for understanding immunity elicited by natural infection, and strategies to develop live attenuated vaccines against flaviviral species. ImportanceThe envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous flavivirus (such as YF) as a delivery vector. This backbone comprises the non-structural (NS) and capsid (C) antigens which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses amongst different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses, and might thus impair vaccine performance.

immunology↗

bioRxiv2019

NNAlign_MA; semi-supervised MHC peptidome deconvolution for accurate characterization of MHC binding motifs and improved T cell epitope prediction

Antigen presentation by Major Histocompatibility Complex (MHC) is a cornerstone of the cellular immune system, and characterization of the rules defining which peptides are suitable for MHC presentation is of key interest for understanding T cell immunogenicity and the development of T cell driven therapeutics. The collective set of peptides presented on a cell surface by MHC molecules is known as the immunopeptidome. Due to the vast MHC polymorphism, this peptidome is unique between individuals. Current state-of-the-art liquid chromatography mass spectrometry (LC-MS) technologies allow the identification of large peptidomes specific for a given host or cell line, and numerous studies have proven this data to be a rich source of information for learning the rules of MHC-mediated antigen presentation. The data obtained with MS techniques is usually poly-specific - i.e. it contains multiple sequence motifs matching the different MHC molecules expressed in the system under investigation. Deconvolution of this poly-specificity has proven a challenge, and imposes a substantial limitation for learning MHC specific rules for antigen presentation from immunopeptidome data. This since each ligand first must be unambiguously associated to its presenting MHC molecule(s) within the haplotype of the cell line. Here, we describe NNAlign_MA, a method that is able to address this challenge. NNAlign_MA is capable of taking a mixed training set composed of single-allele (SA) data (peptides assigned to single MHCs) and multi-allele (MA) data (peptides with multiple options for MHCs assignments) as input, and in a fully automated manner deconvolute the individual MHC restriction of all MA sequences while learning the binding specificities of all the MHCs present in the training set. The NNAlign_MA method was benchmarked on a large and diverse dataset, covering human MHC class I and class II, and bovine class I (BoLA) data. For all cases, the method was demonstrated to have unprecedented performance compared to state-of-the-art methods, achieving a complete deconvolution of binding motifs contained within poly-specificity MS eluted ligand data and an improved predictive performance for identification of both eluted ligands and T cell epitopes. Given its very high flexibility and ease of use, we expect NNAlign_MA to serve as an effective tool to increase our understanding of the rules for MHC antigen presentation and guide the development of novel T cell-based therapeutics.

bioinformatics↗

bioRxiv2018

Footprints of antigen processing boost MHC class II natural ligand binding predictions

Major Histocompatibility complex class II (MHC-II) molecules present peptide fragments to T cells for immune recognition. Current predictors for peptide:MHC-II binding are trained on binding affinity data, generated in-vitro and therefore lacking information about antigen processing. For the first time, we here describe prediction models of peptide:MHC-II binding trained directly on naturally eluted peptides, and show that these, in addition to peptide binding to the MHC, incorporate identifiable rules of antigen processing. In fact, we observed detectable signals of protease cleavage at defined positions of the peptides. We also hypothesize a role of the length of the terminal ligand protrusions for trimming the peptide to the epitope presented. The results of integrating binding affinity and eluted ligand data in a combined model demonstrate improved performance for the prediction of MHC-II ligands, and foreshadow a new generation of improved peptide:MHC-II prediction tools of considerable importance for understanding and manipulating immune responses.

immunology↗