The SysteMHC Atlas project.

Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts.

Development of a strategy and computational application to select candidate protein analogues with reduced HLA binding and immunogenicity.

Unwanted immune responses against protein therapeutics can reduce efficacy or lead to adverse reactions. T-cell responses are key in the development of such responses, and are directed against immunodominant regions within the protein sequence, often associated with binding to several allelic variants of HLA class II molecules (promiscuous binders). Herein, we report a novel computational strategy to predict 'de-immunized' peptides, based on previous studies of erythropoietin protein immunogenicity. This algorithm (or method) first predicts promiscuous binding regions within the target protein sequence and then identifies residue substitutions predicted to reduce HLA binding. Further, this method anticipates the effect of any given substitution on flanking peptides, thereby circumventing the creation of nascent HLA-binding regions. As a proof-of-principle, the algorithm was applied to Vatreptacog α, an engineered Factor VII molecule associated with unintended immunogenicity. The algorithm correctly predicted the two immunogenic peptides containing the engineered residues. As a further validation, we selected and evaluated the immunogenicity of seven substitutions predicted to simultaneously reduce HLA binding for both peptides, five control substitutions with no predicted reduction in HLA-binding capacity, and additional flanking region controls. In vitro immunogenicity was detected in 21·4% of the cultures of peptides predicted to have reduced HLA binding and 11·4% of the flanking regions, compared with 46% for the cultures of the peptides predicted to be immunogenic. This method has been implemented as an interactive application, freely available online at http://tools.iedb.org/deimmunization/.

Development of a novel clustering tool for linear peptide sequences.

Epitopes identified in large-scale screens of overlapping peptides often share significant levels of sequence identity, complicating the analysis of epitope-related data. Clustering algorithms are often used to facilitate these analyses, but available methods are generally insufficient in their capacity to define biologically meaningful epitope clusters in the context of the immune response. To fulfil this need we developed an algorithm that generates epitope clusters based on representative or consensus sequences. This tool allows the user to cluster peptide sequences on the basis of a specified level of identity by selecting among three different method options. These include the 'clique method', in which all members of the cluster must share the same minimal level of identity with each other, and the 'connected graph method', in which all members of a cluster must share a defined level of identity with at least one other member of the cluster. In cases where it is not possible to define a clear consensus sequence with the connected graph method, a third option provides a novel 'cluster-breaking algorithm' for consensus sequence driven sub-clustering. Herein we demonstrate the tool's clustering performance and applicability using (i) a selection of dengue virus epitopes for the 'clique method', (ii) sets of allergen-derived peptides from related species for the 'connected graph method' and (iii) large data sets of eluted ligand, major histocompatibility complex binding and T-cell recognition data captured within the Immune Epitope Database (IEDB) with the newly developed 'cluster-breaking algorithm'. This novel clustering tool is accessible at http://tools.iedb.org/cluster2/.

Deciphering the MHC-associated peptidome: a review of naturally processed ligand data.

INTRODUCTION: The availability of big data sets ('OMICS') has greatly impacted fundamental and translational science. High-throughput analysis of HLA class I and II associated peptidomes by mass spectrometry (MS) has generated large datasets, with the last decade witnessing tremendous growth in the breadth and number of studies. Areas covered: For this, we first analyzed naturally processed peptide (NP) data captured within the IEDB to survey and characterize the current state of NP data. We next asked to what extent the NP data overlap with existing T cell epitope and MHC binding data. Expert commentary: The current collection of NP data represents a large and diverse set of class I/II peptides mostly derived from self-antigens. These data overlap only marginally with existing immunogenicity and binding data and it is thus difficult to ascertain the correspondence between the different assay methodologies. This highlights a need for unbiased studies benchmarking in model antigen systems how well MHC binding and NP data predicts immunogenicity. Going forward, efforts at generating an integrated process for capturing all NP, curating associated metadata and accessing NP data from an immunological viewpoint will be important for development of novel methods for identifying optimal target antigens and for class I and II epitope prediction.

The Use of the Immune Epitope Database to Study Autoimmune Epitope Data Related to Alopecia Areata.

The Immune Epitope Database (IEDB) is a repository of published epitope data for infectious diseases, allergy, transplantation and autoimmunity. Herein we provide an introduction to the IEDB search interface, focusing on data related to autoimmune diseases, including alopecia areata (AA). We demonstrate how common questions related can be answered, such as how to search for specific autoantigens, epitope sequences, response types (B- and/or T-cell assays), or host, as well as how to search for epitopes of known major histocompatibility complex restriction and for data related to a specific disease. Our survey of the data found that while as a whole Autoimmunity-specific records represent a significant portion (∼30%); epitopes reported for AA are remarkably few, just 23 epitopes from six antigens. This reveals a significant knowledge gap for AA, and suggests that additional mapping of epitopes and identification of novel AA-associated autoantigens is warranted. Citing recently published examples, we show how bioinformatic, proteomic, and technological advances make it now increasingly feasible to identify epitopes and novel antigens in human disease. The goal herein was to increase awareness of the IEDB as a free resource for the scientific community and to demonstrate its use in finding (existing) and analyzing (prediction) epitope data.

Strategies to query and display allergy-derived epitope data from the immune epitope database.

The recognition of specific epitopes on allergens by antibodies and T cells is a key element in allergic processes. Analysis of epitope data may be of interest for basic immunopathology or for potential application in diagnostics or immunotherapy. The Immune Epitope Database (IEDB) is a freely available repository of epitope data from infectious disease agents, as well as epitopes defined for allergy, autoimmunity, and transplantation. The IEDB curates the experiments associated with each epitope and thus provides a variety of different ways to search the data. This review aims to demonstrate the utility of the IEDB and its query strategies, including searching by epitope structure (peptidic/nonpeptidic), by assay methodology, by host, by the allergen itself, or by the organism from which the allergen was derived. Links to tools for visualization of 3-D structures, epitope prediction, and analyses of B and T cell reactivity by host response frequency score are also highlighted.

Pre-existing immunity against swine-origin H1N1 influenza viruses in the general human population.

A major concern about the ongoing swine-origin H1N1 influenza virus (S-OIV) outbreak is that the virus may be so different from seasonal H1N1 that little immune protection exists in the human population. In this study, we examined the molecular basis for pre-existing immunity against S-OIV, namely the recognition of viral immune epitopes by T cells or B cells/antibodies that have been previously primed by circulating influenza strains. Using data from the Immune Epitope Database, we found that only 31% (8/26) of B-cell epitopes present in recently circulating H1N1 strains are conserved in the S-OIV, with only 17% (1/6) conserved in the hemagglutinin (HA) and neuraminidase (NA) surface proteins. In contrast, 69% (54/78) of the epitopes recognized by CD8(+) T cells are completely invariant. We further demonstrate experimentally that some memory T-cell immunity against S-OIV is present in the adult population and that such memory is of similar magnitude as the pre-existing memory against seasonal H1N1 influenza. Because protection from infection is antibody mediated, a new vaccine based on the specific S-OIV HA and NA proteins is likely to be required to prevent infection. However, T cells are known to blunt disease severity. Therefore, the conservation of a large fraction of T-cell epitopes suggests that the severity of an S-OIV infection, as far as it is determined by susceptibility of the virus to immune attack, would not differ much from that of seasonal flu. These results are consistent with reports about disease incidence, severity, and mortality rates associated with human S-OIV.

Comprehensive Review of Human Plasmodium falciparum-Specific CD8+ T Cell Epitopes.

Control of malaria is an important global health issue and there is still an urgent need for the development of an effective prophylactic vaccine. Multiple studies have provided strong evidence that Plasmodium falciparum-specific MHC class I-restricted CD8+ T cells are important for sterile protection against Plasmodium falciparum infection. Here, we present an interactive epitope map of all P. falciparum-specific CD8+ T cell epitopes published to date, based on a comprehensive data base (IEDB), and literature search. The majority of the described P. falciparum-specific CD8+ T cells were directed against the antigens CSP, TRAP, AMA1, and LSA1. Notably, most of the epitopes were discovered in vaccine trials conducted with malaria-naïve volunteers. Only few immunological studies of P. falciparum-specific CD8+ T cell epitopes detected in patients suffering from acute malaria or in people living in malaria endemic areas have been published. Further detailed immunological mappings of P. falciparum-specific epitopes of a broader range of P. falciparum proteins in different settings and with different disease status are needed to gain a more comprehensive understanding of the role of CD8+ T cell responses for protection, and to better guide vaccine design and to study their efficacy.

Investigation of Outbreak-Specific Nonsynonymous Mutations on Ebolavirus GP in the Context of Known Immune Reactivity.

The global response to the most recent EBOV outbreak has led to increased generation and availability of data, which can be globally analyzed to increase our understanding of immune responses to EBOV. We analyzed the published antibody epitope data to identify regions immunogenic for humans on the main GP antigenic target and determine sequence variance/nonsynonymous mutations between historical isolates and variants from the 2013-2016 outbreak. Approximately half of the GP sequence has been reported as targeted by antibody responses. Our results show an enrichment of nonsynonymous mutations (NSMs) within epitopic regions on GP (70%, p = 0.0133). Mapping NSMs to human epitope reactivity may be useful for future therapeutic and prophylaxis development as well as for our general understanding of immunity against EBOV.

Predicting T cell recognition of MHC class I restricted neoepitopes.

Epitopes that arise from a somatic mutation, also called neoepitopes, are now known to play a key role in cancer immunology and immunotherapy. Recent advances in high-throughput sequencing have made it possible to identify all mutations and thereby all potential neoepitope candidates in an individual cancer. However, most of these neoepitope candidates are not recognized by T cells of cancer patients when tested in vivo or in vitro, meaning they are not immunogenic. Especially in patients with a high mutational load, usually hundreds of potential neoepitopes are detected, highlighting the need to further narrow down this candidate list. In our study, we assembled a dataset of known, naturally processed, immunogenic neoepitopes to dissect the properties that make these neoepitopes immunogenic. The tools to use and thresholds to apply for prioritizing neoepitopes have so far been largely based on experience with epitope identification in other settings such as infectious disease and allergy. Here, we performed a detailed analysis on our dataset of curated immunogenic neoepitopes to establish the appropriate tools and thresholds in the cancer setting. To this end, we evaluated different predictors for parameters that play a role in a neoepitope's immunogenicity and suggest that using binding predictions and length-rescaling yields the best performance in discriminating immunogenic neoepitopes from a background set of mutated peptides. We furthermore show that almost all neoepitopes had strong predicted binding affinities (as expected), but more surprisingly, the corresponding non-mutated peptides had nearly as high affinities. Our results provide a rational basis for parameters in neoepitope filtering approaches that are being commonly used. Abbreviations: SNV: single nucleotide variant; nsSNV: nonsynonymous single nucleotide variant; ROC: receiver operating characteristic; AUC: area under ROC curve; HLA: human leukocyte antigen; MHC: major histocompatibility complex; PD-1: Programmed cell death protein 1; PD-L1 or CTLA-4: cytotoxic T-lymphocyte associated protein 4.

Enhancing Chemotherapy Capabilities in Rural Hospitals: Implementation of a Telechemotherapy Model (QReCS) in North Queensland, Australia.

INTRODUCTION: The Queensland Remote Chemotherapy Supervision (QReCS) model enables rural nurses to administer chemotherapy in smaller rural towns under supervision by health professionals from larger centers using telehealth. Its implementation began in North Queensland, Australia (population, 650,000), in 2014 between two regional cancer centers (Townsville and Cairns as primary sites) and six rural sites (125 to 1,000 kilometers from primary sites). Our study examined the implementation processes, feasibility, and safety of this model. METHODS: Details of implementation and patients' clinical details for the period of 2014 to 2016 for descriptive analysis were extracted from telechemotherapy project notes and oncology information systems of North Queensland, respectively. RESULTS: After a successful pilot study in Townsville Cancer Centre, statewide rural and cancer networks of Queensland Health, in collaboration with clinicians and managers across the state of Queensland, developed the QReCS model and a guide for operationalizing it. QReCS was implemented at six sites from 2014 to 2016. Main enablers across North Queensland included collaboration among clinicians and managers, availability of common electronic medical records, funding from Queensland Health, and installation of telehealth infrastructure by statewide telehealth services. Main barriers included turnover of senior management and nursing staff at two rural towns. Sixty-two patients received 327 cycles of low- to medium-risk chemotherapy agents. Rates of treatment delays, adverse events, and hospital admissions were similar to those in face-to-face care. CONCLUSION: Implementation of the QReCS model across a large geographic region is feasible with acceptable safety profiles. Leadership by and collaboration among clinicians and managers, adequacy of resources and common governance are key enablers.

A DNA vaccine against dolphin morbillivirus is immunogenic in bottlenose dolphins.

The immunization of exotic species presents considerable challenges. Nevertheless, for facilities like zoos, animal parks, government facilities and non-profit conservation groups, the protection of valuable and endangered species from infectious disease is a growing concern. The rationale for immunization in these species parallels that for human and companion animals; to decrease the incidence of disease. The U.S. Navy Marine Mammal Program, in collaboration with industry and academic partners, has developed and evaluated a DNA vaccine targeting a marine viral pathogen - dolphin morbillivirus (DMV). The DMV vaccine consists of the fusion (F) and hemagglutinin (H) genes of DMV. Vaccine constructs (pVR-DMV-F and pVR-DMV-H) were evaluated for expression in vitro and then for immunogenicity in mice. Injection protocols were designed for application in Atlantic bottlenose dolphins (Tursiops truncatus) to balance vaccine effectiveness with clinical utility. Six dolphins were inoculated, four animals received both pDMV-F and pDMV-H and two animals received a mock vaccine (vector alone). All animals received an inoculation week 0, followed by two booster injections weeks 8 and 14. Vaccine-specific immune responses were documented in all four vaccinated animals. To our knowledge, this is the first report of pathogen-specific immunogenicity to a DNA vaccine in an aquatic mammal species.

The Immune Epitope Database: How Data Are Entered and Retrieved.

Easy access to a vast collection of experimental data on immune epitopes can greatly facilitate the development of therapeutics and vaccines. The Immune Epitope Database and Analysis Resource (IEDB) was developed to provide such a resource as a free service to the biomedical research community. The IEDB contains epitope and assay information related to infectious diseases, autoimmune diseases, allergic diseases, and transplant/alloantigens for humans, nonhuman primates, mice, and any other species studied. It contains T cell, B cell, MHC binding, and MHC ligand elution experiments. Its data are curated primarily from the published literature and also include direct submissions from researchers involved in epitope discovery. This article describes the process of capturing data from these sources and how the information is organized in the IEDB data. Different approaches for querying the data are then presented, using the home page search interface and the various specialized search interfaces. Specific examples covering diverse applications of interest are given to highlight the power and functionality of the IEDB.

Curation of complex, context-dependent immunological data.

BACKGROUND: The Immune Epitope Database and Analysis Resource (IEDB) is dedicated to capturing, housing and analyzing complex immune epitope related data http://www.immuneepitope.org. DESCRIPTION: To identify and extract relevant data from the scientific literature in an efficient and accurate manner, novel processes were developed for manual and semi-automated annotation. CONCLUSION: Formalized curation strategies enable the processing of a large volume of context-dependent data, which are now available to the scientific community in an accessible and transparent format. The experiences described herein are applicable to other databases housing complex biological data and requiring a high level of curation expertise.

Identifying Candidate Targets of Immune Responses in Zika Virus Based on Homology to Epitopes in Other Flavivirus Species.

INTRODUCTION: The current outbreak of Zika virus has resulted in a massive effort to accelerate the development of ZIKV-specific diagnostics and vaccines. These efforts would benefit greatly from the definition of the specific epitope targets of immune responses in ZIKV, but given the relatively recent emergence of ZIKV as a pandemic threat, few such data are available. METHODS: We used a large body of epitope data for other Flaviviruses that was available from the IEDB for a comparative analysis against the ZIKV proteome in order to project targets of immune responses in ZIKV. RESULTS: We found a significant level of overlap between known antigenic sites from other Flavivirus proteins with residues on the ZIKV polyprotein. The E and NS1 proteins shared functional antibody epitope sites, whereas regions of T cell reactivity were conserved within NS3 and NS5 for ZIKV.  Discussion: Our epitope based analysis provides guidance for which regions of the ZIKV polyprotein are most likely unique targets of ZIKV-specific antibodies, and which targets in ZIKV are most likely to be cross-reactive with other Flavivirus species. These data may therefore provide insights for the development of antibody- and T cell-based ZIKV-specific diagnostics, therapeutics and prophylaxis.

Analysis of Human RSV Immunity at the Molecular Level: Learning from the Past and Present.

Human RSV is one of the most prevalent viral pathogens of early childhood for which no vaccine is available. Herein we provide an analysis of RSV epitope data to examine its application to vaccine design and development. Our objective was to provide an overview of antigenic coverage, identify critical antibody and T cell determinants, and then analyze the cumulative RSV epitope data from the standpoint of functional responses using a combinational approach to characterize antigenic structure and epitope location. A review of the cumulative data revealed, not surprisingly, that the vast majority of epitopes have been defined for the two major surface antigens, F and G. Antibody and T cell determinants have been reported from multiple hosts, including those from human subjects following natural infection, however human data represent a minority of the data. A structural analysis of the major surface antigen, F, showed that the majority of epitopes defined for functional antibodies (neutralizing and/or protective) were either shown to bind pre-F or to be accessible in both pre- and post-F forms. This finding may have has implications for on-going vaccine design and development. These interpretations are in agreement with previous work and can be applied in the larger context of functional epitopes on the F protein. It is our hope that this work will provide the basis for further RSV-specific epitope discovery and investigation into the nature of antigen conformation in immunogenicity.

Cytotoxic T lymphocyte activity and cytokine expression in calves vaccinated with formalin-inactivated bovine respiratory syncytial virus prior to challenge.

The development of effective, safe vaccines for human and bovine respiratory syncytial virus (RSV) has been problematic. Inactivated RSV vaccines are of variable efficacy; poor efficacy may be related to induction of ineffective cell-mediated immunity (CMI). To characterize CMI in calves vaccinated with formalin inactivated (FI) BRSV, 11 calves were vaccinated twice with FI-BRSV (n=5) or mock vaccine (n=6) at a 2 week interval and challenged 1 month later. Prior to challenge a cannula was placed in the efferent lymphatic of the caudal mediastinal lymph node of each calf; lymph derived lymphocytes (LDL) were collected for analysis of CMI. Cytotoxic T lymphocyte (CTL) activity by LDL and/or peripheral blood mononuclear cells (PBMC) was measured by 51Cr release on days 5, 7, 9, and 10 post-challenge. Messenger RNA for interferon gamma (IFN-gamma), interleukin 2 (IL-2) and IL-4 was measured on days 0-10 by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of RNA of LDL. BRSV-specific IFN-gamma production by PBMC was measured on days 0 and 10 by ELISA. Clinical signs and postmortem changes following challenge were evaluated. There was no difference between groups in clinical signs, postmortem changes, CTL activity, cytokine message expression, or IFN-gamma production. For both groups, percentage lysis by CTL peaked on days 7-10 and ranged from 11 to 25%. Failure of vaccination to prevent disease following challenge was likely associated with failure to prime for improved CMI responses.

Substantial gaps in knowledge of Bordetella pertussis antibody and T cell epitopes relevant for natural immunity and vaccine efficacy.

The recent increase in whooping cough in vaccinated populations has been attributed to waning immunity associated with the acellular vaccine. The Immune Epitope Database (IEDB) is a repository of immune epitope data from the published literature and includes T cell and antibody epitopes for human pathogens. The IEDB conducted a review of the epitope literature, which revealed 300 Bordetella pertussis-related epitopes from 39 references. Epitope data are currently available for six virulence factors of B. pertussis: pertussis toxin, pertactin, fimbrial 2, fimbrial 3, adenylate cyclase and filamentous hemagglutinin. The majority of epitopes were defined for antibody reactivity; fewer T cell determinants were reported. Analysis of available protective correlates data revealed a number of candidate epitopes; however few are defined in humans and few have been shown to be protective. Moreover, there are a limited number of studies defining epitopes from natural infection versus whole cell or acellular/subunit vaccines. The relationship between epitope location and structural features, as well as antigenic drift (SNP analysis) was also investigated. We conclude that the cumulative data is yet insufficient to address many fundamental questions related to vaccine failure and this underscores the need for further investigation of B. pertussis immunity at the molecular level.

Conservancy of mAb Epitopes in Ebolavirus Glycoproteins of Previous and 2014 Outbreaks.

BACKGROUND: Several monoclonal antibodies (mAb) are being evaluated as treatment options for the current 2014 Ebola outbreak. But they were derived from and tested for protection against the older 1976 Mayinga or 1995 Kikwit Zaire Ebolaviruses (EBOV). The EBOV sequences reported for the current outbreak contain several mutations whose significance remained to be established. METHODS: We analyzed sequence and structural conservation of the Ebolavirus glycoprotein (GP) epitopes for all experimentally identified protective mAbs published to date. RESULTS: The conservancy analysis of protective mAb epitopes in the Ebolavirus glycoprotein sequences spanning all Ebola virus lineages since 1976 showed that conservancy within the Zaire EBOV lineage was high, with only one immunodominant epitope of mAb 13F6-1-2 acquiring two novel mutations in the 2014 outbreak that might potentially change the antibody specificity and neutralization activity. However, the conservation to other Ebola viruses was unexpectedly low. CONCLUSION: Low conservancy of Zaire EBOV mAb epitopes to other EBOV lineages suggests that these epitopes are not indispensable for viral fitness, and that alternative mAbs could be developed to broadly target all EBOV. However, average percent sequence identity of the epitopes for mAbs used in current cocktails to the Zaire EBOV is high with only one epitope differing in the 2014 outbreak. These data bode well for general usefulness of these antibodies in the context of the current outbreak.

A molecular view of multiple sclerosis and experimental autoimmune encephalitis: what can we learn from the epitope data?

An analysis to inventory all immune epitope data related to multiple sclerosis (MS) was performed using the Immune Epitope Database (IEDB). The analysis revealed that MS related data represent >20% of all autoimmune data, and that studies of EAE predominate; only 22% of the references describe human data. To date, >5800 unique peptides, analogs, mimotopes, and/or non-protein epitopes have been reported from 861 references, including data describing myelin-containing, as well as non-myelin antigens. This work provides a reference point for the scientific community of the universe of available data for MS-related adaptive immunity in the context of EAE and human disease.