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Zika virus (ZIKV) is a flavivirus primarily transmitted by Aedes species mosquitoes, first discovered in Africa in 1947, that disseminated through Southeast Asia and the Pacific Islands in the 2000s. The first ZIKV infections in the Americas were identified in 2014, and infections exploded through populations in Brazil and other countries in 2015/16. ZIKV infection during pregnancy can cause severe brain and eye defects in offspring, and infection in adults has been associated with higher risks of Guillain-Barre syndrome. We initiated a study to describe the natural history of Zika (the disease) and the immune response to infection, for which some results have been reported. In this paper, we identify ZIKV-specific CD4+ and CD8+ T cell epitopes that induce responses during infection. Two screening approaches were utilized: an untargeted approach with overlapping peptide arrays spanning the entire viral genome, and a targeted approach utilizing peptides predicted to bind human MHC molecules. Immunoinformatic tools were used to identify conserved MHC class I supertype binders and promiscuous class II binding peptide clusters predicted to bind 9 common class II alleles. T cell responses were evaluated in overnight IFN-g ELISPOT assays. We found that MHC supertype binding predictions outperformed the bulk overlapping peptide approach. Diverse CD4+ T cell responses were observed in most ZIKV-infected participants, while responses to CD8+ T cell epitopes were more limited. Most individuals developed a robust T cell response against epitopes restricted to a single MHC class I supertype and only a single or few CD8+ T cell epitopes overall, suggesting a strong immunodominance phenomenon. Noteworthy is that many epitopes were commonly immunodominant across persons expressing the same class I supertype. Nearly all of the identified epitopes are unique to ZIKV and are not present in Dengue viruses. Collectively, we identified 31 immunogenic peptides restricted by the 6 major class I supertypes and 27 promiscuous class II epitopes. These sequences are highly relevant for design of T cell-targeted ZIKV vaccines and monitoring T cell responses to Zika virus infection and vaccination. [ABSTRACT FROM AUTHOR]

Background: Prediction of T cell immunogenicity is a topic of considerable interest, both in terms of basic understanding of the mechanisms of T cells responses and in terms of practical applications. HLA binding affinity is often used to predict T cell epitopes, since HLA binding affinity is a key requisite for human T cell immunogenicity. However, immunogenicity at the population it is complicated by the high level of variability of HLA molecules, potential other factors beyond HLA as well as the frequent lack of HLA typing data. To overcome those issues, we explored an alternative approach to identify the common characteristics able to distinguish immunogenic peptides from non-recognized peptides. Methods: Sets of dominant epitopes derived from peer-reviewed published papers were used in conjunction with negative peptides from the same experiments/donors to train neural networks and generate an “immunogenicity score.” We also compared the performance of the immunogenicity score with previously described method for immunogenicity prediction based on HLA class II binding at the population level. Results: The immunogenicity score was validated on a series of independent datasets derived from the published literature, representing 57 independent studies where immunogenicity in human populations was assessed by testing overlapping peptides spanning different antigens. Overall, these testing datasets corresponded to over 2,000 peptides and tested in over 1,600 different human donors. The 7-allele method prediction and the immunogenicity score were associated with similar performance [average area under the ROC curve (AUC) values of 0.703 and 0.702, respectively] while the combined methods reached an average AUC of 0.725. This increase in average AUC value is significant compared with the immunogenicity score (p = 0.0135) and a strong trend toward significance is observed when compared to the 7-allele method (p = 0.0938). The new immunogenicity score method is now freely available using CD4 T cell immunogenicity prediction tool on the Immune Epitope Database website ( http://tools.iedb.org/CD4episcore ). Conclusion: The new immunogenicity score predicts CD4 T cell immunogenicity at the population level starting from protein sequences and with no need for HLA typing. Its efficacy has been validated in the context of different antigen sources, ethnicities, and disparate techniques for epitope identification. [ABSTRACT FROM AUTHOR]

One of the primary tasks in vaccine design and development of immunotherapeutic drugs is to predict conformational B-cell epitopes corresponding to primary antibody binding sites within the antigen tertiary structure. To date, multiple approaches have been developed to address this issue. However, for a wide range of antigens their accuracy is limited. In this paper, we applied the transfer learning approach using pretrained deep learning models to develop a model that predicts conformational B-cell epitopes based on the primary antigen sequence and tertiary structure. A pretrained protein language model, ESM-1v, and an inverse folding model, ESM-IF1, were fine-tuned to quantitatively predict antibody-antigen interaction features and distinguish between epitope and non-epitope residues. The resulting model called SEMA demonstrated the best performance on an independent test set with ROC AUC of 0.76 compared to peer-reviewed tools. We show that SEMA can quantitatively rank the immunodominant regions within the SARS-CoV-2 RBD domain. SEMA is available at https://github.com/AIRI-Institute/SEMAi and the web-interface http://sema.airi.net. [ABSTRACT FROM AUTHOR]

Infectious diseases continue to pose significant global health challenges. In addition to the enduring burdens of ailments like malaria and HIV, the emergence of nosocomial outbreaks driven by antibiotic-resistant pathogens underscores the ongoing threats. Furthermore, recent infectious disease crises, exemplified by the Ebola and SARS-CoV-2 outbreaks, have intensified the pursuit of more effective and efficient diagnostic and therapeutic solutions. Among the promising options, antibodies have garnered significant attention due to their favorable structural characteristics and versatile applications. Notably, nanobodies (Nbs), the smallest functional single-domain antibodies of heavychain only antibodies produced by camelids, exhibit remarkable capabilities in stable antigen binding. They offer unique advantages such as ease of expression and modification and enhanced stability, as well as improved hydrophilicity compared to conventional antibody fragments (antigen-binding fragments (Fab) or single-chain variable fragments (scFv)) that can aggregate due to their low solubility. Nanobodies directly target antigen epitopes or can be engineered into multivalent Nbs and Nb-fusion proteins, expanding their therapeutic potential. This review is dedicated to charting the progress in Nb research, particularly those derived from camelids, and highlighting their diverse applications in treating infectious diseases, spanning both human and animal contexts. [ABSTRACT FROM AUTHOR]

The collection, cryopreservation, thawing, and culture of peripheral blood mononuclear cells (PBMCs) can profoundly influence T cell viability and immunogenicity. Gold-standard PBMC processing protocols have been developed by the Office of HIV/AIDS Network Coordination (HANC); however, these protocols are not universally observed. Herein, we have explored the current literature assessing how technical variation during PBMC processing can influence cellular viability and T cell immunogenicity, noting inconsistent findings between many of these studies. Amid the mounting concerns over scientific replicability, there is growing acknowledgement that improved methodological rigour and transparent reporting is required to facilitate independent reproducibility. This review highlights that in human T cell studies, this entails adopting stringent standardised operating procedures (SOPs) for PBMC processing. We specifically propose the use of HANC's Cross-Network PBMC Processing SOP, when collecting and cryopreserving PBMCs, and the HANC member network International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) PBMC Thawing SOP when thawing PBMCs. These stringent and detailed protocols include comprehensive reporting procedures to document unavoidable technical variations, such as delayed processing times. Additionally, we make further standardisation and reporting recommendations to minimise and document variability during this critical experimental period. This review provides a detailed overview of the challenges inherent to a procedure often considered routine, highlighting the importance of carefully considering each aspect of SOPs for PBMC collection, cryopreservation, thawing, and culture to ensure accurate interpretation and comparison between studies. [ABSTRACT FROM AUTHOR]

Background: Respiratory syncytial virus (RSV) is the most common cause of viral lower respiratory tract infections (LRTIs) in young children around the world and an important cause of LRTI in the elderly. The available treatments and FDAapproved vaccines for RSV only lessen the severity of the infection and are recommended for infants and elderly people. Methods: We focused on developing a broad-spectrum vaccine that activates the immune system to directly combat RSV. The objective of this study is to identify CD4+ and CD8+ T-cell epitopes using an immunoinformatics approach to develop RSV vaccines. The efficacy of these peptides was validated through invitro and in-vivo studies involving healthy and diseased animal models. Results: For each major histocompatibility complex (MHC) class-I and II, we found three epitopes of RSV proteins including F, G, and SH with an antigenic score of >0.5 and a projected SVM score of <5. Experimental validation of these peptides on female BALB/c mice was conducted before and after infection with the RSV A2 line 19f. We found that the 3RVMHCI (CD8+) epitope of the F protein showed significant results of white blood cells (19.72 × 103 cells/ml), neutrophils (6.01 × 103 cells/ml), lymphocytes (12.98 × 103 cells/ml), IgG antibodies (36.9 µg/ml), IFN-g (86.96 ng/L), and granzyme B (691.35 pg/ml) compared to control at the second booster dose of 10 µg. Similarly, 4RVMHCII (CD4+) of the F protein substantially induced white blood cells (27.08 × 103 cells/ml), neutrophils (6.58 × 103 cells/ml), lymphocytes (16.64 × 103 cells/ml), IgG antibodies (46.13 µg/ml), IFN-g (96.45 ng/L), and granzyme B (675.09 pg/ml). In-vitro studies showed that 4RVMHCII produced a significant level of antibodies in sera on day 45 comparable to mice infected with the virus. 4RVMHCII also induced high IFN-g and IL-2 secretions on the fourth day of the challenge compared to the preinfectional stage. Conclusion: In conclusion, epitopes of the F protein showed considerable immune response and are suitable for further validation. [ABSTRACT FROM AUTHOR]

Introduction: Outer membrane vesicles (OMVs) of Neisseria meningitidis in the group B-directed vaccine MenB-4C (BexseroR) protect against infections with Neisseria gonorrhoeae. The immunological basis for protection remains unclear. N. meningitidis OMV vaccines generate human antibodies to N. meningitidis and N. gonorrhoeae lipooligosaccharide (LOS/endotoxin), but the structural specificity of these LOS antibodies is not defined. Methods: Ten paired human sera obtained pre- and post-MenB-4C immunization were used in Western blots to probe N. meningitidis and N. gonorrhoeae LOS. Post-MenB-4C sera (7v5, 19v5, and 17v5), representing individual human variability in LOS recognition, were then used to interrogate structurally defined LOSs of N. meningitidis and N. gonorrhoeae strains and mutants and studied in bactericidal assays. Results and discussion: Post-MenB-4C sera recognized both N. meningitidis and N. gonorrhoeae LOS species, ~10% of total IgG to gonococcal OMV antigens. N. meningitidis and N. gonorrhoeae LOSs were broadly recognized by post-IgG antibodies, but with individual variability for LOS structures. Deep truncation of LOS, specifically a rfaK mutant without a-, b-, or g-chain glycosylation, eliminated LOS recognition by all post-vaccine sera. Serum 7v5 IgG antibodies recognized the unsialyated L1 a-chain, and a 3-PEA-HepII or 6-PEA-HepII was part of the conformational epitope. Replacing the 3-PEA on HepII with a 3-Glc blocked 7v5 IgG antibody recognition of N. meningitidis and N. gonorrhoeae LOSs. Serum 19v5 recognized lactoneotetrose (LNT) or L1 LOSexpressing N. meningitidis or N. gonorrhoeae with a minimal a-chain structure of Gal-Glc-HepI (L8), a 3-PEA-HepII or 6-PEA-HepII was again part of the conformational epitope and a 3-Glc-HepII blocked 19v5 antibody binding. Serum 17v5 LOS antibodies recognized LNT or L1 a-chains with a minimal HepI structure of three sugars and no requirement for HepII modifications. These LOS antibodies contributed to the serum bactericidal activity against N. gonorrhoeae. The MenB-4C vaccination elicits bactericidal IgG antibodies to N. gonorrhoeae conformational epitopes involving HepI and HepII glycosylated LOS structures shared between N. meningitidis and N. gonorrhoeae. LOS structures should be considered in next-generation gonococcal vaccine design. [ABSTRACT FROM AUTHOR]

The application of B-cell epitope identification to develop therapeutic antibodies and vaccine candidates is well established. However, the validation of epitopes is time-consuming and resource-intensive. To alleviate this, in recent years, multiple computational predictors have been developed in the immunoinformatics community. Brewpitopes is a pipeline that curates bioinformatic B-cell epitope predictions obtained by integrating different state-of-the-art tools. We used additional computational predictors to account for subcellular location, glycosylation status, and surface accessibility of the predicted epitopes. The implementation of these sets of rational filters optimizes in vivo antibody recognition properties of the candidate epitopes. To validate Brewpitopes, we performed a proteome-wide analysis of SARS-CoV-2 with a particular focus on S protein and its variants of concern. In the S protein, we obtained a fivefold enrichment in terms of predicted neutralization versus the epitopes identified by individual tools. We analyzed epitope landscape changes caused by mutations in the S protein of new viral variants that were linked to observed immune escape evidence in specific strains. In addition, we identified a set of epitopes with neutralizing potential in four SARS-CoV-2 proteins (R1AB, R1A, AP3A, and ORF9C). These epitopes and antigenic proteins are conserved targets for viral neutralization studies. In summary, Brewpitopes is a powerful pipeline that refines B-cell epitope bioinformatic predictions during public health emergencies in a high-throughput capacity to facilitate the optimization of experimental validation of therapeutic antibodies and candidate vaccines. [ABSTRACT FROM AUTHOR]

Introduction: Tuberculosis (TB) remains the first cause of death from infection caused by a bacterial pathogen. Chemotherapy does not eradicate Mycobacterium tuberculosis (Mtb) fromhuman lungs, andthe pathogen causes a latent tuberculosis infectionthatcannotbepreventedbythecurrently availableBacilleCalmetteGuerin (BCG) vaccine, which is ineffective inthe prevention of pulmonary TBinadults. HLAE-restricted CD8+ T lymphocytes are essential players in protective immune responses against Mtb. Hence, expanding this population in vivo or ex vivo may be crucial for vaccination or immunotherapy against TB. Methods: The enzymatically inactive Bordetella pertussis adenylate cyclase (CyaA) toxoid is an effective tool for delivering peptide epitopes into the cytosol of antigen-presenting cells (APC) for presentation and stimulation of specific CD8+ T-cell responses. In this study, we have investigated the capacity of the CyaA toxoid to deliver Mtb epitopes known to bind HLA-E for the expansion of human CD8+ T cells in vitro. Results: Our results show that the CyaA-toxoid containing five HLA-E-restricted Mtb epitopes causes significant expansion of HLA-E-restricted antigen-specific CD8+ T cells, which produce IFN-g and exert significant cytotoxic activity towards peptide-pulsed macrophages. Discussion: HLA-E represents a promising platform for the development of new vaccines; our study indicates that the CyaA construct represents a suitable delivery system of the HLA-E-binding Mtb epitopes for ex vivo and in vitro expansion of HLA-E-restricted CD8+ T cells inducing a predominant Tc1 cytokine profile with a significant increase of IFN-γ production, for prophylactic and immunotherapeutic applications against Mtb. [ABSTRACT FROM AUTHOR]

Antibodies are the largest class of biotherapeutics. However, in recent years, single-domain antibodies have gained traction due to their smaller size and comparable binding affinity. Antibodies (Abs) and single-domain antibodies (sdAbs) differ in the structures of their binding sites: most significantly, singledomain antibodies lack a light chain and so have just three CDR loops. Given this inherent structural difference, it is important to understand whether Abs and sdAbs are distinguishable in how they engage a binding partner and thus, whether they are suited to different types of epitopes. In this study, we use non-redundant sequence and structural datasets to compare the paratopes, epitopes and antigen interactions of Abs and sdAbs. We demonstrate that even though sdAbs have smaller paratopes, they target epitopes of equal size to those targeted by Abs. To achieve this, the paratopes of sdAbs contribute more interactions per residue than the paratopes of Abs. Additionally, we find that conserved framework residues are of increased importance in the paratopes of sdAbs, suggesting that they include non-specific interactions to achieve comparable affinity. Furthermore, the epitopes of sdAbs are only marginally less accessible than those of Abs: we posit that this may be explained by differences in the orientation and compaction of sdAb and Ab CDR-H3 loops. Overall, our results have important implications for the engineering and humanization of sdAbs, as well as the selection of the best modality for targeting a particular epitope. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a flavivirus primarily transmitted by Aedes species mosquitoes, first discovered in Africa in 1947, that disseminated through Southeast Asia and the Pacific Islands in the 2000s. The first ZIKV infections in the Americas were identified in 2014, and infections exploded through populations in Brazil and other countries in 2015/16. ZIKV infection during pregnancy can cause severe brain and eye defects in offspring, and infection in adults has been associated with higher risks of Guillain-Barré syndrome. We initiated a study to describe the natural history of Zika (the disease) and the immune response to infection, for which some results have been reported. In this paper, we identify ZIKV-specific CD4+ and CD8+ T cell epitopes that induce responses during infection. Two screening approaches were utilized: an untargeted approach with overlapping peptide arrays spanning the entire viral genome, and a targeted approach utilizing peptides predicted to bind human MHC molecules. Immunoinformatic tools were used to identify conserved MHC class I supertype binders and promiscuous class II binding peptide clusters predicted to bind 9 common class II alleles. T cell responses were evaluated in overnight IFN-γ ELISPOT assays. We found that MHC supertype binding predictions outperformed the bulk overlapping peptide approach. Diverse CD4+ T cell responses were observed in most ZIKV-infected participants, while responses to CD8+ T cell epitopes were more limited. Most individuals developed a robust T cell response against epitopes restricted to a single MHC class I supertype and only a single or few CD8+ T cell epitopes overall, suggesting a strong immunodominance phenomenon. Noteworthy is that many epitopes were commonly immunodominant across persons expressing the same class I supertype. Nearly all of the identified epitopes are unique to ZIKV and are not present in Dengue viruses. Collectively, we identified 31 immunogenic peptides restricted by the 6 major class I supertypes and 27 promiscuous class II epitopes. These sequences are highly relevant for design of T cell-targeted ZIKV vaccines and monitoring T cell responses to Zika virus infection and vaccination.

Glycan masking is a novel technique in reverse vaccinology in which sugar chains (glycans) are added on the surface of immunogen candidates to hide regions of low interest and thus focus the immune system on highly therapeutic epitopes. This shielding strategy is inspired by viruses such as influenza and HIV, which are able to escape the immune system by incorporating additional glycosylation and preventing the binding of therapeutic antibodies. Interestingly, the glycan masking technique is mainly used in vaccine design to fight the same viruses that naturally use glycans to evade the immune system. In this review we report the major successes obtained with the glycan masking technique in epitopefocused vaccine design. We focus on the choice of the target antigen, the strategy for immunogen design and the relevance of the carrier vector to induce a strong immune response. Moreover, we will elucidate the different applications that can be accomplished with glycan masking, such as shifting the immune response from hyper-variable epitopes to more conserved ones, focusing the response on known therapeutic epitopes, broadening the response to different viral strains/sub-types and altering the antigen immunogenicity to elicit higher or lower immune response, as desired. [ABSTRACT FROM AUTHOR]

Background: Almost half of severe hemophilia A (HA) is caused by an intron 22 inversion mutation (Int22Inv), which disrupts the 26-exon F8 gene. Inverted F8 mRNA exons 1-22 are transcribed, while F8B mRNA, containing F8 exons 23-26, is transcribed from a promoter within intron 22. Neither FVIII activity nor FVIII antigen (cross-reacting material, CRM) are detectable in plasma of patients with an intron-22 inversion. Objectives: To test the hypothesis that (putative) intracellular synthesis of FVIII proteins encoded by inverted F8 and F8B mRNAs confers T-cell tolerance to almost the entire FVIII sequence, and to evaluate the immunogenicity of the region encoded by the F8 exon 22-23 junction sequence. Patients/Methods: Peripheral blood mononuclear cells (PBMCs) from 30 severe or moderate HA subjects (17 with an Int22Inv mutation) were tested by ELISPOT assays to detect cytokine secretion in response to FVIII proteins and peptides and to map immunodominant T-cell epitopes. Potential immunogenicity of FVIII sequences encoded by the F8 exon 22-23 junction region was also tested using peptide-MHCII binding assays. Results: Eight of the Int22Inv subjects showed robust cytokine secretion from PBMCs stimulated with FVIII proteins and/or peptides, consistent with earlier publications from the Conti-Fine group. Peptide ELISPOT assays identified immunogenic regions of FVIII. Specificity for sequences encoded within F8 mRNA exons 1-22 and F8B mRNA was confirmed by staining Int22Inv CD4+ T cells with peptide-loaded HLA-Class II tetramers. FVIII peptides spanning the F8 exon 22-23 junction (encoding M2124-V2125) showed limited binding to MHCII proteins and low immunogenicity, with cytokine secretion from only one Int22Inv subject. Conclusions: PBMCs from multiple subjects with an Int22Inv mutation, with and without a current FVIII inhibitor, responded to FVIII epitopes. Furthermore, the FVIII region encoded by the exon 22-23 junction sequence was not remarkably [ABSTRACT FROM AUTHOR]

The immunologic significance of cross-reactivity of TCR recognition of peptide:MHC complexes is still poorly understood. We have described TCR cross-reactivity in a system involving polyclonal CD8 T cell recognition of the well characterized influenza viral M158-66 epitope. While M158-66 is generally conserved between influenza A isolates, error-prone transcription generates stable variant RNA during infection which could act as novel epitopes. If packaged and viable, variant genomic RNA generates an influenza quasispecies. The stable RNA variants would generate a new transmissible epitope that can select a specific repertoire, which itself should have crossreactive properties. We tested two candidate peptides in which Thr65 is changed to Ala (A65) or Ser (S65) using recall responses to identify responding T cell clonotypes. Both peptides generated large polyclonal T cell repertoires of their own with repertoire characteristics and cross-reactivity patterns like that observed for the M158-66 repertoire. Both substitutions could be present in viral genomes or mRNA at sufficient frequency during an infection to drive immunity. Peptides from the resulting protein would be a target for CD8 cells irrespective of virus viability or transmissibility. These data support the hypothesis that cross-reactivity is important for immunity against RNA virus infections. [ABSTRACT FROM AUTHOR]

The role of the indirect T-cell recognition pathway of allorecognition in acute T cell-mediated rejection (aTCMR) is not well defined. The amount of theoretical T-cell epitopes available for indirect allorecognition can be quantified for donor-recipient combinations by the Predicted Indirectly ReCognizable HLA Epitopes algorithm (PIRCHE-II). The PIRCHE-II score was calculated for 688 donor kidney-recipient combinations and associated with the incidence of first-time diagnosed cases of TCMR. A diagnosis of TCMR was made in 182 cases; 121 cases of tubulo-interstitial rejection cases (79 cases of borderline TCMR, 42 cases of TCMR IA-B) and 61 cases of vascular TCMR (TCMR II-III). The PIRCHE-II score for donor HLA-DR/DQ (PIRCHE-II DR/DQ) was highly associated with vascular rejection. At one year after transplantation, the cumulative percentage of recipients with a vascular rejection was 12.7%, 8.6% and 2.1% within respectively the high, medium and low tertile of the PIRCHE-II DR/DQ score (p<0.001). In a multivariate regression analysis this association remained significant (p<0.001 for PIRCHE-II DR/DQ tertiles). The impact of a high PIRCHE-II DR/DQ score was mitigated by older recipient age and a living donor kidney. In conclusion, indirect antigen presentation of donor HLApeptides may significantly contribute to the risk for acute vascular rejection. [ABSTRACT FROM AUTHOR]

One of the primary tasks in vaccine design and development of immunotherapeutic drugs is to predict conformational B-cell epitopes corresponding to primary antibody binding sites within the antigen tertiary structure. To date, multiple approaches have been developed to address this issue. However, for a wide range of antigens their accuracy is limited. In this paper, we applied the transfer learning approach using pretrained deep learning models to develop a model that predicts conformational B-cell epitopes based on the primary antigen sequence and tertiary structure. A pretrained protein language model, ESM-1v, and an inverse folding model, ESM-IF1, were fine-tuned to quantitatively predict antibody-antigen interaction features and distinguish between epitope and non-epitope residues. The resulting model called SEMA demonstrated the best performance on an independent test set with ROC AUC of 0.76 compared to peer-reviewed tools. We show that SEMA can quantitatively rank the immunodominant regions within the SARS-CoV-2 RBD domain. SEMA is available at https://github.com/AIRI-Institute/SEMAi and the web-interface http://sema.airi.net.

TCR-epitope pair binding is the key component for T cell regulation. The ability to predict whether a given pair binds is fundamental to understanding the underlying biology of the binding mechanism as well as developing T-cell mediated immunotherapy approaches. The advent of large-scale public databases containing TCR-epitope binding pairs enabled the recent development of computational prediction methods for TCR-epitope binding. However, the number of epitopes reported along with binding TCRs is far too small, resulting in poor out-of-sample performance for unseen epitopes. In order to address this issue, we present our model ATM-TCR which uses a multi-head self-attention mechanism to capture biological contextual information and improve generalization performance. Additionally, we present a novel application of the attention map from our model to improve out-of-sample performance by demonstrating on recent SARS-CoV-2 data. [ABSTRACT FROM AUTHOR]

B-cell epitopes (BCEs) are a set of specific sites on the surface of an antigen that binds to an antibody produced by B-cell. The recognition of BCEs is a major challenge for drug design and vaccines development. Compared with experimental methods, computational approaches have strong potential for BCEs prediction at much lower cost. Moreover, most of the currently methods focus on using local information around target residue without taking the global information of the whole antigen sequence into consideration. We propose a novel deep leaning method through combing local features and global features for BCEs prediction. In our model, two parallel modules are built to extract local and global features from the antigen separately. For local features, we use Graph Convolutional Networks (GCNs) to capture information of spatial neighbors of a target residue. For global features, Attention-Based Bidirectional Long Short-Term Memory (Att- BLSTM) networks are applied to extract information from the whole antigen sequence. Then the local and global features are combined to predict BCEs. The experiments show that the proposed method achieves superior performance over the state-of-the-art BCEs prediction methods on benchmark datasets. Also, we compare the performance differences between data with or without global features. The experimental results show that global features play an important role in BCEs prediction. Our detailed case study on the BCEs prediction for SARS-Cov-2 receptor binding domain confirms that our method is effective for predicting and clustering true BCEs. [ABSTRACT FROM AUTHOR]

We generated CD4+ T cell lines (TCLs) reactive to either SARS-CoV-2 spike (S) or membrane (M) proteins from unexposed naïve T cells from six healthy donor volunteers to understand in fine detail whether the S and M structural proteins have intrinsic differences in driving antigen-specific CD4+ T cell responses. Having shown that each of the TCLs were antigen-specific and antigen-reactive, single cell mRNA analyses demonstrated that SARS-CoV-2 S and M proteins drive strikingly distinct molecular signatures. Whereas the S-specific CD4+ T cell transcriptional signature showed a marked upregulation of CCL1, CD44, IL17RB, TNFRSF18 (GITR) and IGLC3 genes, in general their overall transcriptome signature was more similar to CD4+ T cell responses induced by other viral antigens (e.g. CMV). However, the M protein-specific CD4+ TCLs have a transcriptomic signature that indicate a marked suppression of interferon signaling, characterized by a downregulation of the genes encoding ISG15, IFITM1, IFI6, MX1, STAT1, OAS1, IFI35, IFIT3 and IRF7 (a molecular signature which is not dissimilar to that found in severe COVID-19). Our study suggests a potential link between the antigen specificity of the SARS-CoV-2-reactive CD4+ T cells and the development of specific sets of adaptive immune responses. Moreover, the balance between T cells of significantly different specificities may be the key to understand how CD4+ T cell dysregulation can determine the clinical outcomes of COVID-19. [ABSTRACT FROM AUTHOR]

Background: Decoy receptor 3 (DcR3) belongs to the tumor necrosis factor (TNF) receptor superfamily and neutralizes TNF ligands, including FasL and TRAIL, to prevent T activation during T-cell priming. However, the cellular mechanisms underlying acute cellmediated rejection (ACMR) remain unknown. Methods: We generated DcR3 transgenic (Tg) mice and mice with high DcR3 expression (HDE) to study both in vivo and in vitro. FasR RNA knockdown in immortalized CD4+CD8+ T-cells was used to survey the role of DcR3 on FasR/Fas-associated protein with death domain (FADD)/caspase 8 pathway and its cross-link to TNF receptor-associated factor 1 (TNFR1)-associated death domain protein (TRADD) in suppressing TNFR1. TNF/TRADD knockout mice were used to show the importance of TNF adaptor protein. Results: DcR3.Fc suppressed C57BL/6 female T-cell activation and transformation into CD4+CD69+, CD4+CD44+, and CD4+CD25+Foxp3+ when compared with isotype IgG1 and its co-treatment with FasL/TRAIL after exposing to bone marrow-derived dendritic cells (BMDCs) that carried alloantigen with male H-Y and minor antigenic determinant. Interleukin-17 and interferon-g productions by BMDC-activated T-cells were lowered after co-treating with DcR3.Fc. DcR3.Fc induced effector T-cells (Teffs) and was susceptible to FasR-mediated apoptosis through the FADD/TRADD/caspase 8 pathway. After exposing to DcR3.Fc, TRADD was silenced, likely turning down the inflammatory response. The systemic effects of DcR3 Tg mice and HDE phenotype induced by the promoter of cytomegalovirus not only attenuated ACMR severity but also ameliorated the high serum creatinine and blood urea nitrogen levels even with high T-cell exposure frequencies. Besides this, DcR3 has minor biological effects on both MHC-matched and MHCmismatched models. Conclusions: High DcR3 doses protect renal tubular epithelial cells from acute T-cell attack during the T-cell priming stage via interfering with TNF ligand-mediated reverse signaling and possibly promoting Teff apoptosis through FasR upregulation. Our findings supported that the decoy receptor is involved in T-cell modulation in kidney transplant rejection. [ABSTRACT FROM AUTHOR]

The interaction between T-cell receptors (TCRs) and peptide epitopes is highly degenerate: a TCR is capable of interacting productively with a wide range of different peptide ligands, involving not only cross-reactivity proper (similar epitopes elicit strong responses), but also polyspecificity (ligands with distinct physicochemical properties are capable of interacting with the TCR). Degeneracy does not gainsay the fact that TCR recognition is fundamentally specific: for the vast majority of ligands, the functional sensitivity of a given TCR is virtually null whereas this TCR has an appreciable functional sensitivity only for a minute fraction of all possible ligands. Degeneracy can be described mathematically as the probability that the functional sensitivity, of a given TCR to a randomly selected ligand, exceeds a set value. Variation of this value generates a statistical distribution that characterizes TCR degeneracy. This distribution can be modelled on the basis of a Gaussian distribution for the TCR/ligand dissociation energy. The kinetics of the TCR and the MHCI molecule can be used to transform this underlying Gaussian distribution into the observed distribution of functional sensitivity values. In the present paper, the model is extended by accounting explicitly for the kinetics of the interaction between the co-receptor and the MHCI molecule. We show that T-cells can modulate the level of degeneracy by varying the density of co-receptors on the cell surface. This could allow for an analogue of avidity maturation during incipient T-cell responses. [ABSTRACT FROM AUTHOR]

Background: The diagnosis of borderline rejection (BLR) ranges from mild inflammation to clinically significant TCMR and is associated with an increased risk of allograft dysfunction. Currently, there is no consensus regarding its treatment due in part to a lack of biomarkers to identify cases with increased risk for immune-mediated injury. Methods: We identified 60 of 924 kidney transplant recipients (KTRs) with isolated and untreated BLR. We analyzed the impact of predicted indirectly recognizable HLA epitopes (PIRCHE) score on future rejection, de novo DSA development, and recovery to baseline allograft function. Additionally, we compared the outcomes of different Banff rejection phenotypes. Results: Total PIRCHE scores were significantly higher in KTRs with BLR compared to the entire study population (p=0.016). Among KTRs with BLR total PIRCHE scores were significantly higher in KTRs who developed TCMR/ABMR in follow-up biopsies (p=0.029). Notably, the most significant difference was found in PIRCHE scores for the HLA-A locus (p=0.010). PIRCHE scores were not associated with the development of de novo DSA or recovery to baseline allograft function among KTRs with BLR (p>0.05). However, KTRs under cyclosporine-based immunosuppression were more likely to develop de novo DSA (p=0.033) than those with tacrolimus, whereas KTRs undergoing retransplantation were less likely to recover to baseline allograft function (p=0.003). Conclusions: High PIRCHE scores put KTRs with BLR at an increased risk for future TCMR/ABMR and contribute to improved immunological risk stratification. The benefit of anti-rejection treatment, however, needs to be evaluated in future studies. [ABSTRACT FROM AUTHOR]

Control of human ascariasis, the most prevalent neglected tropical disease globally affecting 450 million people, mostly relies on mass drug administration of anthelmintics. However, chemotherapy alone is not efficient due to the high re-infection rate for people who live in the endemic area. The development of a vaccine that reduces the intensity of infection and maintains lower morbidity should be the primary target for infection control. Previously, our group demonstrated that immunization with crude Ascaris antigens in mice induced an IgG-mediated protective response with significant worm reduction. Here, we aimed to develop a multipeptide chimera vaccine based on conserved B-cell epitopes predicted from 17 common helminth proteomes using a bioinformatics algorithm. More than 480 B-cell epitopes were identified that are conserved in all 17 helminths. The Ascaris -specific epitopes were selected based on their reactivity to the pooled sera of mice immunized with Ascaris crude antigens or infected three times with A. suum infective eggs. The top 35 peptides with the strongest reactivity to Ascaris immune serum were selected to construct a chimeric antigen connected in sequence based on conformation. This chimera, called ASCVac-1, was produced as a soluble recombinant protein in an Escherichia coli expression system and, formulated with MPLA, was used to immunize mice. Mice immunized with ASCVac-1/MPLA showed around 50% reduced larvae production in the lungs after being challenged with A. suum infective eggs, along with significantly reduced inflammation and lung tissue/function damage. The reduced parasite count and pathology in infected lungs were associated with strong Th2 immune responses characterized by the high titers of antigen-specific IgG and its subclasses (IgG1, IgG2a, and IgG3) in the sera and significantly increased IL-4, IL-5, IL-13 levels in lung tissues. The reduced IL-33 titers and stimulated eosinophils were also observed in lung tissues and may also contribute to the ASCVac-1-induced protection. Taken together, the preclinical trial with ASCVac-1 chimera in a mouse model demonstrated its significant vaccine efficacy associated with strong IgG-based Th2 responses, without IgE induction, thus reducing the risk of an allergic response. All results suggest that the multiepitope-based ASCVac-1 chimera is a promising vaccine candidate against Ascaris sp. infections. [ABSTRACT FROM AUTHOR]

Whole genome sequencing of Epstein-Barr virus (EBV) isolates from around the world has uncovered pervasive strain heterogeneity, but the forces driving strain diversification and the impact on immune recognition remained largely unknown. Using a data mining approach, we analyzed more than 300 T-cell epitopes in 168 published EBV strains. Polymorphisms were detected in approximately 65% of all CD8+ and 80% of all CD4+ T-cell epitopes and these numbers further increased when epitope flanking regions were included. Polymorphisms in CD8+ T-cell epitopes often involved MHC anchor residues and resulted in changes of the amino acid subgroup, suggesting that only a limited number of conserved T-cell epitopes may represent generic target antigens against different viral strains. Although considered the prototypic EBV strain, the rather low degree of overlap with most other viral strains implied that B95.8 may not represent the ideal reference strain for T-cell epitopes. Instead, a combinatorial library of consensus epitopes may provide better targets for diagnostic and therapeutic purposes when the infecting strain is unknown. Polymorphisms were significantly enriched in epitope versus non-epitope protein sequences, implicating immune selection in driving strain diversification. Remarkably, CD4+ T-cell epitopes in EBNA2, EBNA-LP, and the EBNA3 family appeared to be under negative selection pressure, hinting towards a beneficial role of immune responses against these latency type III antigens in virus biology. These findings validate this immunoinformatics approach for providing novel insight into immune targets and the intricate relationship of host defense and virus evolution that may also pertain to other pathogens. [ABSTRACT FROM AUTHOR]

Background: Somatic mutations or post-translational modifications of proteins result in changes that enable immune recognition. One such post-translational modification is citrullination, the conversion of arginine residues to citrulline. Citrullinated peptides are presented on MHC class II (MHCII) via autophagy which is upregulated by cellular stresses such as tumourigenesis. Methods: Peptides were eluted from B16 melanoma expressing HLA-DP4 and analysed by mass spectrometry to profile the presented citrullinated repertoire. Initially, seven of the identified citrullinated peptides were used in combination to vaccinate HLA-DP4 transgenic mice. Immune responses were characterised from the combination and individual vaccines by ex vivo cytokine ELISpot assay and assessed for tumour therapy. Results: The combination vaccine induced only weak anti-tumour therapy in the B16cDP4 melanoma model. Immune phenotyping revealed a dominant IFNγ response to citrullinated matrix metalloproteinase-21 peptide (citMMP21) and an IL-10 response to cytochrome p450 peptide (citCp450). Exclusion of the IL-10 inducing citCp450 peptide from the combined vaccine failed to recover a strong anti-tumour response. Single peptide immunisation confirmed the IFNγ response from citMMP21 and the IL-10 response from citCp450 but also showed that citrullinated Glutamate receptor ionotropic (citGRI) peptide stimulated a low avidity IFNγ response. Interestingly, both citMMP21 and citGRI peptides individually, stimulated strong anti-tumour responses that were significantly better than the combined vaccine. In line with the citGRI T cell avidity, it required high dose immunisation to induce an anti-tumour response. This suggests that as the peptides within the combined vaccine had similar binding affinities to MHC-II the combination vaccine may have resulted in lower presentation of each epitope and weak anti-tumour immunity. Conclusion: We demonstrate that tumours present citrullinated peptides that can stimulate Th1 and regulatory responses and that competition likely exists between similar affinity peptides. Characterisation of responses from epitopes identified by peptide elution are necessary to optimise selection for tumour therapy. [ABSTRACT FROM AUTHOR]

Since their discovery, antibodies capable of broad neutralisation have been at the forefront of HIV-1 research and are of particular interest due to in vivo passive transfer studies demonstrating their potential to provide protection. Currently an exact definition of what is required for a monoclonal antibody to be classed as a broadly neutralising antibody (bnAb) has not yet been established. This has led to hundreds of antibodies with varying neutralisation breadth being studied and has given insight into antibody maturation pathways and epitopes targeted. However, even with this knowledge, immunisation studies and vaccination trials to date have had limited success in eliciting antibodies with neutralisation breadth. For this reason there is a growing need to identify factors specifically associated with bnAb development, yet to do this a set of criteria is necessary to distinguish bnAbs from non-bnAbs. This review aims to define what it means to be a HIV-1 bnAb by comparing neutralisation breadth, genetic features and epitopes of bnAbs, and in the process highlights the challenges of comparing the array of antibodies that have been isolated over the years. [ABSTRACT FROM AUTHOR]

The four serotypes of Dengue virus (DENV1-4) are arboviruses (ar thropod- bo rne viruses) that belong to the Flavivirus genus, Flaviviridae family. They are the causative agents of an infectious disease called dengue, an important global public health problem with significant social-economic impact. Thus, the development of safe and effective dengue vaccines is a priority according to the World Health Organization. Only one anti-dengue vaccine has already been licensed in endemic countries and two formulations are under phase III clinical trials. In this study, we aimed to compare the main anti-dengue virus vaccines, DENGVAXIA®, LAV-TDV, and TAK-003, regarding their antigens and potential to protect. We studied the conservation of both, B and T cell epitopes involved in immunological control of DENV infection along with vaccine viruses and viral isolates. In addition, we assessed the population coverage of epitope sets contained in each vaccine formulation with regard to different human populations. As main results, we found that all three vaccines contain the main B cell epitopes involved in viral neutralization. Similarly, LAV-TDV and TAK-003 contain most of T cell epitopes involved in immunological protection, a finding not observed in DENGVAXIA®, which explains main limitations of the only licensed dengue vaccine. In summary, the levels of presence and absence of epitopes that are target for protective immune response in the three main anti-dengue virus vaccines are shown in this study. Our results suggest that investing in vaccines that contain the majority of epitopes involved in protective immunity (cellular and humoral arms) is an important issue to be considered. [ABSTRACT FROM AUTHOR]

HI, a fusion protein that consists of the alpha-toxin (Hla) and the N2 domain of iron surface determinant B (IsdB), is one of the antigens in the previously reported S. aureus vaccine rFSAV and has already entered phase II clinical trials. Previous studies revealed that HI is highly immunogenic in both mice and healthy volunteers, and the humoral immune response plays key roles in HI-mediated protection. In this study, we further investigated the protective efficacy of immunization with HI plus four different adjuvants in a mouse bacteremia model. Results showed that HI-mediated protection was altered in response to different adjuvants. Using antisera from immunized mice, we identified seven B-cell immunodominant epitopes on Hla and IsdB, including 6 novel epitopes (Hla1-18, Hla84-101, Hla186-203, IsdB342-359, IsdB366-383, and IsdB384-401). The immunodominance of B-cell epitopes, total IgG titers and the levels of IFN-γ and IL-17A from mice immunized with HI plus different adjuvants were different from each other, which may explain the difference in protective immunity observed in each immunized group. Thus, our results indicate that adjuvants largely affected the immunodominance of epitopes and the protective efficacy of HI, which may guide further adjuvant screening for vaccine development and optimization. [ABSTRACT FROM AUTHOR]

Autoantigen-directed tolerance can be induced by certain nucleosomal histone peptide epitope/s in nanomolar dosage leading to sustained remission of disease in mice with spontaneous SLE. By contrast, lupus is accelerated by administration of intact (whole) histones, or whole nucleosomes in microparticles from apoptotic cells, or by post-translationally acetylated histone-peptides. Low-dose therapy with the histone-peptide epitopes simultaneously induces TGFβ and inhibits IL-6 production by DC in vivo , especially pDC, which then induce CD4+CD25+ Treg and CD8+ Treg cells that suppress pathogenic autoimmune response. Both types of induced Treg cells are FoxP3+ and act by producing TGFβ at close cell-to-cell range. No anaphylactic adverse reactions, or generalized immunosuppression have been detected in mice injected with the peptides, because the epitopes are derived from evolutionarily conserved histones in the chromatin; and the peptides are expressed in the thymus during ontogeny, and their native sequences have not been altered. The peptide-induced Treg cells can block severe lupus on adoptive transfer reducing inflammatory cell reaction and infiltration in the kidney. In Humans , similar potent Treg cells are generated by the histone peptide epitopes in vitro in lupus patients' PBMC, inhibiting anti-dsDNA autoantibody and interferon production. Furthermore, the same types of Treg cells are generated in lupus patients who are in very long-term remission (2-8 years) after undergoing autologous hematopoietic stem cell transplantation. These Treg cells are not found in lupus patients treated conventionally into clinical remission (SLEDAI of 0); and consequently they still harbor pathogenic autoimmune cells, causing subclinical damage. Although antigen-specific therapy with pinpoint accuracy is suitable for straight-forward organ-specific autoimmune diseases, Systemic Lupus is much more complex. The histone peptide epitopes have unique tolerogenic properties for inhibiting Innate immune cells (DC), T cells and B cell populations that are both antigen-specifically and cross-reactively involved in the pathogenic autoimmune response in lupus. The histone peptide tolerance is a natural and non-toxic therapy suitable for treating early lupus, and also maintaining lupus patients after toxic drug therapy. The experimental steps, challenges and possible solutions for successful therapy with these peptide epitopes are discussed in this highly focused review on Systemic Lupus. [ABSTRACT FROM AUTHOR]

Non-natural modifications are widely introduced into peptides to improve their therapeutic efficacy, but their impact on immunogenicity remains largely unknown. As the CD4 T-cell response is a key factor in triggering immunogenicity, we investigated the effect of introducing D-amino acids (Daa), amino isobutyric acid (Aib), N-methylation, Cα-methylation, reduced amide, and peptoid bonds into an immunoprevalent T-cell epitope on binding to a set of HLA-DR molecules, recognition, and priming of human T cells. Modifications are differentially accepted at multiple positions, but are all tolerated in the flanking regions. Introduction of Aib and Daa in the binding core had the most deleterious effect on binding to HLA-DR molecules and T-cell activation. Their introduction at the positions close to the P1 anchor residue abolished T-cell priming, suggesting they might be sufficient to dampen peptide immunogenicity. Other modifications led to variable effects on binding to HLA-DR molecules and T-cell reactivity, but none exhibited an increased ability to stimulate T cells. Altogether, non-natural modifications appear generally to diminish binding to HLA-DR molecules and hence T-cell stimulation. These data might guide the design of therapeutic peptides to make them less immunogenic. [ABSTRACT FROM AUTHOR]

The adaptive immune system in vertebrates has evolved to recognize non-self antigens, such as proteins expressed by infectious agents and mutated cancer cells. T cells play an important role in antigen recognition by expressing a diverse repertoire of antigen-specific receptors, which bind epitopes to mount targeted immune responses. Recent advances in high-throughput sequencing have enabled the routine generation of T-cell receptor (TCR) repertoire data. Identifying the specific epitopes targeted by different TCRs in these data would be valuable. To accomplish that, we took advantage of the ever-increasing number of TCRs with known epitope specificity curated in the Immune Epitope Database (IEDB) since 2004. We compared seven metrics of sequence similarity to determine their power to predict if two TCRs have the same epitope specificity. We found that a comprehensive k -mer matching approach produced the best results, which we have implemented into TCRMatch, an openly accessible tool (http://tools.iedb.org/tcrmatch/) that takes TCR β-chain CDR3 sequences as an input, identifies TCRs with a match in the IEDB, and reports the specificity of each match. We anticipate that this tool will provide new insights into T cell responses captured in receptor repertoire and single cell sequencing experiments and will facilitate the development of new strategies for monitoring and treatment of infectious, allergic, and autoimmune diseases, as well as cancer. [ABSTRACT FROM AUTHOR]

CD8+ T cell immune monitoring aims at measuring the size and functions of antigen-specific CD8+ T cell populations, thereby providing insights into cell-mediated immunity operational in a test subject. The selection of peptides for ex vivo CD8+ T cell detection is critical because within a complex antigen exists a multitude of potential epitopes that can be presented by HLA class I molecules. Further complicating this task, there is HLA class I polygenism and polymorphism which predisposes CD8+ T cell responses towards individualized epitope recognition profiles. In this study, we compare the actual CD8+ T cell recognition of a well-characterized model antigen, human cytomegalovirus (HCMV) pp65 protein, with its anticipated epitope coverage. Due to the abundance of experimentally defined HLA-A*02:01-restricted pp65 epitopes, and because in silico epitope predictions are most advanced for HLA-A*02:01, we elected to focus on subjects expressing this allele. In each test subject, every possible CD8+ T cell epitope was systematically covered testing 553 individual peptides that walk the sequence of pp65 in steps of single amino acids. Highly individualized CD8+ T cell response profiles with aleatory epitope recognition patterns were observed. No correlation was found between epitopes' ranking on the prediction scale and their actual immune dominance. Collectively, these data suggest that accurate CD8+ T cell immune monitoring may necessitate reliance on agnostic mega peptide pools, or brute force mapping, rather than electing individual peptides as representative epitopes for tetramer and other multimer labeling of surface antigen receptors. [ABSTRACT FROM AUTHOR]

Infections are most common and most severe at the extremes of age, the young and the elderly. Vaccination can be a key approach to enhance immunogenicity and protection against pathogens in these vulnerable populations, who have a functionally distinct immune system compared to other age groups. More than 50% of the vaccine market is for pediatric use, yet to date vaccine development is often empiric and not tailored to molecular distinctions in innate and adaptive immune activation in early life. With modern vaccine development shifting from whole-cell based vaccines to subunit vaccines also comes the need for formulations that can elicit a CD8+ T cell response when needed, for example, by promoting antigen cross-presentation. While our group and others have identified many cellular and molecular determinants of successful activation of antigen-presenting cells, B cells and CD4+ T cells in early life, much less is known about the ontogeny of CD8+ T cell induction. In this review, we summarize the literature pertaining to the frequency and phenotype of newborn and infant CD8+ T cells, and any evidence of induction of CD8+ T cells by currently licensed pediatric vaccine formulations. In addition, we review the molecular determinants of antigen cross-presentation on MHC I and successful CD8+ T cell induction and discuss potential distinctions that can be made in children. Finally, we discuss recent advances in development of novel adjuvants and provide future directions for basic and translational research in this area. [ABSTRACT FROM AUTHOR]

Somatic mutations in cancers affecting protein coding genes can give rise to potentially therapeutic neoepitopes. These neoepitopes can guide Adoptive Cell Therapies and Peptide- and RNA-based Neoepitope Vaccines to selectively target tumor cells using autologous patient cytotoxic T-cells. Currently, researchers have to independently align their data, call somatic mutations and haplotype the patient's HLA to use existing neoepitope prediction tools. We present ProTECT, a fully automated, reproducible, scalable, and efficient end-to-end analysis pipeline to identify and rank therapeutically relevant tumor neoepitopes in terms of potential immunogenicity starting directly from raw patient sequencing data, or from pre-processed data. The ProTECT pipeline encompasses alignment, HLA haplotyping, mutation calling (single nucleotide variants, short insertions and deletions, and gene fusions), peptide:MHC binding prediction, and ranking of final candidates. We demonstrate the scalability, efficiency, and utility of ProTECT on 326 samples from the TCGA Prostate Adenocarcinoma cohort, identifying recurrent potential neoepitopes from TMPRSS2-ERG fusions, and from SNVs in SPOP. We also compare ProTECT with results from published tools. ProTECT can be run on a standalone computer, a local cluster, or on a compute cloud using a Mesos backend. ProTECT is highly scalable and can process TCGA data in under 30 min per sample (on average) when run in large batches. ProTECT is freely available at https://www.github.com/BD2KGenomics/protect. [ABSTRACT FROM AUTHOR]

Immunoglobulin E (IgE) is pivotal for manifestation and persistence of most immediate-type allergies and some asthma phenotypes. Consequently, IgE represents a crucial target for both, diagnostic purposes as well as therapeutic approaches. In fact, allergen-specific immunotherapy – aiming to re-route an IgE-based inflammatory response into an innocuous immune reaction against the allergen – is the only curative approach for IgE-mediated allergic diseases known so far. However, this requires the cognate allergen to be known. Unfortunately, even in well-characterized allergics or asthmatics, often just a small fraction of total IgE can be assigned to specific target allergens. To overcome this knowledge gap, we have devised an analytical platform for unbiased IgE target epitope detection. The system relies on chemically produced random peptide libraries immobilized on polystyrene beads ("one-bead-one-compound (OBOC) libraries") capable to present millions of different peptide motifs simultaneously to immunoglobulins from biological samples. Beads binding IgE are highlighted with a fluorophore-labeled anti-IgE antibody allowing fluorescence-based detection and isolation of positives, which then can be characterized by peptide sequencing. Setting-up this platform required an elaborate optimization process including proper choice of background suppressants, secondary antibody and fluorophore label as well as incubation conditions. For optimal performance our procedure involves a sophisticated pre-adsorption step to eliminate beads that react nonspecifically with anti-IgE secondary antibodies. This step turned out to be important for minimizing detection of "false positive" motifs that otherwise would erroneously be classified as IgE epitopes. In validation studies we were able to retrieve artificial test-peptide beads spiked into our library by using IgE directed against those test-peptides at physiological concentrations (≤20 IU/ml of specific IgE), and disease-relevant bead-bound epitopes of the major peanut allergen Ara h 2 by screening with sera from peanut allergics. Thus, we established a platform with which one can find and validate new immunoglobulin targets using patient material which displays a largely unknown immunoglobulin repertoire. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an outbreak of a pandemic worldwide. For better understanding the viral spike (S) protein variations and its potential effects on the interaction with the host immune system and also in vaccine development, the cell epitopes, glycosylation profile and their changes during the global transmission course were characterized and compared with SARS-CoV for their glycosylation profile. We analyzed totally 7,813 sequences screened from 8,897 whole genome sequences on GISAID database up to April 26, and 18 S protein amino acid variations with relatively high frequency (≥10−3) were identified. A total of 228 sequences of variants had multiple variations, of note, most of them harboring the D614G mutation. Among the predicted 69 linear B cell epitopes, 175 discontinuous B cell epitopes and 41 cytotoxic T lymphocyte epitopes in the viral S protein, we found that the protein structure and its potential function of some sites changed, such as the linear epitope length shortened and discontinuous epitope disappeared of G476S. In addition, we detected 9 predicted N-glycosylation sites and 3 O-glycosylation sites unique to SARS-CoV-2, but no evidently observed variation of the glycan sites so far. Our findings provided an important snapshot of temporal and geographical distributions on SARS-CoV-2 S protein cell epitopes and glycosylation sites, which would be an essential basis for the selection of vaccine candidates. [ABSTRACT FROM AUTHOR]

The development of allergic disease involves the production of IgE antibodies upon allergen exposure in a process called sensitization. IgE binds to receptors on the surface of mast cells and basophils, and subsequent allergen exposure leads to cross-linking of IgE antibodies and release of cell mediators that cause allergy symptoms. Although this process is quite well-understood, very little is known about the epitopes on the allergen recognized by IgE, despite the importance of the allergen-antibody interaction for the allergic response to occur. This review discusses efforts to analyze allergen-antibody interactions, from the original epitope mapping studies using linear peptides or recombinant allergen fragments, to more sophisticated technologies, such as X-ray crystallography and nuclear magnetic resonance. These state-of-the-art approaches, combined with site-directed mutagenesis, have led to the identification of conformational IgE epitopes. The first structures of an allergen (egg lysozyme) in complex with Fab fragments from IgG antibodies were determined in the 1980s. Since then, IgG has been used as surrogate for IgE, due to the difficulty of obtaining monoclonal IgE antibodies. Technical developments including phage display libraries have contributed to progress in epitope mapping thanks to the isolation of IgE antibody constructs from combinatorial libraries made from peripheral blood mononuclear cells of allergic donors. Most recently, single B cell antibody sequencing and human hybridomas are new breakthrough technologies for finally obtaining human IgE monoclonal antibodies, ideal for epitope mapping. The information on antigenic determinants will facilitate the design of hypoallergens for immunotherapy and the investigation of the fundamental mechanisms of the IgE response. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) induces strong T and B cell responses upon infection. Hence, it is difficult to determine the contribution of cell-mediated immunity alone in the long lasting protection against DENV infection and disease. Numerous CD4+ and CD8+ T cell epitopes have been identified, mainly in the non-structural proteins of DENV. Taking into account the immunogenicity and peptide sequence conservation among the different DENV serotypes, a minimal DENV antigen, called DENV1-NS, has been designed. This antigen is enriched in conserved and highly antigenic epitopes located in the NS3, NS4B, and NS5 regions of DENV1. To evaluate the ability of the DENV1-NS poly-epitope to express the antigenic peptides in the context of different HLA class I molecules, we established its in vivo immunogenicity by measuring, after DNA immunization and electroporation, the activation of DENV-specific CD8 T cells in transgenic mice expressing the human HLA-A*0201, -A*2402, -B*0702, and -B*3502 class I alleles. We then engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding DENV1-NS and tested immunogenicity and protection in these human HLA class I transgenic mice, after transient blockade of the interferon (IFN) type I receptor. Significant protection was observed, after two injections of the mRNA vaccine. Collectively, these data strongly support the development of T cell-based vaccines targeting immunodominant T cell epitopes that generate potent virus-specific T cell responses conferring immunity against DENV infection. [ABSTRACT FROM AUTHOR]

Bullous pemphigoid (BP) is a common autoimmune blistering disease in which autoantibodies target the hemidesmosomal components BP180 and/or BP230 in basal keratinocytes. In BP, 80 to 90% of autoantibodies target the juxtamembranous extracellular non-collagenous 16th A (NC16A) domain of BP180. Recently, the administration of dipeptidyl peptidase-IV inhibitors (DPP4i), which are widely used as antihyperglycemic drugs, has been recognized to be a causative factor for BP. DPP4i-associated BP (DPP4i-BP) autoantibodies tend to target epitopes on non-NC16A regions of BP180, and the pathomechanism for the development of the unique autoantibodies remains unknown. To address the characteristics of DPP4i-BP autoantibodies in detail, we performed epitope analysis of 18 DPP4i-BP autoantibodies targeting the non-NC16A domains of BP180 using various domain-specific as well as plasmin-digested polypeptides derived from recombinant BP180. Firstly, Western blotting showed that only one DPP4i-BP serum reacted with the epitopes on the intracellular domain of BP180, and no sera reacted with the C-terminal domain of the molecule. In addition, only 2 DPP4i-BP sera reacted with BP230 as determined by enzyme-linked immunosorbent assay. Thus, DPP4i-BP autoantibodies were found to mainly target the non-NC16A mid-portion of the extracellular domain of BP. Interestingly, Western blotting using plasmin-digested BP180 as a substrate revealed that all of the DPP4i-BP sera reacted more intensively with the 97-kDa processed extracellular domain of BP180, which is known as the LABD97 autoantigen, than full-length BP180 did. All of the DPP4i-BP autoantibodies targeting the LABD97 autoantigen were IgG1, and IgG4 was observed to react with the molecule in only 7 cases (38.9%). In summary, the present study suggests that IgG1-class autoantibodies targeting epitopes on the processed extracellular domain of BP180, i.e., LABD97, are the major autoantibodies in DPP4i-BP. [ABSTRACT FROM AUTHOR]

Immunodominant T cell epitopes preferentially targeted in multiple individuals are the critical element of successful vaccines and targeted immunotherapies. However, the underlying principles of this "convergence" of adaptive immunity among different individuals remain poorly understood. To quantitatively describe epitope immunogenicity, here we propose a supervised machine learning framework generating probabilistic estimates of immunogenicity, termed "immunogenicity scores," based on the numerical features computed through sequence-based simulation approximating the molecular scanning process of peptides presented onto major histocompatibility complex (MHC) by the human T cell receptor (TCR) repertoire. Notably, overlapping sets of intermolecular interaction parameters were commonly utilized in MHC-I and MHC-II prediction. Moreover, a similar simulation of individual TCR-peptide interaction using the same set of interaction parameters yielded correlates of TCR affinity. Pathogen-derived epitopes and tumor-associated epitopes with positive T cell reactivity generally had higher immunogenicity scores than non-immunogenic counterparts, whereas thymically expressed self-epitopes were assigned relatively low scores regardless of their immunogenicity annotation. Immunogenicity score dynamics among single amino acid mutants delineated the landscape of position- and residue-specific mutational impacts. Simulation of position-specific immunogenicity score dynamics detected residues with high escape potential in multiple epitopes, consistent with known escape mutations in the literature. This study indicates that targeting of epitopes by human adaptive immunity is to some extent directed by defined thermodynamic principles. The proposed framework also has a practical implication in that it may enable to more efficiently prioritize epitope candidates highly prone to T cell recognition in multiple individuals, warranting prospective validation across different cohorts. [ABSTRACT FROM AUTHOR]

Leishmania (V.) braziliensis is the etiological agent of Cutaneous (CL) and Mucocutaneous leishmaniasis (ML) in the New World. CL can be more benign but ML can be severe and disfiguring. Immunity to these diseases include hypersensitivity, an enhanced inflammatory response with strong IFN-γ and TNF-α secretion. Additionally, the production of IL-10 which down modulates the immune response is reduced. The Nucleoside hydrolase (NH36) of Leishmania (L.) donovani is the main antigen of the Leishmune veterinary vaccine and its F3 domain induces a CD4+ T cell-mediated protection against L. (L.) infantum chagasi infection. Prevention of L. (L.) amazonensis infection requires in contrast an additional CD8+ T cell mediated response induced by the F1 domain. Consequently, the F1F3 recombinant chimera, which contains both domains cloned in tandem, optimized the vaccine efficacy against L. (L.) amazonensis mouse infection. We compared the efficacies of NH36, F1, F3, and the FIF3 chimera against L. (V.) braziliensis mouse infection. The F1F3 chimera increased the NH36 specific IgA and response before and after infection and the IgG and IgG3 levels after challenge. It also induced a 49% stronger intradermal response to leishmanial antigen (IDR) than NH36 that was positively correlated to the levels of IFN-γ and TNF-α, IgG, IgG2a, IgG2b, and IgG3 anti-NH36 antibodies. However, stronger Th1 responses with elevated IFN-γ / IL-10 and TNF-α / IL-10 ratios were promoted by the F3 and F1 vaccines and detected in infected controls while the F1F3 chimera promoted the highest IL-10 secretion, which reduced the pathological Th1 response, and characterized the induction of a mixed and/or T-cell regulatory response. We identified the epitopes responsible for these immune responses. The F3 vaccine induced the earliest immunity and after challenge, the F1F3 chimera promoted the highest CD4+ and CD8+ cytokine-secreting T cell responses, and the predominant frequencies of multifunctional CD4+ and CD8+IL-2+TNF-α+IFN-γ+ T cells. Also as observed against L. (L.) amazonensis infection, the F1F3 chimera showed the strongest reduction of the ear lesions sizes induced by L. (V.) braziliensis. Our results confirm the potential use of the F1F3 chimera in a multi-species cross-protective vaccine against L. (V.) braziliensis. [ABSTRACT FROM AUTHOR]

B-cells can neutralize pathogenic molecules by targeting them with extreme specificity using receptors secreted or expressed on their surface (antibodies). This is achieved via molecular interactions between the paratope (i.e., the antibody residues involved in the binding) and the interacting region (epitope) of its target molecule (antigen). Discerning the rules that define this specificity would have profound implications for our understanding of humoral immunogenicity and its applications. The aim of this work is to produce improved, antibody-specific epitope predictions by exploiting features derived from the antigens and their cognate antibodies structures, and combining them using statistical and machine learning algorithms. We have identified several geometric and physicochemical features that are correlated in interacting paratopes and epitopes, used them to develop a Monte Carlo algorithm to generate putative epitopes-paratope pairs, and train a machine-learning model to score them. We show that, by including the structural and physicochemical properties of the paratope, we improve the prediction of the target of a given B-cell receptor. Moreover, we demonstrate a gain in predictive power both in terms of identifying the cognate antigen target for a given antibody and the antibody target for a given antigen, exceeding the results of other available tools. [ABSTRACT FROM AUTHOR]

BackgroundPrediction of T cell immunogenicity is a topic of considerable interest, both in terms of basic understanding of the mechanisms of T cells responses and in terms of practical applications. HLA binding affinity is often used to predict T cell epitopes, since HLA binding affinity is a key requisite for human T cell immunogenicity. However, immunogenicity at the population it is complicated by the high level of variability of HLA molecules, potential other factors beyond HLA as well as the frequent lack of HLA typing data. To overcome those issues, we explored an alternative approach to identify the common characteristics able to distinguish immunogenic peptides from non-recognized peptides.MethodsSets of dominant epitopes derived from peer-reviewed published papers were used in conjunction with negative peptides from the same experiments/donors to train neural networks and generate an “immunogenicity score.” We also compared the performance of the immunogenicity score with previously described method for immunogenicity prediction based on HLA class II binding at the population level.ResultsThe immunogenicity score was validated on a series of independent datasets derived from the published literature, representing 57 independent studies where immunogenicity in human populations was assessed by testing overlapping peptides spanning different antigens. Overall, these testing datasets corresponded to over 2,000 peptides and tested in over 1,600 different human donors. The 7-allele method prediction and the immunogenicity score were associated with similar performance [average area under the ROC curve (AUC) values of 0.703 and 0.702, respectively] while the combined methods reached an average AUC of 0.725. This increase in average AUC value is significant compared with the immunogenicity score (p = 0.0135) and a strong trend toward significance is observed when compared to the 7-allele method (p = 0.0938). The new immunogenicity score method is now freely available using CD4 T cell immunogenicity prediction tool on the Immune Epitope Database website (http://tools.iedb.org/CD4episcore).ConclusionThe new immunogenicity score predicts CD4 T cell immunogenicity at the population level starting from protein sequences and with no need for HLA typing. Its efficacy has been validated in the context of different antigen sources, ethnicities, and disparate techniques for epitope identification.

Helminth infections, by nematodes, trematodes, or cestodes, can lead to the modulation of host immune responses. This allows long-duration parasite infections and also impacts responses to co-infections. Surface, secreted, excreted, and shed proteins are thought to play a major role in modulation. A commonly reported feature of such immune modulation is the role of T regulatory (Treg) cells and IL-10. Efforts to identify helminth proteins, which cause immunomodulation, have identified candidates but not provided clarity as to a uniform mechanism driving modulation. In this study, we applied a bioinformatics systems approach, allowing us to analyze predicted T-cell epitopes of 17 helminth species and the responses to their surface proteins. In addition to major histocompatibility complex (MHC) binding, we analyzed amino acid motifs that would be recognized by T-cell receptors [T-cell-exposed motifs (TCEMs)]. All the helminth species examined have, within their surface proteins, peptides, which combine very common TCEMs with predicted high affinity binding to many human MHC alleles. This combination of features would result in large cognate T cell and a high probability of eliciting Treg responses. The TCEMs, which determine recognition by responding T-cell clones, are shared to a high degree between helminth species and with Plasmodium falciparum and Mycobacterium tuberculosis , both common co-infecting organisms. The implication of our observations is not only that Treg cells play a significant role in helminth-induced immune modulation but also that the epitope specificities of Treg responses are shared across species and genera of helminth. Hence, the immune response to a given helminth cannot be considered in isolation but rather forms part of an epitope ecosystem, or microenvironment, in which potentially immunosuppressive peptides in the helminth network via their common T-cell receptor recognition signals with T-cell epitopes in self proteins, microbiome, other helminths, and taxonomically unrelated pathogens. Such a systems approach provides a high-level view of the antigen-immune system signaling dynamics that may bias a host’s immune response to helminth infections toward immune modulation. It may indicate how helminths have evolved to select for peptides that favor long-term parasite host coexistence. [ABSTRACT FROM AUTHOR]

Current schistosomiasis control strategies are mainly based on chemotherapy, but the development of a vaccine against this parasitic disease would contribute to a long-lasting decrease in disease spectrum and transmission. When it comes to vaccine candidates, several genes encoding Schistosoma mansoni proteins expressed at the mammalian host–parasite interface have been tested. Among the most promising molecules are the proteins present on the tegument and digestive tract of the parasite. In this study, we evaluate the potential of Sm KI-1, the first Kunitz-type protease inhibitor functionally characterized in S. mansoni , as a vaccine candidate. Bioinformatic analysis points to the C-terminal fragment as the main region of the molecule responsible for the development of a potential protective immune response induced by Sm KI-1. Therefore, for the vaccine formulations, we produced the recombinant (r) Sm KI-1 and two different fragments, its Kunitz (KI) domain and its C-terminal tail. First, we demonstrate that mice immunized with recombinant SmKI-1 (r Sm KI-1) or its fragments, formulated with Freund’s adjuvant, induced the production of IgG-specific antibodies. Further, all vaccine formulations tested here also induced a Th1-type of immune response, as suggested by the production of IFN-γ and TNF-α by protein-stimulated cultured splenocytes. However, the protective effect conferred by vaccination was only observed in groups which received rSmKI-1 or C-terminal domain vaccines. Mice administered with rSmKI-1 demonstrated reduction of 47% in worm burden, 36% in egg number in mouse livers, and 33% in area of liver granulomas. Additionally, mice injected with C-terminal domain showed reduction of 28% in worm burden, 38% in egg number in liver, and 25% in area of liver granulomas. In contrast, KI domain immunization was unable to reduce worm burden and ameliorate liver pathology after challenge infection. Taken together, our data demonstrated that Sm KI-1 is a potential candidate for use in a vaccine to control schistosomiasis, and its C-terminal tail seems to be the main region of the molecule responsible for protection conferred by this antigen. [ABSTRACT FROM AUTHOR]

A single epitope of Leishmania analog of the receptors for activated C kinase (LACK) from Leishmania major, the polypeptide LACK156–173, is recognized by Vβ4+/Vα8+ T cells, and activate these cells that drives the subsequent T helper (Th)2 response. This study was undertaken to investigate the therapeutic potential of the LACK156–173 epitope in murine autoimmune arthritis models. To explore the influence of the LACK156–173 epitope on murine collagen antibody-induced arthritis, as well as its immunological mechanism, we vaccinated or treated mice with a LACK156–173 epitope expression plasmid or polypeptide. The effect of LACK156–173 epitope was then evaluated by clinical scores, histopathology, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Using flow cytometry, we measured the subsets and maturity of CD11c+ dendritic cells (DCs), as well as T cell polarization, in co-culture experiments. We also measured cytokine gene expression and production. The murine macrophage-like cell line RAW264.7 was used to identify the receptor for the epitope. Vaccination or treatment of the mice with the LACK156–173 epitope expression plasmid or polypeptide ameliorated the severity of arthritis. qRT-PCR analysis revealed that the LACK156–173 epitope improved the balance of effector T cells in synovial tissue compared to that in untreated arthritis controls. Toll-like receptor (TLR) 4 expression was diminished by LACK156–173. The epitope also influenced T cell polarization by regulating the differentiation, maturation, and functions of CD11c+ DCs and upregulating Jagged1 ligand expression. Blocking the mannose receptor (MR) significantly attenuated LACK156–173 epitope-induced macrophage activation. Our data indicate that vaccination or treatment with a single microorganism epitope, LACK156–173, is a highly efficient therapy for murine autoimmune arthritis. The therapeutic effects are mediated by the regulation of the differentiation, maturation, and functions of DCs via MR, resulting in the upregulation of Jagged1 expression and Th2 cell polarization. Our results demonstrate the therapeutic potential of the LACK156–173 epitope in rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Marburg virus (MARV) is a highly lethal hemorrhagic fever virus that is increasingly re-emerging in Africa, has been imported to both Europe and the US, and is also a Tier 1 bioterror threat. As a negative sense RNA virus, MARV has error prone replication which can yield progeny capable of evading countermeasures. To evaluate this vulnerability, we sought to determine the epitopes of 4 llama single-domain antibodies (sdAbs or VHH) specific for nucleoprotein (NP), each capable of forming MARV monoclonal affinity reagent sandwich assays. Here, we show that all sdAb bound the C-terminal region of NP, which was produced recombinantly to derive X-ray crystal structures of the three best performing antibody-antigen complexes. The common epitope is a trio of alpha helices that form a novel asymmetric basin-like depression that accommodates each sdAb paratope via substantial complementarity-determining region (CDR) restructuring. Shared core contacts were complemented by unique accessory contacts on the sides and overlooks of the basin yielding very different approach routes for each sdAb to bind the antigen. The C-terminal region of MARV NP was unable to be crystallized alone and required engagement with sdAb to form crystals suggesting the antibodies acted as crystallization chaperones. While gross structural homology is apparent between the two most conserved helices of MARV and Ebolavirus, the positions and morphologies of the resulting basins were markedly different. Naturally occurring amino acid variations occurring in bat and human Marburgvirus strains all mapped to surfaces distant from the predicted sdAb contacts suggesting a vital role for the NP interface in virus replication. As an essential internal structural component potentially interfacing with a partner protein it is likely the C-terminal epitope remains hidden or "cryptic" until virion disruption occurs. Conservation of this epitope over 50 years of Marburgvirus evolution should make these sdAb useful foundations for diagnostics and therapeutics resistant to drift. [ABSTRACT FROM AUTHOR]

The task of epitope discovery and vaccine design is increasingly reliant on bioinformatics analytic tools and access to depositories of curated data relevant to immune reactions and specific pathogens. The Immune Epitope Database and Analysis Resource (IEDB) was indeed created to assist biomedical researchers in the development of new vaccines, diagnostics, and therapeutics. The Analysis Resource is freely available to all researchers and provides access to a variety of epitope analysis and prediction tools. The tools include validated and benchmarked methods to predict MHC class I and class II binding. The predictions from these tools can be combined with tools predicting antigen processing, TCR recognition, and B cell epitope prediction. In addition, the resource contains a variety of secondary analysis tools that allow the researcher to calculate epitope conservation, population coverage, and other relevant analytic variables. The researcher involved in vaccine design and epitope discovery will also be interested in accessing experimental published data, relevant to the specific indication of interest. The database component of the IEDB contains a vast amount of experimentally derived epitope data that can be queried through a flexible user interface. The IEDB is linked to other pathogen-specific and immunological database resources. [ABSTRACT FROM AUTHOR]