Subset Of Epitopes Research Articles

Discovery and characterization of cross-reactive single-domain antibodies targeting key virulence factors of Salmonellaand Shigella

Abstract Study Objective: Here, we utilize a nanobody yeast display library to identify nanobodies that target virulence factors in Shigellaspp. and Salmonella enterica typhimuriumand determine if these nanobodies (Nbs) can inhibit host cell invasion in vitro. Brief Statement of Methods: Using a synthetic yeast display library containing camelid Nbs with randomized CDR loops, we selected for binders to select Enterobacteriaceae virulence proteins, including IpaD/SipD, IcsA, and IcsP by MACS and FACS. Lead Nbs were cloned and expressed as monomeric VHH domains or Nb-Fc fusions. ELISA and flow cytometry surface staining was used to estimate affinity, and functional activity was assayed via a host cell invasion assay with Shigella flexneri, Shigella sonnei, and Salmonella enterica typhimurium. Summary of Results: Several high affinity Nbs were isolated with specificity for the targe proteins, and a subset of these could bind whole bacterial cells. Lead Nbs were binned into epitopes by competitive binding assays. The efficacy of Nbs against specific epitopes of specific virulence proteins in invasion assays were directly compared. Statement of Conclusions: A direct comparison of high-affinity Nb binders against various epitopes of IpaD/SipD, IcsA, and IcsP reveals that only a subset of epitopes are accessible on whole bacteria and able to inhibit bacterial invasion. These results will inform the development of virulence factor-based vaccines or other immunotherapies. Wellcome Trust (REISTANT Consortium)

Abstract S03-02: Sequence-based prediction of SARS-CoV-2 vaccine targets using a mass spectrometry-based bioinformatics predictor identifies immunogenic T-cell epitopes

Abstract The ongoing COVID-19 pandemic has created an urgency to identify novel vaccine targets for protective immunity against SARS-CoV-2. Consistent with observations for the closely related SARS-CoV, early reports identify a protective role for both humoral and cell-mediated immunity for SARS-CoV-2. In this study, we leveraged our bioinformatics binding prediction tools for human leukocyte antigen (HLA)-I and HLA-II alleles that cover nearly the entire population and were developed using mass spectrometry-based profiling of 74 individual HLA-I and 83 individual HLA-II alleles. We applied these binding predictors, initially developed to predict tumor neoantigen presentation, to identify T-cell epitopes from SARS-CoV-2 proteins. To determine the ability of our tools to identify viral T-cell epitopes, we validated HLA-I and HLA-II predictions on Coronaviridae family epitopes deposited in the Virus Pathogen Database and Analysis Resource (ViPR) database. We then applied our HLA-I and HLA-II predictors to 13 open reading frames (ORFs) of SARS-CoV-2 and identified 11,897 HLA-I and 8,046 HLA-II candidate peptides that were highly ranked for binding. From our SARS-CoV-2 predicted peptide-HLA-I allele pairs, 374 pairs identically matched previously reported pairs in the ViPR database, originating from other coronaviruses with homologous sequences. Of these pairs, 333 (89 %) had a positive HLA-binding assay result, reinforcing the validity of our predictions. Furthermore, we assayed a subset of epitopes with highly predicted binding scores for their ability to be recognized by specific CD8+ T cell in human donor PBMCs. These epitopes were chosen from four structural proteins (S, N, M, E) and one nonstructural protein (ORF1ab) from SARS-CoV-2, and epitopes from all five proteins were found to be immunogenic. Finally, it was important to address the expression of SARS-CoV-2 proteins within cells since their subsequent processing is necessary for MHC presentation and the generation of specific epitopes. We utilized publicly available proteomic data to infer the relative expression of SARS-CoV-2 proteins from infected cell lines and determined that the different proteins vary significantly in their expression levels, with the nucleocapsid being the most highly expressed viral protein across these studies. Our predictions identify few epitopes from each SARS-CoV-2 protein, which are predicted to bind multiple HLA-I or HLA-II alleles, potentially covering over 99% of the USA, European, and Asian populations. Finally, using our bioinformatic platform, we identify multiple putative epitopes that are potential targets for CD4+ and CD8+ T cells whose predicted HLA binding properties cover nearly the entire population. We further propose that when considering the protein expression levels of these epitopes and their ability to elicit a T-cell response, these epitopes may be effective when included in vaccines against SARS-CoV-2 to induce broad cellular immunity. Citation Format: Asaf Poran, Dewi Harjanto, Matt Malloy, Christina M. Arieta, Daniel A. Rothenberg, Divya Lenkala, Marit M. van Buuren, Terri A. Addona, Michael S. Rooney, Lakshmi Srinivasan, Richard B. Gaynor. Sequence-based prediction of SARS-CoV-2 vaccine targets using a mass spectrometry-based bioinformatics predictor identifies immunogenic T-cell epitopes [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S03-02.

Diverse evolutionary patterns of pneumococcal antigens identified by pangenome-wide immunological screening

Characterizing the immune response to pneumococcal proteins is critical in understanding this bacterium's epidemiology and vaccinology. Probing a custom-designed proteome microarray with sera from 35 healthy US adults revealed a continuous distribution of IgG affinities for 2,190 potential antigens from the species-wide pangenome. Reproducibly elevated IgG binding was elicited by 208 "antibody binding targets" (ABTs), which included 109 variants of the diverse pneumococcal surface proteins A and C (PspA and PspC) and zinc metalloprotease A and B (ZmpA and ZmpB) proteins. Functional analysis found ABTs were enriched in motifs for secretion and cell surface association, with extensive representation of cell wall synthesis machinery, adhesins, transporter solute-binding proteins, and degradative enzymes. ABTs were associated with stronger evidence for evolving under positive selection, although this varied between functional categories, as did rates of diversification through recombination. Particularly rapid variation was observed at some immunogenic accessory loci, including a phage protein and a phase-variable glycosyltransferase ubiquitous among the diverse set of genomic islands encoding the serine-rich PsrP glycoprotein. Nevertheless, many antigens were conserved in the core genome, and strains' antigenic profiles were generally stable. No strong evidence was found for any epistasis between antigens driving population dynamics, or redundancy between functionally similar accessory ABTs, or age stratification of antigen profiles. These results highlight the paradox of why substantial variation is observed in only a subset of epitopes. This result may indicate only some interactions between immunoglobulins and ABTs clear pneumococcal colonization or that acquired immunity to pneumococci is an accumulation of individually weak responses to ABTs evolving under different levels of functional constraint.

Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein

BackgroundPlasmodium falciparum apical membrane antigen-1 (AMA1) is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1.MethodsA recombinant adenovirus serotype 5 vector expressing P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software) were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes.ResultsFourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure.ConclusionsThis study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human populations.

Single residue within the antigen translocation complex TAP controls the epitope repertoire by stabilizing a receptive conformation

The recognition of virus infected or malignantly transformed cells by cytotoxic T lymphocytes critically depends on the transporter associated with antigen processing (TAP), which delivers proteasomal degradation products into the endoplasmic reticulum lumen for subsequent loading of major histocompatibility complex class I molecules. Here we have identified a single cysteinyl residue in the TAP complex that modulates peptide binding and translocation, thereby restricting the epitope repertoire. Cysteine 213 in human TAP2 was found to be part of a newly uncovered substrate-binding site crucial for peptide recognition. This residue contacts the peptide in the binding pocket in an orientated manner. The translocation complex can be reversibly inactivated by thiol modification of this cysteinyl residue. As part of an unexpected mechanism, this residue is crucial in complementing the binding pocket for a given subset of epitopes as well as in maintaining a substrate-receptive conformation of the translocation complex.

Functional mapping of protective domains and epitopes in the rotavirus VP6 protein.

The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed in Escherichia coli was examined. Mice (BALB/c) immunized with two 9-microg doses of each of the chimeras and 10 microg of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively; P </= 0.01) following challenge. Because CD produced almost complete protection, we prepared four E. coli-expressed, MBP-fused chimeras containing overlapping fragments of the CD region (i.e., CD1, CD2, CD3, and CD4) whose lengths ranged from 61 to 67 amino acid residues. Following i.n. immunization, CD1, CD2, and CD4 induced significant (P </= 0.004) protection (88, 84, and 92% reduction, respectively). In addition, 11 peptides (18 to 30 residues) of the CD region with between 0 and 13 overlapping amino acids were synthesized. Two 50-microg doses of each peptide with LT(R192G) were administered i.n. to BALB/c mice. Five peptides were found to elicit significant (P </= 0.02) protection. Moreover, a 14-amino-acid region within peptide 6 containing a putative CD4(+) T-cell epitope was found to confer nearly complete protection, suggesting a protective role for CD4(+) T cells. Mice that were protected by fragments BC and CD1 and four of the five protective synthetic peptides did not develop measurable rotavirus antibodies in serum or stool, implying that protection induced by these domains was not dependent on antibody. Together, these observations suggest that multiple regions of VP6 can stimulate protection, a region of VP6 as small as 14 amino acids containing a CD4(+) T-cell epitope can stimulate nearly complete protection, and protection mediated by a subset of epitopes in the VP6 protein does not require antibodies in BALB/c mice.