Identification of three immunodominant motifs with atypical isotype profile scattered over the Onchocerca volvulus proteome.
ABSTRACT: Understanding the immune response upon infection with the filarial nematode Onchocerca volvulus and the mechanisms that evolved in this parasite to evade immune mediated elimination is essential to expand the toolbox available for diagnostics, therapeutics and vaccines development. Using high-density peptide microarrays we scanned the proteome-wide linear epitope repertoire in Cameroonian onchocerciasis patients and healthy controls from Southern Africa which led to the identification of 249 immunodominant antigenic peptides. Motif analysis learned that 3 immunodominant motifs, encompassing 3 linear epitopes, are present in 70, 43, and 31 of these peptides, respectively and appear to be scattered over the entire proteome in seemingly non-related proteins. These linear epitopes are shown to have an atypical isotype profile dominated by IgG1, IgG3, IgE and IgM, in contrast to the commonly observed IgG4 response in chronic active helminth infections. The identification of these linear epitope motifs may lead to novel diagnostic development but further evaluation of cross-reactivity against common co-infecting human nematode infections will be needed.
Project description:In our previous study, a proteome-wide screen was conducted to identify linear epitopes in this parasite's proteome, resulting in the discovery of three immunodominant motifs. Here, we investigated whether such antigenic peptides were found in proteins that were already known as vaccine candidates and excretome/secretome proteins for Onchocerca volvulus This approach led to the identification of 71 immunoreactive stretches in 46 proteins. A deep-dive into the immunoreactivity profiles of eight vaccine candidates that were chosen as most promising candidates for further development (Ov-CPI-2, Ov-ALT-1, Ov-RAL-2, Ov-ASP-1, Ov-103, Ov-RBP-1, Ov-CHI-1, and Ov-B20), resulted in the identification of a poly-glutamine stretch in Ov-RAL-2 that has properties for use as a serodiagnostic marker for O. volvulus infection. A peptide ELISA was developed, and the performance of this assay was evaluated. Based on this assessment, it was found that this assay has a sensitivity of 75.0% [95% CI: 64.9%-83.5%] and a specificity of 98.5% [95% CI: 94.6%-99.8%]. Furthermore, 8.7% reactivity in Asian parasite-infected individuals (8 out of 92) was observed. Besides this identification of a linear epitope marker, the information on the presence of linear epitopes in vaccine candidate proteins might be useful in the study of vaccines for river blindness.
Project description:Current diagnostic tools to determine infection with the helminth parasite Onchocerca volvulus have limited performance characteristics. In previous studies, a proteome-wide screen was conducted to identify linear epitopes in this parasite's proteome, resulting in the discovery of 1110 antigenic peptide fragments. Here, we investigated three of these peptides using peptide ELISA's and evaluated their sensitivity and specificity. Epitope mapping was performed, and peptides were constructed that contained only the minimal epitope, flanked by a linker. Investigation of the performance of these minimal epitope peptides demonstrated that all three of them have a specificity (as defined by lack of response in non-helminth-infected individuals) of 100%, low cross-reactivity (5.6%, 5.6%, and 9.3%, respectively), but low sensitivity (36.9%, 46.5%, and 41.2%, respectively). Some cross-reactivity was observed in samples from individuals infected with soil-transmitted helminths or Brugia malayi. Combining these three minimal epitopes in a single peptide, called OvNMP-48, resulted in a performance that exceeded the sum of the individual epitopes, with a sensitivity of 76.0%, a specificity of 97.4%, and a cross-reactivity of 11.1%. Cross-reactivity was observed in some STH and Brugia malayi-infected individuals. This work opens the opportunity to start exploring how these novel linear epitope markers might become part of the O. volvulus diagnostic toolbox.
Project description:This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/ Onchocerca volvulus is a filarial nematode parasite of humans, causing Onchocerciasis, or River Blindness, which affects over 37 million people, mainly in Africa. It is a severely debilitating disease, which is transmitted to humans by black fly. This project aims to undertake high-throughput sequencing of Onchocerca volvulus transcriptome for de novo assembly of transcripts. The main objective of this project is to recognize genes expressed in given life stages.
Project description:Immunodominant T-cell responses are important for virus clearance. However, the identification of immunodominant T-cell peptide + HLA glycoprotein epitopes has been hindered by the extent of HLA polymorphism and the limitations of predictive algorithms. A simple, systematic approach has been used here to screen for immunodominant CD8(+) T-cell specificities. The analysis targeted healthy HLA-A2(+) donors to allow comparison with responses to the well-studied influenza matrix protein 1 epitope. Although influenza matrix protein 1 was consistently detected in all individual samples in our study, the response to this epitope was only immunodominant in three of eight, whereas for the other five, prominent CD8(+) T-cell responses tended to focus on various peptides from the influenza nucleoprotein that were not presented by HLA-A2. Importantly, with the four immunodominant T-cell epitopes identified here, only one would have been detected by the current prediction programs. The other three peptides would have been either considered too long or classified as not containing typical HLA binding motifs. Our data stress the importance of systematic analysis for discovering HLA-dependent, immunodominant CD8(+) T-cell epitopes derived from viruses and tumors. Focusing on HLA-A2 and predictive algorithms may be too limiting as we seek to develop targeted immunotherapy and vaccine strategies that depend on T cell-mediated immunity.
Project description:Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium's pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is desirable to simplify the identification of structural epitopes, as this would extend the spectrum of novel epitopes to be detected.
Project description:Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors.
Project description:Human onchocerciasis is a serious neglected tropical disease caused by the filarial nematode Onchocerca volvulus that can lead to blindness and chronic disability. Control of the disease relies largely on mass administration of a single drug, and the development of new drugs and vaccines depends on a better knowledge of parasite biology. Here, we describe the chromosomes of O. volvulus and its Wolbachia endosymbiont. We provide the highest-quality sequence assembly for any parasitic nematode to date, giving a glimpse into the evolution of filarial parasite chromosomes and proteomes. This resource was used to investigate gene families with key functions that could be potentially exploited as targets for future drugs. Using metabolic reconstruction of the nematode and its endosymbiont, we identified enzymes that are likely to be essential for O. volvulus viability. In addition, we have generated a list of proteins that could be targeted by Federal-Drug-Agency-approved but repurposed drugs, providing starting points for anti-onchocerciasis drug development.
Project description:We performed epitope mapping studies on the major surface glycoprotein (GP) of Ebola virus (EBOV) using Chemically Linked Peptides on Scaffolds (CLIPS), which form linear and potential conformational epitopes. This method identified monoclonal antibody epitopes and predicted additional epitopes recognized by antibodies in polyclonal sera from animals experimentally vaccinated against or infected with EBOV. Using the information obtained along with structural modeling to predict epitope accessibility, we then constructed 2 DNA vaccines encoding immunodominant and subdominant epitopes predicted to be accessible on EBOV GP. Although a construct designed to produce a membrane-bound oligopeptide was poorly immunogenic, a construct generating a secreted oligopeptide elicited strong antibody responses in mice. When this construct was administered as a boost to a DNA vaccine expressing the complete EBOV GP gene, the resultant antibody response was focused largely toward the less immunodominant epitopes in the oligopeptide. Taken together, the results of this work suggest a utility for this method for immune focusing of antibody responses elicited by vaccination.
Project description:Using phage display and IgG of a goat infected with Caprine Arthritis Encephalitis Virus (CAEV) we obtained families of 7 mer constrained peptides with consensus motifs LxSDPF/Y and SWN/KHWSY and mapped the epitopes mimicked by them at the Env 6-LISDPY-11 and 67-WNTYHW-72 sites of the mature gp135 amino acid sequence. The first epitope fell into the N-terminal immunogenic aa1-EDYTLISDPYGFS- aa14 site identified previously with a synthetic peptide approach; the second epitope has not been described previously. The first epitope is mostly conserved across CAEV isolates whereas the second newly described epitope is extremely conserved in Small Ruminant Lentiviruses env sequences. As being immunodominant, the epitopes are candidate targets for mimotope-mediated diagnosis and/or neutralization.
Project description:The porcine epidemic diarrhea virus (PEDV) spike (S) protein is the major target of neutralizing antibodies against PEDV. Here immunodominant neutralizing epitopes of PEDV were identified using a panel of S-specific monoclonal antibodies (mAbs). Ten of eleven S-specific mAbs successfully neutralized PEDV infectivity in vitro. Notably, epitope mapping by peptide ELISAs revealed that nine of these mAbs recognized linear neutralizing epitopes located in the N-terminus of the S2 glycoprotein subunit (amino acids [aa] 744-759, 747-774 and/or 756-771). Additionally, one mAb recognized a neutralizing epitope located in the C-terminus of S2 (aa 1371-1377), while only one neutralizing mAb reacted against a region of the S1 glycoprotein subunit (aa 499-600). Notably, mAbs that recognized epitopes within the S2 subunit presented the highest neutralizing activity against PEDV. Together these results indicate that the S2 glycoprotein subunit contains major antigenic determinants and, perhaps, the immunodominant neutralizing epitopes of PEDV.