Out of Sight, but Not Out of Mind: Aspects of the Avian Oncogenic Herpesvirus, Marek’s Disease Virus

Marek’s disease virus is an economically important avian herpesvirus that causes tumors and immunosuppression in chickens and turkeys. The virus, disease, and vaccines have been known for more than 50 years, but as knowledge gaps still exists, intensive research is still ongoing. The understanding of MDV complexity can provide scientific insight in topics that cannot be experimented in humans, providing a unique model that is dually useful for the benefit of the poultry industry and for studying general herpesvirology. The present review presents the following topics: the MDV biology, the vaccine’s and virulent virus’ peculiar presence in feathers, protection by vaccination. In addition, two relatively behind the scenes topics are reviewed; first, the meq MDV oncogene and its recent implication in molecular epidemiology and in the MDV virulence determination, and second, the functionality of conformational epitopes of the MDV immunodominant protein, glycoprotein B. Our studies were particular, as they were the only ones describing three-dimensional MDV gB oligomers. MDV gB (glycoprotein B) continuous and discontinuous epitopes were shown to possess distinctive neutralization activities. In contrast, the significance of oligomerization of the viral membrane proteins for the creation of discontinuous epitopes in other herpesviruses was explored extensively.

In silico analysis of a major allergen from Rattus norvegicus, Rat n 1, and cross-reactivity with domestic pets

Background: Lipocalins play a role in the cellular trafficking of pheromones and are involved in allergic responses to domestic pets. However, the cross-reactivity among allergens of this group has been poorly explored, and the pheromone linking capacity is not well characterized. The aim of this study was to explore cross-reactive epitopes and pheromone linking capacity among Rat n 1 and homologues in domestic pets through an in silico approach. Methods: ElliPro and BepiPred in silico tools were used to predict B cell linear and cross-reactive epitopes. The pheromone linking capacity was explored by docking virtual screening with 2-ethylhexanol, 2,5-dimethylpyrazine, 2-sec-butyl-4,5-dihydrothiazole, and 2-heptanone ligands. Results: According to the analysis, Rat n 1 shares 52% identity with Equ c 1, Can f 6, Fel d 4, and Mus m 1 allergens. The overlapping structures analysis revealed high structural homology (root mean square deviation < 1). Four lineal and three discontinuous epitopes were predicted on Ra t n 1. A lineal epitope located between amino acids residues 24 and 36 was highly conserved on all allergens explored. A cross-reactive discontinuous epitope (T142, K143, D144, L145, S146, S147, D148, K152, L170, T171, T173, D174) was also found. Docking molecular simulations revealed the region involved in linking ligands, and we identified the properties of the binding of four pheromones and the binding potential of Rat n 1. Critical residues for interactions are reported in this study. Conclusions: We identified some possible allergens from Rattus norvegicus, and those allergens could have cross-reactivity with allergens from some animals. The results need to be confirmed with in vitro studies and could be utilized to contribute to immunotherapy and reduce allergic diseases related to lipocalins.

Benchmarking the PEPOP methods for mimicking discontinuous epitopes

Background Computational methods provide approaches to identify epitopes in protein Ags to help characterizing potential biomarkers identified by high-throughput genomic or proteomic experiments. PEPOP version 1.0 was developed as an antigenic or immunogenic peptide prediction tool. We have now improved this tool by implementing 32 new methods (PEPOP version 2.0) to guide the choice of peptides that mimic discontinuous epitopes and thus potentially able to replace the cognate protein Ag in its interaction with an Ab. In the present work, we describe these new methods and the benchmarking of their performances. Results Benchmarking was carried out by comparing the peptides predicted by the different methods and the corresponding epitopes determined by X-ray crystallography in a dataset of 75 Ag-Ab complexes. The Sensitivity (Se) and Positive Predictive Value (PPV) parameters were used to assess the performance of these methods. The results were compared to that of peptides obtained either by chance or by using the SUPERFICIAL tool, the only available comparable method. Conclusion The PEPOP methods were more efficient than, or as much as chance, and 33 of the 34 PEPOP methods performed better than SUPERFICIAL. Overall, “optimized” methods (tools that use the traveling salesman problem approach to design peptides) can predict peptides that best match true epitopes in most cases.

In silico analysis of a major allergen from Rattus norvegicus, Rat n 1, and cross-reactivity with domestic pets

Background: Lipocalins play a role in the cellular trafficking of pheromones and are involved in allergic responses to domestic pets. However, the cross-reactivity among allergens of this group has been poorly explored, and the pheromone linking capacity is not well characterized. The aim of this study was to explore cross-reactive epitopes and pheromone linking capacity among Rat n 1 and homologues in domestic pets through an in silico approach. Methods: ElliPro and BepiPred in silico tools were used to predict B cell linear and cross-reactive epitopes. The pheromone linking capacity was explored by docking virtual screening with 2-ethylhexanol, 2,5-dimethylpyrazine, 2-sec-butyl-4,5-dihydrothiazole, and 2-heptanone ligands. Results: According to the analysis, Rat n 1 shares 52% identity with Equ c 1, Can f 6, Fel d 4, and Mus m 1 allergens. The overlapping structures assay revealed high structural homology (root mean square deviation < 1). Four lineal and three discontinuous epitopes were predicted on Ra t n 1. A lineal epitope located between amino acids residues 24 and 36 was highly conserved on all allergens explored. A cross-reactive discontinuous epitope (T142, K143, D144, L145, S146, S147, D148, K152, L170, T171, T173, D174) was also found. Docking molecular simulations revealed the active site, and we identified the properties of the binding of four pheromones and the binding potential of Rat n 1. Critical residues for interactions are reported in this study. Conclusions: We identified some possible allergens from Rattus norvegicus, and those allergens could have cross-reactivity with allergens from some animals. The results need to be confirmed with in vitro studies and could be utilized to contribute to immunotherapy and reduce allergic diseases related to lipocalins.

Benchmarking the PEPOP methods for mimicking discontinuous epitopes

Computational methods provide approaches to identify epitopes in protein antigens to help characterizing potential biomarkers identified by high-throughput genomic or proteomic experiments. PEPOP version 1.0 was developed as an antigenic or immunogenic peptide prediction tool. We have now improved this tool by implementing 32 new methods (PEPOP version 2.0) to guide the choice of peptides that mimic discontinuous epitopes and thus potentially able to replace the cognate protein antigen in its interaction with an antibody. In the present work, we describe these new methods and the benchmarking of their performances.Benchmarking was carried out by comparing the peptides predicted by the different methods and the corresponding epitopes determined by X-ray crystallography in a dataset of 75 antigen-antibody complexes. The Sensitivity (Se) and Positive Predictive Value (PPV) parameters were used to assess the performance of these methods. The results were compared to that of peptides obtained either by chance or by using the SUPERFICIAL tool, the only available comparable method.The PEPOP methods were more efficient than, or as much as chance, and 33 of the 34 PEPOP methods performed better than SUPERFICIAL. Overall, “optimized” methods (tools that use the traveling salesman problem approach to design peptides) can predict peptides that best match true epitopes in most cases.

PEPOP: new approaches to mimic non-continous epitopes

Bioinformatics methods are helpful to identify new molecules for diagnostic or therapeutic applications. For example, the use of peptides capable of mimicking binding sites has several benefits as replacing a protein difficult to produce, or toxic. Using peptides is less expensive. Peptides are easier to manipulate, and can be used as drugs. Continuous epitope predicted by bioinformatics tools are commonly used and these sequential epitopes are used as such in further experiments. Numerous discontinuous epitope predictors have been developed but only two bioinformatics tools proposed so far to predict peptide sequences: Superficial and PEPOP. PEPOP can generate series of peptide sequences that can replace continuous or discontinuous epitopes in their interaction with their cognate antibody. We have developed an improved version of PEPOP dedicated to answer to the experimentalists’ need for a tool able to handle proteins and to turn them into peptides. The PEPOP web site has been reorganized by peptide prediction category and is therefore better formulated to experimental designs. Since the first version of PEPOP, 32 new methods of peptide design were developed. In total, PEPOP proposes 35 methods in which 34 deal specifically with discontinuous epitopes, the most represented epitope type in nature.We present the user-friendly, well-structured web-site of PEPOP and its validation through the use of predicted immunogenic or antigenic peptides mimicking discontinuous epitopes in different experimental ways. PEPOP proposes 35 methods of peptide design to guide experimentalists in using peptides potentially capable of replacing the cognate protein in its interaction with an Ab.

In silicoepitope prediction and 3D model analysis of Peste des petits ruminants virus nucleoprotein (PPRV N)

Peste des petits ruminants virus (PPRV) is an acute, highly contagious viral disease of small ruminants. It is endemic to sub-Saharan Africa, Asia and the Arabian Peninsula. The disease is a major constraint to food security, causing significant economic losses to subsistence farmers in affected areas.The nucleoprotein of morbilliviruses is highly immunogenic and produced in large quantities in virus infected cells. This makes it a suitable target for the host’s immune response. In this study, B-cell and T-cell epitopes of PPRV Nig/75/1 nucleoprotein were predicted using a suite ofin silicotools. Forty-six T-cell epitopes were predicted, of which 38 were MHC-I binding while eight were MHC-II binding. Of the 19 B-cell epitopes predicted, 15 were linear epitopes while four were discontinuous epitopes. Homology modelling of PPRV-N was done to elucidate the 3D structure of the protein and conformational epitopes. Conservation analysis of the discontinuous epitopes gave an indication into the similarity of the selected epitopes with other isolates of PPRV.Predicted epitopes may form an important starting point for serological screening and diagnostic tools against PPRV. Experimental validation of the predicted epitopes will assist in selection of promising candidates for consideration as antigen-based diagnostic tools. Such diagnostic tools would play a role in the global fight and possible eradication of PPR.

Prediction of B-Cell Epitopes in Listeriolysin O, a Cholesterol Dependent Cytolysin Secreted by Listeria monocytogenes

Listeria monocytogenes is a gram-positive, foodborne bacterium responsible for disease in humans and animals. Listeriolysin O (LLO) is a required virulence factor for the pathogenic effects of L. monocytogenes. Bioinformatics revealed conserved putative epitopes of LLO that could be used to develop monoclonal antibodies against LLO. Continuous and discontinuous epitopes were located by using four different B-cell prediction algorithms. Three-dimensional molecular models were generated to more precisely characterize the predicted antigenicity of LLO. Domain 4 was predicted to contain five of eleven continuous epitopes. A large portion of domain 4 was also predicted to comprise discontinuous immunogenic epitopes. Domain 4 of LLO may serve as an immunogen for eliciting monoclonal antibodies that can be used to study the pathogenesis of L. monocytogenes as well as develop an inexpensive assay.