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Introduction
EpiQuest-B is a program designed to predict and rank domains of linear protein sequences based on their ability to trigger a humoral immune response. The program enables in silico prediction of immunodominant epitopes by evaluating their potential antigenicity. Importantly, it performs this assessment equally for linear peptides, regardless of their exposure on the surface of the mature protein molecule and without considering post-processing modifications such as glycosylation or phosphorylation.
Antigenicity and Immune dominance of B-epitopes
To be antigenic, meaning capable of eliciting a humoral response, a peptide sequence should possess specific structural features that determine its ability to strongly bind to IgG/B-cell receptors. It's important to understand that not every peptide sequence may have an antibody capable of binding to it with high affinity, even after the extensive process of B-cell clonal selection and mutation of paratope sequences (the part of IgG that binds to the epitope). Occasionally, IgGs can be raised against a "weak" epitope due to the individual response of a particular animal. However, immunodominant epitopes, which elicit a strong, specific response with high-affinity IgG in most antigen recipients, can provide insight into the sequence's structural basis for strong antibody binding.
The Scheme on the right demonstrates that the stronger the binding of the antigen to IgG on follicular dentritic calls, the stronger the stimulation of the clonal expansion of the respective B-cell clone will be.
The scheme is presented after: El Shikh, M.E.M., El Sayed, R.M., Sukumar, S., Szakal, A.K., Tew, J.G., 2010. Activation of B cells by antigens on follicular dendritic cells. Trends Immunol 31, 205–211. https://doi.org/10.1016/j.it.2010.03.002
The Algorithm
The algorithm employed in EpiQuest-B operates under the assumption that the antigenicity of an epitope is determined by the neighboring amino acids in the sequence. This algorithm is grounded in data derived from over 40,000 individual amino acids'context within epitope sequences (categorized as strong, weak, and negative), and its efficacy has been validated using over 2,000 individual epitopes with known immunization data from immunization studies conducted at Aptum Biologics or its predecessor companies.
The algorithm of EpiQuest B computes the probability of an amino acid being part of an antigenic peptide sequence based on the amino acids comprising its context. The analysis frame determines the width of the context taken into account, with the best results observed for Frames ranging from 6 to 9 amino acids.
EpiQuest-B and other prediction programs
To illustrate what EpiQuest-B can predict, consider the NS1 protein of Dengue virus 2, which serves as an excellent example. The immunodominant epitopes of NS1 have been thoroughly investigated with high precision and detail (as documented in Clin. Vaccine Immunol. 14, 493–504). In total, there are 8 immunodominant epitopes covering 3 distinct areas in the N-terminal part of the molecule. As depicted in the figure below, all 3 areas (highlighted by red frames) were accurately detected by EpiQuest-B, evidenced by 3 main peaks in the antigenicity profile of the molecule. It's worth noting that the sensitivity of the program can be adjusted using the Threshold function, with values of T=0 or T=2 used in the analysis depicted below.
We conducted a comparison of the plots generated for NS1 using EpiQuest-B and other prominent epitope/antigenicity defining programs available on the market, namely Antigenicity by Kolaskar & Tongaonkar, BepiPred, and COBEPro. In the figure above (left), we present the antigenicity propensity plot obtained by the current versions of these programs at their default settings. The positions of the known immunodominant epitopes are indicated for reference.
It is evident from the comparison that EpiQuest (Fig. A) is the only program that detects all three immunodominant epitope areas as the regions with the highest values. On the other hand, both algorithms of Kolaskar & Tongaonkar and the antigenicity algorithm of COBEPro either miss the epitopes entirely or detect only part of them, assigning the other part to low antigenicity areas (Fig. B, D). While BepiPred focuses solely on epitope probability, it correctly predicts one of the immunodominant epitopes but does not assign it a high value (Fig. C).
Antigenicity Index (AGI)
The algorithm of EpiQuest-B assigns an Antigenicity Index (AGI) that characterizes the potential of a sequence to elicit a humoral immune response and produce strong, high-affinity antibodies. A higher AGI indicates a greater likelihood of eliciting a strong humoral response. Remarkably, this capability sets EpiQuest-B apart from existing epitope prediction software, as none of them are capable of performing this task with high statistical significance, as demonstrated below.
Two strains of mice (represented by red dots or blue dots), each with different haplotypes, were immunized with various types of antigens (including NS1 peptide, native NS1, or live DV2). Their pooled sera were then tested against a set of overlapping peptides. We defined AGI values for these fragments and constructed correlation graphs for AGI versus ELISA data. The graphs above clearly demonstrate the correlation between the observed ELISA titers and the predicted AGI for the sequences.
Furthermore, statistical analysis revealed that such correlation was not only significant but also highly biologically significant, with an R-squared value exceeding 0.5.
Program output
New layout of EpiQuest-B (EpiQuest v4.1 and later) allows to perform the antigenicity profiling of the protein sequences along with other types of analysis (accessibility, complexity, and the other ones).
The results can be presented in the form of a plot or as bars (above) indicating the sequences above a predefined level of the peptide feature .
The data is also presented in tabular form (left), both types of the result data can be easily exported into MS Office or other types of office files.
Analysis of Demo Sequences
For a full overview of the program's capabilities you can use Demo Mode. The Manual for EpiQuest-B explains how to modify settings. You can also see the examples of how to analyse the sequences and interpret the results by visiting the Demo Sequences analysis page.
Scanning epitope collections with B-Scanner
When analyzing large numbers of epitopes, whether obtained from your data collection, or isolated from protein sequences using other programs, or selected based on other criteria, B-scanner proves to be invaluable. This tool ranks peptide epitopes according to their relative antigenicity using the same algorithm and matrices as EpiQuest-B.
EpiQuest Suite and site www.epiquest.co.uk belongs to Aptum Biologics Ltd.
EpiQuest® is a registered Trademark of Aptum Biologics Ltd.
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