EpiQuest-B | EpiQuest-A | EpiQuest-IM | EpiQuest-C |EpiQuest-T | EpiQuest-H | EpiQuest-M | In Charge | EpiStat
Antigenicity and Immunogenicity
Whether the immune system will respond to an antigen, and if this response will be strong, is defined by a whole spectrum of factors:
1) Antigenicity of the protein domains
2) Accessibility of antigenic domains, to re-stimulate the response via BCR
3) Copy number, which may compensate for lower antigenicity
4) Presence of strong T-helper epitopes and other factors.
When it comes to linear B-epitopes (they represent the overwhelming majority of all epitopes) we should discriminate between their antigenicity, as the potential of the epitope to elicit strong specific antibodies when presented to the immune system, and immunogenicity, which is the true ability of an epitope to elicit such response when present within the complete sequence of the properly folded native protein.
For a peptide antigen that is coupled to a carrier, antigenicity and immunogenicity will be identical. However, when it comes to a folded native protein, the factor of epitope accessibility becomes critical. This distinction is especially important for the epitopes that will be a target of immune response against viral proteins, complex allergens etc. When the goal is to predict the epitopes in silico that will detect an immune response against the target, preferably - immunodominant epitopes across a whole population of individuals/animals that were exposed to the target protein.
While EpiQuest-B builds an antigenicity profile for a protein sequence, allowing the discovery of potentially immunogenic linear B-epitopes, the new program - EpiQuest-IM - predicts the immunogenicity of such epitopes in real contact of the target protein with the immune system. It compensates for epitopes being hidden or semi-hidden in the mature protein.
The algorithm
The program combines algorithms used in EpiQuest-B (prediction of antigenicity) and EpiQuest-A (prediction of surface exposure). The settings allow you to vary the importance of both parameters in the prediction of protein immunogenic domains when using whole protein immunization.
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The program settings and output
EpiQuest-IM is most valuable when looking for strong linear epitopes, capable of eliciting a strong stable response. This analysis is especially helpful when you plan to use the resulting peptide epitope as an antigen in antibody assay (to detect the response of the host to the native conformation of the infectious agent) or to immunize the animal in order to obtain the antibody recognizing the native molecule, the source of the epitope.
For antigenic linear peptide sequence, to become a real antigen there are two requirements for this potential to be realized: 1) it should be accessible for the B-cell receptor at the surface of the native or intracellularly processed molecule, 2) the level of exposure may contribute to the immunogenicity of the epitope (even if its antigenicity is not very high). The latter usually happens to well exposed epitopes that can be recognized by antibodies of non-specific repertoire: multiple signals from weak but well exposed (or presented with large numbers of copies) epitopes compensates for the weakness of actual binding.
Therefore, when using EpiQuest-IM, you can settle for the Default parameters for the analysis (when looking for the most immunogenic epitopes), or increase the K to increase the contribution of surface presence for the overall immunogenicity. This is useful when you are analyzing the general immunogenicity profile of the sequence. To make it more clear - if you have a serum raised against some native protein, you will see different patterns of reactivity of this serum with peptide arrays representing the molecule in relation to the serum dilution you use: for lower dilution you will observe reactivity with weak epitopes, and only some very strong epitopes will show an enhanced reaction. At high titres you will see mainly the reaction with only the most prominent and immunogenic epitopes.
Here we show an example of the actual correlation between the values for 9-mers produced by EpiQuest-M and actual serum reactivity with these peptides. Overall, the data well correlates with the predicted immunogenicity of the sequences.
It should be noted that the results are shown for the relatively low dilution of serum, allowing to see its reactivity with the best, but also the weak epitopes (an example is for the NS1 protein of Dengue virus 2).
The histogram above and the tabular output on the left show that the program correctly identifies all four main epitopes in NS1 sequence of Dengue virus 2 (88-97, 29-40, 133-148, 230 - 252 - marked by stars in the histogram).
If you look at the denoted immunogenicity indexes (table), you will see the dramatic different between IMI and IPB (compensated for the length of the epitope) of the four best epitopes and the rest of the sequence.
For particular details of how to operate the program, please refer to the Manual for EpiQuest-IM.
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