How to identify homologous epitopes

Epitope mapping is a critical component of therapeutic monoclonal antibody (mAb) development as it provides vital insights into mechanisms of action. It has also been used successfully to identify epitopes that sustain the effects of immunization, thereby accelerating the development of vaccines against several viral pathogens, such as chikungunya, dengue, Ebola, and Zika. More recently, proteome-wide epitope mapping techniques have helped identify antibodies vital to the detection and neutralization of SARS-CoV-2.

Techniques for epitope mapping

Much of the diagnostic and therapeutic potential of epitopes still remains unexplored as there are millions of antibodies and a severe lack of epitope information about most of them. As a result, the development of cost-effective, high-throughput, high-resolution epitope mapping techniques is an urgent priority in the field of bioinformatics.

There are several techniques, such as X-ray co-crystallography, cryogenic electron microscopy, high-throughput shotgun mutagenesis epitope mapping, cross-linking-coupled mass spectrometry, array-based oligo-peptide scanning, and hydrogen-deuterium exchange, that are currently used for epitope mapping.

However, there are still several limitations that make most approaches either inaccessible, inefficient, or inadequate. For instance, both X-ray co-crystallography and cryogenic electron microscopy are extremely effective in generating high-resolution maps of antibody-antigen interactions. But they are expensive and technically complex, making them largely inaccessible to the broader research community.

Similarly, techniques that facilitate high-throughput monitoring may be constrained by lack of resolution or their inability to compare millions of sequences, traverse multiple omics dimensions, or scale disparate biological datasets.

The limitations of conventional epitope mapping techniques translate into consequential challenges for epitope researchers and slow down epitope research.

The BioStrand R&R platform provides an accessible and cost-effective solution that can seamlessly scale across millions of sequences, all omics layers, and multiple databases without compromising speed, efficiency, or accuracy.

Research Objective

The aim of this workflow is to demonstrate how the BioStrand platform enables researchers to start with a specific isotope and then proceed to identify all homolog epitopes using its intuitive features and capabilities.

For the purposes of this demonstration, we will start with a particular antigen expressed in humans and then qualify the steps involved in:

• Listing all homologous epitopes
• Assessing specificity of epitope and potential cross reactivity to a specific antigen.

The BioStrand epitope homology workflow

For this walkthrough, we start with this particular antigen:

Description: bcr_3d_assays: antigen name =shiga-like toxin II B subunit encoded by bacteriophage BP-933W [Escherichia coli O157:H7 str. EDL933] organism name =Escherichia coli O157:H7 str. EDL933

Top Concepts: antigen name, bacteriophage bp-933w, bcr_3d_assays, edl933, escherichia coli o157:h7 str, organism name, Shiga-like toxin ii b subunit

Sequence: MKKMFMAVLFALASVNAMAADCAKGKIEFSKYNEDDTFTVKVDGKEYWTSRWNLQPLLQSAQLTGMTVTIKSSTCESGSGFAEVQFNND

STEP 1: Antigen Sequence Search

We start by simply copying and pasting the antigen sequence mentioned above into the BioStrand Search Bar and hit return.

• As we can see in the Quick Filter view above, the solution returns results tabulated by omics layer, data source, GO definition, etc. We also get a detailed Taxonomy view towards the bottom of the screen that provides us with the primary confirmation of the presence of homologies in Genus Escherichia and Species Escherichia coli.
• Next, we click on Alignment View in the top bar to enable visual assessment and confirmation of potential epitopes.
• We can then use the Filter button in the top right to access a range of fine filters to quickly home in on data most relevant to the objectives of the research.

STEP 2: Choosing and Applying Relevant Filters

In the Alignment View, we now use the Filter feature to apply the relevant filters that will allow us to drill down to data related exclusively to epitopes.

• We click on the Filter button to focus our search on Epitopes and more specifically to epitopes in the Immune Epitope Database (IEDB).

• By focusing our search, we arrive at a shortlist of epitope-only results comprising 8 pertinent and 3 unique candidate epitopes.

STEP 3: Searching Within the Results

Thus far, we have used BioStrand’s simple Search/Filter functionality to significantly narrow down the list of potential epitopes that are relevant to our research. We can now drill further down into any of the potential candidates. In this case, we want to explore the sixth sequence in the list of eight retrieved from the last search.

• We select the relevant sequence, AADCAKGKIEFSKYNEND (hold shift, click the first letter and drag to choose the entire sequence) and press the loupe to launch a new search.
• We can use the Quick Filter view to reconfirm that the chosen sequence is indeed specific to E. coli.

Next, we will further focus our search on finding matches only from the Protein Data Bank (PDB)

• To do this, we shift to the Quick Filter view and apply Data Source filters to remove all datasets except those from PDB. (Quick Tip: You can automatically exclude all other sources by double-clicking on the dataset you need, in this case, PDB).

• We then shift to List View so that we can add relevant data fields like Geneontology and Taxonomy and get a more focused overview of functions and taxonomy.

• We can now click on Alignment View to see all related PDB sequences on the bottom half and all relevant filters at the top.
• By clicking on the GO Definition filter from the list, we also get an overview of all related GO terms.

As we can see, “modulation of host virulence by virus” is one of the main features revealed by applying the Go Definition filter.

• By clicking on “modulation of host virulence by virus,” we can now extract all related sequences from the PDB database.

STEP 4: Relaunch Search with Another Epitope Candidate from the Shortlist

Using Search History on the R&R home page, we navigate to the candidate epitopes generated in Step 2. We then select the next epitope candidate we want to investigate, launch a search focused on the PDB data source.

• In Alignment View, we type in the previous epitope to highlight it in the new results list of sequences. The BioStrand R&R platform uses an asterisk (*) symbol for ease of input so that only a few characters are required to strictly match. (Quick Tip: While researching a list of potential epitope candidates, you can use the Highlight Pattern function to quickly identify all candidates that align to the same results. Or, you can paste the epitope sequence in the filter to view only the sequences that strictly contain both epitopes.)
• As we can see from the highlighted sequences, both the previous and the current potential candidate epitopes point to the same sequences, indicating that both have the capacities pertinent to the research objectives.

Conclusions based on the BioStrand epitope homology workflow output

• Using this easy-to-use workflow, we have been able to conclusively establish that all three epitopes are specific to Escherichia coli and are particularly related to Shiga toxin-producing E. coli.
• We have also determined that:
  • The antigen and epitopes in this example correspond to no other organisms.
  • The antigen has homologous epitope sequences pointing to epitopes that are not specifically associated with this antigen.
  • There is no cross reactivity.

This workflow demonstrates how the BioStrand R&R platform vastly simplifies and streamlines complex multi-omics homology searches.

A comprehensive array of built-in filters empowers researchers to click and filter through complex multilayered datasets, including metadata, to focus on pathways pertinent to their study without having to wrangle with a single line of code.

The process is as simple as a Google search and all relevant results are delivered instantly in an intuitive, interactive, and clickable dashboard that is simple, easy to understand, and visually informative.

The BioStrand R&R Advantage

BioStrand R&R addresses many of the limitations associated with conventional epitope mapping techniques.

It is the only solution that facilitates high-level integrated analysis across 440 million pre-indexed sequences and across all omics layers. With the functionality to scale across multiple databases, the platform can concurrently look up disparate topic- and domain-centric data sets, thereby generating a universal set of relevant results for any epitope.

The solution provides researchers with the flexibility to launch their homology search with either a small epitope or a longer antigen, without having to account for differences in local and global alignment.

By automatically translating all corresponding DNA and RNA into protein code, the platform ensures that all DNA-RNA are integrated into the analysis even if they are not explicitly denoted as a protein sequence. This significantly expands the scope of the search and returns a more exhaustive selection of pertinent sources of information.

Since the solution takes all cross-domain translations into account, researchers can now compare all cross-species DNA sequences that produce the same protein.

Most importantly, the BioStrand platform’s modern, intuitive, digital user interface allows even non-technical users to access all these sophisticated capabilities, and the SaaS delivery model enables anytime-anywhere pay-per-use access that maximizes productivity as well as cost-effectiveness.

Let us know of your epitope-related research challenges so that we can demonstrate how the BioStrand R&R platform can help.

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