2. Use the advanced query to identify the subset of peptides that elicit an IFN- g response and do not require in vitro restimulation

Transcription

1 HCV diagnostic development This exercise demonstrates how the IEDB can be utilized to select sets of epitopes with desired characteristics. As an example, we will study the selection of epitopes to be included in a pool of peptides for the development of a diagnostic IFN- gamma ELISPOT assay to identify individuals infected with HCV type. In detail you shall 1. Use the homepage query to identify HCV peptides recognized in human T cell responses 2. Use the advanced query to identify the subset of peptides that elicit an IFN- g response and do not require in vitro restimulation 3. Select the subset of peptides that are conserved across multiple HCV strains Use the homepage query to identify HCV peptides recognized in human T cell responses Go to the IEDB homepage ( What query parameters should you set to retrieve HCV peptides recognized in human T cell responses? As a general recommendation for any kind of database queries: start general, and make the query more restrictive if many of the results are not what you expect. Here is one reasonable translation of the query: HCV as the source organism of epitopes uncheck all immune response types except for T cell response select human as the host organism mounting the response The completed form should like like this:

2 After clicking the search button you will get a summary of the information available in the IEDB. Q1: How many different journal articles and direct submissions are available in the IEDB that characterize T cell responses in humans against HCV? à In the search result summary table, look up the number of references Q2: Produce an Excel sheet with the peptides sequences giving a positive response according to your query à In the row indicating peptidic epitopes click on the number in the column labeled positives. à On top of the table that appears listing peptidic epitopes, click on the Excel symbol to the right of export all results

3 2. Use the advanced query to identify the subset of peptides that elicit an IFN- g response and do not require in vitro restimulation The IEDB homepage query only offers the most commonly used query parameters. To further narrow the set of epitopes, please proceed to the advanced query, by selecting T cell search from the Query menu on the IEDB homepage. Q3: On the advanced search, set the query parameters for epitope source organism and host organism as you did in the previous section, and confirm that the results are the same. Revise your query to further limit the epitopes to only retrieve those for which no in vitro expansion is necessary for detection (à under immunization, check that there is no administration in vitro ) for which IFN-g responses can be detected (à under assay use the assay finder to select those that measure cytokine production of interferon gamma) Q4: Produce an Excel sheet with the HCV peptides sequences that elicit an IFN-g response in humans and do not require in vitro expansion. 3. Identify the subset of peptides from 2 that are conserved in HCV serotype II HCV is a highly mutable virus, and different isolates are categorized into types based on their antibody reactivity and genetic similarity. To determine conservation of the epitopes we identified across different HCV types, we first have to retrieve the protein sequences from Genbank: Strain NCBI GI b b a b a a a a

4 Q5: Save at least 2 (ideally all) of the sequences above in fasta format to a text file Next, go to the tools homepage of the IEDB ( and choose the Epitope conservancy analysis tool from the Analysis Tools tab: Follow the steps on the conservancy tool page: In step 1, paste in the list peptides sequences that elicit an IFN-g response in humans and do not require in vitro expansion (Q4).

5 In step 2, paste in the list of representative HCV strains retrieved from genbank (Q5) Submit the data to the tool, and sort the resulting list according to the percent of proteins matching at column Q6: What epitopes are most conserved across the representative HCV strains?