ImmunoBiology
  Binf I: HIV-I Gag responses
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Goal: The purpose of this exercise is to demonstrate the value of MHC-peptide binding predictions in exploring HLA-disease associations.
Background: Read Kiepiela et al  and Kosmrlj et al (especially the first page) beforehand. Think about the following issues as a preparation for the exercise:
  1. Which CTL responses seem to be associated with slow disease progression in HIV-1 infection?
  2. Which HLA alleles are associated with slow and fast progression to AIDS?
Main research question: What is the mechanism behind the fact that certain MHC molecules are associated with slow progression to AIDS? Try to answer this question (the subquestions below will help you to do that) as much as possible using your own analysis. If you are stuck, refer to the hints at the end of the page.
HLA-disease associations
1. Study Figure 2 of Kiepiela et al again. What does this result tell you about the relationship between CD8 responses to different HIV-1 proteins and viral loads? Design and perform an analysis that can show that B*57 and B*18 target different HIV-1 proteins, ie. they have different number (or density) of epitopes from different HIV-1 proteins using the peptide binding predictions ( NetMHC-3.2 server , hints 1&2).
2.
Why do you think it might be beneficial to present HIV-1 Gag?

3. For HLA-B*57, Kosmrlj et al proposes a different mechanism to explain its protective effect. Try to estimate the binding fraction of B*57 for other viruses than HIV-1 (proteome sequences of viruses are available from EBI web page ). How close are the numbers you get to the number estimated for self by Kosmrlj et al (Supplementary Material)? Is self the only proteome that would be presented poorly by B*5701 molecule? Study the binding motif of B*5701 (a database of known MHC ligands is IEDB, hint 3). Can the amino acid preference at the anchor positions explain the observed phenomenon? (hint 4)
4.
Which one of the two explanations why B*5701 is protective in HIV-1 infection do you think is more likely?



Links

budding HIV (source: Wikipdia)

Scanning electron micrograph of HIV-1 budding from cultured lymphocyte
Hints
  1. To find which alleles target which HIV-1 proteins, it is useful to use a data set that contains all HIV-1 proteins. We have prepared such a file for you here. Alternatively, you can generate your HIV-1 protein data set in many ways, e.g. you can search for the HIV-1 genome at NCBI and via the protein coding regions download all protein sequences (use the "Protein FASTA" option under DISPLAY at the bottom of the page). Make sure you don't use concatenated proteins (e.g. Gag-Pol) for the analysis.
     
  2. To analyze the output of NetMHC-3.2 use the "download output sheet" option, save the output (by using right mouse button and "save as" option), and view it in Excel. You can sort the affinities to find which peptides are the best binders. The sorting can be done by selecting all columns that contain data and then in the pop-up menu choose for the column that has the predicted affinity values to sort. Remember smaller the predicted binding value, the better the binding. Your fasta files should contain the protein names as the first word of the identifier line. As  NetMHC-3.2 does not offer predictions for B*5703 (the allele mentioned in Kiepiela et al), please use B*5701 in this exercise.

  3. You can  find binding peptides and eluted ligands for a particular MHC molecule in the IEDB. The "Search" menu offers short-cuts to "MHC binding search" and "MHC ligand elution search". Download your results using the "Epitopes" link instead of "Assays" link to get unique ligands. Search for HLA-B5701 explicitly, and download only 9mer ligands, i.e., the peptides of length 9. When you export the data to your excel, sometimes it can go wrong and all your data ends up in one column. In this case use "text to column" option, select the column you want to split, ise comma as a deliminator and it will format nicely. Alternatively, in the  SYFPEITHI database use the Find motif, Ligand or epitope option to find the ligands of a specific MHC molecule. Make sequence logos of the ligands/binders you find using  WebLogo. WebLogo takes a list of peptides as input. You can change the logo dimensions to a suitable size (e.g. 8x15 cm). Study which positions are anchor positions and what amino acids are found at the anchor positions. Alternatively, you can have a look at predicted binding motifs at the MHC motif viewer.

  4. You can find information on amino acid frequencies in the Swissprot statistics (scroll down to the bottom of the page).