Conservation of influenza A T cell epitope

To illustrate the utility of BlockLogo, we analyzed a block of peptides in the influenza virus HA protein. All peptides in the block of 10-mers, starting at position 232 were predicted to bind to the HLA A*02:01 with similar affinities. The frequency of individual peptides in the viral population cannot be determined from the standard sequence logo produced with WebLogo, but is clear from the BlockLogo that the most frequent peptide in this block is present in 64.65% of the viral population. The combination of MHC binding prediction and BlockLogo visualization reveals this particular region to be highly antigenic, and potentially valuable in polyvalent vaccine designs. This peptide is not a known T-cell epitope and it is of potential interest since it is a predicted binder of high affinity to HLA-A*02:01 and it is highly conserved among HA.


Above: Sequence logo plot of the residues in the 10-residue block starting at position 232 of the Influenza virus HA protein generated using WebLogo and BlockLogo of the peptides in the 232-241 block. The residue position in the MSA is shown on the X-axis, and the information content is shown on the Y-axis. The colors of the amino acids correspond to their chemical properties; polar amino acids (G, S, T, Y, C, Q, and N) are shown in green, basic amino acids (K, R, and H) are shown in blue, acidic amino acids (D and E) are shown in red, and hydrophobic amino acids (A, V, L, I, P, W, F, and M) are shown in black.
Below: HLA binding predictions of each peptide present in the block of 10-mer peptides starting at position 232 in an MSA of influenza HA proteins. Prediction were performed for HLA A*02:01 allele, but can be done for a number of alleles (see materials and methods). The table is also included in the BlockLogo web server output if HLA binding predictions are selected upon submission.

Conservation of influenza A B cell epitope

BlockLogo can also be used to display virtual peptides composed from a selection of discontinuous sites within proteins. This function can be applied to visualize conservation of B-cell epitopes, which can, for example, be used for representation and characterization of cross-neutralizing viral B-cell epitopes (Xu et al., 2010). The discontinuous BlockLogo displays the diversity of residues forming conformational epitope of the broadly neutralizing antibody F10 (Sui, et al., 2009). This BlockLogo shows the conservation of F10 epitopes within 29,113 complete influenza HA proteins. The description of the ten most frequent epitopes (“discontinuous peptides”) is shown in the table below.
Above: Sequence logo of neutralizing epitopes for neutralizing antibody F10 on influenza virus HA proteins and BlockLogo of the discontinuous residues in F10 neutralizing epitope.
Below: Ten most frequent influenza A HA discontinuous peptides on neutralizing epitope region recognized by neutralizing antibody F10 in FluKB (29,113 complete HA protein sequences). The table shows the amino acids of the epitope, HA subtype, frequency within the data set, and validation status - escape variants are those strains not neutralized by the F10.
Table of conservation

Signatures of allergenic and hypoallergenic Bet v 1 allergens

Modulation of IgE-mediated responses to allergens such as birch pollen allergen, Bet v 1 can be achieved by site-directed mutagenesis (Ferreira et al. 1998). BlockLogo can be used to distinguish allergenic and hypoallergenic variants of Bet v 1 by visualizing diversity of the antibody binding site such as that of BIP1 monoclonal antibody (Jarolim et al. 1989). The discontinuous BlockLogo of the sites predicted by Ferreira et al. (Ferreira et al. 1998) to influence IgE binding revealed that even though that of 72 discontinuous motifs are theoretically possible from the positional variability, 28 were found in the data set consisting of 217 sequences of Bet v 1 allergen, and only 13 of these were observed more than once. This analysis revealed four allergenic variants (TFSSID, TFNSID, TVSSID, TFSSIN) present in 27% of the analyzed sequences, while hypoallergenic variants account for 5% of all analyzed sequences. The majority of known Bet v 1 sequences have not been analyzed for allergenicity related to the motif defined by positions 11-31-58-114-115-127 in the multiple sequence alignment.


Above: Sequence logo and BlockLogo for signatures of the six critical amino acid positions predicted to influence IgE binding to Bet v 1.
Below: Frequency and allergenic response to discontinuous epitope of Bet v 1 allergen variants. In total, 28 variants exist, but only 10 are required for an accumulated frequency of 90%.
Table of conservation

Variability of HLA-DRB1 binding pocket P1

The usage of BlockLogo can easily be extended beyond T and B cell epitopes to predict and visualize other peptide-protein interactions, structural and functional motifs. For example, BlockLogo can be used to visualize variation in known structural motifs such as HLA class II binding pocket 1 (P1) of HLA-DR defined by variable β1 chain and invariant HLA-DR α chain. Pocket P1 accommodates the primary anchor of class-II HLA-DR binding peptides. Positions that define binding pockets for large number of HLA-DR molecules were described earlier (Chelvanayagam 1997). These sequence motifs can be used to determine preferences for the primary anchor residue of binding peptides and shared specificities. Six variable positions (positions 81, 82, 85, 86, 89 and 90 in the alignment) contain the pocket P1. Three motifs (HNVVFT, HNVGFT, and HNAVFT) account for 97% of the HLA-DRB1 sequences, seven motifs are represented by 2-5 sequences, and seven motifs are represented by a single sequence. The vast majority of alleles from a particular serogroup contain major motif (approximately 90% of the alleles) and small number (approximately 10%) have minor motif. Motif HNVVFT is a major signature of DRB1*03, 13, 14, and 15 serogroups; HNVGFT is a major signature for DRB1*01, 04, 07, 08, 09, 10, 11, and 16 serogroups; and HNAVFT is a major signature for DRB1*12 serogroup. In addition, motif HNVVFT is a minor signature of DRB1*04 and 11 serogroups; HNVGFT is a minor signature for DRB1*03, 14 and 15 serogroups; and HNAVFT is a minor signature for the DRB1*1 serogroup. Other motifs are observed in HLA alleles that are extremely rare in general population (less than 1%). These results show that the fine specificity of primary anchor binding is determined by three major structural motifs and these motifs are unequally distributed between the serogroups.


Above: Visualization of diversity of binding pocket 1 βchain in DRB1 alleles using sequence logo (A) and BlockLogo (B). See Table 4 for frequencies and alleles for each motif.
Below: Frequency and allele distribution of discontinuous motifs in binding pocket 1 β chain of DRB1 protein.
Table of conservation

Consensus pattern in IL-7 and IL-9 signature

Interleukin-7 (IL-7) is a hematopoietic growth factor affecting early T and B cell development (Henney 1989). IL-9 supports development of T helper cells (Renauld et al. 1990). Phylogenetic studies of these two cytokines have suggested evolutionary relationship based on a signature pattern conserved between them. This signature, N-x-[LAP]-[SCT]-F-L-K-x-L-L (prosite.expasy.org/PDOC00228), can be used for identification of members of IL-7/IL-9 family. BlockLogo can be readily used to study this pattern across species in order to elucidate the variation pattern of this signature. The analysis of 34 sequences has shown that the signature reported in the PDOC00228 is actually made of two similar, but distinct signatures: [NG]-x-[LAP]-C-F-L-K-x-L-L (IL-7), and N-x-L-[TS]-F-L-K-x-L-L (IL-9).


Above: Sequence logo and BlockLogo visualization of Interleukin-7 and -9 signature pattern indicating a common evolutionary ancestry between these two cytokines.
Below: Frequency and Interleukin type of IL-7 and -9 consensus pattern.
Table of conservation


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Xu, R., Ekiert, D. C., Krause, J. C., Hai, R., Crowe, J. E., & Wilson, I. A. (2010). Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science (New York, N.Y.), 328(5976), 357–60. doi:10.1126/science.1186430
Developed by Bioinformatics Core at Cancer Vaccine Center, Dana-Farber Cancer Institute.

Version 1.2, August 2012.