HLA class I allele promiscuity revisited.

The peptide repertoire presented on human leukocyte antigen (HLA) class I molecules is largely determined by the structure of the peptide binding groove. It is expected that the molecules having similar grooves (i.e., belonging to the same supertype) might present similar/overlapping peptides. However, the extent of promiscuity among HLA class I ligands remains controversial: while in many studies T cell responses are detected against epitopes presented by alternative molecules across HLA class I supertypes and loci, peptide elution studies report minute overlaps between the peptide repertoires of even related HLA molecules. To get more insight into the promiscuous peptide binding by HLA molecules, we analyzed the HLA peptide binding data from the large epitope repository, Immune Epitope Database (IEDB), and further performed in silico analysis to estimate the promiscuity at the population level. Both analyses suggest that an unexpectedly large fraction of HLA ligands (> 50%) bind two or more HLA molecules, often across supertype or even loci. These results suggest that different HLA class I molecules can nevertheless present largely overlapping peptide sets, and that "functional" HLA polymorphism on individual and population level is probably much lower than previously anticipated.

A comparative study of HLA binding affinity and ligand diversity: implications for generating immunodominant CD8+ T cell responses.

Conventional CD8(+) T cell responses against intracellular infectious agents are initiated upon recognition of pathogen-derived peptides presented at the cell surface of infected cells in the context of MHC class I molecules. Among the major MHC class I loci, HLA-B is the swiftest evolving and the most polymorphic locus. Additionally, responses restricted by HLA-B molecules tend to be dominant, and most associations with susceptibility or protection against infectious diseases have been assigned to HLA-B alleles. To assess whether the differences in responses mediated via two major HLA class I loci, HLA-B and HLA-A, may already begin at the Ag presentation level, we have analyzed the diversity and binding affinity of their peptide repertoire by making use of curated pathogen-derived epitope data retrieved from the Immune Epitope Database and Analysis Resource, as well as in silico predicted epitopes. In contrast to our expectations, HLA-B alleles were found to have a less diverse peptide repertoire, which points toward a more restricted binding motif, and the respective average peptide binding affinity was shown to be lower than that of HLA-A-restricted epitopes. This unexpected observation gives rise to new hypotheses concerning the mechanisms underlying immunodominance of CD8(+) T cell responses.

Primer3Plus, an enhanced web interface to Primer3.

Here we present Primer3Plus, a new web interface to the popular Primer3 primer design program as an enhanced alternative for the CGI- scripts that come with Primer3. Primer3 consists of a command line program and a web interface. The web interface is one large form showing all of the possible options. This makes the interface powerful, but at the same time confusing for occasional users. Primer3Plus provides an intuitive user interface using present-day web technologies and has been developed in close collaboration with molecular biologists and technicians regularly designing primers. It focuses on the task at hand, and hides detailed settings from the user until these are needed. We also added functionality to automate specific tasks like designing primers for cloning or step-wise sequencing. Settings and designed primer sequences can be stored locally for later use. Primer3Plus supports a range of common sequence formats, such as FASTA. Finally, primers selected by Primer3Plus can be sent to an order form, allowing tight integration into laboratory ordering systems. Moreover, the open architecture of Primer3Plus allows easy expansion or integration of external software packages. The Primer3Plus Perl source code is available under GPL license from SourceForge. Primer3Plus is available at http://www.bioinformatics.nl/primer3plus.

HLA Preferences for Conserved Epitopes: A Potential Mechanism for Hepatitis C Clearance.

Hepatitis C virus (HCV) infections affect more than 170 million people worldwide. Most of these individuals are chronically infected, but some clear the infection rapidly. Host factors seem to play a key role in HCV clearance, among them are the human leukocyte antigen (HLA) class I molecules. Certain HLA molecules, e.g., B*27 and B*57, are associated with viral clearance. To identify potential mechanisms for these associations, we assess epitope distribution differences between HLA molecules using experimentally verified and in silico predicted HCV epitopes. Specifically, we show that the NS5B protein harbors the largest fraction of conserved regions among all HCV proteins. Such conserved regions could be good targets for cytotoxic T-cell (CTL) responses. We find that the protective HLA-B*27 molecule preferentially presents cytotoxic T-cell (CTL) epitopes from NS5B and, in general, presents the most strongly conserved epitopes among the 23 HLA molecules analyzed. In contrast, HLA molecules known to be associated with HCV persistence do not have similar preferences and appear to target the variable P7 protein. Overall, our analysis suggests that by targeting highly constrained - and thereby conserved - regions of HCV, the protective HLA molecule HLA-B*27 reduces the ability of HCV to escape the cytotoxic T-cell response of the host. For visualizing the distribution of both experimentally verified and predicted epitopes across the HCV genome, we created the HCV epitope browser, which is available at theory.bio.uu.nl/ucqi/hcv.

Complementarity of Binding Motifs is a General Property of HLA-A and HLA-B Molecules and Does Not Seem to Effect HLA Haplotype Composition.

Different human leukocyte antigen (HLA) haplotypes (i.e., the specific combinations of HLA-A, -B, -DR alleles inherited together from one parent) are observed in different frequencies in human populations. Some haplotypes, like HLA-A1-B8, are very frequent, reaching up to 10% in the Caucasian population, while others are very rare. Numerous studies have identified associations between HLA haplotypes and diseases, and differences in haplotype frequencies can in part be explained by these associations: the stronger the association with a severe (autoimmune) disease, the lower the expected HLA haplotype frequency. The peptide repertoires of the HLA molecules composing a haplotype can also influence the frequency of a haplotype. For example, it would seem advantageous to have HLA molecules with non-overlapping binding specificities within a haplotype, as individuals expressing such an haplotype would present a diverse set of peptides from viruses and pathogenic bacteria on the cell surface. To test this hypothesis, we collect the proteome data from a set of common viruses, and estimate the total ligand repertoire of HLA class I haplotypes (HLA-A-B) using in silico predictions. We compare the size of these repertoires to the HLA haplotype frequencies reported in the National Marrow Donor Program (NMDP). We find that in most HLA-A and HLA-B pairs have fairly distinct binding motifs, and that the observed haplotypes do not contain HLA-A and -B molecules with more distinct binding motifs than random HLA-A and HLA-B pairs. In addition, the population frequency of a haplotype is not correlated to the distinctness of its HLA-A and HLA-B peptide binding motifs. These results suggest that there is a not a strong selection pressure on the haplotype level favoring haplotypes having HLA molecules with distinct binding motifs, which would result the largest possible presented peptide repertoires in the context of infectious diseases.

HLA-B molecules target more conserved regions of the HIV-1 proteome.

BACKGROUND: HLA-B alleles of HIV-infected individuals have been shown to have a major impact on their rate of progression toward AIDS, and the T-cell responses they restrict are immunodominant. OBJECTIVE: We sought to identify whether the association of HLA-B alleles with rate of progression toward AIDS is due to targeting of more restricted and thus more conserved regions of the HIV-1 proteome. METHODS: Each residue of the HIV-1 consensus subtype B sequence was coded according to the presence/absence of an epitope, using the compiled epitope data available in the HIV-LANL immunology database. The Shannon entropy for each HXB2 position was calculated using pre-aligned HIV-1 clade B sequences as a measure of its degree of conservation. We then compared the entropy of empty versus epitope-containing positions and HLA-B-restricted versus HLA-A-restricted positions. RESULTS: Positions containing CD8 epitopes were significantly more conserved than corresponding empty positions. Moreover, residues targeted by HLA-B alleles in the HIV-1 proteome were significantly more conserved than the ones targeted by HLA-A alleles. Analysing a recent dataset, we found that B epitope regions contain significantly more escape mutations and reversions, which might be the reason why we find them to be more conserved. CONCLUSION: Our results suggest that epitopes in HIV-1 targeted by HLA-B alleles lie in more constrained regions of its proteins, in which mutations might have a higher fitness cost and tend to revert. Consequently, HLA-B-restricted cytotoxic T-lymphocyte (CTL) responses may persist longer. This may be one of the factors contributing to the immunodominance and impact of HLA-B-restricted CTL responses on disease progression.