Gustav Røder

NetMHCpan, a Method for Quantitative Predictions of Peptide Binding to Any HLA-A and -B Locus Protein of Known Sequence

Background Binding of peptides to Major Histocompatibility Complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC class I system (HLA-I) is extremely polymorphic. The number of registered HLA-I molecules has now surpassed 1500. Characterizing the specificity of each separately would be a major undertaking. Principal Findings Here, we have drawn on a large database of known peptide-HLA-I interactions to develop a bioinformatics method, which takes both peptide and HLA sequence information into account, and generates quantitative predictions of the affinity of any peptide-HLA-I interaction. Prospective experimental validation of peptides predicted to bind to previously untested HLA-I molecules, cross-validation, and retrospective prediction of known HIV immune epitopes and endogenous presented peptides, all successfully validate this method. We further demonstrate that the method can be applied to perform a clustering analysis of MHC specificities and suggest using this clustering to select particularly informative novel MHC molecules for future biochemical and functional analysis. Conclusions Encompassing all HLA molecules, this high-throughput computational method lends itself to epitope searches that are not only genome- and pathogen-wide, but also HLA-wide. Thus, it offers a truly global analysis of immune responses supporting rational development of vaccines and immunotherapy. It also promises to provide new basic insights into HLA structure-function relationships. The method is available at http://www.cbs.dtu.dk/services/NetMHCpan.

Peptide‐MHC class I stability is a better predictor than peptide affinity of CTL immunogenicity

Efficient presentation of peptide‐MHC class I (pMHC‐I) complexes to immune T cells should benefit from a stable peptide‐MHC‐I interaction. However, it has been difficult to distinguish stability from other requirements for MHC‐I binding, for example, affinity. We have recently established a high‐throughput assay for pMHC‐I stability. Here, we have generated a large database containing stability measurements of pMHC‐I complexes, and re‐examined a previously reported unbiased analysis of the relative contributions of antigen processing and presentation in defining cytotoxic T lymphocyte (CTL) immunogenicity [Assarsson et al., J. Immunol. 2007. 178: 7890–7901]. Using an affinity‐balanced approach, we demonstrated that immunogenic peptides tend to be more stably bound to MHC‐I molecules compared with nonimmunogenic peptides. We also developed a bioinformatics method to predict pMHC‐I stability, which suggested that 30% of the nonimmunogenic binders hitherto classified as “holes in the T‐cell repertoire” can be explained as being unstably bound to MHC‐I. Finally, we suggest that nonoptimal anchor residues in position 2 of the peptide are particularly prone to cause unstable interactions with MHC‐I. We conclude that the availability of accurate predictors of pMHC‐I stability might be helpful in the elucidation of MHC‐I restricted antigen presentation, and might be instrumental in future search strategies for MHC‐I epitopes.

Peptide Binding to HLA Class I Molecules : Homogenous, High-Throughput Screening, and Affinity Assays

The Human MHC Project aims at large-scale description of peptide-HLA binding to a wide range of HLA molecules covering all populations of the world and the accompanying generation of bioinformatics tools capable of predicting binding of any given peptide to any given HLA molecule. Here, the authors present a homogenous, proximity-based assay for detection of peptide binding to HLA class I molecules. It uses a conformation-dependent anti-HLA class I antibody, W6/32, as one tag and a biotinylated recombinant HLA class I molecule as the other tag, and a proximity-based signal is generated through the luminescent oxygen channeling immunoassay technology (abbreviated LOCI and commercialized as AlphaScreen™). Compared with an enzyme-linked immunosorbent assay—based peptide-HLA class I binding assay, the LOCI assay yields virtually identical affinity measurements, although having a broader dynamic range, better signal-to-background ratios, and a higher capacity. They also describe an efficient approach to screen peptides for binding to HLA molecules. For the occasional user, this will serve as a robust, simple peptide-HLA binding assay. For the more dedicated user, it can easily be performed in a high-throughput screening mode using standard liquid handling robotics and 384-well plates. We have successfully applied this assay to more than 60 different HLA molecules, leading to more than 2 million measurements. (Journal of Biomolecular Screening 2009:173-180)

Real-time, high-throughput measurements of peptide-MHC-I dissociation using a scintillation proximity assay.

Efficient presentation of peptide-MHC class I complexes to immune T cells depends upon stable peptide-MHC class I interactions. Theoretically, determining the rate of dissociation of a peptide-MHC class I complexes is straightforward; in practical terms, however, generating the accurate and closely timed data needed to determine the rate of dissociation is not simple. Ideally, one should use a homogenous assay involving an inexhaustible and label-free assay principle. Here, we present a homogenous, high-throughput peptide-MHC class I dissociation assay, which by and large fulfill these ideal requirements. To avoid labeling of the highly variable peptide, we labeled the invariant β2m and monitored its dissociation by a scintillation proximity assay, which has no separation steps and allows for real-time quantitative measurement of dissociation. Validating this work-around to create a virtually label-free assay, we showed that rates of peptide-MHC class I dissociation measured in this assay correlated well with rates of dissociation rates measured conventionally with labeled peptides. This assay can be used to measure the stability of any peptide-MHC class I combination, it is reproducible and it is well suited for high-throughput screening. To exemplify this, we screened a panel of 384 high-affinity peptides binding to the MHC class I molecule, HLA-A*02:01, and observed the rates of dissociation that ranged from 0.1h to 46h depending on the peptide used.

SARS CTL vaccine candidates; HLA supertype-, genome-wide scanning and biochemical validation.

Abstract:  An effective Severe Acute Respiratory Syndrome (SARS) vaccine is likely to include components that can induce specific cytotoxic T‐lymphocyte (CTL) responses. The specificities of such responses are governed by human leukocyte antigen (HLA)‐restricted presentation of SARS‐derived peptide epitopes. Exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information (Lauemoller et al., Rev Immunogenet 2001: 2: 477–91). The latter was recently established when a causative coronavirus (SARS‐CoV) was isolated and full‐length sequenced (Marra et al., Science 2003: 300: 1399–404). Here, we have combined advanced bioinformatics and high‐throughput immunology to perform an HLA supertype‐, genome‐wide scan for SARS‐specific CTL epitopes. The scan includes all nine human HLA supertypes in total covering >99% of all individuals of all major human populations (Sette & Sidney, Immunogenetics 1999: 50: 201–12). For each HLA supertype, we have selected the 15 top candidates for test in biochemical binding assays. At this time (approximately 6 months after the genome was established), we have tested the majority of the HLA supertypes and identified almost 100 potential vaccine candidates. These should be further validated in SARS survivors and used for vaccine formulation. We suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design.

The peptide-binding specificity of HLA-A*3001 demonstrates membership of the HLA-A3 supertype

Human leukocyte antigen class I (HLA-I) molecules are highly polymorphic peptide receptors, which select and present endogenously derived peptide epitopes to CD8+ cytotoxic T cells (CTL). The specificity of the HLA-I system is an important component of the overall specificity of the CTL immune system. Unfortunately, the large and rapidly increasing number of known HLA-I molecules seriously complicates a comprehensive analysis of the specificities of the entire HLA-I system (as of June 2008, the international HLA registry holds >1,650 unique HLA-I protein entries). In an attempt to reduce this complexity, it has been suggested to cluster the different HLA-I molecules into “supertypes” of largely overlapping peptide-binding specificities. Obviously, the HLA supertype concept is only valuable if membership can be assigned with reasonable accuracy. The supertype assignment of HLA-A*3001, a common HLA haplotype in populations of African descent, has variously been assigned to the A1, A3, or A24 supertypes. Using a biochemical HLA-A*3001 binding assay, and a large panel of nonamer peptides and peptide libraries, we here demonstrate that the specificity of HLA-A*3001 most closely resembles that of the HLA-A3 supertype. We discuss approaches to supertype assignment and underscore the importance of experimental verification.

Viral Proteins Interfering with Antigen Presentation Target the Major Histocompatibility Complex Class I Peptide-Loading Complex

The adaptive immune system is responsible for the final clearance and long lasting immunological memory of invading pathogens. Major histocompatibility complex class I (MHC-I) molecules play a central role in this defense as reporters of cellular content by presenting peptides derived from interior proteins in the cell. When recirculating cytotoxic T lymphocytes recognize MHC-I-presented peptides as foreign (e.g., derived from viral proteins), the presenting cell is killed by cytotoxic T lymphocytes, thereby hindering the spread of the virus. Thus, the key to efficient viral clearance by cytotoxic T lymphocytes lies within both the quality of the T-cell-receptor repertoire and the efficient processing and presentation of MHC-I-bound peptides. From the initial synthesis and folding to the final presentation on the cell surface, the MHC-I molecule gradually matures through multiple steps, most of which take place inside the endoplasmic reticulum (ER). The final stage of maturation for most MHC-I molecules takes place in the peptide-loading complex. The immune system and pathogens have evolved side by side for millions of years, and invading pathogens have developed several escape mechanisms to cripple the immune system. Among them are viral proteins that interfere with antigen presentation (VIPRs), which target both MHC-I and MHC-II antigen processing in order to skew or totally inhibit a functional immune response toward the virus. In this review, we discuss the main discoveries and latest developments concerning VIPRs that target the MHC-I peptide-loading complex.

The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I

Tapasin (Tpn) is an ER chaperone that is uniquely dedicated to MHC‐I biosynthesis. It binds MHC‐I molecules, integrates them into peptide‐loading complexes, and exerts quality control of the bound peptides; only when an “optimal peptide” is bound will the MHC‐I be released and exported to the cell surface for presentation to T cells. The exact mechanisms of Tpn quality control and the criteria for being an optimal peptide are still unknown. Here, we have generated a recombinant fragment of human Tpn, Tpn1–87 (representing the 87 N‐terminal and ER‐luminal amino acids of the mature Tpn protein). Using a biochemical peptide–MHC‐I‐binding assay, recombinant Tpn1–87 was found to specifically facilitate peptide‐dependent folding of HLA‐A*0201. Furthermore, we used Tpn1–87 to generate a monoclonal antibody, αTpn1–87/80, specific for natural human Tpn and capable of cellular staining of ER localized Tpn. Using overlapping peptides, the epitope of αTpn1–87/80 was located to Tpn40–44, which maps to a surface‐exposed loop on the Tpn structure. Together, these results demonstrate that the N‐terminal region of Tpn can be recombinantly expressed and adopt a structure, which at least partially resembles that of WT Tpn, and that this region of Tpn features chaperone activity facilitating peptide binding of MHC‐I.

Crystal structures of two peptide-HLA-B*1501 complexes; structural characterization of the HLA-B62 supertype.

MHC class I molecules govern human cytotoxic T cell responses. Their specificity determines which peptides they sample from the intracellular protein environment and then present to human cytotoxic T cells. More than 1100 different MHC class I proteins have been found in human populations and it would be a major undertaking to address each of these specificities individually. Based upon their peptide binding specificity, they are currently subdivided into 12 supertypes. Several of these HLA supertypes have not yet been described at the structural level. To support a comprehensive understanding of human immune responses, the structure of at least one member of each supertype should be determined. Here, the structures of two immunogenic peptide-HLA-B*1501 complexes are described. The structure of HLA-B*1501 in complex with a peptide (LEKARGSTY, corresponding to positions 274-282 in the Epstein-Barr virus nuclear antigen-3A) was determined to 2.3 A resolution. The structure of HLA-B*1501 in complex with a peptide (ILGPPGSVY) derived from human ubiquitin-conjugating enzyme-E2 corresponding to positions 91-99 was solved to 1.8 A resolution. Mutual comparisons of these two structures with structures from other HLA supertypes define and explain the specificity of the P2 and P9 peptide anchor preferences in the B62 HLA supertype. The P2 peptide residue binds to the B-pocket in HLA-B*1501. This pocket is relatively large because of the small Ser67 residue located at the bottom. The peptide proximal part of the B-pocket is hydrophobic, which is consistent with P2 anchor residue preference for Leu. The specificity of the B-pocket is determined by the Met45, Ile66 and Ser67 residues. The apex of the B-pocket is hydrophilic because of the Ser67 residue. The P9 peptide residue binds to the F-pocket in HLA-B*1501. The residues most important for the specificity of this pocket are Tyr74, Leu81, Leu95, Tyr123 and Trp147. These residues create a hydrophobic interior in the F-pocket and their spatial arrangement makes the pocket capable of containing large, bulky peptide side chains. Ser116 is located at the bottom of the F-pocket and makes the bottom of this pocket hydrophilic. Ser116, may act as a hydrogen-bonding partner and as such is a perfect place for binding of a Tyr9 peptide residue. Thus, based on structure information it is now possible to explain the peptide sequence specificity of HLA-B*1501 as previously determined by peptide binding and pool sequencing experiments.

Tapasin Facilitation of Natural HLA-A and -B Allomorphs Is Strongly Influenced by Peptide Length, Depends on Stability, and Separates Closely Related Allomorphs

Despite an abundance of peptides inside a cell, only a small fraction is ultimately presented by HLA-I on the cell surface. The presented peptides have HLA-I allomorph-specific motifs and are restricted in length. So far, detailed length studies have been limited to few allomorphs. Peptide–HLA-I (pHLA-I) complexes of different allomorphs are qualitatively and quantitatively influenced by tapasin to different degrees, but again, its effect has only been investigated for a small number of HLA-I allomorphs. Although both peptide length and tapasin dependence are known to be important for HLA-I peptide presentation, the relationship between them has never been studied. In this study, we used random peptide libraries from 7- to 13-mers and studied binding in the presence and absence of a recombinant truncated form of tapasin. The data show that HLA-I allomorphs are differentially affected by tapasin, different lengths of peptides generated different amounts of pHLA-I complexes, and HLA-A allomorphs are generally less restricted than HLA-B allomorphs to peptides of the classical length of 8–10 aa. We also demonstrate that tapasin facilitation varies for different peptide lengths, and that the correlation between high degree of tapasin facilitation and low stability is valid for different random peptide mixes of specific lengths. In conclusion, these data show that tapasin has specificity for the combination of peptide length and HLA-I allomorph, and suggest that tapasin promotes formation of pHLA-I complexes with high on and off rates, an important intermediary step in the HLA-I maturation process.