The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.

Structure of human T-cell receptors specific for an immunodominant myelin basic protein peptide: positioning of T-cell receptors on HLA-DR2/peptide complexes.

T-cell receptors (TCRs) recognize peptide bound within the relatively conserved structural framework of major histocompatibility complex (MHC) class I or class II molecules but can discriminate between closely related MHC molecules. The structural basis for the specificity of ternary complex formation by the TCR and MHC/peptide complexes was examined for myelin basic protein (MBP)-specific T-cell clones restricted by different DR2 subtypes. Conserved features of this system allowed a model for positioning of the TCR on DR2/peptide complexes to be developed: (i) The DR2 subtypes that presented the immunodominant MBP peptide differed only at a few polymorphic positions of the DR beta chain. (ii) TCR recognition of a polymorphic residue on the helical portion of the DR beta chain (position DR beta 67) was important in determining the MHC restriction. (iii) The TCR variable region (V) alpha 3.1 gene segment was used by all of the T-cell clones. TCR V beta usage was more diverse but correlated with the MHC restriction--i.e., with the polymorphic DR beta chains. (iv) Two clones with conserved TCR alpha chains but different TCR beta chains had a different MHC restriction but a similar peptide specificity. The difference in MHC restriction between these T-cell clones appeared due to recognition of a cluster of polymorphic DR beta-chain residues (DR beta 67-71). MBP-(85-99)-specific TCRs therefore appeared to be positioned on the DR2/peptide complex such that the TCR beta chain contacted the polymorphic DR beta-chain helix while the conserved TCR alpha chain contacted the nonpolymorphic DR alpha chain.

Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove.

Sequence analysis of peptides naturally presented by major histocompatibility complex (MHC) class I molecules has revealed allele-specific motifs in which the peptide length and the residues observed at certain positions are restricted. Nevertheless, peptides containing the standard motif often fail to bind with high affinity or form physiologically stable complexes. Here we present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2Kb molecule at 2.5-A resolution. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2Kb in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water molecule that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2Kb has at least two submotifs: one with Tyr at P5 (or P6 for nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residue at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallographic and immunological studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.

Immunization with a circumsporozoite epitope fused to Bordetella pertussis adenylate cyclase in conjunction with cytotoxic T-lymphocyte-associated antigen 4 blockade confers protection against Plasmodium berghei liver-stage malaria

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8+ T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8+ T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-gamma) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.

Complete protection against P. berghei malaria upon heterologous prime/boost immunization against circumsporozoite protein employing Salmonella type III secretion system and Bordetella adenylate cyclase toxoid

Sterile immunity against malaria can be achieved by the induction of IFNgamma-producing CD8(+) T cells that target infected hepatocytes presenting epitopes of the circumsporozoite protein (CSP). In the present study we evaluate the protective efficacy of a heterologous prime/boost immunization protocol based on the delivery of the CD8(+) epitope of Plasmodium berghei CSP into the MHC class I presentation pathway, by either a type III secretion system of live recombinant Salmonella and/or by direct translocation of a recombinant Bordetella adenylate cyclase toxoid fusion (ACT-CSP) into the cytosol of professional antigen-presenting cells (APCs). A single intraperitoneal application of the recombinant ACT-CSP toxoid, as well as a single oral immunization with the Salmonella vaccine, induced a specific CD8(+) T cell response, which however conferred only a partial protection on mice against a subsequent sporozoite challenge. In contrast, a heterologous prime/boost vaccination with the live Salmonella followed by ACT-CSP led to a significant enhancement of the CSP-specific T cell response and induced complete protection in all vaccinated mice.

Mutations in TAP genes are common in cervical cancinomas

Objective. To determine whether squamous cervical cancers exhibit mutations or deletions in MHC class I genes or transport-associated protein (TAP) genes. Methods. Polymerase chain reaction based protocols were used to examine HLA class I and TAP genes in a panel of cervical tumours, using DNA from corresponding blood samples as controls. SSP-PCR protocols were similarly used for examination of all TAP alleles in tumour and blood samples. Results. In a series of cervical carcinomas, 7 of 27 (26%) exhibited mutations in HLA-A genes, while 12 of 23 (52%) exhibited mutations in TAP genes. HLA gene mutations were detected in 2 of 14 CIN2-3 lesions, and TAP gene mutations in none of 14, a frequency significantly less than observed in the cervical carcinoma samples (P < 0.01). The TAP 2A/2B heterozygous genotype was observed with increased frequency in patients with cervical cancer compared to population controls (P < 0.02). Conclusion. These data suggest that TAP genes may be relevant to evolution of cervical cancer from precursor lesions. (C) 2004 Elsevier Inc. All rights reserved.

Despite differences between dendritic cells and Langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime T cells

As human papillomavirus-like particles (HPV-VLP) represent a promising vaccine delivery vehicle, delineation of the interaction of VLP with professional APC should improve vaccine development. Differences in the capacity of VLP to signal dendritic cells (DC) and Langerhans cells (LC) have been demonstrated, and evidence has been presented for both clathrin-coated pits and proteoglycans (PG) in the uptake pathway of VLP into epithelial cells. Therefore, we compared HPV-VLP uptake mechanisms in human monocyte-derived DC and LC, and their ability to cross-present HPV VLP-associated antigen in the MHC class I pathway. DC and LC each took up virus-like particles (VLP). DC uptake of and signalling by VLP was inhibited by amiloride or cytochalasin D (CCD), but not by filipin treatment, and was blocked by several sulfated and non-sulfated polysaccharides and anti-CD16. In contrast, LC uptake was inhibited only by filipin, and VLP in LC were associated with caveolin, langerin, and CD1a. These data suggest fundamentally different routes of VLP uptake by DC and LC. Despite these differences, VLP taken up by DC and LC were each able to prime naive CD8(+) T cells and induce cytolytic effector T cells in vitro. (C) 2004 Elsevier Inc. All rights reserved.

CTL recognition of a bulged viral peptide involves biased TCR selection

MHC class I molecules generally present peptides of 8-10 aa long, forming an extended coil in the HLA cleft. Although longer peptides can also bind to class I molecules, they tend to bulge from the cleft and it is not known whether the TCR repertoire has sufficient plasticity to recognize these determinants during the antiviral CTL response. In this study, we show that unrelated individuals infected with EBV generate a significant CTL response directed toward an HLA-B*3501-restricted, 11-mer epitope from the BZLF1 Ag. The 11-mer determinant adopts a highly bulged conformation with seven of the peptide side chains being solvent-exposed and available for TCR interaction. Such a complex potentially creates a structural challenge for TCR corecognition of both HLA-B*3501 and the peptide Ag. Surprisingly, unrelated B*3501 donors recognizing the 11-mer use identical or closely related alpha beta TCR sequences that share particular CDR3 motifs. Within the small number of dominant CTL clonotypes observed, each has discrete fine specificity for the exposed-side chain residues of the peptide. The data show that bulged viral peptides are indeed immunogenic but suggest that the highly constrained TCR repertoire reflects a limit to TCR diversity when responding to some unusual MHC peptide ligands.

Lipid based particulate formulations for the delivery of antigen

Particulate adjuvant systems are largely classified according to their functional characteristics, such as the nature of the typical immune response they induce, or their perceived mode of action. From a formulation science perspective, it is practical to classify antigen delivery systems according to the physical nature of the formulations. This article discusses lipid based particulate systems, grouped according to the nature of their predominant lipid constituent.

Overcoming original antigenic sin to generate new CD8 T cell IFN-gamma responses in an antigen-experienced host

The failure to mount effective immunity to virus variants in a previously virus-infected host is known as original antigenic sin. We have previously shown that prior immunity to a virus capsid protein inhibits induction by immunization of an IFN-gamma CD8(+) T cell response to an epitope linked to the capsid protein. We now demonstrate that capsid protein-primed CD4(+) T cells secrete IL-10 in response to capsid protein presented by dendritic cells, and deviate CD8+ T cells responding to a linked MHC class I-restricted epitope to reduce IFN-gamma production. Neutralizing IL-10 while delivering further linked epitope, either in vitro or in vivo, restores induction by immunization of an Ag-specific IFN-gamma response to the epitope. This finding demonstrates a strategy for overcoming inhibition of MHC class I epitopes upon immunization of a host already primed to Ag, which may facilitate immunotherapy for chronic viral infection or cancer.

Novel visualization methods for protein data

Visualization of high-dimensional data has always been a challenging task. Here we discuss and propose variants of non-linear data projection methods (Generative Topographic Mapping (GTM) and GTM with simultaneous feature saliency (GTM-FS)) that are adapted to be effective on very high-dimensional data. The adaptations use log space values at certain steps of the Expectation Maximization (EM) algorithm and during the visualization process. We have tested the proposed algorithms by visualizing electrostatic potential data for Major Histocompatibility Complex (MHC) class-I proteins. The experiments show that the variation in the original version of GTM and GTM-FS worked successfully with data of more than 2000 dimensions and we compare the results with other linear/nonlinear projection methods: Principal Component Analysis (PCA), Neuroscale (NSC) and Gaussian Process Latent Variable Model (GPLVM).

Integrating in silico and in vitro analysis of peptide binding affinity to HLA-Cw*0102:a bioinformatic approach to the prediction of new epitopes

Predictive models of peptide-Major Histocompatibility Complex (MHC) binding affinity are important components of modern computational immunovaccinology. Here, we describe the development and deployment of a reliable peptide-binding prediction method for a previously poorly-characterized human MHC class I allele, HLA-Cw*0102.

Multi-level visualisation using Gaussian process latent variable models

Projection of a high-dimensional dataset onto a two-dimensional space is a useful tool to visualise structures and relationships in the dataset. However, a single two-dimensional visualisation may not display all the intrinsic structure. Therefore, hierarchical/multi-level visualisation methods have been used to extract more detailed understanding of the data. Here we propose a multi-level Gaussian process latent variable model (MLGPLVM). MLGPLVM works by segmenting data (with e.g. K-means, Gaussian mixture model or interactive clustering) in the visualisation space and then fitting a visualisation model to each subset. To measure the quality of multi-level visualisation (with respect to parent and child models), metrics such as trustworthiness, continuity, mean relative rank errors, visualisation distance distortion and the negative log-likelihood per point are used. We evaluate the MLGPLVM approach on the ‘Oil Flow’ dataset and a dataset of protein electrostatic potentials for the ‘Major Histocompatibility Complex (MHC) class I’ of humans. In both cases, visual observation and the quantitative quality measures have shown better visualisation at lower levels.

EpiJen:a server for multistep T cell epitope prediction

Background - The main processing pathway for MHC class I ligands involves degradation of proteins by the proteasome, followed by transport of products by the transporter associated with antigen processing (TAP) to the endoplasmic reticulum (ER), where peptides are bound by MHC class I molecules, and then presented on the cell surface by MHCs. The whole process is modeled here using an integrated approach, which we call EpiJen. EpiJen is based on quantitative matrices, derived by the additive method, and applied successively to select epitopes. EpiJen is available free online. Results - To identify epitopes, a source protein is passed through four steps: proteasome cleavage, TAP transport, MHC binding and epitope selection. At each stage, different proportions of non-epitopes are eliminated. The final set of peptides represents no more than 5% of the whole protein sequence and will contain 85% of the true epitopes, as indicated by external validation. Compared to other integrated methods (NetCTL, WAPP and SMM), EpiJen performs best, predicting 61 of the 99 HIV epitopes used in this study. Conclusion - EpiJen is a reliable multi-step algorithm for T cell epitope prediction, which belongs to the next generation of in silico T cell epitope identification methods. These methods aim to reduce subsequent experimental work by improving the success rate of epitope prediction.

Visualisation of bioinformatics datasets

Analysing the molecular polymorphism and interactions of DNA, RNA and proteins is of fundamental importance in biology. Predicting functions of polymorphic molecules is important in order to design more effective medicines. Analysing major histocompatibility complex (MHC) polymorphism is important for mate choice, epitope-based vaccine design and transplantation rejection etc. Most of the existing exploratory approaches cannot analyse these datasets because of the large number of molecules with a high number of descriptors per molecule. This thesis develops novel methods for data projection in order to explore high dimensional biological dataset by visualising them in a low-dimensional space. With increasing dimensionality, some existing data visualisation methods such as generative topographic mapping (GTM) become computationally intractable. We propose variants of these methods, where we use log-transformations at certain steps of expectation maximisation (EM) based parameter learning process, to make them tractable for high-dimensional datasets. We demonstrate these proposed variants both for synthetic and electrostatic potential dataset of MHC class-I. We also propose to extend a latent trait model (LTM), suitable for visualising high dimensional discrete data, to simultaneously estimate feature saliency as an integrated part of the parameter learning process of a visualisation model. This LTM variant not only gives better visualisation by modifying the project map based on feature relevance, but also helps users to assess the significance of each feature. Another problem which is not addressed much in the literature is the visualisation of mixed-type data. We propose to combine GTM and LTM in a principled way where appropriate noise models are used for each type of data in order to visualise mixed-type data in a single plot. We call this model a generalised GTM (GGTM). We also propose to extend GGTM model to estimate feature saliencies while training a visualisation model and this is called GGTM with feature saliency (GGTM-FS). We demonstrate effectiveness of these proposed models both for synthetic and real datasets. We evaluate visualisation quality using quality metrics such as distance distortion measure and rank based measures: trustworthiness, continuity, mean relative rank errors with respect to data space and latent space. In cases where the labels are known we also use quality metrics of KL divergence and nearest neighbour classifications error in order to determine the separation between classes. We demonstrate the efficacy of these proposed models both for synthetic and real biological datasets with a main focus on the MHC class-I dataset.