Methods and protocols for prediction of immunogenic epitopes

T-cell recognition of peptide/major histocompatibility complex (MHC) is a prerequisite for cellular immunity. Recently, there has been an influx of bioinformatics tools to facilitate the identification of T-cell epitopes to specific MHC alleles. This article examines existing computational strategies for the study of peptide/MHC interactions. The most important bioinformatics tools and methods with relevance to the study of peptide/MHC interactions have been reviewed. We have also provided guidelines for predicting antigenic peptides based on the availability of existing experimental data.     

High non-protective, long-lasting antibody levels in malaria are associated with haplotype shifting in MHC-peptide-TCR complex formation: a new mechanism for immune evasion

An effective malarial vaccine must contain multiple immunogenic, protection-inducing epitopes able to block and destroy the P. falciparum malaria parasite, the most lethal form of this disease in the world. Our strategy has consisted in using conserved peptides blocking parasite binding to red blood cells; however, these peptides are non-immunogenic and non-protection-inducing. Modifying their critical residues can make them immunogenic. Such peptides induced antibody titers (determined by immunofluorescence antibody test, IFA) and made the latter reactive (determined by Western blot) and protection inducing against experimental challenge with a highly infective Aotus monkey adapted P. falciparum strain. Modified peptides also induce highly non-protective long-lasting antibody levels. Modifications performed might allow them to bind specifically to different HLA-DRbeta purified molecules. These immunological and biological activities are associated with modifications in their three-dimensional structure as determined by (1)H-NMR. It was found that modified, high non-protective long-lasting antibody level peptides bound to HLA-DR molecules from a different haplotype (to which immunogenic, protection-inducers bind) and had 4.6 +/- 1.4 A shorter distances between residues fitting into these molecules' Pocket 1 to Pocket 9, suggesting fitting into an inappropriate HLA-DR molecule. A multi-component, subunit-based, malarial vaccine is therefore feasible if modified peptides are suitably modified for an appropriate fit into the correct HLA-DRbeta1* molecule in order to form a proper MHC-II-peptide-TCR complex.     

Quantitative immunoproteomics analysis reveals novel MHC class I presented peptides in cisplatin-resistant ovarian cancer cells

Platinum-based chemotherapy is widely used to treat various cancers including ovarian cancer. However, the mortality rate for patients with ovarian cancer is extremely high, largely due to chemo-resistant progression in patients who respond initially to platinum based chemotherapy. Immunotherapy strategies, including antigen specific vaccines, are being tested to treat drug resistant ovarian cancer with variable results. The identification of drug resistant specific tumor antigens would potentially provide significant improvement in effectiveness when combined with current and emerging therapies. In this study, using an immunoproteomics method based on iTRAQ technology and an LC-MS platform, we identified 952 MHC class I presented peptides. Quantitative analysis of the iTRAQ labeled MHC peptides revealed that cisplatin-resistant ovarian cancer cells display increased levels of MHC peptides derived from proteins that are implicated in many important cancer pathways. In addition, selected differentially presented epitope specific CTL recognize cisplatin-resistant ovarian cancer cells significantly better than the sensitive cells. These over-presented, drug resistance specific MHC class I associated peptide antigens could be potential targets for the development of immunotherapeutic strategies for the treatment of ovarian cancer including the drug resistant phenotype.     

Computational binding assays of antigenic peptides

Computer models can be combined with laboratory experiments for the efficient determination of (i) peptides that bind MHC molecules and (ii) T-cell epitopes. For maximum benefit, the use of computer models must be treated as experiments analogous to standard laboratory procedures. This requires the definition of standards and experimental protocols for model application. We describe the requirements for validation and assessment of computer models. The utility of combining accurate predictions with a limited number of laboratory experiments is illustrated by practical examples. These include the identification of T-cell epitopes from IDDM-, melanoma- and malaria-related antigens by combining computational and conventional laboratory assays. The success rate in determining antigenic peptides, each in the context of a specific HLA molecule, ranged from 27 to 71%, while the natural prevalence of MHC-binding peptides is 0.1–5%.     

Hydrophobicity as a driver of MHC class I antigen processing

The forces that drive conversion of nascent protein to major histocompatibility complex (MHC) class I-restricted peptides remain unknown. We explored the fundamental property of overt hydrophobicity as such a driver. Relocation of a membrane glycoprotein to the cytosol via signal sequence ablation resulted in rapid processing of nascent protein not because of the misfolded luminal domain but because of the unembedded transmembrane (TM) domain, which serves as a dose-dependent degradation motif. Dislocation of the TM domain during the natural process of endoplasmic reticulum-associated degradation (ERAD) similarly accelerated peptide production, but in the context of markedly prolonged processing that included nonnascent species. These insights into intracellular proteolytic pathways and their selective contributions to MHC class I-restricted peptide supply, may point to new approaches in rational vaccine design.     

Computational binding assays of antigenic peptides

Summary Computer models can be combined with laboratory experiments for the efficient determination of (i) peptides that bind MHC molecules and (ii) T-cell epitopes. For maximum benefit, the use of computer models must be treated as experiments analogous to standard laboratory procedures. This requires the definition of standards and experimental protocols for model application. We describe the requirements for validation and assessment of computer models. The utility of combining accurate predictions with a limited number of laboratory experiments is illustrated by practical examples. These include the identification of T-cell epitopes from IDDM-, melanoma-and malaria-related antigens by combining computational and conventional laboratory assays. The success rate in determining antigenic peptides, each in the context of a specific HLA molecule, ranged from 27 to 71%, while the natural prevalence of MHC-binding peptides is 0.1–5%.     

Adoptive transfer of autologous, HER2-specific, cytotoxic T lymphocytes for the treatment of HER2-overexpressing breast cancer

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated antigen by immunotherapeutical approaches based on HER2-directed monoclonal antibodies and cancer vaccines. We describe the adoptive transfer of autologous HER2-specific T-lymphocyte clones to a patient with metastatic HER2-overexpressing breast cancer. The HLA/multimer-based monitoring of the transferred T lymphocytes revealed that the T cells rapidly disappeared from the peripheral blood. The imaging studies indicated that the T cells accumulated in the bone marrow (BM) and migrated to the liver, but were unable to penetrate into the solid metastases. The disseminated tumor cells in the BM disappeared after the completion of adoptive T-cell therapy. This study suggests the therapeutic potential for HER2-specific T cells for eliminating disseminated HER2-positive tumor cells and proposes the combination of T cell-based therapies with strategies targeting the tumor stroma to improve T-cell infiltration into solid tumors.     

Studies of simian virus 40 DNA. VII. A cleavage map of the SV40 genome

A physical map of the Simian virus 40 genome has been constructed on the basis of specific cleavage of Simian virus 40 DNA by bacterial restriction endonucleases. The 11 fragments produced by enzyme from Hemophilus influenzae have been ordered by analysis of partial digest products and by analysis of an overlapping set of fragments produced by enzyme from Hemophilus parainfluenzae. In addition, the single site in SV40 DNA cleaved by the Escherichia coli R_I restriction endonuclease has been located. With this site as a reference point, the H. influenzae cleavage sites and the H. parainfluenzae cleavage sites have been localized on the map.     

Social discrimination by quantitative assessment of immunogenetic similarity

Genes of the major histocompatibility complex (MHC) that underlie the adaptive immune system may allow vertebrates to recognize their kin. True kin-recognition genes should produce signalling products to which organisms can respond. Allelic variation in the peptide-binding region (PBR) of MHC molecules determines the pool of peptides that can be presented to trigger an immune response. To examine whether these MHC peptides also might underlie assessments of genetic similarity, we tested whether Xenopus laevis tadpoles socially discriminate between pairs of siblings with which they differed in PBR amino acid sequences. We found that tadpoles (four sibships, n = 854) associated preferentially with siblings with which they were more similar in PBR amino acid sequence. Moreover, the strength of their preference for a conspecific was directly proportional to the sequence similarity between them. Discrimination was graded, and correlated more closely with functional sequence differences encoded by MHC class I and class II alleles than with numbers of shared haplotypes. Our results thus suggest that haplotype analyses may fail to reveal fine-scale behavioural responses to divergence in functionally expressed sequences. We conclude that MHC–PBR gene products mediate quantitative social assessment of immunogenetic similarity that may facilitate kin recognition in vertebrates.     

Class I and class II MHC bind self peptide sets that are strikingly different in their evolutionary characteristics

 Comparison of peptides eluted from human class I and class II major histocompatibility complex (MHC) molecules and the proteins from which they are derived (source proteins) revealed that class I MHC bind peptides derived from proteins that are highly conserved, hydrophilic, and universally expressed, while the peptides themselves are hydrophobic and even more conserved than their source proteins. In contrast, source proteins for class II-bound peptides were not significantly more conserved than a random sample of proteins. Class II-bound peptides were generally more conserved than their source proteins but were significantly less conserved than class I-bound peptides. The characteristics of class I-bound peptides can probably be explained by the selectivity of processing and transport of peptides for binding by class I, while the relative lack of selectivity of peptide binding for class II may explain the high incidence of autoimmune diseases associated with alleles of these molecules. Received: 17 May 1999 / Revised: 5 August 1999     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

Characterization of a rearrangement in viral DNA: mapping of the circular simian virus 40-like DNA containing a triplication of a specific one-third of the viral genome

Serial passage of the non-defective form of a simian virus 40-like virus (DAR) isolated from human brain results in the appearance of three distinct classes of supercoiled DNAs: R_I resistant, R_I sensitive (one cleavage site) and R_I “supersensitive” (three cleavage sites). The R_I cleavage product of the “super sensitive” form is one-third the physical size of simian virus 40 DNA (10.4 S) and reassociates about three times more rapidly than “standard” viral DNA. To identify the portions of the DAR genome present in the 10.4 S segment, the plus strand of each of the 11 fragments of ³²P-labeled simian virus 40 DNA, produced by cleavage with the Hemophilus influenzae restriction endonuclease, was hybridized in solution with the sheared R_I cleavage product of the “supersensitive” class of viral DNA. Reaction was observed with fragments located in two distinct regions of the simian virus 40 genome: (1) Hin-A and C; (2) Hin-G, J, F and K.Further studies indicated that simian virus 40 complementary RNA transcribed in vitro with Escherichia coli RNA polymerase from one strand of simian virus 40 DNA reacts with both strands of the denatured 10.4 S cleavage product when hybridization is monitored with hydroxyapatite. Treatment of the 10.4 S DNA-simian virus 40 cRNA hybrid with the single-strand spcific nuclease, S₁, converted approximately 50% of the radioactive counts to an acid-soluble product. These results indicate that the 10.4 S product contains a transposition of sequences originally present on one of the DAR DNA strands to the other strand. Examination of heteroduplexes formed between the 10.4 S segment and unique linear forms of DAR DNA produced with the R · Eco RI restriction endonuclease have confirmed these observations. Thus it appears that a molecular rearrangement(s) has resulted in the recombination and inversion of viral DNA sequences from two separate loci on the parental DAR genome. This 1.1 × 10⁶ dalton segment is reiterated three times in a supercoiled molecule equivalent in physical size to parental DAR DNA.