Positional Bias of MHC Class I Restricted T-Cell Epitopes in Viral Antigens Is Likely due to a Bias in Conservation

The immune system rapidly responds to intracellular infections by detecting MHC class I restricted T-cell epitopes presented on infected cells. It was originally thought that viral peptides are liberated during constitutive protein turnover, but this conflicts with the observation that viral epitopes are detected within minutes of their synthesis even when their source proteins exhibit half-lives of days. The DRiPs hypothesis proposes that epitopes derive from Defective Ribosomal Products (DRiPs), rather than degradation of mature protein products. One potential source of DRiPs is premature translation termination. If this is a major source of DRiPs, this should be reflected in positional bias towards the N-terminus. By contrast, if downstream initiation is a major source of DRiPs, there should be positional bias towards the C-terminus. Here, we systematically assessed positional bias of epitopes in viral antigens, exploiting the large set of data available in the Immune Epitope Database and Analysis Resource. We show a statistically significant degree of positional skewing among epitopes; epitopes from both ends of antigens tend to be under-represented. Centric-skewing correlates with a bias towards class I binding peptides being over-represented in the middle, in parallel with a higher degree of evolutionary conservation.     

Distinct pathways generate peptides from defective ribosomal products for CD8+ T cell immunosurveillance

To understand better the endogenous sources of MHC class I peptide ligands, we generated an antigenic reporter protein whose degradation is rapidly and reversibly controlled with Shield-1, a cell-permeant drug. Using this system, we demonstrate that defective ribosomal products (DRiPs) represent a major and highly efficient source of peptides and are completely resistant to our attempts to stabilize the protein. Although peptides also derive from nascent Shield-1-sensitive proteins and "retirees" created by Shield-1 withdrawal, these are much less efficient sources on a molar basis. We use this system to identify two drugs--each known to inhibit polyubiquitin chain disassembly--that selectively inhibit presentation of Shield-1-resistant DRiPs. These findings provide the initial evidence for distinct biochemical pathways for presentation of DRiPs versus retirees and implicate polyubiquitin chain disassembly or the actions of deubiquitylating enzymes as playing an important role in DRiP presentation.     

PAProC: a prediction algorithm for proteasomal cleavages available on the WWW

The first version of PAProC (Prediction Algorithm for Proteasomal Cleavages) is now available to the general public. PAProC is a prediction tool for cleavages by human and yeast proteasomes, based on experimental cleavage data. It will be particularly useful for immunologists working on antigen processing and the prediction of major histocompatibility complex class I molecule (MHC I) ligands and cytotoxic T-lymphocyte (CTL) epitopes. Likewise, in cases in which proteasomal protein degradation has been indicated in disease, PAProC can be used to assess the general cleavability of disease-linked proteins. On its web site (http://www.paproc.de), background information and hyperlinks are provided for the user (e.g., to SYFPEITHI, the database for the prediction of MHC I ligands).     

Binding of human cytomegalovirus US2 to major histocompatibility complex class I and II proteins is not sufficient for their degradation

Human cytomegalovirus (HCMV) glycoprotein US2 causes degradation of major histocompatibility complex (MHC) class I heavy-chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. In US2-expressing cells, MHC proteins present in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. It appears that US2 binding triggers a normal cellular pathway by which misfolded or aberrant proteins are translocated from the ER to cytoplasmic proteasomes. To better understand how US2 binds MHC proteins and causes their degradation, we constructed a panel of US2 mutants. Mutants truncated from the N terminus as far as residue 40 or from the C terminus to amino acid 140 could bind to class I and class II proteins. Nevertheless, mutants lacking just the cytosolic tail (residues 187 to 199) were unable to cause degradation of both class I and II proteins. Chimeric proteins were constructed in which US2 sequences were replaced with homologous sequences from US3, an HCMV glycoprotein that can also bind to class I and II proteins. One of these US2/US3 chimeras bound to class II but not to class I, and a second bound class I HC better than wild-type US2. Therefore, US2 residues involved in the binding to MHC class I differ subtly from those involved in binding to class II proteins. Moreover, our results demonstrate that the binding of US2 to class I and II proteins is not sufficient to cause degradation of MHC proteins. The cytosolic tail of US2 and certain US2 lumenal sequences, which are not involved in binding to MHC proteins, are required for degradation. Our results are consistent with the hypothesis that US2 couples MHC proteins to components of the ER degradation pathway, enormously increasing the rate of degradation of MHC proteins.     

MHC Class I Antigen Presentation of DRiP-Derived Peptides from a Model Antigen Is Not Dependent on the AAA ATPase p97

CD8⁺ T cells are responsible for killing cells of the body that have become infected or oncogenically transformed. In order to do so, effector CD8⁺ T cells must recognize their cognate antigenic peptide bound to a MHC class I molecule that has been directly presented by the target cell. Due to the rapid nature of antigen presentation, it is believed that antigenic peptides are derived from a subset of newly synthesized proteins which are degraded almost immediately following synthesis and termed Defective Ribosomal Products or DRiPs. We have recently reported on a bioassay which can distinguish antigen presentation of DRiP substrates from other forms of rapidly degraded proteins and found that poly-ubiquitin chain disassembly may be necessary for efficient DRiP presentation. The AAA ATPase p97 protein is necessary for efficient cross-presentation of antigens on MHC class I molecules and plays an important role in extracting mis-folded proteins from the endoplasmic reticulum. Here, we find that genetic ablation or chemical inhibition of p97 does not diminish DRiP antigen presentation to any great extent nor does it alter the levels of MHC class I molecules on the cell surface, despite our observations that p97 inhibition increased the levels of poly-ubiquitinated proteins in the cell. These data demonstrate that inhibiting poly-ubiquitin chain disassembly alone is insufficient to abolish DRiP presentation.     

Gamma-herpesvirus latency requires T cell evasion during episome maintenance

The gamma-herpesviruses persist as latent episomes in a dynamic lymphocyte pool. Their consequent need to express a viral episome maintenance protein presents a potential immune target. The glycine-alanine repeat of the Epstein-Barr virus episome maintenance protein, EBNA-1, limits EBNA-1 epitope presentation to CD8(+) T lymphocytes (CTLs). However, CTL recognition occurs in vitro, so the significance of such evasion for viral fitness is unclear. We used the murine gamma-herpesvirus-68 (MHV-68) to define the in vivo contribution of cis-acting CTL evasion to host colonisation. Although the ORF73 episome maintenance protein of MHV-68 lacks a glycine-alanine repeat, it was equivalent to EBNA-1 in conferring limited presentation on linked epitopes. This was associated with reduced protein synthesis and reduced protein degradation. We bypassed the cis-acting evasion of ORF73 by using an internal ribosome entry site to express in trans-a CTL target from the same mRNA. This led to a severe, MHC class I-restricted and CTL-dependent reduction in viral latency. Thus, despite MHV-68 encoding at least two trans-acting CTL evasion proteins, cis-acting evasion during episome maintenance was essential for normal host colonisation.     

Epitope-Based Peptides Prediction from Proteome of Nipah Virus

The identification of MHC class II epitope-based peptides are urgently needed for appropriate vaccination against Nipah virus (NiV) because there are currently no approved vaccines for human NiV infection. In the present study, prediction and modeling of T cell epitopes of NiV antigenic proteins nucleocapsid, phosphoprotein, matrix, fusion, glycoprotein, L protein, W protein, V protein and C protein followed by the binding simulation studies of predicted highest binding scores with their corresponding MHC class II alleles were done. Immunoinformatic tool ProPred was used to predict the promiscuous MHC class II epitopes of viral antigenic proteins. PEPstr server did the 3D structure models of the epitopes and Modeller 9.10 did alleles. We docked epitope with allele structure using the AutoDock 4.2 Tool. The docked peptide–allele complex structure was optimized using molecular dynamics simulation for 5 ps with the CHARMM-22 force field using NAnoscale Molecular Dynamics program incorporated in visual molecular dynamics (VMD 1.9.2) and then evaluating the stability of complex structure by calculating RMSD values. Epitope MKLQFSLGS of Matrix protein has considerable binding energy and score with DRBI*0421 MHC class II allele. This predicted peptide has potential to induce T cell-mediated immune response and is expected to useful in designing epitope-based vaccines against NiV after further testing by wet lab studies.     

Dendritic cell aggresome-like induced structures are dedicated areas for ubiquitination and storage of newly synthesized defective proteins

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. One of these mechanisms is the sorting of polyubiquitinated proteins in large cytosolic aggregates called dendritic cell aggresome-like induced structures (DALIS). DALIS formation and maintenance are tightly linked to protein synthesis. Here, we took advantage of an antibody recognizing the antibiotic puromycin to follow the fate of improperly translated proteins, also called defective ribosomal products (DRiPs). We demonstrate that DRiPs are rapidly stored and protected from degradation in DALIS. In addition, we show that DALIS contain the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E225K, and the COOH terminus of Hsp70-interacting protein ubiquitin ligase. The accumulation of these enzymes in the central area of DALIS defines specific functional sites where initial DRiP incorporation and ubiquitination occur. Therefore, DCs are able to regulate DRiP degradation in response to pathogen-associated motifs, a capacity likely to be important for their immune functions.     

MHC class II-restricted presentation of native protein antigen by B cells is inhibitable by cycloheximide and brefeldin A

The presentation of protein Ag with MHC class II proteins involves the uptake of the protein Ag by endocytosis followed by processing, probably proteolysis, in an intracellular acidic compartment. However, there remains considerable controversy as to the precise route taken by the antigen and the MHC class II protein during this process. The unusual stability of Ag-MHC class II protein complexes has led to speculation that antigen can only associate with newly synthesized MHC class II molecules. An alternate possibility is that the MHC class II binding site can be regenerated within the cell during internalization and recycling of MHC class II proteins. To address these possibilities, three different murine B lymphoma lines were tested for their ability to process and present native protein Ag in the presence of the protein synthesis inhibitor cycloheximide or the protein synthesis inhibitor cycloheximide or the protein export inhibitor, Brefeldin A. Both agents blocked the presentation of native OVA or native hen egg lysozyme to Ag-specific T cell hybridomas. No effect was seen on peptide presentation or on presentation to allo- or autoreactive T cells. Inasmuch as Brefeldin A has been previously shown to block protein export without affecting protein internalization or protein degradation in the endocytic pathway, the simplest interpretation of these data is that antigenic fragments generated in the APC after uptake by the endocytic pathway, preferentially associate with newly synthesized rather than mature MHC class II proteins.     

RNA polymerase II inhibitors dissociate antigenic peptide generation from normal viral protein synthesis: a role for nuclear translation in defective ribosomal product synthesis?

Following viral infection, cells rapidly present peptides from newly synthesized viral proteins on MHC class I molecules, likely from rapidly degraded forms of nascent proteins. The nature of these defective ribosomal products (DRiPs) remains largely undefined. Using inhibitors of RNA polymerase II that block influenza A virus neuraminidase (NA) mRNA export from the nucleus and inhibit cytoplasmic NA translation, we demonstrate a surprising disconnect between levels of NA translation and generation of SIINFEKL peptide genetically inserted into the NA stalk. A 33-fold reduction in NA expression is accompanied by only a 5-fold reduction in K(b)-SIINFEKL complex cell-surface expression, resulting in a net 6-fold increase in the overall efficiency of Ag presentation. Although the proteasome inhibitor MG132 completely blocked K(b)-SIINFEKL complex generation, we were unable to biochemically detect a MG132-dependent cohort of NA DRiPs relevant for Ag processing, suggesting that a minute population of DRiPs is a highly efficient source of antigenic peptides. These data support the idea that Ag processing uses compartmentalized translation, perhaps even in the nucleus itself, to increase the efficiency of the generation of class I peptide ligands.     

Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin

The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degradation. The ability to do so is hampered by the small number of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I molecules. We used this change in signal intensity as a readout to screen a chemical library of 16,320 compounds and identified two structurally related compounds (eeyarestatin I and II) that interfered with the degradation of both EGFP-heavy chain and its endogenous unmodified class I MHC heavy chain counterpart. Eeyarestatin I also inhibited degradation of a second misfolded type I membrane protein, T-cell receptor alpha. Both compounds stabilize these dislocation substrates in the ER membrane, without preventing proteasomal turnover of cytosolic substrates. The new inhibitors must therefore interfere with a step that precedes proteasomal degradation. The use of eeyarestatin I thus allows the definition of a new intermediate in dislocation.     

Antigen processing by proteasomes: insights into the molecular basis of crypticity

Eight to eleven amino acid residues are the sizes of predominant peptides found to be associated with MHC class I molecules. Proteasomes have been implicated in antigen processing and generation of such peptides. Advanced methodologies in peptide elution together with sequence determination have led to the characterisation of MHC class I binding motifs. More recently, screening of random peptide phage display libraries and synthetic combinatorial peptide libraries have also been successfully used. This has led to the development and use of predictive algorithms to screen antigens for potential CTL epitopes. Not all predicted epitopes will be generated in vivo and the emerging picture suggests differential presentation of predicted CTL epitopes ranging from cryptic to immunodominant. The scope of this review is to discuss antigen processing by proteasomes, and to put forward a hypothesis that the molecular basis of immunogenicity can be a function of proteasomal processing. This may explain how pathogens and tumours are able to escape immunosurveillance by altering sequences required by proteasomes for epitope generation. Abbreviations: CTL – cytotoxic T lymphocytes; DRiPs – defective ribosomal products; ER – endoplasmic reticulum; Hsps – heat shock proteins; LMP – low molecular weight peptide; MHC – major histocompatibility complex; TAP – transporter associated with antigen processing.     

Proteasomal Degradation of Proinsulin Requires Derlin-2, HRD1 and p97

Patients with type 1 diabetes (T1D) suffer from beta-cell destruction by CD8⁺ T-cells that have preproinsulin as an important target autoantigen. It is of great importance to understand the molecular mechanism underlying the processing of preproinsulin into these CD8⁺ T-cell epitopes. We therefore studied a pathway that may contribute to the production of these antigenic peptides: degradation of proinsulin via ER associated protein degradation (ERAD). Analysis of the MHC class I peptide ligandome confirmed the presentation of the most relevant MHC class I-restricted diabetogenic epitopes in our cells: the signal peptide-derived sequence A15-A25 and the insulin B-chain epitopes H29-A38 and H34-V42. We demonstrate that specific silencing of Derlin-2, p97 and HRD1 by shRNAs increases steady state levels of proinsulin. This indicates that these ERAD constituents are critically involved in proinsulin degradation and may therefore also play a role in subsequent antigen generation. These ERAD proteins therefore represent interesting targets for novel therapies aiming at the reduction and possibly also prevention of beta-cell directed auto-immune reactions in T1D.     

Prediction and molecular modeling of T-cell epitopes derived from placental alkaline phosphatase for use in cancer immunotherapy

In our ongoing efforts to combat cancer, peptide-based tumor vaccines are promising as one of the several alternatives used for cancer immunotherapy and immunoprevention. We have attempted to identify T-cell epitopes suitable for the development of a peptide-based cancer vaccine directed towards placental isozyme of alkaline phosphatase (PLAP), an oncofetal antigen. After identifying amino acid residues specific to PLAP and distinct from other close PLAP homologs, we have used sequence-based immunoinformatics tools (BIMAS and SYFPEITHI) and conducted molecular modeling studies using InsightII to investigate the binding affinity of the epitopes containing the unique residues with respective MHC class I molecules. Promiscuous epitopes binding to different alleles of different class I HLA loci were analyzed to get a population coverage that is widespread. Binding affinity deduced from the modeling studies corroborated the status of most of the epitopes scoring high in BIMAS and SYFPEITHI. We have thus identified specific epitopes from PLAP that have a potential for binding to their respective MHC class I alleles with high affinity. These peptides would be analysed in experiments to demonstrate their involvement in the induction of primary cytotoxic T-cell responses in vitro, using respective HLA-restricted T-cells in our way towards the development of an effective anti-cancer vaccine in a background of diverse MHC haplotypes.     

The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion

Background

Cytotoxic T cells detect intracellular pathogens by surveying peptide loaded MHC class I molecules (pMHC I) on the cell surface. Effective immune surveillance also requires infected cells to present pMHC I promptly before viral progeny can escape. Rapid pMHC I presentation apparently occurs because infected cells can synthesize and present peptides from antigenic precursors called defective ribosomal products (DRiPs). The molecular characteristics of DRiPs are not known.

Methodology/Principal Findings

Here, using a novel method for detecting antigenic precursors and proteolytic intermediates, we tracked the synthesis and processing of Epstein-Barr Virus encoded nuclear antigen 1 (EBNA1). We find that ribosomes initiated translation appropriately, but rapidly produced DRiPs representing ∼120 amino acid truncated EBNA1 polypeptides by premature termination. Moreover, specific sequences in EBNA1 mRNA strongly inhibited the generation of truncated DRiPs and pMHC I presentation.

Significance

Our results reveal the first characterization of virus DRiPs as truncated translation products. Furthermore, production of EBNA1-derived DRiPs is down-regulated in cells, possibly limiting the antigenicity of EBNA1.     

Quantitative analysis of prion-protein degradation by constitutive and immuno-20S proteasomes indicates differences correlated with disease susceptibility

The main part of cytosolic protein degradation depends on the ubiquitin-proteasome system. Proteasomes degrade their substrates into small peptide fragments, some of which are translocated into the endoplasmatic reticulum and loaded onto MHC class I molecules, which are then transported to the cell surface for inspection by CTL. A reliable prediction of proteasomal cleavages in a given protein for the identification of CTL epitopes would benefit immensely from additional cleavage data for the training of prediction algorithms. To increase the knowledge about proteasomal specificity and to gain more insight into the relation of proteasomal activity and susceptibility to prion disease, we digested sheep prion protein with human constitutive and immuno-20S proteasomes. All fragments generated in the digest were quantified. Our results underline the different cleavage specificities of constitutive and immunoproteasomes and provide data for the training of prediction programs for proteasomal cleavages. Furthermore, the kinetic analysis of proteasomal digestion of two different alleles of prion protein shows that even small changes in a protein sequence can affect the overall efficiency of proteasomal processing and thus provides more insight into the possible molecular background of allelic variations and the pathogenicity of prion proteins.     

Prediction and Molecular Modeling of T-cell Epitopes Derived from Placental Alkaline Phosphatase for use in Cancer Immunotherapy

Abstract

In our ongoing efforts to combat cancer, peptide-based tumor vaccines are promising as one of the several alternatives used for cancer immunotherapy and immunoprevention. We have attempted to identify T-cell epitopes suitable for the development of a peptide-based cancer vaccine directed towards placental isozyme of alkaline phosphatase (PLAP), an oncofetal antigen. After identifying amino acid residues specific to PLAP and distinct from other close PLAP homologs, we have used sequence-based immunoinformatics tools (BIMAS and SYF- PEITHI) and conducted molecular modeling studies using InsightII to investigate the binding affinity of the epitopes containing the unique residues with respective MHC class I molecules. Promiscuous epitopes binding to different alleles of different class I HLA loci were analyzed to get a population coverage that is widespread. Binding affinity deduced from the modeling studies corroborated the status of most of the epitopes scoring high in BIMAS and SYFPEITHI. We have thus identified specific epitopes from PLAP that have a potential for binding to their respective MHC class I alleles with high affinity. These peptides would be analysed in experiments to demonstrate their involvement in the induction of primary cytotoxic T-cell responses in vitro, using respective HLA-restricted T-cells in our way towards the development of an effective anti-cancer vaccine in a background of diverse MHC haplotypes.     

Monoclonal antibodies specific for the empty conformation of HLA-DR1 reveal aspects of the conformational change associated with peptide binding

Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4+ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptide-induced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the beta subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change.     

The impact of misfolding versus targeted degradation on the efficiency of the MHC class I-restricted antigen processing

Evidence suggests that most epitopes presented by MHC class I molecules are derived from those newly synthesized proteins that are defective due to errors during manufacture. We examined epitope production from model cytosolic and exocytic proteins modified in various ways. Substrates containing a degradation targeting sequence demonstrated very rapid turnover and enhanced epitope production, as was the case for substrate retargeted from endoplasmic reticulum to cytosol. For less radical alterations, including point mutation and deletion and elimination of glycosylation sites, despite detectable changes in folding, half-life was only moderately decreased and there were no significant increases in epitope production. Puromycin, which causes premature termination of protein synthesis, also had no impact upon epitope production. It appears that most defective proteins are not rapidly dispensed with and the targeting of most nascent proteins for Ag processing is not tied to quality control.     

Immune surveillance of intracellular pathogens via autophagy

MHC class II molecules are thought to present peptides derived from extracellular proteins to CD4+ T cells, which are important mediators of adaptive immunity to infections. In contrast, autophagy delivers constitutively cytosolic material for lysosomal degradation and has so far been recognized as an efficient mechanism of innate immunity against bacteria and viruses. Recent studies, however, link these two pathways and suggest that intracellular cytosolic and nuclear antigens are processed for MHC class II presentation after autophagy.