Epstein Barr Virus-Encoded EBNA1 Interference with MHC Class I Antigen Presentation Reveals a Close Correlation between mRNA Translation Initiation and Antigen Presentation

Viruses are known to employ different strategies to manipulate the major histocompatibility (MHC) class I antigen presentation pathway to avoid recognition of the infected host cell by the immune system. However, viral control of antigen presentation via the processes that supply and select antigenic peptide precursors is yet relatively unknown. The Epstein-Barr virus (EBV)-encoded EBNA1 is expressed in all EBV-infected cells, but the immune system fails to detect and destroy EBV-carrying host cells. This immune evasion has been attributed to the capacity of a Gly-Ala repeat (GAr) within EBNA1 to inhibit MHC class I restricted antigen presentation. Here we demonstrate that suppression of mRNA translation initiation by the GAr in cis is sufficient and necessary to prevent presentation of antigenic peptides from mRNAs to which it is fused. Furthermore, we demonstrate a direct correlation between the rate of translation initiation and MHC class I antigen presentation from a certain mRNA. These results support the idea that mRNAs, and not the encoded full length proteins, are used for MHC class I restricted immune surveillance. This offers an additional view on the role of virus-mediated control of mRNA translation initiation and of the mechanisms that control MHC class I restricted antigen presentation in general

Genetic Deletion of a Single Immunodominant T-cell Response Confers Susceptibility to Virus-induced Demyelination

An important question in neuropathology involves determining the antigens that are targeted during demyelinating disease. Viral infection of the central nervous system (CNS) leads to T-cell responses that can be protective as well as pathogenic. In the Theiler’s murine encephalomyelitis virus (TMEV) model of demyelination it is known that the immune response to the viral capsid protein 2 (VP2) is critical for disease pathogenesis. This study shows that expressing the whole viral capsid VP2 or the minimal CD8-specific peptide VP2(121-130) as “self” leads to a loss of VP2-specific immune responses. Loss of responsiveness is caused by T cell-specific tolerance, as VP2-specific antibodies are generated in response to infection. More importantly, these mice lose the CD8 T-cell response to the immunodominant peptide VP2(121-130), which is critical for the development of demyelinating disease. The transgenic mice fail to clear the infection and develop chronic demyelinating disease in the spinal cord white matter. These findings demonstrate that T-cell responses can be removed by transgenic expression and that lack of responsiveness alters viral clearance and CNS pathology. This model will be important for understanding the mechanisms involved in antigen-specific T-cell deletion and the contribution of this response to CNS pathology

Glycosylation Focuses Sequence Variation in the Influenza A Virus H1 Hemagglutinin Globular Domain

Antigenic drift in the influenza A virus hemagglutinin (HA) is responsible for seasonal reformulation of influenza vaccines. Here, we address an important and largely overlooked issue in antigenic drift: how does the number and location of glycosylation sites affect HA evolution in man? We analyzed the glycosylation status of all full-length H1 subtype HA sequences available in the NCBI influenza database. We devised the “flow index” (FI), a simple algorithm that calculates the tendency for viruses to gain or lose consensus glycosylation sites. The FI predicts the predominance of glycosylation states among existing strains. Our analyses show that while the number of glycosylation sites in the HA globular domain does not influence the overall magnitude of variation in defined antigenic regions, variation focuses on those regions unshielded by glycosylation. This supports the conclusion that glycosylation generally shields HA from antibody-mediated neutralization, and implies that fitness costs in accommodating oligosaccharides limit virus escape via HA hyperglycosylation

Clusters versus Affinity-Based Approaches in F. tularensis Whole Genome Search of CTL Epitopes

Deciphering the cellular immunome of a bacterial pathogen is challenging due to the enormous number of putative peptidic determinants. State-of-the-art prediction methods developed in recent years enable to significantly reduce the number of peptides to be screened, yet the number of remaining candidates for experimental evaluation is still in the range of ten-thousands, even for a limited coverage of MHC alleles. We have recently established a resource-efficient approach for down selection of candidates and enrichment of true positives, based on selection of predicted MHC binders located in high density “hotspots" of putative epitopes. This cluster-based approach was applied to an unbiased, whole genome search of Francisella tularensis CTL epitopes and was shown to yield a 17–25 fold higher level of responders as compared to randomly selected predicted epitopes tested in Kb/Db C57BL/6 mice. In the present study, we further evaluate the cluster-based approach (down to a lower density range) and compare this approach to the classical affinity-based approach by testing putative CTL epitopes with predicted IC50 values of <10 nM. We demonstrate that while the percent of responders achieved by both approaches is similar, the profile of responders is different, and the predicted binding affinity of most responders in the cluster-based approach is relatively low (geometric mean of 170 nM), rendering the two approaches complimentary. The cluster-based approach is further validated in BALB/c F. tularensis immunized mice belonging to another allelic restriction (Kd/Dd) group. To date, the cluster-based approach yielded over 200 novel F. tularensis peptides eliciting a cellular response, all were verified as MHC class I binders, thereby substantially increasing the F. tularensis dataset of known CTL epitopes. The generality and power of the high density cluster-based approach suggest that it can be a valuable tool for identification of novel CTLs in proteomes of other bacterial pathogens

Proteome Sampling by the HLA Class I Antigen Processing Pathway

The peptide repertoire that is presented by the set of HLA class I molecules of an individual is formed by the different players of the antigen processing pathway and the stringent binding environment of the HLA class I molecules. Peptide elution studies have shown that only a subset of the human proteome is sampled by the antigen processing machinery and represented on the cell surface. In our study, we quantified the role of each factor relevant in shaping the HLA class I peptide repertoire by combining peptide elution data, in silico predictions of antigen processing and presentation, and data on gene expression and protein abundance. Our results indicate that gene expression level, protein abundance, and rate of potential binding peptides per protein have a clear impact on sampling probability. Furthermore, once a protein is available for the antigen processing machinery in sufficient amounts, C-terminal processing efficiency and binding affinity to the HLA class I molecule determine the identity of the presented peptides. Having studied the impact of each of these factors separately, we subsequently combined all factors in a logistic regression model in order to quantify their relative impact. This model demonstrated the superiority of protein abundance over gene expression level in predicting sampling probability. Being able to discriminate between sampled and non-sampled proteins to a significant degree, our approach can potentially be used to predict the sampling probability of self proteins and of pathogen-derived proteins, which is of importance for the identification of autoimmune antigens and vaccination targets

Influenza nucleoprotein delivered with aluminium salts protects mice from an influenza virus that expresses an altered nucleoprotein sequence

Influenza virus poses a difficult challenge for protective immunity. This virus is adept at altering its surface proteins, the proteins that are the targets of neutralizing antibody. Consequently, each year a new vaccine must be developed to combat the current recirculating strains. A universal influenza vaccine that primes specific memory cells that recognise conserved parts of the virus could prove to be effective against both annual influenza variants and newly emergent potentially pandemic strains. Such a vaccine will have to contain a safe and effective adjuvant that can be used in individuals of all ages. We examine protection from viral challenge in mice vaccinated with the nucleoprotein from the PR8 strain of influenza A, a protein that is highly conserved across viral subtypes. Vaccination with nucleoprotein delivered with a universally used and safe adjuvant, composed of insoluble aluminium salts, provides protection against viruses that either express the same or an altered version of nucleoprotein. This protection correlated with the presence of nucleoprotein specific CD8 T cells in the lungs of infected animals at early time points after infection. In contrast, immunization with NP delivered with alum and the detoxified LPS adjuvant, monophosphoryl lipid A, provided some protection to the homologous viral strain but no protection against infection by influenza expressing a variant nucleoprotein. Together, these data point towards a vaccine solution for all influenza A subtypes

Stage-Specific Inhibition of MHC Class I Presentation by the Epstein-Barr Virus BNLF2a Protein during Virus Lytic Cycle

gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (ΔBNLF2a) and compared the ability of ΔBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by ΔBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and ΔBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet ΔBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle

Networks link antigenic and receptor-binding sites of influenza hemagglutinin: Mechanistic insight into fitter strain propagation

Influenza viral passaging through pre-vaccinated mice shows that emergent antigenic site mutations on the viral hemagglutinin (HA) impact host receptor-binding affinity and, therefore, the evolution of fitter influenza strains. To understand this phenomenon, we computed the Significant Interactions Network (SIN) for each residue and mapped the networks of antigenic site residues on a representative H1N1 HA. Specific antigenic site residues are ‘linked’ to receptor-binding site (RBS) residues via their SIN and mutations within “RBS-linked” antigenic residues can significantly influence receptor-binding affinity by impacting the SIN of key RBS residues. In contrast, other antigenic site residues do not have such “RBS-links” and do not impact receptor-binding affinity upon mutation. Thus, a potential mechanism emerges for how immunologic pressure on RBS-linked antigenic residues can contribute to evolution of fitter influenza strains by modulating the host receptor-binding affinity

Identification of Class I HLA T Cell Control Epitopes for West Nile Virus

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity. © 2013 Kaabinejadian et al

Protective Efficacy of Serially Up-Ranked Subdominant CD8+ T Cell Epitopes against Virus Challenges

Immunodominance in T cell responses to complex antigens like viruses is still incompletely understood. Some data indicate that the dominant responses to viruses are not necessarily the most protective, while other data imply that dominant responses are the most important. The issue is of considerable importance to the rational design of vaccines, particularly against variable escaping viruses like human immunodeficiency virus type 1 and hepatitis C virus. Here, we showed that sequential inactivation of dominant epitopes up-ranks the remaining subdominant determinants. Importantly, we demonstrated that subdominant epitopes can induce robust responses and protect against whole viruses if they are allowed at least once in the vaccination regimen to locally or temporally dominate T cell induction. Therefore, refocusing T cell immune responses away from highly variable determinants recognized during natural virus infection towards subdominant, but conserved regions is possible and merits evaluation in humans