Class I HLA molecules mark infected cells for immune targeting by presenting pathogen-encoded peptides on the cell surface. Characterization of viral peptides unique to infected cells is important for understanding CD8(+) T cell responses and for the development of T cell-based immunotherapies. Having previously reported a series of West Nile virus (WNV) epitopes that are naturally presented by HLA-A*02:01, in this study we generated TCR mimic (TCRm) mAbs to three of these peptide/HLA complexes-the immunodominant SVG9 (E protein), the subdominant SLF9 (NS4B protein), and the immunorecessive YTM9 (NS3 protein)-and used these TCRm mAbs to stain WNV-infected cell lines and primary APCs. TCRm staining of WNV-infected cells demonstrated that the immunorecessive YTM9 appeared several hours earlier and at 5- to 10-fold greater density than the more immunogenic SLF9 and SVG9 ligands, respectively. Moreover, staining following inhibition of the TAP demonstrated that all three viral ligands were presented in a TAP-dependent manner despite originating from different cellular compartments. To our knowledge, this study represents the first use of TCRm mAbs to define the kinetics and magnitude of HLA presentation for a series of epitopes encoded by one virus, and the results depict a pattern whereby individual epitopes differ considerably in abundance and availability. The observations that immunodominant ligands can be found at lower levels and at later time points after infection suggest that a reevaluation of the factors that combine to shape T cell reactivity may be warranted.

Aim Advances in mass spectrometry and HLA protein availability have allowed for very a detailed look at the self-peptides naturally presented by HLA. To date, there is considerable variation among studies such that a comparison of the ligands presented by HLA-A and HLA-B has been difficult. Here we set out to characterize the self-ligandome of 5 HLA-A and 3 HLA-B molecules representing various peptide binding supertypes with highly controlled methods - varying only the HLA molecule. Methods HeLa cells were separately transfected with 5 different HLA-A (A1, A2, A3, A11, A24) and 3 HLA-B (B7, B27, B51) as soluble class I gene constructs. HLA-complexes were affinity purified and the eluted ligands were characterized by two-dimensional LCMS. Sequences were identified from the fragment spectra using PEAKS v7.0 at a 1% FDR. Results We find that nonamers are the preferred length for all alleles, however there is wide diversity in the ligand length distributions between allomorphs. For example, HLA-B51 shows a high tolerance for 8-mers while HLA-A11 equally prefers 11mers and nonamers. However there is no overall length bias when comparing HLA-A to HLA-B. For both HLA-A and HLA-B, one or two ligands are typically sampled per protein. However, proteins like GAPDH, alpha-enolase 1, carbamoyl-phosphate synthase 1, and dynenin provide on average approximately 20 ligands per allomorph. For all allomorphs there is an uneven sampling of proteins in the genome with biases shown towards particular proteins. Especially noteworthy is that proteins involved in translation, particularly ribosomal RNA binding proteins, are extensively represented by HLA-A and HLA-B. For both HLA-A and HLA-B there is also a significant enrichment of proteins localized to the lumen of the nucleus including histones, proteins involved in RNA splicing, and factors for biogenesis of ribosomes in the nucleolus. Conclusion The ligands sampled by HLA-A and HLA-B vary in length between the allomorphs in a manner that is highly diverse and independent of locus. Overall, there is little to no difference in the source proteins sampled by HLA-A and HLA-B with all allomorphs showing a ligand sampling bias in RNA binding proteins and nuclear proteins. W. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure Protein LLC. 5. Employee; Company/Organization; Pure Protein LLC.

Aim Class I human leukocyte antigens alert the cellular immune system of infections by presenting peptides derived from viral proteins on the surface of infected cells. Viruses are capable of encoding alternative reading frame (ARF) proteins, and such proteins have only been partially characterized for existence and function. West Nile virus (WNV) encodes an ARF protein (NS1’) and possibly other ARF proteins. Here we use high throughput ligand analysis to characterize the class I HLA of WNV infected cells in order to identify peptides that originate from WNV ARF proteins. Methods Epithelial cells expressing different soluble HLA-A and HLA-B class I molecules were cultured in bioreactors and infected with WNV. HLA/peptide complexes were harvested from WNV infected cells, peptides were eluted from affinity-purified HLA, and two-dimensional LC/MS produced spectra were searched against a WNV ARF database using PEAKS proteomics software. The sequence identity of each WNV ARF peptide was confirmed by comparing the MS2 fragmentation of the eluted experimental ARF peptide with a corresponding synthetic peptide. Results We were able to identify and confirm 4 NS1’ derived peptides presented by HLA-A*01:01 and HLA-A∗24:02. Interestingly we were also able to discover a peptide, from a novel ARF WNV protein presented by HLA-B*35:01. This protein is likely a 21 amino acid long C-terminal extension of NS5 and is presumed to be the result of a +1 frame shift, 38 base pair upstream from the polyprotein stop codon. This viral protein/peptide is of unknown function. Conclusions We demonstrate that different HLA class I molecules present peptides from the NS1’ protein that is created as a result of ribosomal frameshifting during viral replication. In addition, we show the first evidence of presence of another ARF protein that is produced after WNV infection. How these ligands impact the immune responses toward infected cells remains to be resolved.

Abstract Class I HLA reveal virus-derived ligands to cytotoxic T lymphocytes (CTL) whose function is to eliminate infected cells. The Los Alamos National Laboratory (LANL) database reports numerous HIV-1 CTL epitopes, none of which have elicited protective immunity in vaccine testing. The goal of this study was to determine the number and nature of HIV-1 ligands available for CTL targeting through presentation by the HLA class I of virus-infected cells. Class I presented peptides were recovered from immunoaffinity purified HLA-A*11:01 gathered from HIV-1 (NL4-3)-infected human CD4+ SUP-T1 T cells. These peptides were fractionated by RP-HPLC and mapped by tandem mass spectrometry (MS/MS). Seven HIV-derived peptides were confirmed by MS/MS as unique to infected cells. Twelve LANL HIV epitopes, including the well-studied Gag p24 epitope ACQGVGGPGHK (AK11), were clearly absent from the HLA-A*11:01 of infected cells. Of the 7 HIV-1 ligands found to be presented by A*11:01, 4 were derived from Nef, 2 from Gag, and 1 from Pol. When these 7 ligands were tested for T cell reactivity in a gamma interferon ELISPOT assay using PBMC from HIV-1 infected individuals (including elite controllers), the Nef-derived peptides were found to be highly reactive. These data demonstrate that the class I HLA of HIV-1 infected CD4+ T cells present a handful of viral peptide ligands for recognition by CTL and that reported HIV-1 CTL epitopes might not be presented by the HLA class I of infected cells.

Abstract Class I Human Leukocyte Antigens reveal intracellular viruses to the immune system by presenting viral epitopes at the cell surface. In this study we hypothesized that during infection particular HLA class I alleles consistently present more virus-derived peptide ligands to virus-specific CD8+ T cells than do other HLA class I. To test this hypothesis, we infected cells with West Nile virus (WNV) and directly identified and enumerated the virus specific peptides presented by various HLA class I molecules. Cells expressing a number of different HLA-A and HLA-B class I were cultured in bioreactors and infected with WNV. HLA/peptide complexes were harvested from infected and uninfected cells, peptides were eluted from affinity-purified HLA, comparatively mapped by mass spectroscopy, and sequenced. Twenty-four peptides, 20 eluted from HLA-A complexes (HLA-A*02:01, A*01:01, A*11:01 and A*24:02) and 4 from HLA-B complexes (HLA-B*27:05, B*07:02 and B*35:01), were identified as unique to infected cells. Ligands represented different parts of the viral polyprotein demonstrating that peptides sampled by class I HLA are distributed widely throughout the WNV proteome. These data indicate that, in WNV-infected cells, HLA-A present more virus-specific peptides than do HLA-B suggesting a potentially different role for HLA class I loci in developing immune responses that control WNV infection whereby HLA-A is responsible for diverse reactivity and HLA-B leads to more focused immunity.

Class I Human Leukocyte Antigens distinguish healthy cells from infected cells by presenting peptides at the cell surface. Viral epitopes confirmed as unique to infected cells can lead to successful development of vaccines and therapeutics, and a tool to validate viral epitope presentation on a variety of cell lineages is essential. Here, comparative mass spectrometry shows that the West Nile virus peptide epitopes SVGGVFTSV and ILRNPGYAL are presented by HLA-A*02:01 and HLA-B*07:02 of infected cells. To generate monoclonal antibodies against these HLA/WNV peptide epitopes, mice were immunized with peptide/HLA complexes, splenocytes were fused to myeloma cells, and single clones were picked and grown. Hybridoma supernatants were screened for recognition of the appropriate HLA/WNV peptide complex on peptide-pulsed cells with irrelevant HLA/peptides acting as a negative control. The T cell receptor mimic (TCRm) monoclonal antibodies RL15A and RL29A were found to be specific for A*02:01/SVG9 and B*07:02/ILR9, respectively. The RL15A and RL29A T cell receptor mimic mAb were then used to track viral epitope presentation on WNV infected cell lines and primary cells. In summary, we demonstrate the implementation of a mass spectrometry system for the direct discovery of class I HLA-presented viral epitopes from infected cells followed by the complementary use of TCRm mAb as a companion diagnostic for tracking epitope presentation during the course of a viral infection.

ABSTRACT Identification of CD8 + cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro . Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4 + SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4 + T cells into strategies designed to enhance T cell immunity. IMPORTANCE HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4 + T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.

Aim CTL responses against M. tuberculosis (Mtb) are known to be restricted primarily by HLA-B and non-classical MHC molecules such as HLA-E, CD1, and MR1. While a few Mtb derived ligands have been identified in the context of these MHC, we do not have a thorough understanding of the proteins that are sampled by HLA molecules following Mtb infection. Here we identify the Mtb proteins sampled by the non-classical class I HLA-E ∗ 01:03 and the classical class I HLA-B ∗ 44:02. Method Soluble HLA-E ∗ 01:03 and HLA-B ∗ 44:01 were harvested from Mtb (strain H37Rv) infected antigen presenting cells U373 glioblastoma cells. Peptide ligand pools from these MHC were isolated and MS spectra collected using 2-dimensional LCMS. Peptide sequences were assigned from fragment spectra analyzed with the PEAKS and Mascot algorithms using the decoy database search at a 1% False Discovery Rate. All sequences were validated with MS fragmentation of the corresponding synthetic peptide. Results In this study we identified a total of 49 Mtb specific ligands presented by HLA. For HLA-E we found 28 ligands from 14 source proteins. In the classical class I HLA-B44, we identified 21 ligands from 8 different proteins. Strikingly, both HLA-E and HLA-B44 frequently sampled esx family proteins with 50% and 66% of the ligands originating from esx proteins for HLA-E and HLA-B44, respectively. Additionally a hypothetical protein lpq1 (Rv0237) was sampled by both HLA-E and HLA-B44. Conclusions Esx family proteins are known virulence factors for Mtb, and are highly antigenic in a natural infection. Further, esx family proteins are absent from widely used the BCG tuberculosis vaccine. Here, we found that both classical and non-classical HLA consistently sample these esx proteins further confirming their role in the adaptive immune response to Mtb.

Event Abstract Back to Event Motifs of the naturally processed peptides presented by HLA-A*24:07 Marsia Gustiananda1*, Darryl Cox2, Curtis McMurtrey2, Kenneth W. Jackson2, Danijela Mojsilovic2, Wilfried Bardet2, Fredda Schafer2, Steven Cate2, Jane Yaciuk2, Saghar Kaabinejadian2 and William H. Hildebrand2* 1 Eijkman Institute for Molecular Biology, Indonesia 2 University of Oklahoma Health Sciences Center, Microbiology and Immunology, United States Class I HLA molecules mediate immune responses by presenting pathogen-derived peptides to CD8+ T-cells. HLA-A*24:07 is a predominant HLA allele of the Indonesian and South East Asian population. At the moment, there is only one T-cell epitope in the Immune Epitope Database reportedly restricted by A*24:07. HLA-A*24:02 is in the A24 supertype and, although HLA-A*24:07 is closely related to HLA-A*24:02, A*24:07 is not a member of the HLA-A*24 supertype. The two molecules share high homology except for amino acid residue 70, which is a histidine in A*24:02 and a glutamine in A*24:07. The side chain of residue 70 lies in the peptide-binding groove, positioned to impact primary and auxiliary peptide anchors. HLA disease association studies show that these two alleles behave differently in dengue infection as well as in type-1 diabetes. Using proteomic approaches, we systematically compared the peptides presented by A*24:07 and A*24:02. These two A24 were harvested from a matched cell line, immunoaffinity purified, and the A24 ligands were compared by Edman sequencing and tandem mass spectroscopy. Edman analysis indicated slight variations in binding pocket preferences and mass spectroscopy confirmed subtle but clear changes in peptide ligand presentation. This dataset of ligands can be used to improve A*24 predictive algorithms while the A*24:07 reagent can be developed as a tool for characterizing Indonesian and South East Asian immune responses. Keywords: HLA-A*24:07, T-cell epitopes, HLA Class-I, HLA-A*24:02, HLA-A*24 supertype, Proteomics Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Gustiananda M, Cox D, McMurtrey C, Jackson KW, Mojsilovic D, Bardet W, Schafer F, Cate S, Yaciuk J, Kaabinejadian S and Hildebrand WH (2013). Motifs of the naturally processed peptides presented by HLA-A*24:07. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01033 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Marsia Gustiananda, Eijkman Institute for Molecular Biology, Jakarta, Indonesia, Mgustiananda@gmail.com Prof. William H Hildebrand, University of Oklahoma Health Sciences Center, Microbiology and Immunology, Oklahoma City, United States, William-Hildebrand@ouhsc.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Marsia Gustiananda Darryl Cox Curtis McMurtrey Kenneth W Jackson Danijela Mojsilovic Wilfried Bardet Fredda Schafer Steven Cate Jane Yaciuk Saghar Kaabinejadian William H Hildebrand Google Marsia Gustiananda Darryl Cox Curtis McMurtrey Kenneth W Jackson Danijela Mojsilovic Wilfried Bardet Fredda Schafer Steven Cate Jane Yaciuk Saghar Kaabinejadian William H Hildebrand Google Scholar Marsia Gustiananda Darryl Cox Curtis McMurtrey Kenneth W Jackson Danijela Mojsilovic Wilfried Bardet Fredda Schafer Steven Cate Jane Yaciuk Saghar Kaabinejadian William H Hildebrand PubMed Marsia Gustiananda Darryl Cox Curtis McMurtrey Kenneth W Jackson Danijela Mojsilovic Wilfried Bardet Fredda Schafer Steven Cate Jane Yaciuk Saghar Kaabinejadian William H Hildebrand Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

There are many factors that are thought to contribute to T-cell immunodominance hierarchies including timing and levels of antigen presentation to T-cells. One of the most potent tools for studying antigen presentation for a specific determinant are monoclonal antibodies that are specific for not only MHC but a specific ligand, similar to a T cell receptor. In this study we utilize these antibodies to study levels and timing of antigen presentation of immunodominant (SVG9), sub-dominant (SLF9), and super sub-dominant (YTM9) T-cell epitopes during West Nile virus (WNV) infection. In this study, we demonstrate that these ligands are differentially expressed on the surface of a cell infected with WNV. Specifically, the super sub-dominant ligand (YTM9) was presented earlier and at 20 times the levels of the immunodominant (SVG9) and subdominant (SLF9) ligands. Transfection of ICP47 in the infected cells show that all three epitopes are TAP dependent. Using these highly specific reagents we show that there is differential ligand presentation within the restrictive allele. High levels of peptide HLA complexes on the surface of an infected do not correspond with immunodominance hierarchies as the super subdominant epitope YTM9 was presented at higher levels than the most dominant epitope SVG9. This differential in presentation was not due the TAP dependence of the ligands.

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.

Aim Class I human leukocyte antigen molecules (A, B and C) reveal intracellular viruses by presenting viral peptide epitopes at the cell surface. At this point the number of viral ligands that decorate the HLA of an infected cell and biases in the targeting of these viral/HLA complexes by cytotoxic T lymphocytes is not understood. Our goal is to determine the number of viral ligands presented by the HLA of infected cells and to assess T cell decisions in responding to available targets. Methods A human epithelial cell line was transfected with three different soluble class I HLA-A and three HLA-B constructs. HLA-producing clones were expanded and grown in bioreactors and infected with West Nile virus (WNV) or were left uninfected. HLA/peptide complexes were purified from WNV-infected and uninfected cells. To identify WNV derived HLA ligands, peptides were isolated, comparatively mapped by mass spectroscopy and sequenced by MSMS. Results Of the 19 ligands identified in this study, 15 were eluted from HLA-A complexes and 4 were eluted from HLA-B. HLA-A ∗ 02:01 and A ∗ 11:01 each presented 6 viral peptides. Viral epitopes sampled by class I HLA were dispersed fairly evenly throughout different viral proteins (C, M, E, NS2b, NS3, NS4b and NS5). T cells from infected individuals tended to respond strongly to 1-2 particular viral ligands with minimal T cell activity to other viral epitopes. Conclusions These data indicate that HLA-A molecules tend to present the majority of WNV-derived ligands to CTL, suggesting that HLA-A and HLA-B may play different roles in the development of viral immunity. Class I HLA was unbiased, sampling peptides from various locations throughout the virus. While T cell activity was detected to most viral ligands, a clear hierarchy demonstrates that particular viral epitopes are key determinants of immune protection. These data will guide the development of vaccines, diagnostics, and therapeutics. Buchli: Pure Protein L.L.C.: Employee.