Native Epitopes Research Articles

Structures of native and affinity-enhanced WT1 epitopes bound to HLA-A*0201: Implications for WT1-based cancer therapeutics

Presentation of peptides by class I or class II major histocompatibility complex (MHC) molecules is required for the initiation and propagation of a T cell-mediated immune response. Peptides from the Wilms Tumor 1 transcription factor (WT1), upregulated in many hematopoetic and solid tumors, can be recognized by T cells and numerous efforts are underway to engineer WT1-based cancer vaccines. Here we determined the structures of the class I MHC molecule HLA-A*0201 bound to the native 126–134 epitope of the WT1 peptide and a recently described variant (R1Y) with improved MHC binding. The R1Y variant, a potential vaccine candidate, alters the positions of MHC charged side chains near the peptide N-terminus and significantly reduces the peptide/MHC electrostatic surface potential. These alterations indicate that the R1Y variant is an imperfect mimic of the native WT1 peptide, and suggest caution in its use as a therapeutic vaccine. Stability measurements revealed how the R1Y substitution enhances MHC binding affinity, and together with the structures suggest a strategy for engineering WT1 variants with improved MHC binding that retain the structural features of the native peptide/MHC complex.

Pseudovirion particles bearing native HIV envelope trimers facilitate a novel method for generating human neutralizing monoclonal antibodies against HIV.

Monomeric HIV envelope vaccines fail to elicit broadly neutralizing antibodies or to protect against infection. Neutralizing antibodies against HIV bind to native functionally active Env trimers on the virion surface. Gag-Env pseudovirions recapitulate the native trimer and could serve as an effective epitope presentation platform for study of the neutralizing antibody response in HIV-infected individuals. To address if pseudovirions can recapitulate native HIV virion epitope structures, we carefully characterized these particles, concentrating on the antigenic structure of the coreceptor binding site. By blue native gel shift assays, Gag-Env pseudovirions were shown to contain native trimers that were competent for binding to neutralizing monoclonal antibodies. In enzyme-linked immunosorbent assay, pseudovirions exhibited increased binding of known CD4-induced antibodies after addition of CD4. Using flow cytometric analysis, fluorescently labeled pseudovirions specifically identified a subset of antigen-specific B cells in HIV-infected subjects. Interestingly, the sequence of one of these novel human antibodies, identified during cloning of single HIV-specific B cells and designated 2C6, exhibited homology to mAb 47e, a known anti-CD4-induced coreceptor binding site antibody. The secreted monoclonal antibody 2C6 did not bind monomeric gp120, but specifically bound envelope on pseudovirions. A recombinant form of the antibody 2C6 acted as a CD4-induced epitope-specific antibody in neutralization assays, yet did not bind monomeric gp120. These findings imply specificity against a quaternary epitope presented on the pseudovirion envelope spike. These data demonstrate that Gag-Env pseudovirions recapitulate CD4 and coreceptor binding pocket antigenic structures and can facilitate identification of B-cell clones that secrete neutralizing antibodies.

Conditional Superagonist CTL Ligands for the Promotion of Tumor-Specific CTL Responses

Abstract Although it has been demonstrated that CTLs can be raised against tumor-associated self-antigens, achieving consistent and effective clinical responses has proven challenging. Superagonist altered peptide ligands (APLs) can often elicit potent antitumor CTL responses where the native tumor-associated epitope fails. Current methods have identified a limited number of superagonist APLs, including the prototypic 27L mutant of MART-1. However, more comprehensive screening strategies would be desirable. In this study, we use a novel genetic screen, involving recombinant technology and class I Ag cross-presentation, to search for supraoptimal superagonists of the 27L MART-1 mutant by surveying the effectiveness of virtually every single amino acid substitution mutant of 27L to activate human Ag-specific CTL clones recognizing the wild-type MART-126–35 epitope. We identify three novel mutant epitopes with superagonist properties that are functionally superior to 27L; however, the ability of a given analogue to act as superagonist varies among patients and suggests that a given superagonist APL may be ideally suited to different patients. These findings endorse the use of comprehensive methods to establish panels of potential superagonist APLs to individualize tumor peptide vaccines among patients.

Neutralization Efficiency Is Greatly Enhanced by Bivalent Binding of an Antibody to Epitopes in the V4 Region and the Membrane-Proximal External Region within One Trimer of Human Immunodeficiency Virus Type 1 Glycoproteins

Most antibodies are multivalent, with the potential to bind with high avidity. However, neutralizing antibodies commonly bind to virions monovalently. Bivalent binding of a monoclonal antibody (MAb) to a virion has been documented only in a single case. Thus, the role of high avidity in antibody-mediated neutralization of viruses has not been defined clearly. In this study, we demonstrated that when an artificial 2F5 epitope was inserted in the gp120 V4 region so that an HIV-1 envelope glycoprotein (Env) trimer contains a natural 2F5 epitope in the gp41 membrane-proximal envelope region (MPER) and an artificially engineered 2F5 epitope in the gp120 V4 region, bivalent 2F5 IgG achieved greatly enhanced neutralization efficiency, with a 50% inhibitory concentration (IC(50)) decrease over a 2-log scale. In contrast, the monovalent 2F5 Fab fragment did not exhibit any appreciable change in neutralization efficiency in the same context. These results demonstrate that bivalent binding of 2F5 IgG to a single HIV-1 Env trimer results in dramatic enhancement of neutralization, probably through an increase in binding avidity. Furthermore, we demonstrated that bivalent binding of MAb 2F5 to the V4 region and MPER of an HIV-1 Env trimer can be achieved only in a specific configuration, providing an important insight into the structure of a native/infectious HIV-1 Env trimer. This specific binding configuration also establishes a useful standard that can be applied to evaluate the biological relevance of structural information on the HIV-1 Env trimer.

Abstract 2397: SSX-2 vaccines with enhanced MHC class I binding epitopes elicit anti-prostate tumor CTL

Abstract The tumor-associated antigen synovial sarcoma X breakpoint 2 (SSX-2) is classified as a cancer-testis antigen (CTA) based upon its tissue-restricted expression to germline cells and its frequent expression in a variety of malignancies. We have previously identified SSX-2 as a potential prostate cancer antigen that can be inducibly expressed in several prostate cancer cell lines. We have also previously identified two nonamer peptides from the amino acid sequence of SSX-2 that have significant affinity for HLA-A2 as assessed by in vitro T2 binding assay. These HLA-2-binding peptides were found to represent HLA-A2-restricted epitopes, able to induce peptide-specific immune responses in peptide- or DNA-immunized HHD-II (HLA-A2.1+/HLA-DR1+ transgenic) mice. Given these observations, the current study was conducted to determine if CTL generated in vaccinated HHD-II mice are capable of lysing HLA-A2+ prostate cancer cells and whether SSX-2 vaccine efficacy could be enhanced using an altered peptide ligand binding strategy. We found that peptides that were designed in silico to have increased or decreased HLA-A2+ affinity had enhanced and reduced HLA-A2 binding by in vitro T2 binding assay, respectively. Additionally, HHD-II mice immunized with modified SSX-2 vaccines developed increased or decreased frequencies of peptide-specific T cells in vivo, which could be detected by IFNγ ELISPOT assay and cytotoxicity assay. Animals immunized with enhancing modified SSX-2 vaccines generated CTL with cross-reactivity to the native SSX-2 epitopes. These CTL were capable of lysing target cells pulsed with both modified and native peptides and could also lyse an HLA-A2-expressing prostate cancer cell line. These results provide support for the further investigation of SSX-2 as an immunotherapeutic target for prostate cancer using vaccines modified to increase MHC class I binding. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2397.

Abstract 4774: Identification and characterization of colon cancer associated antigens which would be key therapeutic targets in the prevention of disease relapse or progression

Abstract Introduction: Colon cancer (CRC) is an immunogenic tumor and adaptive immunity may play a role in inhibiting disease relapse. A vaccine which could boost cellular immunity in CRC patients would prevent disease recurrence. However, there have been few defined immunogenic proteins, implicated in driving the malignant phenotype, that have been identified as vaccine candidates. Our group has shown that overexpressed tumor associated proteins, abundant in the cancer state but expressed at basal levels in normal cells, can be immunogenic. We hypothesized that ideal candidate antigens would be ones already validated as prognostic markers in multivariate analysis. Methods: We performed a directed literature review using search words e.g. colon cancer, prognosis, biomarker to identify such proteins in CRC. We selected a panel of eight proteins based on (1) incidence of expression, (2) independent predictor of poor prognosis and (3) early disease recurrence, and (4) known biologic function in CRC pathogenesis. Using our algorithm program we identified peptides predicted to be high affinity binders across multiple HLA DR alleles; characteristics shown to be associated with native epitopes of self tumor antigens. We synthesized the 19 peptides that were predicted to be the highest binders. We evaluated the potential immunogencity of the peptides with 1. IFN-g ELISPOT assays using PBMC's from CRC patients and controls and 2. specific IgG antibody response with ELISA using serum from CRC patients and controls. We quantitated antigen gene expression using qPCR in cell lines representing microsatellite unstable (MSI), chromosome unstable (CIN) CpG island methylator phenotype (CIMP) CRC. Summary: The eight proteins are: CDC25B, COX-2, EBAG9, EGFR, Fascin-1, IGF-1R, PRL-3 and VCP. For all candidate antigens there was a greater IFN-g response in CRC patients than in controls; for three proteins, this difference reached statistical significance (CDC25B p=.013, COX-2 p=.013, PRL-3 p=.018). For ELISA positive response, the cut-off point was set at values of the mean of control serum plus two standard deviations. All six proteins were positive. All seven antigen genes were overexpressed relative to normal colon. Conclusions: 1. biologically relevant CRC associated proteins can be identified that are associated with prognosis and potentially expressed in the majority of colorectal cancers, 2. epitopes predicted to bind multiple DR alleles, derived from candidate antigens, elicit T cell responses that are greater in CRC patients than in normal controls, 3. there was an overexpression of candidate genes across three different CRC phenotypes, and 4. these antigens may represent novel immunologic targets for CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4774.

The residue 96Gln of chicken CD132 is critical to bind to chicken IL-2 molecule (43.9)

Abstract The common cytokine receptor γ chain (γc) is shared by at least six cytokine receptors and plays a critical role in the regulation of immune responses. In this study, the functional domains of chicken γc (chCD132) were mapped with monoclonal antibodies (mAb), a synthetic peptide, and a phage display peptide library. One functional mAb to chCD132 was produced using recombinant rchCD132 ectodomain protein expressed in prokaryotic cells as an immunogen and used to finely map the functional domain of the chCD132 protein. The mimotope of anti-chCD132 mAb, QEHQNL, was identified by phage display. The corresponding native epitope in the chCD132 molecule, Q84E94L95Q96N97L98, was identified by peptide-competitive ELISA. Lymphocyte proliferation and inhibition assays further confirmed that the binding of chIL-279-96 to chCD132 molecule was inhibited by chCD13282-101. Site-specific mutation indicates that chCD13282-101 changed the residue Q96 lacked the ability binding to chIL-2 molecule. These findings demonstrates that chIL-279-96 and chCD13282-101 are the functional domains of the interaction between chIL-2 and chCD132 molecules, and that the residue Q96 within chCD13282-101 is the critical site for its bioactivity and chIL-2-binding.

Contribution of Myelin Autoantigen Citrullination to T Cell Autoaggression in the Central Nervous System

Breakdown in immunological self tolerance, leading to autoimmune diseases such as multiple sclerosis, might arise from immune recognition of self proteins that have undergone heightened posttranslational modification under pathophysiological conditions. A posttranslational modification of particular interest is the deimination of Arg to citrulline, catalyzed by peptidylarginyl deiminase (PAD) enzymes. As a CD4(+) T cell-driven model of multiple sclerosis, we used experimental autoimmune encephalomyelitis (EAE) induced with the immunodominant 35-55 peptide of myelin oligodendrocyte glycoprotein (pMOG) in C57BL/6 mice to test whether citrullination of a T cell epitope can contribute to disease etiopathology. Immunization with an altered peptide ligand (APL) of pMOG with an Arg-->citrulline conversion at a TCR contact (residue 41) led to the activation of two populations of APL-responsive T cells that either did, or did not cross-react with the native pMOG peptide. This APL could induce EAE. However, this reflected the activation of T cells that cross-reacted with the native pMOG epitope, because prior tolerization of these T cells using pMOG prevented APL-induced EAE. Using a passive transfer model, we found that T cells that responded specifically to the citrullinated form of pMOG were neither necessary, nor sufficient to initiate the EAE lesion. Nevertheless, these cells could provoke exacerbation of pathology if transferred into mice with ongoing EAE. The PAD2 and PAD4 enzymes were markedly upregulated in the inflamed CNS. Therefore, once inflammation is established, citrullination of target autoantigens can allow an expanded repertoire of T cells to contribute to CNS pathology.

Induction of cross-reactive antibodies against mimotopes of H5N1 hemagglutinin

A sub-library based on peptide mimic 125 was designed and constructed, and 18 phagotopes specifically binding 8H5mAb were isolated. Antisera against three phagotopes, containing peptide 12MH-1, 12MH-5 and 12MH-8 reacted with 3 different H5N1 virus strains, but not with 2 H1N1 and 2 H3N2 viruses by Dot blots. The affinity of 12MH-8 was approximately eight times more than 12MH-1 or 12MH-5 or parent peptide 125. Furthermore, synthesized 12MH-1 and 12MH-8 could block the 8H5mAb binding with 4 H5N1 virus strains via hemagglutinin inhibition. These results suggest that these 3 mimotopes closely mimics the native 8H5 epitopes.

Stimulation of Naive CD8+ T Cells by a Variant Viral Epitope Induces Activation and Enhanced Apoptosis

Classically, naive T cells recognize a specific peptide-MHC complex resulting in their activation and differentiation. However, it is known that T cells also have the ability to interact productively with variant ligands, indicating a flexibility in TCR Ag recognition. These altered peptide ligands have been shown to trigger responses ranging from complete activation to full inhibition of T cell responses, and thus may play an important role in initiating or sustaining T cell-mediated immunity. We have found that influenza virus-specific CD8(+) TCR transgenic T cells differentially respond to a native (agonist) and variant viral epitope, differing in two amino acids that are thought to alter TCR recognition. In response to stimulation with the agonist epitope, these cells activate, proliferate, and differentiate into effector CTLs. Conversely, stimulation with the variant epitope results in activation, proliferation, and development of effector activity followed by rapid and extensive apoptotic cell death. Stimulation of the T cells with the altered ligand results in an inability to sustain the expression of the prosurvival molecules, Bcl-2 and Bcl-xL. These data suggest that the response to the agonist and variant epitopes may reflect TCR avidity-dependent differential signaling through the TCR, resulting either in activation-dependent T cell proliferative expansion and survival or in the accelerated death of acutely activated differentiating T cells. This process of CD8(+) T cell activation, proliferation, and differentiation followed by rapid cell death may represent a novel mechanism of altered peptide ligand-induced apoptosis programmed by initial Ag receptor engagement.

Creating a mosaic in HIV

U.S. researchers have shown that engineered mosaic HIV vaccines could offer a better cellular immune response than vaccines based on natural HIV epitopes. An international consortium plans to begin a Phase I trial, which will help determine whether there is a robust antibody response as well.

Targeting a Novel N-terminal Epitope of Death Receptor 5 Triggers Tumor Cell Death

Tumor necrosis factor-related apoptosis-inducing ligand receptors death receptor (DR) 4 and DR5 are potential targets for antibody-based cancer therapy. Activation of the proapoptotic DR5 in various cancer cells triggers the extrinsic and/or intrinsic pathway of apoptosis. It has been shown that there are several functional domains in the DR5 extracellular domain. The cysteine-rich domains of DR5 have a conservative role in tumor necrosis factor-related apoptosis-inducing ligand-DR5-mediated apoptosis, and the pre-ligand assembly domain within the N1-cap contributes to the ligand-independent formation of receptor complexes. However, the role of the N-terminal region (NTR) preceding the N1-cap of DR5 remains unclear. In this study, we demonstrate that NTR could mediate DR5 activation that transmits an apoptotic signal when bound to a specific agonistic monoclonal antibody. A novel epitope in the NTR of DR5 was identified by peptide array. Antibodies against the antigenic determinant showed high affinities for DR5 and triggered caspase activation in a time-dependent manner, suggesting the NTR of DR5 might function as a potential death-inducing region. Moreover, permutation analysis showed that Leu(6) was pivotal for the interaction of DR5 and the agonistic antibody. Synthetic wild-type epitopes eliminated the cytotoxicity of all three agonistic monoclonal antibodies, AD5-10, Adie-1, and Adie-2. These results indicate that the NTR of DR5 could be a potential target site for the development of new strategies for cancer immunotherapy. Also, our findings expand the current knowledge about DR5 extracellular functional domains and provide insights into the mechanism of DR5-mediated cell death.

Natural epitope variants of the hepatitis C virus impair cytotoxic T lymphocyte activity

To understand how interactions between hepatitis C virus (HCV) and the host's immune system might lead to viral persistence or effective elimination of HCV. Nucleotides 3519-3935 of the non-structural 3 (NS3) region were amplified by using reverse transcription polymerase chain reaction (PCR). PCR products of the HCV NS3 regions were integrated into a PCR((R)) T7TOPO((R)) TA vector and then sequenced in both directions using an automated DNA sequencer. Relative major histocompatibility complex binding levels of wild-type and variant peptides were performed by fluorescence polarization-based peptide competition assays. Peptides with wild type and variant sequences of NS3 were synthesized locally using F-moc chemistry and purified by high-performance liquid chromatography. Specific cytotoxic T lymphocytes (CTLs) clones toward HCV NS3 wild-type peptides were generated through limiting dilution cloning. The CTL clones specifically recognizing HCV NS3 wild-type peptides were tested by tetramer staining and flow cytometry. Cytolytic activity of CTL clones was measured using target cells labeled with the fluorescence enhancing ligand, DELFIA EuTDA. The pattern of natural variants within three human leukocyte antigen (HLA)-A2-restricted NS3 epitopes has been examined in one patient with chronic HCV infection at 12, 28 and 63 mo post-infection. Results obtained may provide convincing evidence of immune selection pressure for all epitopes investigated. Statistical analysis of the extensive sequence variation found within these NS3 epitopes favors a Darwinian selection model of variant viruses. Mutations within the epitopes coincided with the decline of CTL responses, and peptide-binding studies suggested a significant impact of the mutation on T cell recognition rather than peptide presentation by HLA molecules. While most variants were either not recognized or elicited low responses, such could antagonize CTL responses to target cells pulsed with wild-type peptides. Cross-recognition of CTL epitopes from wild-type and naturally-occurring HCV variants may lead to impaired immune responses and ultimately contribute to viral persistence.

Exploring peptide mimics for the production of antibodies against discontinuous protein epitopes

Peptide “mimics” (mimotopes) of linear protein epitopes and carbohydrate epitopes have been successfully used as immunogens to elicit cross-reactive antibodies against their cognate epitopes; however, immunogenic mimicry has been difficult to achieve for discontinuous protein epitopes. To explore this, we developed from phage-displayed peptide libraries optimized peptide mimics for three well-characterized discontinuous epitopes on hen egg lysozyme and horse cytochrome c. The peptides competed with their cognate antigens for antibody binding, displayed affinities in the nM range, and shared critical binding residues with their native epitopes. Yet, while immunogenic, none of the peptides elicited antibodies that cross-reacted with their cognate antigens. We analyzed the 3-D structure of the site within each discontinuous epitope that shared critical binding residues with its peptide mimic, and observed that in each case it formed a ridge-like patch on the epitope; in no case did it cover most or all of the epitope. Thus, the peptides’ lack of immunogenic mimicry could be attributed to their inability to recapitulate the topological features of their cognate epitopes. Our results suggest that direct peptide immunizations are not a practical strategy for generating targeted antibody responses against discontinuous epitopes.

Antibody Binding Site Mapping of SARS-CoV Spike Protein Receptor-Binding Domain by a Combination of Yeast Surface Display and Phage Peptide Library Screening

The receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein plays an important role in viral infection, and is a potential major neutralizing determinant. In this study, three hybridoma cell lines secreting specific monoclonal antibodies against the RBD of the S protein were generated and their exact binding sites were identified. Using yeast surface display, the binding sites of these antibodies were defined to two linear regions on the RBD: S(337-360) and S(380-399). Using these monoclonal antibodies in phage peptide library screening identified 10 distinct mimotopes 12 amino acids in length. Sequence comparison between native epitopes and these mimotopes further confirmed the binding sites, and revealed key amino acid residues involved in antibody binding. None of these antibodies could neutralize the murine leukemia virus pseudotyped expressing the SARS-CoV spike protein (MLV/SARS-CoV). However, these mAbs could be useful in the diagnosis of SARS-CoV due to their exclusive reactivity with SARS-CoV. Furthermore, this study established a feasible platform for epitope mapping. Yeast surface display combined with phage peptide library screening provides a convenient strategy for the identification of epitope peptides from certain antigenic proteins.

P12-05. Using combinatorial phage-display libraries as a source of mimetic peptides of three HIV-1 envelope epitopes

Results Related to CSGKLIC immunodominant epitope, a library of 10E9 sequences were converted into a homogeneous population in which 79% of peptides contained only the CxxKxxC-motif (x represent a non-epitope amino acid). The mimotopes displayed structural, antigenic and immunogenic features of the HIV-1 immnudominant loop epitope. In the case of GPGR epitope, two motifs were most interesting G(P/Q)GP from linear and PxxxGPG from constrained library. After four immunizations with both peptide groups, anti-sera showed a strong reactivity in ELISA with synthetic peptides containing V3 GPGR epitope sequences from different HIV-1 strains, as contrast to the narrow specificity of the patient sera. The rabbit anti-sera were able to decrease the fusion of gp120/ gp41-CD4 complex in two Jurkat cell lines. Finally, when mAb 2F5 screened for structures resembling its natural epitope, it retrieved preferentially peptides with sequences longer than DKW as in previous reports. Nevertheless, in constrained 7 mer library the selection was restricted only to C-DKWAxx-C or C-xxLDKWA-C sequences, rejecting every amino acid variation. The selected mimotopes indicated that two additional residues, the Ala (DKWA) and Leu (LDKWA) residues are involved in the minimal epitope. The data imply that the 7 mer constrained peptide forming a loop represents an adequate structural context for the natural epitope core-like conformation.

P12-06. A 'non-self' mimic of the natural epitope of anti-HIV antibody 2G12 shows enhanced antigenicity

P12-06. A 'non-self' mimic of the natural epitope of anti-HIV antibody 2G12 shows enhanced antigenicity

Vaccine Potentials of an Intrinsically Unstructured Fragment Derived from the Blood Stage-Associated Plasmodium falciparum Protein PFF0165c

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan

The potent oxidants hypochlorous acid (HOCl) and hypobromous acid (HOBr) are produced extracellularly by myeloperoxidase, following release of this enzyme from activated leukocytes. The subendothelial extracellular matrix is a key site for deposition of myeloperoxidase and damage by myeloperoxidase-derived oxidants, with this damage implicated in the impairment of vascular cell function during acute inflammatory responses and chronic inflammatory diseases such as atherosclerosis. The heparan sulfate proteoglycan perlecan, a key component of the subendothelial extracellular matrix, regulates important cellular processes and is a potential target for HOCl and HOBr. It is shown here that perlecan binds myeloperoxidase via its heparan sulfate side chains and that this enhances oxidative damage by myeloperoxidase-derived HOCl and HOBr. This damage involved selective degradation of the perlecan protein core without detectable alteration of its heparan sulfate side chains, despite the presence of reactive GlcNH 2 residing within this glycosaminoglycan. Modification of the protein core by HOCl and HOBr (measured by loss of immunological recognition of native protein epitopes and the appearance of oxidatively-modified protein epitopes) was associated with an impairment of its ability to support endothelial cell adhesion, with this observed at a pathologically-achievable oxidant dose of 425 nmol oxidant/mg protein. In contrast, the heparan sulfate chains of HOCl/HOBr-modified perlecan retained their ability to bind FGF-2 and collagen V and were able to promote FGF-2-dependent cellular proliferation. Collectively, these data highlight the potential role of perlecan oxidation, and consequent deregulation of cell function, in vascular injuries by myeloperoxidase-derived HOCl and HOBr.

Rational optimization of tumor epitopes using in silico analysis‐assisted substitution of TCR contact residues

Altered peptide ligands with increased affinity of the peptide-MHC complex for the TCR provide an alternative strategy to natural T-cell epitopes for cancer immunotherapy, as they can recruit and stimulate stronger T-cell repertoires. However, it remains unclear how alterations of the TCR contact residues improve the interaction between the peptide-MHC complex and the TCR molecule. In this study, we introduced a molecular simulation strategy to optimize a tumor immunodominant epitope NY-ESO-1(157-165) by the substitution of the potential TCR contact residues. We correlated molecule simulation with T-cell activation capacity assay and detected the effect of modifications of TCR contact residues on T-cell recognition. An agonist peptide W5F with substitution at Trp5 with Phe was identified and it exhibits a stronger ability to induce a cross-reactive CTL response with the WT peptide. Additionally, the W5F-induced CTL could be maintained with the WT peptide and possess higher capacity in lysing native NY-ESO-1-expressing tumor cells. These results provide important insights into the enhanced immunogenicity of epitopes through substitution at the TCR contact sites and revealed a novel molecular simulation approach for rational therapeutic peptide vaccine design.