In Silico Characterization of B Cell and T Cell Epitopes for Subunit Vaccine Design of Salmonella typhi PgtE: A Molecular Dynamics Simulation Approach.

Typhoid fever is an acute illness in humans, caused by Salmonella typhi, a gram-negative bacterium. Outer membrane proteins of S. typhi have strong potential for its use in the development of subunit vaccine against typhoid. In the current study, peptide-based subunit vaccine was constructed from outer membrane protease E (PgtE) against S. typhi. B cell and T cell epitopes were identified at fold level with a validated three-dimensional modeled structure. T cell epitopes from PgtE (IHPDTSANY) have 99.5% binding to a maximum number of major histocompatibility complex class I and class II alleles. They also bind to the typhoid-resistant human leukocyte antigen (HLA) alleles DRB1*0401. PgtE epitopes were docked with HLA-DR4 (PDB ID: 1D5M) and a contact map was constructed. A simulation search for the binding site for full flexibility of the peptide from CABS- (Cα, Cß, side-chain)-dock shows stable interactions. Molecular dynamics simulation studies revealed that the PgtE-epitope complex structure was more stable throughout the simulation (20 ns) and interaction did not change the radius of gyration. In conclusion, computational analysis, molecular docking, and molecular dynamics (MD) simulation of PgtE-epitope complex were used to elucidate the binding mode, and the dynamical changes of epitopes were more suitable for vaccine development against typhoid.

Dynamics Determine Signaling in a Multicomponent System Associated with Rheumatoid Arthritis.

Strategies that target multiple components are usually required for treatment of diseases originating from complex biological systems. The multicomponent system consisting of the DR4 major histocompatibility complex type II molecule, the glycopeptide CII259-273 from type II collagen, and a T-cell receptor is associated with development of rheumatoid arthritis (RA). We introduced non-native amino acids and amide bond isosteres into CII259-273 and investigated the effect on binding to DR4 and the subsequent T-cell response. Molecular dynamics simulations revealed that complexes between DR4 and derivatives of CII259-273 were highly dynamic. Signaling in the overall multicomponent system was found to depend on formation of an appropriate number of dynamic intramolecular hydrogen bonds between DR4 and CII259-273, together with the positioning of the galactose moiety of CII259-273 in the DR4 binding groove. Interestingly, the system tolerated modifications at several positions in CII259-273, indicating opportunities to use analogues to increase our understanding of how rheumatoid arthritis develops and for evaluation as vaccines to treat RA.

How MHC molecules grab citrullinated peptides to foster rheumatoid arthritis.

The molecular immunologist's dream is to elucidate a fundamental biochemical process that explains the basis of an affliction that affects millions of people, and that, precisely understood, might yield a rational approach to diagnosis, prevention, or therapy. In this issue of JBC, Ting et al. report proteomic, biochemical, and structural analyses that better explain how the antigen-presenting HLA-DR4 molecules bind citrullinated peptides to provoke rheumatoid arthritis (RA), a chronic autoimmune disease that affects 0.5-1% of the population.

Effects of type II collagen epitope carbamylation and citrullination in human leucocyte antigen (HLA)-DR4(+) monozygotic twins discordant for rheumatoid arthritis.

The aim of this study is to investigate the effect of the native, citrullinated or carbamylated type II human collagen T cell- and B cell-epitopes on the adaptive immune response in rheumatoid arthritis (RA). Peripheral blood T and B cells obtained from a human leucocyte D4-related (antigen DR4(-) HLA-DR4)(+) woman with early RA, her healthy monozygotic twin and an unrelated HLA-DR3(+) woman with early RA were analysed for activation (CD154/CD69), apoptosis (annexin/7-aminoactinomycin), cytokine production [interferon (IFN)γ/interleukin (IL)-17/IL-4/IL-10/IL-6] and functional phenotype (CD45Ra/CCR7) after stimulation with the collagen native T cell epitope (T261-273), the K264 carbamylated T cell epitope (carT261-273), the native B cell epitope (B359-369) or the R360 citrullinated B cell epitope (citB359-369), and the combinations of these. The T cell memory compartment was activated by T cell epitopes in both discordant DR4(+) twins, but not in the DR3(+) RA. The collagen-specific activation of CD4(+) T cells was induced with both the native and carbamylated T cell epitopes only in the RA twin. Both T cell epitopes also induced IL-17 production in the RA twin, but a greater IL-4 and IL-10 response in the healthy twin. The citrullinated B cell epitope, particularly when combined with the carbamylated T cell epitope, induced B cell activation and an increased IL-6/IL-10 ratio in the RA twin compared to a greater IL-10 production in the healthy twin. Our data suggest that circulating collagen-specific T and B cells are found in HLA-DR4(+) subjects, but only RA activated cells express co-stimulatory molecules and produce proinflammatory cytokines. Carbamylation and citrullination further modulate the activation and cytokine polarization of T and B cells.

T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex.

Central to adaptive immunity is the interaction between the αß T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and ß-chain are overlaid with the α-chain and ß-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition.

A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis.

Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine-containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01(+) individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4(+) T cells in the peripheral blood of HLA-DRB1*04:01(+) RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.

A novel transport mechanism for MOMP in Chlamydophila pneumoniae and its putative role in immune-therapy.

Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE(-/-) mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane ß-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events.

Minimal conformational plasticity enables TCR cross-reactivity to different MHC class II heterodimers.

Successful immunity requires that a limited pool of αß T-cell receptors (TCRs) provide cover for a vast number of potential foreign peptide antigens presented by 'self' major histocompatibility complex (pMHC) molecules. Structures of unligated and ligated MHC class-I-restricted TCRs with different ligands, supplemented with biophysical analyses, have revealed a number of important mechanisms that govern TCR mediated antigen recognition. HA1.7 TCR binding to the influenza hemagglutinin antigen (HA(306-318)) presented by HLA-DR1 or HLA-DR4 represents an ideal system for interrogating pMHC-II antigen recognition. Accordingly, we solved the structure of the unligated HA1.7 TCR and compared it to both complex structures. Despite a relatively rigid binding mode, HA1.7 T-cells could tolerate mutations in key contact residues within the peptide epitope. Thermodynamic analysis revealed that limited plasticity and extreme favorable entropy underpinned the ability of the HA1.7 T-cell clone to cross-react with HA(306-318) presented by multiple MHC-II alleles.

Design of glycopeptides used to investigate class II MHC binding and T-cell responses associated with autoimmune arthritis.

The glycopeptide fragment CII259-273 from type II collagen (CII) binds to the murine A(q) and human DR4 class II Major Histocompatibility Complex (MHC II) proteins, which are associated with development of murine collagen-induced arthritis (CIA) and rheumatoid arthritis (RA), respectively. It has been shown that CII259-273 can be used in therapeutic vaccination of CIA. This glycopeptide also elicits responses from T-cells obtained from RA patients, which indicates that it has an important role in RA as well. We now present a methodology for studies of (glyco)peptide-receptor interactions based on a combination of structure-based virtual screening, ligand-based statistical molecular design and biological evaluations. This methodology included the design of a CII259-273 glycopeptide library in which two anchor positions crucial for binding in pockets of A(q) and DR4 were varied. Synthesis and biological evaluation of the designed glycopeptides provided novel structure-activity relationship (SAR) understanding of binding to A(q) and DR4. Glycopeptides that retained high affinities for these MHC II proteins and induced strong responses in panels of T-cell hybridomas were also identified. An analysis of all the responses revealed groups of glycopeptides with different response patterns that are of high interest for vaccination studies in CIA. Moreover, the SAR understanding obtained in this study provides a platform for the design of second-generation glycopeptides with tuned MHC affinities and T-cell responses.

Identification of small-molecule inhibitors against human leukocyte antigen-death receptor 4 (HLA-DR4) through a comprehensive strategy.

Rheumatoid arthritis (RA) is an autoimmune disease mediated by T-lymphocytes and associated with the human leukocyte antigen-death receptor 4 (HLA-DR4). The HLA-DR4 protein selectively interacts with the antigenic peptides on the cell surface and presents them to the T cell receptor (TCR) on CD4+ T cells. The HLA-DR4-antigen-TCR complex initiates the autoimmune response and eventually causes the chronic inflammation within patients bodies. To inhibit HLA-DR4-restricted T cell activation, an ideal approach is to discover non-T cell stimulating substrates that specifically bind to HLA-DR4. In this paper, a comprehensive structure-based design strategy involved de novo design approach, pharmacophore search, and dock method was presented and applied to "simplify" the known binding peptide ligand of HLA-DR4 and identified specific small-molecule inhibitors for HLA-DR4. The designed three-step strategy successfully identified five nonpeptide ligands with novel scaffolds from a chemical library containing 4 × 10(6) commercially available compounds within a tolerable computing time. The identified five chemicals, BAS-0219606, T0506-2494, 6436645, 3S-71981, and KM 11073, are all non-T cell stimulators and are able to significantly inhibit HLA-DR4-restricted T cell activation induced by type II collagen (CII) 263-272 peptide. IC(50) for the best two potentials, BAS-0219606 and T0506-2494, was 31 and 17 µM, respectively, which is equivalent or better than the known peptide ligands. It is hopeful that they can be used as effective therapeutic means for further treatment of RA patients. In addition, the comprehensive strategy presented in this paper exhibited itself to be an effective flow line from peptide ligands to small-molecule inhibitors and will have applications to other targets.

An expanded self-antigen peptidome is carried by the human lymph as compared to the plasma.

BACKGROUND: The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. A comprehensive qualitative and quantitative investigation of the self-antigenic repertoire transported by the human lymph is still missing. METHODOLOGY/PRINCIPAL FINDINGS: A major difference between lymph and plasma could be visualized by FPLC and 2D gel in the amount of low molecular weight products corresponding to peptide fragments. Naturally processed peptides in normal pre-nodal human lymph were then fractionated by HPLC and characterized by multidimensional mass spectrometry. Analysis of more then 300 sequences identified self-peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration. CONCLUSIONS/SIGNIFICANCE: The peptidome, generated by physiological tissue catabolism and transported by the pre-nodal lymph, is in addition to the self-peptidome generated in endosomal compartment. Unlike self antigen processed by local or nodal APC, which mostly produce epitopes constrained by the endosomal processing activity, self antigens present in the lymph could derived from a wider variety of processing pathways; including caspases, involved in cellular apoptosis, and ADAM and other metalloproteinases involved in surface receptor editing, cytokines processing and matrix remodeling. Altogether, expanding the tissue-specific self-repertoire available for the maintenance of immunological tolerance.

The rheumatoid arthritis-associated allele HLA-DR10 (DRB1*1001) shares part of its repertoire with HLA-DR1 (DRB1*0101) and HLA-DR4 (DRB*0401).

OBJECTIVE: To identify the peptide anchor motif for the rheumatoid arthritis (RA)-related HLA allele, DR10, and find shared natural ligands or sequence similarities with the other disease-associated alleles, DR1 and DR4. METHODS: The HLA-DR10-associated peptides were purified, and a proportion of these natural ligands were de novo sequenced by mass spectrometry. Based on crystallographic structures, the complexes formed by peptide influenza virus hemagglutinin HA306-318 with DR1, DR4, and DR10 were modeled, and binding scores were obtained. RESULTS: A total of 238 peptides were sequenced, and the anchor motif of the HLA-DR10 peptide repertoire was defined. A large proportion of the DR10-associated peptides had the structural features to bind DR1 and DR4 but were theoretical nonbinders to the negatively associated alleles DR15 and DR7. Among the sequenced ligands, 10 had been reported as ligands to other RA-associated alleles. Modeling data showed that peptide HA306-318 can bind DR1, DR4, and DR10 with similar affinities. CONCLUSION: The data show the presence of common peptides in the repertoires of RA-associated HLA alleles. The combination of the shared epitope present in DR1, DR4, and DR10 together with common putative arthritogenic peptide(s) could influence disease onset or outcome.

Ligand design by a combinatorial approach based on modeling and experiment: application to HLA-DR4.

Combinatorial synthesis and large scale screening methods are being used increasingly in drug discovery, particularly for finding novel lead compounds. Although these "random" methods sample larger areas of chemical space than traditional synthetic approaches, only a relatively small percentage of all possible compounds are practically accessible. It is therefore helpful to select regions of chemical space that have greater likelihood of yielding useful leads. When three-dimensional structural data are available for the target molecule this can be achieved by applying structure-based computational design methods to focus the combinatorial library. This is advantageous over the standard usage of computational methods to design a small number of specific novel ligands, because here computation is employed as part of the combinatorial design process and so is required only to determine a propensity for binding of certain chemical moieties in regions of the target molecule. This paper describes the application of the Multiple Copy Simultaneous Search (MCSS) method, an active site mapping and de novo structure-based design tool, to design a focused combinatorial library for the class II MHC protein HLA-DR4. Methods for the synthesizing and screening the computationally designed library are presented; evidence is provided to show that binding was achieved. Although the structure of the protein-ligand complex could not be determined, experimental results including cross-exclusion of a known HLA-DR4 peptide ligand (HA) by a compound from the library. Computational model building suggest that at least one of the ligands designed and identified by the methods described binds in a mode similar to that of native peptides.

Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining.

HLA class I tetramers have revolutionized the study of Ag-specific CD8+ T cell responses. Technical problems and the rarity of Ag-specific CD4+ Th cells have not allowed the potential of HLA class II tetramers to be fully realized. Here, we optimize HLA class II tetramer staining methods through the use of a comprehensive panel of HIV-, influenza-, CMV-, and tetanus toxoid-specific tetramers. We find rapid and efficient staining of DR1- and DR4-restricted CD4+ cell lines and clones and show that TCR internalization is not a requirement for immunological staining. We combine tetramer staining with magnetic bead enrichment to detect rare Ag-specific CD4+ T cells with frequencies as low as 1 in 250,000 (0.0004% of CD4+ cells) in human PBLs analyzed directly ex vivo. This ultrasensitive detection allowed phenotypic analysis of rare CD4+ T lymphocytes that had experienced diverse exposure to Ag during the course of viral infections. These cells would not be detectable with normal flow-cytometric techniques.

Analysis of eluted peptides from type 1 diabetes-susceptible HLA class II molecules identified novel islet protein, heparin/heparan sulfate-interacting protein.

Identification of peptides derived from pancreatic islet and presented by type 1 diabetes-susceptible MHC class II molecules has great significance to elucidate the pathogenesis of type 1 diabetes. A bulk culture of Epstein-Barr virus-transformed B-cells, which were established from a 22-year-old type 1 diabetic woman with HLA-DR4 and -DQw8, was pulsed with the homogenate of a human embryonic pancreas-derived cell line 1B2C6, and another culture was not pulsed with antigen. Peptide fractions were obtained by treatment of affinity-purified HLA-DR and -DQ molecules with 0.1% trifluoroacetic acid, and were subjected to reverse-phase high performance liquid chromatography (RP-HPLC). The RP-HPLC profiles of peptides derived from DR molecules revealed three peaks that specifically appeared after pulsing, but no such peaks were obtained from DQ molecules. From one of these three peaks, a peptide that consisted of 14 amino acids (AKSXNHTXXNQXRK, where X represents the undetermined amino acids) was identified. This peptide was derived from heparin/heparan sulfate-interacting protein (HIP). Immunostaining of pancreatic sections using antiserum for HIP peptide revealed exclusive staining of the islets. Thus, HIP was identified as an islet protein naturally processed and presented by HLA-DR4 molecules.

Disease-stage variance in functional CD4(+) T-cell responses against novel pan-human leukocyte antigen-D region presented human papillomavirus-16 E7 epitopes.

Given the anticipated clinical importance of helper and regulatory CD4(+) T cells reactive against human papillomavirus-16 E7 in the cervical carcinoma setting, we performed this study to identify novel E7-derived T helper (Th) epitopes and to characterize functional anti-E7 Th responses in normal donors and patients with cervical intraepithelial neoplasia I-III or cervical cancer. Candidate pan-HLA-DR (D region) binding peptides were identified and synthesized based on results obtained using a predictive computer algorithm, then applied in short-term in vitro T-cell sensitization assays. Using IFN-gamma/IL-5 (interleukin 5) enzyme-linked immunospot assays as readouts for Th1-type and Th2-type CD4(+) T-cell responses, respectively, we identified three E7-derived T helper epitopes (E7(1-12), E7(48-62), and E7(62-75)), two of which are novel. Normal donor CD4(+) T cells failed to react against these E7 peptides, whereas patients with premalignant cervical intraepithelial neoplasia I-III lesions displayed preferential Th1-type responses against all three E7 epitopes. Th1-type responses were still observed to the E7(48-62) but not to the E7(1-12) and E7(62-75) peptides in cancer patients, where these latter two epitopes evoked Th2-type responses. Notably all responders to the E7(1-12) and E7(62-75) peptides expressed the HLA-DR4 or -DR15 alleles, whereas all responders to the E7(48-62) peptide failed to express the HLA-DR4 allele. Our results are consistent with a model in which cervical cancer progression is linked to an undesirable Th1- to Th2-type shift in functional CD4(+) T cell responses to two novel E7-derived epitopes. These peptides may prove important in vaccines to promote and maintain protective Th1-type antihuman papillomavirus immunity and in the immune monitoring of treated patients harboring HPV-16(+) malignancies.

A chaperone-assisted high yield system for the production of HLA-DR4 tetramers in insect cells.

MHC tetramers have become essential tools for the analysis of antigen specific responses of CD8+ and CD4+ T cells. However, the use of MHC class II tetramers is hampered by the relatively low yields of most current expression systems. We have devised an insect cell/baculovirus expression system in which yields of 50-70 mg of recombinant HLA-DR4 molecules, with or without covalently linked peptide, per liter of insect cell supernatant, are routinely obtained. These yields are rendered possible by an optimized design and use of DRalpha and DRbeta expression cassettes and by co-expression of a housekeeping chaperone of the endoplasmic reticulum, calreticulin, which, due to its co-secretion, increases secretion of HLA-DR molecules two- to threefold. A tetramer produced in the system specifically was shown to stain an HLA-DR4 restricted T cell line obtained from a healthy donor by in vitro priming, but which recognizes a type I diabetes autoantigen. Co-expression of chaperones may represent a general strategy for enhancing yields of recombinant proteins expressed in insect cells and facilitate production of MHC class II tetramers in the future.

[The inhibitory effect of hyporesponsive peptide on human leukocyte antigen-DRbeta1 specific T cell activation].

OBJECTIVE: To evaluate the inhibitory effect of human leukocyte antigen (HLA)-DRbeta1 specific collagen II (CII) peptides with substitutions of TCR binding residues on T cell activation, and explore a new therapeutic strategy for T cell mediated autoimmune diseases by interfering with antigen recognition of T Cell receptor (TCR). METHODS: Non-TCR binding peptides were designed by computer modeling based on interaction of HLA DR1. The modified CII263-272. Intracellular transfer of the modified CII peptide and its binding to HLA DR1 were studied using confocal microscopy and fluorescence-activated cell sorter (FACS). The effects of altered peptides on T cell activation were evaluated using an antigen presenting system consisting of HLA-DR1 transgenic APC and CII specific T cells. RESULTS: Computer modeling showed the side chains of 263 (F), 266 (E) fit in the peptide binding groove, and form hydrogen bond with alpha1, beta1 chain of HLA-DR1. The side chains of TCR specific 267 (Q) and 270 (K) protruded out of the groove, which might be TCR recognizing residues. The modified CII peptides with intact HLA-DR1 binding residues were bound to intracellular HLA-DR1 and expressed on cell surface. The modified peptides with single residue substitution of 267-270 and consecutive substitution of 268-270 showed a hyporesponsive T cell activation. Altered peptides 270A, sub268-270 could significantly suppress the T cell activation induced by CII263-272. CONCLUSION: The altered peptides with substitution of TCR binding residues are hyporesponsive in T cell activation, and may competitively inhibit the T cell activation in T cell mediated autoimmune diseases.

Low-avidity recognition by CD4+ T cells directed to self-antigens.

Self-reactive T cells populate the peripheral immune system, and likely form the reservoir from which autoreactive cells are derived. We analyzed a panel of self and non-self peptides presented by HLA-DR4, a class II molecule associated with autoimmunity, by immunization of mice transgenic for HLA-DR4. Significant structural avidity for T cell recognition, as measured by MHC class II tetramer binding to CD4(+) T cells was only observed in mice immunized with the non-self antigens. T cell hybridomas were generated from mice immunized with the naturally processed self-peptide hGAD65 (552-572) and also from mice immunized with an influenza-derived non-self epitope (HA 306-318). T cells specific for the self peptide failed to bind tetramers and exhibited low functional avidity as measured by the peptide concentration required to reach half-maximum proliferation values. In contrast, T cells specific for the non-self HA (306-318) peptide exhibited high structural and functional avidity profiles. As recently described in studies of murine CD8(+) T cell function, the predominance of low avidity recognition of self-peptide epitopes may be a characteristic feature of CD4(+) T cells responding to autoantigens.