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Using a recombinant protein antigen for antibody testing shows a sum of antibody responses to multiple different immune epitopes existing in the protein antigen. In contrast, the antibody testing to an immunogenic peptide epitope reflects a singular antibody response to the individual peptide epitope. Therefore, using a panel of peptide epitopes provides an advantage for profiling multiple singular antibody responses with potential to estimate recent malaria exposure in human infections. However, transitioning from malaria immune epitope peptide-based ELISA to an all peptide bead-based multiplex Luminex assay presents some challenges including variation in the ability of different peptides to bind beads. The aim of this study was to develop a peptide coupling method while demonstrating the utility of these peptide epitopes from multiple stage antigens of Plasmodium falciparum for measuring antibodies. Successful coupling of peptide epitopes to beads followed three steps: 1) development of a peptide tag appended to the C-terminus of each peptide epitope consisting of beta-alanine-lysine (x 4)--cysteine, 2) bead modification with a high concentration of adipic acid dihydrazide, and 3) use of the peptide epitope as a blocker in place of the traditional choice, bovine serum albumin (BSA). This new method was used to couple 12 peptide epitopes from multiple stage specific antigens of P. falciparum, 1 Anopheles mosquito salivary gland peptide, and 1 Epstein-Barr virus peptide as an assay control. The new method was applied to testing of IgG in pooled samples from 30 individuals with previously repeated malaria exposure in western Kenya and IgM and IgG in samples from 37 U.S. travelers with recent exposure to malaria. The new peptide-bead coupling method and subsequent multiplex Luminex assay showed reliable detection of IgG to all 14 peptides in Kenyan samples. Among 37 samples from U.S. travelers recently diagnosed with malaria, IgM and IgG to the peptide epitopes were detected with high sensitivity and variation. Overall, the U.S. travelers had a much lower positivity rates of IgM than IgG to different peptide epitopes, ranging from a high of 62.2% positive for one epitope to a low of only 5.4% positive for another epitope. In contrast, the travelers had IgG positive rates from 97.3% to 91.9% to various peptide epitopes. Based on the different distribution in IgM and IgG positivity to overall number of peptide epitopes and to the number of pre-erythrocytic, erythrocytic, gametocytic, and salivary stage epitopes at the individual level, four distinct patterns of IgM and IgG responses among the 37 samples from US travelers were observed. Independent peptide-bead coupling and antibody level readout between two different instruments also showed comparable results. Overall, this new coupling method resolves the peptide-bead coupling challenge, is reproducible, and can be applied to any other immunogenic peptide epitopes. The resulting all peptide bead-based multiplex Luminex assay can be expanded to include other peptide epitopes of P. falciparum, different malaria species, or other diseases for surveillance, either in US travelers or endemic areas., (Published by Elsevier B.V.)

Background: Although DAAs hold promise to significantly reduce rates of chronic HCV infections, its eradication still requires development of an effective vaccine. Prolonged T cell responses and cross neutralizing antibodies are ideal for vaccination against the infection. We aimed to design and synthesize a 6 multi epitope peptide vaccine candidate and provide evidence for production of extended cellular and neutralizing Abs in mice., Methods: Six peptides derived from conserved epitopes in E1, E2 (n = 2),NS4B, NS5A and NS5B were designed, synthesized in a multiple antigenic peptide (MAP) form and administered w/o adjuvant to BALB/c mice as HCVp6-MAP at doses ranging from 800 ng to 16 μg. Humoral responses to structural epitopes were assayed by ELISA at different times after injection. ELISpot assay was used to evaluate IFN ɣ producing CD4 + / CD8 + T- lymphocytes at extended durations i.e. > 20 weeks. Viral neutralization by mice sera was tested for genotypes 2a (JFH1) and a chimeric 2a/4a virus (ED43/JFH1) in HCVcc culture., Results: HCVp6-MAP confers potent viral neutralization and specific cellular responses at > 1600 ng/ animal for at least 20 weeks., Conclusion: We report on a promising anti HCV vaccine for future studies on permissive hosts and in clinical trials.

The design of nanoparticles exposing a high density of antigens constitutes a promising strategy to address safety concerns of conventional life-attenuated vaccines as well as to increase the immunogenicity of subunit vaccines. In this study, we developed a fully synthetic nanovaccine based on an amyloid peptide sequence with high self-assembling properties. The immunogenic epitope E2EP3 from the E2 glycoprotein of the Chikungunya virus was used to evaluate the potential of a 10-mer peptide derived from an endogenous amyloidogenic polypeptide as a novel vaccine platform. Chimeric peptides, comprising the peptide antigen attached to the amyloid core by a short flexible linker, were prepared by solid phase synthesis. As observed using atomic force microscopy, these polypeptides self-assembled into linear and unbranched fibrils with a diameter ranging from 6 to 8 nm. A quaternary conformation rich in cross-β-sheets characterized these assemblies, as demonstrated by circular dichroism spectroscopy and thioflavin T fluorescence. ELISA assays and transmission electronic microscopy of immunogold labeled-fibrils revealed a high density of the Chikungunya virus E2 glycoprotein derived epitope exposed on the fibril surface. These amyloid fibrils were cytocompatible and were efficiently uptaken by macrophages. Mice immunization revealed a robust IgG response against the E2EP3 epitope, which was dependent on self-assembly and did not require co-injection of the Alhydrogel adjuvant. These results indicate that cross-β-sheet amyloid assemblies constitute suitable synthetic self-adjuvanted assemblies to anchor antigenic determinants and to increase the immunogenicity of peptide epitopes.

We previously identified human phosphatidylethanolamine-binding protein 4 (hPEBP4) as an antiapoptotic protein with increased expression levels in breast, ovarian and prostate cancer cells, but low expression levels in normal tissues, which makes hPEBP4 an attractive target for immunotherapy. Here, we developed hPEBP4-derived immunogenic peptides for inducing antigen-specific cytotoxic T lymphocytes (CTLs) targeting breast cancer. A panel of hPEBP4-derived peptides predicted by peptide-MHC-binding algorithms was evaluated to characterize their HLA-A2.1 affinity and immunogenicity. We identified a novel immunogenic peptide, P40-48 (TLFCQGLEV), that was capable of eliciting specific CTL responses in HLA-A2.1/K b transgenic mice, as well as in peripheral blood lymphocytes from breast cancer patients. Furthermore, amino-acid substitutions in the P40-48 sequence improved its immunogenicity against hPEBP4, a self-antigen, thus circumventing tolerance. We designed peptide analogs by preferred auxiliary HLA-A*0201 anchor residue replacement, which induced CTLs that were crossreactive to the native peptide. Several analogs were able to stably bind to HLA-A*0201 and elicit specific CTL responses better than the native sequence. Importantly, adoptive transfer of CTLs induced by vaccination with two analogs more effectively inhibited tumor growth than the native peptide. These data indicate that peptide analogs with high immunogenicity represent promising candidates for peptide-mediated therapeutic cancer vaccines.

Hepatitis B virus (HBV) infection has persisted as a major public health problem due to the lack of an effective treatment for those chronically infected. Therapeutic vaccination holds promise, and targeting HBV polymerase is pivotal for viral eradication. In this research, a computational approach was employed to predict suitable HBV polymerase targeting multi-peptides for vaccine candidate selection. We then performed in-depth computational analysis to evaluate the predicted epitopes' immunogenicity, conservation, population coverage, and toxicity. Lastly, molecular docking and MHC-peptide complex stabilization assay were utilized to determine the binding energy and affinity of epitopes to the HLA-A0201 molecule. Criteria-based analysis provided four predicted epitopes, RVTGGVFLV, VSIPWTHKV, YMDDVVLGA and HLYSHPIIL. Assay results indicated the lowest binding energy and high affinity to the HLA-A0201 molecule for epitopes VSIPWTHKV and YMDDVVLGA and epitopes RVTGGVFLV and VSIPWTHKV, respectively. Regions 307 to 320 and 377 to 387 were considered to have the highest probability to be involved in B cell epitopes. The T cell and B cell epitopes identified in this study are promising targets for an epitope-focused, peptide-based HBV vaccine, and provide insight into HBV-induced immune response.

We have designed a cysteine-rich β-hairpin peptide which dimerises spontaneously to the antiparallel double β-hairpin motif C1-C12', C1'-C12, C5-C8, C5'-C8'-tricyclo-(CHWECCitGCRLVC) 2 . The highly regioselective oxidation of eight cysteines yields an intermolecular bi-disulfide 24mer hinge peptide from two individual 12mer β-hairpins, each rigidified by an additional intramolecular disulfide bond - all in all a tetra-disulfide. The reaction kinetics of air-oxidation were followed by HPLC and the constitutional isomer was identified by mass spectrometry. The hairpin conformation was characterised in detail by NMR spectroscopy and the opening angle of the antiparallel hinge was estimated from drift times obtained by ion-mobility spectrometry. Based on a set of investigated disulfide motifs, we are able to rationalise how the unbalanced number of bonded and non-bonded hydrogen pairs in a 12 mer hairpin causes their dimerisation. The unique dimeric bi-/tetra-disulfides provide systematic insights into β-hairpin formation. They can serve as a standalone structural element for the oligomerisation of peptide epitopes where structural diversity is generated from a minimal number of amino acids.

Respiratory syncytial virus (RSV) infection is a serious health problem in young children, immunocompromised patients, and the elderly. The development of novel prevention strategies, such as a vaccine to RSV, is a high priority. One strategy is to design a peptide-based vaccine that activates appropriate CD8(+) T-cell responses. However, this approach is limited by the low number of RSV peptide epitopes defined to date that activate CD8(+) T cells. We aimed to identify peptide epitopes that are presented by common human leukocyte antigen types (HLA-A*01, -A*02, and -B*07). We identify one novel HLA-A*02-restricted and two novel HLA-A*01-restricted peptide epitopes from RSV polymerase. Peptide-HLA multimer staining of specific T cells from healthy donor peripheral blood mononuclear cell, the memory phenotype of such peptide-specific T cells ex vivo, and functional IFNγ responses in short-term stimulation assays suggest that these peptides are recognized during RSV infection. Such peptides are candidates for inclusion into a peptide-based RSV vaccine designed to stimulate defined CD8(+) T-cell responses.

Peptides are attracting increasing interest from the pharmaceutical industry because of their specificity and ability to address novel targets, including protein-protein interactions. However, typically they require stabilization for therapeutic applications owing to their susceptibility to degradation by proteases. Advances in the ability to chemically synthesize peptides and the development of new side-chain and backbone ligation strategies provide new tools to stabilize bioactive peptide epitopes. Two such epitopes are LyP1, a nine residue peptide that localizes to tumor cells and has potential as an anticancer therapeutic, and RGDS, a tetrapeptide shown to bind to survivin and induce apoptosis. Here we applied a variety of strategies for the stabilization of LyP1 and RGDS, including side-chain cyclization using "click" chemistry and "grafting" the epitopes into two naturally occurring cyclic peptide scaffolds, i.e., θ-defensins and cyclotides. NMR data showed that the three-disulfide θ-defensin and cyclotide scaffolds accommodated the LyP1 and RGDS epitopes but that scaffolds with fewer disulfide bonds were structurally compromised by inclusion of the LyP1 epitope. LyP1, LyP1-, and RGDS-grafted peptides that were largely unstructured also had reduced resistance to degradation in human serum, showing that grafting into a stable cyclic scaffold is an effective strategy for increasing the stability of a bioactive peptide epitope. Overall, the study demonstrates several methods for stabilizing peptide epitopes using side-chain or backbone cyclization and illustrates their potential in peptide drug design., (© 2015 Wiley Periodicals, Inc.)

The role of pathologic auto-antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35-55). In this scenario, we analyzed the anti-MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1-117). To assess the presence of a B-cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1-117 sequence of MOG, including MOG(35-55). For this purpose, we cloned, expressed in Escherichia coli and on-column refolded MOG(1-117), and we applied an optimized microwave-assisted solid-phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid-phase immunoenzymatic assay testing both naïve and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35-55), we hypothesize the presence of both linear and conformational epitopes on MOG(35-55) sequence., (Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.)

The characteristic pentaglycyl cross-bridge of the Staphylococcus aureus peptidoglycan (PG) cell wall component is an attractive epitope to raise specific antibodies against this microorganism. Based on this approach, we report here for the first time a competitive ELISA able to detect S. aureus down to 10(4) CFU mL(-1), without pre-enrichment on cell culture. The antibodies were raised against peptide-protein bioconjugates prepared by covalently coupling peptide haptens (PSau6 and PSau8) designed and synthesized taking into consideration the complex tridimensional structure in the PG polymer. Deglycosylation of the PG under acidic conditions has found to increase assay detectability. Assay performance has been evaluated in clinical samples such as bronchoalveolar lavage (BAL) and bronchoalveolar endotracheal aspirates (BAS) showing promising results for further implementation of this immunoassay as a daily routine diagnostic tool. Cross-reactivity studies have demonstrated that the immunoassay is specific for S. aureus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and has an occluded structure, which prevents direct inhibition of its active site before it enters the cytosol. Target-guided synthesis by in situ click chemistry is combined with synthetic epitope targeting to exploit the tertiary structure of the BoNT protein as a landscape for assembling a competitive inhibitor. A substrate-mimicking peptide macrocycle is used as a direct inhibitor of BoNT. An epitope-targeting in situ click screen is utilized to identify a second peptide macrocycle ligand that binds to an epitope that, in the folded BoNT structure, is active-site-adjacent. A second in situ click screen identifies a molecular bridge between the two macrocycles. The resulting divalent inhibitor exhibits an in vitro inhibition constant of 165 pM against the BoNT/A catalytic chain. The inhibitor is carried into cells by the intact holotoxin, and demonstrates protection and rescue of BoNT intoxication in a human neuron model., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Cat-PAD, the first in a new class of synthetic peptide immuno-regulatory epitopes (SPIREs), was shown to significantly improve rhinoconjunctivitis symptoms in subjects with cat allergy up to 1 year after the start of a short course of treatment., Objective: To evaluate the long-term effects of Cat-PAD on rhinoconjunctivitis symptoms following standardized allergen challenge 2 years after treatment., Methods: In a randomized, double-blind, placebo-controlled, parallel group study, subjects were exposed to cat allergen in an environmental exposure chamber (EEC) before and after treatment with two regimens of Cat-PAD (either eight doses of 3 nmol or four doses of 6 nmol) given intradermally over a 3-month period. In this follow-up study, changes from baseline in rhinoconjunctivitis symptoms were reassessed 2 years after the start of treatment., Results: The primary endpoint showed a mean reduction in total rhinoconjunctivitis symptom scores of 3.85 units in the 4 × 6 nmol Cat-PAD group compared to placebo 2 years after the start of treatment (P = 0.13), and this difference was statistically significant in the secondary endpoint at the end of day 4 when the cumulative allergen challenge was greatest (P = 0.02). Consistent reductions in nasal symptoms of between 2 and 3 units were observed for 4 × 6 nmol Cat-PAD compared to placebo between the 2 and 3 h time points on days 1-4 of EEC challenge at 2 years (P < 0.05). The 8 × 3 nmol dose did not show a meaningful effect in this study., Conclusion and Clinical Relevance: A persistent, clinically meaningful reduction in rhinoconjunctivitis symptoms was observed on EEC challenge 2 years after the start of a short course of treatment with 4 × 6 nmol Cat-PAD. This study is the first to provide evidence of a long-term therapeutic effect with this new class of SPIREs., (© 2015 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.)

Development of antibodies (Abs) against factor VIII (FVIII) is a severe complication of haemophilia A treatment. Recent publications suggest that domain specificity of anti-FVIII antibodies, particularly during immune tolerance induction (ITI), might be related to the outcome of the treatment. Obtaining suitable tools for a fine mapping of discontinuous epitopes could thus be helpful. The aim of this study was to map discontinuous epitopes on FVIII A2 domain using a new epitope prediction functionality of the PEPOP bioinformatics tool and a peptide inhibition assay based on the Luminex technology. We predicted, selected and synthesized 40 peptides mimicking discontinuous epitopes on the A2 domain of FVIII. A new inhibition assays using Luminex technology was performed to identify peptides able to inhibit the binding of anti-A2 Abs to A2 domain. We identified two peptides (IFKKLYHVWTKEVG and LYSRRLPKGVKHFD) able to block the binding of anti-A2 allo-antibodies to this domain. The three-dimensional representation of these two peptides on the A2 domain revealed that they are localized on a limited region of A2. We also confirmed that residues 484-508 of the A2 domain define an antigenic site. We suggest that dissection of the antibody response during ITI using synthetic peptide epitopes could provide important information for the management of patients with inhibitors., (© 2014 John Wiley & Sons Ltd.)

Celiac disease (CD) patients usually present high levels of circulating IgA antibodies directed to different antigens, in particular tissue transglutaminase (tTG), gliadin (Glia), and endomysium. A series of synthetic peptide constructs containing cross-linked tTG and Glia deamidated peptides have been synthesized. Peptides were tested in enzyme-linked immunosorbent assays against celiac disease patients' sera versus normal blood donors, and their conformational features were evaluated by molecular modeling techniques. Four peptides were recognized as epitopes by autoantibodies (IgG class) circulating in CD patients' sera before gluten-free diet. The peptide II, containing Ac-tTG(553-564)-NH2 sequence cross-linked with deamidated Ac-α2-Glia(63-71)-NH2, was able to identify specific disease antibodies with a sensitivity of 50% and a specificity of 94.4%. Structural conformations of the linear fragments Ac-tTG(553-564)-NH2 and Ac-α2-Glia(63-71)-NH2 and the corresponding cross-linked peptide II were calculated by molecular modeling. Results showed that cross-linking is determinant to assume conformations, which are not accessible to the linear fragments.

Antibody production using synthetic peptides has been investigated extensively to develop therapeutic antibodies and prophylactic vaccines. Previously, we reported that a complex of CpG-DNA and synthetic peptides corresponding to B cell epitopes, encapsulated in a phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS) complex, significantly enhanced the synthetic peptide-specific IgG production. Here, we describe synthetic peptide-based epitope screening and antibody production without conventional carriers.

MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were characterized in competitive ELISA experiments, and their solution secondary structure was studied by circular dichroism spectroscopy in water and in the ordered structure promoting trifluoroethanol. Our results show that glycosylation in position 19 (peptide (16) PTPT(GalNAcα)GTQ(22) ) resulted in enhanced antibody recognition and significantly altered secondary structure, while glycosylation in position 21 completely demolished the binding. These findings could be useful in determining the nature of antigen-antibody interaction, and perhaps designing synthetic peptide vaccines for tumor therapy., (© 2014 Wiley Periodicals, Inc.)

The translation of cell-based therapies for ischemic tissue repair remains limited by several factors, including poor cell survival and limited target site retention. Advances in nanotechnology enable the development of specifically designed delivery matrices to address these limitations and thereby improve the efficacy of cell-based therapies. Given the relevance of integrin signaling for cellular homeostasis, we developed an injectable, bioactive peptide-based nanofiber matrix that presents an integrin-binding epitope derived from fibronectin, and evaluated its feasibility as a supportive artificial matrix for bone marrow-derived pro-angiogenic cells (BMPACs) used as a therapy in ischemic tissue repair. Incubation of BMPACs with these peptide nanofibers in vitro significantly attenuated apoptosis while enhancing proliferation and adhesion. Pro-angiogenic function was enhanced, as cells readily formed tubes. These effects were, in part, mediated via p38, and p44/p42 MAP kinases, which are downstream pathways of focal adhesion kinase. In a murine model of hind limb ischemia, an intramuscular injection of BMPACs within this bioactive peptide nanofiber matrix resulted in greater retention of cells, enhanced capillary density, increased limb perfusion, reduced necrosis/amputation, and preserved function of the ischemic limb compared to treatment with cells alone. This self-assembling, bioactive peptide nanofiber matrix presenting an integrin-binding domain of fibronectin improves regenerative efficacy of cell-based strategies in ischemic tissue by enhancing cell survival, retention, and reparative functions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Bovine anaplasmosis is a hemoparasitic disease that causes considerable economic loss to the dairy and beef industries. Cattle immunized with the Anaplasma marginale MSP1 outer membrane protein complex presents a protective humoral immune response; however, its efficacy is variable. Immunodominant epitopes seem to be a key-limiting factor for the adaptive immunity. We have successfully demonstrated that critical motifs of the MSP1a functional epitope are essential for antibody recognition of infected animal sera, but its protective immunity is yet to be tested. We have evaluated two synthetic vaccine formulations against A. marginale, using epitope-based approach in mice. Mice infection with bovine anaplasmosis was demonstrated by qPCR analysis of erythrocytes after 15-day exposure. A proof-of-concept was obtained in this murine model, in which peptides conjugated to bovine serum albumin were used for immunization in three 15-day intervals by intraperitoneal injections before challenging with live bacteria. Blood samples were analyzed for the presence of specific IgG2a and IgG1 antibodies, as well as for the rickettsemia analysis. A panel containing the cytokines' transcriptional profile for innate and adaptive immune responses was carried out through qPCR. Immunized BALB/c mice challenged with A. marginale presented stable body weight, reduced number of infected erythrocytes, and no mortality; and among control groups mortality rates ranged from 15% to 29%. Additionally, vaccines have significantly induced higher IgG2a than IgG1 response, followed by increased expression of pro-inflammatory cytokines. This is a successful demonstration of epitope-based vaccines, and protection against anaplasmosis may be associated with elicitation of effector functions of humoral and cellular immune responses in murine model.

Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform (PrP(Sc)) of a normal cellular protein (PrP(C)). Shared sequence identity of PrP(Sc) with PrP(C) has limited the detection sensitivity of immunochemical assays, as antibodies specific for the disease-causing PrP(Sc) isoform have not been developed. Here we report the generation of three new monoclonal antibodies (MAbs) to PrP, which were isolated following immunization of Prnp(0/0) Balb/cJ mice with highly purified PrP(Sc) isolated from brain lipid rafts. Epitope mapping using synthetic PrP peptides revealed that the three MAbs bind different epitopes of PrP. The DRM1-31 MAb has a conformational epitope at the proposed binding site for the putative prion conversion co-factor "protein X." The DRM1-60 MAb binds a single linear epitope localized to the β2-α2 loop region of PrP, whereas DRM2-118 binds an epitope that includes sequences within the octarepeat region and near the site of N-terminal truncation of PrP(Sc) by proteinase K. Our novel anti-PrP MAbs with defined PrP epitopes may be useful in deciphering the conformational conversion of PrP(C) into PrP(Sc).

Paramyosin of the pig-human parasite Taenia solium (TPmy) is a α-helical protein located on the worm surface that is suggested to fulfill an immunomodulatory role protecting the parasite against host immune system. Besides, in challenging experiments the protein shows a vaccine potential. These observations imply that TPmy harbors antigenic determinants for each of these contrasting actions. However the suggestion was not given a support from experimental data because respective epitopes have not been described thus far. To circumvent this difficulty, we use synthetic peptides with sequences of regions composed of α-helical or linear structure to induce rabbit antibody responses for phage-display mapping of epitope core amino-acid sets. Antibodies to α-helical regions were weak binders and M13 phage-displayed peptides selected by them from two different libraries exhibited no amino-acid similarities with the original protein site. In contrast, the antibodies produced in response to non-helical segment within α-helical structure were better binders and selectors of perfect structural mimics of the protein site. This first phage display epitope analysis of TPmy supports the notion that the rod-like α-helix, which encompasses over 90% of the total amino acids, may serve as an immunomodulatory shield that protects the parasite. Further, the seven non-helical segments of the TPmy molecule may represent the only anti-parasite discrete immunogenic epitopes whose representative mimotopes can be utilized in development of pure epitope vaccines., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The occurrence of alloantibodies against Factor VIII (FVIII) is the main iatrogenic complication in haemophilia A (HA). Anti-FVIII autoantibodies may also spontaneously appear in non-HA patients, leading to acquired haemophilia A. In both contexts, the antibody response against FVIII is complex and difficult to analyse due to the lack of suitable tools. Our purpose was to comprehensively map, at the amino acid level, discontinuous epitopes of the C2 domain of FVIII targeted by patients' antibodies. We synthesized 33 synthetic peptides, which were predicted by the bioinformatic algorithm PEPOP to mimic C2 domain discontinuous epitopes. Using an inhibition assay based on the x-MAP technology, we evaluated their ability to block the binding to the C2 domain of anti-C2 domain antibodies from pooled plasma samples. Nine peptides were thus selected and tested again in individual plasma samples. Our results support the view that C2 domain epitopes are organized as an epitopic mosaic distributed around the molecule, showed that each patient displayed a specific anti-C2 epitopic profile, and confirmed the complexity and variability of the immune response against the C2 domain of FVIII. This ability to finely map epitopes could be further used to follow the antibody specificity modifications over time., (© 2011 Blackwell Publishing Ltd.)

Dendrimeric platforms such as MAPs can be synthesized either entirely by solid-phase methods (SPPS, direct approach) or by conjugation in solution of preformed, SPPS-made building blocks (indirect approach). Although MAPs and MAP-like constructs have been extensively and successfully used for various biological (mainly immunological) applications, experimental reports are most often lacking in chemical detail about their preparation and characterization. Here, we provide complete accounts of the synthesis and analytical documentation of MAPs and similar dendrimers by either all-SPPS (direct) or chemoselective thioether ligation (indirect) methods. We have chosen as model epitopes a 24-residue sequence of the ectodomain of protein M2 from influenza virus (M2e), which is found to be a rather challenging peptide epitope, and a far more manageable, shortened (12-residue) version of the same peptide. The advantages and shortcomings of both direct and indirect methods are discussed., (Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.)

Lymphatic filariasis is a tropical disease, with over 40 million people seriously incapacitated due to lymphangitis and elephantiasis. Over 99% of infections are caused by the nematode Wuchereria bancrofti. Expressed sequence tag (EST) analysis of filarial genome identified novel infective larval (L3) stage-specific antigen, abundant larval transcript (ALT-2), which was shown to be highly essential for parasite establishment and survival in the host. The unique structural features and immunological characteristics of ALT-2 indicate the presence of critical motifs that needs to be explored in natural human infection for understanding and management of the disease. In order to dissect the epitopes of ALT protein recognized in humans, eight peptides spanning the entire protein sequence were selected based on in-silico epitope prediction and synthesized. Analysis of the reactivity of W. bancrofti ALT-2 synthetic peptides with clinical sera (n = 40) from endemic areas identified epitopes recognized by putatively immune sera, of which two comprise the highly variable acidic domain (21-60). Interestingly, our study also revealed crucial epitopes recognized by microfilaremic (MF) sera with significantly high IgG4 isotype (p < 0.001), implicated in immunomodulation. The epitope peptides identified were highly specific for MF sera and, thus, have the potential to be exploited as diagnostic markers.

One of the main challenges of Pseudomonas aeruginosa vaccine development is the design of an antigen that elicits cross-reactive antibodies against multiple virulent strains. Using a rational design approach, we have developed a single 17-residue peptide immunogen that generates antibodies that target the receptor-binding domain of the type IV pilus of more than one strain of P. aeruginosa. Using the receptor-binding domain sequence, of native strain PAO as a template, we have systematically changed up to five residues in the PAO sequence of the peptide immunogen into that of the PAK sequence. We show by indirect and competitive ELISA that the mutant peptide immunogens elicit the development of polyclonal sera that is cross-reactive to both native strain PAO and PAK pilin. We further show that there are at least two separate antibody populations in the polyclonal sera that possess closely related epitopes but which are each strain specific. Moreover, part of the epitope for the PAO-specific antibodies consists of several residues outside the disulfide loop of the receptor-binding domain. This allows us to create two unique epitopes within the same receptor-binding domain sequence., (© 2010 John Wiley & Sons A/S.)

M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca(2+) concentrations [(Ca(2+) )i] were measured. Antibodies to the N-terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca(2+) )i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca(2+) )i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes., (© 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.)

The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or X4 phenotypes and use the CCR5 or CXCR4 chemokine coreceptor, respectively. Previous NMR studies revealed that both R5- and X4-V3 peptides bound to antibodies 0.5beta and 447-52D form beta-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3(JRFL) and V3(IIIB) peptides bound to 447-52D and to 0.5beta, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross-strand interactions suggestive of beta-hairpin-like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized beta-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity, and in one case, a structure close to that of V3(MN) peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3(IIIB) peptide bound to antibody 0.5beta was assumed.

Background: The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity., Results: In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31-121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding., Conclusion: Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.

The reactions of neutralizing monoclonal antibodies (mAbs) that defined dengue virus (DENV) complex, flavivirus subgroup or group neutralizing epitopes were tested against synthetic peptide sequences from domains I, II and III of the envelope (E) glycoproteins of different DENV-2 genotypes/strains. The DENV complex-reactive mAb identified the surface-exposed 304-GKFKV/IVKEIA-313 peptides and the DENV complex-conserved 393-KKGSSIGQ/KM-401 peptides in domain III, which were located adjacently in the native glycoprotein. Both flavivirus group-reactive mAbs reacted most strongly with fusion sequence peptides from domain II when they contained a cysteine (C) by glycine (G) substitution (underlined) (101-WGNGGGLFG-109) to represent the native rotated C side chain. The 393-401 sequence represents a newly identified epitope, present as a highly flexible coil located between the 385 and 393 cell-binding sequence and the 401 and 413 sequence involved in the E glycoprotein homo-trimer formation. The 101-109 sequence containing 105-C by G substitution and the 393-401 sequence are good candidates for diagnostic assays and cross-protection experiments.

The hantavirus nucleocapsid (N) protein fulfills several key roles in virus replication and assembly and is the major antigen in humoral immune responses in humans and mice. Here we report on epitopes involved in serotype-specific and cross-reactive recognition of the N proteins of hantaviruses using monoclonal antibodies (mAbs) against the N proteins of Andes virus (ANDV) and Sin Nombre virus (SNV). The mAbs define at least twelve different epitopic patterns which span eight sequences, including amino acids 17-59, 66-78, 79-91, 157-169, 222-234, 244-263, 274-286 and 326-338 on the SNV and ANDV N proteins. Studies on the cross-reactivity of these mAbs with different hantavirus N proteins indicated that epitopes located within amino acids 244-286 are related to serotype specificity. We analyzed further the location of epitopes with available three-dimensional structure information including the N-terminal coiled-coil and derived exposed and hidden residues of these epitopes. The generated recombinant N proteins and the characterized mAbs are functional tools being now available for hantavirus diagnostics and replication studies.

In this study we evaluate the uptake by murine dendritic cells (DCs) of different synthetic, branched cationic peptide structures with a view to facilitating peptide epitope delivery. The level of cell uptake by fluorescenated peptides was measured by flow cytometry following quenching of extracellular fluorescence with trypan blue. Branched peptides containing either N-terminal arginine or N-terminal lysine residues were able to mediate cell entry but the peptide containing four arginine residues in a branching configuration (R4) was found to be superior not only to other branched peptides in translocating to the cell interior and also to a peptide containing four arginine residues arranged linearly. Fluorescenated R4 was found to be localized within intracellular vesicle-like compartments as well as being distributed throughout the cell cytoplasm. Uptake of R4 utilized an energy-dependent process that appeared to involve phosphatidylinositol-3-kinase and could induce intermediate levels of DC maturation. R4 when conjugated to a T-helper cell and CTL epitope construct was able to induce antigen-specific CD8+ T-cell mediated immune responses in mice when administered in adjuvant as were DCs that were pulsed with this construct and then matured with LPS. Fluorescenated R4 was also found to translocate into the interior of other cell types indicating that it may be useful for the delivery of peptide cargo into other specialized cells.

Ric c 1 and Ric c 3 are the major castor bean allergens. In order to identify continuous IgE-epitopes in Ric c 1 and Ric c 3, pools of sera from rats immunized with a pool of 2S albumin from these seeds, Ric c 1 and Ric c 3 overlapping synthetic peptides, were used to screen for IgE-binding epitopes. The allergenic properties were monitored by mast cell degranulation assays, histamine quantification and human-IgE binding. Large and small chains isolated from these proteins present allergenic properties. Four continuous epitopes were identified in Ric c 3 and two in Ric c 1. This knowledge may allow the induction of protective antibody responses to antagonize the IgE recognition.

Fifty-six Newcastle disease virus strains collected from 2000 to 2006 could be grouped into subgenotype VIId. However, they displayed cumulative mutations in and around the linear epitope of hemagglutinin-neuraminidase (residues 345 to 353) with time. The antigenicities of the variants that became predominant in Korea differ from each other and from the wild type.

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8(+) T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

Previously, we prepared monoclonal antibodies (mAbs) by immunizing rats with the recombinant fusion proteins of mouse Langerin/CD207, which contained a flexible linker sequence from E. coli OmpF and a FLAG epitope. We found many of new rat mAbs were not reactive to mouse Langerin, and here we identify the epitopes of two of these IgG mAbs, L2 and L5, and assess their efficacy in various immunodetection methods. MAb L5 is a rat IgG mAb against the FLAG epitope, which detected both N-terminal and C-terminal FLAG tagged protein 2 to 8 times better than the conventional anti-FLAG mAb M2 by Western blot. For mAb L2, we found its epitope to be a 14 amino acid sequence SGFANELGPRLMGK which consisted of both sequences from the OmpF derived linker and mouse Langerin. This epitope sequence was named OLLAS (E. coliOmpF Linker and mouse Langerin fusion Sequence), and mAb L2 as mAb OLLA-2. When the OLLAS sequence was inserted into recombinant proteins at N-terminal, C-terminal, or internal sites, the OLLAS tag was detected by mAb OLLA-2 with very high sensitivity compared to other conventional epitope tags and anti-tag mAbs. MAb OLLA-2 recognized OLLAS tagged proteins with at least 100-fold more sensitivity than anti-FLAG M2 and anti-V5 mAbs in Western blot analyses. We also find the OLLAS epitope to be superior in immunoprecipitation and other immunodetection methods, such as fluorescent immunohistochemistry and flow cytometry. In the process, we successfully utilized the OLLAS epitope sequence as an internal linker for fusion between the engineered mAb and the antigen, and thus achieved improved immunodetection.

Background: The delineation of allergenic (i.e. IgE-binding) epitopes in cow's milk proteins and the amino acids (AAs) critical for IgE-binding is necessary to understand better the structural properties of an allergen and to develop more efficacious immunotherapeutic reagents. Furthermore, this information may enable us to understand better cross-sensitivity between different allergens., Methods: Eleven peptides, 10-14 AAs in length, representing the IgE-binding epitopes of kappa-casein were synthesized on a derivatized cellulose membrane with single AA substitutions at each position. Membranes were incubated with pooled sera from 15 milk-allergic patients and individual sera from 10 of the patients included in the pool., Results: For 10/11 allergenic peptides, one to five different single AA substitutions resulted in elimination of IgE-binding of pooled patient sera. Overall at least one mutated peptide could be found for these 10 IgE-binding sites that resulted in a reduction of IgE-binding in at least 80% of the patients who recognized the native protein. Furthermore, the IgE-binding region at AA104-112 on bovine kappa-casein showed a high degree of similarity with the human kappa-casein, respectively, including the AAs critical for IgE-binding., Conclusion: This finding suggests that critical AAs should be assessed with both pooled and individual patient sera to account for the B-cell epitope heterogeneity between patients, with cow's milk allergy. In addition, we identified two potentially cross-reactive peptides between bovine and human caseins of unknown clinical relevance.

Aim: To synthesize B cell dominant epitopes on N-terminal part of bactericidal/permeability increasing protein (BPI) and prepare the corresponding antisera., Methods: The antigenicity, hydrophilicity, flexibility, surface probability and secondary structure of N-terminal amino acids 1-199 on BPI were predicted by bioinformatics applications. Two antigen peptides TA/IK were designed and synthesized on the basis of the above analysis. Then the TA/IK were respectively conjugated to keyhole limpet hemocyanin (KLH) and injected into rabbits to prepare corresponding antisera. Indirect ELISA was performed to analyze the antigenicity of TA/IK and to test the titer of the antisera. And Western blot was used to identify the specificity of the antisera., Results: (1) Two B cell epitope-based peptides TA/IK were successfully synthesized; (2) the peptides could bind to commercial polyclonal antibody, anti-BPI(55); (3) titers of the antisera against TA/IK were up to 1:51,200, 1:25,600, respectively; (4) Western blot analysis revealed that these antisera could specifically react with the standard sample of BPI(55)., Conclusion: The two synthetic antigen peptides TA/IK are indeed dominant epitopes of BPI N-terminal part, and the corresponding antisera are competent for detecting and identifying the N-terminal fragments of BPI.

Virus-specific neutralizing antibodies present an obstacle to the effective use of adenovirus vectors for gene therapy and vaccination. The specific sites recognized by neutralizing antibodies have not been identified for any adenovirus, but they have been proposed to reside within the hexon, in small regions of the molecule that are exposed on the capsid surface and possess sequences that vary among serotypes. We have mapped the epitopes recognized by a panel of seven hexon-specific monoclonal antibodies that neutralize the chimpanzee adenovirus 68 (AdC68). Surface plasmon resonance experiments revealed that the antibodies compete for a single hexon binding site, and experiments with synthetic peptides indicated that this site resides within just one small surface loop. Mutations within this loop (but not in other surface loops) permitted virus to escape neutralization by all seven monoclonal antibodies and to resist neutralization by polyclonal antisera obtained from animals immunized against AdC68. These results indicate that a single small surface loop defines a major neutralization site for AdC68 hexon.

For reliable diagnosis of porcine teschovirus (PTV) infection we created an RT-PCR-based molecular strategy for serotyping that encompassed the dominant neutralizing antigenic site of PTV, followed by phylogenetic analyses of amplicons. We identified neutralizing antigenic sites of PTV-1 Talfan strain through epitope mapping of neutralizing monoclonal antibodies (MAbs), using synthetic peptides spanning the capsid proteins. All 11 MAbs obtained recognized peptides in the EF loop ("puff") of VP2 protein. Two MAbs concurrently reacted to peptides, one in the GH loop of VP1 and one in the VP1 C terminus. Three-dimensional modeling of Talfan capsid protein predicted exposure of all these sites on the virion surface in a close line centered around puff. We then designed a single pair of degenerate primers to VP2 and amplified the region of approximately 320 bp encompassing puff in 8 PTV prototype strains and 6 field isolates. Phylogenetic analyses of the puff sequences of 11 prototype strains and 34 field isolates obtained from databanks showed that all homotypic strains (both field and prototype) were always monophyletic, except for one 'untypable' Japanese strain. This RT-PCR-based strategy appears to be a reliable surrogate for serotyping and could facilitate the diagnosis and epidemiological study of PTV infection.

The T-cell receptor of a CD8(+) T-cell recognises peptide epitopes bound by class I major histocompatibility complex (MHC) glycoproteins presented in a groove on their upper surface. Within the groove of the MHC molecule are 6 pockets, two of which mostly display a high degree of specificity for binding amino acids capable of making conserved and energetically favourable contacts with the MHC. One type of MHC molecule, HLA-B*2705, preferentially binds peptides containing an arginine at position 2. In an effort to increase the affinity of peptides for HLA-B*2705, potentially leading to better immune responses to such a peptide, we synthesised two modified epitopes where the amino acid at position 2 involved in anchoring the peptide to the class I molecule was replaced with the alpha-methylated beta,gamma-unsaturated arginine analogue 2-(S)-amino-5-guanidino-2-methyl-pent-3-enoic acid. The latter was prepared via a multi-step synthetic sequence, starting from alpha-methyl serine, and incorporated into dipeptides which were fragment-coupled to resin-bound heptameric peptides yielding the target nonameric sequences. Biological characterisation indicated that the modified peptides were poorer than the native peptides at stabilising empty class I MHC complexes, and cells sensitised with these peptides were not recognised as well by cognate CD8(+) T-cells, where available, compared to those sensitised with the native peptide. We suggest that the modifications made to the peptide have decreased its ability to bind to the peptide binding groove of HLA-B*2705 molecules which may explain the decrease in recognition by cytotoxic T-cells when compared to the native peptide.

The outer surface protein C (OspC) of the Lyme disease agent, Borrelia burgdorferi, is an immunoprotective antigen in laboratory models of infection. However, to understand its protective effects, it is important to identify the key epitopes of this protein. We produced a borreliacidal anti-OspC monoclonal antibody specific to the B31 strain and identified its binding site. The specificity of MAb 16.22 was determined by Western blot reactivity using OspC derived from different Borrelia isolates which had varying amino acid sequences. Comparison of the OspC sequences and binding data suggested that MAb 16.22 binds to amino acids 133-147 of the OspC protein. To test this hypothesis, we synthesized a 15-amino acid peptide containing the target sequence and, using competition enzyme-linked immunosorbent assay (ELISA), we found that this peptide included the epitope of MAb 16.22. In addition, we determined that MAb 16.22 is able to kill of B. burgdorferi in a complement-independent fashion.

The preparation of three different 2-alkoxy-8-hydroxyadenylpeptide conjugates has been accomplished by solid-phase synthesis combined with 'on-resin' Cu(I) catalyzed Huisgen cycloaddition. The immunogenicity of the compounds has been evaluated in IL-12 production and antigen presentation assays.

CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in the immune control of Hepatitis C Virus (HCV) infection. In the current study, a number of HLA-A*0201-restricted CTL epitopes derived from HCV were evaluated by examining the peptide-binding affinity for major histocompatibility complex (MHC) class I molecules, the stability of peptide-MHC complexes, killing activities of peptide-induced CTLs, and frequencies of intracellular interferon (IFN)-gamma-positive CD8+ T cells. Among 24 peptides tested, 15 peptides induced high or medium killing activities of peptide-specific CTLs. Thirteen of the 15 peptides exhibited high or medium binding affinities for HLA-A*0201 molecules, indicating that the high binding affinity for MHC class I molecules is an important factor for immunogenicity. In contrast, the stability of peptide-MHC class I complexes was not correlated with killing activities of peptide-induced CTLs. Furthermore, only a limited number of peptides could induce high or medium frequencies of IFN-gamma-producing CD8+ T cells, which were generally considered to play a crucial role for the clearance of HCV. Analyses of the immunogenicity of CTL epitopes such as in the current study should provide important information about the design of an efficient HCV vaccine that induces vigorous, sustained, and broad HCV-specific CTL responses.

A LigandFit shape-directed docking methodology was used to identify the best position at which the melanoma-derived MHC class-I HLA-A2-binding antigenic peptide ELAGIGILTV could be modified by attaching a small molecule capable of fitting at the interface of complementary determining regional (CDR) loops of a T-cell receptor (TCR) while triggering T-cell responses. The small molecule selected here for determining the feasibility of this alternative track to chemical alteration of antigenic peptides was the electrophilic quinone methide (+)-puupehenone (), a natural product that belongs to a family of marine metabolites capable of expressing immunomodulatory activities. A preliminary chemical reactivity model study revealed the efficacy of the thiol group of a cysteine (C) side-chain in its nucleophilic addition reaction with in a regio- and diastereoselective manner. The best TCR/HLA-A2 ligand [i.e., ELAGCGILTV-S-puupehenol ()] then identified by the LigandFit docking procedure was synthesized and used to pulse HLA-A2(+) T2 cells for T-cell stimulation. Among the ELAGIGILTV-specific T-cell clones we tested, five of them recognized the conjugate in spite of its low binding affinity for the HLA-A2 molecules. The resulting T-cell stimulation was determined through the intracytoplasmic secretion of IFN-gamma and the percentage of T-cells thus activated. These highly encouraging results indicate that small non-peptidic natural product-derived molecules attached onto the central part of an antigenic peptide can fit at the TCR/HLA-A2 interface with induction of T-cell responses.

Phage display is a powerful method for selecting peptides with novel binding functions. Synthetic peptidomimetic chemistry is a powerful tool for creating structural diversity in ligands as a means to establish structure-activity relationships. Here we illustrate a method of bridging these two methodologies, by starting with a disulfide bridged phage display peptide which binds a human antibody Fc fragment (Delano et al. Science 2000, 287, 1279) and creating a backbone cyclic beta-hairpin peptidomimetic with 80-fold higher affinity for the Fc domain. The peptidomimetic is shown to adopt a well-defined beta-hairpin conformation in aqueous solution, with a bulge in one beta-strand, as seen in the crystal structure of the phage peptide bound to the Fc domain. The higher binding affinity of the peptidomimetic presumably reflects the effect of constraining the free ligand into the conformation required for binding, thus highlighting in this example the influence that ligand flexibility has on the binding energy. Since phage display peptides against a wide variety of different proteins are now accessible, this approach to synthetic ligand design might be applied to many other medicinally and biotechnologically interesting target proteins.

As a prelude to development of broader-spectrum vaccines for dental caries, we explored the immune potential of constructs combining epitopes from mutans streptococcal glucosyltransferases (GTF) and glucan binding protein B (GbpB). Two diepitopic peptide constructs were synthesized in a multiple antigenic peptide (MAP) format. Both constructs contained SYI, a 20-mer GbpB peptide that included a sequence having major histocompatibility complex class II binding characteristics. One diepitopic construct (SYI-CAT) also contained a 22-mer sequence from the catalytic domain of GTF. Another diepitopic construct (SYI-GLU) contained a 22-mer sequence from the glucan binding domain of GTF. To assess the ability of each construct to induce antibody reactive with GbpB and GTF native proteins, rats were injected subcutaneously with SYI-CAT, SYI-GLU, or the constituent monoepitopic constructs. Only the SYI-CAT construct induced significant levels of serum immunoglobulin G (IgG) and IgA antibody to both pathogenesis-associated proteins. Also, immunization with SYI-CAT significantly (P < 0.001) enhanced the antibody response to the CAT peptide. Experiments then compared experimental dental caries after immunization with SYI-CAT, SYI, or CAT MAP constructs, followed by infection with Streptococcus mutans strain SJr. Dental caries were lower in each peptide-immunized group than in the sham-injected group. The level of protection after SYI-CAT immunization was similar to that after immunization with constituent MAP constructs. In another experiment, rats were infected with Streptococcus sobrinus strain 6715 under an identical protocol. Significant protection was observed on buccal surfaces in both SYI-CAT and CAT construct-immunized, but not in the SYI construct-immunized, groups. Thus, addition of the GbpB-derived SYI peptide to the GTF-derived CAT peptide construct not only enhanced the immunological response to CAT and GTF epitopes, but also extended the protective effect of the construct to include both S. mutans and S. sobrinus.

Aim: To prepare and characterize anti-human beta-Netrin antibodies., Methods: B cell dominant epitopes of human beta-Netrin C-terminal 114 amino acid sequences were predicated by the GoldKey software. One of the epitopes was synthesized and coupled with bovine serum album (BSA) by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The BALB/c mice were immunized with the coupled protein. The splenocytes of immunized mice were fused with Sp2/0 cells by routine method and the hybridomas were selected in HAT medium. The hybridoma cells secreting specific antibody were detected by ELISA and cloned by limiting dilution. The titer specificity, and Ig subclass of anti-beta-Netrin mAbs were characterized by ELISA, Western blot and immunocytochemical staining. In addition, New Zealand rabbits were immunized with the coupled protein to prepare polyclonal antibody against beta-Netrin. The specificity of the antiserum was verified by Western blot., Results: A 16-mer peptide NH2-FRGKRTLYPES-WTDRG-COOH was the dominant epitope of the B cells. Synthesized peptide coupled with BSA was used as the immunogen to immunize BALB/c mice. Three hybridoma cell lines that stably secrete specific mAbs were obtained. The result of immunocytochemical staining showed that prepared mAb specifically recognize the antigen in the neuronal cells. The polyclonal antibody against beta-Netrin had high specificity. Western blot analysis showed that the antiserum bind with the prokaryotically expressed beta-Netrin specifically., Conclusion: Using the synthesized peptides as hapten, we have prepared epitope-specific mAbs and pAb against beta-Netrin successfully.

Aim: To develop a new method for antibody detection based on fluorescence polarization (FP) technique., Methods: BALB/c mice were immunized with single epitope synthetic 8 branches peptide antigen (SVEVGKVADL)8 of Helicobacter pylori (Hp) UreB protein. FP values of the corresponding linear peptide antigen labeled with FITC in different concentration were measured to determine the optimal concentration of the antigen. The antigenicity of the synthetic linear peptide was identified by FP assay. Then, the antibody-positive and negative murine sera were used in FP assay to determine the optimal dilution factor of serum samples. To apply FP technique in Hp antibody detection, 126 human serum samples were detected either by FPIA (fluorescence polarized immunoassay) using the single epitope linear synthetic peptide as antigen or by a commercial ELISA kit. Then, receiver operating characteristic (ROC) curve analysis was performed on the FPIA results by MedCalc software., Results: The UreB single epitope peptide had strong antigenicity. 1.0 nmol/L of the synthetic linear peptide labeled with FITC was the optimal concentration of the antigen and the optimal dilution factor of serum sample was 1:25. In 77 Hp antibody-positive serum samples detected by ELISA, 66 samples were positive by FP detection method. The sensitivity and specificity of the FPIA assay was 85.7% and 98%, respectively., Conclusion: The single epitope synthetic peptide antigen can be used in FP method for rapid detection of serum antibody against Hp UreB. The established FP assay for antibody detection may be used in clinical diagnosis in the future.

Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal beta-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation.

Peptide based vaccines offer practical advantages, but unmodified peptides usually require an adjuvant or delivery vehicle to promote immunogenicity. When peptides containing ovalbumin (OVA) derived CD4 and CD8 T cell epitopes were conjugated to 0.05 microm nano-beads, they gave strong immune responses and inhibition of growth of tumour cells expressing the CD8 T cell epitope with MHC class I. These responses were inducible with both high (50 microg) and low (5 microg) peptide doses after a single immunisation. The helper CD4 T cell epitope was unnecessary for induction of CD8 T cell or tumour challenge responses. However, the CD4 T cell epitope contained a B cell epitope and triggered strong antibody responses. This simple approach offers a convenient experimental tool and a potentially useful clinical method for peptide immunisation.