Academic literature on the topic 'Discontinuous epitope'

Although discontinuous epitopes make up 90% of total number of B-cell epitopes, however, because of difficulties in the development of method for their prediction, most of the B-cell epitope prediction methods today focus on continuous epitopes. To serve for the development of vaccine against H5N1 virus, we have been studying on in silico prediction of T- and B-cell continuous as well as B-cell discontinuous epitopes on H5N1 viral antigens. In this study, using the homology modeling method, we have generated structures of matrix protein of the H5N1 virus and predicted B-cell discontinuous epitopes. 60 out of 72 predicted residues were similar with those reported by the CEP method (Conformational Epitope Prediction). All predicted aminoacid residues were hydrophilic, polar, electrically charged and located on the surface of the antigen structures.

Abstract Abstract 1120 Context: The development of alloantibodies (AlloAbs) directed against the factor VIII (FVIII) is actually the main iatrogenic complication in hemophilia A (HA). Immune tolerance induction (ITI) is the only validated treatment to eradicate inhibitors. ITI is based on the infusion of high doses of therapeutic FVIII. It has been recently suggested that epitope specificity during ITI could be related to the outcome of this therapy. Anti-FVIII immune response is polyclonal, complex, dynamic over time and preferentially directed against C2 and A2 domains of FVIII. Tools for fine epitope mapping are useful to identify new epitopes and follow the over time evolution of epitope specificity. The aim of our work was to identify new epitopes on FVIII A2 domain using computer-designed peptides mimicking discontinuous epitopes. We used a multiplex assay based on Luminex™ technology to select the most reactive peptide (1). Patients and methods: All the peptides were predicted by a bioinformatical tool named PEPOP using a new protocol of prediction. Starting from the tridimensional structure of the FVIII, PEPOP were able to work out the sequence of 40 peptides mimicking discontinuous epitopes distributed on the A2 domain surface. Seven negative control peptides were predicted in unaccessible solvant area. A previously published peptide mimicking a linear epitope (residues 484–508) were also synthetized and tested (2). The capacity of peptides to block the binding of the anti-A2 inhibitors on beads coated with the A2 domain obtained by thrombin cleavage of FVIII was assessed. This multiplexed inhibition assay were based on Luminex™ technology. We used a pool of 10 plasma with anti-A2 domain inhibitors as a source of antibodies. Results: The inhibition assays realized with pool of anti-A2 antibodies made it possible to identify 2 peptides mimicking discontinuous epitopes on the A2 domain. The inhibition rate of the two new identified peptides reaches to 50% and 26% at the maximal concentration. We also confirm that the previously published peptide is able to block the binding of anti-A2 inhibitors in our population. The localization of the discontinuous epitopes mimicked by peptides within the tridimensional structure of the A2 domain showed than these epitopes are only represented on a restricted area. Conclusion: We identified two peptides mimicking new discontinuous epitopes on the A2 domain and predicted by a new algorithm of PEPOP. According to the literature, immune response against A2 domain seems to be more restricted. Several residues involved in the new identified discontinuous epitopes are crucial for thrombin, protein S, FIXa and low-density lipoprotein receptor-related protein (LRP) interactions. Tools for fine epitope mapping are essential to identify epitopes on FVIII domains, with a particular concern for discontinuous epitopes. Further studies are needed to validate the concept that fine epitope mapping at the epitope level could be useful to predict ITI outcome. Disclosures: No relevant conflicts of interest to declare.

To develop vaccine with stable efficiency against easily transforming virus such as influenza A/H5N1 virus, bioinformatic tools have been used to predict conserved epitopes from viral antigens to be used as materials for the development of polyvalent vaccine against this virus. Using this approach, we have successfully predicted one discontinuous B-cell epitope, a peptide of 14 aminoacids on conserved domains of HA antigen from H5N1 influenza A virus. In this study, we report results of the preparation of this discontinuous B-cell epitope as the recombinant fusion form with H:1,2 flagellin from Salmonella typhimurium to increase the immunogenicity of this epitope using genetic manipulating techniques with Escherichia coli as host cell. The immunogenicity of the chemically synthesized predicted epitope and the recombinant epitope was examined by immunization of each of these epitopes to mice. Using hemagglutination inhibition (HI) method, we successfully demonstrated that both anti-sera from mice immunized either with chemically synthesized peptide or with recombinant flagellin H:1,2 fused epitope could recognize and inhibit the agglutination of inactivated influenza H5N1 virus strains, such as A/H5N1/ Chicken13/ Vietnam/LA/2006 and A/Vietnam/CL26/2004 (H5N1) isolated from infected chicken and human in Vietnam.

Cancer is one of the most lethal diseases. Recently, cancer immunotherapy has a tremendous achievement in cancer treatment. A certain number of cancer based epitope vaccines with different moiety have been discovered. In japan, several clinical tests of cancer based epitope vaccine derived from tumor associated antigens (TAAs) are now ongoing or have recently been completed. a novel of TAAs potentially as cancer vaccine have been retrieved from a fragment weighed 48kDa derived from human DNA-topoisomerase 1 (TOP1) called Topo48. Therefore, it is still critical to discover a derived Topo48 epitope based cancer vaccine. Immuno-informatics considered as a methods noted to have better accuracy to design promising vaccine candidates. Here, continuous and discontinuous B-cell epitopes following with CTL epitopes and their docking interaction to major histocompatibility complex (MHC) class I Human Leukocyte Antigens (HLA)- A0201 were predicted. Kolaskar-Tongaonkar’s, Emini’s, Karpus-Schulz’s, and Parker’s methods were used to predict continuous B-cell epitopes while ElliPro was used for prediction of discontinued B-cell epitopes. Those considered methods marked to have better accuracy to design promising vaccine candidates. Similarly, CTL epitopes was also predicted by using NetCTL server and the best candidates were further investigated their binding affinity by mean of PEP-FOLD3, PatchDock rigid-body docking server, and FireDock server. Total 27 continuous epitopes and 7 discontinuous B-cell epitopes were predicted. In the other hand, 9 peptides were predicted as CTL epitopes. Whereas, three predicted CTL epitope in range 263MLDHEYTTK27, 755AIDMADEDY763, 715ALGTSKLNY724) exhibited good interactions to HLA-A0201. Moreover, we also found residues His266, Thr270, Ala755, Tyr723, Thr718, Ser719, Lys720 from Topo48 and residues Thr163, Asp757, His70, Glu63 from HLA- A0201 were indicated to be antigenic. Ultimately, our proposed continuous/discontinuous B-cell epitopes, and also CTL epitopes can be potential vaccines for cancer immunotherapy.

The hormone human chorionic gonadotrophin (hCG) shows extensive sequence homology with LH. Thus, many of the antigenic epitopes on hCG are shared with LH, leading to immunological cross-reaction between these two hormones. Anti-fertility and anti-cancer vaccines based upon hCG should ideally target only the hCG-specific epitopes. The hCG-unique linear epitopes located in the C-terminal peptide of the hCG beta-chain are well characterised. In contrast, the hCG-specific discontinuous epitopes, termed beta1, beta6 and beta7, have remained poorly defined. By generating hCG beta-chain molecules containing single amino acid substitutions we have identified R10, N13, R60 and Q89 as being important in the formation of the beta1 epitope, with R60 providing a particularly dominant residue. We also show that the amino acid residue Q89 contributes to the beta7 epitope, whilst D61 plays a role in both the beta6 and beta7 epitopes.

ABSTRACT We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.

In pregnant women, Plasmodium falciparum-infected red blood cells adhere to the placenta via the parasite protein VAR2CSA. Two vaccine candidates based on VAR2CSA are currently in clinical trials; however, these candidates failed to elicit strain-transcending antibody responses. We previously showed that a cross-reactive monoclonal antibody (3D10) raised against the P. vivax antigen PvDBP targets epitopes in VAR2CSA. We now aim to design a peptide vaccine against VAR2CSA based on the epitope that generated 3D10. We mapped the epitope to subdomain 1 (SD1) of PvDBP and identified a peptide that contained the minimal sequence. However, this peptide did not elicit cross-reactive VAR2CSA antibodies in mice. When tested against a broader, overlapping peptide array spanning SD1, 3D10 in fact recognized a discontinuous epitope consisting of three segments of SD1. These findings presented the challenge to generate this larger structural epitope as a synthetic peptide since it is stabilized by two pairs of disulfide bonds. We overcame this using a synthetic scaffold to conformationally constrain the SD1 peptide and coupled it to keyhole limpet hemocyanin (KLH). The SD1-KLH conjugate elicited antibodies in mice that cross-reacted with VAR2CSA. This strategy successfully recapitulated a discontinuous epitope with a synthetic peptide and represents the first heterologous vaccine candidate against VAR2CSA.

ABSTRACT We have characterized a conformational epitope on capsids of hepatitis B virus (HBV) by cryo-electron microscopy and three-dimensional image reconstruction of Fab-labeled capsids to ∼10-Å resolution, combined with molecular modeling. The epitope straddles the interface between two adjacent subunits and is discontinuous, consisting of five peptides—two on one subunit and three on its neighbor. Together, the two icosahedral forms of the HBV capsid—T=3 and T=4 particles—present seven quasiequivalent variants of the epitope. Of these, only three bind this Fab. Occupancy ranges from ∼100 to ∼0%, reflecting conformational variations in the epitope and steric blocking effects. In the former, small shifts of the component peptides have large effects on binding affinity. This approach appears to hold general promise for elucidating conformational epitopes of HBV and other viruses, including those of neutralizing and diagnostic significance.

Epitopes involved in a protective immune response to Hendra virus (HeV) (Henipavirus, Paramxyoviridae) were investigated by generating five neutralizing monoclonal antibodies (mAbs) to the virus attachment protein (G) of HeV (HeV G) and sequencing of the G gene of groups of neutralization-escape variants selected with each mAb. Amino acid substitutions occurred at eight distinct sites on HeV G. Relationships between these sites were investigated in binding and neutralization assays using heterologous combinations of variants and mAbs. The sites were also mapped to a proposed structural model for the attachment proteins of Paramyxoviridae. Their specific locations and the nature of their interactions with the mAb panel provided the first functional evidence that HeV G in fact resembled the proposed structure. Four sites (aa 183–185, 417, 447 and 570) contributed to a major discontinuous epitope, on the base of the globular head, that was similar to immunodominant virus neutralization sites found in other paramyxoviruses. Amino acid similarity between HeV and Nipah virus was relatively highly conserved at these sites but decreased significantly at the other sites identified in this study. These included another discontinuous epitope on the base of the head region defined by sites aa 289 and 324 and well separated epitopes on the top of the head at sites aa 191–195 and 385–356. The latter epitope corresponded to immunodominant neutralization sites found in Rinderpest virus and Measles virus.

The synthesis of IH1, a peptide designed to mimic a discontinuous epitope on HIV-I gp120, is reported. The aspartimide rearrangement inherent in this sequence, and in the peptide GC1, has been studied and reduced to low levels. The syntheses of variants of peptide GC1, containing differing number of residues found to be important for CD4 binding, have also been achieved. Thus peptides containing one, two, three and four residues necessary for high affinity binding have been synthesised. In addition a peptide has been synthesised which incorporates a synthetic β turn moiety other than the Cys-Val-Cys bridge present in GC1. Polyclonal sera raised to these peptides and their CD4 binding properties have been investigated. IH1, the peptide containing four residues responsible for high affinity binding to CD4, has also been shown to interact with receptors on the surface of CD4^- cells. This non-CD4 recognition has been investigated utilising a photolabelled chemokine, MIP-1α. Results indicate that this binding involves interaction with CC-CKR5, a MIP-1α binding site thought to be involved in HIV-cell fusion.

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Member of the Herpesviridae family, subfamily Alphaherpesvirinae, gender Varicellovirus, the bovine herpesvirus 1 (BoHV-1) has been associated with different clinical conditions (respiratory and genital/reproductive diseases) in cattle. There is no standard procedure to control or prevent infections caused by herpesviruses. In this sense, phage display was used to select new glycoprotein mimotopes antigen of BoHV-1 that has potential for use in vaccines and diagnostics. The phage display technique was performed using a linear random peptide library consisting of 12 amino acid residues fused to the protein III of M13 phage (no peptide) against BoHV-1 specific IgGs, purified by affinity chromatography. After three cycles of selection (biopanning) and amplification, 44 clones were isolated and their amino acid sequences were determined by sequencing generating 16 different sequences. ELISA, demonstrating the efficiency of selection from the specific antibodies, confirmed the reactivity of pooled clones. Another ELISA evaluated the individual specificity of the most frequent clones, the M13 phage was used as a negative control. We selected three peptides (B, C and E) with affinity for anti-BoHV-1 antibodies, and the E peptide (pepE), showed to have potential as antigen for antibody detection in a serological test for BoHV-1. Immunization of rabbits with the peptides induced specific production of serum antibodies, but they were not able do neutralize BoHV-1 cell lysis. The in silico analysis of the dodecapeptide E (1DRALYGPTVIDH12) enabled the identification of a new discontinuous epitope on the envelope glycoprotein B (gB Env) of bovine herpesvirus type 1 (BoHV-1). There is a short motif (338YKRD341) within a region of the env gB BoHV-1 with high similarity to motifs shared by dodecapeptide the N-terminal region (5YxARD1) of gB and HSV-1 (326YARD329), wherein the 328Arg residue is described as a target for neutralizing monoclonal antibodies (mAb) for HSV-1 gB. Besides the characterization of an antibody-binding site of the BoHV-1 Env gB, we have demonstrated that the phage-fused peptide has potential use as a reagent for virus diagnosis by phage-ELISA assay, discriminating BoHV-1 positive serum samples from negative ones.
Membro da família Herpesviridae, subfamília Alphaherpesvirinae, gênero Varicellovirus, o herpesvírus bovino 1 (BoHV-1) tem sido associado a diferentes condições clínicas em bovinos (doenças respiratórias, genitais e falhas reprodutivas). Não existe um procedimento padrão para medidas de controle e profilaxia das infecções causadas por herpesvírus. Nesse sentido, o phage display foi utilizado com o objetivo de selecionar novos antígenos mimetopos de glicoproteínas do herpesvírus bovino 1 (BoHV-1) e que apresentam potencial para uso em vacinas e diagnóstico. A técnica de phage display foi realizada com a utilização de uma biblioteca de peptídeos randômicos e lineares composta de 12 resíduos de aminoácidos fusionada à pIII de fagos M13 (sem peptídeo), contra anticorpos anti-BoHV-1, purificados em coluna de cromatografia por afinidade. Após três ciclos de seleção (biopanning) e amplificação, 44 clones foram isolados e as sequências de aminoácidos dos peptídeos foram determinadas pelo sequenciamento gerando 16 sequencias diferentes. A reatividade do pool de clones foi confirmada por ELISA, demonstrando a eficiência da seleção a partir dos anticorpos específicos. Para avaliação da especificidade individual, realizou-se o ELISA dos clones mais frequentes, tendo como controle negativo o fago M13. Foram selecionados três peptídeos (B, C e E) com afinidade por anticorpos anti-BoHV-1, e um destes, o peptídeo E (pepE), apresentou potencial antigênico na detecção de anticorpos para o diagnóstico sorológico do BoHV-1. Nos testes de imunização em coelhos, os três peptídeos induziram a produção de anticorpos específicos, porém, estes não foram capazes de neutralizar a lise celular ocasionada pelo BoHV-1 em placa. A análise in silico do dodecapeptídeo E (1DRALYGPTVIDH12) possibilitou a identificação de um novo epitopo descontínuo na glicoproteína B de envelope (Env gB) do BoHV-1. Há um curto motivo (338YKRD341) dentro de uma região do gene Env gB do BoHV-1, com alta similaridade com os motivos compartilhados pelo dodecapeptídio da região N-terminal (5YxARD1) da gB e do Herpesvirus Humano 1 (HSV-1) (326YARD329), em que o resíduo 328Arg é descrito como um alvo para anticorpos monoclonais neutralizantes (mAb) para a gB do HSV-1. Concluindo, além da caracterização de um sítio de ligação ao anticorpo na Env gB do BoHV-1, o pepE expresso pelo fago tem potencial de utilização como reagente para o diagnóstico virológico por ensaio ELISA-fago, que discrimina amostras de soro positivas e negativas para o BoHV-1.

Among the genetically encoded amino acid residues, methionine (Met) and cysteine (Cys) are special because they each contain an atom of sulphur. The present chapter describes how these residues are incorporated into peptides in the context of an Fmoc/tBu solid-phase synthesis strategy, as well as further considerations once the synthetic peptide is released from the support. Of added interest, some manipulations of Cys are advantageously performed at the level of the assembled peptide-resin, prior to cleavage. Many of the aspects discussed here also carry over to the preparation of peptides using a Boc/Bzl strategy. The major problems associated with management of Met reflect the susceptibility of the thioether to alkylation and oxidation. One of the merits of the Fmoc/tBu strategy, in contrast to Boc/Bzl, is that in the former strategy Met is usually introduced without recourse to a protecting group for the thioether side-chain. As documented in this chapter, a proper understanding of acidolytic cleavage conditions and the availability of selective procedures to reverse any inadvertent oxidation are likely to lead to success in obtaining homogeneous peptides containing Met. Management of Cys provides additional significant challenges. For some targets, Cys is required with its side-chain in the free thiol form, whereas for other targets, an even number of Cys residues pair with each other via disulphide linkage(s) to provide cystine residue(s). Disulphide bridges play an important role in the folding and structural stabilization of many natural peptides and proteins, and their artificial introduction into natural or designed peptides is a useful approach to improve biological activities/specificities and stabilities. Furthermore, use of a disulphide bridge is a preferred method to conjugate peptides to protein carriers for increasing the response in immuno-logical studies, to link two separate chains for developing discontinuous epitopes, and to generate active site models. This chapter describes Cys protecting groups, how they are removed to provide either free thiols or disulphides directly, and various strategies and practical considerations to minimize side reactions and maximize formation of the desired products. The thioether side-chain of Met is subject to alkylation and oxidation side reactions, either during the synthetic process or during subsequent handling of the Met-containing peptide.

Five individuals from a single kindred and 3 other unrelated patients with type IID vWD have been studied. Multimeric analysis patterns obtained using discontinuous gel electrophoresis were identical both in major and minor band positions and distribution. In 2 where lysed platelets were studied, platelet and plasma multimeric analysis patterns were identical. EDTA plasma when compared with citrate plasma had more intense bands but there was no increase in intermediate or high molecular weight multimers. When fresh, frozen and thawed plasma or plasma left at 4°C for 24 hours were studied using multimeric analysis with autoradiography using 2 monoclonals (ESVWF 2 and 10) against vWF epitopes, significantly more intense autoradiographs were obtained with non-fresh rather than fresh plasma. These results suggest that freezing and thawing or storing plasma initiate a conformational change with the exposure of more epitope sites in these variants.Despite bleeding times in excess of 20 minutes ristocetin and botrocetin induced platelet aggregation and ristocetin and botrocetin cofactor assays were normal on fresh plasma. Both cofactor activities fell by 25-30% when fresh plasma was frozen and thawed once, whereas vWF:Ag fell by 20%.These results suggest that in IID vWD vWF:Ag is unstable and easily undergoes conformational change with loss of ristocetin and botrocetin cofactor activities and exposure of increased numbers of some epitopes.

Two new murine monoclonal antibodies to human vonWitlebrand factor (vWF), designated as SZ - 29 (IgG2a) and SZ-34 (IgG1), were utilized for observing the multimeric structure of vWF. These two monoclonal antibodies reacted specifically with vWF whilethey had no effects on fibringen, fibronectin and thrombospondin. In S DS-agarose electrophoresis and autoradiography experiments SZ _ 29 and SZ-34 were found binding to all multimers of vWF. 5mg of purified monoclonal antibodies were coupled to 4mg of horseradish peroxidase (HRP).Four to ten microlitres of citrated normal plasma or cryoprecipitate were applied to SDS-agarose gel plate (get concentration was 1.7%), and the electrophoresis was performed in the discontinuous buffer system. When the marker reached the anodic terminal, the plate was assembled with a nitrocellulose film, then the electroblot was carried out.The film was washed and followed by incubationfor 2 hours with HRP-con- jugated monoclonal antibodies which were diluted in 10% bovine serum albumin. After being thoroughly washed, the film was developed in substrate solution(4~chloro-1-naphthoI,0.5mg/mI) in the dark for fifteen minutes. In some experiments, the film was then incubated with HRP-conjugatedrabbit-anti mouse IgG F(ab´ )2 secondary antibodiesfor another 2 hours and followed by the same development procedure so as to increase the staining intensity.The whole procedure took about thirty-six hours.With the aid of this technique, we have obtained thedistinct patterns of multimeric structure of normalvWF.Plasma from a patient with 11A variant of von Willebrand's disease(vWD) gave a typical multimeric composition pattern as that observed by using autoradiography. These results suggested that the use of peroxidase-conjugated monoclonal antibodies was more convenient and timesaving, despite its relatively lower sensitivity which could be improved by mixing some monoclonal antibodies to recognize different epitope of vWF in the same system. We have used both SZ_ 29 and SZ-34 simul-taneously in one experiment andfound the sensitivity markedly increased. Thus, thismethod might be useful for analysis of the multimeric structure of vWF and differentiation of vWD.

Thirty four IIA vWD patients (16 from kindred I, 2 from kindred II and 17 unrelated patients) from 19 families were studied to compare multimer patterns using discontinuous SDS gel electrophoresis on a variety of agarose gels. Platelet multi-mers and effect of EDTA on plasma multimers were also studied in some.The large kindred and 9 other patients showed identical multimer and triplet abnormalities. The 11 other patients showed different multimer patterns either by having intermediate multimers or different triplet patterns. The second kindred had a similar triplet abnormality to kindred I but had intermediate multimers. Two other patients showed similar patterns except on 2% agarose gels when differences in the lowest multimer was seen. Of the 3 patients of YS, one showed the common IIA pattern but also had intermediate multimers, another had an unusually faint upper triplet band, while the third in addition to a faint upper triplet band with ESVWF 2+ had no identification of minor or major bands with ESVWF 10+ Another patient lacked high and some intermediate multimers but had a normal triplet pattern. The pattern we have seen in Kernoff's patient (1) still appears unique. In kindred II abnormal triplets persisted and high multimers appeared in EDTA plasma. In kindred I (and similar patients) intermediate multimers and a change in triplet pattern was observed in EDTA while lysed platelets showed an abnormal pattern different to the plasma one.This emphasizes the heterogeneity of IIA vWD and the need to consider multimer deletion, triplet pattern, platelet multimers, effect of EDTA in trying to subclassify in order to study structure function relationships of vWF.1. Kernoff PBA, Gruson R, Rizza CR. (1974) A variant of factor VIII related antigen. Br. J. Haematol. 26: 435.+ESVWF 2 and 10 are monoclonal antibodies to vWF epitopes.