A Transmissible Gastroenteritis Coronavirus Nucleoprotein Epitope Elicits T Helper Cells That Collaborate in the in Vitro Antibody Synthesis to the Three Major Structural Viral Proteins

AbstractFour strong T cell epitopes have been identified studying the blastogenic response of lymphocytes from haplotype-defined transmissible gastroenteritis virus (TGEV) immune miniswine to sixty-one 15-mer synthetic peptides. Three of these epitopes are located on the nucleoprotein (N48 amino acids 46 to 60; N272, amino acids 272 to 286; and N321 amino acid 321 to 335), and one on the membrane protein (M196, amino acids 196 to 210). N321, peptide induced the highest T cell response and was recognized by immune miniswine lymphocytes with haplotypes dd, aa , and cc. T lymphocytes from peptide N321-immune miniswine reconstituted the in vitro synthesis of TGEV-specific antibodies by complementing CD4- TGEV-immune cells. This response was directed at least against the three major structural proteins. The synthesized antibodies specific for S protein preferentially recognized discontinous epitopes and neutralized TGEV infectivity. These results show that peptide N321 defines a functional T helper epitope eliciting T cells capable of collaborating with B cells specific for different proteins of TGEV

Antigenic Profile of African Horse Sickness Virus Serotype 4 VP5 and Identification of a Neutralizing Epitope Shared with Bluetongue Virus and Epizootic Hemorrhagic Disease Virus

AbstractAfrican horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned inEscherichia coliusing the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151–200) and II (residues 83–120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85–92 (PDPLSPGE) for MAb 10AE12 and at 179–185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes

A fast mutagenesis procedure to recover soluble and functional scFvs containing amber stop codons from synthetic and semisynthetic antibody libraries

8 p.-4 fig.The selection and production of scFvs from phage display synthetic antibody libraries are frequently delayed by the presence of amber (TAG) stop codons within the sequences corresponding to the variable CDRs. This is due to the use of randomised oligonucleotides for library design and amber mutations for joining the scFv to the phage protein pIII. The screening of such libraries may lead to the selection of scFvs containing stop codons. Then, multiple site-directed mutagenesis is required for their removal or, alternatively, the proteins must be expressed as scFv-pIII fusions, which are not suitable for many functional assays. We describe here an alternative procedure to express soluble scFvs, despite the presence of TAG stop codons, in the currently used Escherichia coli suppressor strain TG1. It is based on a simple mutagenesis protocol that replaces the amber codon between the scFv and the pIII gene by a different stop codon (TAA), functional in E. coli TG1. The expression of soluble scFvs in the suppressor strain TG1 permits their fully functional characterization including the determination of affinity constants, which are critical for selecting the right scFvs for further studies.This project was partially supported by an EU grant COOP-CT-2004-512691 and the grant B102003-01481 from the Ministry of Science and Technology (MCYT) of Spain.Peer reviewe

Residues involved in the antigenic sites of transmissible gastroenteritis coronavirus S glycoprotein

The S glycoprotein of transmissible gastroenteritis virus (TGEV) has been shown to contain four major antigenic sites (A, B, C, and D). Site A is the main inducer of neutralizing antibodies and has been previously subdivided into the three subsites Aa, Ab, and Ac. The residues that contribute to these sites were localized by sequence analysis of 21 mutants that escaped neutralization or binding by TGEV-specific monoclonal antibodies (MAbs), and by epitope scanning (PEPSCAN). Site A contains the residues 538, 591, and 543, which are essential in the formation of subsites Aa, Ab, and Ac, respectively. In addition, mar mutant 1B.H6 with residue 586 changed had partially altered both subsite Aa and Ab, indicating that these subsites overlap in residue 586; i.e. this residue also is part of site A. The peptide 537-MKSGYGQPIA-547 represents, at least partially, subsite Ac which is highly conserved among coronaviruses. This site is relevant for diagnosis and could be of interest for protection. Other residues contribute to site B (residues 97 and 144), site C (residues 50 and 51), and site D (residue 385). The location of site D is in agreement with PEPSCAN results. Site C can be represented by the peptide 48-P-P/S-N-S-D/E-52 but is not exposed on the surface of native virus. Its accessibility can be modulated by treatment at pH >11 (at 4°) and temperatures >45°. Sites A and B are fully dependent on glycosylation for proper folding, while sites C and D are fully or partially independent of glycosylation, respectively. Once the S glycoprotein has been assembled into the virion, the carbohydrate moiety is not essential for the antigenic sites

Residues involved in the antigenic sites of transmissible gastroenteritis coronavirus S glycoprotein

The S glycoprotein of transmissible gastroenteritis virus (TGEV) has been shown to contain four major antigenic sites (A, B, C, and D). Site A is the main inducer of neutralizing antibodies and has been previously subdivided into the three subsites Aa, Ab, and Ac. The residues that contribute to these sites were localized by sequence analysis of 21 mutants that escaped neutralization or binding by TGEV-specific monoclonal antibodies (MAbs), and by epitope scanning (PEPSCAN). Site A contains the residues 538, 591, and 543, which are essential in the formation of subsites Aa, Ab, and Ac, respectively. In addition, mar mutant 1B.H6 with residue 586 changed had partially altered both subsite Aa and Ab, indicating that these subsites overlap in residue 586; i.e. this residue also is part of site A. The peptide 537-MKSGYGQPIA-547 represents, at least partially, subsite Ac which is highly conserved among coronaviruses. This site is relevant for diagnosis and could be of interest for protection. Other residues contribute to site B (residues 97 and 144), site C (residues 50 and 51), and site D (residue 385). The location of site D is in agreement with PEPSCAN results. Site C can be represented by the peptide 48-P-P/S-N-S-D/E-52 but is not exposed on the surface of native virus. Its accessibility can be modulated by treatment at pH >11 (at 4°) and temperatures >45°. Sites A and B are fully dependent on glycosylation for proper folding, while sites C and D are fully or partially independent of glycosylation, respectively. Once the S glycoprotein has been assembled into the virion, the carbohydrate moiety is not essential for the antigenic sites

Immunogenicity of peptides simulating a neutralization epitope of transmissible gastroenteritis virus

Previously, an epitope recognized by a set of neutralizing monoclonal antibodies directed against the S protein of transmissible gastroenteritis has been identified. This neutralization epitope can be simulated by a single peptide combining residues 380 to 387 and 1176 to 1184 of the S protein; this combination peptide (SFFSYGEI-QLAKDKVNE) was more antigenic than its single constituents. Here we describe the immunogenicity of this combination peptide, in comparison with monomer and tandem peptides of both constituents, and with a cyclic peptide consisting of residues 373 to 398. All antisera, raised in rabbits, bound to the peptide used as immunogen. Only sera that recognized the residues 380 to 387 bound to whole virus. Three of the four antisera with the highest binding titers to whole virus also had neutralization activity. Analysis of the fine-specificity of the antisera with PEPSCAN peptides indicated that the spectrum of antibodies induced by the 380 to 387 sequence depended on the presentation of this sequence in a peptide to the immune system. The nonbinding and nonneutralizing anti-(380 to 387)-sera appeared to contain a limited spectrum of antipeptide antibodies. Furthermore, the lack of neutralization of the antiserum against the combination peptide could be explained by the immunodominance in rabbits of the 1176 to 1184 sequence over the 380 to 387 sequence. These findings demonstrate a few fundamental problems of simulating discontinuous epitopes by single synthetic peptides

Immunogenicity of peptides simulating a neutralization epitope of transmissible gastroenteritis virus

Previously, an epitope recognized by a set of neutralizing monoclonal antibodies directed against the S protein of transmissible gastroenteritis has been identified. This neutralization epitope can be simulated by a single peptide combining residues 380 to 387 and 1176 to 1184 of the S protein; this combination peptide (SFFSYGEI-QLAKDKVNE) was more antigenic than its single constituents. Here we describe the immunogenicity of this combination peptide, in comparison with monomer and tandem peptides of both constituents, and with a cyclic peptide consisting of residues 373 to 398. All antisera, raised in rabbits, bound to the peptide used as immunogen. Only sera that recognized the residues 380 to 387 bound to whole virus. Three of the four antisera with the highest binding titers to whole virus also had neutralization activity. Analysis of the fine-specificity of the antisera with PEPSCAN peptides indicated that the spectrum of antibodies induced by the 380 to 387 sequence depended on the presentation of this sequence in a peptide to the immune system. The nonbinding and nonneutralizing anti-(380 to 387)-sera appeared to contain a limited spectrum of antipeptide antibodies. Furthermore, the lack of neutralization of the antiserum against the combination peptide could be explained by the immunodominance in rabbits of the 1176 to 1184 sequence over the 380 to 387 sequence. These findings demonstrate a few fundamental problems of simulating discontinuous epitopes by single synthetic peptides

Designing antibodies for the inhibition of gastrin activity in tumor development

9 páginas, 7 figuras, 3 tablas -- PAGS nros. 2351-2359Gastrin and its derivatives are becoming important targets for immunotherapy of pancreatic, gastric and colorectal tumors. This study was conducted to design antibodies able to block gastrin binding to the gastrin/cholecystokinin-2 (CCK-2) receptor in order to delay tumor growth. The authors have used different gastrin molecules, combined with the diphtheria toxoid, to generate and select human single chain variable fragments (scFvs) as well as mouse monoclonal antibodies and scFvs against different regions of gastrin. There was a remarkable conservation in the antibody repertoire against gastrin, independently of the approach and the species. The germlines most frequently used in gastrin antibody formation were identified. Three different epitopes were identified in the gastrin molecule. The resulting mouse monoclonal antibodies and scFvs were analyzed for gastrin neutralization using Colo 320 WT cells, which overexpress the CCK-2 receptor. The gastrin neutralizing activity assay showed that N-terminal specific mouse monoclonal antibodies were more efficient to inhibit proliferation of Colo 320 WT cells than the anti-C terminal antibodies. Moreover, the human antigastrin scFvs obtained in this study inhibited significantly the proliferation of Colo 320 tumoral cells. These findings should contribute to a more rational design of antibody-based antigastrin therapies in cancer, including passive administration of human antibodies with blocking activityThe authors thank the Monoclonal Antibody Unit staff of the CNIO for their collaboration in the production of the mouse Mabs. They also thank Mr. Ronald Boshuizen (Pepscan) for his practical assistance in peptide synthesis and Dr. Frank Schmitz (St. Josef-Hospital, Ruhr-University of Bochum) for kindly providing the Colo 320 WT cells. The authors thank Dr. Juan Carlos Murciano (CNIC, Madrid, Spain) for his assistance with the G17 and CCK-2 receptor binding experiments. Dr. Rodrigo Barderas is recipient of a Postdoctoral Contract of the FIS supported by Spanish Ministry of HealthPeer reviewe