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Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus
No full textThe amino acid sequences recognized by monoclonal antibodies (MAbs) specific for the antigenic site IV of the spike protein S of transmissible gastroenteritis virus were analyzed by PEPSCAN. All MAbs of group IV recognized peptides from the S region consisting of residues 378 to 390. In addition, the neutralizing MAbs (subgroup IV-A) also bound to peptides from the region consisting of residues 1173 to 1184 and to several other peptides with a related amino acid composition. The contribution of the individual residues of both sequences to the binding of a MAb was determined by varying the length of the peptide and by a consecutive deletion or replacement of parental residues by the 19 other amino acids. The sequence consisting of residues 326 to 558, tested as part of a cro-β-galactosidase hybrid protein, was antigenic, but the sequence consisting of residues 1150 to 1239 was not. Furthermore, antibodies raised in rabbits against the peptide SDSSFFSYGEIPFGN (residues 377 to 391), but not those raised against the peptide VRASRQLAKDKVNEC (residues 1171 to 1185), recognized the virus and had neutralizing activity. We infer that the epitope of the neutralizing MAbs is composite and consists of the linear sequence SFFSYGEI (residues 380 to 387) with contributions of A, D, K, N, Q, or V residues from other parts of the S molecule. The complex epitope was simulated by sythesizing peptides in which the sequences consisting of residues 380 to 387 and 1176 to 1184 were combined. MAbs of subgroup IV-A recognized the combination peptides two to six times better than the individual sequences. These results may offer prospects for the development of an experimental vaccine
Immunogenicity of peptides simulating a neutralization epitope of transmissible gastroenteritis virus
No full textPreviously, 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
Isolation of sequences from a random-sequence expression library that mimic viral epitopes
No full textWe describe the use of random peptide sequences for the mapping of antigenic determinants. An oligonucleotide with a completely degenerate sequence of 17 or 23 nucleotides was inserted into a bacterial expression vector. This resulted in an expression library producing random hexa- or octapeptides attached to a β-galactosidase hybrid protein. Mimotopes, or antigenic sequences that mimic an epitope, were selected by immunoscreening of colonies with monoclonal antibodies, which were specific for antigenic sites on the spike protein of the coronavirus transmissible gastroenteritis virus. We report one mimotope for antigenic site II, eight for site III and one for site IV. The site III and site IV mimotopes were closely similar to the corresponding linear epitopes, localized previously in the amino acid sequence of the S protein. An alignment of the site II mimotope and the sequence of the S protein around Trp97, which is substituted in escape mutants, suggests that this mimotope mimics a conformational epitope located around residues 97-103. Applications of mimotopes to epitope mapping, serodiagnosis and vaccine development are discussed
Analysis of an immunodominant region of infectious bronchitis virus
No full textWe analyzed the antigenic fine-structure of an immunodominant region in the peplomer protein of infectious bronchitis virus. This region near the N-terminus of the S2 subunit is recognized by polyclonal antisera and by the majority of mAb that cross-react with denatured protein. Despite their involvement in neutralization, epitopes in this region were conserved in different serotypes. Epitopes of four mAb and two chicken antisera were localized by using prokaryotic expression of cDNA fragments, and overlapping peptides with lengths increasing from 3 to 12 residues (PEPSCAN). We found overlapping epitopes with lengths of 6, 9, 11, and more than 17 residues. The results indicate that the expression products are antigenically equivalent to denatured protein fragments. This suggests a general strategy for the localization of sequential epitopes in large proteins. We propose that the immunodominance of the N-terminal region of S2 is explained by features of the protein structure. Presumably, this region is a protruding protein segment of about 20 residues with a high local mobility, as indicated by the antigenicity of the peptides. The conservation of the sequence points to an involvement in a molecular recognition process during infection
Location of antigenic sites of the S-glycoprotein of transmissible gastroenteritis virus and their conservation in coronaviruses
No full textTransmissible gastroenteritis virus (TGEV) has a single-stranded, positive-sense RNA genome of more than 20 kb (Brian et al., 1980; Rasschaert et al., 1987) and three structural proteins: S, N and M, with 1447, 382 and 262 amino acids, respectively (Kapke and Brian, 1986; Laude et al., 1987; Rasschaert and Laude, 1987; Jacobs et al., 1987)
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Residues involved in the antigenic sites of transmissible gastroenteritis coronavirus S glycoprotein
No full textThe 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
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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