Search

We 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 beta-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.

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 greater than 11 (at 4 degrees) and temperatures greater than 45 degrees. 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.

The antigenic homology of 26 coronavirus isolates, of which 22 were antigenically related to transmissible gastroenteritis virus (TGEV), was determined with 42 monoclonal antibodies. Type, group, and interspecies specific epitopes were defined. Two group specific MAbs distinguished the enteric TGEV isolates from the respiratory variants. An antigenic subsite involved in neutralization was conserved in porcine, feline, and canine coronavirus. The classification of the human coronavirus 229E in a taxonomic cluster distinct from TGEV group is suggested.

Four antigenic sites of the E2 glycoprotein of transmissible gastroenteritis virus were defined by competitive radioimmunoassays of monoclonal antibodies (MAbs). Here, we describe the localization of these sites by testing the antigenicity of protein fragments and prokaryotic expression products of E2 gene fragments, and by sequencing of MAb-resistant (mar) mutants. Partial proteolysis of purified E2 protein allowed the isolation of a 28K fragment recognized by both site A- and site C-specific MAbs. An antiserum against this fragment bound to a synthetic peptide containing residues 1 to 18 and to an expression product containing residues 1 to 325. The same expression product was recognized by site C-specific MAbs. These data indicate that residues within the sequence 1 to 325 contribute to site C and possibly also to site A. Sequencing of mar mutants that escaped neutralization by site A-specific MAbs indicated that residues 538 and 543 also belong to site A. The binding of site-specific MAbs to expression products led directly to the localization of sites B and D, between residues 1 to 325 and 379 to 529, respectively. The first 37% of the polypeptide chain of E2 appears to be more immunogenic than the rest of the sequence.

Monoclonal antibodies (Mabs) specific for the E1 and E2 surface glycoproteins of the transmissible gastroenteritis virus (TGEV) of swine were examined either alone or in combination to evaluate their potential value in protecting neonatal pigs against a lethal dose of TGEV. Cesarean-delivered colostrum-deprived (CDCD) piglets were given one pre-challenge dose of Mab and an equal dose of the same Mab at each successive feeding after challenge. In vivo challenge results demonstrated that neither Mabs given individually nor combinations of the Mabs were able to protect neonatal pigs against a lethal dose of TGEV. However, in parallel experiments, polyclonal antibodies from immune colostrum or serum were protective.

Purified transmissible gastroenteritis (TGE) virus was found to be composed of three major structural proteins having relative molecular weights of 200,000, 48,000, and 28,000. The peplomer glycoprotein was purified by affinity chromatography with the monoclonal antibody (MAb) 1D.G3. A collection of 48 MAbs against TGE virus was developed from which 26, 10, and 3 were specific for proteins E2, N, and E1, respectively. A total of 14 neutralizing MAbs of known reactivity were E2 protein specific. In addition, MAb 1B.C11, of unknown specificity, was also neutralizing. These MAbs reduced the virus titer 10(2)- to 10(9)-fold. Six different epitopes critical in TGE virus neutralization were found, all of which were conformational based on their immunogenicity and antigenicity. Only the epitope defined by MAb 1G.A7 was resistant to sodium dodecyl sulfate treatment, although it was destroyed by incubation in the presence of both the detergent and beta-mercaptoethanol. The frequency of MAb-resistant (mar) mutants selected with four MAbs (1G.A7, 1B.C11, 1G.A6, and 1E.F9) ranged from 10(-6) to 10(-7), whereas the frequency of the putative mar mutant defined by MAb 1B.B11 was lower than 10(-9). Furthermore, the epitopes defined by these MAbs and by MAbs 1H.C2 and 1A.F10, were present in 11 viral isolated with different geographical locations, years of isolation, and passage numbers (with the exception of two epitopes absent or modified in the TOY 56 viral isolate), suggesting that the critical epitopes in TGE virus neutralization were highly conserved.

The antigenic relationship between a recently isolated porcine respiratory coronavirus (TLM 83) and transmissible gastroenteritis (TGE) virus of swine was studied by neutralization, immunoblotting and radioimmunoassay (RIA) using TGE virus-specific monoclonal antibodies (MAbs) and polyclonal antibodies specific for both viruses. A complete two-way neutralization activity between the two viruses was found. Immunoblotting revealed cross-reactions between TLM 83 and TGE virus antigens at the level of the envelope protein (E1), the nucleoprotein (N) and the peplomer protein (E2). By virus neutralization assays and RIA with TGE virus-specific MAbs, the presence of similar epitopes in the E1 and N proteins and in the neutralization-mediating antigenic site of the E2 protein were demonstrated. E2 protein-specific MAbs, without neutralizing activity and reacting with antigenic sites B, C and D (previously defined), failed to recognize TLM 83. These results indicated a close antigenic relationship and structural similarity between TLM 83 and TGE viruses and also suggested potential ways of differentiating between the two viruses.

The antigenic structure of transmissible gastroenteritis (TGE) virus E2 glycoprotein has been defined at three levels: antigenic sites, antigenic subsites and epitopes. Four antigenic sites (A, B, C and D) were defined by competitive radioimmunoassay (RIA) using monoclonal antibodies (MAbs) selected from 9 fusions. About 20% (197) of the hybridomas specific for TGE virus produced neutralizing MAbs specific for site A, which was one of the antigenically dominant determinants. Site A was differentiated in three antigenic subsites: a, b and c, by characterization of 11 MAb resistant (mar) mutants, that were defined by 8, 3, and 3 MAbs, respectively. These subsites were further subdivided in epitopes. A total of 11 epitopes were defined in E2 glycoprotein, eight of which were critical for virus neutralization. Neutralizing MAbs were obtained only when native virus was used to immunize mice, although to produce hybridomas mice immunizations were made with antigen in the native, denatured, or mixtures of native and denatured form. All neutralizing MAbs reacted to conformational epitopes. The antigenic structure of the E2-glycoprotein has been defined with murine MAbs, but the antigenic sites were relevant in the swine, the natural host of the virus, because porcine sera reacted against these sites. MAbs specific for TGE virus site C reacted to non-immune porcine sera. This reactivity was not directed against porcine immunoglobulins. These results indicated that TGE virus contains epitope(s) also present in some non-immunoglobulin component of porcine serum.