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L Enjuanes

Publications and source records attributed to L Enjuanes.

At least 73 records · Page 4Linked to original sources
PubMed1992

Epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus.

The epitope specificity of the protective immune response against swine transmissible gastroenteritis (TGE) has been investigated by using circulating and secretory antibodies. This study was carried out with sows vaccinated with TGEV or the antigenically related porcine respiratory coronavirus (PRCV). TGEV vaccination of sows resulted in greater lactogenic protection of suckling piglets against TGEV challenge and a higher secretory immune response than PRCV vaccination did. These differences in the immune response were conditioned by the route of antigen presentation as a result of the different tropism of each virus. Epitopes on S protein, and in particular those contained in its antigenic site. A, were more immunogenic than epitopes on N and M proteins in both groups of vaccinated sows, as determined by a competitive radioimmunoassay. Minor differences in antibody response against the previously defined antigenic subsites Aa, Ab, and Ac were also detected, with subsite Ab being the most antigenic in both TGEV- and PRCV-immune sows. These findings suggest that antigenic site A on S protein, involved in virus neutralization, is the immunodominant site in pregnant sows that confer lactogenic protection. They also validate, in experiments with secretory antibodies, the antigenic maps made with murine monoclonal antibodies. Therefore, this antigenic site should be considered for vaccine or diagnostic development.

Animals↗
PubMed1991

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 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.

Amino Acid Sequence↗
PubMed1991

A conserved coronavirus epitope, critical in virus neutralization, mimicked by internal-image monoclonal anti-idiotypic antibodies.

Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.

Animals↗
PubMed1990

Mechanisms of transmissible gastroenteritis coronavirus neutralization.

Transmissible gastroenteritis virus (TGEV) was neutralized more than 10(9)-fold with antibodies of a single specificity [monoclonal antibodies (MAbs)]. Most of the virus was neutralized in the first 2-3 min of a reversible reaction, which was followed by a second phase with a decreased neutralization rate and, in some cases, by a persistent fraction, which was a function of the MAb and of the antibody-to-virus ratio. Neutralization of TGEV is a specific event that requires the location of the epitope involved in the neutralization in the appropriate structural context, which is present in the wild-type virus but not in certain MAb escaping mutants. In neutralization of TGEV by binary combinations of MAbs specific for the same or for different antigenic sites, either no cooperation or a synergistic effect, respectively, was observed. Mechanisms of TGEV neutralization by MAbs were characterized at high, intermediate, and low antibody-to-virus ratios. Under these conditions, mainly three steps of the replication cycle were inhibited: binding of virus to the cell, internalization, and a step that takes place after internalization. In addition, virus aggregation could be responsible for the neutralization of 10 to 20% of virus infectivity.

Animals↗
PubMed1990

Expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed.

The peplomer protein (S) and the transmembrane protein (M) of transmissible gastroenteritis virus (TGEV) of swine were identified by iodination and serologically on the surface of infected cells. Of a total of 4 monoclonal antibodies (mAb) directed against four antigenic sites of S protein (Correa et al., 1988), 3 specific for sites A, B and D attached to the plasma membrane of infected cells, as disclosed by indirect immunofluorescence and by complement-mediated cytolysis. Four of the mAbs assayed were specific for the viral protein M and two of them gave plasma membrane immunofluorescence and mediated cytolysis in the presence of complement. The viral nucleoprotein N could not be demonstrated on the surface of infected cells either by iodination or employing 3 mAbs against this protein. Finally, a time course infection experiment demonstrated that S and M proteins were expressed on the surface of infected cells at 4 h after infection, before infective virus was released from infected cells.

Animals↗
PubMed1990

Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus.

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.

Amino Acid Sequence↗
PubMed1990

Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus.

The 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-beta-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 synthesizing 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.

Amino Acid Sequence↗
PubMed1989

Inter-specific analysis of Drosophila alcohol dehydrogenase by an immunoenzymatic assay using monoclonal antibodies.

Three Drosophila alcohol dehydrogenase monoclonal antibodies have been prepared and characterized. These antibodies cross-react with alcohol dehydrogenase from different species as revealed by immunoblotting assay. An enzyme-linked immunosorbent assay has been devised to quantify alcohol dehydrogenase in several species, different strains and individual larval organs. The assay detects alcohol dehydrogenase via a double-antibody sandwich assay technique giving strictly proportional values for antigen concentration and optical densities in the range of 3-30 ng of antigen per 100 microliters of sample. When alcohol dehydrogenase specific activity is compared in different larval organs a remarkable similarity is observed, whereas protein distribution varies substantially. Larval fat body and larval alimentary canal contribute 63% and 26% respectively to recovered alcohol dehydrogenase.

Alcohol Dehydrogenase↗
PubMed1989

Induction of transmissible gastroenteritis coronavirus-neutralizing antibodies in vitro by virus-specific T helper cell hybridomas.

Three transmissible gastroenteritis (TGE) virus-specific T helper (Th) cell hybridomas have been generated from virus-primed BALB/c mice, by fusion with the thymoma BW5147. The hybridomas responded to purified u.v.-inactivated TGE virus with interleukin production and growth inhibition. TGE virus recognition by the hybridomas was restricted by the major histocompatibility complex: only splenocytes from syngeneic or semi-syngeneic mice were able to recognize the antigen. The three hybridomas were Thy 1.2+, but did not express detectable levels of Lyt 1 or Lyt 2 antigens by fluorescent cell sorting analysis. Only one hybridoma (T.1J.B5) expressed the L3T4 marker. These hybridomas had helper activity, as they were able to reconstitute in vitro the synthesis of TGE virus-specific antibodies by Th cell-depleted spleen cells from immune BALB/c mice. The antibodies that they induced specifically neutralized by 10(3)- to 10(4)-fold the infectivity of TGE virus, ruling out the possibility of inhibition of virus replication by interferon. These hybridomas could be very useful for identifying antigenic domains in TGE virus recognized by Th cells, which cooperate with B cells in the synthesis of neutralizing antibodies.

Animals↗
PubMed1988

Extensive antigenic heterogeneity of foot-and-mouth disease virus of serotype C.

The antigenic behavior of 46 field isolates of foot-and-mouth disease virus (FMDV) of serotype C has been studied with a panel of 24 monoclonal antibodies (MAbs) prepared against FMDV C1 or FMDV C3 Indaial. Reactivities were assayed by immunodot, immunoelectrotransfer blot, and neutralization of infectivity. The epitopes recognized by the 10 nonneutralizing MAbs are conserved in all isolates analyzed. In contrast, extreme antigenic heterogeneity is documented with regard to reactivity with 14 MAbs that, on this basis, define at least 12 epitopes involved in neutralization of FMDV of serotype C. The 31 isolates from South America were divided into 17 distinct antigenic groups and the 15 isolates from Europe into 7 groups. Lack of correspondence between antigenic composition and the origin--date and place of isolation--of the viruses was noted in several instances. Antigenic heterogeneity is shown among epidemiologically closely related FMDVs. In most--but not all--cases tested, a good correlation was found between binding of a neutralizing MAb to virions and its ability to neutralize infectivity. It is concluded that variation of epitopes involved in neutralization of FMDV is extensive among subtypes of serotype C and also among individual isolates of one subtype.

Animals↗
PubMed1988

Antigenic structure of the E2 glycoprotein from transmissible gastroenteritis coronavirus.

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.

Animals↗
PubMed1988

Lack of protection in vivo with neutralizing monoclonal antibodies to transmissible gastroenteritis virus.

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.

Animals↗
PubMed1988

Antigenic differentiation between transmissible gastroenteritis virus of swine and a related porcine respiratory coronavirus.

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.

Antibodies, Monoclonal↗