INGENASA, Hermanos Garcia Noblejas 41, 28037 Madrid, Spain1
Author for correspondence: J. Ignacio Casal. Fax +34 91 4087598. e-mail icasal{at}ingenasa.es
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
GP5 contains most of the epitopes involved in virus neutralization (Pirzadeh & Dea, 1997 ). Previous studies have reported the production of neutralizing MAbs specific for PRRSV GP5 (Pirzadeh & Dea, 1997
; Wieczorek-Krohmer et al., 1996
; Zhang et al., 1998
) by using recombinant proteins or pre-induction of tolerance against non-infected porcine alveolar macrophages. Also, pigs vaccinated with plasmid DNA encoding GP5 (Pirzadeh & Dea, 1998
) or baculovirus-expressed GP5 plus GP3 (Plana Duran et al., 1997
) were partially protected.
From a structural point of view, GP5 contains two N-glycosylation sites that are highly conserved among strains from Europe and North America and a very hydrophobic region in the N terminus, which could act as a leader sequence. GP5 also contains several hydrophobic transmembrane regions. These hydrophobic domains probably provoke retention of the protein in the ER, causing inhibition of the synthesis of GP5 in expression systems such as baculovirus (Plana Duran et al., 1997 ). However, removal of the transmembrane region has improved considerably the solubility of similar proteins such as gp55 of classical swine fever virus (Hulst et al., 1993
). A similar approach was also used for epitope mapping of the GL protein of equine arteritis virus (EAV) (Chirnside et al., 1995
).
In general, proteins containing hydrophobic or membrane-associated domains are insoluble and difficult to recover. In order to study whether deletion of these anchoring hydrophobic regions could enhance expression of PRRSV GP5, we tested the expression of several deletion mutants of GP5 in E. coli. The same fragments were used to immunize mice in order to produce MAbs, which were then used to define an important antigenic domain in GP5.
Transmembrane regions in the amino acid sequence of GP5 were identified by using the program PredictProtein (EMBL) (Rost, 1996 ; Rost et al., 1995
). There are two major transmembrane domains, regions 1431 and 64134, of a total of 201 residues. Based on these data, we prepared three GP5 mutants: GP5
Ns, spanning residues 28201, which has the signal peptide deleted, and GP5[3067] (residues 3067) and GP5[30201] (residues 3067 plus 130201), which have respectively two and one transmembrane region deleted (Fig. 1
).
|
Briefly, 20 ml cell cultures were harvested 3 h after induction by centrifugation at 4000 r.p.m. for 5 min, washed twice with PBS and analysed on an 11% SDSpolyacrylamide gel. Complete GP5 was expressed at low levels (0·08 mg/ml), as expected from its structural properties and its toxicity for cells. The remaining fragments, Ns, 3067 and 30201, yielded recombinant truncated fusion proteins of the expected sizes (51·2, 36·8 and 45·2 kDa), including the 260 amino acids of the gene 10 leader peptide of phage T7 expressed as the fused protein. They were expressed at much higher levels (1·5, 1·6 and 2·6 mg/ml). The identity of the proteins was confirmed by immunoblotting with PRRSV-specific pig antisera. Recombinant proteins were partially purified by solubilization of inclusion bodies with Triton X-100 and 4 M guanidinium chloride. The purity of the proteins recovered was around 6070%.
In order to study the antigenicity of the different GP5 fragments and to learn whether the different domains of GP5 were recognized equally well by sera from PRRSV-infected pigs, the reactivity of the different fragments was analysed by immunoblotting. We used a collection of 13 European, two Canadian and two PRRSV-negative pig sera. Complete GP5 was recognized weakly, probably because of its lower level of expression. However, all but one of the sera reacted well with fragments GP5 Ns and GP5[30201]. In contrast, fragment GP5[3067] was recognized by only two pig sera. Reactivity was isolate-specific, as none of the Canadian pig sera recognized the European GP5 mutants, and was focused on the C-terminal part of GP5, residues 130201.
To determine the immunogenicity of the recombinant truncated fragments of GP5 in mice, partially purified PRRSV GP5 and fragments were used for immunization of BALB/c mice. Each mouse received three doses of 50 µg protein in Freunds adjuvant at 15-day intervals and mice were bled for serological analysis 7 days after the last immunization. Complete GP5 and fragment GP5 Ns were less immunogenic, as respectively no mice or only one mouse elicited antibodies that reacted against the virus in ELISA or immunoblotting. In the case of GP5[30201], antibodies from three of four mice were able to recognize the virus. GP5[3067] showed intermediate immunogenicity; two of four mice developed PRRSV-specific antibodies. The four recombinant proteins were tested for the production of MAbs, without success. Several factors could explain these poor results: low immunogenicity of PRRSV proteins in mice, insufficient purification of the recombinant proteins or interference of the fused protein. In fact, there is only one previous report (Wieczorek-Krohmer et al., 1996
) describing the production of MAbs against a European PRRSV GP5.
In order to determine whether the purity of the proteins influenced these results, a new fragment, GP5[30201]H, was subcloned in pET28c (Novagen). This construct allowed expression of fragment GP5[30201] with a polyhistidine tail at the N terminus. GP5[30201]H (17·1 kDa) was expressed at an intermediate level and reacted well with the pig antisera by immunoblotting. The fragment was further purified by using Ni2+NTA columns (Qiagen). Briefly, the recombinant material was solubilized in TSG buffer (20 mM TrisHCl, pH 7·9, 0·5 M NaCl, 5 mM imidazole, 6 M guanidineHCl) and clarified by centrifugation. Elution of GP5[30201]H was achieved with 200 mM imidazole in TSG buffer. The recovery of final product, after purification, was 3 mg/l, with 90% purity. This highly purified fragment was much more efficient in immunization of mice. All immunized mice developed high ELISA titres against PRRSV virions (>1/8000). Thus, it was selected for the production of MAbs.
Protocols for immunization and the preparation of MAbs have been described previously (Rodriguez et al., 1997 ). After immunization of mice with GP5[30201]H, two MAbs specific for PRRSV GP5 by ELISA were produced. The isotypes of the MAbs were IgG2b for 3AH9 and IgG2a for 4BE12. The protein specificity of the two MAbs was confirmed by immunoblotting analysis. Both MAbs reacted with the 25 kDa band corresponding to viral GP5 (Fig. 2
). Also, the MAbs recognized the native virus protein by indirect immunofluorescence and immunoperoxidase assays, suggesting that they recognized linear epitopes. In contrast, no reactivity was detected with North American strains of PRRSV by ELISA or immunoblotting (data not shown).
|
The antigenic domain recognized by the MAbs in PRRSV GP5 was identified by probing the reactivity of the two MAbs with the four truncated GP5 fragments. The MAbs reacted with fragments Ns and GP5[30201], but not with GP5[3067] (Fig. 2
). For fine mapping of MAbs 3AH9 and 4BE12, the C-terminal region (aa 130201) was split in two fragments, as shown in Fig. 1
. Fragments were prepared by PCR with the indicated primers (Fig. 1
). Fragment 130170 was cloned in pET3Xa and fragment 170201 was cloned in pET3Xb. The fragments were expressed and transferred to Immobilon-P membranes for immunoblotting analysis. The results are shown in Fig. 3
(A
, B
). The two MAbs recognized different epitopes, one in each fragment. MAb 4BE12 reacted with fragment 130170 and 3AH9 reacted with fragment 170201, as well as with the virus and fragment 30201.
|
An alignment analysis showed that region 170201 is highly conserved in all European isolates and shows 60% identity to American isolates in the last 25 residues. This reinforces the significance of this epitope as a diagnostic tool. A potentially antigenic region was identified close to the C terminus on the basis of theoretical predictions (Goldberg et al., 2000 ). Interestingly, some predictions assigned fragment 170190 as a transmembrane region (Dea et al., 2000
). This prediction, together with our results, might suggest that only the last 11 residues (190201) are accessible to antibodies on the surface of the virus.
Previous expression of PRRSV GP5 in the baculovirus system was inefficient (Plana Duran et al., 1997 ; Kreutz & Mengeling, 1997
), making the preparation of large amounts of the protein for characterization and immunogenicity studies difficult. Removal of the predicted transmembrane region has resulted in a significant improvement in expression levels, probably by redirecting the protein to inclusion bodies, from where they can be recovered by regular solubilization procedures. Subsequently, we have used these fragments to characterize the antigenic structure of GP5. The definition of antigenic regions of GP5 is very important for vaccine development and diagnostic purposes.
Our results indicate that, despite good antigenicity, the C-terminal region may not actually be involved in virus neutralization. Thus, either the hypervariable ectodomain of the protein is the region involved in virus neutralization or the neutralizing epitopes are mainly conformational. Considering that neutralizing epitopes were defined in the ectodomain for EAV GL (Balasuriya et al., 1997 ), it is easy to speculate that something similar might occur in PRRSV. However, preliminary results obtained after expressing a fragment containing the first 40 residues of GP5 indicate that a much larger region is necessary to form the epitope. In that case, if the neutralizing epitopes require several sequences from different regions, it would make production of a subunit vaccine based on GP5 very difficult. In any case, the immunogenicity of these GP5 mutants should be tested in pigs to provide a definitive assessment of the relevance of these epitopes in virus neutralization.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Benfield, D. A., Nelson, E., Collins, J. E., Harris, L., Goyal, S. M., Robison, D., Christianson, W. T., Morrison, R. B., Gorcyca, D. & Chladek, D. (1992). Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332). Journal of Veterinary Diagnostic Investigation 4, 127-133.[Medline]
Chirnside, E. D., de Vries, A. A. F., Mumford, J. A. & Rottier, P. J. M. (1995). Equine arteritis virus-neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein GL. Journal of General Virology 76, 1989-1998.[Abstract]
Conzelmann, K. K., Visser, N., Van Woensel, P. & Thiel, H. J. (1993). Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group. Virology 193, 329-339.[Medline]
Dea, S., Gagnon, C. A., Mardassi, H., Pirzadeh, B. & Rogan, D. (2000). Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates. Archives of Virology 145, 659-688.[Medline]
Goldberg, T. L., Hahn, E. C., Weigel, R. M. & Scherba, G. (2000). Genetic, geographical and temporal variation of porcine reproductive and respiratory syndrome virus in Illinois. Journal of General Virology 81, 171-179.
Hulst, M. M., Westra, D. F., Wensvoort, G. & Moormann, R. J. (1993). Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera. Journal of Virology 67, 5435-5442.[Abstract]
Kreutz, L. C. & Mengeling, W. L. (1997). Baculovirus expression and immunological detection of the major structural proteins of porcine reproductive and respiratory syndrome virus. Veterinary Microbiology 59, 1-13.[Medline]
Loemba, H. D., Mounir, S., Mardassi, H., Archambault, D. & Dea, S. (1996). Kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus. Archives of Virology 141, 751-761.[Medline]
Martinez-Torrecuadrada, J. L. & Casal, J. I. (1995). Identification of a linear neutralization domain in the protein VP2 of African horse sickness virus. Virology 210, 391-399.[Medline]
Meulenberg, J. J., Hulst, M. M., de Meijer, E. J., Moonen, P. L., den Besten, A., de Kluyver, E. P., Wensvoort, G. & Moormann, R. J. (1993). Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology 192, 62-72.[Medline]
Meulenberg, J. J., Petersen-den Besten, A., De Kluyver, E. P., Moormann, R. J., Schaaper, W. M. & Wensvoort, G. (1995). Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus. Virology 206, 155-163.[Medline]
Morrison, R. B., Collins, J. E., Harris, L., Christianson, W. T., Benfield, D. A., Chladek, D. W., Gorcyca, D. E. & Joo, H. S. (1992). Serologic evidence incriminating a recently isolated virus (ATCC VR-2332) as the cause of swine infertility and respiratory syndrome (SIRS). Journal of Veterinary Diagnostic Investigation 4, 186-188.[Medline]
Pirzadeh, B. & Dea, S. (1997). Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants. Journal of General Virology 78, 1867-1873.[Abstract]
Pirzadeh, B. & Dea, S. (1998). Immune response in pigs vaccinated with plasmid DNA encoding ORF5 of porcine reproductive and respiratory syndrome virus. Journal of General Virology 79, 989-999.[Abstract]
Pirzadeh, B., Gagnon, C. A. & Dea, S. (1998). Genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope GP5 glycoprotein. Canadian Journal of Veterinary Research 62, 170-177.[Medline]
Plana Duran, J., Climent, I., Sarraseca, J., Urniza, A., Cortes, E., Vela, C. & Casal, J. I. (1997). Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91. Involvement of ORF3 and ORF5 proteins in protection. Virus Genes 14, 19-29.[Medline]
Pont-Kingdon, G. (1994). Creation of chimeric junctions, deletions and insertions by PCR. Methods in Molecular Biology 67, 167-172.
Rodriguez, M. J., Sarraseca, J., Garcia, J., Sanz, A., Plana-Durán, J. & Casal, J. I. (1997). Epitope mapping of the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus. Journal of General Virology 78, 2269-2278.[Abstract]
Rost, B. (1996). PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods in Enzymology 266, 525-539.[Medline]
Rost, B., Casadio, R., Fariselli, P. & Sander, C. (1995). Transmembrane helices predicted at 95% accuracy. Protein Science 4, 521-533.
Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology 185, 60-89.[Medline]
Wensvoort, G., Terpstra, C., Pol, J. M., ter Laak, E. A., Bloemraad, M., de Kluyver, E. P., Kragten, C., van Buiten, L., den Besten, A., Wagenaar, F. and others (1991). Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Veterinary Quarterly 13, 121130.[Medline]
Wieczorek-Krohmer, M., Weiland, F., Conzelmann, K., Kohl, D., Visser, N., van Woensel, P., Thiel, H. J. & Weiland, E. (1996). Porcine reproductive and respiratory syndrome virus (PRRSV): monoclonal antibodies detect common epitopes on two viral proteins of European and US isolates. Veterinary Microbiology 51, 257-266.[Medline]
Zhang, Y., Sharma, R. D. & Paul, P. S. (1998). Monoclonal antibodies against conformationally dependent epitopes on porcine reproductive and respiratory syndrome virus. Veterinary Microbiology 63, 125-136.[Medline]
Received 10 October 2000;
accepted 30 January 2001.