Institute of Veterinary Virology, University of Berne, Länggass-Str. 122, CH-3012 Berne, Switzerland1
Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-7040, USA2
Génétique des Virus (ICGM-CNRS UPR 0415), Institut Cochin de Génétique moléculaire, 75014 Paris, France3
Author for correspondence: Giuseppe Bertoni. Fax +41 31 631 25 34. e-mail bertoni{at}ivv.unibe.ch
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Abstract |
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Introduction |
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Caprine arthritisencephalitis virus (CAEV) is a lentivirus that causes persistent infection in goats. Between 20% and 30% of naturally infected goats show symptoms of progressive arthritis. A relationship between anti-Env response and arthritis has been proposed (Knowles et al., 1990 ). In particular, goats with progressive arthritis have been found to have high serum titres to the transmembrane envelope subunit TM (McGuire et al., 1992
). We previously described two immunodominant epitopes of CAEV TM that elicit an antibody response associated with the appearance of disease (Bertoni et al., 1994
). In the present study we performed an extensive analysis of the immunogenic domains of the CAEV Env in order to identify the targets of the natural antibody response to CAEV infection. In addition to the epitopes previously described (Bertoni et al., 1994
) we identified five antigenic sites in the SU and six in the TM (two new epitopes) subunit by screening a
gt11 random Env expression library and recombinant proteins. We followed the development of the antibody response to the SU epitopes in experimentally infected goats. Although we observed a strong and rapid response to three epitopes of the C-terminal part of the SU domain, we found no correlation between serum reactivity and neutralizing activity. Finally, screening of a panel of sera from naturally infected goats with recombinant peptides revealed a restricted, type-specific response to the immunodominant epitopes of the SU subunit consistent with their localization on variable regions. Our results will permit a further functional dissection of the humoral response to the continuous B-cell epitopes of CAEV Env to address the role played by antibodies in protection against CAEV infection or in its pathogenesis.
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Methods |
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Twelve Saanen goats between 6 and 24 months of age were included in the experimental infection. Six goats were infected with 1 ml supernatant (107 TCID50/ml) of goat synovial membrane (GSM) cells transfected with the CAEV-CO molecular clone (Pyper et al., 1986 ); 0·5 ml was injected intracarpally, 0·5 ml intravenously. The remaining six goats were mock-infected with culture supernatant of non-transfected GSM cells.
Env expression library.
Construction of the random Env expression library used for epitope mapping was described previously (Bertoni et al., 1994 ).
The env gene was derived from an infectious clone of CAEV-CO (Pyper et al., 1986 ). The positions of the nucleotides (bp) and amino acids (aa) defining different Env regions in this paper refer to the sequences published by Saltarelli et al. (1990)
.
Briefly, random DNA fragments of an average length of 200 bp were isolated, blunt-ended, ligated with EcoRI linkers and subsequently cloned in EcoRI-digested gt11 DNA arms. Ligations were packaged and plated onto E. coli Y1090 to express the Env fragments as
-galactosidase fusion proteins from the recombinant phages. All phages contained inserts of different sizes, ranging from 93 to 285 bp. The representativeness of the library was demonstrated by hybridization with radioactively labelled fragments covering the entire sequence of CAEV-CO Env.
Screening procedure to detect immunoreactive plaques.
Our previous mapping strategy (Bertoni et al., 1994 ) was aimed at detecting group-specific epitopes and had been performed with sera obtained from goats infected with field strains of CAEV. In contrast, in this study we used sera of animals experimentally infected with the CAEV strain (CAEV-CO) that we had used to create the Env expression library. This strategy was chosen to warrant the detection of type-specific epitopes that can be missed when heterologous sera are used. The library was screened using serum 89G38 and a pool of four sera (91G27, 91G31, 91G33, 91G34) obtained from animals experimentally infected with a biologically cloned CAEV-CO. These sera and their neutralizing activity have previously been described (Lichtensteiger et al., 1991
). Approximately 3x104 p.f.u. from the original, unamplified library was plated onto E. coli Y1090 (5000 p.f.u. per plate) and incubated for 4 h at 42 °C. Plates were then overlaid with nitrocellulose filters saturated with 10 mM IPTG and further incubated for another 3 h at 37 °C to induce
-galactosidase fusion protein expression. Immunological screening of the filters was performed as described previously (Pancino et al., 1993
). Briefly, the filters were processed and incubated with the two sera, and the immunoreactive plaques were recovered from agar plugs and purified for two additional runs. The positive plaques were isolated and the PCR-amplified inserts were sequenced.
PCR and sequencing.
A total of 10 µl of phage lysate was diluted with 60 µl of water and boiled for 10 min. After microcentrifugation, 15 µl of supernatant was added to 85 µl of PCR mixture containing 50 pM of each of the primers mentioned below, 10 nM of each deoxynucleoside triphosphate and 0·5 U of Taq polymerase (Perkin Elmer) in a solution of 10 mM TrisHCl, 50 mM KCl and 2 mM MgCl2. The gt11 primers used were complementary to the
-galactosidase portion of the
gt11 template (forward, 5' GGTGGCGACGACTCCTGGAGCCCG 3'; reverse, 5' TTGACACCAGACCAACTGGTAATG 3'). PCR was carried out for 32 cycles (cycle 1: 94 °C, 2 min; cycles 231: 94 °C, 15 s/55 °C, 30 s/72 °C, 30 s; cycle 32: 72 °C, 3 min). For sequencing, the PCR products were run on a 1·8% agarose gel (FMC) and purified with QIAEX beads (QIAGEN) according to the manufacturers protocol. The fragments were sequenced using the ABI PRISM fluorescent sequencing kits on an ABI PRISM 310 Genetic Analyser (Perkin Elmer) according to the manufacturers protocol.
Synthetic peptide.
A synthetic peptide of 17 aa (Rev-1: aa 2137, bp 60726122) included in the first CAEV-Rev exon (60126123) was synthesized and purified by Neosystem, Strasbourg, France. This peptide contains numerous charged amino acids and was therefore predicted to be hydrophilic and exposed at the surface of the molecule. Sera of rabbit immunized with KLH-coupled Rev-1 peptide developed high titres of antibody to the peptide and were used in a Western blot to detect the recombinant proteins SU1 and SU1-signal.
Expression of recombinant proteins.
The SU antibody-binding domains SU1 to SU5, identified by screening the Env library, and two shorter peptides in the SU1 domain (SU1-signal and SU1-mature), as well as the unreactive regions located between the SU2 and SU3 (SU23) and the SU3 and SU5 (SU35) domains were expressed as recombinant proteins.
Two expression systems were used. Seven recombinant proteins (SU1, SU1-signal, SU2, SU23, SU3, SU4, SU5) were expressed using the pET14b vector, which allows the expression of proteins fused at the amino terminus either with 19 additional aa for the constructs cloned in the NdeI site, or with 23 aa for the constructs cloned in the BamHI site. This amino-terminal tail contains six histidine residues in a row, which allow the expressed proteins to be purified using nickel-coupled Sepharose beads.
Three proteins (SU35, SU35 amino terminus and SU1-mature) could not be expressed in this vector but were successfully expressed using the pGEX-4T-1 plasmid, which allows the expression of proteins fused at their amino terminus with the 29 kDa glutathione S-transferase (GST) protein.
Fragments corresponding to the CAEV-CO Env regions to be expressed were amplified using a standard PCR technique, with primers containing the cloning sites (NdeI or BamHI added to the 5' primer, BamHI added to the 3' primer for the pET14b vector and EcoRI or XhoI for the pGEX-4T-1 vector), and were purified and cloned using standard techniques (Sambrook et al., 1989 ).
The wild-type SU5-1163M, SU5-615 and SU5-1355 fragments were cloned by RTPCR starting from total RNA isolated from infected GSM cells using TRIzol reagent (Gibco BRL) according to the manufacturers protocol. cDNA synthesis was performed using SuperScript RNase H- RT (Gibco BRL) at 200 U/50 µl according to the manufacturers protocol. The SU5 fragments were amplified by nested PCR using the external primer pairs #423 forward, 5' GGAGCAGAAATAATMCCWGAARSTATG 3', and #425 reverse, 5' TGCRGCAGCKAYTATTGCCATGAT 3', followed by amplification with the internal primer pairs containing in the 5' extensions (in bold), the required BamHI cloning sites and, in the reverse primer, a stop codon (TAA): #442 forward 5' TATGGATCCGGGTAGGGTAAAGGCACAATATAGT 3' and #443 reverse 5' TATAGGATCCTTAAAGCACCATTACTAACCCTATTC 3' for the isolate 1163M, and #444 forward 5' TATGGATCCGACAAGAGTGAAAGCACAGTACAGC 3' and #445 reverse 5' TATAGGATCCTTAGAGCATTATGACCAAGCCAACGC 3' for the 615 and 1355 isolates. The heat-soaked PCR protocol described by Ruano et al. (1992) was applied. Finally, these wild-type SU5 peptides were expressed using the pET14b vector as described above. The different plasmids were electroporated in the appropriate host cells [BL21(DE3) (Novagen) for pET14b and DH5
(Gibco BRL) for pGEX-4T-1 constructs] and immediately used for protein expression according to the manufacturers protocol. Purification of the recombinant proteins was performed using protocols suggested by the manufacturers for the purification of denatured proteins.
Western blot analysis.
Western blot was used to follow the development of antibodies against the SU epitopes in experimentally infected goats and to test this antibody response in naturally infected goats. Purified SU1, SU1-signal, SU2, SU23, SU3, SU4 and SU5 peptides expressed as 6xHis fusion proteins and SU1-mature and SU35 peptides expressed as GST fusion proteins were migrated onto 12% SDSpolyacrylamide gels and transferred to nitrocellulose filters. The nitrocellulose strips were incubated overnight at 4 °C with 1:50 to 1:800 diluted goat sera and further processed as described elsewhere (Bertoni et al., 1994 ). Sera collected before experimental infection and a pool of sera from CAEV-negative goats were used as antibody controls. Additionally, the co-localization of the immunoreactive bands with the fusion proteins was verified with alkaline phosphatase-labelled nickel (NiNTAAP, QIAGEN) for the 6xHis recombinant proteins, and a murine monoclonal antibody to GST for the GST fusion proteins. A polyclonal rabbit serum to CAEV-Rev, obtained by immunizing two rabbits with a synthetic peptide of 17 aa (aa 2137, bp 60726122) included in the first CAEV-Rev exon (60126123), was used for the recombinant SU1 and SU1-signal peptides.
Neutralization assay.
Neutralization assays were performed using the virus reduction neutralization assay described previously (McGuire et al., 1988 ). Neutralizing antibodies were detected by mixing 0·4 ml of goat serum heat-inactivated for 30 min at 56 °C with 0·4 ml of MEM supplemented with 2% FCS and containing 104·5 or 103·5 TCID50, respectively. To determine serum neutralizing antibody titres a constant quantity of virus (101·5 TCID50) was incubated with a fourfold dilution of serum. Non-neutralized virus was detected on GSM cells (McGuire et al., 1988
).
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Results |
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None of the sera reacted with the SU23 peptide, which confirms the results of library screening, suggesting that this region does not contain any continuous B-cell epitopes. Conversely, three out of six sera from the experimentally infected goats reacted with the SU35 peptide (Table 3 and Fig. 3
). The other three sera could not be scored on this peptide because of their reactivity with the GST fusion partner. The sequence comparison of several CAEV and maedivisna virus (MVV) isolates revealed the carboxy-terminal region of this peptide to contain a highly variable region (Knowles et al., 1991
; Valas et al., 1997
; Leroux et al., 1997
; Zanoni, 1998
). We expressed this region as a recombinant protein using the pET14b expression vector (SU4: aa 491575, bp 74857736) and tested its reactivity in Western blot. The sera from all six experimentally infected animals and six sera from naturally infected goats recognized this peptide (Table 3
and data not shown). Thus, this peptide contains the dominant epitope of the SU35 region. By expressing the remaining amino-terminal portion of the SU35 (SU35 amino terminus: aa 346497, bp 70477502) as a GST-fusion protein we observed a weak reactivity with sera from two of six experimentally infected animals, with serum 89G50 from one animal immunized with a CAEV-CO Env-expressing vaccinia virus (Cheevers et al., 1994
) and with serum #4663 from a naturally infected animal. The additional 15 sera tested, which were selected on the basis of their strong reactivity to at least one of the SU epitopes, did not react with this peptide (data not shown). Serum 89G38, which was used to screen the peptide library, was negative on the SU4 peptide as well as on the SU35 and SU35 amino terminus peptides (data not shown), which explains why we were unable to detect this epitope in our expression library. The pool of sera used, in parallel with 89G38, to screen the Env library, was positive on the SU4 peptide. The higher background found with these sera, however, allowed us to select only the most reactive clones during the library screening.
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Development of antibody response to SU epitopes in CAEV infection
Recombinant proteins corresponding to the following regions of the SU were expressed and purified, SU1: aa 21112, bp 60726348; SU2: aa 114184, bp 63496564; SU23: aa 184254, bp 65616773; SU3: aa 246351, bp 67487065; SU35: aa 346569, bp 70477718; SU4: aa 491575, bp 74857736; SU5: aa 565635, bp 77207907. We used rather large peptides and not the epitopes defined by the minimal overlapping sequences described in the first section of the results because, in contrast to the phage expression library, these SU peptides are expressed fused to a minimal fusion partner (6xHis) and not to -galactosidase. The addition of residues flanking the epitopes would preserve the reactivity of antibody binding to the edges of the epitopes. The kinetics of seroconversion to the epitopes contained in these recombinant proteins was monitored by Western blot from 2 weeks to 3·5 years post-infection using the sera of six goats experimentally infected with the CAEV-CO molecular clone whose Env was used to generate the epitope library. The results obtained at 4 weeks and at 3·5 years post-infection with sera of goat #3 are presented in Fig. 3
and the kinetics of seroconversion for all six animals are summarized in Table 3
. ELISA was also used in order to detect potential additional reactivity to peptides in non-denaturing conditions. Although ELISA could not be performed on all the sera due to strong background reactivity to E. coli proteins contaminating the recombinant peptides, the results obtained in ELISA matched the Western blot data (data not shown).
These data can be summarized as follows. (1) SU1 appears to be a minor epitope inducing late seroconversions. Indeed, only five of six animals seroconverted to the SU1 epitope and this occurred a long time after infection. (2) SU2 reactivity was detected only in goat #6 and then only several months after infection. Three of six sera clearly reacted with the SU35 peptide, whereas the remaining three sera could not be scored due to their reactivity with the GST fusion partner (data not shown). (3) Seroconversion to the SU4 peptide was observed in all six goats, albeit at different time-points: goat #5 seroconverted 2 weeks post-infection, goats #1, 2, 4 and 6 seroconverted 4 weeks post-infection and goat #3 more than 1 year post-infection. The SU3 and SU5 epitopes were the most immunogenic. Five animals had antibody against SU3 and SU5 peptides as early as 2 weeks after experimental infection and all six goats developed a strong and sustained response to both epitope regions.
Sera from the six experimentally infected goats taken at 3·5 years post-infection were tested for neutralizing activity in order to establish a correlation between the response against the SU epitopes and the development of neutralizing antibodies. Sera were tested for neutralizing activity using the virus reduction assay. Four goats did not show any neutralizing activity and two goats (#1 and #5) showed low titre neutralizing antibody (1:4). These results confirm that in CAEV-infected animals the overall neutralizing activity is very low and that the strong reactivity observed with some SU epitopes (SU3, SU4 and SU5) in sera from experimentally infected goats is unlikely to be associated with neutralization.
Reactivity of field sera to SU3, SU4 and SU5
Thirty-two sera from Swiss and Italian goats naturally infected with CAEV, selected for their strong reactivity to at least one Gag protein (p28, p18 and p15) in Western blot (Zanoni et al., 1989 ), were tested for reactivity to the SU3, SU4 and SU5 peptides. In summary, four sera reacted to SU3, 11 sera to SU4, only two sera reacted to SU5 and 20 sera did not react with any of these epitopes (data not shown). This suggests that these epitopes are type-specific, albeit at different stringency levels. SU5 showed the most restricted pattern of reactivity, whereas SU4 displayed a broader reactivity with this panel of sera.
The restricted pattern of reactivity with these SU epitopes may be due either to infections with viruses phylogenetically distant from the CAEV-CO isolate or to different genetic backgrounds of the naturally infected goats.
To distinguish between these hypotheses we concentrated on the carboxy-terminal SU5 epitope, which showed the most restricted pattern of reactivity with the sera of the naturally infected goats tested. Four SU5-recombinant proteins derived from the CAEV-CO molecular clone and from three CAEV field isolates, CH-1355, CH-615 and CH-1163M, were cloned and expressed as recombinant peptides. Sixteen sera of naturally infected goats (Fig. 4a) were tested in Western blot on SU5-CO, -1355, -615 and -1163M peptides, respectively. As shown in Fig. 4(a)
, all control sera (P1 to P4) reacted with their specific epitopes. With few exceptions, the sera sampled from different geographical regions (Fig. 4a
, AG) showed a type-specific reactivity. Interestingly, the three Swedish sera (Fig. 4a
, E) showed a weak but specific reactivity only to the SU51163M peptide.
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Discussion |
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An analysis of the antibody response to the SU epitopes in experimentally infected animals revealed that the most immunogenic epitopes of SU (SU3, SU4 and SU5) are located in the C-terminal part of the molecule. These results are in agreement with those obtained in the analysis of the antibody response to SU epitopes in goats infected with field isolates (Valas et al., 2000 ). While serum reactivity against the correspondent peptides developed early post-infection and was sustained during the 42 months of observation in infected goats, no significant neutralizing activity appeared to be associated with this response. Indeed, of the six experimentally infected goats, only goats #1 and #5 developed weak titres of neutralizing antibody against the homologous virus. The sera from these two goats showed antibody responses to the SU peptides that were indistinguishable from those of the animals that did not develop neutralizing antibody at all (Table 3
). In particular, the reactivity to the SU4 peptide, corresponding to a highly variable region of the Env of small ruminant lentiviruses (Wain-Hobson et al., 1995
; Leroux et al., 1997
; Turelli et al., 1997
; Valas et al., 1997
) that was proposed to contain the putative counterpart of the V3 neutralizing domain of HIV-1 (Knowles et al., 1991
), did not appear to be the target of a neutralizing response. Recently, Skraban et al. (1999)
described a type-specific, conformation-dependent neutralizing epitope in MVV. This epitope is localized between aa 525 and 552 and mapped in the MVV counterpart of our CAEV-SU4 peptide (aa 491575). The authors proposed a model consistent with the existence of a conformation-dependent epitope centred on a cysteine loop highly conserved in MVV and in CAEV, which are closely related lentiviruses of sheep and goats, respectively. In MVV, this loop is flanked by two immunodominant linear epitopes not directly involved in neutralization which mapped to the CAEV-SU4 region described in this paper. Our results do not exclude the presence of such a conformation-dependent neutralizing epitope in the CAEV-SU4 region. However, as in MVV, the reactivity to linear epitopes in SU4 is unlikely to be involved in neutralization. Indeed, the sera of goats #2 and #4, which showed a strong reactivity to the SU4 peptide, did not have any neutralizing activity. Moreover, serum 89G38, which displays a strong neutralization activity in relation to other anti-CAEV sera (Lichtensteiger et al., 1991
; data not shown), did not bind to the homologous SU4 peptide.
Serum reactivity to the SU3, SU4 and SU5 domains appeared type-specific. Indeed, these antigenic domains correspond to variable regions of the SU with high rates of amino acid substitutions (Fig. 5; Leroux et al., 1997
; Turelli et al., 1997
; Valas et al., 1997
). Although this indicates that selective pressure for change exists, the absence of a consistent neutralizing response suggests that neutralizing antibody may not be the driving force accounting for the variability of these regions. This differs from other lentivirus infections, such as HIV-1 or EIAV infections, in which the neutralizing response drives the appearance of antigenic escape variants and, at least partly, accounts for the variability of immunodominant domains, like the principal neutralizing domains of HIV-1 gp120 or EIAV gp90 (Goudsmit et al., 1991
; Zheng et al., 1997
). In CAEV infection neutralizing antibody develops slowly, in only a minority of the infected individuals and at low titres. Although neutralization escape mutants have been described (Ellis et al., 1987
; McGuire et al., 1988
), neutralizing antibody does not appear to control virus replication in vivo: indeed, cell free virus was isolated from the synovial fluid of goats that had neutralizing antibody to the isolated virus (Cheevers et al., 1993
). Therefore, variability in these SU antigenic regions can depend on selection by immune mechanisms other than neutralizing antibody or it can occur independently of the immune response, as has been suggested for different RNA viruses (Domingo et al., 1993
). Small ruminant lentiviruses and especially CAEV are transmitted from the mothers to the kids predominantly via infected colostrum. Vertical transmission and animal breeding in a relatively restricted pool of animals, as shown by pedigree analysis, favour the creation of geographical and/or breeding niches for variant viruses. Accordingly, the variability observed in certain Env regions, such as SU5, of CAEV isolates of different geographical provenance may be the result of the adaptation of viruses to genetically relatively homogeneous populations. The uniform reactivity to a region-specific SU5 peptide observed in the experiments performed on three field isolates from goats of different Swiss cantons (Ticino, Fribourg and Berne) (Fig. 4 a
) supports this hypothesis, which has yet to be confirmed by molecularepidemiological studies of the circulating viruses.
The relatively strict type-specific reactivity of the SU3SU5 immunodominant domains (Fig. 4 and data not shown) suggests that peptides corresponding to these epitopes and particularly to different SU5 sequences may be used to serotype field viruses. Using peptides corresponding to the highly variable V3 loop region of HIV-SU, investigators have shown that in some instances serotyping can substitute the more cumbersome genotyping of virus strains (Barin et al., 1996
; Plantier et al., 1999
). The development of new methods for serotyping field isolates of small ruminant lentiviruses would be a useful tool to support the eradication programs of these viruses. Furthermore, the rapid seroconversion observed with the SU3 and SU5 peptides in experimentally infected animals makes these epitopes interesting candidates for a serological diagnosis of recent infections. This would complement a diagnostic method based on the broadly reacting epitope TM3, which induces a slower seroconversion in some infected goats (Bertoni et al., 1994
; unpublished results). Taking into account the type-specific reactivity of the SU epitopes, a diagnostic use would require a further definition of the reactive sequences and mixtures of different peptides (Baillou et al., 1993
).
The biological significance of the anti-Env antibody response in CAEV infection is unclear: the sustained non-neutralizing response against the immunodominant linear epitopes of the CAEV Env observed here could simply deflect the antibody response from domains more critical for virus replication. However, in CAEV infection non-neutralizing antibodies may be directly involved in the pathogenic mechanisms of chronic inflammation in infected goats, as suggested by the association of anti-Env antibodies with the degree of severity of arthritis (Knowles et al., 1990 ; McGuire et al., 1992
). The identification of Env continuous epitopes represents a valuable tool for investigating the contribution of epitope-specific antibody to the pathogenesis of CAEV-induced arthritis. We are now in the process of immunizing goats with the recombinant peptides described here in order to produce monospecific sera for analysing the effect on the in vitro replication of CAEV and also to study the impact of the antibody response on CAEV infection of goats.
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Acknowledgments |
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Footnotes |
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c Present address: Unité de Biologie des Rétrovirus Département de Virologie, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.
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Received 25 May 2000;
accepted 7 August 2000.