INGENASA, Hnos García Noblejas 41, 28037 Madrid, Spain1
Institute for Animal Science and Health (ID-Lelystad), Department of Molecular Recognition, Edelhertweg 15, 8219 PH Lelystad, The Netherlands2
Author for correspondence: Jorge Martínez-Torrecuadrada. Fax +34 91 4087598. e-mail jmartinez{at}ingenasa.es
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Abstract |
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Introduction |
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The virion consists of an outer layer composed of proteins VP2 and VP5, which surround an icosahedral core containing two major proteins, VP3 and VP7, three minor proteins, VP1, VP4 and VP6, and ten segments of double-stranded RNA (Roy et al., 1994 ). VP2 is the most variable protein of the virion, sharing only 1924% of identical amino acids with the VP2 species of other orbiviruses, such as bluetongue virus (BTV) and epizootic haemorrhagic disease virus (Iwata et al., 1992
). The overall identity index score rises to 33·4% when the VP2 sequences of different AHSV serotypes sequenced to date (AHSV-3, -4, -6 and -9) are compared (Venter et al., 2000
). VP2 is the main determinant of a serotype-specific immune response (Bremer et al., 1990
) and, together with VP5, is the major target of the neutralizing response of the host (Burrage et al., 1993
; Martínez-Torrecuadrada et al., 1994
, 1999
; Vreede & Huismans, 1994
). In BTV and AHSV VP5, most of the neutralization epitopes have been described to be probably conformational (Martínez-Torrecuadrada et al., 1999
; Rossitto & MacLachlan, 1992
).
Since there is no known therapy for the disease, vaccination is essential to protect horses against AHSV and to avoid new outbreaks. Current vaccines for AHSV include attenuated and inactivated virus vaccines. In general, these vaccines are quite effective. However, the use of attenuated vaccines does not allow the differentiation of infected animals from vaccinated animals and therefore affects the international trade of horses, and could contribute to the spread of the disease by insect vectors. Subunit vaccines would potentially avoid most of these concerns.
The efficacy of synthetic peptides is usually lower than that of conventional vaccines, since B cell epitopes are generally conformational structures and mimicking these epitopes with short peptides is difficult. Nevertheless, in other diseases, anti-peptide antibodies were protective and, therefore, make a peptide vaccine feasible (Dalsgaard et al., 1997 ; Langeveld et al., 1994
). Thus, it is valuable to identify antigenic and neutralizing regions on VP2 and to test whether synthetic peptides representing these regions can induce neutralizing antibodies. The precise location of antigenic determinants, especially neutralizing epitopes, on AHSV VP2 would be a prerequisite for the development of efficacious synthetic peptide vaccines to control AHSV infection.
A previous study using overlapping fragments of AHSV-4 VP2 expressed in Escherichia coli and tested with a collection of antibodies demonstrated that the major antigenic domain of VP2 was located in a central region (aa 199414) and that neither the N-terminal (aa 1199) nor the C-terminal (aa 4141060) domains were immunogenic (Martínez-Torrecuadrada & Casal, 1995 ). A region comprising aa 254414 (fragment H) was able to elicit consistently high titres of neutralizing antibodies. The capability of several subfragments of this region to evoke neutralizing antibodies suggested the presence of several sites inside this domain.
The present study aimed to define more precisely the location of linear neutralizing epitopes in AHSV-4 VP2. The sequence of VP2 from aa 199 to 689, which was previously found to harbour antigenic determinants (Martínez-Torrecuadrada & Casal, 1995 ), was used as the template for the synthesis of overlapping peptides. To this end, PEPSCAN analysis was carried out using a panel of sera from different sources. Reactive peptides were then selected and tested for their capability to induce neutralizing antibodies in rabbits. As will be shown, enhancement of the neutralization effect was observed when combinations of peptides were used for immunization.
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Methods |
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Peptides and conjugation to carrier proteins.
Peptides were synthesized with an N-terminal cysteine and an amidated C terminus as well as an acetylated N terminus following Meryl procedures using Fmoc amino acids and Rink resin. Peptides were released from the resins and protecting side chains using trifluoroacetic acidwater, in the presence of thioanisole and phenol. Subsequently, precipitation was performed in diethyletherpentane (1:1) for 15 min at -20 °C and 30 min at 4 °C. After centrifugation, pellets were washed and centrifuged in the presence of diethyletherpentane. Pellets were dried under a stream of air, dissolved in acetonitrilewater (1:1) and finally lyophilized.
Purified peptides were conjugated to KLH using m-maleimidobenzoic acid N-hyroxysuccinimide ester as the divalent linking agent (Langeveld et al., 1994 ). In general, 1 mg of peptide was coupled to 1 mg of KLH. To remove free peptides, the final product was dialysed against PBS, pH 7·2. Coupling yields varied between 50 and 350 µg/mg of KLHpeptide, as determined by amino acid analysis using the PicoTag method (Waters).
Immunization of animals with synthetic peptides.
Rabbit immunizations were carried out by injections of 50350 µg of peptide through intramuscular and subcutaneous routes at days 0 (complete Freunds adjuvant) and 42 (incomplete Freunds adjuvant). Blood samples were collected at day 0 and 10 days after the second immunization. All sera were checked for anti-peptide antibodies by indirect ELISA using the corresponding peptide as antigen source. Sera for testing of in vitro AHSV neutralization activity were taken on day 56.
ELISA.
An indirect ELISA was used to detect anti-peptide antibodies. ELISA procedures for testing antibody responses to peptide-related epitopes were described previously (Langeveld et al., 1994 ). Titres were estimated at serum dilutions yielding an absorbance value three times above the blank (pre-immune serum).
The accessibility of the antigenic sequences in the AHSV virion was also studied by indirect ELISA. Purified AHSV-4 was used as antigen source (Martínez-Torrecuadrada & Casal, 1995 ). Briefly, polystyrene plates (Labsystem) were coated with 0·5 µg of AHSV-4 diluted in 50 mM carbonate buffer, pH 9·6, overnight at 4 °C. Plates were incubated at 37 °C for 1 h with the corresponding anti-peptide rabbit serum diluted in blocking buffer (350 mM NaCl, 0·05% Tween 20 in PBS). Peroxidase-labelled protein A (Sigma) was diluted 1:1000 in blocking buffer and incubated at room temperature for 1 h. Washes between incubations were performed with PBS containing 0·05% Tween 20. Peroxidase activity was detected by adding 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) as substrate and stopped after 10 min with 2% SDS. The absorbance values of the samples were determined at 405 nm (Bio-Tek Instruments).
In vitro neutralization assays.
To determine the ability of the specific anti-peptide rabbit sera to neutralize the virus in vitro, a monolayer protection assay on Vero cells was carried out as described previously (Martínez-Torrecuadrada & Casal, 1995 ). End-point titration was calculated as the value of the highest serum dilution that caused a 50% reduction in the cell monolayer.
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Results |
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By using sera from animals immunized with the whole protein or with fragment D, two antigenic regions were clearly identified. One antigenic region stretched from aa 223 to 400 and the other one stretched from aa 568 to 681. Sera raised against fragments L or H gave similar profiles (Fig. 1, mouse sera). However, some differences were observed in the chromatograms from rabbit sera immunized with recombinant VP2 alone or in combination with VP5. When VP5 was included in the immunizing mixture, the resulting serum recognized three extra VP2 antigenic sites (5, 10 and 13) that the serum against VP2 alone was not able to recognize, suggesting that the combination of both proteins created new antigenic sites.
Finally, sera from immunized, vaccinated or experimentally infected horses with a high neutralizing activity (titres >1000) were also tested. Only one serum sample, from a horse immunized with recombinant VP2, was able to react with the dodecapeptides (antigenic site 15) (Fig. 1, horse sera). Neither sera from infected horses nor sera from vaccinated horses showed any reactivity, suggesting that the natural host sera are recognizing either conformational or longer epitopes.
Immunogenicity of PEPSCAN-mapped VP2 peptides
The potential of these 15 antigenic sites to elicit neutralizing antibodies was analysed by immunization of rabbits with the peptides indicated in Fig. 2 and Table 2
. Each peptide was synthesized, coupled to KLH and injected into two rabbits. All rabbits developed antibodies to the peptides with which they were immunized (data not shown). Three peptides, corresponding to antigenic sites 8, 11 and 12, were able to elicit neutralizing antibodies with titres ranging from 1/4 to 1/8. These titres are lower than those obtained with rabbit sera against fragment H (1/1280) (Martínez-Torrecuadrada & Casal, 1995
), which contains the sequences of the three neutralizing peptides. Only two neutralization sites were identified, since the two peptides corresponding to antigenic sites 11 and 12 overlapped each other by 14 amino acid residues. These two sites, named a and b, were located in the sequence of VP2 at positions 321339 and 377400, respectively (Fig. 2
).
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Discussion |
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A total of 15 antigenic sites was identified and these could be grouped in two clusters. The first, covering aa 223400, contains 12 of the 15 antigenic sites, confirming that this region of VP2 is a hot antigenic spot. The second covers aa 568681 and is defined by three reacting peptides. In other serotypes, other antigenic domains have been identified. Most of them were also in the N-terminal half of the molecule. In AHSV-3 VP2, several antigenic regions were identified by phage display, scattered mainly between residues 175 and 563 (Bentley et al., 2000 ). By analysing the reactivity of baculovirus-expressed truncated VP2 peptides with AHSV-9-specific serum, a linear epitope was located between residues 369 and 403 in serotype 9 (Venter et al., 2000
). Homologous sites are also present in VP2 of BTV (Gould & Eaton, 1990
; Gould et al., 1988
; Jewell & Mecham, 1994
).
Of the 14 MAbs tested, only six were reactive with peptides, indicating that most of the MAbs were recognizing conformational epitopes. In contrast, mouse sera raised against E. coli-derived fragments were extraordinarily effective at defining linear antigenic sites. An interesting observation was that the VP2 PEPSCAN profiles obtained with rabbit sera elicited against baculovirus-expressed VP2 alone, or in combination with VP5, were slightly different. This result is consistent with a model in which VP5 would interact with VP2 and lead to a better exposition of the neutralizing epitopes of VP2 and, therefore, enhance the immune response (Marshall & Roy, 1990 ; Martínez-Torrecuadrada et al., 1996
; Roy et al., 1990
).
The fact that the equine sera do not bind to small linear peptides was also observed previously in the AHSV-4 VP5 epitope mapping, carried out with horse sera and dodecameric synthetic peptides (Martínez-Torrecuadrada et al., 1999 ). Here, only one serum out of ten gave a slightly positive reaction with a set of peptides (antigenic site 14). This failure supports the hypothesis given previously that horses preferably recognize discontinuous epitopes or linear sequences longer than 12 residues.
Of the 15 synthetic peptides, three were able to elicit reproducibly neutralizing antibodies as KLHpeptide conjugates, albeit at low levels insufficient to guarantee protection. Two neutralizing epitopes (a and b) were defined. The location of both sites is in agreement with those results obtained previously, in which the region spanning residues 254414 (fragment H) would contain several neutralizing sites (Martínez-Torrecuadrada & Casal, 1995 ), since subfragments of this domain (fragments L and M) were able to induce neutralizing responses. Neutralizing site a maps within fragment L (aa 284380), whereas site b corresponds to the N terminus of fragment M (aa 380414) (Fig. 2
).
No assignment of neutralizing epitopes in other AHSV serotypes has been carried out to date. However, sites a and b have been described as antigenic in AHSV-3 VP2 due to their reactivity with virus-specific chicken IgY (Bentley et al., 2000 ) and site b in AHSV-9 VP2 mapped with guinea pig anti-AHSV-9 (Venter et al., 2000
). Therefore, these antigenic regions mapped previously in AHSV-3 and AHSV-9 could contain neutralizing determinants. Also, neutralizing epitopes located in these domains have been described in BTV, specifically between residues 328 and 335 (Gould & Eaton, 1990
), and 327 and 402 (DeMaula et al., 1993
). Altogether, these results confirm this region to be a major antigenic determinant in orbiviruses and an important target for neutralizing antibodies.
The immunogenic epitopes identified here would be expected to be on the exposed surface of the AHSV capsid, since neutralizing epitopes generally bind to the virus surface to prevent virus binding to cellular receptors and virus uptake into the cell. The hydrophobicity profile indicates that neutralizing sites a and b are present in the most hydrophilic areas of the protein, with a high probability of being in the surface (Fig. 5). Indeed, all neutralizing antisera reacted with AHSV by ELISA, confirming the exposed location of the neutralizing domains. Antigenic site 11 and consequently neutralizing site b was defined by two neutralizing MAbs, 10BC12 and 10BG7, as well as other mouse sera. However, the antisera against antigenic site 3, recognized by neutralizing MAb 8DH3, did not neutralize the virus. This observation may reflect the lack of exposure of the 8DH3 epitope on the surface of virion. On the other hand, antigenic site 4 seems to be well-exposed on the virus surface but neutralizing activity is absent in the antisera. Although exposure does not necessarily have to coincide with the inhibition of virus infectivity, this site might not be represented fully by peptide 279297 and, therefore, it could be optimized to achieve some neutralizing activity.
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We cannot rule out better results if other peptides from site a, such as peptide 323341, are tested for neutralization, together with peptides of site b. Also, combinations with other peptides representing neutralizing epitopes on other capsid proteins, e.g. VP5 (Martínez-Torrecuadrada et al., 1999 ), should be analysed. Studies to evaluate these hypotheses are currently under way. While neutralization induction has been investigated with synthetic peptides from VP5, including the neutralizing epitopes published previously (Martínez-Torrecuadrada et al., 1999
), this did not lead to any candidate peptides. These findings would be the starting point for the production of a synthetic peptide vaccine against AHSV, although chances of success seem to be very limited, probably due to the complex proteinprotein interactions in the outer shell of the virion. This would include intermolecular interactions between VP2 and itself and between VP2 and VP5, plus the VP7 trimer spikes that are accessible for possible antibody binding (Basak et al., 1996
; Grimes et al., 1995
; Hewat et al., 1992
). These complex interactions would explain the absence of reactivity of the horse sera with single peptides of individual proteins.
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Acknowledgments |
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Footnotes |
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References |
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Received 7 March 2001;
accepted 8 June 2001.