Definition of neutralizing sites on African horse sickness virus serotype 4 VP2 at the level of peptides

Jorge L. Martínez-Torrecuadrada1, Jan P. M. Langeveld2, Rob H. Meloen2 and J. Ignacio Casalb,1

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The antigenic structure of African horse sickness virus (AHSV) serotype 4 capsid protein VP2 has been determined at the peptide level by PEPSCAN analysis in combination with a large collection of polyclonal antisera and monoclonal antibodies. VP2, the determinant for the virus serotype and an important target in virus neutralization, was found to contain 15 antigenic sites. A major antigenic region containing 12 of the 15 sites was identified in the region between residues 223 and 400. A second domain between residues 568 and 681 contained the three remaining sites. These sites were used for the synthesis of peptides, which were later tested in rabbits. Of the 15 synthetic peptides, three were able to induce neutralizing antibodies for AHSV-4, defining two neutralizing epitopes, ‘a’ and ‘b’, between residues 321 and 339, and 377 and 400, respectively. A combination of peptides representing both sites induced a more effective neutralizing response. Still, the relatively low neutralization titres make the possibility of producing a synthetic vaccine for AHSV unlikely. The complex protein–protein interaction of the outer shell of the viral capsid would probably require the presence of either synthetic peptides in the correct conformation or peptide segments from the different proteins VP2, VP5 and VP7.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
African horse sickness virus (AHSV) belongs to the Orbivirus genus within the family Reoviridae (Mayo & Pringle, 1998 ). AHSV is transmitted to vertebrates by Culicoides midges and causes an acute disease in horses and other equids, with a high mortality rate. The disease is confined to sub-Saharan Africa and the Middle East, although periodic epizootics have caused severe outbreaks of the disease outside enzootic regions. To date, nine serotypes of AHSV have been defined on the basis of serum cross-neutralization tests (Howell, 1962 ). AHSV serotype 4 was the strain responsible for the last outbreaks in Spain and Portugal from 1987 to 1990.

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 19–24% 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 199–414) and that neither the N-terminal (aa 1–199) nor the C-terminal (aa 414–1060) domains were immunogenic (Martínez-Torrecuadrada & Casal, 1995 ). A region comprising aa 254–414 (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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Monoclonal antibodies and polyclonal sera.
The antibodies used in PEPSCAN analysis and their neutralizing abilities are shown in Table 1. Murine monoclonal antibodies (MAbs) were developed as described previously (Martínez-Torrecuadrada et al., 1996 ). Mouse and rabbit sera were prepared by immunizing animals with E. coli-derived VP2-based fragments (Martínez-Torrecuadrada & Casal, 1995 ) and from rabbits immunized with baculovirus-derived VP2 and VP2/VP5 (Martínez-Torrecuadrada et al., 1994 ). Horse sera were obtained from animals vaccinated with recombinant proteins and experimentally challenged or vaccinated with inactivated virus (Martínez-Torrecuadrada et al., 1996 ).


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Table 1. Antisera and MAbs used in PEPSCAN analyses

 
{blacksquare} PEPSCAN analysis.
Based on the sequence described for AHSV-4 VP2 (Iwata et al., 1992 ) and the location of neutralizing regions (Martínez-Torrecuadrada & Casal, 1995 ), a complete set of 480 overlapping dodecapeptides covering the VP2 region between residues 199 and 689 was prepared. Peptides were linked through the C terminus to polyethylene rods and immunoscreening was carried out by ELISA, as described previously (Geysen et al., 1984 ). A site was assigned as antigenic if the absorbance values measured at 405 nm of two or more consecutive peptides were at least twice the background. Background was calculated as the average of absorbance values measured for 20 consecutive peptides with a variation coefficient of <=20%.

{blacksquare} 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 acid–water, in the presence of thioanisole and phenol. Subsequently, precipitation was performed in diethylether–pentane (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 diethylether–pentane. Pellets were dried under a stream of air, dissolved in acetonitrile–water (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 KLH–peptide, as determined by amino acid analysis using the PicoTag method (Waters).

{blacksquare} Immunization of animals with synthetic peptides.
Rabbit immunizations were carried out by injections of 50–350 µg of peptide through intramuscular and subcutaneous routes at days 0 (complete Freund’s adjuvant) and 42 (incomplete Freund’s 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.

{blacksquare} 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).

{blacksquare} 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.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Definition of AHSV-4 VP2 antigenic determinants by PEPSCAN analysis
Mapping of antigenic determinants was carried out at the peptide level by analysing the reactivity of 480 overlapping 12-mer peptides, derived from the VP2 sequence comprising aa 199–689. A complete panel of sera and MAbs (Table 1) was used to identify reacting peptides by PEPSCAN analyses. Summarized in eight scans (Fig. 1), 15 antigenic sites were defined and numbered according to their locaton with regard to the N terminus. The amino acid sequences of each site and their exact positions in AHSV-4 VP2, together with the antibody samples that recognized every site, are presented in Table 2. All antigenic sites were recognized by various antibody samples, except site 12, which was only weakly recognized by one mouse serum sample against fragment H (aa 254–414). Antigenic site 8 showed the highest reactivity and was recognized by most mouse and rabbit sera tested.



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Fig. 1. Mapping of linear antigenic sites in AHSV-4 VP2 by PEPSCAN analysis. ELISA results obtained using antibody samples from mice, rabbits and horses with overlapping peptides covering the VP2 region comprising aa 199–689 are shown. The 15 antigenic sites are indicated and numbered according to their location with regard to the N- and C-terminus. Sequences involved in these antigenic sites are displayed in Table 2. The code for MAbs and the antigen source used to generate the corresponding polyclonal sera are situated on the left above each scan. Dark lines represent the length and position of the VP2 fragments used for animal immunization.

 

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Table 2. Antigenic sequences derived from AHSV-4 VP2 after PEPSCAN analysis

 
Of the 14 MAbs tested, six were able to react with sets of dodecapeptides, identifying several linear epitopes. The neutralizing MAb 8DH3 and the non-neutralizing MAb 8BC2 recognized peptides with common sequences: 272ERDDLSRETI281 and 273RDDLSR278, respectively, located within antigenic site 3. MAb 10BB4 reacted with the sequence 339WVKGMP344 in antigenic site 9. Likewise, peptides of antigenic site 11 were bound by neutralizing MAbs 10BC12 and 10BG7: the minimum antibody-reactive sequence was 383KGKWKE388. Neutralizing MAb SMAA reacted with peptides located within antigenic sites 7, 13 and 14, suggesting that the epitope recognized by this MAb would be discontinuous and composed of relatively distant residues that were brought together by the folding of the protein.

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 321–339 and 377–400, respectively (Fig. 2).



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Fig. 2. Relative locations of the PEPSCAN-defined antigenic sites and neutralizing epitopes on the sequence of AHSV-4 VP2. The relative positions of neutralizing sites ‘a’ and ‘b’ are indicated by white blocks within the VP2 protein (black bar) and the numbers of antigenic sites (triangles) defining neutralizing regions are boxed. White bars A, C, D and F–M represent the E. coli-expressed fragments of VP2 that were used to define antigenic regions (Martínez-Torrecuadrada & Casal, 1995 ). Numbering at the ends of each bar indicates the region of VP2 covered by fragments. Fragment D (grey bar) denotes the VP2 region covered by overlapping 12-mer peptides used in PEPSCAN analyses. Locations of antigenic sites are depicted by lines with the relative amino acid positions at the ends. Peptides with neutralizing activity are represented by thick black lines and the corresponding sequences defining neutralizing sites ‘a’ and ‘b’ are boxed. The number on the right of each neutralizing peptide shows the respective neutralizing titre as the serum dilution causing a 50% reduction in cell monolayer in a monolayer protection assay.

 
Accessibility of antigenic sites on the virion surface
The reactivity of peptide antisera with AHSV-4 virions was investigated to obtain experimental evidence for the exposure of the VP2 antigenic sites on the virion surface. Antibody samples were assayed by indirect ELISA on purified AHSV-4. The results are shown in Fig. 3. All neutralizing sera against antigenic sites 8, 11 and 12 reacted with the virion, indicating that the corresponding antigenic sites were accessible for antibody binding in the infectious virus. Other sera, as those against sites 4, 6 and 15, gave a positive reaction with the virion, but they did not show neutralizing activity. Site 4 is unusual in that it is properly exposed on the virion surface but does not elicit neutralizing antibodies. The remaining sera did not bind the virion, suggesting that the sequences were probably buried or exposed on the virion surface but in a conformation that is not recognized by the antibody.



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Fig. 3. Exposure of antigenic sites on the surface of the virion measured as reactivity of peptide-immunized rabbit sera against AHSV-4 by indirect ELISA. The absorbance values measured at 405 nm of antisera against the corresponding antigenic sites are represented as black bars.

 
Optimization of neutralizing responses
To improve the neutralizing response, an optimization of the amino acid sequences was attempted. A collection of systematically overlapping 19-mer peptides for neutralizing site ‘a’ (aa 318–342) was synthesized. Also, the peptide 374–403, comprising site ‘b’ plus some extra amino acids at each end to ensure that the whole site was represented, was generated. A cocktail of two peptides representing sites ‘a’, from aa 321 to 339, and ‘b’, from aa 374 to 403, was investigated as well. Every peptide was coupled to KLH and used to immunize rabbits. Results are shown in Fig. 4. All rabbits induced anti-peptide antibodies that reacted with AHSV by ELISA in all cases (data not shown). In the case of site ‘a’, an enhancement of the neutralizing capability was achieved, since peptides 322–340 and 323–341 gave higher neutralizing responses than those obtained with peptide 321–339, with titres of 1/32 and 1/64, respectively. No improvement of neutralizing activity was obtained when a long peptide covering the whole neutralizing site ‘b’ was used. However, the combination of peptides from both sites (321–339 and 377–400) induced the best neutralizing response, with a titre of 1/128, suggesting a cumulative effect.



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Fig. 4. Optimization of neutralizing activity of sites ‘a’ and ‘b’. Sequences and positions of peptides used to optimize neutralizing sites ‘a’ and ‘b’ are displayed. Neutralizing peptides defined by PEPSCAN are underlined and the corresponding sites are indicated with vertical dotted lines. The neutralizing titres of the respective anti-peptide sera are represented to the right of each peptide as a black bar.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Antibodies against the outer capsid protein VP2 of AHSV and other orbiviruses are able to neutralize virus infectivity, suggesting that antigenic determinants located on this protein are critical to provoke a protective immune response (Burrage et al., 1993 ; Grubman et al., 1983 ; Martínez-Torrecuadrada et al., 1994 ; Purdy et al., 1985 ). In previous work, we demonstrated that a strong neutralization domain was located between residues 254 and 414, which included various neutralization sites (Martínez-Torrecuadrada & Casal, 1995 ). In this report, we have carried out a systematic search of the neutralization determinants of AHSV-4 VP2 at the peptide level. Due to the technical difficulties of generating a complete set of overlapping peptides from as large a protein as VP2 (1060 aa, 1049 peptides) and given that the N- and C-terminal regions are not immunogenic (Martínez-Torrecuadrada & Casal, 1995 ), solid-phase synthetic peptides were prepared only for the central part of the protein, spanning from residue 199 to 689.

A total of 15 antigenic sites was identified and these could be grouped in two clusters. The first, covering aa 223–400, contains 12 of the 15 antigenic sites, confirming that this region of VP2 is a hot antigenic spot. The second covers aa 568–681 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 KLH–peptide 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 254–414 (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 284–380), whereas site ‘b’ corresponds to the N terminus of fragment M (aa 380–414) (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 279–297 and, therefore, it could be optimized to achieve some neutralizing activity.



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Fig. 5. Hydrophobicity profile of AHSV-4 VP2 region comprising aa 199–689. The distribution of the hydrophilic and hydrophobic areas along the amino acid sequence of VP2 was determined by using the algorithm of Kyte & Doolittle (1982) . The hydrophobic regions are shown above zero and the hydrophilic regions are shown below zero. The precise locations of neutralizing sites ‘a’ and ‘b’ are indicated.

 
A significant improvement of the neutralizing ability of site ‘a’ was achieved when peptides 322–340 and especially 323–341 were used for immunization. Interestingly, a combination of peptide 321–339 of site ‘a’ and the long peptide 374–403 of site ‘b’ induced a more effective neutralizing activity, but it is not clear if this effect is synergistic or additive. A plausible explanation for this result is that the binding of antibodies to one epitope would produce a conformational change in the virus particle such that a second set of antibodies with different epitope specificities would bind more easily, leading to a more potent neutralization. Other likely explanations could be the neutralization of two sites with a different role in virus infection (e.g. virus–cell attachment, virus translocation, etc.) or that sites ‘a’ and ‘b’ constitute continuous parts of a relevant discontinuous epitope. Enhanced neutralization has been also described in other viruses, such as human immunodeficiency virus (Buchbinder et al., 1992 ; Davis et al., 1993 ; Laal et al., 1994 ; Vijh-Warrier et al., 1996 ), herpes simplex virus (Weijer et al., 1988 ) or Newcastle disease virus (Iorio & Bratt, 1984 ).

We cannot rule out better results if other peptides from site ‘a’, such as peptide 323–341, 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 protein–protein 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.


   Acknowledgments
 
This work was carried out as a collaborative project supported in part by the EU, FAIR program, contract no. FAIR-CT95-0720. With thanks to Drohpati Parohi for skilful assistance in PEPSCAN analyses, Wouter Puijk for the PEPSCAN syntheses and Ronald Boshuizen for peptide–conjugate preparations and rabbit vaccination experiments.


   Footnotes
 
b Present address: Najeti Ventures, Zurbaran 28, 28010 Madrid, Spain.


   References
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Discussion
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Received 7 March 2001; accepted 8 June 2001.