1 Centre for Equine Virology, School of Veterinary Science, The University of Melbourne, Victoria 3010, Australia
2 Department of Microbiology and Immunology and the Co-operative Research Centre for Vaccine Technology, The University of Melbourne, Victoria 3010, Australia
3 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia
Correspondence
Carol Hartley
carolah{at}unimelb.edu.au
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ABSTRACT |
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MAIN TEXT |
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Picornavirus capsid proteins VP1, VP2 and VP3 share structural homology and are composed of wedge-shaped, eight-stranded, -barrels, which differ in the size and conformation of the connecting loops between their strands and the extensions of their N- and C-termini (Rueckert, 2001
). The amino acid sequences of the loops that connect the
-strands and the N- and C-terminal regions that extend from the
-barrel domain give each picornavirus its distinct morphology and antigenicity (Mateu, 1995
; Rueckert, 2001
). For example, the surface loops of VP1, VP2 and VP3 of poliovirus type 1 (PV-1) and human rhinovirus type 14 capsid structures protrude from the virion surface to form the receptor-binding canyon, as well as the major antigenic sites of these viruses. In comparison, the VP1 protein within the relatively smooth surface of FMDV particles contains a linear peptide, the
G
H loop, which is both the dominant neutralization epitope and is also involved in receptor binding to various integrins via an RGD motif (Berinstein et al., 1995
; Danen et al., 1995
; Jackson et al., 2000
; Mateu et al., 1995
; Stanway, 1990
; Strohmaier et al., 1982
).
In marked contrast to FMDV and human rhinoviruses, the predicted amino acid sequence of ERAV P1 and, in particular, VP1 has remained remarkably stable over time (Varrasso et al., 2001). Amino acid variations that do occur among naturally occurring strains of ERAV locate mostly to the proposed
E
F loop of VP1 and the
A2
Z loop of VP2, although some variation also occurs at the N terminus and at the
C
D and
G
H loops of VP1 (Varrasso et al., 2001
). Amino acid sequence variation in these regions suggests that the regions may contain epitopes that elicit neutralizing antibodies. We have shown that the ERAV capsid protein VP1 contains B cell epitopes that elicit neutralizing antibodies and has receptor-binding activity (Warner et al., 2001
). It was anticipated, therefore, that the major antigenic sites for ERAV would be located within the surface structures of VP1. In particular, in the loop regions, which are not predicted to play an essential role in stabilizing the capsid structure and are, therefore, likely to be exposed to antibodies. In ERAV, the
G
H loop of VP1 is smaller than that in FMDV and does not contain an identifiable integrin-binding motif. In this study, using panels of glutathione S-transferase (GST) fusion proteins comprising overlapping segments of VP1, we define further linear epitopes of ERAV VP1 that elicit antibodies in the natural host following infection and in rabbits and mice following immunization.
To map the location of ERAV VP1 B cell epitopes, two sets of GST fusion proteins were prepared. The first set comprised four overlapping fragments (GSTNT, GSTVP1.2, GSTVP1.3 and GSTVP1.4) designed to encompass the complete VP1 protein, where each fragment contained one or more of the predicted surface loops (Fig. 1). The second set of GST fusion proteins (GSTCA, GSTDE, GSTEF and GSTCT) was designed to contain the individual loop regions between the predicted
-sheet and
-helical structural elements of VP1 (Fig. 1
).
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To determine if the individual antigenic fusion proteins (GSTNT, GSTEF, GSTGH and GSTCT) elicit ERAV antibodies in rabbits, two rabbits were immunized with 75 µg fusion protein in Freund's complete adjuvant (FCA) per rabbit, and sera collected after twice boosting with 25 µg of the same fusion protein in Freund's incomplete adjuvant (FIA). Antisera from these rabbits were used to probe purified ERAV virion proteins in Western blot. VP1-specific antibodies were detected in sera from rabbits immunized with each of the fusion proteins (Fig. 3a), confirming the presence of authentic VP1 epitopes within the fusion proteins. As described by Warner et al. (2001)
, rabbits immunized with the full-length recombinant VP1 (GSTVP1) produced neutralizing antibodies at a level comparable to rabbit ERAV antisera. Neutralizing antibodies were not detected in sera from rabbits immunized with any of the fusion proteins GSTNT, GSTEF, GSTGH or GSTCT (SN titres <10).
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To investigate further the production of antibodies to ERAV, rabbit fusion protein antisera were also tested for their ability to bind to purified whole virions in ELISA. ELISA was carried out as described previously (Crabb et al., 1995), with the exception that wells were coated with 1 µg ml-1 purified ERAV.393/76 and that bound rabbit antibodies were detected using a 1 : 1000 dilution of horseradish peroxidase (HRP)-conjugated swine anti-rabbit IgG (Dako). Despite the finding that antibodies elicited to each of these fusion proteins bound to VP1 in Western blot, only antibodies to GSTNT reacted strongly to whole ERAV.393/76 in ELISA, although full-length VP1 (GSTVP1) and the C-terminal fragment (GSTCT) elicited somewhat lower antibody titres to ERAV (Fig. 3b
). Neither the rabbit GSTEF antisera nor the rabbit GSTGH antisera showed any significant reactivity.
In this study, ERAV VP1 B cell epitopes were mapped to regions at the N- and C-termini and to the predicted E
F and
G
H loop regions. Loop regions are not predicted to play an essential role in the formation of the capsid structure and are predicted to project from the capsid surface. They are, therefore, also likely to elicit antibodies. Direct comparison of the known structure of FMDV VP1 with that predicted for ERAV VP1 (Wutz et al., 1996
) shows that most of the predicted loops of ERAV VP1 are larger than those of FMDV. In particular, the
E
F loop of ERAV, which contains a strong B cell epitope, is more than double the size of that in FMDV (32 compared to 14 aa in FMDV) and in the case of FMDV is not reported to contain any antigenic sites. Antigenicity of the
E
F loop of VP1 is not described commonly amongst picornaviruses but has been demonstrated for hepatitis A viruses and PV-2 and -3 (Luo et al., 1988
; Mateu, 1995
; Page et al., 1988
; Ping & Lemon, 1992
). The
G
H loop of ERAV VP1 is considerably shorter than that of FMDV (23 compared to 37 aa) and does not contain a recognized integrin-binding motif. We have shown that the
G
H loop contains B cell epitopes that elicit antibodies in horses following infection but not in rabbits following immunization. The presence of the strongest B cell epitope of ERAV VP1 within the N-terminal peptide and the strong binding of rabbit GSTNT antisera to whole virus particles in ELISA suggests that at least part of this region may be oriented more towards the virus surface. In support of this is the fact that, in comparison to FMDV, the N terminus of VP1 in ERAV is highly hydrophilic (Varrasso et al., 2001
). In FMDV, the N terminus of VP1 is confined to the interior of the capsid, within a deep and predominantly hydrophobic cleft formed by the interface between VP2 and VP3 (Acharya et al., 1989
; Curry et al., 1997
; Lea et al., 1994
), although N-terminal residues of PV-1, which are known also to be located internally in the capsid, have been shown to elicit virus-neutralizing antibodies (Fricks & Hogle, 1990
; Roivainen et al., 1994
).
Immunization of rabbits and mice with each of the fusion proteins (GSTNT, GSTEF, GSTGH and GSTCT) resulted in antibodies that reacted specifically with VP1, in addition to being recognized by sera from ERAV-infected and -immunized animals. Taken together, these results confirm the presence of authentic viral VP1 epitopes within the fusion proteins. However, while each of these fusion proteins elicited the production of antibodies that bound to reduced and denatured viral VP1, only GSTNT and GSTCT (and full-length GSTVP1) induced antibodies that bound to whole virus particles in ELISA and none of the polyclonal sera were neutralizing. Neutralization epitopes may exist as a linear sequence within VP1, as has been shown for FMDV, or as conformational epitopes comprising a combination of different surface loops (Xie et al., 1987). The induction of virus-neutralizing antibodies would require the presentation of authentic, conformationally intact epitopes, which may not be present when these individual loop fragments are expressed in Escherichia coli as GST fusion proteins. The correct conformation must exist on the surface of intact virus or when the complete VP1 is expressed as a GST fusion protein, since both these immunogens have been shown to induce the production of neutralizing antibodies (Hartley et al., 2001
; Warner et al., 2001
).
An enhanced response could result from combining the epitopes from the small fusion proteins into a single peptide or fusion protein to represent conformational epitopes. Proteins in whole virions would be folded in such a way that various regions of the capsid would lie in close proximity to one another, despite appearing distant in a linear representation of the sequence. In FMDV, the VP1 C terminus is reported to lie close to the G
H loop region (Acharya et al., 1989
). This provides structural evidence for the enhanced immunogenicity that has been reported for a FMDV VP1 hybrid peptide containing
G
H loop residues and C-terminal residues 200213 (Brown, 1992
; DiMarchi et al., 1986
). Peptides containing both regions induced antibodies that provided complete protection against infection with approximately one-hundredth the dose compared to that of a
G
H loop peptide that did not contain any C-terminal residues (DiMarchi et al., 1986
).
In summary, this work shows that the N-terminal region of ERAV VP1 contains strong B cell epitopes. Other small regions of ERAV VP1, namely the E
F and
G
H loops and the C-terminal region, also contain epitopes recognized by sera from ERAV-infected horses.
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ACKNOWLEDGEMENTS |
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Received 24 September 2002;
accepted 4 February 2003.