The Danish Veterinary Institute for Virus Research, Lindholm, 4771 Kalvehave, Denmark1
Author for correspondence: M. B. Oleksiewicz. Present address: Symphogen A/S, Elektrovej, Building 375, 2800 Lyngby, Denmark. Fax +45 4526 5060. e-mail mbo{at}symphogen.com
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Abstract |
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Introduction |
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We are exploring phage display for large-scale epitope mapping of PRRSV proteins, on the assumption that an understanding of the antigenicity of all viral proteins might aid in the interpretation of the consequences of PRRSV sequence variability for vaccine and diagnostic test performance and might perhaps also guide the development of recombinant antigens for use in vaccines or serological tests. Previous studies have demonstrated the rapid identification of multiple PRRSV epitopes by phage display (Oleksiewicz et al., 2000 , 2001b
) and the use of phage-displayed PRRSV epitopes for novel diagnostic tests (Oleksiewicz et al., 2001a
). While several epitopes were identified in the PRRSV replicase polyprotein, the number of epitopes found in the envelope glycoproteins of PRRSV was disappointingly low (Oleksiewicz et al., 2000
, 2001b
). Therefore, in the current study, we repeated the epitope screen of the PRRSV envelope glycoproteins, modifying the phage display method to enhance the detection of weak antigenic sites.
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Methods |
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Phage ELISA.
Phages for ELISA were plaque-purified and phage particles were purified and concentrated by two rounds of precipitation with PEG and NaCl (Oleksiewicz et al., 2001a , b
). Particle concentrations were normalized by spectrophotometry and 3·8x1010 phage particles were used per well to coat ELISA plates. Indirect ELISA with porcine serum and rabbit anti-porcine HRP-conjugated immunoglobulin was done as described (Oleksiewicz et al., 2001a
, b
). Each porcine serum sample was assayed in parallel on a well coated with peptide-displaying phage and a well coated with a library phage without peptide. The specific reactivity against the phage-displayed peptide was calculated as the ratio between the two wells (Oleksiewicz et al., 2001a
, b
).
Discrimination between positive and negative reactions was done using cut-off values determined by analysis of ten known negative sera (sera that were negative for antibody to PRRSV in our routine diagnostic assays). The maximal and mean+2SD values were determined for each phage antigen using the negative serum panel and the larger of these two values was employed as a cut-off value to ensure 100% specificity.
Protein sequence analysis.
Protein sequence analysis was made using HMOMENT (http://bioweb.pasteur.fr/intro-uk.html#phylo) and other related prediction tools (http://www.expasy.ch/). Signal peptidase cleavage sites were predicted using SIGNAL P-HMM (Nielsen et al., 1997 ). Transmembrane regions were predicted using the combined output of TMHMM (Sonnhammer et al., 1998
) and PREDICTPROTEIN (Rost, 1996
). Secondary structure was predicted with PREDICTPROTEIN using the high-accuracy SUB output subset. Eisenberg hydrophobic moment plots (Eisenberg et al., 1984
) were generated with HMOMENT using a 20-residue sliding window. Amphipathic helices in ORF2 and 3 proteins were identified by combining PREDICTPROTEIN secondary structure prediction and HMOMENT Eisenberg hydrophobic moment plots (Eisenberg et al., 1984
). Amphipathic helices were modelled with software written by E. K. ONeil and C. M. Grisham, available directly at http://cti.itc.Virginia.EDU/
c mg/Demo/wheelApp.html or with explanation through http://www.dkfz-heidelberg.de/tbi/bioinfo/Individual/HelicalWheel/index.html.
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Results and Discussion |
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The size of the PRRSV sequence amplified by RTPCR was 1·31·4 kb (Fig. 1); these amplicons were cloned in the pCR-XL-Topo vector (3·5 kb size) prior to DNase treatment and cloning of the DNase-treated products in the M13 KE gIII display vector. Thus, the total sequence used for library construction was 1·4+3·5 kb (4·9 kb) in length. The library size required to identify an epitope encoded by
number of nucleotides in a target that is
number of nucleotides long, with
certainty, is ln(1-
)/ln(1-
/
) (Clarke & Carbon, 1992
). Thus, the library size required if one is to be 99% certain to identify epitopes of 80 nt size contained in a sequence of 4·9 kb is ln0·01/ln(1-80/4800)=274 primary transformants. Similarly, the library size required if one is to be 95% certain to identify epitopes of 80 nt size contained in a sequence of 4·9 kb is 178 primary transformants. Because (i) the DNase-treated inserts had to be in frame with the phage pIII sequence at both the 5' and the 3' end, (ii) only 50% of the DNase-treated fragments were expected to be cloned in the correct orientation and (iii) stop codons must not occur in the DNase-treated fragments [the chance for no stop codons in a fragment of 80 nt is 61/6480/3=0·29 (New England Biolabs, technical information)], we used a correction factor of 3x3x3x2=54, leading to a calculated required library size of 178x54=9·6x103 primary transformants (95% certainty of identifying 80 nt large epitopes in a 4·8 kb target) to 274x54=1·5x104 primary transformants (99% certainty of identifying 80 nt large epitopes in a 4·8 kb target). Similarly, the required library sizes to identify epitopes encoded by 40 nt in a 4·9 kb large target are 1·3x104 (99% certainty) and 2x104 (95% certainty) primary transformants. Therefore, the libraries used in the present study were of sufficient size to identify epitopes in the PRRSV structural proteins targeted (Fig. 1
) at 4080 nt resolution, with 9599% certainty. In our opinion, this represents an optimal epitope resolution, as current evidence suggests that the minimal size of a porcine linear B-cell epitope is around 13 amino acids in length (Oleksiewicz et al., 2001b
).
Libraries were selected with pooled sera from experimentally PRRSV-infected pigs, as described in Methods. In this study (as in our previous studies), 10 µl library or approximately 1011 p.f.u. of phage was used as input for selection (Oleksiewicz et al., 2000 , 2001b
). Due to the library construction strategy used by us (see Methods), the libraries contained M13 phages displaying random fragments of PRRSV proteins as well as non-PRRSV peptides derived from pCR-XL-Topo sequence or out-of-frame or minus-sense PRRSV sequence. The displayed peptides were fused to the M13 pIII protein, which is present at 35 copies at one end of the M13 virion. Assuming that minimal loss of diversity occurred during library amplification, the percentage of phages in the final library that displayed peptides derived from real ORFs (as opposed to ORFs from, for example, minus-sense sequence or cryptic ORFs in the plus-sense sequence) was up to 54 times lower (see above for calculation of the 54x correction factor) than the number of electroporated bacteria that contained M13KE gIII DNA with foreign inserts at the EagI site (see PCR results above). Thus, the 10 µl library used for selection represented a minimum of 7·4x108 peptides derived from real ORFs and the complexity of this library was sufficient to identify PRRSV epitopes with a resolution of 1326 amino acids, as described above.
The libraries underwent two to three rounds of selection with immune sera, after which the displayed inserts were sequenced for a total of 190 plaque-purified phages. Deduced amino acid sequences of the phage-displayed peptides were then matched against the sequence of PRRSV strain 111/92 (GenBank accession number AJ223078) (data not shown). Of the phages from selected populations, 3090% displayed peptides matching PRRSV proteins (data not shown). Specifically, phages displaying peptides matching PRRSV ORF2, 3, 5 and 6 proteins were identified. These phages were then tested as antigen in an ELISA that specifically assayed the reactivity of porcine sera against the phage-displayed peptides (see Methods). The phage-displayed peptides were not recognized by preinfection sera and PRRSV-negative field sera (Fig. 2 and data not shown) but were recognized by sera collected 2156 days p.i. (Fig. 2
), indicating that the phage-displayed peptides represented naturally antigenic segments of PRRSV ORF2, 3, 5 and 6 proteins. A phage displaying 14 residues of the PRRSV helicase, which is not an epitope (Oleksiewicz et al., 2001b
), was not recognized by any serum, ruling out the possibility that sera taken late post PRRSV-infection produced unspecific reactions (Fig. 2
, hel). The sequences of the phage-displayed peptides and the titres observed in sera collected 42 days p.i. are presented in Table 1
. As a further negative control, 23 plaque-purified phages from parallel selections with preinfection sera from the same pigs were sequenced; none of these phages displayed peptides matching PRRSV proteins (data not shown).
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Epitopes in the EU-type ORF2 protein
The ORF2 protein of EU-type PRRSV is most likely incorporated in virions in monomeric form, anchored in the envelope by a C-terminal transmembrane segment and exposing a large, glycosylated N-terminal ectodomain at the virion surface (Meulenberg & Petersen-den Besten, 1996 ; Meulenberg et al., 1995
). Despite its status as the next-largest envelope glycoprotein (Fig. 3
), the antigenicity of the ORF2 protein is completely unexplored. Two epitope sites (ES) were identified in the ORF2 protein in the present study (Fig. 2
and Table 1
). ES10a was situated in the extreme N terminus of the mature ORF2 protein (Fig. 3
and Table 1
), with the peptide displayed by phage 1309 containing the last two amino acids of the predicted ORF2 protein signal peptide (Fig. 1
, signal peptidase cleavage site indicated by an asterisk).
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The other ORF2 epitope identified in the present study (Table 1, ES10b) was neighbouring a hitherto unrecognized heptad motif, a single copy of which was found to be completely conserved in the ORF2 and 3 proteins of EU-type PRRSV (Fig. 3
, black box).
Epitopes in the EU-type ORF3 protein
The ORF3 protein of EU-type PRRSV, but not US-type PRRSV, has been described to be virion-associated (Gonin et al., 1998 ; Mardassi et al., 1998
; Meulenberg et al., 1995
; van Nieuwstadt et al., 1996
). However, it seems unclear whether this discrepancy is due to the methodology used for virion purification or whether it reflects ORF3 protein properties that are genuinely different. The ORF3 protein contains a predicted signal sequence (Fig. 3
) (Meulenberg et al., 1995
) and a hydrophobic segment spanning residues 80100 in the mature protein (data not shown). The mechanism of its association with virions is unknown (Faaberg & Plagemann, 1997
; Hedges et al., 1999
). The antigenic nature of the ORF3 protein is of interest, because vaccination with baculovirus-produced ORF3 protein was partly protective against PRRSV-induced abortion in sows (Durand et al., 1997
). Two epitopes have been described previously in the ORF3 protein: RKASLSTS in the extremely variable C terminus, which appears to be dispensable for ORF3 protein function (Oleksiewicz et al., 2000
), and ES11 in the conserved N terminus (Fig. 3
) (Oleksiewicz et al., 2001b
). Previously, ES11-displaying phages were found only at very low frequency, resulting in imprecise mapping of ES11 (Oleksiewicz et al., 2001b
). In the present study, exclusion of an immunodominant ORF4 epitope from the phage libraries and possibly the other changes in the selection protocol outlined above led to phages expressing ES11 being isolated at a much higher frequency (Table 1
and data not shown). The availability of several, overlapping ES11 peptides resulted in slightly quicker seroconversion kinetics (Fig. 2
) and fivetenfold higher titres (Table 1
) than reported in our previous study (Oleksiewicz et al., 2001b
).
Epitopes in the EU-type ORF4 protein
The ORF4 protein contains a predicted signal sequence, a C-terminal predicted transmembrane segment that probably mediates the observed virion association and most likely has a large, N-terminal, glycosylated ectodomain (Meulenberg et al., 1995 , 1997
; van Nieuwstadt et al., 1996
). A single linear epitope has been described in the ORF4 protein, which is immunodominant in mice (Meulenberg et al., 1997
) as well as in pigs (Fig. 3
, ES12) (Oleksiewicz et al., 2001b
). The phage libraries used in the present study were designed to exclude ES12 precisely (Fig. 1
) and it is perhaps noteworthy that, while this improved the epitope mapping of the ORF2 and 3 proteins (see above), no new epitopes were discovered in the ORF4 protein. This suggests that the conformation of the ORF4 protein may favour the presentation of a single, highly immunogenic linear sequence to porcine B-cells. mAbs to the ORF4 epitope are neutralizing in vitro (Meulenberg et al., 1997
) but mAbs against the ORF5 protein have been described to be much more efficient than mAbs against the ORF4 protein in neutralizing virus (Weiland et al., 1999
) and we have previously presented indirect evidence that suggests that ES12 might be a decoy epitope (Oleksiewicz et al., 2001b
). Interestingly, mAbs against US-type ORF4 protein are not neutralizing (Dea et al., 2000
).
Epitopes in the EU-type ORF5 and 6 proteins
The ORF5 and 6 proteins are present in the virion at high levels and are thought to be triple-spanning membrane proteins with very small ectodomains and large endodomains (Dea et al., 2000 ; Mardassi et al., 1996
; Snijder & Meulenberg, 1998
). Current evidence suggests that the ORF5 and 6 proteins are present in the PRRSV envelope as heterodimers, which are disulfide-linked through N-terminal ectodomain cysteines (Fig. 3
) (Dea et al., 2000
; Mardassi et al., 1996
; Snijder & Meulenberg, 1998
). The ORF5 protein is a very promising vaccine candidate, because anti-ORF5 mAbs are neutralizing in vitro (Pirzadeh & Dea, 1997
) and vaccination with baculovirus-produced ORF5 protein as well as DNA expression constructs confers partial protection against PRRSV-induced disease (Durand et al., 1997
; Pirzadeh & Dea, 1998
). For the related equine arteritis virus (EAV), it has been found that the ORF5 protein contains some, but probably not all, of the neutralizing determinants of the virion (Balasuriya et al., 1995
). We found no epitopes in the putative ORF5 and 6 ectodomains. This might be due to the glycosylation and tertiary structure of the putative ORF5/6 heterodimer N-terminal ectodomain, because the peptides displayed by our current phage libraries were probably too short to form complex tertiary structure and were not glycosylated. However, epitopes were found in the large putative endodomains of both proteins (Fig. 3
). While the ORF5 and 6 epitopes were recognized very late in infection, and only by a fraction of the experimentally infected animals, they were not recognized by preinfection and PRRSV-negative sera, thus ruling out non-specific reactions (Fig. 2
and Table 1
). In support of this, others have also reported that the C terminus of the ORF5 protein is antigenic (Dea et al., 2000
; Rodriguez et al., 2001
).
Application of peptides
For any practical application of peptide antigen, knowledge of the viral sequence variability and the inter-pig variability in antibody responses are important considerations. We explored these issues by analysing selected phage-displayed peptides against panels of field sera, with the same indirect ELISA used for experimental sera above. All the phage-displayed peptides were derived from PRRSV strain 111/92, an EU-type field isolate from 1992 which is typical of the PRRSV strains present at the start of the Danish PRRSV epidemic (Oleksiewicz et al., 2000 ). The field sera were all collected in 2001, from farms infected with current PRRSV strains that are quite different from those that were present in 1991 (Oleksiewicz et al., 2000
). The type of the PRRSV (EU or US) circulating on the farms was determined from herd samples by routine diagnostic assays at our institute (Bøtner et al., 1994
; Sørensen et al., 1997
, 1998
). Additionally, prior to their use in phage ELISA, individual field sera were confirmed as positive for antibodies to EU-type PRRSV (20 serum samples from 20 different farms), positive for antibodies to US-type PRRSV (21 serum samples from 21 different farms) or negative for anti-PRRSV antibodies by routine diagnostic assays at our institute (Bøtner et al., 1994
; Sørensen et al., 1997
, 1998
). A low percentage of the EU-positive sera recognized the ORF2, 5 and 6 peptides (Table 2
). None of the positive sera recognized a negative-control phage-displayed peptide (Table 2
, phage 1905) and none of the negative sera recognized the ORF2, 5 and 6 peptides, ruling out the possibility that the low percentage of positive reactions was a non-specific phenomenon. These results expanded those obtained using experimental sera (Table 1
), by showing that reactivity against the ORF2, 5 and 6 peptides was not idiosyncratic to the experimental sera, which had also been used for library selection. We believe that the infrequent recognition of the ORF2, 5 and 6 peptides by field sera was unlikely to be due to PRRSV sequence variability, as these epitopes were highly conserved, to the extent that they cross-reacted significantly with US-positive field sera (Table 2
). Instead, the poor reactivity of the ORF2, 5 and 6 peptides with field sera were more likely to be due to the inherent low antigenicity of these epitope sites. For example, slow seroconversion towards the ORF2, 5 and 6 peptides, as demonstrated by the experimental animals (Fig. 2
), could account for the low percentage of positive reactions in the unselected field sera (Table 2
), where acutely infected animals might well have been over represented. Alternatively, because some epitope sites were defined based on a single phage clone (Table 1
, ES10a, ES13 and ES14), it is possible that imprecision in epitope mapping led to an underestimation of the antigenicity of these ES. Based on the findings for ES11, which was originally defined based on a single phage clone (Oleksiewicz et al., 2001b
) and which was mapped to a higher precision in the current study (Tables 1
, 2
and Fig. 2
), we anticipate that identification of epitopes based on a single phage clone may be erroneous by up to eight amino acids (data not shown in detail). Thus, fine mapping of ES10a, ES13 and ES14 using phage display or synthetic peptides is required to confirm whether these epitope sites are naturally of low antigenicity. In contrast, ES10b was mapped by two independent phage clones displaying different but overlapping peptides (Table 1
, phages 326 and 802) and low antigenicity of ES10b, as opposed to imprecise mapping, is therefore the most likely explanation for the infrequent recognition of phages 326 and 802 by field and experimental sera (Tables 1
, 2
and Fig. 2
).
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ES12 in ORF4 is the strongest linear PRRSV epitope we have identified (Meulenberg et al., 1997 ; Oleksiewicz et al., 2001b
), but phage 62 displaying the ES12 sequence of PRRSV strain 111/92 was not recognized by any of the field sera (Table 2
). This was expected, as we have reported previously that ES12 is deleted in more than 60% of current Danish field isolates (Oleksiewicz et al., 2000
) and we and others have also shown that ES12 is hypervariable in non-deleted isolates (Drew et al., 1997
; Katz et al., 1995
; Meulenberg et al., 1997
; Oleksiewicz et al., 2000
). We believe that the very high natural antigenicity and hypervariability of ES12 (Drew et al., 1997
; Katz et al., 1995
; Meulenberg et al., 1997
; Oleksiewicz et al., 2000
, 2001b
) might allow the design of a peptide antigen that is specific for a very narrow range of PRRSV isolates. This could be exploited to monitor the spread of live EU-type PRRSV vaccines by simple, serological means.
Colocalization of naturally antigenic and putative functional sequence in the minor envelope glycoproteins of PRRSV
Viral protein sequence that is antigenic in the natural host is relevant for recombinant protein (subunit) vaccine development. Naturally antigenic sequence that also has functional importance for the virus is particularly interesting: antibody responses against functional sites might be extra effective in quenching infection and, because functional sequence is generally well conserved, might provide broad protection against diverse PRRSV types. In this context, the minor envelope glycoproteins (ORF2, 3 and 4 glycoproteins) are particularly interesting, because a recent study indicated that the major ORF5 envelope glycoprotein of the related EAV may not, as was hitherto assumed, be involved in receptor recognition (Dobbe et al., 2001 ). Additionally, amongst the minor envelope glycoproteins, we consider the ORF2 and 3 proteins to be particularly interesting, because, as mentioned above, there is some indirect evidence that the ORF4 protein may contain a decoy epitope (Fig. 3
, ES12). For these reasons, we subjected the epitope sites identified in the PRRSV ORF2 and 3 proteins to closer scrutiny.
Based on sequence analysis as well as the observed cross-reaction with US-positive sera (Table 2), ES10a, ES10b and ES11 were highly conserved, which indicated that these sites are important for PRRSV protein function. For ES10b, functionality was further supported by its colocalization with the hitherto unrecognized VSRRIYQ motif (Fig. 3
). We found that the VSRRIYQ motif is completely conserved in the ORF2 and 3 proteins of EU-type PRRSV (Fig. 3
and data not shown). The motif contained a predicted phosphorylation site for protein kinase C (SRR). In the ORF3 protein, the motif is flanked by conserved prolines [PVSRRIYQP, the C-terminal proline would be expected to act as a strong helix breaker (Chou & Fasman, 1978
)] and was predicted to form a localized, amphipathic minihelix (Fig. 3
and data not shown). In the ORF2 protein, the VSRRIYQ motif was predicted to form the start of a longer helix, which stretched into ES10b (Figs 3
and 4b
). Eisenberg hydrophobic moment analysis indicated that this longer ORF2 helix was also amphipathic (Fig. 4a
). A putative amphipathic helix was also present in the ORF2 protein of US-type PRRSV (Fig. 4a
) but, in contrast to EU-type PRRSV, there are no sequence repeats between the US-type ORF2 and 3 proteins (data not shown). This is perhaps not unexpected, as the C-terminal part of the ORF3 protein, which harbours the VSRRIYQ motif in EU-type PRRSV (Fig. 3
), is truncated in US-type PRRSV (data not shown) (Dea et al., 2000
).
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ES that are exposed to the surface would be expected to be of particular value in designing recombinant proteins for vaccine use. The topology of PRRSV envelope glycoproteins has not been determined and it is not known whether ES10a, 10b and 11 localize to the surface of folded ORF2 and 3 protein ectodomains. However, amphipathic helices, as the one contained in ES10b (Fig. 4), have been suggested to be more likely to occur at protein surfaces (Eisenberg et al., 1984
). Further studies are needed to determine whether antibodies against the ORF2 and 3 sites are neutralizing in vitro and protective in vivo.
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Acknowledgments |
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Received 19 July 2001;
accepted 1 February 2002.