Porcine B-cells recognize epitopes that are conserved ... - CiteSeerX

Journal of General Virology (2002), 83, 1407–1418. Printed in Great Britain ...................................................................................................................................................................................................................................................................................

Porcine B-cells recognize epitopes that are conserved between the structural proteins of American- and European-type porcine reproductive and respiratory syndrome virus M. B. Oleksiewicz, A. Bøtner and P. Normann The Danish Veterinary Institute for Virus Research, Lindholm, 4771 Kalvehave, Denmark

By selecting phage display libraries with immune sera from experimentally infected pigs, porcine B-cell epitopes in the open reading frame (ORF) 2, 3, 5 and 6 proteins of European-type porcine reproductive and respiratory syndrome virus (PRRSV) were identified. The sequences of all the epitopes were well conserved in European-type PRRSV and even between European- and American-type PRRSV. Accordingly, sera from pigs infected with American-type PRRSV crossreacted with the European-type epitopes. Thus, this study showed, for the first time, the presence of highly conserved epitopes in the matrix protein and envelope glycoproteins of PRRSV. ORF5 and 6 epitopes localized to protein parts that are predicted to be hidden in PRRSV virions. In contrast, ORF2 and 3 epitopes localized to putative protein ectodomains. Due to the interesting localization, the sequence surrounding the ORF2 and 3 epitopes was subjected to closer scrutiny. A heptad motif, VSRRIYQ, which is present in a single copy in ORF2 and 3 proteins, was identified ; this arrangement is completely conserved in all European-type PRRSV sequences available. The VSRRIYQ repeat motif colocalized closely with one of the ORF2 epitopes and secondary structure modelling showed that this segment of the ORF2 protein could form an amphipathic helix. Intriguingly, a mutation associated with virulence/attenuation of an American vaccine strain of PRRSV also localized to this ORF2 protein segment and affected the hydrophobic face of the predicted amphipathic helix. Further work is needed to determine whether these findings delineate a functional domain in the PRRSV ORF2 protein.

Introduction Disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) was first recognized in the late 1980s. Now, PRRSV is an economically important pathogen of domesticated swine worldwide (Dea et al., 2000 ; Meng, 2000 ; Meulenberg et al., 1993 ; Snijder & Meulenberg, 1998). Two PRRSV genotypes exist, American (US) and European (EU), which are genetically only very distantly related (Nelsen et al., 1999). Within both EU and US genotypes, a considerable genetic variability exists in field PRRSV isolates (Meng, 2000 ; Oleksiewicz et al., 2000). A correlation has been observed between the porcine antibody response and PRRSV sequence variability (Drew et al., 1997 ; Katz et al., 1995 ; Meulenberg et al., 1997 ; Oleksiewicz et al., 2000). This suggests that PRRSV evolution could provide an escape from the Author for correspondence : M. B. Oleksiewicz. Present address : Symphogen A/S, Elektrovej, Building 375, 2800 Lyngby, Denmark. Fax j45 4526 5060. e-mail mbo!symphogen.com

0001-7984 # 2002 SGM

porcine immune response, which, in turn, suggests that continued PRRSV evolution might impact the efficacy of current diagnostic tests and vaccines. Examples of this have already been reported : recent outbreaks of ‘ atypical\acute ’ PRRSV in North America (Lager et al., 1998 ; Mengeling et al., 1998) have been suggested to be due to the emergence of more pathogenic PRRSV strains, against which current vaccines are apparently not effective (Meng, 2000 ; Mengeling et al., 1998). Also, PRRSV sequence variability is known to affect the reactivity even of pan-PRRSV-reactive monoclonal antibodies (mAbs) directed against the highly conserved nucleocapsid protein (Dea et al., 2000 ; Wootton et al., 1998 ; Yoon et al., 1995). 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 BEAH

M. B. Oleksiewicz, A. Bøtner and P. Normann

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.

Methods

Manufacture and selection of phage libraries. Open reading frames (ORFs) 2–3 and 4–6 were amplified by RT–PCR from the Danish European-type PRRSV strain 111\92 (Fig. 1) (Bøtner et al., 1994 ; Madsen et al., 1998). RT–PCR amplicons were designed to exclude an immunodominant epitope site in ORF4 identified previously (Fig. 1) (Meulenberg et al., 1997 ; Oleksiewicz et al., 2001b). Amplicons were cloned into the pCR-XL-Topo vector (Invitrogen). The constructs were confirmed by sequencing, fragmented by DNase treatment to an average size of 30–150 nt and blunt-end cloned using the EagI site of the M13KE gIII display vector (New England Biolabs). Ligation reactions were used to electroporate TOP10Fh Escherichia coli (Invitrogen). All steps of the library construction have been described previously in detail (Oleksiewicz et al., 2001b). Library sizes (the number of transformed bacteria) were estimated from an aliquot of electroporated bacteria by plating serial dilutions of bacteria, in agarose overlay, on LB plates with IPTG and XGal and counting the number of blue foci. The percentage of transformed bacteria that harboured the M13KE gIII phage DNA containing inserts at the EagI site, and the size of the inserts, were determined by PCR analysis of the DNA from the bacteria resulting in blue foci using primers that span the M13KE gIII EagI site (Oleksiewicz et al., 2001b). Selection of M13 libraries with porcine sera was done essentially as described previously (Oleksiewicz et al., 2001b), with the following modification to enhance detection of weak epitopes : because not all infected pigs produce detectable antibodies to PRRSV glycoproteins, we selected the libraries with pools of sera obtained 42–56 days postinfection (p.i.). Sera were from the same group of six pigs, experimentally infected with PRRSV strain 111\92 at 4 weeks of age, used in our previous study (Oleksiewicz et al., 2001b). Also, the serum concentration used for selection was increased to 1 : 40 in early rounds of selection and 1 : 50 in later rounds of selection. Finally, we reduced the detergent concentration (0n05 % Tween 20 in PBS) for washing immunoprecipitated phages. Plaque purification and DNA sequencing of phages from selected populations were done as described previously (Oleksiewicz et al., 2000, 2001b).

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 3n8i10"! 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). BEAI

Fig. 1. PCR strategy for library construction. The approximately 3 kb righthand part of the PRRSV genome encoding the structural proteins is shown. ORFs 2 and 3 were amplified by PCR, as a single piece, from random hexamer-primed cDNA. The ORF4–6 segment was amplified in two equal, overlapping pieces (denoted left and right PCR), which were fused by overlap extension PCR prior to cloning. ES12 and the black bar in ORF4 marks a 60 nt stretch encoding an immunodominant epitope described previously (Meulenberg et al., 1997 ; Oleksiewicz et al., 2001b), which was excluded from these libraries. The same 60 nt stretch encodes an epitope indicated by RKASLSTS and a black bar in the overlapping ORF3 (Oleksiewicz et al., 2000).

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 meanj2SD 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  (http :\\bioweb.pasteur.fr\intro-uk.htmlFphylo) and other related prediction tools (http :\\www.expasy.ch\). Signal peptidase cleavage sites were predicted using  P-HMM (Nielsen et al., 1997). Transmembrane regions were predicted using the combined output of  (Sonnhammer et al., 1998) and  (Rost, 1996). Secondary structure was predicted with  using the highaccuracy ‘ SUB ’ output subset. Eisenberg hydrophobic moment plots (Eisenberg et al., 1984) were generated with  using a 20-residue sliding window. Amphipathic helices in ORF2 and 3 proteins were identified by combining  secondary structure prediction and  Eisenberg hydrophobic moment plots (Eisenberg et al., 1984). Amphipathic helices were modelled with software written by E. K. O’Neil 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.

Results and Discussion We made two phage display libraries covering the ORF2 –3 and ORF4–6 segments of the PRRSV genome (Fig. 1). The number of transformed bacteria in the ORF2–3 library was 1n7i10% (determined by titrating electroporated bacteria), 70 % of which contained M13KE gIII DNA with foreign inserts at the EagI site (determined by PCR across the EagI site). The number of transformed bacteria in the ORF4–6 library was

PRRSV epitopes (a)

(b)

(c)

Fig. 2. Seroconversion of experimentally PRRSV-infected pigs to phagedisplayed peptides. Sera were from a group of six pigs experimentally infected with PRRSV strain 111/92 at 4 weeks of age (Oleksiewicz et al., 2001b). Sera were assayed at a 1 : 100 dilution in an indirect ELISA described previously, which was designed to correct for unspecific reactivity against the M13 virion and to measure the specific reactivity of sera against phage-displayed peptides (Oleksiewicz et al., 2001a, b). All six animals were bled at 0–42 days but only four animals were available at 56 days p.i. The fraction of seropositive animals (y-axis) was thus determined by dividing the number of seropositive animals by either six (days 0–42) or four (day 56). The ‘ hel ’ phage (black bar) is a negative control antigen.

1i10% (determined by titrating electroporated bacteria), 40 % of which contained M13KE gIII DNA with foreign inserts at the EagI site (determined by PCR across the EagI site). For both libraries, the inserts were 40–150 nt in length, as determined

by PCR. The primary libraries were amplified briefly at low temperature (5 h at 32 mC) to preserve diversity and phages were concentrated by PEG –NaCl precipitation (Oleksiewicz et al., 2001b). The final libraries had titres of 1n1i10"% p.f.u.\ml (ORF2–3 library) and 1n9i10"$ p.f.u.\ml (ORF4–6 library) (data not shown). The size of the PRRSV sequence amplified by RT–PCR was 1n3–1n4 kb (Fig. 1) ; these amplicons were cloned in the pCRXL-Topo vector (3n5 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 1n4j3n5 kb (4n9 kb) in length. The library size required to identify an epitope encoded by α number of nucleotides in a target that is β number of nucleotides long, with γ certainty, is ln(1kγ)\ln(1kα\β) (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 4n9 kb is ln0n01\ln(1k80\4800) l 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 4n9 kb is 178 primary transformants. Because (i) the DNase-treated inserts had to be in frame with the phage pIII sequence at both the 5h and the 3h 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\64)!/$ l 0n29 (New England Biolabs, technical information)], we used a correction factor of 3i3i3i2 l 54, leading to a calculated required library size of 178i54 l 9n6i10$ primary transformants (95 % certainty of identifying 80 nt large epitopes in a 4n8 kb target) to 274i54 l 1n5i10% primary transformants (99 % certainty of identifying 80 nt large epitopes in a 4n8 kb target). Similarly, the required library sizes to identify epitopes encoded by 40 nt in a 4n9 kb large target are 1n3i10% (99 % certainty) and 2i10% (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 40–80 nt resolution, with 95–99 % 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 10"" 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 3–5 copies at one end of BEAJ


Table 1. Deduced amino acid sequences of phage-displayed, PRRSV strain 111/92-derived peptides and titres of sera from 42 days post experimental PRRSV strain 111/92 infection Epitope sites (ES) were assigned in ascending, consecutive numbers in the 5h 3h direction in the PRRSV genome to fit in with the scheme started in our previous study (Oleksiewicz et al., 2001b). The PRRSV ORFs to which the ES localize are indicated. Peptides were identified by selection of phage display libraries of the structural genes of the EU-type PRRSV strain 111\92 with porcine sera. Assignation of phage-displayed peptides to PRRSV ORFs was done by alignment. The amino acid numbering in the phage-displayed peptides (superscript numbers) proceeds from the starting methionine of the indicated PRRSV ORF. The context in which the peptides are expressed in the M13 pIII protein has been described previously (Oleksiewicz et al., 2001b). The terminal sequence of the phage-displayed peptides that does not match the sequence of PRRSV strain 111\92, probably due to ligation and cloning artefacts, is in parentheses. The asterisk in the peptide displayed by phage 1309 (ES10a) indicates the predicted signal peptidase cleavage site in the ORF2 protein. Sera were from a group of six pigs experimentally infected with PRRSV strain 111\92 at 4 weeks of age (Oleksiewicz et al., 2001b). The same sera were used to select the phage-display libraries. Titres were determined on threefold serum dilutions from 1 : 100–1 : 2700 and are expressed as the dilution range that spanned half-maximal ELISA reactivity. All six serum samples had high titres in routine diagnostic assays employing complex PRRSV antigen. Seronegative means that no reaction was detected against the indicated phage-displayed peptide. Note that for some phage-displayed peptides, animals that were seronegative at 42 days p.i. (Table 1) became seropositive at 56 days p.i. (Fig. 2). Titre of sera 42 days p.i. (n l 6) Epitope site

Phagedisplayed peptide

ES10a (ORF2) ES10b (ORF2) ES10b (ORF2) ES11 (ORF3) ES11 (ORF3) ES11 (ORF3) ES11 (ORF3) ES11 (ORF3) ES11 (ORF3) ES13 (ORF5) ES14 (ORF6)

1309 326 802 31 34 315 318 302 308 553 512

1 : 100– 1 : 300– Seronegative 1 : 300 1 : 900 L$'G*SPSQDGYWSFFSGW&" E""(HSGQAAWKQVVGEATLTKLSDL"$* (E) G"#!QAAWKQVVGEATLTKLSDLDIV"%# N(#MWCKIGHTRCEER)& S'!QATQQRLEPGRNMWCKIGHTRCEERDH)( (E) Q'%QRLEPGRNMWCKIGHTRCEER)& S'!QATQQRLEPGRNMWCKIGHTRCEE)% C&(PTSQATQQRLEPGRNMWCKIG() (Q) L'(EPGRNMWCKIGHTRCEERD)' (Q) I"()KHVVLEGVKAQPLTRTSAEQW"** (PVKIVGQPVGPIQLA)A"$$VRKPGLTSVNGTL VPGLRSLVLGGKR"&*

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 54i 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 7n4i10) peptides derived from real ORFs and the complexity of this library was sufficient to identify PRRSV epitopes with a resolution of 13–26 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, 30–90 % displayed peptides matching PRRSV proteins (data not shown). Specifically, phages displaying peptides matching PRRSV ORF2, 3, BEBA

4 5 5

6 1

1 1

1 : 900– 1 : 2700

1

2 2 2

1 3 3 4

1 1

5 1

4

1

6 6

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 21–56 days p.i. (Fig. 2), indicating that the phagedisplayed 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 PRRSVinfection 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). Except for the ORF3 peptides, the peptides identified in the present study were poorly antigenic. Only a fraction of experimentally PRRSV-infected pigs recognized the phage-

PRRSV epitopes

deletions

deletions

Fig. 3. Correlation between linear epitope sites and conserved structural features in EU-type PRRSV ORF2, 3, 4, 5 and 6 proteins. PRRSV strain 111/92 (GenBank accession number AJ223078) and Lelystad virus (GenBank accession number M96262), two distantly related EU-type PRRSV isolates (Oleksiewicz et al., 2000), were analysed and features conserved between these two isolates are shown. The polypeptide backbone (vertical bold line) and features (boxes) are drawn to scale (scale bar corresponds to 20 residues). The N- and C-termini are indicated. The number in parentheses at the C terminus indicates the total amino acid number, including any leader peptide. For the ORF3 and 4 proteins, the total number of residues is given as a span, reflecting the deletions observed in field isolates (Oleksiewicz et al., 2000). The deletion-prone segments of the ORF3 and 4 proteins are indicated by shaded ovals. Arrowheads indicate predicted signal peptidase cleavage sites. Open boxes indicate epitope sites (ES) in PRRSV strain 111/92. Hatched boxes indicate predicted transmembrane regions. Loop and helix indicate predicted secondary structure. Cys denotes conserved cysteines, whereas Glyc denotes conserved N-linked glycosylation sites. Virtually all of the glycosylation sites shown are assumed to be used in vivo (Dea et al., 2000 ; Meulenberg & Petersen-den Besten, 1996 ; Meulenberg et al., 1995, 1997 ; van Nieuwstadt et al., 1996). The black box indicates a conserved seven-residue motif (VSRRIYQ), which was found in this study to be conserved in a single copy in the ORF2 and 3 proteins of EU-type PRRSV. RKASLSTS and ES12 are epitopes that have been described in previous studies (Meulenberg et al., 1997 ; Oleksiewicz et al., 2000, 2001b).

displayed peptides (Fig. 2) and titres were low (Table 1). This result was expected based on results from our previous study where stronger epitopes were mapped (Oleksiewicz et al., 2001b). The current study was, in fact, specially designed to identify weak epitopes, as described in Methods. We believe that weak epitopes are of interest for several reasons. Neutralizing antibodies against PRRSV are known to arise relatively late in infection and be of low titre (Loemba et al., 1996 ; Nelson et al., 1994 ; Yoon et al., 1994). Hence, we believe that knowledge of weak epitope sites may be highly relevant for subunit vaccine development. Additionally, we have shown

that combining weak and strong epitopes as peptide antigen for serological tests may allow monitoring of the progression of PRRSV infection at the herd level (Oleksiewicz et al., 2001b). 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 BEBB


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). Assuming that the ORF2 protein leader peptide cleavage occurs as predicted, this provided an estimate of the precision with which epitopes were mapped : for an epitope based on a single phage clone (Table 1, ES10a, phage 1309), at least the Nterminal border was apparently mapped with a precision of about two amino acids. This strongly supported the fact that our phage display libraries were of sufficient complexity for precise epitope mapping of the structural PRRSV genes targeted in the present study (Fig. 1). Also, the concurrence between the N terminus of peptide 1309 and the predicted ORF2 protein leader peptide cleavage site (Table 1) suggested that epitope mapping by phage display might, in some cases, provide information about PRRSV protein structure. The potential usefulness of epitope mapping data for protein topology prediction has also been noted by others (Jesaitis et al., 1999). 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 80–100 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 BEBC

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 five–tenfold 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 baculovirusproduced 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

PRRSV epitopes

Table 2. Reactivity of phage-displayed PRRSV strain 111/92-derived peptides with field porcine sera The serum samples used for study were a non-selected sample set from the most recent submissions to our institute. By routine diagnostic tests at our institute, herds were characterized as infected by EU-type PRRSV, US-type PRRSV or as being free from PRRSV. All PRRSV-positive sera were from different farms and, therefore, probably derived from pigs infected by different PRRSV isolates, thus ensuring maximal sampling of the present-day PRRSV diversity. All sera were assayed by ELISA using the indicated peptide-displaying phages as antigen. Discrimination between positive and negative reactions against the phage-displayed peptides was done using cut-off values established by examination of a panel of ten negative sera, as described in Methods. The 15 negative sera for which results are shown in the table were different from the ten negative sera used to establish ELISA cut-off values. Amino acid identities were calculated by dividing the number of identical residues by the total length of the ES. PRRSV strains Lelystad and 111\92 are distantly related EU-type viruses (Oleksiewicz et al., 2000). PRRSV strain VR2332 is a US-type PRRSV isolate. Number of field sera which reacted with phage-displayed peptides Phage

Antigen

Anti-EU sera (n l 20)

Anti-US sera (n l 21)

PRRSV-negative sera (n l 15)

1309 326 802 31 34 308 6–2* 553 512 1905†

ES10a (ORF2) ES10b (ORF2) ES10b (ORF2) ES11 (ORF3) ES11 (ORF3) ES11 (ORF3) ES12 (ORF4) ES13 (ORF5) ES14 (ORF6) hel

4 2 2 8 13 13 0 3 2 0

6 1 2 6 7 7 0 1 4 0

0 0 0 0 0 0 0 0 0 0

Amino acid identity (%) 111/92 versus Lelystad 88 96 96 93 89 95 42 96 96 

111/92 versus VR2332 56 69 69 64 64 70 40 64 77 

* Phage 6–2, representing the previously described epitope ES12 displays the peptide sequence CARPHGASEESQSVTFNKPLNTPQ (Oleksiewicz et al., 2001b). † Phage 1905 (hel) is a previously described negative-control phage displaying a sequence from the PRRSV helicase (DSSQGATFDVVTLQ) that is not an epitope (Oleksiewicz et al., 2001a, b). , Not relevant.

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 EUpositive 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 BEBD


(a)

(b)

(c)

Fig. 4. The VSRRIYQ motif and ES10b may participate in the formation of an amphipathic helix in the ORF2 protein. (a) Eisenberg hydrophobic moment plots for PRRSV strain 111/92 (EU type) and PRRSV strain VR2332 (US-type, GenBank accession number U87392) ORF2 proteins. uH(100deg) is a measure of 100-degree periodicity in hydrophobic residues ; i.e. the tendency of hydrophobic residues to cluster at one face of a helix (Eisenberg et al., 1984). The plots demonstrated a central region in both types of ORF2 protein with maximal 100-degree periodicity of hydrophobic residues, indicative of amphipathic helix formation (Eisenberg et al., 1984). The predicted leader peptide and transmembrane regions (hatched boxes), which are expected to form purely lipophilic (hydrophobic) helices, did not show a similar hydrophobic residue periodicity. Strings of 25 residues responsible for the indicated uH peaks were confirmed by hydrophobic moment analysis of virtual ORF2 protein sequences with internal deletions. Numbering in the 25-residue strings, as well as indicated total ORF2 protein length, is relative to the starting methionine. The VSRRIYQ motif of EU-type PRRSV (underlined), and a similar sequence in US-type PRRSV, formed the start of these 25-residue strings. (b) ORF2 protein sequence spanning the Eisenberg uH maximum was aligned for EU-type PRRSV strain 111/92 and US-type PRRSV strain VR2332. This sequence stretch also

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PRRSV epitopes

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 crossreacted 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). A high proportion of EU-positive field sera recognized the ORF3 peptides (Table 2). ES11 was highly conserved, as shown by the significant cross-reactivity observed with USpositive sera (Table 2). Conserved sequence appeared less likely to be antigenic in PRRSV (Oleksiewicz et al., 2001b). Yet, 65 % of the EU-positive field sera reacted with the 20-residue peptide displayed by phage 308 (Table 2) and titres against ES11 were relatively high (Table 1). This indicated that ES11 exhibited a unique combination of sequence conservation and antigenicity, which may be attractive for specialized diagnostic

applications, such as peptide arrays to simultaneously screen sera for reactivity to many different pathogens. 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 crossreaction with US-positive sera (Table 2), ES10a, ES10b and ES11 were highly conserved, which indicated that these sites

contained the novel VSRRIYQ motif (boxed) and ES10b (boxed). Secondary structure conserved between the two PRRSV types is indicated. Secondary structure prediction was uncertain for the two positions numbered 9 and 10 in the alignment. Because these two positions do not contain helix-breaking residues (Chou & Fasman, 1978), we considered it likely that they participate in the flanking helices. (c) An 18-residue string with a high helix probability [numbered 1–18 in the alignment in (b)] is shown as a helical wheel (graphics adapted from http ://cti.itc.Virginia.EDU/img/Demo/wheelApp.html), with grey circles indicating hydrophobic residues, (j) and (k) indicating charged residues and white circles indicating hydrophilic and other residue types. An amino acid substitution in the ORF2 protein of a live vaccine (GenBank accession nos AF159149 and AF066183) made by cell culture attenuation of PRRSV strain VR2332 is indicated. This substitution is predicted to change a hydrophobic residue (Ala) in PRRSV strain VR2332 to a hydrophilic residue (Ser) in the vaccine. A sequence from the human immunodeficiency virus V3 loop (RAFYTTGEIIGDIRNAHC), which has been shown experimentally to form an amphipathic helix (strongest amphipathic helix-forming residues underlined), is shown for comparison (Vranken et al., 2001).

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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). Amphipathic helices are commonly found in viral envelope glycoproteins (Auger & Lawrence, 1990) and viral amphipathic helices as short as ten residues may be involved in functional interactions with cellular lipid membranes (Oess & Hildt, 2000 ; Vranken et al., 2001). The relevance of the amphipathic helix predicted in the PRRSV ORF2 protein is unknown, but a functional role is supported by the observation that a live vaccine made by cell culture attenuation of PRRSV strain VR2332 exhibited an Ala Ser mutation, which was predicted to weaken the hydrophobic face of the helix (Fig. 4c), and that reversion to virulence is sometimes accompanied by a mutation back to an Ala (H. S. Nielsen, personal communication ; Allende et al., 1999 ; Nielsen et al., 2001). 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. Henriette S. Nielsen is thanked for sharing and explaining own sequence data regarding the ORF2 mutations associated with reversion to virulence of a US-type live PRRSV vaccine. Poul Toft and Bertel Strandbygaard are thanked for providing characterized experimental and field sera. Torben Storgaard is thanked for critical reading of the manuscript. BEBG

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Received 19 July 2001 ; Accepted 1 February 2002