An Epitope Located at the C Terminus of Isolated VP1 of Foot-and-Mouth Disease Virus Type O Induces Neutralizing Activity but Poor Protection

J. gen. Virol. (1986), 67, 289-294. Printed in Great Britain Key words: FMD V/neutralizing activity/vp1 289 An Epitope Located at the C Terminus of Isolated VP1 of Foot-and-Mouth Disease Virus Type O Induces Neutralizing Activity but Poor Protection By R. H. MELOEN* AND S. J. BARTELING Central Veterinary Institute, Edelhertweg 15, 8219 PH Lelystad, The Netherlands (Accepted 9 October 1985) SUMMARY Both whole virus particles and isolated VP 1 of foot-and-mouth disease virus type O 1 induce neutralizing antibodies. Results obtained with pigs vaccinated with either isolated VP1 or intact particles and subsequently challenged show that neutralizing activity induced by intact virus correlates well with protection in pigs, whereas neutralizing activity induced by isolated VP1 confers little or no protection. Further evidence suggests that the epitope responsible for the induction of neutralizing antibodies by VP1 is located at the C-terminal end of the protein between residues 200 and 210. INTRODUCTION Foot-and-mouth disease virus (FMDV) belongs to the aphthovirus genus of the Picornaviridae family. Picornaviruses are small RNA viruses with icosahedral capsid symmetry, a diameter of about 25 nm and a particle weight of 8.6 106. FMDV has four structural proteins; VP1, VP2 and VP3 have mol. wt. of approximately 24000 and VP4 has a mol. wt. of approximately 7000 (Rueckert, 1976; Boothroyd et al., 1982). FMDV occurs as seven distinct serotypes. Serotypes are defined as isolates that show no cross-protection; within serotypes, subtypes occur which show cross-protection to a variable extent (Pereira, 1977). On the complete virion, VP 1 is mainly exposed and probably carries the epitopes responsible for the induction of neutralizing antibody and protection. It has repeatedly been shown that a correlation exists between neutralizing activity and protection, suggesting that neutralizing activity by itself would be a sufficient measure. When the virus is incubated with trypsin (Wild & Brown, 1967; Rowlands eta[., 1971 ; Strohmaier & Adam, 1974) only VP1 is cleaved and the virus particle remains intact. Isolated VP1 induces neutralizing antibody and protection (Laporte et al., 1973; Bachrach et al., 1975; Meloen et al., 1979; Kleid et al., 1981). However, even when antigen doses as high as 1250 ~tg were applied insufficient protection was found in pigs and cattle after just one vaccination (McKercher et al., 1983). At least two injections are needed with either isolated 'natural' VP1 or VP 1 produced by recombinant DNA techniques. In contrast, a single dose of vaccine containing a few ~tg of intact virus particles is sufficient to induce protection. A possible clue to this observation was provided by earlier results indicating that the neutralizing ability of anti-vp1 sera differed qualitatively from that of sera induced by intact virus (Meloen & Briaire, 1980; Cartwright et al., 1982). Moreover anti-vp1 type O1 sera were shown to neutralize both serotypes O1 and A10 in contrast to anti-140s sera which do not cross-neutralize heterologous serotypes. This suggests that the epitope on VP1 inducing neutralization differs from the epitope(s) on intact virus. In this report, this suggestion was tested in a challenge experiment. Pigs were repeatedly vaccinated with isolated 'natural' VP1 to obtain serum neutralizing titres similar to those usually produced in pigs by one vaccination with intact virus. Subsequently both groups of pigs were challenged. The results showed that for given neutralizing antibody titres obtained by vaccination with VP1 there was less protection than for animals with similar titres obtained after vaccination with intact virus. Analysis of existing data and those obtained with neutralizing antibody assay (MNT), a radioimmunoassay (RIA) and ELISA with virus and 0000-6601 i986 SGM

290 R. H. MELOEN AND S. J, BARTELING subviral particles, anti-peptide sera, a selected monoclonal antibody and a peptide scanning method, suggest that the 'neutralizing epitope' of VP1 is located on the C-terminal end of VP1 approximately between residues 200 and 210 and can be classified as a minor one with respect to protection. METHODS Antigen preparation. FMDV type O, subtype 1 (strain BFS 1860) and type A, subtype 10 were grown in BHK cell suspension cultures (Barteling, 1974). Virus purification, trypsin treatment, 12S subunit and VP1 preparation were done as described by Meloen & Briaire (1980). Antisera. Antisera were raised in rabbits against purified 140S virion preparations and against isolated VP1 in the presence of 0.1 ~/o SDS as described previously (Meloen & Briaire, 1980). Monoclonal antibodies were prepared against purified 140S as described (Meloen et al., 1983). Antisera against peptides 146 to 152 and 204 to 213 of VP 1 (O1 type) were raised in rabbits as described elsewhere (Geysen et al., 1985). Serological tests. The MNT, RIA and ELISA were essentially the same as those applied previously (Meloen & Briaire, 1980). In the RIA, [35S]methionine.labelled virus particles, trypsin-treated virus (140S tryp) and 12S were used; isolated VP1 was prepared as described by Meloen & Briaire (1980). When VP1 was used in the ELISA, plates were coated with isolated VP1 in the presence of 0.01 ~ SDS. Pig experiment. Sixteen piglets were vaccinated once with 2 ~tg of acetylethyleneimine (0-5~, 20 C, 16 h)- inactivated FMDV type O1 ; 15 other animals each received 100 gg of isolated VP1 (Meloen & Briaire, 1980) and were revaccinated after 4 and 8 weeks. Vaccines were prepared by emulsifying the antigens in incomplete Freund's adjuvant and injected intramuscularly in the neck. All animals were challenged with virus of type O1 4 weeks after the last (which was for the FMDV-injected animals only) vaccination. The challenge was performed by injecting 0.05 ml of the virus suspension, containing 105 p.f.u./ml, intracutaneously into the bulb of the heel of one hind claw. The experiment included control animals and the results were read as described (De Leeuw et al., 1979). Peptide scan. Overlapping heptapeptides of the VPIs of FMDV used were synthesized and tested as described previously (Geysen et al., 1984). In short, 'scanning' for antibody-reactive hexapeptides required the synthesis of every overlapping heptapeptide in the relevant protein sequence. For example, a protein of n residues can be read as (n - 6) overlapping heptapeptides, in which peptide l represents residues l to 7, peptide 2 represents residues 2 to 8 and so on. Oligopeptides were synthesized according to the amino acid sequences derived from the respective nucleotide sequence: FMDV type O1 (Kurz et al., 1981). The peptides still coupled to their solid supports were then tested against the appropriate serum in an ELISA. Absorbances were plotted at the position of the first N-terminal amino acid in the sequence of the oligopeptide. Immunoadsorption. One mg of the oligopeptide Cys-Arg-His Lys-Gln-Lys Ileu-Val-Ala-Pro-Val-Lys corresponding to the sequence of VP1-OK between positions 200 and 210 (for coupling reasons Cys was added to the N terminus and Lys to the C terminus) was covalently coupled to 5 mg CH-Sepharose with the aid of carbodiimide according to the procedures of the manufacturer (Pharmacia). The peptide CH-Sepharose was incubated with 100 to 200 gl serum for 3 h at room temperature. The unadsorbed serum components were removed with phosphate-buffered saline (PBS) and the adsorbed antibody was eluted with 3~o acetic acid in 0.1 M-NaCI. Both fractions were concentrated by freeze-drying, redissolved in PBS to approximately twice the original volume and tested in the microneutralization test. RESULTS AND DISCUSSION A group of pigs was injected and re-injected with VP1-O1 until the neutralizing activity came within the range of that expected for pigs vaccinated once with intact virus. Both groups were challenged at the same time under identical conditions. The results are shown in Table 1. From these data it is clear that the relationship between neutralizing activity and protection in pigs vaccinated with isolated VP1 is not identical with that of pigs vaccinated with intact virus. Thus, the protective effect of neutralizing activity raised by VP1 seems inferior to that of intact virus, suggesting that the appropriate epitope(s) on VP! differ from those on intact virus. We were interested in locating this epitope on VP1. It was shown by Bachrach et al (1975) and Meloen et al. (1983) that anti-vp1 does not react with trypsin-treated virus. Strohmaier et at. (1982) showed that trypsin treatment removes residues 136 to 158 and 199 to 213 from the virion and that the ability of isolated VP1 to induce neutralizing activity probably resides in these areas. Tests with anti-peptide sera against sequence 146 to 152 confirmed that this part of VP 1 is not exposed on the isolated protein (Table 2) in accordance with the observation of Bittle et al (1982), indicating that anti-peptide 141 to 160 antiserum reacts only poorly with isolated VP1.

FMDV neutralizing epitope on VP1 291 Table 1. Protection after vaccination with (a) VP1 or (b) FMDV type O1 Neutralizing Protection Neutralizing Protection antibody a~er antibody a~er (a) titre after challenge (b) titre a~er challenge Pig no. three vaccinations with Ol virus Pig no. one vaccination with O1 virus 818 0.75* -t 808 1.20" +t 803 0.90-814 1.20 + 807 0.90-817 1.35 + 804 0.90-824 1.35 + 802 0.90-825 1.35 + 801 0.90-79l 1.35 + 822 1.05-795 1.50 + 819 1.05-826 1.50 + 794 1.35 + 792 1-65 + 800 1.35-813 1.65 + 816 1.35-820 1.80 + 796 1.50-805 1.80 + 809 1.50 + 793 1.95 + 797 1.65 + 798 1.95 + 823 1.95-821 2.10 + 799 2.10 + *log10 Endpoint dilution of the serum in a MNT. f -,No protection: +, protection. Table 2, Serological reactions of different FMDV type 01 antisera and a monoclonal antibody raised against type AIO RIA~- A ( Antiserum* MNT 140S (O1) 12S (O1) ELISA (VP1) a-146-152 --:~ 2.2 1.9 -- a-204-213 3.1 2.8 2.5 2-8 MCA a-a 0.6 4.8 4.8 6-1 a-vp1 1.7 2.9 1.6 4.4 * a-146-152 is an anti-peptide serum raised against an oligopeptide corresponding to residues 146 to 152 of VP 1-O 1. a-204 213 is an anti-peptide serum raised against an oligopeptide corresponding to residues 204 to 213 of VP1-O1. MCA a-a is a monoclonal antibody raised against intact virus particles of type AI0.? In the RIA none of the sera used reacted with trypsin-treated 140S. :~ Serum titre is given as logj o endpoint dilution; indicates < 0.3 in the MNT, < 1.0 in the RIA and < 2.0 in the ELISA. Furthermore, results obtained with several anti-140s sera showed that antibody-binding oligopeptides starting at residue 146 or 147 did not react after these sera were absorbed with intact virus. All other peptides in this area, even those oligopeptides containing residues 146 and 147 were not absorbed by whole virus, suggesting that only peptides 146 and 147 are involved in raising neutralizing activity (Geysen et al., 1984; Meloen & Barreling, 1986). These peptides were never found to react with anti-vp 1 sera. Typical examples of both are shown in Fig. 1 (a, b). Therefore, this area on whole virus seems an unlikely candidate for the epitope on isolated VP1 which induces neutralizing activity. On the other hand the peptide 204 to 213 antiserum reacted well with isolated VP1, indicating that this sequence is exposed on isolated VP1. In addition some of these latter anti-peptide sera did neutralize the virus (Table 2). All these data suggest that residues located between positions 204 and 213 could be responsible for the induction of 'VP1 neutralizing activity'. Additional evidence was obtained with a neutralizing monoclonal antibody raised against FMDV type A10 that showed some cross-neutralizing activity with type O1 virus and reacted in a RIA equally well with intact virus and isolated VP1 of both serotypes (Table 2). This

292 (a) 2 I- R. H. MELOEN AND S. J. BARTELING (b) e- (c) 0 aiihmnalhhihanlll~lhiil mnlmldhhimlnnllhlaiiiibiallihihlilhiln~mmhmmlmmhihihm 50 100 150 200 Residue number Fig. 1. Peptide scans of VPI-OI with (a) anti-140s serum, (b) anti-vp1 serum and (c) neutralizing monoclonal antibody against heterologous FMDV type A10. Peptides in this scan are seven amino acids long. monoclonal antibody did not react with trypsin-treated virus. Furthermore, in a peptide scan of seven amino acid-long oligopeptides it reacted with peptides 200 to 204, involving residues 200 to 210 (Fig. 1 c). This latter observation may explain the observed cross-reactive neutralizing activity induced by isolated VP1 and can be easily explained on the basis of partial sequence homologies in this

FMD V neutralizing epitope on VP1 293 Table 3. Absorption of neutralizing activity from anti-vp1 serum by H2N-CRHKQKIVAPVK- COOH coupled to a solid support Titre Neutralizing activity bound 1.8" Neutralizing activity eluted 0.3 Neutralizing activity of untreated serum 1.8 * Serum titre is given as log~0 endpoint dilution. area between viruses of types O and A (Boothroyd et al., 1982; Cheung et al., 1983). Apparently, antibodies present in anti-140s sera reacting with oligopeptides starting at residue 205 and 206 do not cross-neutralize. This suggests that the core of the cross-reactive neutralizing activity of VP1 is located to the left of residue 205. The C terminus of VP1 might be located on whole virus near amino acids 141 to 160 (Parry et al., 1985; R. H. Meloen & S. J. Barteling, unpublished observations). Since it has been suggested that peptides located in this latter area might be missed due to unreactivity when coupled to solid supports (Parry et al., 1985) the peptide corresponding with the sequence of VP1 from type O1 between positions 200 and 210 was covalently coupled to an immunoadsorbent. All detectable neutralizing activity from an anti- VP1 serum was retained by the immunoadsorbent (Table 3). Thus, we think that we may have identified the neutralizing epitope present on isolated VP1, and have shown that it is not a main one (a main epitope being defined as one that induces serotype-specific neutralizing activity and solid protection). Obviously this epitope located between residues 200 and 210 is not the preferred candidate for a peptide vaccine because of the inferior protecting activity that it induces. Furthermore, it is clear that these data do not favour the use of isolated or recombinant VP1 as a vaccine. Whether or not this epitope on VP1 is related to a main one, for instance by being a part of a discontinuous epitope that loses its integrity upon disruption of the virus (Parry et al., 1985), or whether it is a member of a whole set of neutralizing epitopes on intact virus remains to be seen. We would like to thank Dr P. W. de Leeuw for his assistance with the challenge test, Dr J. G. van Bekkum for critical reading of the manuscript, Jan Briaire and Wouter Puyck for their excellent technical assistance and Ella Fanoy for doing the microneutralization tests. REFERENCES BACHRACH, H. L., MOORE, D. M., McKERCHER, P. D. & POLATNICK, J. (1975). Immune and antibody responses to an isolated capsid protein of FMDV. Journal of Immunology 115, 1636-1641. BARTELIYG, S. J. (1974). Use of polyethylene glycol treated serum for the production of foot-and-mouth disease virus growing in BHK-suspended cell cultures. Bulletin. Office international des kpizooties 81, 1243-1254. BITTLE, J. L., HOUGHTEN, R. A., ALEXANDER, H., SHINNICK, T. M., SUTCLIFEE, J. G,, LERNER, R. A., ROWLANDS, D. J. & BROWN, r. (1982). Protection against FMDV by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence. Nature. London 298, 30-34. BOOTHROYtg, L C., HARRIS, T. ~. R., ROWLANDS, D. J. & LOWE, P. A. (1982). The nucleotide sequence of edna coding for the structural proteins of foot-and-mouth disease virus. Gene 17, 153 161. CARTWRIGHT, B., MORRELL, D. J. & BROWN, F. (1982). Nature of the antibody response to the foot-and-mouth disease virus particle, its 12S protein subunit and the isolated immunizing polypeptide VP1. Journal of General Virology 63, 375-381. CHEUYG, A., DELAMARTER, J., WEISS, S. & KUPPER, n. (1983). Comparison of the major antigenic determinants of different serotypes of foot-and-mouth disease virus. Journal of Virology 48, 451~459. DE EEEUW, P. W., TIESSlNK, J. W. a. & VAN BEKKUM, J. G. (1979). The challenge of vaccinated pigs with foot-andmouth disease virus. Zentralblattfi;tr Veterinfirmedizin, reihe B, 26, 98-109. GEYSEN, H. M., MELOEN, R. H. & BARTELING, S. J. (1984). Peptide synthesis used to probe viral antigens for epitopes to a resolution of a single amino acid. Proceedings of the National Academy of Sciences, U.S.A. 81, 3998 4002. GEYSEN, n. M., BARTELING, S. J. & MELOEN, R. H. (1985). Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein. Proceedings of the National Academy of Sciences, U.S.A. 82, 178-182. KLEID, D. G., YANSURA, D., SMALL, B., DOWBENKO, D., MOORE, D. M., GRUBMAN, J. M., MORGAN, D. O., ROBERTSON, B. H. & BACHRACn, H. L. (1981). Cloned viral protein vaccine for foot-and-mouth disease. Responses in cattle and swine. Science 214, 1125-1128.

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