Department of Virology, Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, U.K. and heterologous virus challenge.

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1 Journal of General Virology (1992), 73, Printed in Great Britain 727 Comparison between in vitro neutralization titres and in vivo protetion against homologous and heterologous hallenge indued by vaines prepared from two serologially distint variants of foot-and-mouth disease virus, serotype A22 C. Bolwell, N. R. Parry and D. J. Rowlands* Department of Virology, Wellome Researh Laboratories, Langley Court, Bekenham, Kent BR3 3BS, U.K. Guinea-pigs were hallenged with homologous or heterologous strains of foot-and-mouth disease virus (FMDV) following vaination with baby hamster kidney (BHK) monolayer ell-adapted or BHK suspension ell-adapted strains of FMDV serotype A22 lraq 24/64. The protetion afforded by these vaines was analysed as a funtion of antigen dose and the in vitro serum virus neutralization titres ahieved. The results show that the level of neutralizing antibody indued that afforded 50% protetion was similar for both vaines in homologous or heterologous hallenge situations. However, although the dose of antigen required to ahieve this titre against homologous virus was similar for the two vaines, approximately 20-fold more of the suspension ell-adapted virus was required to eliit a protetive titre against heterologous hallenge ompared to the dose of monolayer ell-adapted virus required. A syntheti peptide representing the amino aid sequene 135 to 167 of VP1, whih is idential in the A22 raq 24/64 variant viruses, was shown to indue protetion against both homologous and heterologous virus hallenge. Antigeni variation is an important feature of foot-andmouth disease virus (FMDV; family Piornaviridae, genus aphthovirus). The virus ours as seven serotypes and there is onsiderable antigeni diversity between viruses within eah serotype. This antigeni variation has impliations for the seletion and use of vaine strains, those whih eliit antibodies apable of neutralizing a broad range of field viruses being preferred to viruses that indue responses of narrow speifiity. For pratial purposes the one-way antigeni relationships between virus strains are determined by in vitro neutralization assays using antivirus antisera and are expressed as r values, whih are the ratios of heterologous to homologous neutralization titres. Following adaptation to growth in baby hamster kidney (BHK) 21 monolayer ells, the virus strain A22 raq 24/64 (Arrowsmith, 1975) eliits antibodies whih neutralize a wide range of field strains of the A serotype (Rweyemamu et al., 1984). However, passage of this virus in BHK suspension ells, whih are required for large-sale vaine prodution and have a different karyotype from BHK monolayer ells (Capstik et al., 1966), leads to the seletion of variants whih indue antibody responses of only very narrow speifiity (Bolwell et al., 1989a). Cell attahment assays suggest that the seletive pressure resulting in these hanges in properties of the virus is related to the poor ability of the monolayer ell-grown virus (MV) to attah to suspension ells (Bolwell et al., 1989a). Cloned progeny from the MV lone , or suspension ell-grown virus (SV) lone had essentially the same antigeni properties as the parent stoks from whih they were derived, showing that these properties were not due to the presene of mixtures of antigeni variants. Furthermore, both monolonal antibodies (MAbs) eliited by MV partiles and polylonal antisera eliited by syntheti peptides, representing the immunodominant sequene of VP1 amino aids 135 to 167 (Bittle et al., 1982) whih is ommon to both viruses, neutralized a wide range of field strains indiating that broad spetrum antigeni properties an reside in a single epitope on the virus (Bolwell et al., 1989b). The potential effiay of a vaine is determined by two major fators, firstly by its immunogeniity in terms of the level of humoral antibody indued and, seondly, by the antigeni relationships between vaine and field isolate strains. n vitro neutralization tests an provide valuable information on both these aspets. However, it is important to onfirm preditions of vaine effiay based on in vitro tests with in vivo protetion data. We have, therefore, examined the ability of vaines prepared from MV, SV or the syntheti peptide VP SGM

2 728 Short ommuniation 100 8O = 60 0 ~ 4o,.d _ (a) P () 7 i i J ~ 1 (d). i i t i Log,0 antigen onentration Fig. 1. Relationship between the perentage protetion against (a) homologous or (b) heterologous virus hallenge and the antigen dose of MV (0, O) or SV (A, ~ used to vainate the animals. Relationship between antigen dose of () MV or (d) SV vaines and the serum neutralization titres (log~0 SNso) ahieved against homologous (O, AiO or heterologous (O, A) virus. The heterologous hallenge virus was A Bangladesh 8/ to protet guinea-pigs against hallenge either with homologous virus or a heterologous virus, A Bangladesh 8/78. The latter virus was hosen for heterologous hallenge as being representative of field virus isolates whih have a high r value with anti-mv antisera, denoting antigeni similarity, and a low r value with anti-sv antisera, indiating antigeni diversity (Bolwell et al, 1989a). i ' T Groups of guinea-pigs were inoulated with dilutions of purified (Brown & Cartwright, 1963) binary ethyleneimine (BE)-inativated (Bahnemann, 1973) preparations of either the MV or SV viruses emulsified in Freund's inomplete adjuvant. For the SV vaines animals reeived 5 ~tg (10), 1 txg (20), 0.2 ~tg (20) or 0.01 ~tg (20) of viral antigen intramusularly and for the MV vaine the doses were 5 ~tg (20), 1 ~tg (20), 0.2 rtg (30), 0-04 xg (10) or ~tg (10) (figures in parentheses denote the number of animals per group). At 43 days postvaination half of the animals in eah group were hallenged with homologous virus (MV or SV) and the other half were hallenged with the heterologous virus A Bangladesh 8/78 by the method desribed by Blak et al. (1985) and Franis et al. (1985). The relationship between antigen dose and perentage protetion is shown in Fig. 1 (a) and 1 (b). Statistial analysis of the data (Cox, 1989; Williams, 1986) indiated that the dose of antigen required to protet 50 % of the animals (PDs0) against homologous hallenge was similar for the two vaines, equating to 0.05 ~tg for MV and 0.08 ~tg for SV (Table 1). A marked differene between vaine groups was observed, however, when the animals were hallenged with the heterologous virus, 50% protetion being ahieved with ~tg of MV ompared to 0.32 xg of SV (Table 1). Sera olleted 1 day prior to hallenge in the above experiment were tested for their neutralizing ativity against the hallenge viruses by a two-dimensional mironeutralization assay (Rweyemamu et al., 1978). Sera from animals vainated with MV neutralized both the homologous and heterologous viruses with equal effiieny; the error bars of the neutralization titres against both viruses overlap throughout the antigen dose range (Fig. 1 ). However, sera from animals vainated with SV readily distinguished between the two viruses, the neutralization titres against the heterologous virus being signifiantly lower than those against the homologous virus (Fig. 1 d). These results are in agreement with the r value determinations from earlier experiments (Bolwell et al., 1989a). The proteted status of individual animals against hallenge with homologous or heterologous virus was Table 1. 50% Protetive dose (PDso) values Vaine Challenge virus 95% Confidene limits Antigen dose ~g) Neutralization titre giving 50% protetion giving 50% protetion Lower Upper (PDso) MV MV 1"17 A Bangladesh 0.74 SV SV 1.28 A Bangladesh "70 0" '57 0"89 0'05 0" '32

3 Short ommuniation 729 Challenge virus z r~ (a) (b) () MV A Bangladesh SV A Bangladesh MV SV A Bangladesh ooo i o! "1 i! " '.3 $ / " "1 Go 0-2 T,,, " 0.6 llu P U P U P U P U P U P U P U Fig. 2. The proteted status (P, proteted; U, unproteted) of guinea-pigs vainated with (a) MV or (b) SV vaines following homologous or heterologous (A Bangladesh 8/78) virus hallenge as related to serum neutralizing antibody titres (loglo SNs0) at the time of hallenge. () Relationship between the proteted status of guinea-pigs vainated with 25 gg (0) or 250 gg (O) of peptide VP Cys following hallenge with MV, SV or A Bangladesh 8/78 and the serum neutralizing antibody titres 0ogl0 SNso) at the time of hallenge. Eah point represents an individual animal. ompared to the serum neutralization titre against the relevant virus at the time of hallenge (Fig. 2a and b). There was generally good orrelation between serum neutralization titre and protetion, in agreement with earlier studies with FMDV (Pay & Hingley, 1986). The neutralization titres that orrelated with 50~o protetion against homologous virus hallenge were similar for MV and SV, being 1.17 and 1.28 loglo units respetively (Table 1). The titres giving 50~ protetion against heterologous virus hallenge were also similar for MV and SV (Table 1). Surprisingly, however, they were signifiantly lower than the titres required for 50~ protetion against homologous hallenge, being 0.74 and 0-71 log10 units respetively. Sine earlier work has shown that syntheti peptides representing the sequene of the G-H loop region of VP1 of A22 virus an indue ross-reative neutralizing antibody responses (Bolwell et al., 1989b), the ability of peptide to indue protetive responses has been investigated. Groups of 15 guinea-pigs were inoulated with either 25 gg or 250 gg of unonjugated peptide Cys in Freund's inomplete adjuvant. At 42 days postvaination, test bleeds were taken and five animals from eah group were hallenged with MV, SV or A Bangladesh 8/78 virus. The individual serum neutralization titres against the hallenge virus and results of hallenge are shown in Fig 2(). Both dose levels of peptide indued good titres of neutralizing antibody against eah of the viruses and only one animal was not proteted. This single failure ould not be orrelated with the level of serum neutralizing antibody. Unfortunately the responses to even the lower dose of peptide were too high to determine the minimum weight of peptide or the minimum level of antipeptide neutralizing antibody required to indue protetive immunity. However the results do emphasize the ability of peptide vaines to indue broad protetive immunity to virus hallenge in this system. The guinea-pig protetion data reported here showed that, in the homologous hallenge situation, the level of serum neutralizing ativity whih orrelated with 50~o protetion was similar for both MV and SV. Furthermore the antigen doses required to eliit this level of response were also similar for both viruses. When animals vainated with MV or SV were hallenged with a heterologous virus strain (A Bangladesh) the level of

4 730 Short ommuniation serum neutralizing ativity giving 509/0 protetion was again similar for the two vaines and, surprisingly, was somewhat lower than the titre affording protetion against homologous hallenge. However, the dose of antigen required to ahieve this level of heterologous neutralizing ativity was approximately 20-fold greater for SV ompared to MV. Despite this, the high immunogeniity per unit mass of the SV variant ompared to serotype O1 virus, for example, explains its effiay as a vaine strain in the field. The distint antigeni and immunogeni harateristis of MV and SV ould not be orrelated with amino aid substitutions within VP1. n fat, MV and SV have no sequene differenes in the VP1 protein. Only three amino aid substitutions were found, all loated in VP2 (Bolwetl et al, 1989a) and, by omparison with the resolved struture of FMDV serotype O1 (Aharya et al., 1989), only two of these are predited to be loated at the surfae of the virus. Despite this finding, the epitopes reognized by nine of 10 MAbs to MV, whih have speifiities similar to that of polylonal antisera, learly mapped to amino aids representing the G-H loop of VP1 (residues approx. 137 to 159) (Bolwell et al., 1989b). Furthermore, syntheti peptides representing this sequene have previously been shown to eliit broadly ross-reative neutralizing antibodies (Bolwell et al., 1989b) and have been shown here to provide protetion against homologous and heterologous hallenge. Unfortunately the doses of peptide used to vainate the animals were too high to allow a quantitative analysis of the amount required to afford protetion. An indiation of the moleular nature of the profound antigeni differenes between MV and SV may be provided by the reent serologial and rystallographi analysis of antigeni variants of serotype O1 virus seleted with MAbs (Parry et al., 1990). n that ase serologial experiments suggested that the VP1 G-H loop region was involved in the antigeni differenes between the parental and variant viruses, but sequene data showed that the single substitution in eah variant whih onferred the differene was situated in the B-C loop of VP 1 (residue 43, 48 or 59). However, rystallographi analysis showed that these substitutions indued profound onformational hanges in the VP1 G-H loop and it was onluded that the altered antigeni properties of the variant viruses were related to the hanged orientation of this feature.. The amino aid differenes between MV and SV are all in VP2 and two of these (amino aids 82 and 88) are predited to underlie the G- H loop of VP1 in one of its onformations by analogy to the O1 virus struture (Parry et al., 1990). Also, substitutions at position 80 of VP2 have been impliated in the resistane of A10 virus to neutralization by NAbs (Thomas et al., 1988). t is therefore possible that the substitutions at these positions affet the onformation and/or orientation of the VP1 G-H loop and thus affet the antigeni properties of the viruses. Suh a possibility may also explain the differenes in the ell attahment properties of MV and SV (Bolwell et al., 1989a) sine an important omponent of the reeptor binding domain of FMDV is also loated in the G-H loop of VP1 (Fox et al., 1989; Surovoi et al., 1989; Baxt et al., 1990). X-ray rystallographi analysis of MV and SV should help to resolve the preise details of the strutural differenes between the two viruses whih aount for their distint biologial properties. We would like to express our thanks to Mr L. PuUen of the nstitute for Animal Health, Pirbright, U.K., who performed the animal experiments, Mr A. Syred for the peptides, Mr G. Layton, Management Servies Department, Wellome Foundation, for the statistial analyses and Ms Lorraine Joye for typing the manusript. Referenes ACHARYA, R., FRY, E., STUART, D., FOX, G., ROWLANDS, D. & BROWN, F. (1989). The three dimensional struture of foot and mouth disease virus at 2.9.~ resolution. Nature, London 327, ARROWSMTH, A. E. M. (1975). Variation among strains of type A footand-mouth disease virus in the Eastern Mediterranean region Journal of Hygiene 75, BAHNEMANN, H. G. (1973). The inativation of FMDV by ethyleneimine and propyleneimine. Zentralblatt far Veterinaermedizin Reihe B 20, BAXT, B. & BECKER, Y. (1990). The effet of peptides ontaining the arginine-glyine-asparti aid sequene on the adsorption of foot and mouth disease virus to tissue ulture ells. Virus Genes 4, B'TLE, J. L., HOUGHTEN, R. A., ALEXANDER, H., SHN'NCK, T. M., SUTCLFFE, J. G., LERNER, R. A., ROWLANDS, D. J. & BROWN, F. (1982). Protetion against foot-and-mouth disease by immunisation with a hemially synthesised peptide predited from the viral nuleotide sequene. Nature, London 298, BLACK, L., PULLEN, L. & BOGE, A. (1985). Guinea pig protetion test as indiator of poteny of oil emulsion foot and mouth disease vaines. Researh in Veterinary Siene 39, BOLWELL, C., BROWN, A. L., BARNETT, P. V., CAMPBELL, R. O., CLARKE, B. E., PARRY, N. R., OULDRDGE, E. J., BROWN, F. & ROWLANDS, D. J. (1989a). Host ell seletion of antigeni variants of foot-and-mouth disease virus. Journal of General Virology 70, BOLWELL, C., CLARKE, B. E., PARRY, N. R., OULDRDGE, E. J., BROWN, F. & ROWLANDS, D. J. (1989b). Epitope mapping of foot and mouth disease virus with neutralizing monolonal antibodies. Journal of General Virology, 70, 59~58. BROWN, F. & CARTWRGn'r, B. (1963). Purifiation of radioative footand-mouth disease virus. Nature, London 199, CAPSTCK, P. B., GARLAND, A. J., MASTERS, R. C. & CHAPMAN, W. G. (1966). Some funtional and morphologial alterations ourring during and after the adaptation of BHK 21 lone 13 ells to suspension ulture. Experimental Cell Researh 44, Cox, D. R. (1989). Analysis of Binary Data. Edited by D. R. Cox & E. J. Snell. London Chapman and Hall. Fox, G., PARRY, N. R., BARNETT, P. V., MGNN, B., ROWLANOS, D. J. & BROW~, F. (1989). The ell attahment site on foot and mouth disease virus inludes the amino aid sequene RGD (arginineglyine-asparti aid). Journal of General Virology 70, FRANCS, M. J., FRY, C. M., ROWLANDS, D. J., BROWN, F., BTTLE, J. L., HOUOHTEN, R. A. & LERNER, R. A. (1985). mmunologial priming with syntheti peptides of foot-and-mouth disease virus. 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5 Short ommuniation 731 PARRY, N., FOX, G., ROWLANDS, D., BROWN, F., FRY, E., ACHARYA, R., LOGAN, D. & STUART, D. (1990). Strutural and serologial evidene for a novel mehanism of antigeni variation in foot and mouth disease virus. Nature, London 347, PAY, T. W. F. & HNGLE'~, P. J. (1986). The use of serum neutralization antibody assay for the determination of the poteny of foot and mouth disease vaines in attle. Developments in Biologial Standardization 64, RWEYEMAMU, M. M., BOOTH, J. C., HEAD, M. & PAY, T. W. F. (1978). Mironeutralization tests for serologial typing and subtyping of foot-and-mouth disease virus strains. Journal of Hygiene $1, RWEYEMAMU, M. M., OULDRDGE, E. J., HEAD, M. & PURSE, F. (1984). Evaluation of the antigeni variation within type A foot-and-mouth disease virus isolates from Asia. Journal of Biologial Standardization 12, SUROVO, A. Y., VANOV, V. T., CHEPURKN, A. V., VANYUSHCHENKOV, V. N. & DRYAGALN, N. N. (1989). s the Arg-Gly-Asp sequene the binding site of foot and mouth disease virus with the ell reeptor? Soviet Journal of BiD-Organi Chemistry 14, THOMAS, A. A. M., WOORTMEJER, R. K., PUJK, W. BARTELNG, S. J. (1988). Antigeni sites on foot and mouth disease virus type A10. Journal of Virology 62, WLLAMS, D. A. (1986). nterval estimation of the median lethal dose Biometris 42, (Reeived 13 Marh 1991; Aepted 18 Otober 1991)