Antigenic Conservation of H1N1 Swine Influenza Viruses

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1 J. gen. Virol. (1989), 70, Printed in Great Britain 3297 Key words: influenza/haernagglutinin/swine Antigenic Conservation of H1N1 Swine Influenza Viruses By M. G. SHEERAR, B. C. EASTERDAY AND V. S. HINSHAW* Department of Pathobiological Sciences, School of Veterinary Medicine, 2015 Linden Drive, University of Wisconsin-Madison, Madison, Wisconsin 53706, U.S.A. (Accepted 23 August 1989) SUMMARY Influenza viruses of the H 1N 1 subtype have been continually circulating in pigs in the U.S.A. for at least 50 years. To examine the level of antigenic variation in these swine viruses, a panel of 60 monoclonal antibodies (MAbs) to the haemagglutinin (HA) of recent swine isolates was prepared. Evaluation of neutralization escape mutants selected with these MAbs defined four antigenic sites on the HA, two of which overlap. Swine viruses isolated over 24 years in an enzootic area in Wisconsin were examined by ELISA and haemagglutination inhibition (HI) with these MAbs and the results indicated that the antigenic sites defined by these MAbs were highly conserved in these viruses. In comparing recent H1N1 viruses from pigs, turkeys, ducks and humans, changes in the antigenic sites were detected on the basis of HI reactivity. However, results of ELISA with these viruses clearly showed that the antigenic sites were still present on almost all H1N1 viruses of swine origin; thus, altered reactivity of these viruses in HI tests with MAbs was not a reflection of changes in the antigenic sites defined by the MAbs. It seems likely that the variation detected in these viruses occurs by a mechanism other than immune selection. INTRODUCTION Influenza viruses of subtype H1 N1 were first detected in pigs in the U.S.A. in 1930 and since that time have become widespread in the pig population and are responsible for substantial disease problems (Hinshaw et al., 1978; Kilbourne, 1987). Although the mechanism by which these viruses are maintained in pigs is not clear, it has been suggested that they are maintained by continual passage in a large population of young, susceptible pigs (Hinshaw et al., 1978). If this is the case, the viruses would be subjected to little immune selection, in contrast with the situation in humans (Kendal et al., 1985). Thus, to examine the level of antigenic variation in swine viruses, we prepared and defined a panel of monoclonal antibodies (MAbs) to the haemagglutinin of recent swine isolates. These MAbs were then used to determine whether there was antigenic variation among swine influenza viruses in enzootic areas, H1N1 viruses of swine origin are transmitted to other species, for example since 1980 they have been responsible for disease problems in the U.S.A. in turkeys (Hinshaw et al., 1983; Ficken et al., 1989) and in humans (Dasco et al., 1984; DeJong et al., 1986; Rota et al., 1989). In 1988 a person in Wisconsin, who was exposed to pigs with an influenza-like illness at a fair, died after infection with an H 1N 1 swine influenza virus (Centers for Disease Control, 1988; Rota et al., 1989). H1N1 viruses of avian origin are also apparently transmitted from birds to pigs, e.g. European swine isolates were shown to be most closely related antigenically and genetically to avian viruses (Scholtissek et al., 1983; Hinshaw et al., 1984). To define the relatedness of these H1N 1 viruses appearing in different species, antigenic comparison with current swine strains was undertaken. A panel of MAbs generated in this study was used to compare current H1N1 isolates from pigs with those from humans, turkeys and ducks. The results of ELISA with these MAbs indicate that the antigenic sites on the haemagglutinin of enzootic swine viruses, as well SGM

2 3298 M.G. SHEERAR, B. C. EASTERDAY AND V. S. HINSHAW as the swine viruses transmitted to other species, are highly conserved. However, a number of H1N1 isolates failed to react with the MAbs in H1 assays, which reflects changes in receptorbinding properties, rather than antigenic differences. METHODS Virus growth and purification. The viruses used in this study were from the influenza repository at The University of Wisconsin-Madison, with the exception of A/WI/3523/88, which was kindly provided by Dr A. Kendal, Centers for Disease Control, Atlanta, Ga., U.S.A. All viruses were grown in 11-day-old embryonated chicken eggs for 48 to 72 h at 35 C; the allantoic fluid was harvested and the virus concentrated with an Amicon hollow fibre filter, then purified by sucrose gradient centrifugation as described (Laver, 1969). Mutants which escaped neutralization with the MAbs (termed escape mutants) of the wild-type A/Sw/IN] 1726/88 were selected in vitro, expanded and cloned twice in eggs as described (Gerhard & Webster, 1978). Mutants were named according to the antibody used for their selection, preceded by the letter 'v' to indicate virus. Serological assays. Haemagglutination (HA) titres and haemagglutination inhibition (HI) tests were performed as described (Palmer et al., 1975). ELISA binding assays were done in microtitre plates using 200 HA units of whole or Sarkosyl-disrupted purified virus per well, diluted in phosphate-buffered saline (PBS) ph 7.2. Ascites fluids were diluted in PBS with 0.05 ~ Tween (PBS-Tween) and 0.5 ~ bovine serum albumin (BSA), added to the wells and incubated for 1 h at 25 C, followed by three washes with PBS-Tween. Horseradish peroxidaseconjugated anti-mouse Ig (Zymed) at a dilution of 1 : 2000 was added to the well and incubated for 1 h at 25 C. Enzyme activity was detected by the hydrolysis of hydrogen peroxide in the presence of azino-di-3-ethylbenzothiazobine-6-sulphuric acid (ABTS) (Boehringer Mannheim) in a 0-4 M-citrate buffer and quantified by measuring the absorbance at 410 nm as described (Kida et al., 1982). Haemagglutinin-specific MAbs were prepared to ether-disrupted purified viruses A/Sw/IN/1726/88 and A/Sw/WI/27/86 as described (Kfhler & Milstein, 1976). Two viruses were used in an effort to increase the diversity of the MAbs. Briefly, BALB/c mice were immunized intraperitoneally with HA units of etherdisrupted virus and boosted intravenously with the same dose of virus 3 days prior to removal of the spleen. Splenocytes were fused with the myeloma line NS-1 (Oi & Herzenberg, 1980) and hybridomas were cloned twice by limiting dilution. The HA specificity of the MAbs was established by radioimmunoprecipitation of HA from 3 ~S-labelled infected cell lysates, followed by PAGE and autoradiography (Kida et al., 1982). Ascitic fluids to each of the cloned hybridomas were prepared in pristane-treated BALB/c mice as previously described (Oi & Herzenberg, 1980). Virus neutralization activity of the monoclonal antibodies was determined by mixing antibody dilutions in Dulbecco's PBS supplemented with 0.5 ~ BSA (Gibco) (DPBS/A) with an equal volume of virus diluted to 1000 EIDso in DPBS/A. The virus-antibody mixtures were incubated for 30 min at 25 C and inoculated into 10-dayold embryonated eggs, which were incubated for 48 h at 35 C and virus growth in the allantoic fluid was detected by haemagglutination of chicken erythrocytes. RESULTS Definition of antigenic sites on the H1 haemagglutinin To define the antigenic sites on the HA of current swine viruses, a panel of 60 MAbs specific for the H1 HA was prepared and characterized. This panel was tested in HI assays against swine, avian and human H 1N 1 viruses and, based on different reactivity patterns in these assays (results not shown), five MAbs were chosen for further study. These five MAbs all neutralized A/Sw/IN/1726/88 (Sw/IN) so they were used for selection of escape mutants. In HI assays (Table 1) and ELISA (Table 2), these escape mutants did not react with the homologous Mab used for their selection, i.e. titres of < 100, but they retained their reactivities with the other MAbs. The sites recognized by MAbs 2-15F1 and 3F2c are clearly distinct since the mutant viruses do not react with the homologous MAbs (HI and ELISA titres of < 100), but react similarly to the parental virus with the other MAbs. The situation with the other variants is not as clear but the analyses suggest that two additional sites are recognized by MAbs 1-6B2 and 7Blb. For example, MAb 1-6B2 reacts with mutant 7Blb in both HI and ELISA but, in the reciprocal reactions, MAb 7Blb does not react with mutant 1-6B2. There is a reduction in the HI and ELISA titres of MAbs 7B 1 b and 1-6B2 with the escape mutants, suggesting that changes in one of these sites can affect the other, but that both are present. In support of this, HI results with A/N J/8/76 (Table 4) indicated that 7Blb reacted to high titres with A/N J/8/76, but 1-6B2 did not. Neutralization assays (not shown) also showed that 7Blb, but not 1-6B2, neutralized

3 Conservation of swine influenza viruses 3299 Table 1. Antigenic comparisons of escape mutants of A/swine/IN/1726/88 with monoclonal antibodies to the HI haemagglutinin in haemagglutination inhibition assays* HI titre with HI-specific monoclonal antibodiest ~k g Virus 2-15 F 1 3 F2c 1-6B2 7B 1 b 4A 12a Sw/IN/1726/ >2048 > Escape mutant v2-15f1 < > v3f2c 128 < < 1 v 1-6B < v7blb < 1 < 1 v4a12a < 1 1 < 1 * HI titres expressed as the reciprocal of the dilution 10 -z of ascitic fluid inhibiting 4 haemagglutinating doses of virus. t MAbs 2-15F1 and 1-6B2 were prepared to the HA of A/Sw/WI/27/86; MAbs 3F2c, 4A12a and 7Blb, to the HA of A/Sw/IN/1726/88. Table 2. Antigenic comparisons of escape mutants of A/swine/IN/1726/88 with monoclonal antibodies to the H1 haemagglutinin in ELISA* ELISA titre with HI-specific monoclonal antibodies Virus 2-15F 1 3F2c 1-6B2 7B 1 b 4A 12a Sw/IN/1726/ Escape mutant v2-15 F 1 < v3f2c < < 1 vl-6b < I < 1 < 1 v7blb < 1 < 1 v4a12a < 1 < 1 < 1 * ELISA titres are expressed as the reciprocal of the dilution x 10-2 of ascitic fluid providing an absorbance of at 410 rim. t MAbs as described in Table 1. A/N J/8/76, which supports this grouping. These results indicate that 7B lb and 1-6B2 recognize different antigenic sites. The results with MAb 4A12a were more difficult to interpret. This MAb did not react with three of the mutants in HI assays and with four of the mutants in ELISA, suggesting that a change in this site affected a number of other sites. However, HI assays with other H 1 NI viruses (Table 4) indicated that this was not always the case; thus, 4A12a did not appear to recognize a distinct antigenic site, but this MAb was included in subsequent assays because of its unusual reactivity. Antigenic comparisons of enzootic swine viruses To define the extent of variation in swine viruses involved in enzootic disease, viruses isolated from swine farms in southern Wisconsin over a period of 20 years were compared with the MAbs described above. These viruses were from seven swine farms located in southern Wisconsin where annual outbreaks typically occur. The results of HI assays (Table 3) indicate that the viruses have changed little over a period of more than 20 years. For example A/Sw/WI/1/65 and A/Sw/WI/48/84 are indistinguishable in these HI tests, even though they are chronologically distant. A/Sw/WI/27/86 reacted poorly in HI tests (HI titre of 1 : 100) with MAb 1-6B2 which was raised using this virus, yet this MAb had high neutralizing (1 : 10000) and ELISA (1: ) titres against A/Sw/WI/27/86. Thus, MAb 1-6B2 actually does react with the virus, even though the HI titre is low. These results suggest that the antigenic sites defined by these MAbs have remained essentially unchanged in the viruses involved in an enzootic disease problem.

4 3300 M.G. SHEERAR, B. C. EASTERDAY AND V. S. HINSI-IAW Table 3. Comparison of enzootic H1N1 swine isolates with monoclonal antibodies to the HI haemagglutinin in haemagglutination-inhibition assays* f HI titre with HI-specific monoclonal antibodies A "~ Virus 2-15F1 3F2c 1-6B2 7BI b 4A12a Sw/Wl/27/ Sw/WI/408/ > 2048 > Sw/WI/48/ >2048 > Sw/WI/3/ > 2048 > Sw/WI/18/ > 2048 > Sw/W 1/46/ Sw/WI/5/ Sw/WI/2/ > Sw/WI/1/ > 128 > 128 > 128 Sw/WI/1/ >2048 > * HI titres expressed as the reciprocal of the dilution x of virus. t MAbs as described in Table of ascitic fluid inhibiting 4 haemagglutinating doses Table 4. Comparison of H1NI viruses with monoclonal antibodies to Sw/IN/1726/88 and Sw/WI/27/86 in haemagglutination inhibition assays* Virus HI titres with HI-specific monoclonal antibodiest A 2-15F1 3F2c 1-6B2 7Blb 4A12a Swine Sw/WI/1915/88 < > Sw/WI/2185/ > 2048 > 2048 > Sw/IN/1726] > 2048 > Sw/WI/27/ Human WI/3523/ >2048 > WI/263/ NJ/8/ > 2048 < 1 Turkey Ty/N C/1780/ Ty/KS/4880/80 < < Duck Mal/Alb/499/83 Dk/Alb/35/76 <1 <1 16 <1 1 < 1 < * HI titres expressed as the reciprocal of the dilution x 10-2 of ascitic fluid inhibiting 4 haemagglutinating doses of virus. t MAbs as described in Table 1. Antigenic variation of H1N1 viruses from different species The five HI-specific MAbs were then used to compare recent H1N1 isolates from pigs, humans, turkeys and ducks in H1 assays (Table 4) and ELISA (Table 5). The most striking finding was that the MAbs reacted to high and equivalent titres with almost all of the viruses examined in ELISA (Table 5), despite substantial differences in HI reactivity of the various viruses (Table 4). For example, the two human isolates from Wisconsin in 1976 and 1988 were indistinguishable from each other and current swine strains. However, the other human isolate, A/N J/8/76, showed little or no reactivity with MAbs 1-6B2 and 4A12a in HI assays (Table 4), but reacted with both in ELISA at a level equal to the parental virus. This was also the case with the viruses from turkeys, i.e., A/Ty/KS/4880/80 failed to react in HI tests with three of the five

5 Conservation of swine influenza viruses 3301 Table 5. Comparison of H1NI viruses with monoelonal antibodies to the H1 haemagglutinin in ELISA* Virus ELISA titre with HI-specific monoclonal antibodies t g A 2-15F1 3F2c 1-6B2 7Blb 4A12a Swine Sw/WI/1915/88 < Sw/IN/1726/ Sw/WI/27/ Human WI/3523/ NJ/8/ Turkey Ty/NC/1780/ Ty/KS/4880/ Duck Dk/Alb/35/76 < 1 < * ELISA titres expressed as the reciprocal of the dilution x 10-2 of the ascitic fluid providing an absorbance of at 410 nm. 1" MAbs as described in Table 1. MAbs, but reacted in ELISA to high titres with all five MAbs. These findings suggested that the viruses of swine origin which have appeared in other species, such as turkeys and humans, are antigenically similar to the current swine viruses, but differ in HI reactivity. Actual variation in the antigenic sites was detected in a few viruses. Isolates in 1988 from pigs from different farms in Wisconsin were not identical, i.e. MAb 2-15F1 reacted with A/Sw/WI/2185/88, but not with A/Sw/WI/1915/88 in either HI or ELISA, indicating a difference in that antigenic site. However, even with the swine viruses a marked difference in HI reactivity did not necessarily indicate a loss of that antigenic site, e.g. A/Sw/WI/27/86 reacts at 1:100 in HI assays with 1-6B2 in comparison to the parental virus titre of > 1:204800, whereas their reactivity in ELISA is only a fourfold difference, indicating that the site is still present. The differences were more dramatic between the duck and swine viruses, in agreement with other antigenic comparisons of these particular viruses (Hinshaw et al., 1984; Austin et al., 1986). For example, A/Dk/ALB/35/76 did not react with MAbs 2-15F1 or 3F2c in ELISA or HI assays, indicating that the swine and avian H1NI viruses did not share these antigenic sites. Comparison of antibody binding to whole and disrupted viruses The above results suggested that, with many viruses, the MAbs failed to inhibit haemagglutination yet bound in ELISA. Since the virus in ELISA was disrupted, it was possible that the MAbs could not bind to the intact virus used in the HI assays. To address this possibility, the viruses that showed the greatest difference in HI and ELISA reactivities were examined in ELISA with and without disruption. The results of these assays (not shown) indicate that the MAbs bound to both whole and disrupted viruses at equivalent titres. This shows that the MAbs do bind to the intact virus yet fail to block its haemagglutinating activity. DISCUSSION This study was conducted to define the antigenic sites on the H1 haemagglutinin of current swine viruses and to examine the relatedness of H1N1 viruses isolated from pigs and other species. The MAbs generated in this study indicate the presence of four antigenic sites on the H 1 haemagglutinin with some overlap, similar to other H1 viruses (Gerhard & Webster, 1978; Laver etal., 1979; Caton et al., 1982; Raymond et al, 1983). The location of these antigenic sites

6 3302 M. G. SHEERAR, B. C. EASTERDAY AND V. S. HINSHAW in the HA molecule of the swine viruses is currently being determined by sequence analyses of the HAs of these MAb-selected variants. The H1N1 viruses isolated from pigs in southern Wisconsin from 1965 to 1988 were remarkably similar, indicating little antigenic variation in these viruses. This differs from the viruses circulating in humans in which antigenic drift is quite marked within a short time (Gerhard & Webster, 1978; Laver et al., 1979; Nakajima et al., 1983 ; Raymond et al., 1983), but is more similar to the conservation reported for avian viruses (Kida et al., 1987). This contrast between viruses maintained in humans and those in pigs may be explained by differences in the host populations. Since pigs in the U.S.A. are raised in close contact and are relatively shortlived, their viruses may be transmitted continually to non-immune, susceptible pigs. Antibody pressure and thus selection of antigenic variants within the population would be limited. The results of this study are consistent with the suggestion that swine influenza viruses are maintained by continual passage to naive hosts, since the antibody-binding sites within the neutralizing epitopes of these viruses are conserved. An additional possibility is that pigs do not make antibodies to these antigenic sites; this is currently being examined by competitive inhibition ELISA of immune swine sera against the individual MAbs. Our initial impression, from the HI results, was that the human and turkey H1N1 viruses of swine origin were antigenically quite different from the current swine viruses. However, the MAbs to the different antigenic sites obviously do bind to all of these viruses. This indicates that these antigenic sites are conserved on these different isolates. In considering why these viruses differ in HI, one explanation is that the human and turkey viruses are adsorption mutants, i.e. they differ in receptor binding affinities. In studies by YewdeU et al. (1986), Rogers et al. (1985) and Daniels et al. (1984, 1987), it was determined that MAb-selected variants which bound the selecting MAb in ELISA but were not inhibited in HI assays were adsorption mutants. These adsorption mutants were antigenically indistinguishable from the parental virus, but differed in their avidity for host cell receptors. It is tempting to speculate that the turkey and human H 1N 1 viruses are also adsorption mutants. Replication of the swine H1N1 viruses in humans and turkeys could involve virus interaction with cell receptors different from those encountered in swine, resulting in selection of receptor-binding mutants. This seems likely since Rogers et al. (1983) determined that virus isolates from different species varied in their receptor recognition. Since the receptor-binding site on the haemagglutinin has been defined (Weis et al., 1988), comparison of the HA sequences of both the escape and adsorption mutants should indicate the location of these changes on the haemagglutinin molecule and their relationship to receptor binding. Thus, the HA sequences of the escape and adsorption mutants are currently being determined. The excellent technical assistance of Tom Ruffalo is gratefully acknowledged. The authors would like to thank Ray Pawlisch and Eric Gonder for providing samples for virus isolation and also Hana Van Campen, Matthew Philpott, Catarina Hioe and Kevin Schultz for their helpful suggestions. This work was supported in part by USDA Biotechnology CRCR , Agricultural Experimental Station grant WIS3101, USDA special grant CRSR and U.S.P.H.S. research grant AI24902 from the National Institutes of Health, Allergy and Infectious Diseases. REFERENCES AUSTIN, F. J. & WEBSTER, R. G. (1986). Antigenic mapping of an avian HI influenza virus haemagglutinin and interrelationships of H1 viruses from humans, pigs and birds. Journal of General Virology 67, CATON, A. J., BROWNLEE, G. G. & YEWDELL, J. W. (1982). The antigenic structure of the influenza virus A/PR/8/34 haemagglutinin (H1 subtype). Cell 31, CENTERS FOR DISEASE CONTROL (1988). Human infection with swine influenza virus - Wisconsin. Morbidity and Mortality Weekly Report 37, DANIELS, R. S., DOUGLAS, A. R., SKEHEL, J. J., WILEY, D. C., NAEVE, C. W., WEBSTER, R. G., ROGERS, G. N. & PAULSON, J. C. (1984). Antigenic analyses of influenza virus hemagglutinins with different receptor-binding specificities. Virology 138, DANIELS, R. S., JEFFRIES, S., YATES, P., SCHILD, G. C., ROGERS, G. N., PAULSON, J. C., WHARTON, S. A., DOUGLAS, A. R., SKEHEL, J. J. & WILEY, D. C. (1987). The receptor-binding and membrane-fusion properties of influenza virus variants selected using anti-haemagglutinin monoclonal antibodies. EMBO Journal 6,

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