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Journal of General Virology (1993), 74, 1311-1316. Printed in Great Britain 1311 Influenza A virus haemagglutinin polymorphism: pleiotropic antigenic variants of A[Shanghai[ll[87 (H3N2) virus selected as high yield reassortants E. D. Kilbourne, ~* B. E. Johansson, 2 T. Moran, 2 S. Wu, 4 B. A. Pokorny, 1 Xiyan Xu 3 and N. C o x 3 1Department of Microbiology and Immunology, New York Medical College, Valhalla, New York 10595, 2Mount Sinai School of Medicine, New York, New York 10029, a W H O Influenza Center, Center for Infectious Diseases, Atlanta, Georgia 30333 and 4Department of Medicine, University of Rochester, Rochester, New York 14642, U.S.A. Genetic reassortment o f the A/Shanghai/11/87 (H3N2) variant of influenza A virus with A/PR8/34 (H1N1) virus [the standard donor of high yield (hy) genes for influenza vaccine viruses] resulted in the isolation o f two reassortants with differing H3 haemagglutinin (HA) phenotypes, X-99 and X-99a. The two H A phenotypes were derived from individual subpopulations of the H3N2 wild-type virus during the reassortment event. The H A mutants and their respectively derived reassortants (identical in RNA genotype) differed in antigenicity, replication characteristics, yield in chick embryos and haemagglutinin gene sequence. Despite antigenic differences in reactions to polyclonal rabbit antisera of 60 %, both X-99 and X-99a, the hy reassortants, were equally immunogenic and protective in BALB/c mice to challenge by parental wild-type virus. Differences in H A phenotype were related to a Ser to Ile change at amino acid position 186. These findings emphasize the polymorphism of influenza virus strains as well as the need for caution in selection of vaccine strains from among antigenicaUy distinct viral subpopulations. Introduction ants, although antigenically and biologically different, were equally immunogenic and protective in a mouse model system. Furthermore, the parental virus comprises at least two haemagglutinin (HA) subpopulations from which the reassortants, differing from each other by only three coding changes in HA1, were derived. Influenza viruses are an outstanding example of viral polymorphism. The genetic non-homogeneity of standard laboratory strains (reviewed by Kilbourne, 1978, 1987), recent isolates (Robertson et al., 1991), or even unpassaged clinical material (Katz & Robertson, 1992), is increasingly recognized. For the most part, intrastrain differences are minor and detectable only through analysis with monoclonal antibody (MAb) panels. However, antigenic differences demonstrated using polyclonal antibodies in hyperimmune sera have been described (Kilbourne, 1978; Both et al., 1983; Johansson & Kilbourne, 1992). Such differences have potential significance in the perennial fabrication of high yield (hy) influenza virus reassortants (Kilbourne, 1969; Baez et al., 1980) for use in vaccines against antigenic variants emerging in nature. We describe here two hy reassortants (X-99 and X99a) derived from different subpopulations of A / S h a n g h a i / l l / 8 7 (H3N2) that differ in antigenicity, binding affinity and yield. These differences introduced a dilemma in vaccine choice in 1989 because X-99a, the highest yielding reassortant, when subjected to initial antigenic analysis with ferret antisera appeared to be less broadly immunogenic. We shall show that both reassort0001-1488 © 1993 SGM Methods Viruses. The hy reassortant viruses X-99 and X-99a were produced by genetic reassortment of A/Shanghai/l 1/87 (H3N2) with A/PR/8/34 (H1N1) influenza A virus variants as described under Results. Hy reassortants X-91, X-97 and X-101 were derived from reassortment of A/PR/8/34 with A/Leningrad/360/86, A/Sichuan/ 2/87 and A/Beijing 4/89 H3N2 viruses, respectively(Table 1). Antisera. Antisera were produced by intravenous (i.v.) injection of rabbits with 3000haemagglutinatingunits of purifiedvirus. The rabbits were bled 42 days after initial injection and 7 days after a booster injection of virus on day 42. Prior to use in neutralization and haemagglutination inhibition (HI) tests, antisera were heated at 56 °C for 30 min and treated with Vibriocholeraereceptor-destroyingenzyme as previouslydescribed (Kilbourne et al., 1990). Serological titrations and antigenic analyses. HI, neuraminidase inhibition and neutralization tests and ELISA were carried out as previously described (Kilbourne et al., 1990). HI tests for antigenic analysis were performed in tubes using large volume transfers and interpolated dilutions. This precise method has a mean S.D. of_+19% (Horsfall & Tamm, 1953).Antibody titre ratios were calculated by the method of Archetti & Horsfall (1950). Homologous and heterologous Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 23:25:42 1312 E. D. Kilbourne and others goat antimouse IgG and the substrate 3-amino-9-ethyl carbazole. Hybridomas positive on X-99a but not PR8 or X-99 were considered X-99a-specific, and this was confirmed by HI testing. T a b l e 1. Biological phenotype o f A / Shanghai/11/87 H A mutants and their reassortants HA titre* HI titter Virus Egg MDCK NHS$ A-9 A-56 A/Sh/87 (ly) A/Sh/87 (hy) X-99 X-99a X-99I-§ X-99aEII 32 128 256 632 512 1264 16 45 8 128 - 11 7 11 7 8 5 < 1 7 < 1 8 < 1 < 1 < 1 7 < 1 8 < 1 < 1 * The reciprocal of endpoint dilution. Geometric mean of six individual eggs. t Expressed as logs of value. NHS, heated at 56 °C, as the source of non-specific ct2-macroglobulin inhibitor. § Inhibitor resistant escape mutant from the passage of X-99 with NHS. II Escape mutant from the passage of X-99a with X-99a-specific MAb A-9. titre ratios were determined for each virus, Va and V2. The following formula was used to calculate R (the coefficient of antigenic relatedness): R = v ~ , where R 1 = homologous titre V1/heterologous titre V~; R~ = homologous titre VJheterologous titre V1. When l / R = 2 , antigenic relatedness is 50%; if l / R = 4 , the antigenic relatedness is 25 % etc. Antigenic differences of 50% or more (1/R >~ 2) are significant (Archetti & Horsfall, 1950). All mathematical calculations were performed on an IBM-XT using a BASIC program written for these calculations (Kilbourne et al., 1990). PAGE. The differential migration of isolated virion proteins was studied by the method of Ritchey et al. (1977) using 7 to 14% gels under reducing conditions. RNA sequencing. RNA sequence analysis was performed by the dideoxynucleotide chain termination method, using synthetic oligodeoxynucleotide primers and reverse transcriptase essentially as described elsewhere (Cox et al., 1988) except that 10 gCi of [aSS]dATP (Amersham; specific activity > 1000Ci/mmol) for each 5.5 ~tl of reaction mixtures was used and reverse transcriptase incubations and chase were done at 42 °C for 20 min. The HA1 domains of the HA genes of the X-99aE and Sh/ly (low yield) viruses (Table 1) were amplified by the PCR method (Saiki et al., 1988; X. Xu et al., unpublished) with primer 7, 5'd ACTATCATTGCTTTG as the forward primer and reverse complement primer 1184, 5'd ATGGCTG CTTGAGTGCTT as the reverse primer. Primers complementary to the mRNA sense strand 5'd CCTGCGATTGCGCCGAAT, 5'd CGATATGTCTCCCGGTTT, 5'd TGGCATAGTCACGTTCAG and 5'd TAAGGGTAACAGTTGCTG beginning at nucleotides 1090, 809, 588 and 379, respectively, were used for sequencing the asymmetrically amplified PCR products. Production of X-99a HA-specific MAbs. Female BALB/c mice (Charles River) were immunized with 25 gg of sucrose-gradient purified X-99a virus by intraperitoneal injection. Animals were immunized on two occasions, 3 weeks apart, rested for 10 weeks and boosted with 10 gg X-99a intravenous injections, 3 days before fusion. The fusions were performed by a standard method (Holmdahl et al., 1989) and positive cultures were cloned by limiting dilution. Screening of MABs by immunostaining. The method of Usuba et al. (1990) was used. Briefly, MDCK cells were infected with virus and, following fixation with paraformaldehyde, hybridoma supernatants were added to each well. Binding was identified with peroxidase-linked Infection of mice. Groups of 20 g female BALB/mice were infected intranasally under light ether anaesthesia (Johansson & Kilbourne, 1991) and pulmonary samples of virus were measured as described previously (Schulman & Kilbourne, 1963). Results Derivation o f X-99 and X-99a hy reassortants f r o m A / S h a n g h a i / l l / 8 7 (H3N2) influenza virus X-99 was p r o d u c e d by r e a s s o r t m e n t o f A / S h a n g h a i / 11/87 ( S h / 8 7 ) a n d A / P R / 8 / 3 4 (PR8) viruses b y the usual p r o c e d u r e o f d u a l infection o f a chick e m b r y o a l l a n t o i c sac ( K i l b o u r n e et al., 1971). Viruses c o n t a i n i n g the H A a n d n e u r a m i n i d a s e ( N A ) antigens o f S h / 8 7 virus were isolated b y p a s s a g e with P R 8 a n t i b o d y . W h e n X-99 p r o v e d relatively p o o r yielding in vaccine p r o d u c t i o n , we screened 26 eggs i n o c u l a t e d with S h / 8 7 virus to o b t a i n the highest yielding w i l d - t y p e virus, then c a r r i e d o u t a second r e a s s o r t m e n t e x p e r i m e n t with the hy m u t a n t a n d PR8. T h e r e a s s o r t a n t r e c o v e r e d (X-99a) consistently p r o d u c e d two to three times m o r e H A t h a n X-99. Like its w i l d - t y p e p a r e n t X-99a differed in o t h e r b i o l o g i c a l p r o p e r t i e s f r o m X-99, i n c l u d i n g b i n d i n g affinity to a n t i b o d y a n d non-specific i n h i b i t o r (see below). F u r t h e r m o r e , p r e l i m i n a r y testing o f X-99 a n d X - 9 9 a w i t h specific ferret a n t i s e r a at the Centers for Disease C o n t r o l , A t l a n t a suggested t h a t there were significant antigenic differences, with X-99a being less like c o n t e m p o r a r y H 3 N 2 isolates a n d hence less suitable as a vaccine c a n d i d a t e . R e p e t i t i o n o f r e c i p r o c a l H I t i t r a t i o n s with the same ferret sera in the M o u n t Sinai l a b o r a t o r y c o n f i r m e d the antigenic difference in the r e a s s o r t a n t s , which b y o u r q u a n t i t a t i v e analysis ( K i l b o u r n e et al., 1990) was 65 % ( d a t a n o t shown). Phenotypic characterization o f X-99 and X-99a T a b l e 1 s u m m a r i z e s the b i o l o g i c a l p h e n o t y p e s o f X-99 a n d X - 9 9 a c o m p a r e d with the p h e n o t y p e s o f their H 3 N 2 p a r e n t a l viruses [Sh/87 a n d S h / 8 7 (hy)]. T h e p h e n o t y p i c characteristics o f an escape m u t a n t X - 9 9 I - , d e r i v e d b y p a s s a g e o f X-99 with n o r m a l horse s e r u m ( N H S ) a n d o f a n o t h e r , X - 9 9 a E , derived b y p a s s a g e o f X - 9 9 a with the X-99a-specific M A b , A-9, are also shown. I t is clear t h a t the r e a s s o r t a n t s X-99 a n d X - 9 9 a have derived their p l e i o t r o p i c serological a n d o t h e r characteristics f r o m their respective S h / 8 7 a n d S h / 8 7 hy p a r e n t s a l o n g w i t h a c q u i s i t i o n o f their surface g l y c o p r o t e i n s . A n a l y s i s o f the escape m u t a n t s also suggests t h a t the H A s o f X-99 a n d X - 9 9 a differ in at least two sites as defined b y r e a c t i o n s with N H S a n d the X-99a-specific M A b s , A-9 a n d A - 5 6 ; Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 23:25:42 Influenza virus haemagglutinin polymorphism 1313 Table 2. Antigenic relatedness of selected H3N2 strains as defined by reciprocal HI tests with polyclonal rabbit antisera (Ai/68)* (Eng/72) (Ln/86) (Sich/87) (Sh/87) (Bj/89) X-31 X-37a X-91 X-975 X-99 X-99a Sh/87 Sh/87(hy) X-101 X-31 X-37a X-91 lOOt 35 35 100 - 3 10 - 8 22 - 100 50 79 43 26 X-97 X-99 50 100 87 34 100 3 10 79 87 100 40 71 43 31 X-99a Sh/87 Sh/87 (hy) 8 22 43 34 40 100 43 89 24 71 43 100 40 - (Bj/89) X-101 43 89 40 100 - 26 100 31 24 100 * Strains from which HA and N A antigens were derived are in parentheses. t Percentage antigenic relatedness as determined by fractional dilution HI (homologous relatedness is 100 %). $ Reassortant virus, X-97, and antiserum to Sich/87 were used in this analysis. i.e. the antigenic and inhibitor phenotype are not linked (co-varying). 100 ~80 Antigenic characterization of X-99 and X-99a viruses and antecedent and succeeding H3N2 variants The same magnitude of antigenic difference between X99 and X-99a (60 %) is found for their respective wildtype antecedents Sh/87 and Sh/87 (hy) (Table 2). Although not completely identical, Sh/87 and X/99 and Sh/87 hy and X/99a were found to be related by 71% and 89%, respectively, by HI analysis. In plaque neutralization tests, X-99 and X-99a and Sh/98 and Sh/87 (hy) share only 25 % identity. Regarding the antigenic relatedness of recent H3N2 strains, summarized in Table 2, there is the significant difference between X-99a and all other strains, and similarity between X-99 and the Leningrad and Sichuan viruses isolated in 1986 and 1987. The scant relationship between both X-99 and X-99a to their antecedents of earlier decades, HK/68 and Eng/72 is apparent. The two Sh/87 HA variants, X-99 and X-99a, differ almost as much from one another as does the first epidemiologically significant H3N2 drift variant (Eng/ 72) from the antecedent 1968 prototype virus (Fig. 1). There are also differences between the reassortants with respect to their reaction in serological (HI) tests using sera of patients recently infected with Sh/87-1ike influenza virus. Geometric mean serum antibody titres was fourfold higher when X-99 was the test antigen and antibody increases after infection averaged 6.5-fold with X-99, and 2.5-fold with X-99a virus (data not shown). Whether indicative of differences amongst the reassortants in antibody binding affinity in the test system, or reflective of preferential infection and/or antigenic stimulation by the X-99-1ike phenotype in man, these results constitute further evidence of significant HA- i60 .R 40 g 20 0 Ai/68 Eng/72 Sh/87 Sh/87 (X-99) (X-99a) Fig. 1. The antigenic relatedness of hy reassortants of A/Aichi/2/68 (Ai/68) and A / E n g l a n d / 4 2 / 7 2 (Eng/72) and of A / S h a n g h a i / l l / 8 7 (Sh/87) reassortants X-99 and X-99a are comparable in degree. (Each pair has been separately compared.) mediated differences in X-99 and X-99a. The differing efficiency of MDCK-adapted and egg-adapted variants in demonstrating human antibody response (Schild et al., 1983) also may be relevant to the present observations. Comparative immunogenicity of X-99 and X-99a viruses in mice We designed an experiment to test the relative efficacies of X-99 and X-99a vaccines in protection of mice from challenge infection by the uncloned Sh/87 virus from which both reassortants had been derived. Groups of five mice were injected intraperitoneally with one or two doses of u.v.-inactivated X-99 or X-99a virus, then infected intranasally with Sh/87 virus 28 or 41 days later, respectively. Just prior to infection, serum HI antibody was measured and 3 days after infection infective murine pulmonary-derived virus was assayed in chick embryos. The virus recovered from infected mice was identical in phenotype (i.e. X-99-1ike) to the original Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 23:25:42 1314 E. D. Kilbourne and others Table 3. Amino acid differences among S h / 8 7 H A mutants and reassortants Amino acid Sh/hy X-99a X-99aE Sh/ly X-99 X-99I- 133 145 157 158 159 186 193 219 247 262 S N S E Y S K S E Y S K S E D N N S E Y S N S E Y S N -* Y S S S I I I N S R T K S S T K S S T N F S N N S S T N S S T * Deletion. infecting virus. The HI antibody responses to identical viruses or to the Sh/87 challenge virus did not differ significantly. When these mouse antisera were employed in cross-HI tests of X-99 and X-99a viruses, as had been done with rabbit and ferret antisera, they demonstrated complete viral antigenic relatedness (103 %). Concordant with this finding, both vaccines were found to be equally protective against the dose of the parent Sh/87 virus used in challenges following either primary or secondary immunization. Molecular basis o f the differences between X-99 and X99a The envelope proteins of X-99 and X-99a were identified by H1 and NI tests as being H3 and N2. The M 1 proteins of both viruses, and by inference RNA 7, were identified as derived from the PR8 virus through the use of PR8 M 1-specific MAb in ELISA (Johansson et al., 1989). The remaining viral proteins were identified as derived from the PR8 virus by PAGE of viral proteins (Ritchey et al., 1977) (results not shown). That the different reactivity of X-99 and X-99a in HI tests with polyclonal antisera was not ascribable to intrinsic differences in their N2 neuraminidases or to different ratios of the HA in their virions was demonstrated by the antigenic identity of their NAs in cross-NI tests and also by the preservation of typical X-99a-like reactivity in an H3N1 (PR8) reassortant derived from X99a (data not shown). Therefore, attention turned to HA as the determinant of the pleiotropic differences in X-99 and X-99a. Sequencing of HA-coding RNA 4 demonstrated nonsynonymous (coding) changes reflected in the amino acid differences shown in Table 3. Viruses reactive with X-99a specific MAb (hy phenotype) differed from non-reactive X-99 (ly phenotype) viruses by having serine rather than isoleucine at amino acid 186 of HA1. X-99aE, an escape mutant from the neutralization of X-99a with MAb, did not, as expected, show the above substitution at position 186, but did differ from all other viruses by an aspartic acid for tyrosine substitution at amino acid 159 which, like 186, is located in the antigenic site B (see Discussion). Discussion The emphasis in these studies is not upon the frequently reported minor antigenic variations detectable only with MAbs, but rather on the antigenic variation that is sufficiently extreme to be of potential epidemiological and immunological significance. The present results add to previous evidence of influenza virus strain heterogeneity (Kilbourne, 1987; de Jong et al., 1988; Robertson et al., 1991; Katz & Robertson, 1992) and confirm that significantly different antigenic variants identifiable with polyclonal serum as well as MAbs may coexist within a given viral strain. Selection of antigenic variants need not be immunological (Kilbourne, 1980; Erickson & Kilbourne, 1980; Dietzschold et al., 1983). Rather, antigenic change may be the consequence of selection for the hy characteristic (Both et al., 1983), the result of host adaptation (Schild et al., 1983), or may be entirely fortuitous. The recovery of 'Czech/89-1ike' or 'Guangdong/89-1ike' antigenically distinct variants from the same isolate has also been described (Johansson & Kilbourne, 1992). Recent studies suggesting a role for the host cultivation system in the selection of HA antigenic variants (Wood et al., 1989; de Jong et al., 1988) are relevant to the suggestion that egg-grown viruses tend to have greater antigenic diversity (Robertson et al., 1991 ; Wang et al., 1989; Katz & Robertson, 1992) and to the identification of certain sites on the HA molecule that appear to characterize egg adaptation of the virus. One study, however, demonstrated the identity of sequences obtained from an egg isolate and its corresponding clinical specimen (Rajakumar et al., 1990). A mutation site identified by Katz et aI. (1990) as a probable determinant of host specificity is position 186, the site that apparently is critical in the distinction of the X-99 and X-99a antigenic phenotypes. Both variants, however, have been cultivated only in the chick embryo host, in which differences in viral yield are demonstrable with both the wild-type (Sh/hy and Sh/ly) and reassortant (X-99 and X-99a) pairs. It is worth noting that serine at position 186 was usual for most egg-grown and two MDCKgrown clones studied by Katz et al. with PCR, yet in the present case, isoleucine at this position characterizes our egg-grown high yield phenotype viruses. Although Sh/hy, Sh/ly, X-99 and X-99a differ at other sites, these pairs have no substitution differences in common other than at position 186. With a panel of anti-H3 MAbs, amino acid 186 maps to the antigenic site B at the top of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 23:25:42 Influenza virus haemagglutinin polymorphism the HA molecule and at the interface of antigenic sites A and B. This site has been associated with frequent antigenic changes in nature (Underwood, 1984). It is not clear whether or not the inhibitor phenotype differences are defined also by changes at position 186. Escape mutants X - 9 9 I - from X-99, and X-99aE from X-99a, selected respectively with NHS and MAb A56, demonstrated no reversion at site 186 coincident with their changes in phenotype. Rather, a deletion of amino acids 157 and 158 occurred with X - 9 9 I - , and a tyrosine to aspartic acid substitution occurred at position 159 with X-99aE. These amino acids also lie within the antigenic site B and might influence binding of either antibody or inhibitor at position 186. The control of influenza is dependent upon a global (WHO) surveillance system that collects and categorizes new viral isolates with respect to their antigenic identity. At present antigenic characterization of new isolates is based principally on HI tests with serum obtained from ferrets 14 days after infection. However, the present experiments demonstrate a lack of concordance of antigenic analysis results when antisera from other species (rabbit and ferret) are compared with mouse antisera. Furthermore, whether or not the X-99 and X99a reassortants differ by 65 % (ferret), 60 % (rabbit) or not at all (mice), vaccines from both viruses were equally effective in protecting mice against infection with the uncloned parental Sh/87 virus. This result suggests that minor variation among vaccine candidate strains can be tolerated, particularly because it is uncertain which variant is truly representative of the original human virus. In this connection, intraisolate antigenic heterogeneity has been shown to exist even in humans by sequencing viral HAs directly from clinical specimens (Katz et al., 1990) or from separate clones derived from a single specimen (Robertson et al., 1991; Katz & Robertson, 1992). Prior studies have attempted to assess the significance of minor HA antigenic variation with respect to crossprotection in animal models. Comparisons among these studies and between these studies and our own are confounded by differences in viruses, host species and experimental design. Our finding that BALB/c mice cannot detect antigenic differences in X-99 and X-99a as measured either by HI antibody response or crossprotection are in accord with the studies in A/J mice by Rota et al. (1989) with vaccinia virus recombinants containing variant influenza B virus HAs. Katz et al. (1987) also demonstrated cross-protection of ferrets immunized by infection with antigenically distinguishable MDCK cell- and egg-grown variants. However, Wood et al. (1989), studying similar H1N1 host-adapted variants found a concordance of antibody response with cross-protection in guinea-pigs, ferrets and hamsters. 1315 Although mapping of HA epitopes with MAbs in combination with RNA sequencing has produced valuable information on virus structure, antigenic variation and evolution, pragmatic consideration of the epidemiological significance of antigenic variation and vaccine choice must depend also on the use of polyclonal antibody for analysis of strain differences. Ideally, whatever animal species is used for strain antigenic characterization, immunization should be carried out with non-replicating virus to forestall the selection of HA antigenic mutants best suited for replication in that particular host, but not necessarily representative of the input immunizing virus. 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