The antigenic structure of a human influenza A (H1N1) virus isolate grown exclusively in MDCK cells

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1 Journal of General Virology (1990), 71, Printed in Great Britain 1683 The antigenic structure of a human influenza A (H1N1) virus isolate grown exclusively in MDCK cells Phil J. Yates, Janet S. Bootman and James S. Robertson* National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, U.K. A panel of monoclonal antibodies has been produced against a 1983 human influenza A (H1N1) virus that has been isolated and grown exclusively in MDCK cells. Several of these antibodies were used to select variants from MDCK-derived virus in cells and their epitopes were then located by determining the HA1 amino acid sequence. The operational antigenic map of the haemagglutinin indicates the presence of two distinct immunodominant antigenic regions which correspond but are not identical to antigenic sites Sa and Sb of the A (H1N1) virus A/PR/8/34. Also during this study, we characterized a unique group of antibodies for which variants require two distinct and specific HA1 amino acid substitutions to escape neutralization. Introduction The haemagglutinin (HA) of influenza virus is the major viral target for immune mechanisms in addition to its role of mediating adsorption and penetration into host cells. The antigenic structure of the HAs of A (H1), A (H3) and B subtypes of influenza virus has been investigated by constructing operational antigenic maps and by amino acid sequence analysis of natural field isolates and laboratory-derived variants (Wiley et al., 1981 ; Gerhard et al., 1981 ; Caton et al., 1982; Hovanec & Air, 1984; Berton & Webster, 1985; Raymond et al., 1986). The antigenic map of an influenza A (H1NI) virus, A/PR/8/34 (PR8), indicates five immunodominant antigenic sites designated Sa, Sb, Ca1, Ca: and Cb (Gerhard et al., 1981 ; Caton et al., 1982) and is comparable to that for A (H3N2) viruses (Wiley et al., 1981). However the PR8 virus analysed was isolated over 50 years ago and undoubtedly has undergone considerable laboratory adaptation and passage especially in the embryonated hen's egg. It has been demonstrated that during egg adaptation antigenic variants of influenza virus are selected (Schild et al., 1983; Oxford et al., 1987), and specific changes in the HA of B, A (H1N1) and A (H3N2) viruses associated with egg adaptation have been characterized (Robertson et al, 1985, 1987; Katz et al., 1987; Katz & Webster, 1988). The aim of this study was to investigate the antigenic structure of a human A (H 1N 1) influenza virus, isolated almost 50 years after the PR8 virus, and which has been isolated and undergone limited passage exclusively in mammalian tissue culture cells (MDCK). An operational antigenic map was constructed and the amino acid substitutions involved in antigenicity have been determined. We also determined that for variants to escape neutralization by a unique group of monoclonal antibodies (MAbs), two simultaneous amino acid substitutions on the HA1 are required either at positions 187 and 225 or at 189 and 225. Methods Viruses. The original viruses used in this study were obtained during an outbreak of influenza at Christ's Hospital Boarding School, Horsham, U.K. in February 1983 (Oxford et al., 1987). Viruses were isolated and propagated in MDCK cells or in the allantoic cavity of 11 day old embryonated hens' eggs. The viruses were collected from culture medium or allantoic fluid by centrifugation and purified on sucrose density gradients (Skehel & Schild, 1971). MAbs. BALB/c mice were immunized intraperitoneally with approximately haemagglutinating units (HAU) of a purified virus, A/Chr/91/83, isolated and grown exclusively in MDCK cells, followed by booster doses at 6 and 8 weeks. Spleens were removed 3 days after the final immunization and fused with NSO myeloma cells using standard techniques (Gerhard, 1980). The hybridoma cell supernates were screened for anti-influenza virus antibody production by ELISA in which plates were sensitized with 256 HAU of whole virus per well. Positive samples were cloned in soft agar and grown as ascites in BALB/c mice. Ascitic fluids were analysed for anti-ha antibodies by the haemagglutination inhibition (HI) assay. All antibodies with no HI activity were subsequently assigned to other viral polypeptides, primarily the nucleoprotein SGM

2 1684 P. J. Yates, J. S. Bootman and J. S. Robertson Selection of antigenic variants. Anti-HA antibodies were used to select antigenic variants from virus A/Chr/157/83. MDCK-derived viruses A/Chr/157/83 and A/Chr/91/83, obtained from the same outbreak of influenza, cannot be distinguished antigenically and have identical HA gene nucleotide sequences (data not shown). For selection of variants on MDCK cells viruses were treated with doubling dilutions ofa MAb and incubated at room temperature for 1 h (Daniels et al., 1987). The antibody virus mixtures were inoculated onto MDCK cells and grown in the presence of the selecting MAb at the same concentration to that used at the adsorption stage. Viruses obtained from cultures containing the minimum dilution of antibody at which virus escaped neutralization were cloned using a plaquing technique (Appleyard & Maber, 1974). Selection of antigenic variants was also carried out in the auantoic cavity of 11 day old embryonated hens" eggs (Daniels et al., 1987). The allantoic fluid was harvested at 3 days post-inoculation and the procedure repeated at a limiting dilution of virus so that a cloned population of virus was obtained. Antigenic analysis. Haemagglutination titrations and HI tests were carried out using microtitre plates, employing 0.7% turkey red blood cells. Ascitic fluids were treated prior to assay with receptor-destroying enzyme. RNA sequencing. Purified virus was disrupted in 10 mm-tris-hcl ph 7.5, 5 mm-edta containing 0-5% SDS and treated with 50 p.g/ml proteinase K (BDH) for 30 rain at 37 C. Total virion RNA was extracted twice with phenol-chloroform, twice with chloroform and then ethanol-precipitated. Nucleotide sequences were analysed directly by the dideoxynucleotide chain determination technique as described by Caton et al. (1982) using synthetic oligonucleotide primers supplied by A. Caton and R.S. Daniels. Results Selection and characterization of MDCK-derived antigenic variants Thirty-six anti-haemagglutinin MAbs derived from four individual fusions were prepared using virus isolated and propagated exclusively on MDCK cells. They were grouped initially according to their HI reactivity with a series of human influenza A (H1NI) viruses obtained from previous epidemics (data not shown) and, using 13 representative antibodies, 26 antigenic variants were selected from a virus which was isolated on MDCK cells and has undergone limited laboratory passage on the same cell substrate. This virus, A/Chr/157/83, is designated 157M. Not all of the antibodies used gave rise to variants and resistant variants could not be selected on MDCK cells against antibodies 336 and 485. An operational antigenic map of the 157M virus HA was constructed by testing each variant against the 36 MAbs in HI assays (Table 1). Thirteen distinct antibody profiles were obtained including a profile exhibited by eight antibodies (Table 1, group C) which had unchanged HI reactivity with all variants. There were 10 unique antigenic phenotypes amongst the 26 variants analysed and variants identical to M/725/V3A were isolated with high frequency and had lost HI reactivity with the greatest number of antibodies. The table shows two distinct, non-overlapping groups of virus-antibody interactions indicating at least two immunodominant antigenic regions on the HA of 157M virus. The nucleotide sequence of the HAl-coding region of the unique antigenic variants was determined by the dideoxynucleotide chain termination procedure using synthetic oligonucleotide primers. For seven of the variants a single base substitution in the HAl-coding region was detected, each of which resulted in an altered amino acid residue (Table 2) [for ease of comparison with the established three-dimensional (3D) structure of an H3 subtype haemagglutinin, H3 numbering is used throughout]. The remainder had two base changes which resulted in a single amino acid substitution in one variant and a double amino acid substitution in two variants. Several variants, antigenically identical to those whose substitutions are described in Table 2, were sequenced and had identical amino acid substitutions (data not shown). Variants resistant to antibodies in reactivity group A arose with amino acid substitutions at residues 30, 82, 159, 163,167 and 171 (Table 2). Variants resistant to antibodies in group B had substitutions at residues 102, 133, 188, 189 and 192 (Table 2). The location of these substitutions on the 3D structure of H3 subtype HA is shown in Fig. 1. The substitution at residue 163 (variant M/578/V21C), resulted in the loss of a glycosylation site. Selection and characterization of antigenic var&nts resistant to antibody 485 No variants resistant to eight of the MAbs (Table 1, group C) were obtained on MDCK cells. Two of these antibodies, 485 and 550, were used unsuccessfully on three occasions to select variants on MDCK cells. In an attempt to produce variants resistant to these antibodies, the selection process was performed in embryonated hens' eggs with virus 157E. This virus was isolated from the same clinical specimen as 157M, but in eggs instead of in tissue culture; 157E differs antigenically from 157M and structurally at HA l residues 163 and 189 (Robertson et al., 1987). Virus 157E did not react in an HI test with most of the antibodies but retained high HI reactivity with the eight antibodies of group C (data not shown). Using antibody 485, antigenic variants were selected both in eggs and on MDCK cells from virus 157E (variants E/485/E2 and E/485/M2, Table 3). Subsequently variants resistant to antibody 485 were obtained from 157M virus in eggs (variant M/485/E3, Table 3). These three variants had lost HI reactivity with all group C antibodies (data not shown). The HAl-coding regions of the antigenic variants

3 Antigenic map of an influenza A (H1N1) virus 1685 Table 1. Construction of the operational antigenic map of A/Chr/157/83 haemagglutinin HI reactivity of MAbs with antigenic variants M373 M373 M578 M339 M339 M740 M740 M725 M725 M721 Reactivity V 1A V2A V21C V1A V6A V3A V2A V3A V4D V6A group MAb (1)* (2) (1) (1) (1) (2) (3) (11) (3) (1) A 373 (2)~- - ~ _ (2) - - B (11) (1) ' _ C 485 (7) * Of the 26 antigenic variants derived directly on MDCK cells from 157M virus, there were 10 unique antigenic phenotypes. Numbers in parentheses indicate the total number of antigenic variants with a particular antigenic phenotype. t Numbers in parentheses indicate the number of additional MAbs with identical HI profiles. :~ Absence of an entry indicates that there was no loss of HI titre of the variant with respect to the parental virus, 157M; (+), indicates > fourfold drop in HI titre; (-), indicates >eightfold drop in HI titre. Table 2. Nucleotide changes in the HAl-coding region of MDCKderived antigenic variants and deduced amino acid substitutions* Variant Group Base change Amino acid substitution M/373/V1A A 549 (G-,A) G--,S (159) M/373/V2A A 574 (C--,T), 782 (G-,A)# S--,F (167) M/578/V21C A 562 (A-,T), 141 (C-,A) N-,I (163), M/339/V1A A 585 (A~G) N-,D (171) M/339/V6A A 303 (A-,G) K-,E (82) M/740/V3A B 637 (T~G) I~R (188) M/740/V2A B 649 (A~G), 363 (T-,C) K-,R (192), M/725/V3A B 639 (G~A) E--,K (189) M/725/V4D B 640 (A-,G) E-,G (189) M/721/V6A B 469 (C~T) T--,I (133) L--,I (30) Y-,H (102) * For ease of comparison with the established 3D structure haemagglutinin, H3 numbering is used. t Silent base change. of an H3 subtype resistant to antibody 485 were subjected to sequence analysis and the deduced amino acid substitutions are shown in Table 3. Each of the three variants had common substitutions at residues 225 (D~G) and 189 (E--,K) compared to 157M. These data suggested that, with certain MAbs viz. MAb 485, variants may require at least two amino acid substitutions, one at residue 225 and another at residue 189, to escape neutralization. Selection of antigenic variants from previously character&ed egg-adapted variants In a separate study (Robertson et al., 1987) a different set of variants were derived from 157M by adapting the virus to growth in eggs. Several of these egg-adapted variants contained substitutions in the HA1 at one of the residues: 187, 189 or 225. The HI reactivity of such variants with antibody 485 and substitutions in their HA1 compared to 157M HA are shown in Table 4. Eggadapted variants 5a, 5b and 21b which had single amino acid mutations at residues 189, 187 and 225 respectively reacted with antibody 485 with titres comparable to 157M. In addition, variant 5a (189, E-,K) had lost reactivity with most of the group B antibodies; variant 5b (187, N-,K) had lost reactivity with several of the group B antibodies and had a considerably reduced HI titre with one of the group C antibodies; whereas variant 21b (225, D--,G) reacted with all antibodies (data not

4 1686 P. J. Yates, J. S. Bootman and J. S. Robertson 159 Table 4. HI reactivity with MAb 485 and HA1 amino acid substitutions of antigenic variants derived from egg-adapted variants 225 T 171 HA1 amino acid HI titre substitution Variant Parent with 485 relative to 157M 157M * a 157M (E~K) t 5b 157M (N~K) t 21b 157M (D~G) t 56c 157M < (N~K), 225 (D~G)t 5a/485/V1A 5a < (E~K), 225 (D--,G) 5b/485/V1A 5b < (N~K), 225 (D~G) 21b/485/V2A 21b < (N~K), 225 (D~G) * Isolated from clinical specimen. t Data from Robertson et al. (1987). Fig. 1. Location of residues implicated in the antigenicity of A/Chr/157/83 in the haemagglutinin 3D structure. The figure shows a side view of the peptide backbone for amino acid residues 58 to 263 of the HA 1 region of the H3 subtype haemagglutinin (Wilson et al., 1981) and was kindly provided by Dr C. Naeve, Memphis, Tenn., U.S.A. (~1,), Residues in site Sa; (O), residues in site Sb; (A), residue 225; (11), residues 102 and 236; (O), glycosylation sites 131, 158 and 163. shown). Significantly, egg-adapted variant 56c which had a double mutation at residues 187 and 225 had no HI reactivity with antibody 485 (Table 4). To test the premise that in order to lose HI reactivity with the group C antibodies, two substitutions are required (one at residue 225 and one in the region of residue 189), egg-adapted variants 5a, 5b and 21b were subjected to selection with antibody 485 on MDCK cells. The resulting variants had lost HI activity with antibody 485 (Table 4) and all other group C antibodies. The variants derived from 5a (189, E~K) and 5b (187, N~K) had a single additional amino acid substitution at position 225 (D--,G) whereas the variants derived from 21b (225, D--,G) had a single additional amino acid substitution at position 187 (N-,K) (Table 4). The full antigenic profiles of egg-adapted variant 56c and the variants selected by antibody 485 from 5b and 21b were identical (data not shown). Discussion The HA of the 1983 MDCK-derived influenza A (H1N1) virus A/Chr/157/83 contains at least two immunodominant antigenic regions (Table 1) which correspond approximately to antigenic sites Sa and Sb defined for A/PR/8/34 (PR8) (Caton et al., 1982). PR8 was isolated almost 50 years previously and is antigenically distinct from 157M due to the evolution of H1N1 viruses since 1934, and presumably also to extensive laboratory passaging of PR8. In PR8, HA sites Sa and Sb are closely linked but separated by a polypeptide loop formed by residues 156 to 160. Site Sb is defined by residues 156 and 159 within this loop and by adjacent residues in a region of the a-helix at the top of the HA molecule. Site Sa is defined by the continuation of the polypeptide loop down Table 3. HI reactivity of MAb 485 with resistant variants and their HA 1 substitutions HI titre Virus Parent with 485 HA1 amino acid substitution relative to 157M 157M * E * 9600 E/485/E2 157E < 50 E/485/M2 157E < 50 M/485/E3 157M < (N--,S), 189 (E-~K) 163 (N--,S), 189 (E~K), 192 (K-*Q), 225 (D--,G) 163 (N-~S), 189 (E-~K), 192 (K~N), 225 (D~G), 236 (L-~M) 189 (E--,K), 225 (D~G), 300 (V~I) * Isolated directly from a single clinical specimen.

5 Antigenic map of an influenza A (H1N1) virus 1687 the side of the HA molecule including most of the residues from 158 to 167. Site Sb of 157M comprises residues 133, 187, 188, 189 and 192, the latter four of which are located in the short a-helix situated on the distal tip of the HA molecule (Fig. l). Site Sa of 157M is defined by residues 82, 159, 163, 167 and 171 (Fig. l). Additional non-unique substitutions were found at residues 30, 102, 236 and 300. Residues 30 and 300 are located in the stem region and are unlikely to affect antigenicity whereas any contribution to antigenicity by residues 102 and 236, which occurred in variants M/740/V2A and E/485/M2 respectively, is unknown. Thus in 157M, site Sa covers a greater area and is not as closely linked to site Sb as in PR8. This is probably due to carbohydrate masking of site Sa. PR8 (Mt Sinai) used in the study by Caton et al. (1982) contains one glycosylation site in the HA1 globular head at Asn (271). In contrast recent H 1 N 1 viruses including 157M have four additional HA1 glycosylation sites at Asn residues 94a, 131, 158 and 163 (Raymond et al., 1986). Glycosylation of residues 131,158 and 163 would considerably mask site Sa and form a barrier between this and site Sb resulting in their increased separation. The activity of some of the group A antibodies, viz. 377, 379 and 578, is potentially influenced by a carbohydrate at AsH (163), and the increased glycosylation of site Sa may also account for the large spread of epitopes within this region. Most of the antibodies raised in this study were against site Sb and residue 225 in site Ca. Similarly, the location of substituted residues in field isolates in the study by Raymond et al. (1986) suggested that sites Sb and Ca are of greatest significance. The absence of immunodominant regions in 157M corresponding to PR8 sites Ca1, Ca 2 and Cb may also be due to the use of whole virus in this study to raise and characterize anti-ha antibodies whereas in Caton et al. (1982) several antibodies were raised against infected cells and all were characterized using disrupted virus. To escape neutralization by the group C antibodies there was an absolute requirement for variants to substitute two specific amino acid residues. These could be either residues 187 and 225 or 189 and 225. In this study variants resistant to antibody 485 could not be obtained directly from the parent 157M virus in MDCK cells presumably because of the low frequency of occurrence of the required double mutant (~10-9'13, Yewdell et al., 1979). Selection of a resistant mutant occurred readily either in MDCK cells or in eggs if one of the two required substitutions was already present, as with isolate 157E or egg-adapted variants 5a, 5b or 2lb. Selection of a resistant mutant directly from parental 157M virus in eggs, apparently involved the initial selection of an egg-adapted virus. Studies on the antigenic structure of influenza HA (Caton et al., 1982; Berton & Webster, 1985), and on the surface proteins of other RNA viruses (Vandepol et al., 1986; Sherry et al., 1986; Coelingh et al., 1987) indicate that in general, a single amino acid substitution is sufficient for virus to escape neutralization. Sherry et al (1986) suggested that some variants selected during a study of rhinovirus neutralization sites required two substitutions to achieve full resistance. In all other cases, there has been no demonstration that more than one substitution within a double mutant is necessary for resistance and the ease of isolation of reported double mutants suggests that only one substitution is required. One possibility for our observation is that the group C antibodies are not monoclonal. This is highly unlikely for the following reasons: isolation of eight MAbs, all of the same isotype (IgG class 2b), from two individual fusions by the agar technique, the stability of the cell lines; and the unaltered characteristics of the antibodies after 4 years of use. These factors argue for a homogeneous antibody preparation. In addition, the hybridoma cell line-secreting antibody 485 has recently been recloned and no change was observed in the HI profile of nine out of nine clones against the viruses listed in Table 4. Thus we have described a unique class of MAbs against which variants must acquire two amino acid substitutions to escape neutralization. We are indebted to Dr Clayton Naeve, Memphis, Tenn., U.S.A. for generating the computer printout of the HA 3D structure. References APPLEYARD, G. & MABER, H. B. (1974). Plaque formation by influenza viruses in the presence of trypsin. Journal of General Virology 25, BERTON, M. T. & WEBSTER, R. G. 0985). 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