Department of Bacteriology, Yamagata University School of Medicine, Iida-Nishi, Yamagata 990-9585, Japan1
Author for correspondence: Emi Tsuchiya. Fax +81 23 628 5250. e-mail etakasit{at}med.id.yamagata-u.ac.jp
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Abstract |
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Introduction |
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The HA glycoprotein is the major surface antigen of influenza virus. Earlier studies of the HA amino acid sequences of natural and laboratory-selected antigenic variants of A/H3N2 viruses identified five distinct antigenic domains (designated AE) on the surface of the H3 molecule (Wiley et al., 1981 ; Daniels et al., 1983
) and are located as follows: site A is centred around a protruding loop containing residues 133 and 137 and 140146; site B is centred on a loop of residues 155160 and an
-helix at residues 186197; site C comprises the bulge around the bonded cysteine residues 52 and 277; site D is located near the interface between monomer subunits; and site E is near the bottom of the globular domain between sites A and C (see Fig. 2A
). Five operationally distinct antigenic sites (designated Sa, Sb, Ca1, Ca2 and Cb) were also identified on the H1 subtype HA molecule (Gerhard et al., 1981
; Caton et al., 1982
), although these sites may form a large contiguous antigenic area on the surface of its globular domain (Caton et al., 1982
). In contrast, very little is known about the antigenic structure of the H2 subtype HA. Yamada et al. (1984)
attempted to establish an operational antigenic map of the H2 subtype HA by selecting escape mutants with anti-H2 monoclonal antibodies (MAbs) and analysing their reactivity patterns, and obtained results that suggested that rather than discrete antigenic regions, the HA may have a continuum of determinants on its surface. However, none of the antigenic determinants has been located on the H2 molecule as yet.
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Here, we investigated the antigenic structure of the HA of A/Kayano/57 (H2N2) virus by using anti-HA MAbs and the escape mutants selected by these antibodies. The results show that the H2 subtype HA has an antigenic structure largely similar to that of the H3 subtype HA, but differs from the latter in having a highly conserved neutralizing epitope in the stem domain. We also discuss the epidemiological significance of the failure of A/H2N2 viruses to increase the number of oligosaccharide chains on the globular head of the HA.
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Methods |
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Monoclonal antibodies.
MAbs against the A/Kayano/57 virus HA were produced, as described previously (Hongo et al., 1986 ; Sugawara et al., 1991
), using purified egg-grown virions for immunization of BALB/c mice. The isotypes of the MAbs were determined by double immunodiffusion using rabbit antisera specific for each immunoglobulin subclass (Sugawara et al., 1986
).
Selection of escape mutants.
Serial 10-fold dilutions of the cloned parental viruses were mixed with an equal volume of a 1:10 dilution of ascites fluid containing MAb. After incubation for 30 min at room temperature, the mixture was inoculated onto a monolayer of MDCK cells and the viruses that escaped neutralization were allowed to grow under the agar overlay medium. Five to six days later, plaques were picked and treated again with a 1:10 dilution of ascites fluid containing MAb and then plaqued again. Escape mutants were used for analysis after growth in eggs. Cloned viruses were found to have greatly decreased reactivity with the MAbs used for selection, confirming that they were escape mutants.
Serological assays.
Enzyme-linked immunosorbent assays (ELISA) were performed according to the method of Kida et al. (1982) with purified virions (2·0 µg per well) as antigens (Sugawara et al., 1988
). ELISA titres are expressed as the highest antibody dilution that showed an absorbance value of >0·2 at 414 nm. Haemagglutination inhibition (HI) tests were carried out in microtitre plates with a 0·5% suspension of chicken erythrocytes (Katagiri et al., 1983
). Neutralization (NT) tests were carried out as described previously (Sugawara et al., 1986
).
Nucleotide sequence analysis.
Viral RNA was extracted from purified virions using the RNeasy Mini kit (Qiagen). The full-length HA gene cDNA was synthesized from viral RNA using AMV reverse transcriptase XL (Life Sciences) and an oligonucleotide primer complementary to positions 125 of RNA segment 4. cDNA was amplified by PCR using a plus-sense primer (positions 125) and a minus-sense primer corresponding to positions 17731751. Nucleotide sequences were determined from the PCR products by cycle sequencing using the BigDye Terminator Cycle Sequencing FS Ready Reaction kit and an automatic sequencer ABI PRISM 310 (Applied Biosystems).
Radioisotope labelling and immunoprecipitation.
Monolayers of MDCK cells were infected with stock virus at a multiplicity of about 10 p.f.u. per cell and labelled with 10 µCi/ml of [35S]methionine (ARC) for 15 min at 5 h post-infection (p.i.). Cells were then disrupted in 0·01 M TrisHCl (pH 7·4) containing 1% Triton X-100, 1% sodium deoxycholate, 0·1% SDS, 0·15 M NaCl and a cocktail of protease inhibitors (Hongo et al., 1997 ), and immunoprecipitated, as described previously (Sugawara et al., 1986
), with an anti-HA MAb. The immunoprecipitates obtained were analysed by SDSPAGE on 13% gels containing 4 M urea and processed for analysis by fluorography (Yokota et al., 1983
).
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Results |
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To delineate the antigenic sites recognized by the group I MAbs, a panel of escape mutants was examined for reactivity with each of the antibodies (including MAbs 32/105 and 33/105) in HI tests. The results (Table 2) show the presence of at least three non-overlapping or partially overlapping antigenic sites (I-A, I-B and I-C) defined by antibodies of this group; sites I-A and I-C are discrete from each other, whereas site I-B overlaps partially both site I-A and site I-C. MAbs 32/105 and 33/105 were both reactive with all of the escape mutants tested. Rather, their HI titres against most of the escape mutants were
10-fold higher than the titres against the parental virus. Thus, these two antibodies seemed to be directed to an antigenic site (tentatively designated I-D) distinct from sites I-A, I-B and I-C. However, the inability to select escape mutants with MAbs 32/105 and 33/105 did not allow reciprocal analysis, making the definition of the epitopes recognized by these antibodies difficult.
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Amino acid substitutions in escape mutants
To identify the amino acid changes responsible for the observed antigenic alterations (Tables 2 and 3
), the HA gene sequences of 25 escape mutants were determined and their predicted HA amino acid sequences were compared with that of the parental virus (Table 5
). Hereafter, all amino acid positions will be given relative to their position on the H3 molecule.
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To examine whether carbohydrate attachment sites produced by amino acid substitutions occurring at positions 131, 162 and 187 are used for glycosylation, MDCK cells infected with each of three mutants (1/119-EM2, 5/77-EM2 and 3/186-EM1) were labelled with [35S]methionine for 15 min at 5 h p.i. in the presence or absence of 1 µg/ml of tunicamycin (TM), a specific inhibitor of N-linked glycosylation (Takatsuki et al., 1971 ). Cells were then immunoprecipitated with MAb 4/68 to site II-B (reactive with all of the mutants selected by group I MAbs) and the resulting precipitates were analysed by SDSPAGE. Fig. 1(A)
shows that the HA molecules of two mutants (1/119-EM2 and 3/186-EM1) synthesized in the absence of TM migrated slightly more slowly than that of the parental virus, whereas non-glycosylated HA (NG-HA) molecules of these mutants synthesized in the presence of TM showed an electrophoretic mobility indistinguishable from that of the parental virus, indicating that the new glycosylation sites at positions 131 and 160 are both used. In contrast to the HA molecules of 1/119-EM2 and 3/186-EM1, the HA of 5/77-EM2 exhibited an electrophoretic mobility virtually identical to that of the parental virus. However, the NG-HA of this mutant migrated slightly faster than that of the parental virus, raising the possibility that the conformational change in the HA caused by the amino acid substitution at position 187 (D
N) may have resulted in an increase in electrophoretic mobility. Thus, it seems likely that the HA of 5/77-EM2, although it has an additional oligosaccharide chain at position 187, co-migrated with the parental virus HA because of its increased mobility caused by a D
N change at position 187.
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In Fig. 2(A), the positions of amino acid changes (except a change at residue 218), which were detected in the 25 escape mutants selected by MAbs to antigenic sites I-A, I-B, I-C, II-A and II-B, are shown on the three-dimensional structure of the H3 molecule. Residue 218 seems unlikely to be related to resistance to neutralization by site I-C MAb 3/179A, since three mutants selected by this antibody contain a mutation at residue 131 in common, but only one of them (3/179A-EM2) contains a mutation at residue 218. Two amino acid substitutions at positions 162 and 248, which were detected in the mutants selected by site I-A MAb 1/119, occurred in the upper part of the globular head of the HA corresponding to antigenic site B in the H3 subtype HA. Residues 137 and 187, whose changes were found in the mutants resistant to neutralization by site I-B MAbs 4/79 and 5/77, were located in a protruding loop corresponding to antigenic site A of the H3 subtype HA and on the N-terminal exterior face of an
-helix at residues 187196 belonging to site B, respectively. The change at residue 131, which was detected in 12 of the 13 escape mutants selected by six site I-C MAbs, occurred in an external loop corresponding to antigenic site A of the H3 molecule. One of the MAbs to site I-C (MAb 4/11) selected a mutant (4/11-EM1) with a change at residue 222, which is remote from residue 131 in the HA monomer and falls into a region corresponding to site D (or site B). Amino acid residue 40, whose substitution occurred in all of the three mutants selected by MAb 1/87 to site II-A, was located in the middle of the stem region where a neutralizing epitope has not been identified in either the H1 subtype HA (Caton et al., 1982
) or the H3 subtype HA (Wiley et al., 1981
). The amino acid change at position 273, detected in all of the three mutants selected by site II-B MAb 4/68, was found in the bulge around the disulfide-bonded cysteine residues 52 and 277, which correspond to site C on the H3 subtype HA.
Further characterization of site I-D MAbs
As mentioned above, the HI and NT activities of MAbs 32/105 and 33/105 to site I-D were very low compared to those of the antibodies to sites I-A, I-B and I-C when the parental virus was used for the assays (Table 1). However, the HI titres of site I-D antibodies against almost all of the escape mutants (except 4/79-EM2) selected by antibodies to sites I-B and I-C were
10-fold higher than the titres against the parental virus (Table 2
). To determine whether site I-D MAbs can neutralize these mutants more efficiently than the parental virus, the NT activity of MAb 32/105 against each of three representative mutants (4/79-EM1, 4/148-EM1 and 4/11-EM1) was compared with that against the parental virus, according to the procedures described in Fig. 3
. The results in Fig. 3
show that although the parental virus was neutralized by MAb 32/105, neutralization was incomplete even at the lowest antibody dilution tested (1:100); the escape mutants, however, were all neutralized completely at antibody dilutions from 1:4000 to 1:10000. This observation raised the possibility that the use of these escape mutants may allow us to obtain antigenic variants resistant to neutralization by site I-D antibodies. Therefore, we attempted to isolate such variants by using MAb 32/105 as the selecting antibody and the seed stock of 4/11-EM1 as a parent virus and succeeded in isolating five escape mutants that were completely resistant to neutralization by MAb 32/105 (data not shown). Comparison of the deduced HA amino acid sequences of these mutants with that of 4/11-EM1 showed that four of the five mutants had a single amino acid change at position 80 (I
F), although the remaining one was a double mutant with an additional change (T
A) at position 200. Residue 80 is located near the bottom of the globular head corresponding to antigenic site E on the H3 molecule (Fig. 2A
).
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Discussion |
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The positions of amino acid substitutions that were found in the HA1 subunits of 20 human A/H2N2 viruses isolated between 1957 and 1968 are shown in Fig. 2(B). Clearly, most of the substitutions occurred in the areas corresponding to antigenic sites A, B and D on the H3 molecule (sites I-A, I-B and I-C on the H2 molecule), as has been first pointed out by Schäfer et al. (1993)
. As Klimov et al. (1996)
reported more recently, however, a few changes also occurred in sites C (corresponding to site II-B) and E (corresponding to site I-D). These observations support again the notion that the H2 molecule has an antigenic structure similar to that of the H3 molecule. Changes were also found at residues 40 and 41 located in antigenic site II-A. However, both of these replacements occurred only in a single strain and were not detected in the subsequently isolated strains.
Most of the escape mutants selected by MAbs to sites I-A, I-B and I-C were found to acquire a new oligosaccharide at position 160, 187 or 131, respectively. It also became clear that a carbohydrate chain added to position 131 limits the antibody recognition of all of the five different epitopes identified in site I-C and that the chain added to position 187 not only masks two distinct epitopes in site I-B completely, but also partially or completely blocks the access of antibodies to five different epitopes in site I-C (Tables 2, 4
and 5
), which confirms the previously documented idea (Daniels et al., 1983
; Schulze, 1997
) that the addition of carbohydrates is much more effective than a single amino acid substitution in altering the antigenic properties of the HA. As described earlier, there is circumstantial evidence that suggests that influenza A (H1N1 and H3N2) and B viruses that have acquired additional oligosaccharide chains on the HA tips are more likely to prevail in the human population (Daniels et al., 1983
; Schulze, 1997
). The presence of an increased number of oligosaccharides on the HA tips of these viruses may promote their growth in humans by masking the antigenic sites efficiently.
The present study demonstrated that the 1957 strain of A/H2N2 virus had the potential to acquire at least one additional carbohydrate chain at position 131, 160 or 187. However, examination of the available HA amino acid sequences of human influenza A/H2N2 viruses showed that A/H2N2 viruses have never acquired a new carbohydrate attachment site on the tip of the HA during 11 years of circulation in humans and have only one conserved glycosylation site at position 169 or 170, which raises the possibility that the H2 subtype HA, unlike the H3 and H1 subtype HA molecules, might have a unique structural characteristic that does not allow the creation of new glycosylation sites on the tip. Although no differences were seen in the ability to grow in either eggs or MDCK cells between the parental virus and the escape mutants that had a new oligosaccharide at position 131, 160 or 187 (data not shown), it is still possible that these mutants may have the decreased ability to replicate in humans. Moreover, our preliminary experiments with site-specific mutagenesis showed that the H2 protein mutated to possess two or three oligosaccharide chains at positions 131, 160 and 187 had drastically decreased receptor-binding and membrane-fusing activities. Here, we observed that site I-D MAbs neutralized mutants with an additional oligosaccharide at position 131 or 187 much more efficiently than the parental virus, although its mechanism remains obscure. This suggests that site I-D antibodies might play a role in limiting the spread of such mutants in the human population by strongly inhibiting their growth. Although the reason why the number of oligosaccharides on the tip of the H2 subtype HA did not increase over a period of 11 years must be studied in the future, the failure of A/H2N2 viruses to employ this effective strategy for evading immune pressures might be one of the causes for their short survival time in humans.
Okuno et al. (1993) demonstrated previously that a neutralizing epitope was present in the middle of the stem region of the A/H2N2 virus HA and showed that the C179 antibody to this epitope cross-reacted with all human influenza virus strains of the H1 and H2 subtypes (but not with strains of the H3 subtype). The present study also showed the existence of an antigenic site (II-A) in the stem region of the H2 subtype HA and demonstrated that antibodies to this site were cross-reactive with all of the A/H2N2 viruses examined, although they did not react with the A/H1N1 viruses (data not shown). Furthermore, we found that site II-B MAbs, like site II-A MAbs, were highly cross-reactive. It should also be noted that three of the 19 MAbs generated in this study were directed to sites II-A and II-B, suggesting that the immunogenicity of these antigenic sites was not very weak, at least in mice. It is possible, therefore, that these cross-reactive antibodies may be produced in considerable amounts in humans, particularly in those infected repeatedly with A/H2N2 virus, which may inhibit significantly the spread of antigenic variants in the human population. This might be another cause for the short life of human A/H2N2 viruses.
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Acknowledgments |
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References |
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Received 30 March 2001;
accepted 7 June 2001.