The D. I. Ivanovsky Institute of Virology, 16 Gamaleya Str., 123098 Moscow, Russia1
Division of Virology, Department of Infectious Diseases, St Jude Childrens Research Hospital, 332 North Lauderdale St, Memphis TN 38105-2794, , USA2
Author for correspondence: Robert Webster. Fax +1 901 523 2622. e-mail robert.webster{at}stjude.org
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Abstract |
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Introduction |
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Only a few of the 15 antigenic subtypes of influenza virus haemagglutinin (HA) have been structurally characterized. The H3 molecule has been well characterized with the use of antigenic drift variants and HA escape mutants, and the antigenic epitopes have been mapped within the molecules three-dimensional structure (Wiley et al., 1981 ). The HA antigenic sites of the H1 and H2 subtypes have been mapped on the H3 three-dimensional structure (Caton et al., 1982
; Tsuchiya et al., 2001
). The H1, H2 and H3 subtypes that circulate (or have circulated) in humans comprise a variety of HA antigenic drift variants (Murphy & Webster, 1990
). In contrast, avian HA subtypes retain a relatively stable antigenic structure because of the brevity of the avian lifespan and the replication of virus in the intestinal tracts of aquatic birds (Webster et al., 1992
). For this reason, selection and characterization of escape mutants is the only available means of identifying antigenic epitopes in these HA subtypes. The antigenic structure of H5 HA of the pathogenic avian A/Turkey/Ontario/7732/66 (H5N9) influenza virus was characterized by this method (Philpott et al., 1989
; 1990
). Five neutralizing epitopes were identified, and their location was mapped on the three-dimensional model of the H3 HA molecule. However, because only six amino acid changes were detected in these five epitopes, additional information is needed to elucidate the location and fine structure of H5 antigenic sites.
Here we describe the selection of escape mutants of a mouse-adapted variant (Smirnov et al., 2000 ) of non-pathogenic avian A/Mallard/Pennsylvania/10218/84 (H5N2) virus. We characterized the escape mutants by their cross-reactions with mAbs and by the amino acid changes in their HA molecules. The extent and fine structure of two antigenic sites in the H5 HA molecule are revealed. We also describe the effect of the amino acid changes in the escape mutants on the glycosylation of HA, sensitivity to normal mouse serum inhibitors and virulence in mice.
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Methods |
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Monoclonal antibodies.
A panel of virus-neutralizing mAbs to the HA of H5 strains was used. The mAbs 24B9 and 77B1 (kindly provided by V. Hinshaw) were obtained by priming mice with A/Turkey/Ontario/7732/66 (H5N9) (Tk/Ontario/66) virus and administering a boost dose of Tk/Ontario/66 (for 77B1) or Mld/PA/84 (for 24B9) (Philpott et al., 1989 ). Monoclonal antibodies to the H5N2 viruses A/Chicken/Pennsylvania/1370/83 (Ck/PA/1370/83) (four mAbs) and A/Chicken/Pennsylvania/8125/83 (Ck/PA/8125/83) (three mAbs) were produced by the Virology Department of St Jude Childrens Research Hospital. The mAbs are shown in Tables 1
and 2
.
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Selection of escape mutants.
Single-step selection was performed as described previously (Webster & Laver, 1980 ). Virus was incubated with an excess of mAb for 1 h at 20 °C and the mixture was inoculated into 10-day-old embryonated chicken eggs. Virus was harvested and used for limiting-dilution cloning in embryonated chicken eggs. The first-generation mutants (one-step escape mutants) were selected from the wild-type Mld/PA/84-MA virus. One-step escape mutants underwent further selection with the mAbs to which each mutant retained sensitivity and the resistant variants (two-step escape mutants) were cloned. The two-step mutants underwent selection with a third mAb to generate three-step escape mutants.
PCR amplification and sequencing.
Viral RNA was isolated from virus-containing allantoic fluid using the RNeasy Mini kit (Qiagen), as specified by the manufacturer. Uni-12 primer was used for reverse transcription. PCR was performed with primers specific for the HA gene segment (primer sequences are available on request). PCR products were purified with QIAquick PCR purification kit (Qiagen). The sequencing reaction was performed by the Hartwell Center for Bioinformatics and Biotechnology at St Jude Childrens Research Hospital. The DNA template was sequenced using rhodamine or dRhodamine dye terminator cycle-sequencing Ready Reaction Kits with AmpliTaq DNA polymerase FS (Perkin-Elmer, Applied Biosystems) and synthetic oligonucleotides. Samples were analysed in a Perkin-Elmer Applied Biosystems model 373 or model 377 DNA sequencer. DNA sequences were completed and edited using the Lasergene sequence analysis software package (DNASTAR). Multiple sequence alignments were performed according to the methods of Nobusawa et al. (1991) and Ha et al. (2001)
with GeneDoc, version 2.3, software (developed by K. B. Nikolas).
Infection of mice.
White female mice weighing 8 g were lightly anaesthetized with diethyl ether and inoculated intranasally with 50 µl of serial 10-fold dilutions of virus (six mice per dilution). The mortality rate was registered for 10 days after inoculation and expressed in 50% mouse lethal doses (MLD50) calculated by the routine method of Reed & Muench (1932) .
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Results |
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The one-step escape mutants selected by mAbs cp55 and cp46 were not cross-resistant to these two mAbs. However, the escape mutants selected by mAbs cp58 and 176/26 were resistant to both cp46 and cp55 (and to mAb 364/1, which reacted similarly to cp55). This finding suggested that the epitopes recognized by cp58 (and 176/26), by cp55 (and 364/1) and by cp46 partially overlap. The epitopes were designated Ia, Iab and Iac, respectively (Table 1).
The mAb 77B1 could not be definitely assigned to any of the groups. It reacted with the wild-type virus at a relatively low HI titre but reacted with some escape mutants at much higher titres. However, because the mutants selected with cp55 did not react with 77B1, it seems likely that the latter is related to group Iab. The mAb 77B1 has been shown to recognize an area equivalent to site B in the H3 subtype (Philpott et al., 1990 ).
The two-step and three-step escape mutants reacted with mAbs in a pattern generally consistent with that of the one-step escape mutants. However, mAb 176/26 reacted to a limited extent with two two-step escape mutants selected either with 176/26 (the mutant m24B9-176/26) or with a similar mAb, cp58 (the mutant m58(1)-24B9). It should be noted that mAb 176/26 also reacted with one-step escape mutants selected by cp58 and 176/26, although to a very slight extent, so that HI titres were too low to be reflected in Table 1 (7 log2 units lower than the titre of the wild-type virus). Possible reasons for the anomalous reactions of mAb 176/26 are discussed with the ELISA results.
The pattern of cross-reaction of the escape mutants with mAbs in ELISA (Table 2) differed from the results of HI in several ways. The mAbs cp58, cp46, 24B9 and 77B1 reacted similarly in ELISA and in HI with one-step, two-step and three-step escape mutants. However, mAbs 176/26, cp55 and 364/1 reacted differently. In ELISA, mAb 176/26 reacted with several escape mutants that it had selected (or that the closely related cp58 had selected). The mAb cp55 also reacted with its own escape mutants, although to a lesser extent. The mAb 364/1 had not been used for the selection of mutants. However, it reacted in ELISA (but not in HI) with the mutants selected by the closely related cp55. The escape mutants that reacted in ELISA with the mAbs used for their selection were confirmed to be genuine escape mutants: they could not be neutralized by these mAbs in egg infectivity neutralization tests (not shown). One three-step escape mutant, m46(7)-55-176/26, unlike other mutants selected by the mAb 176/26, did not bind the mAb in ELISA (Table 2
). This mutant also exhibited no residual HI reactivity with mAb 176/26, unlike the two-step escape mutants m24B9-176/26 and m58(1)-24B9 (Table 1
). On the whole, the results of ELISA confirmed the grouping based on HI cross-reactions, although the continued binding of some mAbs to the neutralization-resistant mutants made interpretation of ELISA results more difficult than interpretation of HI results.
Sequence analysis of the escape mutants
The HA genes of the cloned Mld/PA/84-MA virus and of all escape mutants were sequenced and the encoded amino acid sequences were compared with the previously reported sequence of the Mld/PA/84-MA HA (Smirnov et al., 2000 ) (GenBank accession no. AF100179). The HA sequence of the cloned Mld/PA/84-MA virus used in our studies differed from the reported HA sequence in only one amino acid, A263T [H5 numbering according to Ha et al. (2001)
is used here and below]. The amino acid changes in escape mutants were grouped mainly in three areas of the HA1 subunit (Table 3
). The mutations conferring resistance to 24B9 (epitope II in the operational mapping) were concentrated in the area 136141 (140145 in the H3 sequence). With the exception of m46(7)-55-176/26, the mutants selected by the mAbs cp58 and 176/26 (epitope Ia) had a D126N change (position 131 in the H3 sequence). The mutants selected by cp55 (epitope Iab) had a change at position 152. Interestingly, the amino acid changes in two one-step escape mutants selected by cp46 (epitope Iac) occurred in different areas: N124D in m46(7) and K153M in m46(8). Fig. 1
shows the positions of the mutations identified in the escape mutants aligned with the three-dimensional structure of H5 HA (Ha et al., 2001
). The epitopes outlined by operational mapping clearly correspond to specific sites in the three-dimensional structure. The mutants selected by 24B9 (epitope II) have amino acid changes located in the loop 136141, whereas the mutants selected by the mAbs recognizing Ia, Iab and Iac epitopes lie in the region of the beta-structure at the top of the HA molecule.
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Several mutants had additional amino acid changes in the regions not usually associated with antigenic sites. The mutant m58(2) had an additional change in the HA1 subunit (R48S), and m176/26 and m46(7)-24B9 had additional changes in the HA2 subunit. There is no evidence that these changes affected the antigenic specificity of the mutants. The mutants m58(1) and m58(2) behaved identically in the immunological tests, although m58(1), unlike m58(2), had no amino acid change at position 48. The location of the amino acid change T185K in the m46(7)-55a mutant corresponds to site B in H3 HA (T189K in H3 numbering) (Wiley et al., 1981 ); however, because no antigenic differences were observed between m46(7)-55a and m46(7)-55, which did not have this mutation, there is no evidence that the T185K affects immune specificity.
To determine whether the potential glycosylation site created by the D126N mutation is glycosylated and, if so, whether the carbohydrate chain blocks mAb binding, we infected MDCK cells with the mutant m58(1) or with the wild-type virus and incubated them with or without tunicamycin (TM) (2 µg/ml) for 2 h before and for 1 h during incubation with [14C]amino acids. Cells were lysed and precipitated with mAb cp58. The mAb 176/26, which selected the same mutation, 176/26, could not be used in the immunoprecipitation studies because it retained the ability to bind the D126N mutants, as revealed by ELISA. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that mAb cp58 precipitated HA in the samples infected with wild-type virus and incubated either with or without TM, but did not precipitate HA in the samples infected with m58(1) and incubated without TM (Fig. 2). The incubation of m58(1)-infected cells with TM restored the reactivity of HA with the mAb to the level observed in cells infected with the wild-type virus. These results suggest that the prevention of glycosylation by TM unmasks the antigenic epitope that reacts with cp58.
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Discussion |
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We observed several unusual reactions between mAbs and escape mutants. The escape mutants that had the amino acid change D126N were resistant to infectivity neutralization by mAb 176/26 but continued to bind the mAb in ELISA (Table 2). H3 mutants with residual binding capacity for the mAb used for selection have been described (Daniels et al., 1987
), but they were receptor-binding variants whose antigenic specificity was not altered. This explanation is unlikely to apply to our D126N mutants, because the mAb cp58 selects such mutants but does not react with them in ELISA. The two-step mutants with the D126N change reacted with mAb 176/26 (but not with cp58) in HI, although to a low titre (Table 1
). It seems plausible that the epitope recognized by 176/26 includes two parts, one of which is important for infectivity neutralization and is recognized by both cp58 and 176/26, and one that is inaccessible to cp58 and is able to bind 176/26 with no effect on infectivity. The retention of antibody-binding ability by escape mutants is probably not exclusive to the mutants selected with mAb 176/26. The mAb cp55 also binds its escape mutants (Table 2
). The mAb 364/1, although not used for the selection of escape mutants, may have similar features, because it behaves like cp55 with respect to the cp55 escape mutants (Tables 1
and 2
). Therefore, H5 escape mutants may have a characteristic tendency to acquire a resistance to infectivity neutralization by a mAb while retaining the ability to bind the antibody molecule.
The mAb 77B1 exhibited higher HI titres with D126N escape mutants than with the wild-type virus. The strain Tk/Ontario/66, against which mAb 77B1 had been raised, has an asparagine residue at this position (Philpott et al., 1990 ). Thus, the increase in titre may reflect the presence in the mutant (but not in the wild-type virus) of an epitope identical to that of the virus used for immunization.
The reduced virulence of the escape mutants in mice, at least of the mutants having D126N and K152N substitutions (Table 4), is most likely due to the mutations selected by the mAbs in the HA rather than to random co-selection of the mutations in other viral genes. First, the loss of virulence associated with the D126N change occurred in two escape mutants selected independently with different mAbs, making a random effect of a co-selected mutation unlikely. Secondly, we previously showed that an amino acid change in this position in a mouse-adapted H1N1 virus eliminated a glycosylation site and correlated with the acquisition of virulence in mice (Gitelman et al., 1986
). A correspondence between reduced virulence in mice and the appearance of new glycosylation sites has also been shown in viruses of the H3 subtype (Reading et al., 1997
). In this case, as with our D126N mutants, the loss of virulence was associated with an enhanced reaction with mouse serum inhibitors. Another amino acid change associated with decreased virulence in mice, K152N (Table 4
), is located at the same position as a substitution that attenuates the virulence of an H5 escape mutant in birds (Philpott et al., 1990
). This fact argues against a random co-selection of mutations in other viral genes as the cause of the reduced virulence of the K152N escape mutant in mice. The mutations in the H5 escape mutants that attenuate their virulence may be regarded as pleiotropic, and their occurrence can presumably influence immune selection under natural conditions.
We were unable to select mutants that had amino acid changes in the regions equivalent to the H3 sites C and E or at position 120 (H3 numbering), a site described by Philpott et al. (1990) . Tsuchiya et al. (2001)
recently reported that mAbs reacting with the sites in the H2 molecule equivalent to the H3 sites C and E neutralized infectivity but did not react in HI. We used exclusively high-titre HI-reactive mAbs, and this choice of mAbs could have limited the diversity of the selected escape mutants. However, our findings provide detailed information about the sites equivalent to the H3 A and B regions. In the studies of Philpott et al. (1989
, 1990
), mAb 24B9 selected a mutant with an S141P change. In our study, this mAb selected a series of mutants with changes at positions 136, 138, 140 and 141. Thus, in H5 HA, the loop forming site A must extend at least from position 136 to position 141. The H5 site that is equivalent to site B of H3 appears to be more complex in H5 than in H3. We found that this site contains not only the region present in the H3 site B but also the region 124129, which partially overlaps site Sa of H1 (Caton et al., 1982
). It is noteworthy that all mAbs obtained by immunization with Ck/PA/1370/83 and Ck/PA/8125/83 viruses reacted with this complex site (Tables 1
and 2
). Therefore, the cells that produce antibodies against this site appear to predominate in the mouse immune repertoire. Interestingly, only one amino acid change in our H5 escape mutants coincided with the position (131 in H3 numbering) of a mAb-selected mutation in the closely related H2 HA (Tsuchiya et al., 2001
). However, amino acid changes in the H5 escape mutants occurred at several positions (131, 144, 156, 157 and 189 in H3 numbering) that coincided with substitutions in H2 drift variants (Tsuchiya et al., 2001
). It seems plausible that the antigenic sites described and analysed here may play an important role in drift variation if the H5 virus appears as a pathogen in human circulation.
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Acknowledgments |
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Footnotes |
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References |
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Bean, W. J., Kawaoka, Y., Wood, J. M., Pearson, J. E. & Webster, R. G. (1985). Characterization of virulent and avirulent A/chicken/Pennsylvania/83 viruses: potential role of defective interfering RNAs in nature. Journal of Virology 54, 151-160.[Medline]
Caton, A. J., Brownlee, G. G., Yewdell, J. M. & Gerhard, W. (1982). The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell 31, 417-427.[Medline]
Cauthen, A. N., Swayne, D. E., Schultz-Cherry, S., Perdue, M. L. & Suarez, D. L. (2000). Continued circulation in China of highly pathogenic avian influenza viruses encoding the hemagglutinin gene associated with the 1997 outbreak in poultry and humans. Journal of Virology 74, 6592-6599.
Centers for Disease Control & Prevention. (1998). Update: isolation of avian influenza A (H5N1) viruses from humans Hong Kong, 19971998. Morbidity and Mortality Weekly Report 46, 12451247.[Medline]
Claas, E. C. J., Osterhaus, A. D. M. E., van Beek, R., de Jong, J. C., Rimmelzwaan, G. F., Senne, D. A., Krauss, S., Shortridge, K. F. & Webster, R. G. (1998). Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472-477.[Medline]
Daniels, R. S., Jeffries, S., Yates, P., Schild, G. C., Rodgers, 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, 1459-1465.[Abstract]
Gitelman, A. K., Kaverin, N. V., Kharotonenkov, I. G., Rudneva, I. A., Sklyanskaya, E. I. & Zhdanov, V. M. (1986). Dissociation of the haemagglutination inhibition and the infectivity neutralization in the reactions of influenza A/USSR/90/77 (H1N1) virus variants with monoclonal antibodies. Journal of General Virology 67, 2247-2251.[Abstract]
Ha, Y., Stevens, D. J., Skehel, J. J. & Wiley, D. C. (2001). X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proceedings of the National Academy of Sciences, USA 98, 11181-11186.
Horimoto, T., Rivera, E., Pearson, J., Senne, D., Krauss, S., Kawaoka, Y. & Webster, R. G. (1995). Origin and molecular changes associated with emergence of a highly pathogenic H5N2 influenza virus in Mexico. Virology 213, 223-230.[Medline]
Laemmly, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Murphy, B. R. & Webster, R. G. (1990). Orthomyxoviruses. In Fields Virology , pp. 1091-1152. Edited by B. N. Fields & D. M. Knipe. New York:Raven Press.
Nobusawa, E., Aoyama, T., Kato, H., Suzuki, Y., Tateno, Y. & Nakajima, K. (1991). Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. Virology 182, 475-485.[Medline]
Palmer, D. F., Dowdle, W. R., Coleman, M. T. & Schild, G. C. (1975). Advanced Laboratory Techniques for Influenza Diagnosis. US Department of Health, Education and Welfare Immunology Series No. 6. Atlanta, GA: Center for Disease Control.
Philpott, M., Easterday, B. C. & Hinshaw, V. S. (1989). Neutralizing epitopes of the H5 hemagglutinin from a virulent avian influenza virus and their relationship to pathogenicity. Journal of Virology 63, 3453-3458.[Medline]
Philpott, M., Hioe, C., Sheerar, M. & Hinshaw, V. S. (1990). Hemagglutinin mutations related to attenuation and altered cell tropism of a virulent avian influenza A virus. Journal of Virology 64, 2941-2947.[Medline]
Reading, P. C., Morey, L. S., Crouch, I. C. & Anders, M. E. (1997). Collectin-mediated antiviral host defence of the lung: evidence from influenza virus infection of mice. Journal of Virology 71, 8204-8212.[Abstract]
Reed, L. J. & Muench, H. (1932). A simple method for estimating 50% endpoints. American Journal of Hygiene 27, 493-497.
Smirnov, Y. A., Lipatov, A. S., van Beek, R., Gitelman, A. K., Osterhaus, A. D. M. E. & Claas, E. C. J. (2000). Characterization of adaptation of an avian influenza A (H5N2) virus to a mammalian host. Acta Virologica 44, 1-8.[Medline]
Smirnov, Y. A., Kaverin, N. V., Govorkova, E. A., Lipatov, A. S., Claas, E. C. J., Makarova, N. V., Gitelman, A. K., Webster, R. G. & Lvov, D. K. (2001). Cross-protection studies with H5 influenza viruses. In Options for the Control of Influenza IV, International Congress Series 1219 , pp. 767-773. Edited by A. D. M. E. Osterhaus, N. Cox & A. W. Hampson. Amsterdam:Excerpta Medica.
Suarez, D. L., Perdue, M. L., Cox, N., Rowe, T., Bender, C., Huang, J. & Swayne, D. E. (1998). Comparisons of highly virulent H5N1 influenza viruses isolated from humans and chickens from Hong Kong. Journal of Virology 72, 6678-6688.
Swayne, D. E. (1997). Pathobiology of H5N2 Mexican avian influenza viruses for chickens. Veterinary Pathology 34, 557-567.[Abstract]
Swayne, D. E., Perdue, M. L., Garcia, M., Rivera-Cruz, E. & Brugh, M. (1997). Pathogenicity and diagnosis of H5N2 Mexican avian influenza viruses in chickens. Avian Diseases 41, 335-346.[Medline]
Tsuchiya, E., Sugawara, K., Hongo, S., Matsuzaki, Y., Muraki, Y., Li, Z.-N. & Nakamura, K. (2001). Antigenic structure of the haemagglutinin of human influenza A/H2N2 virus. Journal of General Virology 82, 2475-2484.
Webster, R. G. & Laver, W. G. (1980). Determination of the number of nonoverlapping antigenic areas on Hong Kong (H3N2) influenza virus hemagglutinin with monoclonal antibodies and the selection of variants with potential epidemiological significance. Virology 104, 139-148.[Medline]
Webster, R. G., Bean, W. J., Gorman, O. T., Chambers, T. M. & Kawaoka, Y. (1992). Evolution and ecology of influenza A viruses. Microbiology and Molecular Biology Reviews 56, 152-179.[Abstract]
Webster, R. G., Guan, Y., Peiris, M., Walker, D., Krauss, S., Zhou, N. N., Govorkova, E. A., Ellis, T. M., Dyrting, K. C., Sit, T., Perez, D. R. & Shortridge, K. F. (2002). Characterization of H5N1 influenza viruses that continue to circulate in geese in southeastern China. Journal of Virology 76, 118-126.
Wiley, D. C., Wilson, I. A. & Skehel, J. J. (1981). Structural identification of the antibody-binding sites of Hong Kong influenza hemagglutinin and their involvement in antigenic variation. Nature 289, 373-378.[Medline]
Wilson, I. A., Skehel, J. J. & Wiley, D. C. (1981). Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 resolution. Nature 289, 366-373.[Medline]
Received 4 March 2002;
accepted 13 May 2002.