Proc. Natl. Acad. Sci. USA Vol. 87, pp. 786-790, January 1990 Evolution

Independent and disparate evolution in nature of influenza A virus hemagglutinin and neuraminidase glycoproteins (mutation/antigenic drift/molecular epidemiology/antigenic analysis)

EDWIN D. KILBOURNE*, BERT E. JOHANSSON,

AND BERNARDO GRAJOWER

Department of Microbiology, Mount Sinai School of Medicine of the City University of New York, New York, NY 10029

Contributed by Edwin D. Kilbourne, October 6, 1989 Table 1. High-yield and antigenically hybrid reassortant viruses used in production of antisera and in antigenic analyses Reassortant viruses* Field strain H1N1 HiN1, H1N7, H6N1 A/USSR/90/77 (HlNl) H1N1, H1N2, H6N1 A/India/6263/80 (HlNl) H1N1, H1N7, H6N1 A/Chile/1/83 (HlNi) t H1N2, H6N1 A/Taiwan/1/86 (HlNl) H3N2 H3N2, H3N1, H7N2 A/Aichi/2/68 (H3N2) H3N2, H3N7, H7N2 A/Victoria/3/75 (H3N2) H3N2, H3N7, H6N2 A/Philippines/2/82 (H3N2) H3N2, H3N1, H6N2t A/Shanghai/11/87 (H3N2) *High-yield reassortants containing non-envelope protein genes from A/PR/8/34. tNo high-yield reassortant available; field strain used. tN2 is from A/Sichuan/2/87 (H3N2); antigenically indistinguishable from the N2 of A/Shanghai/11/87.

The hemagglutinin (HA) and neuraminidase ABSTRACT (NA) external glycoprotein antigens ofHlNl and H3N2 subtypes of epidemiologically important influenza A viruses prevalent during recent decades were subjected to intensive antigenic analysis by four different methods. Prior to serological analysis with polyclonal rabbit antisera, HA and NA antigens of four viruses of each subtype were segregated by genetic reassortment to forestall nonspecific steric hindrance during antigen-antibody combination. This analysis has demonstrated that with respect to antigenic phenotype, HA and NA proteins have evolved at different rates. With HlNl viruses, an arrest of signfcant evolution of the NA discordant with the continuing antigenic drift of HA was found in the 1980-1983 period. It is probable that the different and independent rates of evolution of HA and NA reflect the greater selective pressure of HA antibodies, which forces the more rapid emergence of HA escape mutants. The slower antigenic change found for NA further supports the potential for NA-specific infection-permissive immunization as a useful stratagem against influenza.

subtype viruses and provides strong evidence for the slower antigenic evolution of the NA protein.

The hemagglutinin (HA) and neuraminidase (NA) glycoproteins of influenza A viruses comprise the major surface proteins and the principal immunizing antigens of the virus. These proteins are inserted into the viral envelope as spikelike projections (1) in a ratio (HA:NA) of about 4:1 (2) but varying in proportion in some mutant or reassortant viruses (2, 3). Neutralization of the virus is mediated through the HA, which is thus subject to major selective pressure by human antibody as mutant viruses emerge to produce new epidemics. Like the genes of other RNA viruses, the genes of influenza virus mutate with high frequency (4, 5). However, in contrast to other viruses, the survival of influenza A viruses in nature seems to depend on the continuing evolution of new antigenic phenotypes. Although the major changes in HA phenotype (antigenic shift) associated with pandemics probably reflect the acquisition of new HA genes from animal influenza viruses (6, 7), epidemiologically significant interpandemic antigenic drift reflects sequential point mutations-principally in the HA gene (8). Not surprisingly, the frequency of amino acid changes in the HA exceeds that of genes coding for internal proteins less subject to immunoselection, although with respect to noncoding silent mutations all influenza virus genes appear to be governed by the same molecular clock (9). Although published evidence suggests a similar mutation rate for HA and NA proteins (9, 10), the lesser number of influenza virus NA subtypes and the lesser immunoselective pressure of NA antibody on virus replication (11-13) suggested to us that the NA protein may evolve at a slower rate in nature. The present paper addresses the comparative rates of change of antigenic phenotype of H3N2 and HlN1 human

Viruses. The viruses used in test assays and for the production of antisera are identified in Table 1. It was necessary to produce antigenically hybrid reassortant viruses for antigenic analyses to avoid reciprocal nonspecific steric hindrance by antibody of these two glycoproteins adjacent on the virus particle (12). All viruses selected for analysis were epidemiologically significant variants that had sufficient public health importance to require the fabrication of high-yield vaccine strains (14, 15) containing their antigens. Antisera. Antisera were produced by intravenous (i.v.) inoculation of New Zealand White rabbits with 3000 HA units of purified virus. Rabbits were bled for antisera 7 days after a second injection of virus at 40 days. For hemagglutination inhibition (HI) tests, sera were treated with Vibrio cholerae receptor-destroying enzyme to destroy nonspecific inhibitor. Serologic Testing. HI tests were performed in test tubes at a final concentration of 0.8 ml (16). Tests for NA inhibition (NI) were performed as described (17). Enzyme-linked immunoabsorbent assays (ELISA) were performed as described (18) using the appropriate whole virus diluted in 0.015 M Na2CO3/0.035 M NaHCO3 (pH 9.6) carbonate buffer. Optimal concentration of antigen was determined by checkerboard titration of the antigen and a known positive (homologous) control serum. Plaque reduction tests were carried out as described (19) using Madin-Darby canine kidney cells with incorporation of antiserum dilutions in agar overlays. In the case of titration of

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Abbreviations: HA, hemagglutinin; NA, neuraminidase; HI, HA inhibition; NI, NA inhibition. *To whom reprint requests should be addressed.

MATERIALS AND METHODS

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Proc. Natl. Acad. Sci. USA 87 (1990)

Table 2. Antigenic analysis by HI Virus

H1USSRN2

Antiserum* Phase 1 H1USSRN1USSR HiTaiwanNiTaiwan 256* 16

HlTaiwanN2

8

% antigen related

128

6

Phase 2

HlUSSRN2 H1ussRN1ussR HlTaiwanNlTaiwan

*AIl

HlTaiwanN2

256

8

16

2048

2 Mean 4 values are the reciprocal of the serum dilution at the end point.

antisera reactive with NA, the number and the size of all individual plaques were scored for all serum dilutions. Separate end points were then calculated on the basis of 50% inhibition of plaque number and reduction of mean plaque size. For definitive analysis the two end points were averaged. Calculation of Antigenic Relatedness. Antibody titer ratios were calculated by the method of Archetti and Horsfall (20). Homologous and heterologous titer ratios were determined for each virus. The following formula was used to calculate R (the coefficient of antigenic relatedness), VR1 x R2, where R, = homologous titer V1/heterologous titer V2; R2 = homologous V2/heterologous V1. All mathematical calculations were performed on an IBM-XT using a BASIC program written for these calculations. As shown in Table 2, homotypic and heterotypic antibody titers were measured in two ways. In phase 1, antisera to viruses of field strain antigenic phenotype were titrated against antigenically hybrid viruses bearing NAs of different-i.e., non-cross-reactive-phenotypes to preclude steric hindrance of HI by NA antibody (21). Phase 2 titrations were done in converse fashion employing antisera to antigenic hybrids against viruses of wild-type phenotype. After determination of the percentage of antigenic relationship by each method the results were averaged as shown. These phase 1 and phase 2 analyses were used in plaque reduction as well as HI and NI tests. Table 3. Summary of end point titers Virus

Aichi Philippines Victoria Shanghai

Aichi 640 48 8 24

RESULTS A summary of titration end points for antigenic analyses by HI, NI, and plaque reduction tests is compiled in Table 3. Each titer shown represents the average of phase 1 and phase 2 analyses of each virus-antiserum pair (Table 2). For clarity of presentation, data have been normalized to percentage expressions of antigenic relatedness in Fig. 1. The primary data are in Table 3. The decreasing antigenic similarity with time of HiN1 and H3N2 influenza A viruses is shown in Fig. 1. Fig. 1 A and C demonstrate the marked drift of H1 and H3 antigens during one and two decades, respectively. A lesser antigenic change in N1 and N2 NA antigens is shown in Fig. 1 B and D. The relationships plotted here used the earliest virus, A/USSR/ 77 (HlNi) or A/Aichi/68 (H3N2), as a base. Antigenic Analysis in a Common (Plaque Reduction) System. Although HI and NI tests are based on inhibition of biologically functional proteins of the virus and therefore have relevance to infection, it was essential to determine whether the different rates of antigenic drift found for the HA and NA reflected only differences in the test systems employed in their analysis. Therefore, we have measured antigenic relationships also in an infection-dependent plaque assay system (see Materials and Methods) in which antigenic analysis is dependent upon limitation of infection by viral-antibody interaction in a common system. Results of the analysis for the HiN1 viruses are shown in Table 3. In this system also, greater antigenic drift is seen for the HA antigens. A phase 1 analysis of the H3N2 viruses in this system yielded similar results (data not shown). Antigenic Analysis by ELISA. Detection of antibody in ELISA is not dependent on the inhibition of biological function, thereby enabling an examination of antigenic relatedness in a system common to both surface glycoproteins. Although ELISA was unable to detect minor antigenic differences among heterovariant viruses, this system was able to demonstrate that there was a significantly greater degree of antigenic similarity between viral NAs separated by the maximum interval (i.e., 1977-1986 for H1; 1968-1987 for H3) than between the HAs of these strains. Specifically, data from ELISA indicate a 39% antigenic relatedness between Antiserum H3N2

Anti-H3 in HI test Philippines Victoria 34 384 18 10

787

36 127

384 80

Shanghai 132 48 40 640

Aichi 1127 131 117 94

Anti-N2 in NI test Philippines Victoria 868 365 200 270

450 400 1345 235

Shanghai 151 190 546 384

HiN1 USSR 256

Anti-H1 in HI test India Chile

Anti-N1 in NI test India Chile

Taiwan USSR Taiwan 32 12 227 114 47 36 144 576 40 74 338 130 210 48 12 40 300 133 371 492 12 72 1088 31 143 124 741 Anti-Hi in plaque inhibition test* Anti-N1 in plaque size reduction test* USSR India Chile Taiwan USSR India Chile Taiwan USSR 58 50 10 10 20 9 11 4 India 24 158 36 1 13 18 8 20 Chile 42 416 38 23 15 18 34 22 Taiwan 2 3 3 36 10 17 22 69 All titers are the means of phase 1 and phase 2 analyses (see Table 2). Homologous titers are underlined. *Reciprocal end point dilution x 10-3. USSR India Chile Taiwan

68 260 640 16

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Proc. Natl. Acad. Sci. USA 87 (1990)

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FIG. 1. Progressive antigenic dissimilarity with time of the HA antigens (A and C) and NA antigens (B and D) of HiNi and H3N2 influenza A viruses prevalent in the years indicated. Antigenic analysis was done by HI and NI tests.

the NAs of A/Hong Kong/68 and A/Philippines/82 and 9% relatedness for their HAs. Similarly, the NAs from the HiNi heterovariants, A/USSR/77 and A/Taiwan/86, were found by ELISA to be 24% antigenically related and their HAs 6.8% related (Table 4). Comparative Evolutionary Rates of HA and NA Antigenic Change. The percent antigenic change for the different intervals bracketed by the viruses studied was calculated, and from these data an annual rate of change was determined for each antigen (Table 5). Significantly different rates of change for HA and NA antigens of HINM and H3N2 subtype viruses were found. In the case of the USSR/77-India/80 and Aichi/ 68-Victoria/75 comparisons, percentage changes of amino acid composition based on comparative cDNA sequences of

the HAs are available (22, 23) (see Table 5). Perhaps not unexpectedly, the number of amino acid changes is not concordant with differences noted by the antigenic analyses shown here. Evolutionary Stasis: A Period of Arrested Drift of the Ni Antigen. The foregoing analysis of the degree of relatedness of sequential viral pairs has revealed a period of arrested drift of the NI antigen. Antigenic change between the NA antigens of A/India/80 and A/Chile/83 (HiNl) viruses was insignificant (8%), but a striking (93%) change in their HA antigens occurred during the 1980-1983 period (Table 5, line 2). Similar NA/HA discordance was demonstrated also in the plaque reduction system (NA 10%, HA 93% difference).

Table 4. Summary of end point titers (ELISA) Virus*

Antiserumt HiNi

Anti-Hi HI Taiwan HI USSR NI Taiwan Ni USSR

Anti-N I

Hi Taiwan

Hi USSR

1407 45

141 948

N1 Taiwan

Ni USSR

3036 1728

4ii

143

H3N2 H3 Aichi H3 H3 N2 N2

Aichi

Philippines

Anti-H3 H3 Philippines

N2 Aichi

Anti-N2 N2 Philippines

105 40

128 310 41

Aichi

Philippines Homologous titers are underlined. *Reactive component. The reassortant viruses used tReciprocal end point dilution x i0-3.

are

identified in Table 1.

23

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Table 5. Evolution rates of HA and NA antigenic change

% antigenic change HA (HI test)

NA (NI test) % change per

% change per

Interval,

Viruses

years

% change

USSR/77-India/80 India/80-Chile/83 Chile/83-Taiwan/86

3 3 3

88 (4)* 93 91

annum

% change

annum

43 8 63

14 3 21

H1N1

29 31 Mean

30 30

13

H3N2

13 68 13 52 60 18 Mean 15 A Student t test indicated significant differences between groups (a = 0.05). *% amino acid change in interval based on cDNA sequencing (22, 23).

Aichi/68-Victoria/75 Victoria/75-Phil./82 Phil./82-Shanghai/87

7 7 5

93 (7)* 89 89

DISCUSSION Critical to our thesis that the HA and NA glycoproteins of influenza A virus evolve at different rates is the precise definition of antigenic phenotype. For this reason we have designed and employed elaborate methods for characterization of these viral antigens that involve three different measures of antibody-epitope binding: (0) in vitro inhibition of function of these two biologically reactive proteins in HI and NI tests; (ii) in vivo inhibition of viral replication in a common plaque reduction system; and (iii) ELISA, an in vitro system not dependent upon functional activity of the tested antigens. In all three test systems we have avoided nonspecific sterically mediated blocking by antibody of relevant antigens by segregating HA and NA into antigenically hybrid (24) reassortant viruses. Furthermore, the probability that antigenic analysis might be influenced by differences in antigenantibody affinity has been reduced by a combined two-phase analysis involving reciprocal antigen-antibody pairing and testing with two different intravirionic representations of each antigen and antibodies to two different reassortant viruses containing the same antigen. Using these methods, we have demonstrated in epidemiologically significant HiN1 and H3N2 influenza A viruses an increasing antigenic dissimilarity with time of HA and NA proteins compared with those in the earliest virus studied. Furthermore, we have shown by all three methods of antigenic analysis the slower evolution of the NA phenotype. Further evidence of the discordant evolution of HA and NA is the stasis of the N1 antigenic phenotype from 1980 to 1983 coincident with a marked drift of the H1 HA in the same time period. A similar retention of N2 antigenic similarity can be inferred for the HA divergent H3N2 strains A/Port Chalmers/1/73 and A/Victoria/i/75 analyzed with a panel of monoclonal antibodies to N2 (25). Analysis of the rate of non-amino acid-changing (silent) nucleotide substitutions suggests that all genes of influenza A virus are governed by the same molecular clock (9). However, the rate of amino acid-changing substitutions varies greatly from gene to gene. Thus, by this measure the external HA and NA proteins are evolving at a greater rate than other proteins of the virus, although in a comparison of a limited number of H2N2 viruses, the rates of HA and NA evolution were found to be comparable (2.94-2.80 x 103 amino acidchanging substitutions per site per year) (9). Selection of antigenic variants with monoclonal antibodies demonstrated that the frequencies of variation in HA and NA were comparable (26). Although we do not presume to calculate definitive per annum evolutionary rates for HA and NA proteins from our analysis of antigenic phenotypes, we have shown that these proteins differ significantly in their

10 7 12 10

rates of change. Further, we would emphasize that changes in phenotype (the traditional measure of evolution) (27) rather than nucleotide or mean rates of amino acid change, per se, are the relevant indicator of viral adaptation and reflect the real differences in the selective constraints imposed by preexisting host antibody. In this regard, the differing effects of HA and NA antibody on viral replication are well documented. Although viral neutralization and prevention of infection are mediated by antibody reactive with the HA, NA antibody does not prevent infection but rather partially suppresses replication of virus under conditions of multicycle infection (28). Therefore, it is reasonable to assume that within virions infecting partially immune hosts selection pressure is substantially less on the NA than the HA protein. Such pressure may be further reduced by antigenic competition between HA and NA that is suppressive to NA antibody synthesis (29). There is, of course, abundant precedent for discordant evolution affecting not only different proteins of an organism but also different regions of the same molecule (30, 31). The functional constraints against evolution of the active site of an enzyme (30) are unlikely to be relevant to the greater preservation of the antigenicity of the NA enzyme because its antigenic and enzymatically active sites, although adjacent, are dichotomous (32, 33). The preservation of NA structure is not restricted to the active site. It is notable that during a 22-year period of N2 drift, 90% of the nucleotide positions and 87% of the amino acid positions remained invariant (34). Dichotomous antigenic drift of HA and NA antigens may not be restricted to influenza A viruses. Curry et al. (35) found cross-reactivity among the NA but not among the HA antigens of influenza B viruses isolated during a 25-year period. The slower antigenic evolution of the NA has important implications for artificial immunization against influenza. Present influenza vaccines must be revised annually or biannually because of the rapid drift of the HA antigens in field strains of virus. However, the recently proposed approach of NA-specific, infection-permissive immunization with purified NA antigens (13, 36) is reinforced by the prospect of greater antigenic stability of the NA in nature. Furthermore, previous studies have shown that significant NA-mediated cross-protection can be expected even among heterovariant viruses (12, 37, 38). We are indebted to Ms. B. A. Pokorny for excellent technical assistance. This work was supported in part by Grant RO1 Al 109304 from the National Institute of Allergy and Infectious Diseases.

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2. Webster, R. G., Laver, W. G. & Kilbourne, E. D. (1968) J. Gen. Virol. 3, 315-326. 3. Mowshowitz, S. & Kilbourne, E. D. (1975) in Negative Strand Viruses, eds. Barry, R. D. & Mahy, B. W. J. (Academic, London), Vol. 2, pp. 127-168. 4. Holland, J., Spindler, K., Horodyski, F., Grabau, E., Nichol, S. & VandePol, S. (1982) Science 215, 1577-1585. 5. Buonagurio, D. A., Nakada, S., Parvin, J. D., Krystal, M., Palese, P. & Fitch, W. M. (1986) Science 232, 980-982. 6. Scholtissek, C., Rohde, W., von Hoyningen, V. & Rott, R. (1978) Virology 87, 13-20. 7. Fang, R., Min Jou, W., Huylebroeck, D., Devos, R. & Fiers, W. (1981) Cell 25, 315-323. 8. Webster, R. G., Laver, W. G. & Air, G. M. (1983) in Genetics of Influenza Viruses, eds. Palese, P. & Kingsbury, D. W. (Springer, New York), pp. 127-168. 9. Hayashida, H., Toh, H., Kikuno, R. & Miyata, T. (1985) Mol. Biol. Evol. 2, 289-303. 10. Huddleston, J. A. & Brownlee, G. G. (1982) Nucleic Acids Res. 10, 1029-1038. 11. Kilbourne, E. D., Laver, W. G., Schulman, J. L. & Webster, R. G. (1968) J. Virol. 2, 281-288. 12. Schulman, J. L., Khakpour, M. & Kilbourne, E. D. (1968) J. Virol. 2, 778-786. 13. Johansson, B. E., Bucher, D. J. & Kilbourne, E. D. (1989) J. Virol. 63, 1239-1246. 14. Kilbourne, E. D. (1969) Bull. World Health Organ. 41, 643645. 15. Johansson, B. E., Bucher, D. J., Pokorny, B. A., Mikhail, A. & Kilbourne, E. D. (1989) Virology 171, 634-636. 16. Salk, J. E. (1944) J. Immunol. 49, 87-98. 17. Kilbourne, E. D. (1976) J. Infect. Dis. 134, 384-394. 18. Khan, M. W., Gallagher, M., Bucher, D., Cerini, C. P. & Kilbourne, E. D. (1982) J. Clin. Microbiol. 16, 115-122. 19. Jahiel, R. I. & Kilbourne, E. D. (1966) J. Bacteriol. 92, 15211534. 20. Archetti, I. & Horsfall, F. L., Jr. (1950) J. Exp. Med. 92, 441-462.

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