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Journal of Clinical Microbiology, April 2006, p. 1564-1566, Vol. 44, No. 4
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.4.1564-1566.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Discovery of the Neutralizing Epitope Common to Influenza B Virus Victoria Group Isolates in Japan

Naoko Nakagawa,1* Jun Suzuoki,2 Ritsuko Kubota,3 Shinzo Kobatake,2 and Yoshinobu Okuno3

Department of Microbiology, Kobe Institute of Health, 4-6, Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan,1 Diagnostics Research Laboratories, Wako Pure Chemical Industries, Ltd., 6-1, Takada-cho, Amagasaki 661-0963, Japan,2 Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, 3-69, 1-Chome, Nakamichi, Higashinari-ku, Osaka 537-0025, Japan3

Received 2 December 2005/ Returned for modification 24 January 2006/ Accepted 13 February 2006


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ABSTRACT
 
Monoclonal antibody 9B2 possesses hemagglutination inhibition activity against all the 2002/2003 influenza B virus Victoria group isolates in Kobe, Japan, as well as representative strains isolated between 1987 and 1997. The 9B2 epitope localizes three-dimensionally in the vicinity of antigenic site A of the hemagglutinin molecule, and amino acid substitutions in this region affected the binding of 9B2.


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TEXT
 
Influenza B virus, along with influenza A virus H1 and H3 subtypes, is a causative agent of influenza epidemics in humans. Influenza B virus strains isolated since the mid-1980s are divided into two large lineages in a phylogenic tree: one group is represented by B/Victoria/2/87, and the other is represented by B/Yamagata/16/88 (2). The B/Victoria group was predominant in the 1980s, while B/Yamagata became predominant in the early 1990s (2, 5, 6, 13, 15, 19). In the 1996/1997 season, there was a B/Victoria epidemic in Japan after almost 10 years' absence of the strain. Then, the next one was in the 2002/2003 season (8, 11, 12). The antigenicities of B/Victoria isolates in these seasons were distinct from those of the isolates in the mid-1980s. The first variants appeared in the 1996/1997 season with an extra oligosaccharide chain near the receptor binding region, and they became predominant in the 2002/2003 season (11). Then, the second variants appeared with variation in the conserved epitope of B/Victoria isolates, at the "tip" of the hemagglutinin (HA) molecule (12). The "tip" consists of the antigenic site of influenza AH3 virus HA, previously defined as site B. Site B and site A (a protruding loop region) of the HA molecule have been reported to be the highly immunodominant regions of influenza A and B viruses (1, 17). We developed monoclonal antibodies (MAbs) 10B8 and 10D7, whose epitopes had been conserved at site B of B/Victoria isolates since the mid-1980s until the 1996/1997 season (7, 9). However, the 2002/2003 isolates in Kobe, Japan, were divided into three groups according to their reactivities to 10B8 and 10D7. The first group, represented by B/Kobe/1/2003, was of the classical variety, which reacted to 10B8 and 10D7. The second group, represented by B/Kobe/2/2003, reacted to 10D7 but not to 10B8 and showed an amino acid substitution at site B (D164E). There was one isolate, B/Kobe/28/2003, which did not react either to 10B8 or to 10D7. It showed another amino acid substitution at site B (N165K) (12) (Fig. 1).


Figure 1
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  		FIG. 1. Comparison of deduced amino acid sequences of the HA1 region and HI titers of the clinical isolates. Sequences of amino acid residues of the 9B2 epitope and the "tip" are shown. These sequences are compared with those of B/Kobe/1/2003. The results of HI tests are expressed as the reciprocal of antibody dilution (14). "del" means an amino acid deletion.

We obtained MAb 9B2, which has hemagglutination inhibition (HI) activity against all the 2002/2003 B/Victoria isolates, and investigated the common neutralizing epitopes among them.

Virus strains B/Kobe/1/2003, B/Kobe/2/2003, and B/Kobe/28/2003 were isolated from clinical specimens. Nucleotide sequences for the HA gene were previously reported (DDBJ accession no. AB081570, AB126836, and AB126841, respectively) (12). B/Victoria/2/87, B/Nagasaki/1/87, B/Aichi/5/88, B/Guandong/5/94, and B/Shangdong/7/97 were utilized as representative B/Victoria strains, while B/HongKong/22/89, B/Mie/1/93, and B/Yamanashi/166/98 were utilized as representative B/Yamagata strains. MAb 9B2 was obtained by immunizing mice with B/Kobe/28/2003. Ascitic fluids of mice injected with hybridoma cells were used as sources of MAbs. Human sera, collected from three individual adults after the 2002/2003 season, were utilized. The results of HI tests are expressed as the reciprocal of antibody dilution (14). Escape mutants were induced by incubating virus with 9B2, by a modification of the method previously described (1, 4, 10). The nucleotide sequences of the escape mutants were analyzed as described previously (8-12).

MAb 9B2 has HI activity against all the 2002/2003 isolates, as well as the representative B/Victoria isolates (Fig. 1). In order to examine the 9B2 epitope, escape mutants were induced. Two mutants (B/Kobe/1/2003-V51 and -V52) (DDBJ accession no. AB232144 and AB232145, respectively) were induced by incubating a classical strain, B/Kobe/1/2003, with 9B2. In HI tests, they reacted to 10B8 and 10D7 as a parental strain but did not react to 9B2. In addition, four mutants (B/Kobe/28/2003-V1 through -V4) (DDBJ accession no. AB232146 through AB232149, respectively) were induced by incubating an N165K variant, B/Kobe/28/2003, with 9B2. They did not react to any of the three MAbs in the HI tests. Between two parental strains, B/Kobe/1/2003 and B/Kobe/28/2003, there was only one amino acid difference (N165K) in the HA1 region (12). B/Kobe/1/2003-V51 and B/Kobe/28/2003-V1 showed the same single amino acid substitution (N75D), while B/Kobe/1/2003-V52 showed another single amino acid substitution (P77T). The others had double amino acid substitutions: B/Kobe/28/2003-V2 had R118G and H122R, V3 had N75D and R118G, and V4 had N75D and H116R. The HI titers of the variants against human sera were four to eight times lower than those of their parental strains. The amino acid substitutions at the 9B2 epitope modulated the viral antigenicities.

The amino acid sequences of the HA1 molecule of the influenza B virus were compared to those of A/Aichi/2/68 and numbered according to the A/Aichi/2/68 sequence for ease of reference to the structure of the AH3 HA molecule (1, 3). Thus, amino acid 75 is referred to as 78, 77 as 80, 116 as 121, 118 as 123, and 122 as 125. Figure 2 shows the three-dimensional structure of the influenza A virus H3 HA molecule, as determined by Wilson et al. (18). Recently, Tung et al. (16) reported with their energy-minimized predicted structure that the overall shape of the influenza A virus HA molecule is preserved in that of influenza B virus. Thus, though the identified amino acid substitutions of influenza B virus are predicted based only on the sequence alignments, it is speculated that all the above amino acid residues are situated three-dimensionally close to site A, though they do not belong to site A. With influenza AH3 virus HA, four antigenic sites (A to D) were reported in the HA1 molecule. In addition, the substitutions of other amino acid residues with naturally occurring variants were reported at the same time. They include the residues 78, 83, 122, and 126. These residues were suggested to form the additional antigenic sites such as site E and affect site A (17). In contrast, only two immunodominant antigenic sites, site A and site B, have been discussed with regard to influenza B virus (1, 9, 10). It is noteworthy that the 9B2 epitope did not belong to either of the above antigenic sites.


Figure 2
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  		FIG. 2. Three-dimensional model of the influenza A virus HA molecule. The data for the secondary structure of the HA molecule of A/Aichi/2/68 (code 2VIU) were obtained through the Internet with Rasmol software. A view of the HA molecule from the front is shown. Residues 78, 80, 121, 123, and 125 are marked. Numbers in parentheses show the residues of influenza B virus which correspond to the above residues of influenza A virus.

Nucleotide sequences of representative strains were obtained from the DDBJ database, and amino acid residues at the 9B2 epitope (75 to 77 and 116 to 122) and the "tip" (163 to 170) are shown in Fig. 1. Though some B/Victoria isolates show one or two amino acid differences at the 9B2 epitope, all reacted to 9B2 to the same degree, as well as to 10B8 and 10D7, while B/Yamagata isolates did not react to any of the antibodies. Thus, the epitopes have been conserved in B/Victoria since the mid-1980s but not in B/Yamagata. Now, it should be noted that D164E variants represented by B/Kobe/2/2003 reacted to 9B2 with four-times-lower HI titers. They showed two amino acid differences (H116R and T121N), and it is suggested that the established human immunity began to induce naturally occurring escape mutants. With another amino acid substitution, viral antigenicities will be further affected. The mutants may appear as new variants in future epidemics. Then, the scale of the epidemics will be positively modulated. This information will be of benefit to the management of public health, especially from the point of view of selecting suitable strains for vaccines.


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ACKNOWLEDGMENTS
 
We thank T. Iwamoto (Kobe Institute of Health) for valuable advice in sequencing.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Kobe Institute of Health, 4-6, Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan. Phone: 81-78-302-6260. Fax: 81-78-302-0894. E-mail: nknkgw{at}h2.dion.ne.jp. Back


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Journal of Clinical Microbiology, April 2006, p. 1564-1566, Vol. 44, No. 4
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.4.1564-1566.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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  • Wang, Q., Cheng, F., Lu, M., Tian, X., Ma, J. (2008). Crystal Structure of Unliganded Influenza B Virus Hemagglutinin. J. Virol. 82: 3011-3020 [Abstract] [Full Text]  

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