Escape mutants of pandemic influenza A/H1N1 2009 virus: Variations in antigenic specificity and receptor affinity of the hemagglutinin

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Abstract

A panel of 6 neutralizing monoclonal antibodies (MAbs) raised against A/Moscow/IIV01/2009 (H1N1) virus isolated during the 2009 pandemic was used for the selection of 26 escape mutants. The mutants were characterized in immune cross-reactions with the panel of MAbs. The sequencing of the mutant HA genes revealed 5 amino acid positions recognized by monoclonal antibodies: 129, 156, 158, 159, and 190 (H3 numbering). The amino acid positions were distributed in two epitopes belonging to antigenic sites Sa and Sb. The mutant HAs exhibited variations in the affinity to synthetic high molecular mass sialic acid-containing receptor analogues. Results are discussed in connection with the antigenic drift potential of the “swine-like” pandemic 2009 influenza virus.

Highlights

► Escape mutants of influenza pandemic A/Moscow/IIV01/2009 (H1N1) strain were selected. ► The mutants were characterized in immune cross-reactions with monoclonal antibodies (MAbs). ► The sequencing of the mutant HA genes revealed 5 amino acid positions recognized by MAbs. ► The amino acid positions were distributed in two epitopes belonging to antigenic sites Sa and Sb. ► The mutant HAs exhibited variations in the affinity to synthetic sialic acid-containing receptors.

Introduction

The targets of anti-virus immunity in influenza are mostly the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The leading role in this respect belongs to the HA. For this reason it is important to reveal the antigenically relevant parts of the HA molecule, where the amino acid substitutions lead to the acquisition of resistance to the neutralizing antibodies. Few of influenza A virus 16 HA subtypes have been characterized with respect to the location and structure of their antigenic sites on the three-dimensional (3D) model of the HA molecule. Three decades ago the H3 HA was characterized by sequencing the HA molecules of antigenic drift variants and escape mutants, and the antigenic epitopes were mapped (Wiley et al., 1981) in the 3D structure of the molecule (Wilson et al., 1981). For many years the 3D structure of HA was available only for the H3 subtype. The H3 structure was used for the antigenic mapping of the H1 (Caton et al., 1982) and H2 (Tsuchiya et al., 2001) HA molecules, and to perform initial studies on the mapping of the H5 HA molecule (Philpott et al., 1990). After the X-ray crystallographic structures of H5 and H9 HA were reported (Ha et al., 2001, Ha et al., 2002), we performed the analysis of the antigenic sites of the HA molecule of H5 and H9 HA subtypes (Kaverin et al., 2002, Kaverin et al., 2004, Kaverin et al., 2007, Rudneva et al., 2010). Besides the mapping of the antigenic sites on 3D structure, we revealed a pleiotropic character of mutations conferring resistance to monoclonal antibodies (MAbs). In several cases, the amino acid substitution in the HA of an escape mutant resulted not only in the escape from the neutralizing effect of the MAb, but also in a change in virus virulence, and/or a change in the affinity to sialic acid-containing polymers mimicking the cellular receptors used by the influenza viruses for the attachment to cells (Kaverin et al., 2004, Rudneva et al., 2005).

The 2009 influenza pandemic was caused by a novel “swine-like” H1N1 influenza A virus (Neumann et al., 2009, Smith et al., 2009) that resulted from a reassortment of two previously circulating swine viruses: an American “triple reassortant” strain and a Euroasiatic swine virus (Smith et al., 2009, Garten et al., 2009). The 2009 pandemic raised a concern about the future appearance of drift variants and the new outbreaks caused by the influenza virus of H1N1 subtype. The first publication on the antigenic mapping of the HA molecule of H1 subtype appeared as early as 1982 (Caton et al., 1982). However, since there exists a broad variability within the H1 subtype, the peculiarities of the antigenic structure of the HA of the new pandemic 2009 strain are of interest.

In the present studies we performed selection of escape mutants using influenza A/Moscow/IIV01/2009 (H1N1) strain isolated during the 2009 pandemic. The strain was isolated in parallel in Madin–Darby canine kidney (MDCK) cell culture and in embryonated chicken eggs (Lvov et al., 2009). The variant isolated in eggs was used in our studies. We used a reassortant having HA and NA genes of A/Moscow/IIV01/2009 (H1N1) virus and the other genes of A/Puerto Rico/8/34 (H1N1) virus (Ignatieva et al., 2011) as the wild-type virus for the selection of escape mutants. The escape mutants were characterized by their cross-reactions with MAbs, by the amino acid changes in their HA molecules, and by their affinity to high molecular mass synthetic sialosides.

Section snippets

Viruses

The variant of the virus strain A/Moscow/IIV01/2009 (H1N1) isolated in the embryonated chicken eggs (designated as A/Moscow/IIV01/2009-E), as well as strain X-31 (H3N2), were obtained from the Virus Collection of D. I. Ivanovsky Institute of Virology, Moscow. The reassortant ReM8 containing HA and NA genes of the A/Moscow/IIV01/2009-E strain and the other 6 genes of A/Puerto Rico/8/34 (H1N1) virus was obtained by crossing of A/Moscow/IIV01/2009-E and X-31 (H3N2) viruses and characterized in our

Sequential selection and sequence analysis of escape mutants

The reassortant strain used as the wild-type virus for the selection of escape mutants, ReM8, was obtained by crossing of A/Moscow/IIV01/2009 (H1N1)-E strain with reassortant X-31 (H3N2) virus containing HA and NA genes of A/Aichi/2/68 (H3N2) virus and the other genes of A/Puerto Rico/8/34 (H1N1) virus (Baez et al., 1980). The virus produced higher yields in the embryonated chicken eggs as compared to the parent A/Moscow/IIV01/2009 (H1N1)-E strain (Ignatieva et al., 2011), which allowed us to

Discussion

The studies on the antigenic epitopes recognized by MAbs in the HA of the H1N1 pandemic 2009 strain are scarce, and only 4 amino acids at positions, 125, 157, 158, and 166, have been revealed as antigenically relevant in the previously described antigenic sites (Manicassamy et al., 2010, Krause et al., 2010). Recently 3 amino acid substitutions in a novel low-variable site were identified (Krause et al., 2011). Overall, the number of amino acid changes in the HA of the escape mutants of the

Acknowledgement

The work was supported by grant 10-04-00023 of the Russian Foundation for Basic Research.

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