Identification of amino acid substitutions supporting antigenic change of influenza A(H1N1)pdm09 viruses

Abstract

The majority of currently circulating influenza A(H1N1) viruses are antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as population immunity increases. Amino acid substitutions in the hemagglutinin protein can result in escape from neutralizing antibodies, affect viral fitness, and change receptor preference. In this study, we constructed mutants with substitutions in the hemagglutinin of A/Netherlands/602/09 in an attenuated backbone to explore amino acid changes that may contribute to emergence of antigenic variants in the human population. Our analysis revealed that single substitutions affecting the loop that consists of amino acid positions 151 to 159 located adjacent to the receptor binding site caused escape from ferret and human antibodies elicited after primary A(H1N1)pdm09 virus infection. The majority of these substitutions resulted in similar or increased replication efficiency in vitro compared to that of the virus carrying the wild-type hemagglutinin and did not result in a change of receptor preference. However, none of the substitutions was sufficient for escape from the antibodies in sera from individuals that experienced both seasonal and pandemic A(H1N1) virus infections. These results suggest that antibodies directed against epitopes on seasonal A(H1N1) viruses contribute to neutralization of A(H1N1)pdm09 antigenic variants, thereby limiting the number of possible substitutions that could lead to escape from population immunity.

Importance: Influenza A viruses can cause significant morbidity and mortality in humans. Amino acid substitutions in the hemagglutinin protein can result in escape from antibody-mediated neutralization. This allows the virus to reinfect individuals that have acquired immunity to previously circulating strains through infection or vaccination. To date, the vast majority of A(H1N1)pdm09 strains remain antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as a result of increasing population immunity. We show that single amino acid substitutions near the receptor binding site were sufficient to escape from antibodies specific for A(H1N1)pdm09 viruses but not from antibodies elicited in response to infections with seasonal A(H1N1) and A(H1N1)pdm09 viruses. This study identified substitutions in A(H1N1)pdm09 viruses that support escape from population immunity but also suggested that the number of potential escape variants is limited by previous exposure to seasonal A(H1N1) viruses.

FIG 1

Position of amino acid substitutions indicated on an A/California/04/09 HA crystal structure. (A) The three HA monomers are indicated in white, gray, and black; the RBS is in yellow. Amino acid positions that were mutated in this study are indicated in orange. (B) Zoom image of the globular head of HA. Amino acid substitutions in mutants that were substantially antigenically different from A/Netherlands/602/09 (escape mutants) are indicated in red; substitutions in mutants that were antigenically similar to A/Netherlands/602/09 and that were included in further antigenic analyses (nonescape mutants) are indicated in blue.

FIG 2

HI titer differences between viruses with wild-type or mutant HAs against an A/Netherlands/602/09 ferret antiserum. Viruses with either wild-type or mutant A/Netherlands/602/09 HAs were tested in HI assays with a ferret antiserum prepared against the A/Netherlands/602/09 wild-type virus. Each point represents the log₂ HI titer difference between a mutant and A/Netherlands/602/09. Mutants with HI titers at least 4-fold (2 log₂) lower than that of A/Netherlands/602/09 (dashed line) were considered substantially antigenically different. The viruses are ordered by the log₂ HI titer difference from A/Netherlands/602/09.

FIG 3

HI titer differences between viruses with wild-type or mutant HAs in ferret antisera. Each point in panel A represents the log₂ HI titer difference between a mutant and A/Netherlands/602/09 for an individual ferret antiserum. The viruses are ordered by the mean log₂ HI titer difference from A/Netherlands/602/09, which is indicated as red horizontal lines. Names of escape and nonescape mutants are shown in black and gray, respectively. Ferret antisera are indicated in the leftmost column of panel B and are ordered from top to bottom by a decreasing ability of the serum to inhibit the test viruses in the HI assay. Two antisera (labeled A and B) were prepared against each virus. HI titers are color-coded for the difference from A/Netherlands/602/09 (NL602): orange, equal to or higher than that of A/Netherlands/602/09; yellow, up to 2-fold lower; green, 2- to 4-fold lower; cyan, 4- to 8-fold lower; blue, 8- to 16-fold lower; purple, 16- to 32-fold lower; and magenta, at least 32-fold lower.

FIG 4

HI titer differences between viruses with wild-type or mutant HAs against human infant sera. (A) Symbols, order, and nomenclature are as in Fig. 3. (B) HI titers are color-coded for the difference from A/Netherlands/602/09: orange, equal to or higher than that of A/Netherlands/602/09; yellow, up to 2-fold lower; green, 2- to 4-fold lower; cyan, 4- to 8-fold lower; blue, 8- to 16-fold lower; purple, 16- to 32-fold lower; and magenta, at least 32-fold lower. NT, the virus-serum combination was not tested. (C) The analysis was repeated with inclusion of only the sera that differentiated between the mutants (sera 6, 7, and 16 were omitted). Nonescape mutants were not tested with serum 25 because of insufficient material available for this serum. Gray horizontal lines indicate the mean log₂ HI titer difference from A/Netherlands/602/09 when this serum was also omitted for the other mutants.

FIG 5

Effects of substitutions on virus replication. MDCK cells were inoculated with viruses containing wild-type or mutant A/Netherlands/602/09 HAs. After 36 h, the plaque sizes were determined as a measure of replication efficiency. Each point indicates the size of a single plaque. The mutants are ordered by increasing median plaque size, which is indicated by red horizontal lines. Escape mutants are in bold.

Fig 6

HI titer differences between viruses with wild-type or mutant HAs against human sera. (A) HI titers of human sera against antigenic variants of seasonal A(H1N1) viruses isolated between 1977 and 2009 (USSR77, A/USSR/90/77; NL78, A/Netherlands/3075/78; TA89, A/Taiwan/1/89; NL87, A/Netherlands/414/87; NC99, A/New Caledonia/20/99; NL99, A/Netherlands/271/99; NL03, A/Netherlands/02/03; NL06, A/Netherlands/364/06; SS06, A/Solomon Islands/03/06; and NL09, A/Netherlands/1005/09). (B) HI titer differences between viruses with wild-type or mutant NL602 HAs against human sera. Symbols, order, and nomenclature are as in Fig. 3. (C) HI titers are color-coded for the difference from A/Netherlands/602/09: orange, equal to or higher than A/Netherlands/602/09; yellow, up to 2-fold lower; green, 2- to 4-fold lower; cyan, 4- to 8-fold lower; and blue, 8- to 16-fold lower. The first two digits of the serum number indicate the age of the individual at the time of sampling.