Deep Sequencing Reveals Potential Antigenic Variants at Low Frequencies in Influenza A Virus-Infected Humans

Abstract

Influenza vaccines must be frequently reformulated to account for antigenic changes in the viral envelope protein, hemagglutinin (HA). The rapid evolution of influenza virus under immune pressure is likely enhanced by the virus's genetic diversity within a host, although antigenic change has rarely been investigated on the level of individual infected humans. We used deep sequencing to characterize the between- and within-host genetic diversity of influenza viruses in a cohort of patients that included individuals who were vaccinated and then infected in the same season. We characterized influenza HA segments from the predominant circulating influenza A subtypes during the 2012-2013 (H3N2) and 2013-2014 (pandemic H1N1; H1N1pdm) flu seasons. We found that HA consensus sequences were similar in nonvaccinated and vaccinated subjects. In both groups, purifying selection was the dominant force shaping HA genetic diversity. Interestingly, viruses from multiple individuals harbored low-frequency mutations encoding amino acid substitutions in HA antigenic sites at or near the receptor-binding domain. These mutations included two substitutions in H1N1pdm viruses, G158K and N159K, which were recently found to confer escape from virus-specific antibodies. These findings raise the possibility that influenza antigenic diversity can be generated within individual human hosts but may not become fixed in the viral population even when they would be expected to have a strong fitness advantage. Understanding constraints on influenza antigenic evolution within individual hosts may elucidate potential future pathways of antigenic evolution at the population level.

Importance: Influenza vaccines must be frequently reformulated due to the virus's rapid evolution rate. We know that influenza viruses exist within each infected host as a "swarm" of genetically distinct viruses, but the role of this within-host diversity in the antigenic evolution of influenza has been unclear. We characterized here the genetic and potential antigenic diversity of influenza viruses infecting humans, some of whom became infected despite recent vaccination. Influenza virus between- and within-host genetic diversity was not significantly different in nonvaccinated and vaccinated humans, suggesting that vaccine-induced immunity does not exert strong selective pressure on viruses replicating in individual people. We found low-frequency mutations, below the detection threshold of traditional surveillance methods, in nonvaccinated and vaccinated humans that were recently associated with antibody escape. Interestingly, these potential antigenic variants did not reach fixation in infected people, suggesting that other evolutionary factors may be hindering their emergence in individual humans.

FIG 1

Phylogenetic relationships of influenza A viruses infecting nonvaccinated and vaccinated subjects in Marshfield, WI. (a) H3N2 phylogenetic tree constructed using 68 hemagglutinin genes from subjects infected during the 2012-13 season and 11 outgroup sequences representing WHO-recommended vaccine strains and representative H3N2 taxonomic clades (alignment length, 1,641 nucleotides). (b) H1N1pdm phylogenetic tree constructed using 46 hemagglutinin genes from subjects infected during the 2013-14 season and nine outgroup sequences representing WHO-recommended vaccine strains and representative H1N1pdm taxonomic clades (alignment length, 1,698 nucleotides). Bootstrap values were determined from 1,000 replicates and are indicated above the corresponding nodes when values are above 50%.

FIG 2

Deep sequencing reveals sequence variation in hemagglutinin genes during human infection. We used deep sequencing to reveal the within-host viral variation from nonvaccinated and vaccinated subjects. Displayed are SNPs detected in H3N2-infected (a, c, and e) and H1N1pdm-infected (b, d, and f) subjects. All sequence reads were mapped against three reference sequences: a vaccine strain, the most abundantly detected HA gene sequence, and against each sample's own autologous consensus sequence. Black circles represent nonsynonymous mutations, and gray squares represent synonymous mutations. The x axis represents the nucleotide position, and the y axis represents the frequency for which each mutation was detected from human biological samples. Above each SNP frequency plot is a cartoon depiction of the linear HA gene with shaded functional domains: HA1 (dark gray), HA2 (light gray), and the receptor-binding domain (RBD, black). Density plots indicate the likelihood for a nonsynonymous (black) or synonymous (gray) SNP to occur, regardless of frequency, in a given position across the HA gene.

FIG 3

Localization of amino acid substitutions identified in this study on the HA structure. The structure of an A/Aichi/2/1968 HA trimer (Protein Data Bank accession 5HMG) (53) is shown. The three monomers are shown in black and gray with the receptor-binding site in light brown, and positions previously defined as responsible for antigenic cluster transitions are shown in dark brown (12). Mutations detected in human subjects infected with H3N2 and H1N1pdm viruses are shown in red. All mutations are shown in H3 numbering. (a) H3N2 antigenic sites, as defined by Wiley et al. (8); antigenic sites A to E are shown in blue, green, magenta, orange, and yellow, respectively. (b and c) Mutations detected in subjects infected with H3N2 viruses. (d) H1N1 antigenic sites, as defined by Caton et al. (9); antigenic sites Sa, Sb, Ca1, Ca2, and Cb are shown in blue, green, magenta, orange and yellow, respectively. (e and f) Mutations detected in subjects infected with H1N1pdm viruses. Images were created with MacPymol (http://www.pymol.org/).