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 virusspecific 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. IMPORTANCEInfluenza 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. S easonal influenza A epidemics cause an estimated 3 to 5 million cases of severe respiratory illness each year, resulting in approximately 250,000 to 500,000 deaths worldwide (1). Available influenza vaccines only provide moderate protection against infection and illness...

Parallel evolution of influenza across multiple spatiotemporal scales

“…For instance, one recent study deep-sequenced HA from several hundred patients but only found a small number of antigenic variants, and mostly at low frequencies (Dinis et al, 2016). But our study suggests that influenza may experience many of the same selective pressures within acute infections as it does globally, even if the short durations of these infections make it difficult for selected mutations to reach frequencies that are detectable with current methods.…”

A novel framework for inferring parameters of transmission from viral sequence data

“…The statistical significance achievable in these studies is limited by the number of animal pairs that can be examined [67][68][69]. Furthermore, the comparison between one genotype and another may be confounded by viral heterogeneity, whereby each population contains a cloud of genetic diversity [24,70]. As we have shown, data from replicate transmission events leads to an improved ability to infer selection, in particular by reducing the false positive rate of inference and by increasing the accuracy in inferred selection coefficients.…”

Respiratory viral infections

“…Influenza evolution at a population level has been studied years, yet, new antigenic variants are initially generated and selected at the level of the individual infected host. Within a host, influenza viruses exist as a "swarm" of genetically distinct viruses [41]. Sanger sequencing defines consensus sequences and cannot resolve minority variants below 20% of the viral population.…”

“…Deep sequencing has been used in natural infection and human challenge studies to characterize between and within host genetic diversity [41,42]. The identification of low frequency mutations in the hemagglutinin (HA) antigenic sites or near the receptor-binding domain in vaccinated and unvaccinated influenza infected persons highlight viral evolution within a host due to selective immune pressure [41]. Similarly, NGS can reveal the rapid evolution of drug resistant variants during therapy [43].…”