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Title: Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs}
Article:
Abstract:
Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin( HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies. Antibody-free propagation following antigenic escape rapidly selected viruses with mutations that modulated receptor binding avidity through the addition of N-linked glycans to the HA globular domain. These findings expand our understanding of the viral mechanisms that maintain fitness during antigenic evolution to include glycan addition, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms.
Introduction:
Influenza A virus( IAV) persists in human populations by continuously evolving to escape herd immunity. Protective immunity is predominantly mediated by neutralizing antibodies( Abs) specific for the viral surface glycoprotein hemagglutinin( HA). HA mediates both target cell attachment, by binding terminal sialic acids( SA) on cellular membrane components, and fusion between viral and cellular membranes following virion internalization. Neutralizing Abs mainly target the five highly variable immunodominant antigenic sites on the globular head domain of HA, blocking either HA-mediated attachment or fusion[1–4]. Viruses can escape neutralization by amino substitutions in HA that reduce antibody affinity and/or function. The fitness costs imposed by Ab escape, and the mechanisms by which the virus compensates remain poorly understood, yet play a central role in governing HA antigenic evolution[5, 6]. Influenza virus escapes from neutralizing antibody via a number of defined mechanisms involving HA mutations. The simplest is an amino acid substitution in a cognate epitope that diminishes antibody affinity[2]. Less commonly, distant substitutions can affect antibody binding via allosteric effects on antibody access to its epitope[7–9]. Amino acid substitutions in the sialic acid receptor site, or other regions of the globular domain that increase affinity for cellular SA receptors, enable Ab-escape by shifting the binding equilibrium towards virus binding to cells versus antibody[10–13]. The most drastic alterations in overall antigenicity typically result from amino acid substitutions that create a N-linked glycosylation site in the globular domain, as the attached glycan can sterically block the binding of Abs to multiple sites[5, 14–19]. The ability of viruses to exploit these escape routes is limited by their costs to viral fitness. The sialic acid receptor binding site is nestled among the immunodominant antigenic sites, and escape substitutions often alter viral fitness by changing HA receptor binding avidity. Amino acid substitutions that change the net charge of the globular domain typically alter cell binding avidity[13, 20]. We previously demonstrated that selection of substitutions that add N-linked glycosylation sites within the globular domain to escape antibody neutralization often reduces receptor avidity[5]. Receptor avidity and HA antigenicity are thus intimately linked, and must be continuously balanced to maintain fitness during antigenic evolution[13, 21]. Here, we use the primerID method for error-correcting virus population sequencing to reveal a surprising new role for N-linked glycosylation in facilitating IAV immune escape. Beyond its canonical role in blocking Ab binding, we show that glycosylation can also compensate for fitness costs imposed by escape substitutions elsewhere on HA, thus increasing the viability and subsequent emergence potential of Ab-escape variants.
Discussion:
IAV has persisted in humans despite widespread vaccination and infection, while vaccination has driven other RNA viruses with similarly high mutation rates, such as measles virus and poliovirus, to near-extinction. Understanding the specific features of IAV that enable effective immune escape is critical for designing more effective vaccines and therapeutics. Our findings reveal a novel role for glycosylation in viral immune evasion, independent of its well-known steric effects on epitope shielding[14–19]. Previous studies clearly showed that glycan addition generally impairs HA function while dramatically increasing fitness in the presence of neutralizing antibodies[5]. Our results demonstrate that glycan addition can also play the opposite role during antigenic drift: increasing the fitness of antigenic escape variants by restoring optimal HA receptor interactions, even in the absence of antibody selective pressure. This raises the intriguing possibility that both mechanisms contribute to the steady evolutionary accretion of N-linked glycans within the globular domain observed during human circulation of H1 and H3 viruses[17]. Importantly, the fact that we selected for glycan addition in the absence of Ab selection does not diminish the profound effects that these glycans have on the antigenicity of HA. In particular, the predominant variant selected, N133S/T, also emerged during the circulation of seasonal H1N1 in humans, and has been demonstrated to have enormous effects on antigenicity and sensitivity to neutralization[17]. These results highlight the self-perpetuating nature of HA antigenic evolution. Hensley et al. found that infection of naïve hosts with antigenic escape variants selected for compensatory mutations that were themselves antigenically significant[13]. Similarly, our results demonstrate that antigenic escape can lead to the emergence of compensatory mutations that push HA even further in antigenic space. These factors make it difficult to infer the nature of selective pressures resulting in given HA sequence changes in human IAV isolates. Defining the specific properties of the HA molecule that facilitate mutational tolerance is an area of intense study. Studies in vitro found that the HA globular head was highly tolerant of both random substitutions and 5-amino acid block insertions[3, 27, 31, 32]. These results suggested that the HA globular head may exhibit a high degree of mutational robustness, such that the median fitness effects of substitutions are relatively small[32–34]. Alternatively, mutations may generally be costly, but are compensated by epistatic mutations that restore protein function and fitness[6, 35]. Our data, along with other studies that demonstrate fitness costs associated with antigenic variation and/or rapid emergence of compensatory mutations following HA antigenic escape, suggest that compensatory epistatic interactions play a critical role during antigenic drift[6, 13, 36, 37]. If rapid compensation of antigenic escape mutations is important for maintaining fitness during antigenic drift, then defining compensatory mechanisms is essential. Many antigenicity-determining residues scattered in the HA globular domain also strongly influence avidity and specificity for sialic acid receptors, and thus Ab escape substitutions at these residues can negatively affect receptor interactions[13]. As a result, many of the compensatory mutations that have been described in association with antigenic escape affect receptor interactions, and likely serve to restore optimal receptor binding properties. The need to accumulate compensatory mutations in order to combine antigenic novelty with fitness and transmissibility has been proposed as an explanation for the discordance between the high in vitro mutation rates and rapid Ab-based selection of influenza viruses in vivo and the relatively slow rates of population-level antigenic evolution seen with influenza viruses[13, 38]. Our results indicate that glycosylation can play an important role in this process. The availability of multiple mutational pathways to restore fitness increases the odds that a fit variant will emerge under selection[39]. Our discovery that glycan addition beneficially tunes receptor interactions adds to previously described mechanisms, including charge transitions in the globular HA domain and alterations in NA activity[13, 21, 36, 40]. While difficult to test experimentally, it is intriguing to consider that IAV may have evolved to maximize the number of compensatory pathways available during antigenic drift. Our unexpected discovery of a new role for glycosylation in compensating for IAV immune evasion highlights the power of virus population sequencing for uncovering novel mechanisms of viral adaptation through high-definition forward genetics screening. The ability to accurately measure changes in the viral population through methods such as primerID can provide a comprehensive, unbiased profile of the genetic pathways to increased fitness under defined selection conditions. The primerID approach offers several advantages over conventional deep sequencing protocols. First, the ability to generate a multi-read consensus of the original cDNA sequence reduces the background nucleotide error rate from ~1% to ~0. 01%, greatly increasing the sensitivity of minor variant detection. In theory, this background represents the bona fide mutation frequency within the population, combined with the mutation rate of the reverse transcriptase used to generate the cDNA pool( ~0. 01%)[23]. Second, assembly of amplicon reads into ancestral cDNA sequences eliminates the distortion of measured variant frequencies by the stochastic PCR re-sampling that occurs during conventional amplicon sequencing. Third, the number of unique primerID barcodes used to generate consensus sequences represents the actual depth of the viral population sequenced. Fourth, the ability to phase substitutions into authentic haplotypes enables quantitation of linkage between substitutions within a given genome segment. Finally, primerID requires far less virus input than other methods of highly accurate population sequencing such as CirSeq, facilitating a wider range of experimental applications, including the analysis of samples collected from infected animals or human subjects[41, 42]. Altogether, we reveal a surprising new role for glycosylation in facilitating IAV immune evasion by compensating for the fitness costs of antigenic escape mutations. These results broaden our understanding of the critical function of glycosylation during antigenic drift and shed light on the unique mutational plasticity of influenza HA. Further, these studies highlight the remarkable potential of highly accurate virus population sequencing and high-definition forward genetics for exploring viral evolution.
Seasonal influenza A viruses ( IAV ) cause tens of thousands of deaths and tens of billions of dollars in economic costs every year in the U . S . alone , despite widespread pre-exposure and vaccination . IAV persists within the human population by evading herd immunity through the continual accumulation of immune escape substitutions in a process known as antigenic drift . Unfortunately , the specific mechanisms that facilitate IAV antigenic drift remain unclear , making it difficult to design more efficacious , escape-proof vaccines . Here , we used a high-definition population sequencing approach to understand how IAV tolerates the accumulation of immune escape substitutions during antigenic drift without losing fitness . We found that the virus rapidly acquired N-linked glycan structures on the hemagglutinin ( HA ) protein following escape from a panel of neutralizing antibodies . Glycosylation of HA alleviated the deleterious effects of immune escape substitutions on interactions with host cell receptors . These results expand our understanding of the mechanisms that facilitate IAV immune evasion , and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms .