Killed viral vaccines are commonly used in the United States to control swine influenza virus. Most vaccines are multivalent, containing genetically and antigenically distinct viruses to elicit broadly cross-reactive antibodies. Sequencing of the hemagglutinin gene is a standard practice for assessing genetic relationships and selection of vaccine strains. Variation in hemagglutinin N-linked glycosylation is a known mechanism employed by influenza virus to evade host immune responses; however, analysis of glycosylation variability is not routinely performed for swine influenza viruses in the swine industry. Additionally, the effects of glycosylation pattern changes (in both the number of sites and location) on antigenicity are not well understood for swine influenza virus.
Routine antigenic analysis in our laboratory, using swine influenza virus isolates from clinical submissions, found evidence for considerable antigenic drift, with 20% of isolates not being neutralized by a broad panel of antisera generated against all genetic clusters of influenza virus circulating in the United States. Genetic analysis of the HA genes of these isolates found that nearly all are within the δ cluster, a seasonal human influenza virus lineage (28
). Hemagglutinin sequence analysis found a conserved T144N mutation (at the predicted glycosylation site at N144) present in all isolates that were not neutralized in the HTSN assay. Analysis of the glycosylation patterns of seasonal human influenza viruses found that a majority of viruses from 1940 to 1985 were glycosylated at N144 (24
). Conversely, nearly all seasonal human influenza viruses from 1986 to the present were glycosylated at N142. Swine influenza viruses containing human seasonal influenza virus HA genes were first identified in 2003, and accordingly, N142 is most common in swine influenza virus isolates with human lineage HA (17
In this study, we confirmed that viruses containing N144, which is located in a consensus glycosylation sequence motif, are not neutralized by antisera generated against genetically similar viruses exhibiting a predicted glycosylation site at N142. Four field isolates with N142 and one with N137 were all neutralized by well-matched antisera in HTSN assays and effectively inhibited in HI and SN assays. Conversely, viruses with the T144N change were not neutralized in the HTSN assay and exhibited much less inhibition in HI and SN assays. N142 is located in the well-characterized Sa epitope, while N144 is immediately adjacent. The Sa epitope is located on the top part of the HA globular head, and mutations in this region have previously been shown to have a pronounced effect on viral antigenicity (6
). To eliminate the possibility of other genetic differences, viruses possessing glycosylation consensus sequences at N142 or N144 were created by reverse genetics and subsequently mutated to migrate the glycosylation consensus sequence to N144 or N142, respectively. Cross-reaction of the engineered viruses was consistent with observations from field isolates with respect to the glycosylation sequence located at either N142 or N144. Antigenic cartography also illustrated that the neutralization-sensitive and -resistant phenotypes were determined by differential glycosylation at N142 and N144.
In this study, we also attempted to address whether the N142 and N144 mutations altered the glycosylation patterns of the HA proteins of both viruses. Examination of purified virions from both viruses by SDS-PAGE and Western blotting revealed a shift in mobility of HA proteins when peptide N
-glycosidase F-treated and nontreated samples were compared directly, indicating that N142- and N144-carrying HA proteins are modified by N-linked glycans (data not shown). A previous study convincingly demonstrated N142-linked glycosylation (8
). No visible differences in HA mobility were observed for viruses bearing N-linked glycosylation sites at N142 or N144. This result was expected, as the numbers of potential glycosylation sites in HA proteins containing N142 or N144 are equivalent. Alternatively, it could be suggested that the low-resolution SDS-PAGE/Western blotting assay using polyclonal antisera may not detect any quantitative difference in glycosylation patterns that may occur in HA proteins with an N142 or N144 mutation. In this regard, a high-resolution mass spectrometry approach will be pursued in our future work.
To elucidate the structural basis for the lack of cross-reactivity of antibodies generated against viruses with N142 to those with N144, models of hemagglutinin in complex with neutralizing antibody were developed. The complex glycan attached to N144 is projected from the hemagglutinin head domain, which may sterically hinder binding of the antibody heavy chain. Conversely, N142-carrying viruses had complex glycans extended away from the antigen-antibody contact interface, which may not interfere with antibody binding to HA protein. Additionally, a recent genetic analysis of influenza virus glycosylation site migrations suggested that glycosylation at N142 better protects the antigenic Sa site, while glycosylation at N144 is more effective at shielding the antigenic Sb site (25
). Thus, the modeling data appear to explain the observed results of our HTSN, HI, and SN assays, showing that antibody is sterically hindered from binding to the head domain of HA for viruses with N144-associated glycans, as well as supporting other predictions based on genetic and structural studies.
Genetic analysis of δ-cluster hemagglutinin sequences in the Newport Laboratories swine influenza virus database found a low prevalence of viruses with N144 prior to 2009 (3.6%). From 2009 to 2011, viruses with N144 represented 18.5% of δ-cluster isolates, and viruses with N144 were identified in each of the five δ subclusters. It can be postulated that these viruses are neutralization resistant, though further experiments are needed to prove this hypothesis. Phylogenetic analysis shows a close relationship between neutralization-resistant and neutralization-sensitive viruses, suggesting that the neutralization-resistant viruses evolved from the neutralization-sensitive δ-cluster viruses circulating in swine since 2003, possibly due to immune selection pressure. Given that swine influenza viruses within the δ cluster contain an HA gene derived from human seasonal influenza virus and that viruses with N-linked glycosylation at position 144 have not circulated in humans since 1985, individuals born thereafter may be more vulnerable to zoonotic transmission of these viruses, as previous work has shown no cross-protection between H1 vaccine strains produced prior to 1986 and vaccines produced in subsequent years (13
). Importantly, vaccines for swine influenza virus may require multiple strains of δ-cluster isolates representing glycosylation variants. This work also highlights the limitations of genetic analysis alone, illustrating the need for coupled antigenic surveillance of influenza virus.
Finally, our observation that relocation of potential N-linked glycosylation sites protects swine influenza virus from rapid neutralization by antibodies is interesting and novel. It suggests a role for immune selection in influenza virus in swine. While this study clearly demonstrates the effect of glycosylation on in vitro antigenicity, little is known about the role of glycosylation on in vivo immunogenicity, which will be a focus of future investigations.