Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains

Abstract

H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014-2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016-2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016-2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016-2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA.

Keywords: antibody; hemagglutinin; influenza; vaccine.

Fig. 1.

Contemporary H3N2 viruses possess a new mutation that introduces a glycosylation site in antigenic site B of HA. (A) The K160T HA mutation rapidly rose to fixation during the 2014–2015 influenza season. Shown are frequencies estimated from viral samples (from GISAID database) collected from December 2011 to March 2017 and divided into 2-mo windows. (B) Putative glycosylated sites on contemporary HAs are shown on the A/Victoria/361/2011 HA trimer (PDB ID code 4O5I). The new putative site introduced by the K160T mutation is shown in blue, while the other putative sites are shown in black. (C) H3 viruses possessing either K160 HA or T160 HA were created by reverse genetics. The molecular weights of the HAs of these viruses were determined using Western blots with an anti-HA antibody, either with or without prior PNGase treatment. PNGase treatment was completed under reducing conditions. On the –PNGase gel, the upper bands correspond to HA trimers and the lower bands correspond to HA monomers.

Fig. 2.

Contemporary H3N2 viruses with T160 HA are antigenically distinct compared with H3N2 viruses with K160 HA. (A) ELISAs were completed to test the binding of 26 anti-H3 human monoclonal antibodies (mAbs) to a 2009 HA, a 2014 HA with T160, and a 2014 HA with K160. All antibodies in this panel were elicited by a 2009 HA following vaccination before the 2010–2011 season. Shown is percent binding of antibodies to the 2014 viruses relative to binding to the 2009 virus. (B) ELISA binding data for an antibody that binds efficiently to virus with K160 HA but not T160 HA is shown. (C) ELISA binding data for an antibody that recognizes a conserved epitope on the HA stalk is shown to verify that ELISA plates were coated with similar amounts of HA antigen.

Fig. 3.

Ferrets elicit different types of antibody responses when exposed to H3 viruses with K160 HA and T160 HA. Ferrets (n = 3 animals per group) were infected with viruses possessing (A) K160 HA or (B) T160 HA and sera were collected 28 d later. FRNTs were completed using viruses that possessed K160 HA or T160 HA. Neutralization titers are expressed as inverse dilution of sera that reduced foci by 90%. We completed three independent experiments with each sera. Shown are geometric means from the three independent experiments. Statistical significance was determined using a paired Student’s t test.

Fig. 4.

Vaccine antigens possessing K160 HA and T160 HA elicit different responses in humans. Donors were vaccinated with seasonal influenza vaccines, and sera were collected before and 28 d after vaccination. FRNTs were completed using viruses that possessed T160 HA or K160 HA. (A) Flublok induced higher fold changes to T160 HA than did Flucelvax and Fluzone (P = 0.01 and P = 0.04 in adjusted analysis, respectively; Table S3; ns, nonsignificant). (B) The vaccine types did not differ in their ability to induce responses to K160 HA (P > 0.1 in adjusted analysis; Table S3). Thick horizontal lines show the median fold changes of the geometric mean titers. Colored rectangles indicate the interquartile range, and whiskers indicate the 150% interquartile ranges. Individual data points are superimposed. See Table S2 for raw titer data.