Potential antigenic mismatch of the H3N2 component of the 2019 Southern Hemisphere influenza vaccine

Here, we find that the egg-adapted H3N2 component of the 2019 Southern Hemisphere influenza vaccine elicits an antibody response in ferrets that is highly focused on antigenic site A of hemagglutinin. This is potentially problematic since most H3N2 viruses currently circulating in the Southern Hemisphere possess antigenic site A substitutions.

3 Human egg-adapted 3c2.A H3N2 influenza vaccine strains possess adaptive substitutions that result in the loss of a key glycosylation site within antigenic site B of hemagglutinin (HA) [1]. We previously demonstrated that the 2016-2017 Northern Hemisphere egg-adapted H3N2 vaccine strain (A/Hong Kong/4801/2014) elicits HA antigenic site B antibodies that bind to the vaccine strain but not to circulating 3c2.A H3N2 strains [1]. This antigenic mismatch has been associated with low vaccine effectiveness (VE) during recent H3N2-dominated influenza seasons [2,3]. 3c2.A viruses recently diversified into several subgroups (Figure 1A), and a new H3N2 vaccine strain, A/Switzerland/8060/2017 (3c2.A2 subgroup), was recommended for the 2019 Southern Hemisphere vaccine formulation [4]. Similar to previous egg-adapted 3c2.A H3N2 vaccine strains, the HA of A/Switzerland/8060/2017 possesses a T160K HA substitution that results in the loss of a glycosylation site in antigenic site B of HA ( Figure 1B). Interestingly, data from the World Health Organization indicate that antibodies elicited by the egg-adapted A/Switzerland/8060/2017 strain are less focused towards HA antigenic site B compared to antibodies elicited by previous 3c2.A H3N2 egg-adapted vaccine strains [4]. To determine if the A/Switzerland/8060/2017 strain is antigenically matched to H3N2 strains currently circulating in the Southern Hemisphere, we completed antigenic analyses using sera isolated from ferrets exposed to either wild-type or egg-adapted A/Switzerland/8060/2017 strains.

Infection of ferrets with influenza virus
We used reverse-genetics [5] to create viruses with the wild-type or egg-adapted

Foci Reduction Neutralization Tests (FRNTs)
Serum samples were treated with receptor-destroying enzyme (Denka Seiken) followed by heatinactivation at 55°C for 30 minutes prior to testing in FRNTs. FRNTs were performed as previously described [1] using ferret sera and 3c2.A2 viruses with wild-type and egg-adapted HAs. We also completed assays with a 3c2.A1 virus (wild-type A/Singapore/INFIMH-16-0019/2016) and 3c2.A2 viruses with single HA substitutions. Each sample was tested in three independent experiments and geometric mean titers of the replicates were used for analysis.

Statistical methods
Log2-transformed antibody titers were compared using RM one-way ANOVA corrected for multiple comparisons (Bonferroni method). Statistical analysis was performed using Prism version 7.

Results
Ferrets infected with the wild-type A/Switzerland/8060/2017 strain mounted antibody responses that neutralized wild-type and egg-adapted 3c2.A2 viruses equivalently ( Figure 1C). Ferrets infected with the egg-adapted A/Switzerland/8060/2017 strain mounted antibody responses that efficiently neutralized egg-adapted 3c2.A2 virus and moderately neutralized wild-type 3c2.A2 virus ( Figure 1D). Importantly, antibodies elicited by the wild-type and egg-adapted A/Switzerland/8060/2017 strains poorly neutralized the 3c2.A1 virus ( Figure 1D). This is important since 3c2.A1 viruses are currently circulating at high levels in parts of the Southern Hemisphere [6]. These data suggest that antibodies elicited by the A/Switzerland/8060/2017 strain are minimally affected by antigenic site B HA substitutions that distinguish wild-type and egg-adapted 3c2.A2 strains. This indicates that most antibodies elicited in ferrets by wild-type and egg-adapted A/Switzerland/8060/2017 strains target other HA epitopes that are mismatched between 3c2.A2 and 3c2.A1 viruses.
To further define the specificity of antibodies elicited by wild-type and egg-adapted  (Figures 1C-D). These data indicate that unlike past 3c2.A egg-adapted strains that elicit a heavily focused antigenic site B HA response [1], the egg-adapted A/Switzerland/8060/2017 strain elicits antibody responses that are more directed towards epitopes involving residue 142 that is located in antigenic site A of HA. There are several limitations of our study. Our study evaluated antibody responses elicited in ferrets, not humans. Studies from our laboratory and others [9] clearly demonstrate that the specificity of influenza virus antibodies elicited in ferrets and humans can differ because most humans have complex immune histories, whereas ferrets do not. It is also worth noting that we tested serum from ferrets that were intranasally infected with virus rather than vaccinated. Ferret serum used for antigenic analyses are typically prepared in this manner [4]