Longitudinal Analysis of Neuraminidase and Hemagglutinin Antibodies to Influenza A Viruses after Immunization with Seasonal Inactivated Influenza Vaccines

Abstract

Neuraminidase (NA)-based immunity could reduce the harmful impact of novel antigenic variants of influenza viruses. The detection of neuraminidase-inhibiting (NI) antibodies in parallel with anti-hemagglutinin (HA) antibodies may enhance research on the immunogenicity and duration of antibody responses to influenza vaccines. To assess anti-NA antibodies after vaccination with seasonal inactivated influenza vaccines, we used the enzyme-linked lectin assay, and anti-HA antibodies were detected in the hemagglutination inhibition assay. The dynamics of the anti-NA antibody response differed depending on the virus subtype: antibodies to A/H3N2 virus neuraminidase increased later than antibodies to A/H1N1pdm09 subtype neuraminidase and persisted longer. In contrast to HA antibodies, the fold increase in antibody titers to NA after vaccination poorly depended on the preexisting level. At the same time, NA antibody levels after vaccination directly correlated with titers before vaccination. A difference was found in response to NA antigen between split and subunit-adjuvanted vaccines and in NA functional activity in the vaccine formulations.

Keywords: antibodies; influenza; neuraminidase-inhibition; persistence; vaccines.

Figure 1

Antibody dynamics throughout 1 year after vaccination with subunit-adjuvanted vaccine (n = 46) or split vaccine (n = 27). Data are represented by a box plot diagram with a solid black line at the median titer. Cross symbol indicates the group geometric mean titer (GMT). Red lines indicate the conventional antibody level threshold adopted in this study: 1:40 for HI antibodies and 1:20 for NI antibodies.

Figure 2

Combined seroconversions of HI and NI antibodies on day 21 after vaccination with split or subunit-adjuvanted IIVs presented by Venn’s diagrams. Numbers in circles present absolute number or responders in one or both tests. The total number of participants was 27 in the split vaccine group and 46 in the subunit-adjuvanted vaccine group. The number of non-responders to both antigens is not shown on Venn’s diagram.

Figure 3

Correlation between post-vaccination HI and NI antibody fold increases. Each dot represents one participant. The results of the Pearson correlation test applied to logarithmic values are presented on graphs. The population in the analyses included all participants, regardless of the vaccine type. The shaded area represents the 95% CI for the regression line. The dashed lines mark the conventional seroconversion threshold (4-fold increase in HI antibody titer and 2-fold increase in NI antibody titer).

Figure 4

Post-vaccination antibody response dependence on the preexisting antibody level. (a) HI antibodies; (b) NI antibodies. Each dot represents one participant. The results of the Pearson correlation test applied to logarithmic values are presented on graphs. The population in the analyses included all participants, regardless of the vaccine type. The shaded area represents the 95% CI for the regression line. Participants with an antibody titer of < 10 are considered seronegative. The dashed lines mark the conventional seroconversion threshold (4-fold increase in HI antibody titer and 2-fold increase in NI antibody titer).

Figure 5

Correlation matrix between HI and NI antibody titers throughout the year after vaccination. The population in the analyses included all participants, regardless of the vaccine type. The heatmap represents the level of correlation between each pair of listed parameters. The color scale shows the Spearman correlation coefficient for positive (blue) or negative (red) correlation; a white color means no correlation. The lower left and upper right corners present the same data (the map is symmetrical about the diagonal), and the squares on the diagonal should not be considered as they represent the “self-correlation.” White-outlined areas can be interpreted as follows: (a,b) show a positive correlation of anti-HA antibody titers early and late after vaccination, except for the baseline titer for A/H1N1pdm09 and A/H3N2 viruses, respectively; (c,d) show positive correlation of anti-NA antibody titers early and late after vaccination, including the baseline titer for A/H1N1pdm09 and A/H3N2 viruses, respectively; (e) shows a low positive correlation between anti-NA antibody titers against two influenza A subtypes; (f) shows an absence of correlation between anti-HA and anti-NA antibody titers.

Figure 6

NA enzyme activity of influenza vaccines and H6NX viruses.