Impact of Pre-Existing Immunity and Age on Antibody Responses to Live Attenuated Influenza Vaccine

Abstract

Live attenuated influenza vaccines (LAIV) typically induce a poor hemagglutination inhibition (HI) response, which is the standard correlate of protection for inactivated influenza vaccines. The significance of the HI response is complicated because the LAIV vaccine primarily induces the local mucosal immune system, while the HI assay measures the circulating serum antibody response. However, age and pre-existing immunity have been identified as important factors affecting LAIV immunogenicity. This study aimed to extend our understanding of LAIV-induced immunity, particularly, the impact age and pre-existing immunity have on eliciting functional and neutralising antibody responses in paediatric and adult populations vaccinated with LAIV. Thirty-one children and 26 adults were immunized with the trivalent LAIV during the 2013-2014 influenza season in Norway. Children under 9 years received a second dose of LAIV 28 days after the first dose. Blood samples were collected pre- and post-vaccination. HI, microneutralization (MN) and enzyme-linked lectin assay for neuraminidase (NA) antibodies were measured against the vaccine strains. IgG antibody avidity against hemagglutinin (HA) and NA proteins from the vaccine strains was also assessed. Significant correlations were observed between HI and MN responses to A/California/7/2009 (A/H1N1)pdm09-like strain and B/Massachusetts/2/2012-like strain, suggesting that MN is a potential immunological correlate for LAIV. However, the relationship between recipient age (or priming status) and serological response varied between vaccine strains. There was a notable increase in HI and MN responses in all cohorts except naive children against the H1N1 strain, where most recipients had responses below the protective antibody threshold. NAI responses were generally weak in naive children against all vaccine strains compared with adults or antigen-primed children. Post-vaccination antibody avidity increased only in primed children below nine years of age against the A/H1N1 strain. Overall, our findings indicate that LAIV elicits functional and neutralizing antibody responses in both naive and antigen experienced cohorts, however, the magnitude and kinetics of the response varies between vaccine strains.

Keywords: adults; children; functional antibodies; immune response; live attenuated influenza vaccine.

Figure 1

Hemagglutination inhibiting titres against the three vaccine strains in children stratified by virus exposure and adults. The virus strains are shown on the top of the figure and the Y-axis shows the HI titres, (A) A/California/7/2009 (pdm09 H1N1), (B) A/Texas/50/2012 (A/H3N2), (C) B/Massachusetts/2/2012. Each symbol indicates an individual HI response. Red circles indicate children under 9 years old who have not been primed, blue triangles represent children under 9 who have been primed, purple squares represent children above 9, and green diamonds represent adults. The dotted horizontal line at 40 refers to the protective HI titre. Significance levels calculated using Dunn’s test indicated as (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.

Figure 2

Microneutralization (MN) titres against the three vaccine strains in children stratified by influenza virus exposure and adults. The virus strains are shown on the top of the figure as (A) A/California/7/2009 (pdm09 H1N1), (B) A/Texas/50/2012 (A/H3N2), or (C) B/Massachusetts/2/2012, while the Y axis is the MN titre. Each symbol indicates an individual MN response. See Figure 1 for figure legends. The dotted horizontal line indicates the MN titre of 80. Horizontal bars indicate geometric mean titres for the group with 95% confidence intervals. Significance levels calculated using the Dunn’s test are indicated as (*) p < 0.05, (***) p < 0.001, (****) p < 0.0001.

Figure 3

Regression analysis of microneutralization (MN) and hemagglutination inhibition (HI) antibody titres. The Deming regression analysis was performed using log₂-transformed HI and MN antibody titres. The vaccine strains are indicated at the top of the figure as A/California/7/2009 (pdm09 H1N1), A/Texas/50/2012 (A/H3N2), or B/Massachusetts/2/2012. MN (y-axis) and HI (x-axis) titres from time points 0, day of tonsillectomy, 28-, and 56-days post-vaccination were plotted. The slope (S), and p-value (P) are shown for each correlation.

Figure 4

Neuraminidase inhibiting (NAI) antibody titres against the three vaccine strains in children were stratified by virus exposure and adults. The virus strains are shown on the top of the figure as (A) A/Equine/Prague/56 (H7) + A/California/07/09 (N1), (B) A/turkey/Massachusetts/3740/1965 (H6) + A/Texas750/2012 (N2), (C) A/turkey/Massachusetts/3740/1965 (H6) + B/Yamagata/16/1988 (NB), while the Y-axis is the NAI titre. Each point indicates an individual response and figure legends are presented in Figure 1. Horizontal bars indicate geometric mean titres for the group with 95% confidence intervals. Significance levels calculated using the Dunn’s test are indicated as (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.

Figure 5

The antibody avidity to haemagglutinin in children stratified by virus exposure and adults. The hemagglutinin proteins are shown on the top of the figure (A) A/ H1, (B) A/ H3, (C) B/ H Mass, while the Y-axis is the avidity index percentage against Haemagglutinin (HA, top). Each symbol indicates an individual response. Red shows naive children below 9 years of age. Blue shows primed children below the age of 9 years. Purple shows primed children above the age of 9. Green shows the adult cohort. Horizontal bars indicate geometric mean titres for the group and 95% confidence intervals. Significance levels calculated using the Dunn’s test are indicated as (**) p < 0.01.

Figure 6

Neuraminidase avidity index changes against the three vaccine strains in children stratified by virus exposure and adults. The neuraminidase strains are shown on the top of the figure (A) A/ N1, (B) A/ N2, (C) B/ N Mass, while the Y-axis is the avidity index percentage against Neuraminidase (NA). Each symbol indicates an individual response. Red shows naive children below 9 years of age. Blue shows primed children below the age of 9 years. Purple shows primed children above the age of 9. Green shows the adult cohort. Horizontal bars indicate geometric mean titres for the group, and 95% confidence intervals.

Figure 7

Assay Spearman R Correlation Matrix in Children and Adults Following LAIV. The fold-change between day 0 and 56 was taken for each vaccine strain, and then averaged to correlate changes between different assays.