B cell response and hemagglutinin stalk-reactive antibody production in different age cohorts following 2009 H1N1 influenza virus vaccination

Abstract

The 2009 pandemic H1N1 (pH1N1) influenza virus carried a swine-origin hemagglutinin (HA) that was closely related to the HAs of pre-1947 H1N1 viruses but highly divergent from the HAs of recently circulating H1N1 strains. Consequently, prior exposure to pH1N1-like viruses was mostly limited to individuals over the age of about 60 years. We related age and associated differences in immune history to the B cell response to an inactivated monovalent pH1N1 vaccine given intramuscularly to subjects in three age cohorts: 18 to 32 years, 60 to 69 years, and ≥70 years. The day 0 pH1N1-specific hemagglutination inhibition (HAI) and microneutralization (MN) titers were generally higher in the older cohorts, consistent with greater prevaccination exposure to pH1N1-like viruses. Most subjects in each cohort responded well to vaccination, with early formation of circulating virus-specific antibody (Ab)-secreting cells and ≥4-fold increases in HAI and MN titers. However, the response was strongest in the 18- to 32-year cohort. Circulating levels of HA stalk-reactive Abs were increased after vaccination, especially in the 18- to 32-year cohort, raising the possibility of elevated levels of cross-reactive neutralizing Abs. In the young cohort, an increase in MN activity against the seasonal influenza virus A/Brisbane/59/07 after vaccination was generally associated with an increase in the anti-Brisbane/59/07 HAI titer, suggesting an effect mediated primarily by HA head-reactive rather than stalk-reactive Abs. Our findings support recent proposals that immunization with a relatively novel HA favors the induction of Abs against conserved epitopes. They also emphasize the need to clarify how the level of circulating stalk-reactive Abs relates to resistance to influenza.

Fig 1

Ab responses to an inactivated pH1N1 vaccine in the 18- to 32-year (circles), 60- to 69-year (diamonds), and ≥70-year (triangles) age cohorts. The titers of pH1N1-specific Abs in serum on days 0 and 28 after vaccination were measured by HAI (A and B) and MN (C and D) assays. The titers on days 0 and 28 (A and C) and fold changes in titers from days 0 to 28 (B and D) are shown for individual subjects. Bars identify geometric mean titers (GMTs) (A and C) or geometric means of fold increases (B and D). Within-cohort statistical analyses represent paired comparisons of day 0 and day 28 titers. Only statistically significant differences are indicated: **, P < 0.01, and ***, P < 0.001.

Fig 2

Virus-specific IgA in nasal secretions after pH1N1 vaccination. Nasal secretions sampled on days 0 and 28 after vaccination of the indicated age cohorts (18 to 32 years, 60 to 69 years, and ≥70 years) were tested by an ELISA for pH1-specific IgA. Titers of pH1-specific IgA (mOD/min/μg total IgA) on day 0 (A) and fold changes in the titers from days 0 to 28 (B) are shown for individual subjects. The median values (bar) and interquartile ranges are identified.

Fig 3

Circulating ASCs induced by pH1N1 vaccination. B cells enriched from blood were analyzed by an ELISpot assay for vaccine-specific ASCs and by flow cytometry for ASC phenotypes. (A to C) Vaccine-specific ASCs. IgG and IgA ASC frequencies on days 6 to 8 after vaccination are shown for individual subjects in the 18- to 32-year (A), 60- to 69-year (B), and ≥70-year (C) age cohorts. Vaccine-specific ASCs were not detected in the day 0 samples. (D and E) Flow cytometric identification of ASCs. Frequencies of CD38^high CD27^high cells (plasmablasts) (D) and CD38^high CD138⁺ cells (plasma cells) (E) represent the percentages of CD3⁻ CD19⁺ cells. Frequencies on days 6 to 8 after vaccination are shown for individual subjects in the indicated age cohorts (18 to 32 years, 60 to 69 years, and ≥70 years). The median values (bars) and interquartile ranges are identified. Day 0 values did not differ between cohorts and are shown as the means (dashed lines) and 95% confidence intervals (CIs) (shaded zones). Only statistically significant differences in the ASC frequencies are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 4

Relationship between vaccine-specific IgG ASCs and the HAI response. A linear regression model assuming different coefficients for different age cohorts was fitted to evaluate the relationship between the frequency (log 10) of circulating vaccine-specific IgG ASCs (per 10⁶ B cells) on days 6 to 8 after pH1N1 vaccination and the fold change (log 2) in the pH1N1-specific HAI titer from days 0 to 28. The likelihood ratio test indicated zero intercepts for all regression lines (P = 0.88). The fitted model explained 87% of the variability of the data (R² ≈ 0.87). For each cohort, a strong positive correlation existed between the virus-specific IgG ASC frequency and the fold change in the HAI titer (P < 0.0001). The slope was significantly greater for the 18- to 32-year cohort than for the 60- to 69-year cohort (P < 0.0001) and the ≥70-year cohort (P = 0.0018); the slopes for the 60- to 69-year and ≥70-year cohorts were not significantly different.

Fig 5

Induction of HA stalk-reactive Abs by pH1N1 vaccination. Serum titers of IgG reactive with the chimeric HA molecule cH6/1 (A and B) or with the intact HA (pH1) of the vaccine virus (C and D) were measured by ELISAs on days 0 and 28 after vaccination of the indicated age cohorts (18 to 32 years [circles], 60 to 69 years [diamonds], and ≥70 years [triangles]). The titers were determined by endpoint titration and are expressed as the reciprocal of the serum dilution. Fold changes in the titer were calculated from OD ratios for dilutions in the linear regions of serum titration curves. The titers on day 0 (A and C) and the fold changes in titer from days 0 to 28 (B and D) are shown for individual subjects. Bars identify geometric mean titers (GMTs) (A and C) or geometric means of the fold increase (B and D). Only statistically significant differences are indicated: **, P < 0.01, and ***, P < 0.001.

Fig 6

HAI and MN titers against the seasonal H1N1 virus Bris/07 after pH1N1 vaccination. Bris/07-specific HAI (A) and MN (B) titers in serum were determined on days 0 and 28 after vaccination of the indicated age cohorts (18 to 32 years, 60 to 69 years, and ≥70 years). Titers are shown for individual subjects; paired titers are connected. The P values for paired comparisons of day 0 and day 28 titers are shown.