FIG 2.
Alternating the influenza B virus lineage in the vaccine magnifies the plasmablast response. (A) An ELISPOT assay was performed on PBMCS at 7 days after influenza vaccination with the 2012-2013 TIV to determine the proportion of IgG+/IgA+ plasmablasts (PB) capable of binding each influenza virus strain. ELISPOT assay plates were coated with the given year's vaccine or influenza virus rHA and incubated with fresh PBMCs overnight, and spots were counted under each condition. Shown is the proportion of total vaccine-positive plasmablasts capable of binding rHA from the vaccinating 2012-2013 influenza B virus lineage (Yamagata) and H3N2 strain or the influenza B virus lineage (Victoria) and H3N2 strain present in the previous year's vaccine. Each line links the proportion of vaccine-positive plasmablasts that can bind rHA of each of the two strains in an individual. Statistical significance was determined using a paired Wilcoxon test. (B) Plasmablasts present in peripheral blood at day 7 after vaccination with the 2012-2013 or 2013-2014 TIV were tested for specificity to the vaccinating H1N1, H3N2, or influenza B virus rHA or whole virus by ELISPOT assay as described for panel A. Shown is the percentage of vaccine-positive antibody-secreting cells that were specific to the 2012-2013 vaccinating influenza B virus strain in each donor who had or had not been vaccinated in the 2011-2012 season or to the 2013-2014 vaccinating B strain after vaccination with the 2013-2014 TIV in donors who had also received the 2012-2013 TIV. (C) Proportion of vaccine-positive plasmablasts generated in response to the 2013-2014 TIV specific to the H1N1 (H1), H3N2 (H3), and influenza B virus strain in the vaccine as measured by ELISPOT assay. Each bar represents the response in one donor. (D) Percentage of CD19+ B cells that were CD27hi CD38hi plasmablasts as detected by flow cytometry in the peripheral blood 7 days after either the 2012-2013 or 2013-2014 TIV. Shown is the percent plasmablasts in each donor, with the median indicated by the horizontal line. Statistical significance was determined with a Mann-Whitney test. (E) The proportion of total antibody-secreting cells specific to the H1N1, H3N2, or B strain was determined by ELISPOT assay and multiplied by the percentage of total plasmablasts detected by flow cytometry to calculate the percentage of total B cells that were influenza virus strain-specific plasmablasts at day 7 after vaccination with the 2012-2013 or 2013-2014 TIV. Shown is the percentage of B cells in each donor specific for each strain, as indicated, with the median represented by the horizontal line. Statistical significance was determined with a Mann-Whitney test. (F and G) Immunoglobulin genes from single-cell sorted plasmablasts were cloned and expressed as MAbs and tested for binding to the vaccinating influenza virus strains by ELISA. Shown is the proportion of total influenza virus-specific MAbs able to bind the vaccinating influenza B virus strain as detected by ELISA in the years indicated. Each dot represents the proportion from a given donor. All donors in the 2007-2008 and 2008-2009 seasons were vaccinated the year prior as well. Shown is the percentage of influenza B virus-specific MAbs (Flu MAbs) comparing all donors as a group between years (F) or a paired comparison of the same individual across years in the subset of donors for whom we tracked the immune response over multiple years (G). Statistical significance was determined using a Mann-Whitney test (F) or paired Wilcoxon test (G).