High Preexisting Serological Antibody Levels Correlate with Diversification of the Influenza Vaccine

Reactivation of memory B cells allows for a rapid and robust immune response upon challenge with the same antigen. Variant influenza virus strains generated through antigenic shift or drift are encountered multiple times over the lifetime of an individual. One might predict, then, that upon vaccination with the trivalent influenza vaccine across multiple years, the antibody response would become more and more dominant toward strains consistently present in the vaccine at the expense of more divergent strains. However, when we analyzed the vaccine-induced plasmablast, memory, and serological responses to the trivalent influenza vaccine between 2006 and 2013, we found that the B cell response was most robust against more divergent strains. Overall, the antibody response was highest when one or more strains contained in the vaccine varied from year to year. This suggests that in the broader immunological context of viral antigen exposure, the B cell response to variant influenza virus strains is not dictated by the composition of the memory B cell precursor pool. The outcome is instead a diversified B cell response.

Vaccine strategies are being designed to boost broadly reactive B cells present in the memory repertoire to provide universal protection to the influenza virus. It is important to understand how past exposure to influenza virus strains affects the response to subsequent immunizations. The viral epitopes targeted by B cells responding to the vaccine may be a direct reflection of the B cell memory specificities abundant in the preexisting immune repertoire, or other factors may influence the vaccine response. Here, we demonstrate that high preexisting serological antibody levels to a given influenza virus strain correlate with low production of antibody-secreting cells and memory B cells recognizing that strain upon revaccination. In contrast, introduction of antigenically novel strains generates a robust B cell response. Thus, both the preexisting memory B cell repertoire and serological antibody levels must be taken into consideration in predicting the quality of the B cell response to new prime-boost vaccine strategies.

A primary immune response induced upon first exposure to a given antigen is characterized by a wave of low-affinity IgM B cells activated from the naive B cell pool with few to no mutations in their immunoglobulin (Ig) genes. This is followed by isotypeswitched, higher-affinity B cells generated from germinal center reactions with greater numbers of genetic mutations (1, 2). Subsequent exposures, or secondary responses, are largely driven by activated and differentiated memory B cells and thus dominated by mutated, isotype-switched, medium- to high-affinity B cells (1, 3, 4). While this profound difference between the primary and secondary immune responses is relatively straightforward upon repeated exposure to the same antigen, it is less clear how the immune system responds to challenge with a varying antigen such as the influenza virus.

Virus and recombinant HA

Due to the evolving nature of the influenza virus, individuals are repeatedly exposed over their lifetimes to viral strains containing both novel and immune-experienced epitopes (5). In keeping with the very definition of immune memory, the memory response to conserved epitopes encountered before should dominate the response to novel epitopes introduced by mutations in divergent influenza virus strains. If the magnitude of the immune response is driven solely by the memory B cell repertoire, then one would expect these repeated exposures to influenza virus to progressively focus the B cell repertoire toward viral strain epitopes encountered multiple times.

Influenza virus was grown in eggs and purified using polyethylene glycol (PEG) virus precipitation (BioVision Research Products, CA) according to the manufacturer’s instructions. Viral hemagglutination activity units (HAU) were measured by incubating serial dilutions of virus with 0.5% turkey red blood cells (Lampire Biological Laboratories); 1 HAU was determined as the minimum virus amount needed to induce red blood cell agglutination. A/Victoria/351/ 2011 and B/Massachusetts/2/2012-like recombinant HA (rHA) proteins were obtained from Protein Sciences Corporation (Meriden, Connecticut). All other rHA proteins were obtained from the NIAID BEI Resources Repository or Influenza Reagent Resource.

Peripheral blood mononuclear cells (PBMCs) were obtained by centrifugation of whole blood through a Ficoll gradient and resuspension in phosphate-buffered saline (PBS)– 0.2% bovine serum albumin (BSA) for staining and sorting, or cells were viably frozen at 80°C in fetal calf serum (FCS)–10% dimethyl sulfoxide (DMSO) for later use. At days 5 to 7 after vaccination, PBMCs were enriched for B cells by incubation of whole blood with a Rosette Sep B cell enrichment cocktail (Stem Cell Technologies), followed by centrifugation through a Ficoll gradient. PBMCs were then stained with fluorescently labeled Abs recognizing CD3 (7D6), CD19 (H1B19), CD27 (O323), CD38 (HIT2), and CD20 (2H7). CD19 CD3 CD27hi CD38hi plasmablasts (80 to 90% CD20lo) were then bulk sorted using a FACSAria followed by single-cell sorting into 96-well plates containing a Tris buffer with RNase inhibitors (Promega).

Freshly isolated PBMCs or thawed viably frozen PBMCs containing 5 to 10% CD19 B cells were resuspended at 3 106 to 4 106 cells/ml in complete medium (RPMI medium supplemented with 4 mM L-glutamine, 10% FCS, 1:1,000 penicillin-streptomycin [Pen/Strep], mM Na pyruvate 50 M -mercaptoethanol, 10 mM HEPES) containing 2 g/ml CpG (InvivoGen), 1:10,000 Staphylococcus aureusCowan I (Sigma-Aldrich) and 1:1,000 pokeweed mitogen (PWM; a kind gift from Shane Crotty, La Jolla Institute for Allergy and Immunology) for 5 days in 24-well plates with 1 106 to 4 106 cells/well. All activated cells were then pooled, washed three times with complete medium, and transferred to antigen-coated ELISPOT assay plates. All cells were 80 to 90% viable as measured by trypan blue staining before activation.

PBMC isolation and single-cell sorting

First vaccination againstinfluenza virus has the highest serological response. If the magnitude of the immune response is driven solely by the memory B cell repertoire, then one would expect that adults vaccinated recently against the influenza virus would have a higher plasmablast response upon subsequent vaccination than those vaccinated for the first time. To test this, we vaccinated individuals who had received an influenza vaccine the year prior or who reported never having been vaccinated before. Interestingly, we observed by flow cytometry a higher overall CD19 CD38hi CD27hi plasmablast response 7 days after vaccination in newly vaccinated donors (Fig. 1A).

This was true when we combined the vaccine response from individuals vaccinated between 2010 and 2013 (Fig. 1A, left panel) and when we looked specifically at the vaccine response to the 2010-2011 TIV (Fig. 1A, right panel). In the 2009-2010 season, two vaccines were available: the seasonal TIV containing drifted variants of H1N1, H3N2, and influenza B virus and a monovalent vaccine that protected against the pandemic 2009 H1N1 strain (A/California/4/2009 [A/Cal]) (Table 1). When we measured the fold change in influenza virusspecific Abs in serum between day 0 and day 14/21 following vaccination with the 2010-2011 TIV, we found that individuals who had received the 2009-2010 TIV had a reduced serum Ab response to the H3N2 and B virus strain present in the vaccine compared to those who had not been vaccinated or had received only the monovalent pandemic H1N1 vaccine the prior year. -Fig 1 Influenza vaccine-induced serological Ab response varies with vaccine history. (A) Percentage of peripheral blood CD19 B cells at day 7 after vaccination that were CD27hiCD38hi plasmablasts as determined by flow cytometry. The left panel compares the percentage of plasmablasts of all donors vaccinated between the 2010-2011 and 2013-2014 influenza vaccine seasons who had or had not been vaccinated the year prior. The right panel compares the plasmablast response to the 2010-2011 vaccine only. (B) The serum antibody EC50 to the three virus strains present in the 2010-2011 vaccine was determined on the day of vaccination (day 0) with the 2010-2011 TIV and at day 14/21 by ELISA. Each dot represents the fold change increase in the binding EC50 to each strain between day 0 and day 14/21 in an individual donor. Donors are divided according which vaccine(s) they received the year prior (2009-2010 season).

The line represents the median fold change within each vaccine group. (C) Fold change in serum Ab EC50 between day 0 and day 14/21 in five individuals to the vaccinating H1N1, H3N2, or influenza B virus strain in the indicated year. Each line represents the yearly serological response to the vaccinating influenza strain of a given donor. None of the donors received the 2009-2010 TIV, while four of them (except 007) received the A/Cal monovalent vaccine. (D) Fold change in serum Ab EC50 as described for panel C to the vaccinating H1N1, H3N2, or influenza B virus strain. As in the data shown in panel C, all 2010-2011 donors had received the A/Cal monovalent vaccine but not the 2009-2010 TIV. After the 2010-2011 season all donors had been vaccinated the year prior as well.

Each dot represents the fold increase in serum Abs in each donor with the median indicated by the line. All serum Ab EC50 data are the average from three independent experiments. Statistical analysis was determined using a Mann-Whitney test. Vacc, vaccine; Yam, Yamagata lineage; Vic, Victoria lineage; n.s., not significant.