Broad Hemagglutinin-Specific Memory B Cell Expansion by Seasonal Influenza Virus Infection Reflects Early-Life Imprinting and Adaptation to the Infecting Virus

Abstract

Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutininspecific antibody production and changes in the size and character of hemagglutininreactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection "imprints" for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus. IMPORTANCE Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs).Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalkreactive, as well as to head-reactive, MBCs. Our findings provide experimental

Application of Volumetric Absorptive Micro Sampling to Measure Multidimensional Anti-Influenza Hemagglutinin Igg Antibodies by MPlex-Flu Assay

“…The mPlex-Flu assay was performed as described previously [7,8,12] [7,12], and the standard curve for each influenza virus strain was generated by 1:4 serially diluting STD01 for each batch of samples. 50µL of the diluted sample was added into each reaction well.…”

“…The influenza HA bead panel used in this study is shown in Table ??, comprising 33 separate rHAs. 50µL of bead mix was added to each well of the plate as previously described [7,8,12]. Plates were then incubated with gentle shaking for 2 hours at room temperature, and then washed (PBS + 1% Bris + 0.1% BSA).…”

“…Thus, developing a solution would require both a simple method for in-the-field collection of small amounts of peripheral blood or serum, coupled with an assay that uses very small sample sizes (10-20 µL). Here we describe and validate such a system, using a combination of volumetric absorptive microsampling (VAMS) [6] coupled with a Luminex-based assay (mPLEX-Flu) [7,8,9,10,11,12] to quantitatively measure IgG antibodies against 33 strains of influenza hemagglutinin.…”

“…We have previously developed a Luminex-based multiplex assay (mPlex-Flu assay) that can simultaneously measure absolute antibody concentrations (IgG, IgM or IgA) against up to 50 influenza strains using ≤ 5µL of serum [7,8,11,12]. The mPLEX-Flu assay has a continuous linear read-out over 4 logs, with low Type-I (false positives, specificity) and Type-II (false negatives, sensitivity) errors [10].…”

Computer Simulations of the Humoral Immune System Reveal How Imprinting Can Affect Responses to Influenza HA Stalk with Implications for the Design of Universal Vaccines

“…Additionally, there was an induction of antibodies towards the conserved stalk region of HA[6,7]. Additionally studies have suggested a role of Memory B cells in these highly cross-reactive antibody responses[8,9].…”