Research paper
Plasmablast-derived polyclonal antibody response after influenza vaccination

https://doi.org/10.1016/j.jim.2010.12.008Get rights and content

Abstract

Conventional measurement of antibody responses to vaccines largely relies on serum antibodies, which are primarily produced by bone marrow plasma cells and may not represent the entire vaccine-induced B cell repertoire, including important functional components such as those targeted to mucosal sites. After immunization or infection, activated B cells differentiate into plasmablasts in local lymphoid organs, then traffic through circulation to the target sites where they further develop into plasma cells. On day 7 after influenza vaccination, a burst of plasmablasts, highly enriched for vaccine-specific antibody secreting cells, appears in the peripheral blood. This provides a unique window to the overall B cell response to the vaccine, without interference of pre-existing cross-reactive serum antibody. In this study we isolated B cells from volunteers on day 7 after immunization with the inactivated influenza vaccine and cultured them ex vivo to collect plasmablast-derived polyclonal antibodies (PPAb). The PPAb contained secreted IgG and IgA, which was approximately 0.2 ng per antibody secreting cell. Influenza-specific IgG and IgA binding activity was detected in PPAb at dilutions up to 105 by ELISA. The ratio of the titers of influenza-specific IgA to IgG by ELISA was 4-fold higher in PPAb than in day 28 post-vaccination sera, suggesting that vaccine-induced IgA is enriched in PPAb compared to sera. Functional activity was also detected in PPAb as determined by microneutralization and hemagglutination inhibition assays. In addition to bulk B cell cultures, we also cultured plasmablast subsets sorted by cell surface markers to generate PPAb. These results suggest that PPAb better reflects the mucosal IgA response than serum samples. Since PPAb are exclusively produced by recently activated B cells, it allows assessing vaccine-induced antibody response without interference from pre-existing cross-reactive serum antibodies and permits an assessment of antibody avidity based on antigen specific binding and antibody quantity. Therefore this assay is particularly useful for studying vaccine/infection-induced antibodies against antigens that might have previously circulated, such as antibody responses to rotavirus, dengue or influenza viruses in which cross-reactive antibodies against different virus serotypes/subtypes play a critical role in immunity and/or pathogenesis.

Introduction

Influenza viruses are respiratory pathogens that cause annual epidemics and intermittent pandemics, resulting in significant disease burden in all age groups. Influenza vaccines, including live attenuated and inactivated vaccines, can effectively protect children and adults against influenza infection. Although the mechanisms for the protective efficacy of these vaccines are not completely understood, antibody response to natural influenza infection and vaccination is believed to be a critical part of the protective immunity (Gerhard, 2001). However, the conventional evaluation of the antibody response to influenza vaccination by serum antibody titer, based on hemagglutination inhibition (HAI) assay or neutralization assay, does not always predict protection, especially in the situations such as live attenuated influenza vaccine (LAIV) (Treanor and Wright, 2003). Therefore better correlates of the protective immune response induced by different vaccines are desirable.

After administration of a vaccine by different routes, naïve or memory B cells are activated at the site of immunization and draining lymph nodes. The activated B cells proliferate and differentiate into plasmablasts in the germinal centers of the local lymph nodes. At around days 6–8 after immunization, a large number of plasmablasts leave the germinal centers to transiently enter the circulation and form a sharp peak in the blood which is highly enriched for vaccine antigen-specific antibody secreting cells (ASC) (Cox et al., 1994, el-Madhun et al., 1998, Sasaki et al., 2008, Wrammert et al., 2008). Depending on local antigen-presenting environmental factors during the immune response (Sigmundsdottir and Butcher, 2008), these plasmablasts express different trafficking receptors. These receptors direct the plasmablast migration to specific sites and tissues of the body (Kunkel and Butcher, 2002, Kunkel and Butcher, 2003). Those cells homing to the bone marrow become plasma cells, which are the primary source of serum antibodies (Benner et al., 1981). Other plasmablasts are targeted to different mucosal sites including the respiratory tract, and secrete musosal antibodies that are directly involved in protection against respiratory or other mucosal infections. Since the bone marrow plasma cells are only derived from a subset of the entire vaccine-activated B cell population, serum antibodies do not necessarily reflect the overall antibody response to the vaccine.

In addition to the conventional serological assays for measuring serum antibody titer before and after vaccination, different assays have been utilized to address the characteristics of the peripheral plasmablast response at day 7 after influenza vaccination. We and others have used ELISPOT assay to determine the number of total and influenza-specific IgG- and IgA-secreting plasmablasts in children and adults immunized with different types of influenza vaccines (Cox et al., 1994, el-Madhun et al., 1998, Sasaki et al., 2007, Sasaki et al., 2008). The plasmablasts can be identified and isolated by flow cytometry based on their surface markers, which allows analysis of the immunoglobulin gene repertoire of vaccine-activated B cell population (Wrammert et al., 2008). In addition, the mRNA encoding the heavy chain and light chain of immunoglobulin genes from individual plasmablasts can be cloned and co-expressed to generate recombinant monoclonal antibodies for functional analysis (Wrammert et al., 2008, Smith et al., 2009).

Instead of generating a panel of recombinant monoclonal antibodies from individual plasmablasts derived from each vaccinee to represent the overall antibody response to the vaccine, it is also possible to analyze polyclonal antibodies secreted ex vivo from the bulk plasmablast population (Ershler et al., 1982, Chang and Sack, 2001). In this study we generated the plasmablast-derived polyclonal antibodies (PPAb) from a group of volunteers at day 7 after immunization with influenza vaccine and compared the PPAb with serum antibody response. Our results suggest that the PPAb analysis might provide new insights to the B cell and antibody responses to influenza vaccination, as well as other viral vaccines and infection.

Section snippets

Human participants, vaccination protocol and blood samples

Five healthy adults 18–30 years of age (donors No. 1–No. 5) were enrolled during the 2008–2009 influenza season for the study. In addition, two healthy adults of the same age group (donors No. 6 and No. 7) were enrolled during the 2009–2010 influenza season for the flow cytometry sorting experiment (Section 3.4). The study protocol was approved by the institutional review board at Stanford University. Informed consent was obtained from each participant. Participants were immunized with one dose

Generation of PPAb with ex vivo culturing of B cells isolated before and after influenza vaccination

We immunized five adults with the 2008–2009 inactivated influenza vaccine. B cells were isolated by negative selection from blood samples collected on day 0 (baseline), day 7, and day 28 after vaccination. The isolated B cells were cultured for one week. The conditioned media containing the secreted PPAb were recovered from the cultures and the concentrations of IgG and IgA determined. Both IgG and IgA were detected in the supernatant of B cell cultures (Fig. 1A), indicating that the

Discussion

Almost three decades ago Ershler et al. (1982) developed a new assay for antibody response after tetanus toxoid immunization, which involved culturing lymphocytes in ELISA plates pre-coated with tetanus toxoid to capture and quantify antigen-specific IgG secreted from ASC. When this assay, named microculture antibody synthesis enzyme linked assay (MASELA), was used to study antibody response after influenza vaccination, the authors reported highly correlated results with serum IgG antibody

Role of the funding source

The project described was supported by the NIH grants AI057229 and DK56339. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Acknowledgements

We thank K. Subbarao for providing the influenza virus vaccine strains, our study subjects for their participation; S. Mackey for coordinating the clinical study; and T. Quan, S. Swope, and C. Walsh for enrolling the subjects and collecting the blood samples.

References (22)

  • R.J. Cox et al.

    An early humoral immune response in peripheral blood following parenteral inactivated influenza vaccination

    Vaccine

    (1994)
  • E.J. Kunkel et al.

    Chemokines and the tissue-specific migration of lymphocytes

    Immunity

    (2002)
  • R.B. Belshe et al.

    Correlates of immune protection induced by live, attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine

    J. Infect. Dis.

    (2000)
  • R. Benner et al.

    The bone marrow: the major source of serum immunoglobulins, but still a neglected site of antibody formation

    Clin. Exp. Immunol.

    (1981)
  • Centers for Disease Control and Prevention

    The 1998–99 WHO Influenza Reagent Kit for the Identification of Influenza Isolates

    (1998)
  • H.S. Chang et al.

    Development of a novel in vitro assay (ALS assay) for evaluation of vaccine-induced antibody secretion from circulating mucosal lymphocytes

    Clin. Diagn. Lab. Immunol.

    (2001)
  • R.J. Cox et al.

    Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines

    Scand. J. Immunol.

    (2004)
  • A.S. el-Madhun et al.

    Systemic and mucosal immune responses in young children and adults after parenteral influenza vaccination

    J. Infect. Dis.

    (1998)
  • W.B. Ershler et al.

    Specific in vivo and in vitro antibody response to tetanus toxoid immunization

    Clin. Exp. Immunol.

    (1982)
  • W.B. Ershler et al.

    Specific antibody synthesis in vitro. IV. The correlation of in vitro and in vivo antibody response to influenza vaccine in rhesus monkeys

    Clin. Exp. Immunol.

    (1988)
  • W. Gerhard

    The role of the antibody response in influenza virus infection

    Curr. Top. Microbiol. Immunol.

    (2001)
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    Present address: Facultad de Salud, Programa de Medicina, Universidad Surcolombiana, Neiva, Colombia.

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