Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells

Abstract

We have previously shown that broadly neutralizing antibodies reactive to the conserved stem region of the influenza virus hemagglutinin (HA) were generated in people infected with the 2009 pandemic H1N1 strain. Such antibodies are rarely seen in humans following infection or vaccination with seasonal influenza virus strains. However, the important question remained whether the inactivated 2009 pandemic H1N1 vaccine, like the infection, could also induce these broadly neutralizing antibodies. To address this question, we analyzed B-cell responses in 24 healthy adults immunized with the pandemic vaccine in 2009. In all cases, we found a rapid, predominantly IgG-producing vaccine-specific plasmablast response. Strikingly, the majority (25 of 28) of HA-specific monoclonal antibodies generated from the vaccine-specific plasmablasts neutralized more than one influenza strain and exhibited high levels of somatic hypermutation, suggesting they were derived from recall of B-cell memory. Indeed, memory B cells that recognized the 2009 pandemic H1N1 HA were detectable before vaccination not only in this cohort but also in samples obtained before the emergence of the pandemic strain. Three antibodies demonstrated extremely broad cross-reactivity and were found to bind the HA stem. Furthermore, one stem-reactive antibody recognized not only H1 and H5, but also H3 influenza viruses. This exceptional cross-reactivity indicates that antibodies capable of neutralizing most influenza subtypes might indeed be elicited by vaccination. The challenge now is to improve upon this result and design influenza vaccines that can elicit these broadly cross-reactive antibodies at sufficiently high levels to provide heterosubtypic protection.

Fig. 1.

Rapid and potent plasmablast and serological responses after vaccination with the subunit pH1N1 2009 vaccine. Healthy adult volunteers were vaccinated with the subunit pH1N1 2009 monovalent vaccine. (A) Fold change in serum antibody titers between day 0 and 28 determined by HAI. (B) The number of vaccine-specific IgG-producing plasmablasts were determined by ELISPOT after vaccination. (C) The number of vaccine-specific plasmablasts correlates with increased serum antibody titers by HAI (Spearman’s correlation). (D) The numbers of influenza-specific IgG-, IgA-, and IgM-producing plasmablasts at day 7 as determined by ELISPOT after immunization with the monovalent pH1N1 2009 vaccine compared with the response elicited by the seasonal 2008/2009 TIV. These TIV data are from a cohort vaccinated in 2008. Dotted lines indicate limit of detection.

Fig. 2.

Stem-binding antibodies are induced following pH1N1 2009 vaccination. Human mAbs were generated from plasmablasts isolated from pH1N1 2009 vaccinees. (A) Binding of mAbs to the pH1N1 2009 virus by ELISA. (B) Binding to pH1N1 2009 HA by ELISA. (C) HA-binding mAbs were tested for HAI and neutralization activity. Three putative stem-binding mAbs are shown in blue. Dotted lines show the highest concentration of mAb tested. Data are representative of two to four repeat experiments. (D) The three putative stem-binding mAbs were tested by competition ELISA with two known stem-binding mAbs (70-1F02 and 70-5B03) (7). The reciprocal stem-binding mAb was used as a positive control, and a previously described HA-head-specific antibody (EM4C04) was used as a negative control. Bars represent means ± SEM for three repeats. The V_H gene use of the individual stem-binding mAbs is shown on the right.

Fig. 3.

The pH1N1 2009 vaccine induces highly cross-reactive HA-specific antibodies. (A) pH1N1 2009 HA-binding mAbs were tested for binding to HAs from the indicated influenza strains by ELISA. Monoclonal antibodies are arranged according to degree of binding by ELISA to pH1N1 2009 HA and grouped according to cross-reactivity by ELISA (blue: stem-binders, bind all H1N1, H5N1, and H3N2; black: bind all H1N1; red: bind A/California/04/09 and A/Brevig Mission/1/18; green: bind A/California/04/09 only). (B) HA head-binding mAbs were tested for neutralizing activity against the indicated panel of H1N1 virus strains. Two mAbs (20-3G06 and 15-1A03) expressed poorly and were not tested for cross-reactivity (ND). (C) Three stem-binding mAbs were tested for neutralizing activity against various influenza virus strains. Influenza strains are arranged from left to right in order of sequence similarity to the pH1N1 2009. Dotted lines represent limits of detection. Data are representative of two to four repeats.

Fig. 4.

Monoclonal antibodies induced following the pH1N1 2009 vaccine display high levels of somatic hypermutation consistent with a recall response. Variable genes from plasmablasts induced following the pH1N1 2009 vaccine were amplified by single-cell RT-PCR and scored for numbers of somatic mutations. (A) The number of mutations per V_H gene following pH1N1 2009 vaccination is compared with published data (, 23–25). The red line shows the mean (P values by Student t tests). (B) The number of mutations per V_H gene in HA-specific mAbs only. Colors represent the degree of cross-reactivity as in Fig 3.

Fig. 5.

Memory B cells reactive to the pH1N1 2009 influenza are detectable even before the emergence of the pandemic strain. Peripheral blood mononuclear cells (PBMCs) obtained before vaccination were tested for the presence of memory B cells reactive against the pH1N1 2009 HA as described (11). The pH1N1 HA-specific IgG memory B cell frequencies are shown in subjects from the year that the pH1N1 2009 emerged (2009/10) (A) and the previous year (2008/09) (B).

Fig. 6.

A model contrasting the antibody response induced after infection or vaccination with seasonal versus pandemic influenza virus strains. The preexisting influenza-specific B-cell pool primarily consists of memory cells that recognize epitopes from recent seasonal strains in the globular head of HA that change relatively little (drift) year to year (shown in green). These memory B cells are repeatedly expanded following infection or vaccination with the seasonal influenza virus strains, whereas the less frequent memory B cells specific for highly conserved epitopes in the stem and head of HA (shown in red) are crowded out. With a pandemic strain, many epitopes in the HA head are replaced with new epitopes (depicted in blue), whereas conserved epitopes in the stem and head remain. Cross-reactive memory B cells specific for the conserved epitopes now have a greater chance of being recruited into the response. Naive responses to the novel epitopes are likely also induced but, based on the Ig isotype and the rapid appearance of the plasmablast response, it is unlikely that these newly induced naive B cells make up a significant proportion of the overall response.