Two Types of Antibodies Are Induced by Vaccination with A/California/2009pdm Virus: Binding near the Sialic Acid-Binding Pocket and Neutralizing Both H1N1 and H5N1 Viruses

<div><p>Many people have a history of catching the flu several times during childhood but no additional flu in adulthood, even without vaccination. We analyzed the total repertoire of antibodies (Abs) against influenza A group 1 viruses induced in such a flu-resistant person after vaccination with 2009 H1N1 pandemic influenza virus. They were classified into two types, with no exceptions. The first type, the products of B cells newly induced through vaccination, binds near the sialic acid-binding pocket. The second type, the products of long-lived memory B cells established before vaccination, utilizes the <i>1-69</i> V_H gene, binds to the stem of HA, and neutralizes both H1N1 and H5N1 viruses with few exceptions. These observations indicate that the sialic acid-binding pocket and its surrounding region are immunogenically very potent and majority of the B cells whose growth is newly induced by vaccination produce Abs that recognize these regions. However, they play a role in protection against influenza virus infection for a short period since variant viruses that have acquired resistance to these Abs become dominant. On the other hand, although the stem of HA is immunogenically not potent, the second type of B cells eventually becomes dominant. Thus, a selection system should function in forming the repertoire of long-lived memory B cells and the stability of the epitope would greatly affect the fate of the memory cells. Acquisition of the ability to produce Abs that bind to the stable epitope could be a major factor of flu resistance.</p></div

Cytokine production in whole-blood cultures following immunization with an influenza vaccine

A clinical trial of a quadrivalent split influenza vaccine was performed in the 2014/15 season. Sixty-four subjects aged 6 months to 18 years were enrolled in order to investigate the relationship between cellular and humoral immune responses. Subjects were categorized into two groups by measuring neutralizing antibodies: non-primed naïve/primed or seroconverted/non-seroconverted groups. Whole-blood cultures were stimulated with the H1N1 split antigen before immunization and one month after the first and second immunizations for subjects < 13 years and before and one month after the first dose for those ≥ 13 years in order to investigate cytokine production. Significant amounts of IL-2, IL-12, IL-13, MCP-1, MIP-1β, and TNF-α were detected from one month after the first dose in the naïve group. In addition to these cytokines, the production of IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17, G-CSF, and IFN-γ was enhanced one month after the second dose. No significant increase was noted in the primed group, except in the production of IL-10. In seroconverted subjects, the production of IL-2, IL-4, IL-8, IL-10, G-CSF, MCP-1, TNF-α, and IFN-γ increased one month after the first dose, which was earlier than in the naïve group, whereas no significant cytokine response was noted in subjects without seroconversion. Subjects ≥ 13 years were primed and the production of G-CSF, IL-4, and IL-1β increased in subjects with seroconversion. Whole-blood cultures were also stimulated with the H3N2 split antigen and similar cytokine profiles were obtained. Many cytokines and chemokines, including inflammatory cytokines, were produced in seroconverted, but not non-seroconverted subjects

HI and virus neutralizing activity of serum against H1N1 and H5N1 virus strains.

a<p>HI activity.</p>b<p>Virus neutralizing activity detected by VN method.</p>c<p>Virus neutralizing activity detected by M-VN method.</p>d<p>Serums before vaccination.</p>e<p>Not determined.</p

Three Types of Broadly Reacting Antibodies against Influenza B Viruses Induced by Vaccination with Seasonal Influenza Viruses

We analyzed the antibody (Ab) repertoire against influenza B viruses induced by vaccination with seasonal influenza viruses in one individual who had never been vaccinated until 2009. The vaccine used in this study comprised B/Massachusetts/2/2012 (Yamagata lineage), A/Texas/50/2012 (H3N2), and A/California/7/2009 (H1N1). One month after the subject received two vaccinations, blood (200 ml) was obtained and peripheral mononuclear cells were prepared, and a large Ab library was constructed using phage display technology. The library was screened with HA-enriched fraction of B/Massachusetts/2/2012 and B/Brisbane/60/2008 (Victoria lineage) virus, and a total of 26 Abs that potentially bound to hemagglutinin (HA) molecules were isolated. Their binding activities to six influenza B viruses, three of Yamagata lineage and three of Victoria lineage, and two influenza A viruses, H1N1 and H3N2, were examined. The Abs showed cross-reactivity at three different levels. The first type bound to all Yamagata lineage viruses. The second type bound to both Yamagata and Victoria lineage viruses. The third type bound to both influenza A and B viruses. These results indicate that common epitopes exist on HA molecules of influenza virus at various levels, and humans have capability to produce Abs that bind to such common epitopes

Competitive inhibition of binding to HA among the Abs newly induced by vaccination.

<p>The binding activity of Fab-PP Ab (indicated at upper side) to Cal09 virus particles was measured by ELISA under the presence of a 10-times greater concentration of Fab-cp3 Ab (indicated at the left side). F008-009 and F033-367 are not anti-HA Abs and were used as controls. The binding inhibition was calculated as follows: the absorbance value under the presence of F008-009cp3 was used as 100% binding, and the degree of reduction in the absorbance value under the presence of Fab-cp3 of Ab was measured and shown as percent inhibition. Percent inhibition is shown as follows: ≥70% (white), 50%–70% (blue), 0%–50% (grey). The experiment was performed at least three times in duplicate.</p

Inhibition of the binding of C179 to HA by type 2 Abs.

<p>Binding of C179 to Bri07 virus particles was examined under the presence of a 10-times greater concentration of various Fab-cp3 Abs by ELISA. F008-009 and F033-367 are not anti-HA Abs and were used as negative controls. The group number is indicated under the name of the clone. The experiment was performed two times in duplicate, and the error bars show standard deviation.</p

Virus neutralizing activity of serum in the presence of K1-18 Ab.

a<p>Serum before vaccination.</p

Activities of representative clones classified into 63 groups.

<p>The first three columns contain the numbers of clones isolated by the screenings. Germline genes were identified by comparing the amino acid sequence of V_H of the representative clone with the sequences of all of the germline V_H genes, and the identity (%) is indicated. The amino acid sequence of CDR3 is shown. The binding activity to four H1N1 (NC99, SI06, Bri07 and Cal09) and one H3N2 virus particles was examined by enzyme-linked immunosorbent assay (ELISA). Absorbance at 492 nm is shown as follows: ≥1.00 (red), 0.50–0.99 (orange), and 0.10–0.49 (yellow). The virus neutralizing activity of 100 or 250 µg/ml Fab-PP Ab against H1N1, H5N1, and H3N2 viruses was examined by the focus reduction test. The reduction rate is shown as a percentage as follows: ≥60% (dark blue), 40%–60% (blue), and 20%–40% (light blue). The HI activity of Fab-PP Ab was measured by using two H1N1 (Bri07 and Cal09) viruses. The lowest concentration (µg/ml) of Fab-PP Abs to inhibit hemagglutination is shown.</p

Schedule of vaccination and blood collection.

<p>In 2009, a donor born in 1947 was vaccinated with A/California/7/2009 pandemic vaccine strain two times (on November 2 and 16). Blood was collected from the donor two times before vaccination (on October 30 and November 2), once after 1st vaccination (on November 9) and 5 times after 2nd vaccination (on November 17, 23, 30, December 7, and 14). Large phage Ab libraries were prepared from blood collected on October 30 (before vaccination) and December 14 (after vaccination).</p