Dataset 2: Secretion of H3 clade cross-reactive antibodies by B cells stimulated with inactivated A/Vic11

Purified B cells were obtained by negative selection and stimulated with CpG ODN alone or together with A/Vic11 or A/SH13 for 6 days. (A) ASCs for H3 HA were examined by ELISpot assay. H3-binding antibodies in supernatants of activated B cells were monitored using beads bound with HA from H3N2 strains. (B) A/Vic11-specific IgG. Each symbol represents an individual donor. One-way AVONA was used to evaluate the difference among different groups (* P<0.05; ** P<0.01). (B) Results were verified by ELISpot after stimulation with A/Vic11. The values represent the number of anti-HA IgG specific antibody-secreting cells in 1.25 x 10^5 stimulated B cells/donor. (C) Correlation between anti-A/Vic11 IgG levels in plasma and secretion of A/Vic11-specific IgG by activated B cells. (D) Antibodies binding to H3 clades. (E) Comparison of A/Vic11-specific IgG and A/Swi13-reactive IgG

Dataset 1: Anti-H3 Stalk Reactive Antibodies in Human Plasma

Plasma was obtained from 13 donors, 12 of which had been previously vaccinated within the past 5 years with trivalent or quadrivalent seasonal influenza vaccines. Plasma baseline anti-influenza IgG was measured by mPlex-Flu assay. (A) Levels of IgG against distinct H3N2 strains. Each column depicts mean fluorescence intensity (MFI), representing an individual donor. (B) Levels of H3 stalk-reactive IgG. Each symbol and line represents one donor. (C) IgG-binding to chimeric cH5/1 and cH5/3 proteins. This analysis was performed at a 1:5000 dilution

Dataset 6: Stalk-reactive antibody responses to H3 viruses enhanced by Il-15

Purified B cells were costimulated with CpG₂₀₀₆, A/Victoria/361/2011 viruses and IL-15. Stalk-reactive IgG in supernatants was detected at day 6. Chimeric molecules cH5/3, cH4/7 and cH5/1 were used for assessing antibodies against H3 stalk, H7 stalk and H1 stalk, respectively. Each symbol and line represents an individual donor. (A) H3 stalk-reactive antibodies. (B) H7 Stalk-reactive antibodies. (C) H1 stalk-reactive antibodies

Dataset 3: Influenza viruses induce cross-reactive antibody responses in vitro

B cells were obtained by negative selection, and then stimulated with CpG ODN 2006 or together with A/Vic11 for 6 days. Cross-reactive antibodies binding to H1, H2, H5, H6, H7 and B influenza subtypes in B cell culture were measured by mPlex-Flu assay. (A) Fold change in cross-reactive antibodies. All values of IgG levels (MFI) were subtracted those of medium before calculating fold change. Only those values of IgG induced by CpG plus A/Vic11 viruses, which were greater than 100, were selected to calculate fold change. Each symbol represents the median of fold change in levels of IgG induced by CpG plus H3 to IgG stimulated by CpG 2006 ODN alone. (B) Correlation between influenza specific antibodies and HA antigenic sequence

Dataset 4 - Induction of HA stalk-reactive antibodies by H3 viruses

B cells from healthy donors were stimulated with CpG 2006 ODN alone or together with inactivated A/Vic11 viruses, A/SH13 (H7N9), and A/Hong Kong/33982/2009 (A/HK09) (H9N2) and pandemic H1N1 viruses. The levels of IgG against H3 HA, H5 head and chimeric molecules cH5/3 are shown for individual honors. (A) Nine of 13 donors displayed increases in stalk-reactive IgG after A/Vic11 stimulation. (B, C) A correlation model assuming different coefficients for different H3N2 strains-specific antibodies was fitted to evaluate the relationship between HA stalk-reactive and strain-specific IgG

Dataset 5: IL-15 increases cross-reactive antibody responses to H3N2 viruses

(A) B cells from healthy donors were co-stimulated with CpG ODN 2006 inactivated A/Vic11 viruses and interleukin 15. Strain-specific and HA stalk-reactive IgG in supernatants of activated B cells were detected after 6 days. (B) Fold change in cross-reactive antibodies. (B) Correlation between influenza specific antibodies and HA antigenic sequence. No correlation between cross-reactive antibodies and HA sequence was detected

Broad cross-reactive IgG responses elicited by adjuvanted vaccination with recombinant influenza hemagglutinin (rHA) in ferrets and mice

<div><p>Annual immunization against influenza virus is a large international public health effort. Accumulating evidence suggests that antibody mediated cross-reactive immunity against influenza hemagglutinin (HA) strongly correlates with long-lasting cross-protection against influenza virus strains that differ from the primary infection or vaccination strain. However, the optimal strategies for achieving highly cross-reactive antibodies to the influenza virus HA have not yet to be defined. In the current study, using Luminex-based mPlex-Flu assay, developed by our laboratory, to quantitatively measure influenza specific IgG antibody mediated cross-reactivity, we found that prime-boost-boost vaccination of ferrets with rHA proteins admixed with adjuvant elicited higher magnitude and broader cross-reactive antibody responses than that induced by actual influenza viral infection, and this cross-reactive response likely correlated with increased anti-stalk reactive antibodies. We observed a similar phenomenon in mice receiving three sequential vaccinations with rHA proteins from either A/California/07/2009 (H1N1) or A/Hong Kong/1/1968 (H3N2) viruses admixed with Addavax, an MF59-like adjuvant. Using this same mouse vaccination model, we determined that Addavax plays a more significant role in the initial priming event than in subsequent boosts. We also characterized the generation of cross-reactive antibody secreting cells (ASCs) and memory B cells (MBCs) when comparing vaccination to viral infection. We have also found that adjuvant plays a critical role in the generation of long-lived ASCs and MBCs cross-reactive to influenza viruses as a result of vaccination with rHA of influenza virus, and the observed increase in stalk-reactive antibodies likely contributes to this IgG mediated broad cross-reactivity.</p></div

The cross-reactive anti-HA IgG secreting plasma cells (ASCs) in murine bone marrow after infection or vaccination with A/HongKong/1/1968 (A/HK68 H3N2) influenza virus.

<p><b>(A)</b> Representative plates of ELISpot assays as described in the methods, which evaluated antibody secreting plasma cells in murine bone marrow specific against rHA of A/California/07/2009 (A/Cal09, H1N1), A/HongKong/1/1968 (A/HK68, H3N2) and B/Bris08 (B strain) influenza viruses induced by vaccination with Addavax adjuvanted rHA of A/HK68 (VaxIII), without adjuvant (Vax Ø) and by A/HK68 virus infection group. <b>(B)</b> Representative numbers of IgG ASCs specific for rHA of A/HK68 and A/Cal09 starting with 500,000 murine bone marrow cells, after subtracting the numbers of ASC spots specific for rHA of B/Bris08 (background control), induced after vaccination (VaxIII, Vax Ø groups) or infection. The values and error bars shown are the mean and the standard deviation (STD) of n = 4–5 mice/time point. Grouped multiple t-tests were used to determine statistically significant differences.</p

Comparison of cross-reactivity of serum IgG from influenza virus infected versus Addavax adjuvanted rHA vaccinated mice.

<p>(<b>A</b>) Multiplex assay comparison of IgG seroreactivity against a panel of 29 influenza rHA resulting from viral infection or the prime-boost-boost vaccination with Addavax (MF59-like) admixed rHAs from either A/California/07/2009 (A/Cal09, H1N1) or A/HongKong/1/1968 (A/HK68, H3N2). Sera were collected 9 weeks post- infection, or 3 weeks after the final vaccine boost (day 63 after the priming vaccination). Sera harvested from six mice per group were pooled for analysis. The IgG binding against influenza HA are reported as the mean MFI minus the baseline of each strain (n = 3). (<b>B</b>) A cross-reactivity plot of the mouse serum IgG binding data for each influenza virus strain versus the protein feature dissimilarity of vaccine HA and each of the rHA proteins used in mPlex-Flu assay.</p

Comparison of antibody mediated cross-reactive immunity induced by influenza infection versus adjuvanted rHA vaccination in ferrets.

<p>Post-vaccination serum from ferrets infected with A/California/07/2009 (A/Cal, H1N1), A/Vietnam/1203/2004 (A/Vie04, H5N1) and A/Brisbane/10/2007 (A/Bris07, H3N2) or multiply vaccinated with rHA with Freund’s adjuvant in a prime-boost-boost series were analyzed for IgG reactivity against 25 influenza rHA by multiplex assay. <b>(A)</b> Comparison of IgG antibody responses of vaccination versus infection. The results are expressed as the average MFI subtracting blank MFI for each strain (n = 3). The strains are colored according to the different groups. <b>(B)</b> A cross-reactivity plot of the IgG binding data for each influenza strain versus the protein sequence-feature dissimilarity of vaccine-homologous HA and each of the vaccine-heterologous rHA proteins used in mPlex-Flu assay. The protein sequence dissimilarities of rHAs were calculated using a protein feature vector approach[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193680#pone.0193680.ref034" target="_blank">34</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193680#pone.0193680.ref035" target="_blank">35</a>] and Euclidean distance (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193680#pone.0193680.s005" target="_blank">S3 Fig</a>). The positive cut-off MFI unit is 100 (based on the average of negative control sera).</p