Table 1.
Ferret polyclonal anti-HA serum from the Influenza Research Resource (IRR).
Table 2.
The HA panel of the mPlex-Flu assay.
Fig 1.
Vaccination strategy and schedule.
Two cohorts of mice were vaccinated with A/California/07/2009 (A/Cal09, H1N1) and A/Hong Kong/1/1968 (A/HK68, H3N2) influenza virus rHA, respectively. A negative control group was included, consisting of vaccination with Addavax alone; the infection group consisted of intranasal infection with of same influenza viral strains at day 0 for both cohorts. rHA vaccinations (Vax) were delivered three weeks apart, with or without Addavax adjuvant as indicated. Individual mice were bled at pre-vaccination (day 0), and at 21, 42, and 63 days post vaccination, and sacrificed at day 63 for collection of spleen and bone marrow cells.
Fig 2.
Freund’s adjuvanted rHA protein vaccination elicits broader antibody mediated cross-reactivity compared to viral infection in ferrets.
The antibody response against homologuous and cross-reactive HA proteins from different influenza virus strains were evaluated by the mPlex-Flu assay. Samples included ferret reference antisera obtained from the Influenza Research Resource collected after viral infection (black) or after a prime-boost-boost vaccination protocol (see Methods) with Freund’s adjuvant admixed rHA (red). Dilutions of polyclonal antisera are provided in Table 1. The results calculated by subtracting the blank from mean MFI for each influenza virus strain with three replicate wells per influenza strain.
Fig 3.
Comparison of antibody mediated cross-reactive immunity induced by influenza infection versus adjuvanted rHA vaccination in ferrets.
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. (A) 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) 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[34, 35] and Euclidean distance (S3 Fig). The positive cut-off MFI unit is 100 (based on the average of negative control sera).
Fig 4.
Comparison of cross-reactivity of serum IgG from influenza virus infected versus Addavax adjuvanted rHA vaccinated mice.
(A) 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) 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.
Fig 5.
Broader cross-reactive antibody responses are induced by prime-boost-boost vaccination with Addavax adjuvanted rHA as compared to infection with A/Hong Kong/1/1968 (A/HK68 H3N2) influenza virus.
(A). The rHA specific IgG concentrations of heterosubtypic and stalk-specific antibodies reaction induced by vaccination with adjuvant alone control, rHA combined with Addavax adjuvant (Vax I, II and III), rHA without adjuvant (Vax Ø), or infection (Inf) with A/HongKong/1/68 (H3N2) virus, were determined using a 29-panel mPlex-Flu assay, and represented as a heatmap. (B). The selected antibody responses against influenza virus HA of A/HK68 were plotted over time. The antibody concentrations (ng/mL) of individual HA were calculated from the standard curves generated in same assay. Serum of individual mice were harvested before priming, as well as before and post-boost 21 day. The specific antibody concentration of IgG is shown as the mean ± standard deviations (STD) of the mean of individual mice (n = 6). The two-way analysis of variance (ANOVA) statistical analysis was conducted including experimental group and time as factors. ** p<0.0001 Vax Ø comparing to Vax III, II and I group; * p<0.05 Vax Ø comparing to Vax I, II, III group; # p<0.0001 Vax III comparing to Inf group; + p<0.0001 Vax comparing to Inf group.
Fig 6.
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.
(A) 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) 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.
Fig 7.
Comparison of anti-HA IgG from mouse spleen memory B cells (MBC) stimulated by infection with A/HongKong/1/1968 (A/HK68 H3N2) virus or vaccinated with adjuvanted rHA.
The anti-HA IgG levels in supernatants of stimulated memory B cells (MBC) cultures were determined by 29-plex panel mPlex-Flu assay. Briefly, mouse spleen MBCs were cultured for 6 days with 0.4 μg LPS (LPS), 10 μg BPL-inactivated A/Cal09 virus (H1) and B/Brisbane/60/2008 virus (B), 2009–2013 H3N2 viral antigen (H3), and medium control (Con), respectively. Influenza virus specific antibody concentrations present in the cell culture supernatants are shown as the mean of IgG concentrations after subtracting the control baseline antibodies in control unstimulated cell cultures (n = 3–4) and represented as a heatmap. The values of IgG concentrations without subtracting the control baseline antibodies in unstimulated cultures (Con), shown in Supplementary material (S7 Fig), demonstrate that only a low level of IgG antibodies produced by murine splenic B cells, after infection and vaccination, without stimulation.
Table 3.
The neutralization titers (GMT) of antisera generated by viral infection or Addavax adjuvanted rHA vaccination of A/Cal09(H1) or A/HK68 (H3) influenza.