Supplementary Figure 1 Infection strategy. To test the antibody responses against influenza viruses, animals were sequentially infected with two divergent strains of the same subtype. For H1N1 infections, animals were primed with a human seasonal strain from 1999 (NC99, A/New Caledonia/20/1999), followed by the 2009 human pandemic H1N1 strain (NL09, A/Netherlands/602/2009) six weeks later. These viruses express divergent HA head domains, but similar HA stalk domains. For H3N2 infection animals were primed with a human seasonal strain from 1982 (Phil82, A/Philippines/2/1982), followed by a 2011 human seasonal strain (Vic11, A/Victoria/361/2011). These viruses are separated by almost 30 years of antigenic drift.
Supplementary Figure 2 Viral titers on day 2 after infection in animal models. Mice (A, n=5/virus), Guinea pigs (B, n=2/virus) and ferrets (C, n=2/virus) were infected with NC99, NL09, Phil82 and Cal09 and viral titers were measured on day 2 post infection from nasal wash (guinea pigs and ferrets) or nasal turbinates (mice).
Supplementary Figure 3 Heat-map presentation of guinea pig antibody titers after infection. A) Anti-HA titers of guinea pigs (n=3) after one H1N1 (NC99) infection. B) Anti-HA titers of guinea pigs (n=3) after sequential infection with two divergent H1N1 (NC99, NL09) strains. C) Anti-HA titers of guinea pigs (n=3) after one H3N2 (Phil82) infection. D) Anti-HA titers of guinea pigs (n=2) after sequential infection with two divergent H3N2 (Phil82, Vic11) strains. E-H) Anti-NA titers of the corresponding sera to panels A-D.
Supplementary Figure 4 Heat-map presentation of ferret antibody titers after infection. A) Anti-HA titers of ferrets (n=2) after one H1N1 (NC99) infection. B) Anti-HA titers of ferrets (n=2) after sequential infection with two divergent H1N1 (NC99, NL09) strains. C) Anti-HA titers of ferrets (n=2) after one H3N2 (Phil82) infection. D) Anti-HA titers of ferrets (n=2) after sequential infection with two divergent H3N2 (Phil82, Vic11) strains. E-H) Anti-NA titers of the corresponding sera to panels A-D.
Supplementary Figure 5 Profiles of the titers of cross-reactive antibodies after a single infection in animal models. Antibody titers measured by ELISA after single influenza virus infection were plotted on the y-axis and the percent amino acid difference to the HA of the strain used for the (later) second infection was plotted on the x-axis. Each point represents the geometric mean titer measured against a single HA (dark blue dot for H1 HAs, light blue dot for other group 1 HAs, dark red triangle for H3 HAs, light red triangle for other group 2 HAs). A non-linear fit (plateau followed by one phase decay) was performed to illustrate the differences in the breadth of the antibody response in all animals. Points for HAs with titers lower than 10 3 are plotted at 10 3. A) Mouse group 1 HA titers after H1N1 (NC99) infection. ELISAs were performed on pooled sera of 10 mice and geometric mean titers of technical duplicates are shown. B) Guinea pig group 1 HA titers after H1N1 (NC99) infection. ELISAs were performed on individual sera of 3 guinea pigs and geometric mean titers are shown. C) Ferret group 1 HA titers after H1N1 (NC99) infection. ELISAs were performed on individual sera of 2 ferrets and geometric mean titers are shown. D) Mouse group 2 HA titers after H3N2 (Phil82) infection. ELISAs were performed on pooled sera of 10 mice and geometric mean titers of technical duplicates are shown. E) Guinea pig group 2 HA titers after H3N2 (Phil82) infection. ELISAs were performed on individual sera of 3 guinea pigs and geometric mean titers are shown. F Ferret group 2 HA titers after H3N2 (Phil82) infection. ELISAs were performed on individual sera of 2 ferrets and geometric mean titers are shown.
Supplementary Figure 6 Glycosylation versus antibody titers after infection. A-C) Antibody titers post 2x infection with either H1N1 (circles) or H3N2 (triangles) against H1 (blue) or H3 (red) HAs were plotted against the number of corresponding glycosylation sites of each HA for all animal models. Mouse samples show the geometric mean of technical duplicates of pooled sera from 10 mice per virus. Guinea pigs show the geometric mean titers of individual animals (H1N1: n=3, H3N2: n=2). For ferrets, the geometric mean titers of individual animals (n=2/virus) is shown. D) Human antibody titers (geometric mean) post ph1n1 (Cal09, n=9) or H3N2 (Vic11, n=10) infection were plotted against the number of glycosylation sites of the corresponding HAs. Circles indicate titers for individuals infected with ph1n1 and triangles indicate titers for individuals infected with H3N2. Symbols for titers measured against H1 HAs are colored red and symbols for titers measured against H3 HAs are colored blue.
Supplementary Figure 7 Profiles of the titers of cross-reactive antibodies after infection of humans with influenza virus. A) Geometric mean antibody titers of human individuals post ph1n1 infection (n=9) were plotted on the y-axis and the percent amino acid difference to the HA of the infection strain was plotted on the x-axis. Each point represents the geometric mean titer of all individuals measured against a single HA. Dots illustrate group 1 HAs, triangles group 2 HAs and squares influenza B HA. The postinfection titers are shown in color (blue for group 1, red for group 2 and green for influenza B) and the corresponding pre-infection titers are plotted in gray. A non-linear fit (plateau followed by one phase decay) was performed to illustrate the breadth of the antibody response. B) Geometric mean antibody titers of human individuals post H3N2 infection (n=10) were plotted in the same manner as panel A.
Supplementary Figure 8 ELISA of the titers of antibody to HA in humans before and after infection. A) Heat map representation of pre-ph1n1 infection ELISA antibody titers against HA (geometric mean of 9 individuals). B) Heat map representation of post-ph1n1 infection ELISA antibody titers against HA (geometric mean of 9 individuals). C) Heat map representation of pre-h3n2 infection ELISA antibody titers against HA (geometric mean of 10 individuals). B) Heat map representation of post-h3n2 infection ELISA antibody titers against HA (geometric mean of 10 individuals).
Supplementary Figure 9 Human reactivity to NA before and after infection. Heat map representations of human geometric mean ELISA antibody titers against NA pre- and post-ph1n1 (A, B, n=9) or H3N2 (C, D, n=10) infections. E-F) Heat map representations of geometric mean fold-induction of antibody titers against NA post H1N1 (E, n=9) or H3N2 (F, n=10) infection in humans. G-H) Geometric mean fold-induction of antibody titers against NA post-ph1n1 (n=9) or post-h3n2 (n=10) infection in humans were sorted by highest induction in a bar graph. Blue bars represent group 1 NAs, red bars group 2 NAs, green influenza B NA and grey the N10 bat isolate. Error bars indicate the 95% confidence intervals.
Supplementary Figure 10 Heat-map presentation of ELISA of titers of human antibodies to HA and NA (geometric mean values), separated by age group. A) Geometric mean anti-ha titers of 18-20 year olds (n=30). B) Geometric mean anti-na titers of 18-20 year olds (n=30). C) Geometric mean anti-ha titers of 33-44 year olds (n=30). D) Geometric mean anti-na titers of 33-44 year olds (n=30). E) Geometric mean anti-ha titers of 49-64 year olds (n=30). F) Geometric mean anti-na titers of 49-64 year olds (n=30).
Supplementary Figure 11 Serum-transfer experiment. For each virus challenge, serum samples of 30 individuals per age group were pooled, sterile filtrated and 250 l of serum intraperitoneally injected into 10 mice for each group. An equal number of mice received immunoglobulin depleted serum as a negative control group. Two hours after the serum transfer, mice were anesthetized and infected with 1 10 5 PFU of virus diluted in PBS (50 l total volume) intranasally. Five mice each were euthanized on days 3 and 6, their lungs extracted and viral titers measured by plaque assay.
Supplementary Tables Suppl. Table 1: Characteristics of the naturally infected individuals Patient Age Gender Subtype* Diagnostic Sera Technique (d.p.i.) P2-2011 46Y M H1N1pdm09 HI/qRT-PCR 1/21 P3-2011 12Y M H1N1pdm09 HI/qRT-PCR 1/21 P9-2011 34Y M H1N1pdm09 qrt-pcr 1/21 P10-2011 73Y F H1N1pdm09 HI 1/21 P12-2011 21Y F H1N1pdm09 HI/qRT-PCR 1/21 P13-2011 58Y F H1N1pdm09 qrt-pcr 1/21 P18-2011 45Y M H1N1pdm09 qrt-pcr 1/21 P3-2012 15Y F H3N2 HI/qRT-PCR 1/21 P4-2012 75Y M H3N2 HI 1/21 P18-2012 72Y F H3N2 RT-PCR 1/21 P20-2012 97Y M H3N2 HI/RT-PCR 1/21 P24-2012 75Y M H3N2 HI 1/28 P25-2012 72Y F H3N2 HI/RT-PCR 1/28 P36-2012 1M M H3N2 Seq 1/28 P38-2012 82Y F H3N2 HI 1/28 P13-2013 34Y M H1N1pdm09 HI/qRT-PCR 1/28 P20-2013 6Y M H1N1pdm09 qrt-pcr 1/28 P27-2013 5Y M H3N2 HI/RT-PCR 1/28 P34-2013 88Y F H3N2 HI 1/28 *Strains were subtyped by RT-PCR and/or HI, Y =Years; M = Months; F =Female; M = Male; d.p.i.: day post infection.
Suppl. Table 2: Virus strains used in this study Abbreviation Subtype HA NA Backbone NL09 H1N1 A/Netherlands/602/09 A/Netherlands/602/09 A/Netherlands/602/09 NC99 H1N1 A/New Caledonia/20/99 A/New Caledonia/20/99 A/New Caledonia/20/99 H1N8 H1N8 A/New A/mallard/Sweden/50/02 A/PR/8/34 Caledonia/20/99 Phil82 H3N2 A/Philippines/2/82 A/Philippines/2/82 A/PR/8/34 Vic11 H3N2 A/Victoria/361/11 A/Victoria/361/11 A/Victoria/361/11 H3N8 H3N8 A/Philippines/2/82 A/mallard/Sweden/50/02 A/PR/8/34 H4N8 H4N8 A/duck/Czech/56 A/mallard/Sweden/50/02 A/PR/8/34 H5N8 H5N8 A/Vietnam/1203/04* A/mallard/Sweden/50/02 A/PR/8/34 H7N8 H7N8 A/Shanghai/1/13 A/mallard/Sweden/50/02 A/PR/8/34 H9N4 H9N4 A/guinea fowl/hk/wf10/99 A/mallard/Sweden/24/02 A/PR/8/34 * multibasic cleavage site was removed, low pathogenicity virus
Suppl. Table 3: Recombinant HA and NA proteins used in this study Subtype Abbreviation Strain Name From subtype N-linked glycosylation sites* H1 PR8 A/PR/8/34 H1N1 6 H1 1918 A/South Carolina/1/18 H1N1 6 H1 NC99 A/New Caledonia/20/99 H1N1 9 H1 Cal09 A/California/04/09 H1N1 7 H1 FM47 A/Fort Monmouth/1/47 H1N1 7 H1 swjian11 A/swine/Jiangsu/40/11 H1N1 6 H2 Jap57 A/Japan/305/57 H2N2 6 H2 maneth99 A/mallard/Netherlands/5/99 H2N9 7 H3 HK68 A/Hong Kong/1/68 H3N2 7 H3 Phil82 A/Philippines/2/82 H3N2 9 H3 Vic11 A/Victoria/361/11 H3N2 13 H3 Indi11 A/Indiana/10/11 H3N2 8 H3 semass11 A/harbor seal/massachusetts/1/11 H3N8 7 H3 cntexa04 A/canine/Texas/1/04 H3N8 7 H4 duczec56 A/duck/Czech/68 H4N6 5 H5 VN04 A/Vietnam/1203/04 H5N1 8 H5 Indo05 A/Indonesia/5/05 H5N1 9 H6 maswed02 A/mallard/Sweden/81/02 H6N1 7 H7 SH13 A/Shanghai/1/13 H7N9 5 H7 ckjali12 A/chicken/Jalisco/12283/12 H7N3 5 H8 maswed02 A/mallard/Sweden/24/02 H8N4 7 H9 gfhoko99 A/guinea fowl/hk/wf10/99 H9N2 9 H10 malnal10 A/mallard/Interior H10N7 6 Alaska/10BM01929/10 H11 shneth99 A/shoveler/Netherlands/18/99 H11N9 7 H12 malnal07 A/mallard/Interior H12N5 9 Alaska/7MP0167/07 H13 guswed99 A/black headed gull/sweden/1/99 H13N6 7 H14 maastr82 A/mallard/Gurjev/263/82 H14N5 7 H15 swweau79 A/shearwater/West Australia/2576/79 H15N9 7 H16 guswed99 A/black headed gull/sweden/5/99 H16N3 7 H17 btguat10 A/yellow shouldered H17N10 6 bat/guatemala/060/10 H18 btperu10 A/bat/Peru/33/10 H18N11 7 N1 Cal09 A/California/04/09 H1N1 N1 VN04 A/Vietnam/1203/04 H5N1 N1 Texa91 A/Texas/36/91 H1N1 N2 Sing57 A/Singapore/1/57 H2N2 N2 HK68 A/Hong Kong/1/68 H3N2 N2 Vic11 A/Texas/50/12 H3N2 N3 swmiss06 A/swine/Missouri/4296424/06 H2N3 N4 maswed02 A/mallard/Sweden/24/02 H8N4 N5 maswed03 A/mallard/Sweden/86/03 H12N5 N6 maneth99 A/mallard/Netherlands/1/99 H4N6 N7 macali10 A/mallard/Interior Alaska/10BM01929/10 H10N7
N8 maswed02 A/mallard/Sweden/50/02 H3N8 N9 Anhu13 A/mallard/Sweden/36/03 H11N9 N10 N10 A/yellow shouldered H17N10 bat/guatemala/060/10 B HA Yam88 B/Yamagata/16/88 B 8 B NA Yam88 B/Yamagata/16/88 B *Predicted for the HA ectodomain using NetNGlyc 1.0 server
Suppl. Table 4: Characteristics of the three age cohorts Age group Sex Race 18-20 33-44 49-64 M 16 (53.3%) Black 9 (30%) F 14 (46.7%) Caucasian 17 (56.7%) Hispanic 4 (13.3%) M 15 (50%) Black 7 (23.3%) F 15 (50%) Caucasian 18 (60%) Hispanic 5 (16.7%) M 15 (50%) Black 6 (20%) F 15 (50%) Caucasian 24 (80%) Hispanic 0 (0%)