Anamnestic broadly reactive antibodies induced by H7N9 virus more efficiently bind to seasonal H3N2 strains

Figures & data

Table 1. Subject characteristics.

Subject ID	Birth year	Gender	Race	Received influenza vaccination
S1	1969	M	White	Yes
S2	1964	M	White	Yes
S3	1979	F	White	Yes
S4	1969	F	Yellow	NO
S5	1982	F	White	Yes
S6	1981	M	Black	Yes
S7	1971	M	White	Yes
S8	1983	F	White	Yes
S9	1963	F	White	Yes
S10	1990	F	White	Yes
S11	1979	F	White	Yes
S12	1953	F	White	Yes
S13	1957	F	White	Yes

F=Female, M=Male.

Table 2. mPLEX-Flu hemagglutinin panel.

Strain	Gene Bank Accession	Full Strain Name	Abbreviation
H1N1	AF117241.1	A/South Carolina/01/1918	A/SC18
H1N1	CY148243.1	A/Puerto Rico/8/1934	A/PR8
H1N1	DQ508897.1	A/USSR/1977	A/USSR77
H1N1	DQ508889.1	A/Texas/36/1991	A/Tex91
H1N1	CY125100.1	A/New Caledonia/20/1999	A/NewCal99
H1N1	FJ966974.1	A/California/07/2009	A/Cali09
H2N2	L20407.1	A/Japan/305/1957	A/Jap57
H3N2	CY112249.1	A/HongKong/1/1968	A/HK68
H3N2	CY009348.1	A/Port Chalmers/1/1973	A/PC73
H3N2	GQ293081.1	A/Perth/16/2009	A/Perth09
H3N2	KM821347	A/Victoria/361/2011	A/Vic11
H3N2	KC892248.1	A/Texas/50/2012	A/Tex12
H3N2	EPI_ISL_164719Ê	A/Switzerland/2013	A/swi13
H4N6	AB295611.1	A/duck/Czech/1956	A/duck/Czech56
H5N1	EF541403.1	A/Vietnam/1204/2004	A/Viet04
H5N1	EF541394.1	A/Indonesia/5/2005	A/Indo05
H6N1	KJ162860.1	A/Taiwan/2/2013	A/Taiw13
H7N1	EF470586	A/rhea/North Carolina/39482/1993	A/rhea/NC93
H7N3	KF695239	A/mallard/Netherlands/12/2000	A/malNet00
H7N9	KF021597	A/Shanghai/1/2013	A/SH13
H9N2	AY206676.1	A/guinea fowl/Hong Kong/F10/1999	A/gfHK99
B	FJ766842.1	B/Brisbane/60/2008	B/Bris08
B	KC892118.1	B/Massachustts/2/2012	B/Mass12
B	EPI_ISL_165882	B/Phuket/2013	B/Phu13
H5	Head	C52-C277 of A/Indonesia/5/2005	H5 Head
cH5/1	Chimera	Head of A/Indo05, stalk of A/PR34	cH5/1PR8
cH5/1	Chimera	Head of A/Indo05, stalk of A/Cali09	cH5/1Cal09
cH5/3	Chimera	Head of A/Indo05, stalk of A/A/Perth09	cH5/3
cH4/7	Chimera	Head of A/duck/Czech/1956, stalk of A/SH13	cH4/7

Figure 1. Stalk-reactive IgG in human plasma. Plasma was obtained from 13 donors, 12 of which had been previously vaccinated within the past 5 years with seasonal influenza vaccines. Plasma baseline anti-influenza IgG was assessed by mPlex-Flu assay (n = 13). (a) levels of IgG against historic outbreak and recently circulating H3N2 and H1N1 strains. Each column depicts median fluorescence intensity (MFI), representing an individual donor. (b) levels of H7N1-, H7N3- and H7N9-reactive IgG. (c) H1 and H3 stalk-reactive IgG. (d) IgG-binding to cH5/3 and cH4/7 proteins. Each symbol and line represents one donor. Mann-Whitney two-tailed test was used to evaluate the difference among different groups. P < 0.05 was considered statistically significant. This analysis was performed at a 1:5,000 dilution.

Figure 2. Anti-H7 IgG secretion stimulated by inactivated A/SH13 viruses. Purified B cells from 13 donors were obtained by negative selection and stimulated with CpG₂₀₀₆ODN alone or together with IL-15 and A/SH13 (H7) BPL inactivated virus for 6 days in vitro. Supernatants were collected and the IgG-binding to A/rheaNC93 H7N1 (a), A/malNet00 H7N3 (b) and A/SH13 H7N9 (c) HA were measured. Each symbol and line represents one donor. Mann-Whitney two-tailed test was used to evaluate the difference among different groups. P < 0.05 was considered statistically significant.

Figure 3. IgG induced by H7N9 viruses in vitro cross-react with A/Vic11 and A/Cali09 HA. Purified B cells were obtained by negative selection and stimulated with CpG₂₀₀₆ODN alone or together with IL-15 and A/SH13 inactivated virus for 6 days. Supernatants were collected, and H1 and H3 HA-reactive IgG were detected using mPlex-Flu assay. (a) levels of IgG against A/SH13 HA. H7-binding antibodies in supernatants of activated B cells were monitored using beads bound with HA from H7N9 strains (n = 13), and compared with CpG+IL-15+H3 group (n = 10). (b) A/Vic11- and (c) A/Cali09-reactive IgG levels in CpG (n = 13), CpG+IL-15 (n = 10) and CpG+IL-15+H7 (n = 10) groups. (d) The levels of H7N9-induced H1, H3 and H7 antibodies were compared. Each symbol represents an individual donor. Mann-Whitney two-tailed test was used to evaluate the difference among different groups. P < 0.05 was considered statistically significant.

Figure 4. Cross-reactive IgG induced by A/SH13 and CpG₂₀₀₆ODN. B cells were obtained by negative selection, and then stimulated with CpG₂₀₀₆ODN alone or together with A/SH13 for 6 days. Cross-reactive antibodies binding to H1, H2, H5, H6 and H7 subtypes induced in the B cell cultures were measured by mPlex-Flu assay. (a) CpG₂₀₀₆ODN with H7 antigen induces a broad recall response to different influenza strains (n = 13). (b) fold change in cross-reactive antibodies. All values of IgG levels (MFI) were subtracted from the culture medium alone values before calculating fold change (n = 13). Each symbol represents the median of fold change in IgG levels. (c) Comparison between seasonal subtypes and the boosting strain.

Figure 5. IL-15 enhanced the cross-reactive IgG induced by A/SH13. (a) B cells from 13 donors were stimulated with CpG₂₀₀₆ODN, inactivated A/SH13 viruses and IL-15. Strain-specific and HA stalk-reactive IgG in supernatants of activated B cells were measured after 6 days (n = 13). (b) correlation between influenza specific antibodies and HA antigenic sequence (n = 13). (c) fold change in cross-reactive antibodies (n = 13). IL-15 increased the concentration of anti-HA reactive IgG.

Figure 6. Stalk-reactive IgG induced by A/SH13 viruses. B cells from healthy donors were stimulated with CpG₂₀₀₆ODN alone or together with inactivated A/SH13 (H7N9) viruses and IL-15 in vitro. The levels of IgG against H5 head and cH5/3 were shown for individual subjects. 12 of 13 subjects displayed increases in stalk-reactive IgG after A/SH13 stimulation. (a) levels of IgG binding to H5 head and cH5/3. (b) levels of cH5/3-reactive IgG after stimulation with CpG₂₀₀₆ODN alone or together with inactivated A/SH13 (H7N9) viruses. (c) Comparation between H3- and H1-, H7-reactive antibodies.