MF59 adjuvant enhances diversity and affinity of antibody-mediated immune response to pandemic influenza vaccines

Abstract

Oil-in-water adjuvants have been shown to improve immune responses against pandemic influenza vaccines as well as reduce the effective vaccine dose, increasing the number of doses available to meet global vaccine demand. Here, we use genome fragment phage display libraries and surface plasmon resonance to elucidate the effects of MF59 on the quantity, diversity, specificity, and affinity maturation of human antibody responses to the swine-origin H1N1 vaccine in different age groups. In adults and children, MF59 selectively enhanced antibody responses to the hemagglutinin 1 (HA1) globular head relative to the more conserved HA2 domain in terms of increased antibody titers as well as a more diverse antibody epitope repertoire. Antibody affinity, as inferred by greatly diminished (≥10-fold) off-rate constants, was significantly increased in toddlers and children who received the MF59-adjuvanted vaccine. Moreover, MF59 also improved antibody affinity maturation after each sequential vaccination against avian H5N1 in adults. For both pandemic influenza vaccines, there was a close correlation between serum antibody affinity and virus-neutralizing capacity. Thus, MF59 quantitatively and qualitatively enhances functional antibody responses to HA-based vaccines by improving both epitope breadth and binding affinity, demonstrating the added value of such adjuvants for influenza vaccines.

Fig. 1

Analysis of antibody repertoires elicited in adults after vaccination with unadjuvanted and adjuvanted subunit H1N1 vaccines. (A) Distribution of phage clones after affinity selection with sera obtained from adults before and after single vaccination with subunit SOIV-H1N1 vaccine (with and without MF59 adjuvant). (B) Schematic alignment of the peptides recognized by post-first H1N1 vaccination sera in the adults, identified by panning with H1N1-GFPDL A/California/07/2009. The amino acid designation is based on the HA protein sequence (fig. S1). Bars indicate identified inserts in HA1 (red bars) and HA2 (blue bars). The thickness of each bar represents the frequencies of repetitively isolated phage inserts (only clones with a frequency of two or more are shown; sequenced clones are shown in table S1). Phage-displaying peptides from sequences within the HA1 receptor binding domain (RBD) are depicted with yellow bars.

Fig. 2

Antibody epitope repertoire elicited in young children after vaccination with unadjuvanted and adjuvanted subunit H1N1 vaccines. (A) Distribution of phage clones after affinity selection on sera obtained from young children (3 to 8 years) before and after two immunizations with subunit SOIV-H1N1 vaccine (with and without MF59 adjuvant). (B) Schematic alignment of the peptides recognized by post-second H1N1 vaccination sera in the young children (3 to 8 years) as identified with H1N1-GFPDL A/California/07/2009 (sequenced clones are shown in table S1). Phage-displaying peptides from sequences within the HA1 RBD are depicted with yellow bars, whereas identified inserts in HA1 are shown as red bars and HA2 as blue bars. The thickness of each bar represents the frequencies of repetitively isolated phage inserts (only clones with a frequency of two or more are shown; sequenced clones are shown in table S1). The amino acid designation is based on the HA protein sequence (fig. S1).

Fig. 3

Antibody epitope profile elicited in toddlers after vaccination with an unadjuvanted or MF59-adjuvanted subunit SOIV-H1N1 vaccine. (A) Distribution of phage clones after affinity selection with sera obtained from toddlers (12 to 35 months) before and after two immunizations with subunit SOIV-H1N1 vaccine (with and without MF59 adjuvant). (B) Schematic alignment of the peptides recognized by post-second H1N1 vaccination sera in the toddlers, identified by panning with H1N1-GFPDL A/California/07/2009. Amino acid designation is based on the HA protein sequence (fig. S1). Bars indicate identified inserts in HA1 (red bars) and HA2 (blue bars). Phage-displaying peptides from sequences within the HA1 RBD are depicted with yellow bars. The thickness of bars represents the frequencies of repetitively isolated phage inserts (only clones with a frequency of two or more are shown; sequenced clones are shown in table S1).

Fig. 4

Binding of post-H1N1 vaccination human serum IgG to properly folded HA1 protein. (A to C) Steady-state equilibrium analysis of the binding of human vaccine serum IgG to properly folded functional HA1 oligomers was measured with SPR and is shown for two representative samples from each group. Tenfold-diluted individual post-H1N1 vaccination sera from the three age groups [adults in (A), young children in (B), and toddlers in (C)] of the H1N1 vaccine trial were injected simultaneously onto HA1 immobilized on a sensor chip through the free amine group and onto a blank flow cell, free of peptide. Prevaccination serum (VN < 20) is included (black) and was used as control in each assay. The VN titers of each serum used in SPR are in parentheses. Binding of the antibodies to the immobilized protein is shown as resonance unit (RU) values. (D) Maximum RU values for HA1 binding by serum antibodies obtained from multiple individuals before and after vaccination with either MF59-adjuvanted (red dots) or unadjuvanted SOIV-H1N1 (blue dots) vaccination for the three age groups.

Fig. 5

Human serum IgG avidity after immunization with an MF59-adjuvanted and unadjuvanted H1N1 subunit vaccine in different age groups. H1N1-HA1–specific affinity of IgG from post-H1N1 vaccination human sera after 7 M urea wash in individuals with either MF59-adjuvanted or unadjuvanted SOIV-H1N1 vaccination for the three age groups is shown (n = 5 to 7). Data are means ± SD of three independent experiments. Differences between groups were examined for statistical significance with Student’s t test. A P value of <0.05 was considered to be significant.

Fig. 6

Affinity measurements and correlation between in vitro neutralizing capacity and off-rate constant in human sera after immunization with an MF59-adjuvanted and unadjuvanted H1N1 subunit vaccine in different age groups. (A) Antibody avidity measurements in polyclonal serum by off-rate constants with SPR. Antibody off-rate constants that describe the stability of the complex, which is the fraction of complexes that decays per second, were determined directly from the serum-plasma sample interaction with rHA1 protein using SPR in the dissociation phase. For accurate measurements, parallel lines in the dissociation phase for the 10- and 100-fold dilution for each post-vaccination human sera were required. The off-rate constants were determined from two independent SPR runs. (B to G) SPR analysis of post-vaccinated human sera with MF59-adjuvanted (C, E, and G) or unadjuvanted SOIV-H1N1 (B, D, and F) vaccine from three age groups of the vaccine trial was performed with properly folded SOIV-H1N1 HA1 (A/California/07/2009) (21). Serum antibody off-rate constants and serum-neutralizing antibody titers for different individual vaccinees (each symbol is one individual) were determined as described in Materials and Methods. Neutralizing titer is expressed as standardized end-point neutralizing antibody titer of post-H1N1 vaccine human sera. Antibody off-rate constants of human sera after vaccination with MF59-adjuvanted or unadjuvanted SOIV-H1N1 vaccine correlated with their in vitro neutralizing capacity. Correlation statistics of the affinity measurement and off-rate constants of the human sera between MF59-adjuvanted and unadjuvanted vaccine groups were statistically significant only for the toddlers’ samples (12 to 35 months) and young children (3 to 8 years), with P < 0.05 (t test).

Fig. 7

Kinetics of antibody affinity maturation to HA after multiple immunizations with an MF59-adjuvanted and unadjuvanted H5N1 subunit vaccine and its correlation with in vitro VN. (A to F) Sequential SPR analysis of vaccine sera (after the first, second, and third vaccination with unadjuvanted or MF59-adjuvanted H5N1 vaccine) was performed with properly folded H5N1 HA1 (A/Vietnam/1203/2204) (13, 47). Tenfold-diluted individual sera from three arms of the NVD vaccine trial at 28 days after each immunization were evaluated. After binding of the sera to the immobilized ligand, antibody off-rate constants were calculated with a heterogeneous sample model as described in Materials and Methods. Values on the x axis denote the end-point VN titers (mean of three replicates) with individual sera in a VN assay performed with A/Vietnam/1203/2004-rgH5N1×PR8 reassorted virus. Data plotted are shown for four individuals from the unadjuvanted arm (A, C, and E) and six individuals from the MF59- adjuvanted H5N1 vaccine arm (B, D, and F) in the NVD trial after first, second, and third immunizations. To follow the serum VN titer and corresponding polyclonal serum off-rate constants after every vaccination, the values for each individual vaccinee are depicted with the same colored symbol after the first, second, and third immunization in the three corresponding figures of each arm. Statistical analysis of the off-rate constants between MF59-adjuvanted and unadjuvanted vaccine groups after each vaccination showed statistical significance betweenMF59-adjuvanted and unadjuvanted vaccine groups only for samples after the third immunization, with P<0.05 (t test). (G)H5N1-HA1–specific affinity of serum IgG after 7 M urea wash in individuals after the third vaccination with either MF59-adjuvanted or unadjuvanted H5N1 subunit vaccine (n = 6). Data are means ± SD of three independent experiments. Differences between groups were examined for statistical significance with Student’s t test. A P value of <0.05 was considered to be significant.