DNA priming prior to inactivated influenza A(H5N1) vaccination expands the antibody epitope repertoire and increases affinity maturation in a boost-interval-dependent manner in adults

Abstract

DNA priming improves the response to inactivated influenza A(H5N1) vaccination. We compared the immunogenicity of an H5 DNA prime (using strain A/Indonesia/5/2005) followed by an H5N1 monovalent inactivated vaccine boost at 4, 8, 12, 16, or 24 weeks to that of 2 doses of H5N1 monovalent inactivated vaccine in adults. Antibody epitope repertoires were elucidated by genome-fragment phage-display library analysis, and antibody avidities for HA1 and HA2 domains were measured by surface plasmon resonance. H5 DNA priming expanded the H5-specific antibody epitope repertoire and enhanced antibody avidity to the HA1 (but not the HA2) domain in an interval-dependent manner. Enhanced HA1 binding and avidity after an interval of ≥12 weeks between prime and boost correlated with improved neutralization of homologous and heterologous H5N1 strains. Clinical trials registration NCT01086657.

Keywords: H5N1; Phage display; SPR; affinity; antibody; epitope; hemagglutinin; immune response; influenza; vaccine; virus.

Figures

Figure 1.

H5 hemagglutinin (HA) DNA priming significantly expands the antibody epitope repertoire in HA1 generated following receipt of an A/Indonesia/5/2005(H5N1) monovalent inactivated vaccine (MIV) boost. A, Schematic alignment of the peptides recognized by sera obtained after H5 DNA priming and H5N1 MIV boosting, as identified by panning of H5 genome-fragment phage-display libraries with A/Indonesia/5/2005(H5N1). The amino acid designation is based on the A/Indonesia/5/2005(H5N1) HA protein sequence (Supplementary Figure 1). Bars indicate identified inserts in HA1 (red bars) and HA2 (blue bars). Phage displaying peptides from sequences in the receptor-binding domain are depicted with green bars within HA1 segment. The thickness of each bar represent the frequencies of repetitively isolated phage inserts (only clones with a frequency of ≥2 are shown). B, Distribution of phage clones in different HA domains after affinity selection with sera obtained from adults after MIV-prime, MIV-MIV boost, DNA-prime, and DNA-MIV boost is shown. Abbreviation: MN, microneutralization.

Figure 2.

H5 DNA priming enhance antibody affinity (slower off rates) to H5N1 HA1 (but not HA2) with an interval of 12–24 weeks between H5 DNA prime and monovalent inactivated vaccine (MIV) boost. Surface plasmon resonance (SPR) analysis of human sera obtained after vaccination from 6 prime-boost groups (10–11 individuals per group) in the vaccine trial was performed with properly folded functional oligomeric H5N1 HA1 (A) and H5N1 HA2 (B) domains from the A/Indonesia/5/2005 strain. Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the serum sample interaction with rHA1 (1–330) and rHA2 (331–480), using SPR in the dissociation phase. Serum antibody off-rate constants (each symbol represents 1 individual) were determined as described in Materials and Methods. The off-rate constants were determined from two independent SPR runs. *P < .05 and **P < .005, by the Student t test, for differences between mean off-rate constants for human sera obtained after vaccination. D-M, H5 DNA prime, MIV boost; M-M, MIV prime, MIV boost. C and D, A/Indonesia/5/2005(H5N1) responders (microneutralization [MN] titer, ≥ 40) have significantly higher antibody affinity to rHA1 (but not rHA2) than nonresponders (MN titer, < 40). Antibody off-rate constants from the serum sample interaction with rHA1 protein (C) and rHA2 (D), determined using SPR analysis, for all of the individuals in 6 prime-boost groups following prime-boost vaccination were analyzed for A/Indonesia/5/2005(H5N1) responders (MN titer, ≥ 40) and compared to the antibody affinity to rHA1 for nonresponders (MN titer, < 40). The mean off-rate constants of human sera obtained after vaccination were statistically significant between responders and nonresponders only in the HA1 domain, not in the HA2 domain (P < .05, by the Student t test). The off-rate constants were determined from 2 independent SPR runs. E and F, Serum antibody off rates for H5 A/Indonesia/5/2005(H5N1) rHA1 but not HA2 after prime-boost are strongly correlated with the in vitro neutralizing capacity against the homologous A/Indonesia/5/2005(H5N1) strain. Antibody off-rate constants were determined directly from the plasma sample interaction with rHA1 (1–330) or rHA2, using SPR in the dissociation phase. SPR analysis of human sera obtained after boosting from all vaccine groups included in the vaccine trial was performed with rHA1 (E) or rHA2 (F) of the A/Indonesia/5/2005(H5N1) strain. Each symbol represents 1 individual. Antibody affinity of human sera obtained after H5N1 vaccination against HA1 of A/Indonesia/5/2005(H5N1) correlated with homologous A/Indonesia/5/2005(H5N1) MN titers.