Figure 1.

Adjuvanted H5N1 vaccine rapidly elicited strain specific and cross-reactive neutralizing antibody responses. (A) An Illustration of the study design. Sixty adults (20 to 49 years old) were enrolled in this study and vaccinated with inactivated virosomal H5N1 vaccine (Crucell| Berna Biotech, Switzerland). The adults were randomized into 4 groups of 15 subjects and received two doses of H5N1 vaccine with 30 μg hemagglutinin (HA) alone (30μg -), or 1.5, 7.5 and 30 μg HA adjuvanted with Matrix M (1.5 μg +, 7.5 μg +, and 30 μg +, respectively), at an interval of 21 days. Serum samples pre- (day 0), 7 days, 21 days, 28 days, and 42 days post-vaccination were collected. One subject withdrew after first dose vaccine in 7.5μg + group. (B, C) Hemagglutination inhibiting titer (HI titer, B) and fold-induction (FI) after vaccination (HI FI,/D0, C) was measured against NIBRG-14 virus (vaccine strain). (D) Phylogenetic tree shows the genetic divergence between H5HA from the vaccine strain and heterologous H5HAs tested in pseudotype-based neutralization (PN) assay. H1HA from A/Puerto Rico/8/1934 was used as a reference. Phylogenetic analyses were performed at ngPhylogeny.fr. (E, F) Vaccine specific neutralizing antibodies were measured in PN assay against pseudotyped virus derived from A/Vietnam/1203/2004 virus (Vietnam). The PN titer (IC₈₀, E) was calculated as the reciprocal dilutions of the sera that gave 80% inhibition. Fold-induction after vaccination (PN FI,/D0, F) is shown. (G) Cross-neutralizing antibody titers (IC₈₀) were measured against 5 strains of pseudotypes expressing heterologous H5HAs listed in D All antibody responses were measured using serum samples before (D0) and after vaccination (D7, D21, D28, and D42). The geometric mean values are shown as bars, and each symbol represents one subject (B, E, G). The geometric mean of fold-inductions in each group ± geometric standard deviation as error bar is shown (C, F). *P < 0.05, **P < 0.01, ***P < 0.001 (Antibody titers and fold-inductions were Ln transformed in statistical analyses. Turkey’s multiple comparisons between pre-prime (D0) and post-prime (D7, D21, D28, D42), and between pre-boost (D21) and post-boost (D28 and D42) in each group were performed in two-way ANOVA in (B, E, G) Turkey’s multiple comparisons between 30 μg + and 1.5 μg + after prime (D7 and D21), and between 30 μg + and 30 μg - after boost (D28 and D42) were performed in two-way ANOVA in C, F). The horizontal dotted lines indicate HI titer of 32 (B), and fold-induction of 1 (C, F). Duplicates were performed in all experiments.