Feasibility of reconstructed ancestral H5N1 influenza viruses for cross-clade protective vaccine development

Abstract

Since the reemergence of highly pathogenic H5N1 influenza viruses in humans in 2003, these viruses have spread throughout avian species in Asia, Europe, and Africa. Their sustained circulation has resulted in the evolution of phylogenetically diverse lineages. Viruses from these lineages show considerable antigenic variation, which has confounded vaccine planning efforts. We reconstructed ancestral protein sequences at several nodes of the hemagglutinin (HA) and neuraminidase (NA) gene phylogenies that represent ancestors to diverse H5N1 virus clades. By using the same methods that have been used to generate currently licensed inactivated H5N1 vaccines, we were able to produce a panel of replication competent influenza viruses containing synthesized HA and NA genes representing the reconstructed ancestral proteins. We identified two of these viruses that showed promising in vitro cross-reactivity with clade 1, 2.1, 2.2, 2.3.4, and 4 viruses. To confirm that vaccine antigens derived from these viruses were able to elicit functional antibodies following immunization, we created whole-virus vaccines and compared their protective efficacy versus that of antigens from positive control, naturally occurring, and broadly reactive H5N1 viruses. The ancestral viruses' vaccines provided robust protection against morbidity and mortality in ferrets challenged with H5N1 strains from clades 1, 2.1, and 2.2 in a manner similar to those based on the control strains. These findings provide proof of principle that viable, computationally derived vaccine seed viruses can be constructed within the context of currently licensed vaccine platforms. Such technologies should be explored to enhance the cross reactivity and availability of H5N1 influenza vaccines.

Fig. 1.

Phylogenetic relationship of influenza A viruses used to calculate putative ancestral sequences. Four nodes on the HA gene (a) were chosen to represent (A) the midpoint root of the tree; (B) the ancestral node common to clades 1, 2.1, 2.2, 2.3, 2.4, 2.5, and 8; (C) the ancestral node to clade 1 viruses; and (D) the ancestral node for all clade 2 viruses. For the NA gene (b), two nodes were predicted, one at the root of the phylogeny (r) and another at the common ancestor for all NA genes with a 20-aa deletion in the stalk region (d). Numbers at branch nodes indicate neighbor-joining bootstrap values of at least 70% for major clades. Analyses were based on nucleotides 1 to 1,707 and 1 to 1,407 of the HA and NA genes, respectively. The HA tree was rooted to A/goose/Guangdong/1/96 and the NA tree was midpoint rooted. Numbers to the right of the figure refer to WHO H5N1 clade designations (5). (Scale bar, 0.001 substitutions per site.) Viruses used in the present study are in red font.

Fig. 2.

Ferrets’ survival rates after infection with VN1203 (A), DKHUN795 (B), and TYEGY7 (C). Control (mock-vaccinated) ferrets are represented by red open squares. Ferrets vaccinated with ancestral strains are represented by closed blue triangles: A in light blue and D in dark blue. Ferrets vaccinated with positive control isolate-based strains are represented by green closed diamonds: VN1203 in light green and DKHUN795 in dark green.

Fig. 3.

Ferrets’ nasal wash titers after infection with VN1203 (A), DKHUN795 (B), and TYEGY7 (C). The mean virus titers of nasal washes for the four animals per group (same vaccine and same challenge virus) are expressed in log₁₀ EID₅₀/mL ± SEM (st er). Titers are grouped by vaccination regimen on the x axis. Black, white, and gray filled bars represent titers 3, 5, and 7 d after infection, respectively. (*P ≤ 0.05 and ***P ≤ 0.001 by ANOVA comparing the five vaccine regimen groups virus titers.) Slashes separate ANOVA P values at days 3, 5, and 7. [^#All mock-vaccinated (control) VN1203-infected ferrets were dead before nasal washes could be collected at days 5 and 7 after infection.]