Broad immunity and protection from challenge in vaccinated mice.
Eight groups of susceptible BALB/c mice (10 mice/group) (
24) were immunized intramuscularly with 5 × 10
10 viral particles of Ad.VNHA, Ad.VNHA1, Ad.HKHA1, or empty vector Ad.ψ5 and boosted 14 days later (experiment 1). Additional groups of mice were similarly vaccinated and boosted with Ad.VNHA, Ad.VNHA1, Ad.VNHA2, or empty vector Ad.ψ5 (experiment 2). Serum samples were obtained from all mice to screen for antibody responses as a marker of immunogenicity (
18). On week 10, 8 weeks after the booster immunization, high titers of H5-specific antibodies were detected in all vaccinated animals except for those in the Ad.VNHA2 group, which had titers more than 3 orders of magnitude lower than those of all other vaccinated groups (Fig.
1a). We then determined the degree to which antibody responses could neutralize homologous VN/1203/04 and heterosubtypic HK/156/97 influenza virus strains by using the horse red blood cell HI assay (
32). Vaccination with full-length HA induced homologous and heterotypic antibody responses, whereas vaccination with Ad.VNHA1 or Ad.HKHA1 induced primarily antibody responses specific to the vaccinating strain (Fig.
1b). The kinetics of serum HI responses suggest that a single immunization may be sufficient to achieve a high-level anti-HA antibody response (Fig.
1c).
Given that vaccination induced various degrees of humoral immunity, with the Ad.VNHA2-immunized group having a markedly reduced H5-specific antibody response, we next analyzed the cellular immune response to vaccination after an additional boost immunization using the IFN-γ ELISPOT assay for two mice per group. Overlapping 15-mer peptides representing the entire VN/1203/04 HA protein and nonconserved sequences of HK/156/97 were pooled to evaluate the strength and breadth of immunity. Individual epitope-containing peptides were then identified through analysis of matrices in which each peptide was represented by two pools (
6). All animals immunized with full-length HA or the HA1 or HA2 subunits developed strong cellular responses to HA peptides, reaching an average peak intensity of one HA-specific T cell per 1,200 freshly isolated splenocytes in the Ad.VNHA group (Fig.
2a). Cumulative cellular immune responses were HA region specific, with only the animals immunized with full-length HA developing T-cell responses spanning both HA1 and HA2 (Fig.
2a). Detailed characterization of vaccine-induced immune responses identified both conserved and unique peptide targets (Fig.
2b). As expected, cellular responses against the conserved HA1 regions VN
213-227 and VN
241-255 were elicited regardless of HA1 immunization strain, whereas responses to peptides spanning the regions from amino acid 145 to amino acid 163, which differed between VN/1203/04 and HK/156/97, were limited to animals immunized with the respective subtype (Fig.
2b). Ad.VNHA2 immunization revealed the presence of an immunodominant epitope within HA2 represented by VN
529-543/VN
533-547 peptides. Immunization with Ad.VNHA induced a subdominant response to the previously identified SFFRNVVWLIKK epitope (
17,
19) contained within the HA1 peptides VN
153-167/VN
157-171. Immunization with Ad.VNHA altered the nature of HA1-specific immune responses seen when Ad.HA1 was the sole immunogen, generating more-modest responses to VN
145-159/VN
149-163, VN
213-227, and VN
241-255 that were subdominant to the responses to VN
529-543/VN
533-547 (Fig.
2b). These data demonstrate that adenovirus-based vaccination generates robust cellular immune responses to HA, which in the case of HA2 vaccination appear to be dominant to the humoral immune response.
Eight weeks after the second immunization, all mice were challenged by intranasal inoculation with 100 LD
50s of VN/1203/04. All animals immunized with control Ad.ψ5 vector experienced substantial weight loss beginning at day 3 postchallenge and death by 6 to 9 days postchallenge. In contrast, all animals immunized with Ad.VNHA, Ad.VNHA1, or Ad.HKHA1 showed only mild and transient loss of body weight and survived the lethal challenge (Fig.
3a and b). All animals immunized with Ad.VNHA2 experienced substantial weight loss, but three out of five animals in this group regained weight after day 8 and recovered fully (Fig.
3a and b). This recovery is notable given that vaccination with HA2 induced primarily cellular immune responses, which previously have been associated only with enhanced viral clearance and recovery from influenza infection (
26). At day 3 or day 6 postchallenge, three animals per group were sacrificed for virus isolation. Infectious virus was isolated from multiple organs in the control-vaccinated group and to various degrees from animals vaccinated with HA1 or HA2 subunits. In contrast, virus was isolated at extremely low levels on day 3 postinfection (log
10 mean virus titer, 0.5; experiment 1) and not at all on day 6 postinfection (log
10 mean virus titer, ≤0.5; experiment 2) from organs from mice vaccinated with full-length HA (Fig.
3c).
Efficacy of vaccination in chickens.
Following the encouraging responses to vaccination and challenge in the murine model, we evaluated the efficacy of adenovirus-based vaccination in domestic chickens, given the critical role that fowl species play in the spread of HPAI in Southeast Asia (
8). The severity of H5N1 infection in chickens differs from that in mice, as chickens rarely survive past the second day after challenge, whereas the median survival period in naive mice is 8 days. We restricted our experiment to vaccination using full-length HA, given the superior protection noted in mice immunized with this vaccine. Four groups of 10 3-week old chickens received one immunization intranasally or subcutaneously with 5 × 10
10 viral particles of Ad.VNHA or empty vector Ad.ψ5 and were challenged with an intranasal inoculation of 10
6 EID
50s of VN/1203/04 21 days later (at 6 weeks of age). This virus dose was 10,000-fold higher than that given to mice and likely represented a challenge significantly greater than that experienced by chickens in a natural outbreak. The vaccination induced HI antibodies to VN/1203/04 in all chickens belonging to the subcutaneous immunization group whose members were boosted upon virus challenge. All animals in this group survived challenge with no detectable clinical signs of disease (Table
1). In contrast, all control-immunized chickens died (median survival period of 1.8 days). Only one of the chickens immunized with Ad.VNHA intranasally had HI antibodies, while, as a group, the chickens experienced 50% morbidity and 50% mortality following challenge (Table
1). The poorer protection afforded by intranasal immunization may reflect an infection by adenovirus serotype 5 via this route that is limited in comparison to that via the subcutaneous route. Oral and cloacal measurements of virus titers showed that subcutaneously administered vaccine greatly reduced replication of the challenge virus such that virus could not be detected in the gastrointestinal tract and levels were reduced by 3 orders of magnitude in the respiratory tract (Table
1).