The Cre-recombinase recombinase-mediated site-specific recombination system17
was used to construct a replication-defective, recombinant human Ad vector expressing H5 (HAd-H5HA). This vector carries the full-length coding region of the H5 gene of the avian H5N1 influenza virus (A/Hong Kong/156/197 [HK/156/97]) inserted into the early region (E) 1 of the human Ad genome (webfigure 1, A
under the control of cytomegalovirus immediate early promoter, and the H5 gene was expressed efficiently in human embryonic kidney cells (webfigure 1, B
). A human Ad genome with deletions of the E1 and E3 regions (HAd-ΔE1E3) served as a negative control.19
To determine whether the Ad vector H5 vaccine was immunogenic and conferred protection against lethal challenge with a homotypic H5N1 virus, 10-week-old BALB/c mice (five per group) were immunized intramuscularly with 1×108
plaque-forming units (pfu) of HAd-H5HA vaccine or HAd-ΔE1E3. This pfu is equivalent to a 1.2×1010
virus particle number, which is an optimum dose for the induction of protective immune response (webfigure 2
Doses were given twice at a 4-week interval. Another group of animals was inoculated with phosphate-buffered saline (PBS) as a negative control. Two additional groups of mice were immunized to compare the human Ad vaccine strategy with a traditional subunit vaccine approach used in a previous clinical trial,10
the findings of which indicated that a baculovirus-expressed recombinant H5 vaccine (rH5HA), based on the HK/156/97 virus, is modestly immunogenic even after two doses of 90 µg of vaccine. These mice were immunized twice at 4 weeks apart with 3 µg of rH5HA, either in the presence or absence of 1% alum adjuvant. Serum samples were obtained 3 weeks after the second intramuscular immunization from all five groups, to monitor the development of haemagglutinin-specific antibodies by haemagglutination inhibition (HI) assays21
using horse red blood cells and virus neutralization assays before challenge.22
These serological assays determine the concentration (titre) of haemagglutinin-specific antibodies that bind to haemagglutinin on the virus and prevent the agglutination of red blood cells (HI assays) or infection of Madin-Darby canine kidney (MDCK) cells (virus neutralization assays) in vitro
Since HK/156/97 virus is not lethal to mice, to assess protection, we have used the A/Hong Kong/483/97 (HK/483/97) virus for challenges since it is not only genetically and antigenically similar to HK/156/97, but also lethal to mice.
The efficacy of vaccine was determined by challenging all five groups of mice with 100×50% lethal dose (LD50
), which is equivalent to 103–7
(egg infectivity dose; the amount of virus needed to kill 50% of fertilized chicken embryos) of H5N1 HK/483/97 virus, 4 weeks after the second immunization.18, 20
Mice were monitored for clinical signs and changes in bodyweight every day for 14 days postchallenge. The maximum mean weight loss was the weight loss on day 6 postchallenge.
We investigated whether the serological responses induced by HAd-H5HA vaccine would cross-react with more recent H5N1 viruses isolated from human beings, and whether the route of inoculation (systemic vs mucosal) would affect vaccine immunogenicity. BALB/c mice (20 per group) were immunized either intramuscularly or intranasally with 1×108 pfu of HAd-H5HA twice every 4 weeks. Other groups of mice (20 per group) were intramuscularly immunized with 108 pfu of HAd- Δ E1E3 or 3 µg of rH5HA with alum. The serum samples were obtained 4 weeks after the second immunization and analyzed by virus neutralization assays to assess their ability to react with a homologous virus (HK/156/97), or with antigenically heterologous viruses (A/Hong Kong/213/2003 [HK/213/03] and A/Vietnam/1203/04 [VN/1203/04]). Compared with HK/156/97, the aminoacid homology in the haemagglutinin subunit is 94.8% for HK/213/03 and 95.5% for VN/1203/04.
In addition to neutralizing antibody responses against haemagglutinin, CD8 T cell responses have been shown to contribute to viral clearance.23,24
Although the genome of influenza A virus has many potential epitopes that could generate CD8 T cell responses, antiviral CD8 T cell responses are actually restricted to only a few epitopes. Furthermore, some epitopes seem to generate a robust response whereas others tend to generate a weaker response leading to a hierarchy, known as immuno-dominance. CD8 T cell responses to nucleoprotein (NP) 147 are dominant followed by haemagglutinin (HA) 518 responses in influenza-virus-infected BALB/c mice.
HA 518 was originally described for the haemagglutinin of an H1N1 virus, A/Puerto Rico/8/34 (PR/8/34). This epitope is conserved in all three H5N1 viruses used in this study, including currently circulating avian and human H5N1 viruses, and some H9N2 viruses (webtable
The HA 518 epitope has also been shown to be the dominant epitope in H5N3-infected animals.26
To determine whether HAd-H5HA vaccine induced functional CD8 T cells, mice (nine per group) were inoculated intramuscularly or intranasally twice every 4 weeks with 108 pfu of HAd-H5HA. Similarly, groups of mice were immunized with 108 pfu of HAd- ΔE1E3 (as a negative control) or 3 µg of rH5HA with alum. An additional group was inoculated intraperitonally with one dose of 500 haemagglutination units (HAU) of HK/213/03 virus as a positive control. Splenocytes from the immunized animals were stained with a murine MHC-encoded allele Kd -specific pentamer for the immunodominant HA 518 epitope conjugated with phycoerythrin (PE). Spleen cells were also stained with anti-CD8 antibodies conjugated with allophycocyanin (APC). PE and APC fluorochrome-conjugated reagents were used to identify the number of HA-518-specific CD8 T cells among all CD8 T cells of different specificities by use of a flow cytometer.
After recognition of peptide epitopes on the virus-infected cells, CD8 T cells from virus-infected or vaccinated animals kill infected cells via cytotoxic mechanisms and aid in viral clearance, as well as secreting interferon γ. To assess the functionality of CD8 T cells, splenocytes from immunized mice were cultured with syngeneic γ-irradiated spleen cells presenting either HA 518 or NP 147 peptides on anti-mouse interferon-γ-coated filter plates for 60 h and developed according to an ELISpot protocol. Splenocytes treated with phorbol myristate acetate and ionomycin were used as positive controls within every group.
To assess the protective efficacy against challenge with the variant H5N1 strains, mice (15 per group) were inoculated intramuscularly or intranasally twice every 4 weeks with 108 pfu of HAd-H5HA or intramuscularly with 108 pfu of HAd-ΔE1E3 (as a negative control). Five animals from every group were challenged 4 weeks after the second immunization with 100×50% LD50 of HK/483/97 (6.3×103 EID50), VN/1203/04 (6.9×103 EID50), or 100×50% mouse infectious dose (MID50; 6.3×103 EID50) of HK/213/03. Mice challenged with HK/483/97 or VN/1203/04 were monitored for clinical signs and changes in bodyweight daily. Since HK/213/03 is not lethal for mice, to assess the vaccine efficacy we measured the viral titres in the lungs on day 4 after challenge. Viral lung titres were expressed as the mean EID50 (log10/mL). Sensitivity of detection was 1.5 log10 EID50.
We used the Kruskall-Wallis test for all calculations of significance in this study.