Bovine adenoviral vector-based H5N1 influenza vaccine overcomes exceptionally high levels of pre-existing immunity against human adenovirus

Abstract

Because of the high prevalence of adenovirus (Ad) infections in humans, it is believed that pre-existing Ad-neutralizing antibodies (vector immunity) may negatively impact the immune response to vaccine antigens when delivered by human Ad (HAd) vectors. In order to evaluate whether bovine Ad subtype 3 (BAd3), a non-HAd vector, can effectively elude high levels of pre-existing vector immunity, naïve and HAd serotype 5 (HAd)-primed mice were immunized with BAd-H5HA [BAd3 vector expressing the hemagglutinin (HA) gene from H5N1 influenza virus]. Even in the presence of very high levels of HAd-specific neutralizing antibody, no significant reductions in HA-specific humoral and cell-mediated immune (CMI) responses were observed in HAd-primed mice immunized with BAd-H5HA. In naïve mice immunized with HAd-H5HA (HAd5 vector expressing H5N1 HA) and boosted with BAd-H5HA, the humoral responses elicited were significantly higher (P < 0.01) than with either HAd-H5HA or BAd-H5HA alone, while the CMI responses were comparable in the groups. This finding underlines the importance of a heterologous prime-boost approach for achieving an enhanced immune response. The immunization of naïve or HAd-primed mice with BAd-H5HA bestowed full protection from morbidity and mortality following a potentially lethal challenge with A/Hong Kong/483/97. These results demonstrate the importance of BAd vectors as an alternate or supplement to HAd vectors for influenza pandemic preparedness.

Figure 1. Replication-defective BAd vector (BAd-H5HA) expresses HA of H5N1 influenza virus (A/Hong Kong/156/97) in vector-infected cells

(a) Diagrammatic representation of replication deficient BAd vectors, BAd-ΔE1E3 [BAd3 with deleted E1 and E3 regions] and BAd-H5HA [BAd-ΔE1E3 with hemaggluttinin (HA) gene from H5N1 influenza virus (A/Hong Kong/156/97)]. ITR, inverted terminal repeat. (b) Expression of H5N1 HA in BHH3 cells infected with BAd-H5HA. Mock (PBS infected), BAd-ΔE1E3, or BAd-H5HA infected BHH3 cells were harvested 36 h post-infection and cell lysates were analyzed by Western blot using a rabbit anti-H5HA hyperimmune serum.

Figure 2. HA-518 epitope-specific CD8+ T cells in naïve or HAd5-primed mice immunized with BAd-H5HA vaccine

Naïve or HAd-primed mice were immunized as described in the materials and methods. Mice were sacrificed 3 weeks after final immunization and the spleens were collected. Single cell suspensions were prepared by passage through screens and 2 × 10⁶ cells were stained with a murine MHC-encoded allele k^d-specific pentamer for immunodominant HA-518 epitope conjugated with phycoerythrin (PE) and also with anti-CD8 antibody-conjugated with flouro-isothiocyanin (FITC). Flow-cytometric analysis was done to identify the number of HA-518 specific CD8+ T cells. (a) Representative sample of only three mice are shown. (b) Data from seven mice/group are expressed as box and whisker plots (interquartile ranges). *; P<0.001.

Figure 3. ELISpot measurements of IFN-γ expression in spleen cells of naïve or HAd5-primed mice immunized with BAd-H5HA vaccine

Naïve or HAd5-primed mice were immunized as described in the materials and methods. Mice were sacrificed at 3 weeks post second immunization and the spleens were collected Single cell suspensions were prepared by passage through screens and 1 × 10⁶ cells were cultured in the presence of HA-518 peptide on anti-interferon-γ antibody-coated 96-well filter plates and developed according to an ELISpot protocol. Splenocytes cultured in presence of NP-147 peptide or phorbol myristate acetate (PMA) and ionomycin served as negative and positive controls, respectively. The error bars represent Mean ± SD from seven animals/group. * ; P<0.001.

Figure 4. Protection of Naïve or HAd-primed mice immunized with BAd-H5HA vaccine from morbidity and mortality following challenge with a homologous lethal H5N1 virus (HK/483/97)

Naïve or HAd-primed mice (10 animals/group) were immunized i.m. twice at 3 weeks interval with 1×10⁸ p.f.u. of BAd-H5HA vaccine. Naïve mice (10 animals/group) were similarly inoculated with HAd-ΔE1E3 (vector control) or HAd-H5HA to serve as negative and positive controls, respectively. All animals were challenged with 100 × 50% lethal dose (LD₅₀) of A/Hong Kong/483/97. Mice were monitored for weight loss for 13 days after challenge to monitor weight loss (a) and mortality (b). Data are shown as Mean ± SD.