A Bovine Adenoviral Vector-Based H5N1 Influenza -Vaccine Provides Enhanced Immunogenicity and Protection at a Significantly Low Dose

Abstract

Several human and nonhuman adenovirus (AdV) vectors including bovine AdV type 3 (BAdV-3) were developed as gene delivery vectors to supplement and/or elude human AdV (HAdV)-specific neutralizing antibodies (vector immunity). Here we evaluated the vaccine immunogenicity and efficacy of BAdV-3 vector (BAd-H5HA) expressing hemagglutinin (HA) of a H5N1 influenza virus in a dose escalation study in mice with the intranasal (IN) or intramuscular (IM) route of inoculation in comparison with the HAdV type C5 (HAdV-C5) vector (HAd-H5HA) expressing HA of a H5N1 influenza virus. Dose-related increases in the immune responses were clearly noticeable. A single IM inoculation with BAd-H5HA resulted in enhanced cellular immune responses compared with that of HAd-H5HA and conferred complete protection following challenge with a heterologous H5N1 virus at the dose of 3 × 10⁷ plaque-forming units (PFUs), whereas a significant amount of influenza virus was detected in the lungs of mice immunized with 1 × 10⁸ PFUs of HAd-H5HA. Similarly, compared with that of HAd-H5HA, a single IN inoculation with BAd-H5HA produced significantly enhanced humoral (HA-specific immunoglobulin [IgG] and its subclasses, as well as HA-specific IgA) and cellular immune responses, and conferred complete protection following challenge with a heterologous H5N1 virus. Complete protection with BAd-H5HA was observed with the lowest vaccine dose (1 × 10⁶ PFUs), but similar protection with HAd-H5HA was observed at the highest vaccine dose (1 × 10⁸ PFUs). These results suggest that at least 30-fold dose sparing can be achieved with BAd-H5HA vector compared with HAd-H5HA vaccine vector.

Keywords: adenoviral vector; avian influenza; bovine adenoviral vector; dose sparing; enhanced immunogenicity and protection; human adenoviral vector; route of immunization; universal influenza vaccine.

Figure 1

HA Expression Levels by HAd-H5HA or BAd-H5HA in Infected Cells BHH3 cells were either mock infected or infected with HAd-ΔE1E3, HAd-H5HA, BAd-ΔE1E3, or BAd-H5HA at an MOI of 10 PFUs per cell. At 48 hr post-infection, the cells were harvested and processed for immunoblotting. HA expression was detected using an HA-specific polyclonal antibody. The expression levels of β-actin were monitored to demonstrate equal loading. The molecular weight markers in kDa are shown on the left.

Figure 2

HA-Specific Serum IgG Antibody Responses in Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFUs of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, serum samples were collected, diluted to 1:500, and the development of HA-specific IgG antibody responses were monitored by ELISA. Data are represented as the mean ± SD of the optical density (OD) readings. Statistically significant responses are shown as compared with the PBS group (+), IN versus IM route of inoculation in the same group (*), or BAd-H5HA vector versus HAd-H5HA vector (ψ). ** or ++, significant at p < 0.01; ***, +++, or ψψψ, significant at p < 0.001; and **** or ++++, significant at p < 0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using GraphPad Prism 6. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus; ns, no significance at p > 0.05.

Figure 3

HA-Specific Serum IgG1 Antibody Responses in Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFUs of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, serum samples were collected, diluted to 1:500, and the development of HA-specific IgG1 antibody responses was monitored by ELISA. Data are represented as the mean ± SD of the optical density (OD) readings. Statistically significant responses are shown as compared with the PBS group (+), IN versus IM route of inoculation in the same group (*), or BAd-H5HA vector versus HAd-H5HA vector (ψ). +++ or ψψψ, significant at p < 0.001; and **** or ++++, significant at p < 0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using GraphPad Prism 6. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus; ns, no significance at p > 0.05.

Figure 4

HA-Specific Serum IgG2a Antibody Responses in Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFUs of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, serum samples were collected, diluted to 1:50, and the development of HA-specific IgG2a antibody responses was monitored by ELISA. Data are represented as the mean ± SD of the optical density (OD) readings. Statistically significant responses are shown as compared with the PBS group (+), IN versus IM route of inoculation in the same group (*), or BAd-H5HA vector versus HAd-H5HA vector (ψ).+, significant at p < 0.05; **, significant at p < 0.01; ***, +++, or ψψψ, significant at p < 0.001; and **** or ++++, significant at p < 0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using GraphPad Prism 6. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus; ns, no significance at p > 0.05.

Figure 5

HA-Specific Serum IgG2b Antibody Responses in Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFUs of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, serum samples were collected, diluted to 1:50, and the development of HA-specific IgG2b antibody responses was monitored by ELISA. Data are represented as the mean ± SD of the optical density (OD) readings. Statistically significant responses are shown as compared with the PBS group (+), IN versus IM route of inoculation in the same group (*), or BAd-H5HA vector versus HAd-H5HA vector (ψ). * or +, significant at p < 0.05; ** or ++, significant at p < 0.01; +++ or ψψψ, significant at p < 0.001; and **** or ++++, significant at p < 0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using GraphPad Prism 6. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus; ns, no significance at p > 0.05.

Figure 6

HA-Specific IgA Antibody Responses in Lung Washes of Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFU of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, nasal wash samples were collected, diluted to 1:5, and the development of HA-specific IgA antibody responses was monitored by ELISA. Data are represented as the mean ± SD of the optical density (OD) readings. Statistically significant responses are shown as compared with the PBS group (+), IN versus IM route of inoculation in the same group (*), or BAd-H5HA vector versus HAd-H5HA vector (ψ). *, significant at p < 0.05; ψψψ, significant at p < 0.001; and **** or ++++, significant at p < 0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using GraphPad Prism 6. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus; ns, no significance at p > 0.05.

Figure 7

HA-Specific IgA Antibody Responses in Nasal Washes of Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFUs of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, lung wash samples were collected, diluted to 1:10, and the development of HA-specific IgA antibody responses was monitored by ELISA. Data are represented as the mean ± SD of the optical density (OD) readings. Statistically significant responses are shown as compared with the PBS group (+), IN versus IM route of inoculation in the same group (*), or BAd-H5HA vector versus HAd-H5HA vector (ψ). ++, significant at p < 0.01; +++ or ψψψ, significant at p < 0.001; and **** or ++++, significant at p < 0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using GraphPad Prism 6. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus; ns, no significance at p > 0.05.

Figure 8

HA518 Epitope-Specific IFNγ-Secreting CD8⁺ T Cells in the Spleens of Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFUs of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, the spleens were collected, and the splenocytes were evaluated for HA-specific cell-mediated immune responses using IFNγ-ELISpot assay. The data represent mean ± SD of the number of spot-forming units (SFUs). Statistically significant responses are shown as compared with the PBS group (+), IN versus IM route of inoculation in the same group (*), or BAd-H5HA vector versus HAd-H5HA vector (ψ). *, significant at p < 0.05; **, significant at p < 0.01; ***, +++, or ψψψ, significant at p < 0.001; and **** or ++++, significant at p < 0.0001. The statistical analysis was done by Bonferroni post-test and two-way ANOVA using GraphPad Prism 6. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus; ns, no significance at p > 0.05.

Figure 9

HA518 Epitope-Specific IFNγ-Secreting CD8⁺ T Cells in the Respiratory Lymph Nodes (RLNs) of Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFUs of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, the RLNs were collected, and the pooled RLN cells were evaluated for HA-specific cell-mediated immune responses using the IFNγ-ELISpot assay. The data represent the individual replicate numbers of spot-forming units (SFUs) from pooled samples. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus.

Figure 10

HA518 Epitope-Specific IFNγ-Secreting CD8⁺ T Cells in the Inguinal Lymph Nodes (ILNs) of Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFUs of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, the ILNs were collected, and the pooled ILN cells were evaluated for HA-specific cell-mediated immune responses using IFNγ-ELISpot assay. The data represent the individual replicate numbers of spot-forming units (SFUs) from pooled samples. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus.

Figure 11

HA518 Epitope-Specific IFNγ-Secreting CD8⁺ T Cells in the Lungs of Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFUs of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after inoculation, the lungs were collected, and the pooled lung lymphocytes were evaluated for HA-specific cell-mediated immune responses using IFNγ-ELISpot assay. The data represent the individual replicate numbers of spot-forming units (SFUs) from pooled samples. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus.

Figure 12

Lung Influenza Virus Titers in Mice Immunized Once with BAd-H5HA or HAd-H5HA Mice were immunized intramuscularly (IM) or intranasally (IN) once with 1 × 10⁶ (A), 3 × 10⁶ (B), 1 × 10⁷ (C), 3 × 10⁷ (D), or 1 × 10⁸ (E) PFUs of BAd-H5HA or HAd-H5HA. For all dose groups, mice inoculated IM or IN with PBS or 1 × 10⁸ PFUs of BAd-ΔE1E3 or HAd-ΔE1E3 served as negative or internal controls, respectively. Four weeks after immunization, mice were challenged with 100 MID₅₀ of A/Vietnam/1203/2004(H5N1)-PR8/CDC-RG influenza virus. Three days after the challenge, mice were euthanized and the lungs were collected to determine lung virus titers. The data are shown as mean Log₁₀ TCID₅₀ ± SD, and the detection limit was 0.5 Log₁₀ TCID₅₀/mL. Statistically significant responses are shown as compared with the PBS group (+) or IN versus IM route of inoculation in the same group (*). +, significant at p < 0.05; ** or ++, significant at p < 0.01; +++, significant at p < 0.001; and **** or ++++, significant at p < 0.0001. The statistical analyses were done by Bonferroni post-test and two-way ANOVA using GraphPad Prism 6. BAd-ΔE1E3, BAd empty vector; BAd-H5HA, bovine adenoviral vector expressing hemagglutinin (HA) of A/Hong Kong/156/97(H5N1) influenza virus; HAd-ΔE1E3, HAd empty vector; HAd-H5HA, human adenoviral vector expressing HA of A/Hong Kong/156/97(H5N1) influenza virus; NA, not applicable; ns, no significance at p > 0.05.