Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine

Abstract

The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines.

Figure 1. Replication-defective HAd vector (HAd-HA-NP) expresses HA and NP of a H5N1 influenza virus in vector-infected cells.

(A) Diagrammatic representation of replication-deficient HAd vectors, HAd-ΔE1E3 [HAd5 with deleted E1 and E3 regions] and HAd-HA-NP [HAd-ΔE1E3 with hemaggluttinin (HA) and nucleoprotein (NP) gene from A/Vietnam/1203/04 (H5N1) influenza virus]. ITR, inverted terminal repeat; CMV, cytomegalovirus immediate early promoter; pA, polyadenylation signal; MCMV, mouse cytomegalovirus immediate early promoter; SV40pA, simian virus polyadenylation signal. (B and C) Expression of H5N1 HA and NP in 293 cells infected with HAd-HA-NP. Mock (PBS-infected), HAd-ΔE1E3-, or HAd-HA-NP-infected 293 cells were harvested 48 h post-infection, and cell lysates were analyzed by Western blot using polyclonal serum against H5 HA or a NP-specific mouse monoclonal antibody.

Figure 2. Development of vector immunity in wild type (WT) HAd5-primed animals.

To induce HAd vector–specific immunity in mice, 6–8 weeks old female BALB/c mice were primed intramuscularly (i.m.) or intranasally (i.n.) with a single dose of 10⁷, 10⁸, or 10⁹ p.f.u. of HAd-WT. The unprimed mice received PBS. Four weeks after priming, mice were bled by retro-orbital puncture to evaluate the development of HAd-specific neutralizing antibodies by virus neutralization assay. Virus neutralization titers were the reciprocal of the highest serum dilution that completely prevented the development of c.p.e. The error bars represent Mean ± SD from five animals/group: *, P≤0.05; ***, P≤0.005.

Figure 3. NP-147 epitope-specific CD8+ T cells in naïve or HAd5-primed mice immunized with HAd-HA-NP.

Naïve or HAd-primed mice were immunized as described in the Materials and Methods. At four weeks after final immunization, animals were euthanized, and the spleens were collected. Single cell suspensions were prepared by passage through screens, and 1×10⁶ cells were stained with a murine MHC-encoded allele k^d–specific pentamer for immunodominant NP-147 epitope-conjugated with phycoerythrin (PE) and also with anti-CD8 antibody-conjugated with allophycocyanin (APC) and anti-CD19 antibody-conjugated with flouro-isothiocyanin (FITC). Flow cytometeric analysis was done to identify the number of NP-147-specific CD8+ T cells. Data were collected using BD FACSCanto II (BD Bioscience, CA) and FACSDiva software was used for analysis. Data from five mice/groups are shown. (A) Percentage of NP-147-specific CD8+ T cells in intranasally primed groups. (B) Percentage of NP-147-specific CD8 T cells in intramuscularly primed groups. The error bars represent Mean ± SD from five animals/group: *, P≤0.05; **, P≤0.01; †, P≤0.001.

Figure 4. ELISpot measurements of IFN-γ expression in spleen cells of naïve or HAd5-primed mice immunized with HAd-HA-NP.

Naïve or HAd5-primed mice were immunized as described in the Materials and Methods. At four weeks after final immunization, animals were euthanized, 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 or NP-147 peptide on anti-interferon-γ antibody-coated 96-well filter plates and developed according to an ELISpot protocol. Splenocytes cultured in presence of phorbol myristate acetate (PMA) and ionomycin served as positive controls in each group. The ELISpot plates were read using a Bioreader 5000 (BIOSYS, Miami, FL). (A) Number of IFN-γ-secreting NP-147-specific CD8 T cells in intranasally (i.n.) primed groups. (B) Number of IFN-γ-secreting NP-147-specific CD8 T cells in intramuscularly (i.m.) primed groups. (C) Number of IFN-γ-secreting HA-518-specific CD8 T cells in i.n. primed groups. (D) Number of IFN-γ-secreting HA-518-specific CD8 T cells in i.m. primed groups. The error bars represent Mean ± SD from five animals/group: *, P≤0.05; **, P≤0.01; ***, P≤0.005; †, P≤0.001; ‡, P≤0.0001.