Adenovirus vector-based multi-epitope vaccine provides partial protection against H5, H7, and H9 avian influenza viruses

Abstract

The emergence of H5, H7, and H9 avian influenza virus subtypes in humans reveals their pandemic potential. Although human-to-human transmission has been limited, the genetic reassortment of the avian and human/porcine influenza viruses or mutations in some of the genes resulting in virus replication in the upper respiratory tract of humans could generate novel pandemic influenza viruses. Current vaccines do not provide cross protection against antigenically distinct strains of the H5, H7, and H9 influenza viruses. Therefore, newer vaccine approaches are needed to overcome these potential threats. We developed an egg-independent, adenovirus vector-based, multi-epitope (ME) vaccine approach using the relatively conserved immunogenic domains of the H5N1 influenza virus [M2 ectodomain (M2e), hemagglutinin (HA) fusion domain (HFD), T-cell epitope of nucleoprotein (TNP). and HA α-helix domain (HαD)]. Our ME vaccine induced humoral and cell-mediated immune responses and caused a significant reduction in the viral loads in the lungs of vaccinated mice that were challenged with antigenically distinct H5, H7, or H9 avian influenza viruses. These results suggest that our ME vaccine approach provided broad protection against the avian influenza viruses. Further improvement of this vaccine will lead to a pre-pandemic vaccine that may lower morbidity, hinder transmission, and prevent mortality in a pandemic situation before a strain-matched vaccine becomes available.

Fig 1. Schematic diagram of adenovirus (Ad) vector constructs used in the study.

(A) Ad-ΔE1E3; human adenovirus type C5 (Ad-C5) empty vector. (B) Ad-H5HA, Ad-H7HA and Ad-H9HA; Ad-C5 vector containing a full-length coding region of H5 hemagglutinin (HA) of A/Vietnam/1203/04(H5N1), H7 HA of A/Netherlands/219/2003(H7N7) or H9 HA of A/chicken/Hong Kong/G9/1997(H9N2), respectively. (C) Ad-H5ME, Ad-C5 vector containing multi-epitope (ME) gene construct of A/Vietnam/1203/ 2004(H5N1) that contains the extracellular domain of M2 (M2e), fusion domain of HA (HFD), T-cell epitope of nucleoprotein [NP] (TNP), and alpha helix domain of HA (HαD).

Fig 2. Influenza antigen-specific antibody responses generated after immunization with an Ad vector-based multi-epitope (ME) vaccine.

Three weeks after the second inoculation, serum samples were collected from all animal groups as described in the material and methods section, and tested by ELISA for IgG antibody responses specific to M2e (A), HαD (B) or HFD (C). Data are represented as the mean ± standard deviation (SD) of the optical density (OD) readings. Based on OD values, M2e-, HαD- and HFD-specific IgG responses induced by H5ME were statistically significant (*P≤ 0.05 as compared to the ΔE1E3 control). H5HA, Ad-H5HA; H7HA, Ad-H7HA; H9HA, Ad-H9HA, H5ME, Ad-H5ME; ΔE1E3, Ad-ΔE1E3.

Fig 3. HA518 and NP147 epitope-specific IFNγ secreting CD8+ T cells in the spleens of vaccinated mice.

Three weeks after the second inoculation, the spleens were collected from all animal groups after euthanizing the animals as described in the material and methods section. The splenocytes were evaluated for HA-specific (A) or NP-Specific (B) cell-mediated immune responses using INFγ-ELISpot assay. The data represent mean ± standard deviation (SD). The number of spot-forming units (SFU) induced by H5ME in NP-specific INFγ-ELISpot, and the number of SFU induced by H5HA or H9HA in HA-specific INFγ-ELISpot were found to be statistically significant (*P≤ 0.05 as compared to the ΔE1E3 control). H5HA, Ad-H5HA; H7HA, Ad-H7HA; H9HA, Ad-H9HA; H5ME, Ad-H5ME; ΔE1E3, Ad-ΔE1E3.