Immunopotentiators Improve the Efficacy of Oil-Emulsion-Inactivated Avian Influenza Vaccine in Chickens, Ducks and Geese

Abstract

Combination of CVCVA5 adjuvant and commercial avian influenza (AI) vaccine has been previously demonstrated to provide good protection against different AI viruses in chickens. In this study, we further investigated the protective immunity of CVCVA5-adjuvanted oil-emulsion inactivated AI vaccine in chickens, ducks and geese. Compared to the commercial H5 inactivated vaccine, the H5-CVCVA5 vaccine induced significantly higher titers of hemaglutinin inhibitory antibodies in three lines of broiler chickens and ducks, elongated the antibody persistence periods in geese, elevated the levels of cross serum neutralization antibody against different clade and subclade H5 AI viruses in chicken embryos. High levels of mucosal antibody were detected in chickens injected with the H5 or H9-CVCA5 vaccine. Furthermore, cellular immune response was markedly improved in terms of increasing the serum levels of cytokine interferon-γ and interleukine 4, promoting proliferation of splenocytes and upregulating cytotoxicity activity in both H5- and H9-CVCVA5 vaccinated chickens. Together, these results provide evidence that AI vaccines supplemented with CVCVA5 adjuvant is a promising approach for overcoming the limitation of vaccine strain specificity of protection.

Fig 1. The HI antibody titers against H5 viral antigen in broilers.

White feather broilers (A), yellow feather broilers (B), or dot feather broilers (C). The chickens (n = 20) vaccinated at 10–15 days old, and serum collected at 2-, 3-, and 4-week post-vaccination (wpv). H5-CVCVA5, commercial vaccine (H5-Re5) mixed with CVCVA5 adjuvants. H5, commercial vaccine (H5-Re5). Control, naïve control group. **, P < 0.05. Error bars indicates SEM.

Fig 2. The HI antibody titers against H5 viral antigens in Mallard duck.

The ducks (n = 10) vaccinated at 14-day old, and serum were collected at 2–3-, and 4-week post-vaccination (wpv). H5-CVCVA5, commercial vaccine (H5-Re5) mixed with CVCVA5 adjuvants. H5, commercial vaccine (H5-Re5). Control, naïve control group. **, P < 0.05.

Fig 3. The HI antibody titers against H5 viral antigen in domestic geese.

Groups of twenty 14-day-old goslings via subcutaneously route received the prime-boost vaccination of H5 or H5-CVCVA5 vaccines (0.5ml), respectively, and the third group was set as blank control. 2^nd vac., the second vaccination. The horizontal dash line is the qualified antibody titer according to the requirements of commercial vaccine. ***, P < 0.01.

Fig 4. The HI antibody titers against H5 or H9 viral antigen in shelf-life test of CVCVA5.

The adjuvants in H5-CVCVA5 were prepared in exclusive water-in-oil form which stored at 4°C for 15-month period, and mixed with the H5 (Re-5) commercial inactivated vaccine (A). The H9-CVCVA5 was prepared as all components of the adjuvants directly addition to the aqueous or oil phase of H9 vaccine during the preparation processes, and preserved for a period of 15-month at 4°C (B). **, P < 0.05.

Fig 5. Serum cytokine levels from SPF chickens at 3 wpv.

Levels of IFN-γ (A) and IL-4 (B) cytokines in SPF chickens serum were determined by ELISA Kit at three-week post vaccination from each group (n = 4). The values were represented mean±S.D. ***, P < 0.01. Ratio was counted as the following, (H5-CVCVA5-control)/ (H5-control).

Fig 6. Mucosal antibodies against H5 or H9 subtype viral antigen at 3 wpv.

SPF chickens were vaccinated with the adjuvanted H5-CVCVA5, H9-CVCVA5, H5 or H9 inactivated vaccine, respectively. The mucus was collected three-week post vaccination (n = 4) from each group. The mucosal antibody derived from scraps of trachea, small intestine and bronchoalveolar lavage fluids (BAL fluids) alone, against H5 (A) or H9 (B) viral antigen tested by HI assay, respectively. **, P < 0.05. ***, P < 0.01.

Fig 7. Mitogen-stimulated proliferation of splenocytes isolated from SPF chicken receiving H5 (Re-5) or H9 (NJ02/01) vaccine with or without CVCVA5 adjuvant.

Splenocytes were prepared after the immunization and cultured with inactivated H5 (Re-5, 5μg/ml) or H9 (NJ02/01, 5μg/ml) viral antigen. Splenocytes proliferation was measured by the MTT method, and shown as a stimulation index. The values were represented mean ± S.D (n = 5) **, P<0.05. ***, P<0.01.

Fig 8. Cytotoxicity of CD8⁺ T cells from inbred chickens (B₁₉/B₁₉) vaccinated H5 (Re5) or H9 (NJ02/01) subtype vaccine with or without adjuvants.

Inbred chicken embryo fibroblasts infected with S (H5N1) or NJ02/01 (H9N2) avian influenza viruses were used as target cells. PBMCs derived from the vaccinated inbred chicken or control group were used as effector cell.