Quick and improved immune responses to inactivated H9N2 avian influenza vaccine by purified active fraction of Albizia julibrissin saponins

Abstract

Background: H9N2 Low pathogenic avian influenza virus (LPAIV) raises public health concerns and its eradication in poultry becomes even more important in preventing influenza. AJSAF is a purified active saponin fraction from the stem bark of Albizzia julibrissin. In this study, AJSAF was evaluated for the adjuvant potentials on immune responses to inactivated H9N2 avian influenza virus vaccine (IH9V) in mice and chicken in comparison with commercially oil-adjuvant.

Results: AJSAF significantly induced faster and higher H9 subtype avian influenza virus antigen (H9-Ag)-specific IgG, IgG1, IgG2a and IgG2b antibody titers in mice and haemagglutination inhibition (HI) and IgY antibody levels in chicken immunized with IH9V. AJSAF also markedly promoted Con A-, LPS- and H9-Ag-stimulated splenocyte proliferation and natural killer cell activity. Furthermore, AJSAF significantly induced the production of both Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines, and up-regulated the mRNA expression levels of Th1 and Th2 cytokines and transcription factors in splenocytes from the IH9V-immunized mice. Although oil-formulated inactivated H9N2 avian influenza vaccine (CH9V) also elicited higher H9-Ag-specific IgG and IgG1 in mice and HI antibody titer in chicken, this robust humoral response was later produced. Moreover, serum IgG2a and IgG2b antibody titers in CH9V-immunized mice were significantly lower than those of IH9V alone group.

Conclusions: AJSAF could improve antigen-specific humoral and cellular immune responses, and simultaneously trigger a Th1/Th2 response to IH9V. AJSAF might be a safe and efficacious adjuvant candidate for H9N2 avian influenza vaccine.

Keywords: Adjuvant; Albizia julibrissin saponin; H9N2 avian influenza vaccine; Immune response.

Fig. 1

Effect of AJSAF on H9–Ag-specific IgG and its isotype antibody titers in the immunized mice. Mice were s.c. injected with 0.2 ml of IH9V (10⁷ TCID₅₀/0.1 ml) alone or containing Quil A or AJSAF, or 0.2 ml of CH9V (10⁷ TCID₅₀/0.1 ml) on days 1 and 15. Sera were collected 14 days after the boosting immunization, and serum H9–Ag-specific IgG, IgG1, IgG2a, and IgG2b antibody titers were measured by ELISA. The values are presented as means ± SD (n = 6). Significant differences with IH9V alone group were designated as ^*P < 0.05, ^†P < 0.01, and ^‡P < 0.001; those with CH9V group as ^§P < 0.05, ^#P < 0.01, and ^¶P < 0.001

Fig. 2

Time-response relationship of H9–Ag-specific IgG and its isotypes in the IH9V-immunized mice induced by AJSAF. Mice were s.c. immunized with 0.2 ml of IH9V (10⁷ TCID₅₀/0.1 ml) alone or containing AJSAF, or 0.2 ml of CH9V (10⁷ TCID₅₀/0.1 ml). Sera were collected at designated time after a single dose of vaccination for measuring H9–Ag-specific IgG, IgG1, IgG2a, and IgG2b antibody titers. The values are presented as means ± SD (n = 5). Significant differences with IH9V alone group were designated as ^*P < 0.05, ^†P < 0.01, and ^‡P < 0.001; those with CH9V group as ^§P < 0.05, ^#P < 0.01, and ^¶P < 0.001

Fig. 3

Effect of AJSAF on haemagglutination inhibition (HI) and H9–Ag-specific IgY antibody levels in the immunized chicken. The chickens were prime- and boost-immunized with 0.4 ml of IH9V (10⁷ TCID₅₀/0.1 ml) alone or containing AJSAF, or 0.4 ml of CH9V (10⁷ TCID₅₀/0.1 ml) at 3-week interval. Sera were collected on designated days post-immunization, and the serum HI antibody titers (a) and H9–Ag-specific IgY antibody (b) levels were measured by HI assay and ELISA, respectively. The values are presented as means ± SD (n = 30). Significant differences with IH9V alone group were designated as ^*P < 0.05, ^†P < 0.01, and ^‡P < 0.001

Fig. 4

Effect of AJSAF on Con-, LPS-, and H9–Ag-stimulated splenocyte proliferation in the immunized mice. Splenocyte proliferation was measured by the MTT method and shown as a stimulation index (SI). The values are presented as means ± SD (n = 6). Significant differences with IH9V alone group were designated as ^*P < 0.05, ^†P < 0.01, and ^‡P < 0.001; those with CH9V group as ^§P < 0.05, ^#P < 0.01, and ^¶P < 0.001

Fig. 5

Effect of AJSAF on NK cell activity in the splenocytes from the immunized mice. Splenocytes were prepared 2 weeks after the boosting immunization for detecting NK cell activity by the MTT assay. The values are presented as means ± SD (n = 6). Significant differences with IH9V alone group were designated as ^†P < 0.01and ^‡P < 0.001; those with CH9V group as ^¶P < 0.001

Fig. 6

Effect of AJSAF on cytokine secretion from splenocytes in the immunized mice. Splenocytes were incubated with Con A (48 h) or H9–Ag (72 h), and the supernatants were collected for detecting IL-2, IFN-γ, and IL-10 levels using ELISA kits. The values are presented as means ± SD (n = 6). Significant differences with IH9V alone group were designated as ^*P < 0.05, ^†P < 0.01, and ^‡P < 0.001; those with CH9V group as ^#P < 0.01 and ^¶P < 0.001

Fig. 7

Effect of AJSAF on mRNA expression of cytokines and transcription factors in Con A (a) - and H9–Ag (b)-stimulated splenocytes from the immunized mice. Splenocytes were incubated with Con A (for 12 h) or H9–Ag (for 16 h), and the mRNA expression levels of cytokines and transcription factors were measured using RT-qPCR. The values are presented as means ± SD (n = 6). Significant differences with IH9V alone group were designated as ^*P < 0.05, ^†P < 0.01, and ^‡P < 0.001; those with CH9V group as ^§P < 0.05, ^#P < 0.01, and ^¶P < 0.001