Modulation of systemic and mucosal immunity against an inactivated vaccine of Newcastle disease virus by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interleukin-18 and interferon-α

Abstract

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to limitations in the efficacy against currently circulating ND viruses, existing vaccination strategies require improvements, and incorporating immunomodulatory cytokines with existing vaccines might be a novel approach. Here, we investigated the systemic and mucosal immunomodulatory properties of oral co-administration of chicken interleukin-18 (chIL-18) and chicken interferon-α (chIFN-α) using attenuated Salmonella enterica serovar Typhimurium on an inactivated ND vaccine. Our results demonstrate that oral administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α provided enhanced systemic and mucosal immune responses, as determined by serum hemagglutination inhibition antibody and NDV Ag-specific IgG as well as NDV Ag-specific IgA in lung and duodenal lavages of chickens immunized with inactivated ND vaccine via the intramuscular or intranasal route. Notably, combined oral administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α significantly enhanced systemic and mucosal immunity in ND-vaccinated chickens, compared to single administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α. In addition, oral co-administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α provided enhanced NDV Ag-specific proliferation of peripheral blood mononuclear cells and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results provide valuable insight into the modulation of systemic and mucosal immunity by incorporation of immunomodulatory chIL-18 and chIFN-α using Salmonella vaccines into existing ND vaccines.

Fig. 1.

Serum NDV-specific HI titers in chickens immunized via systemic and mucosal routes following co-administration of live attenuated Salmonella Typhimurium expressing chIFN-α and chIL-18. Serum samples were collected from immunized chickens 7 days after primary vaccination and 7 & 14 days after booster vaccination and subjected to the HI test. (A and B) HI antibody titers in chickens immunized with inactivated NDV vaccine via i.m. route. (C and D) HI antibody titers in chickens immunized with inactivated NDV vaccine via i.n. route. Data are expressed as reciprocal log2 of the geometric average and standard error of HI titers obtained from six chickens per group. *P<0.05; **P<0.01; ***P<0.001 compared to vehicle group that was treated with control bacteria. ^¶¶¶P<0.001 compared to chIL18 and ^†††P<0.001 compared to chIFN-α.

Fig. 2.

Serum NDV-specific IgG levels in chickens immunized via the systemic and mucosal routes following oral co-administration of live attenuated Salmonella Typhimurium expressing chIFN-α and chIL-18. (A and B) IgG levels in chickens immunized with inactivated NDV vaccine via i.m. route. (C and D) IgG levels in chickens immunized with inactivated NDV vaccine via i.n. route. Data represent the mean and standard error derived from six chickens per group. *P<0.05; **P<0.01; ***P<0.001 compared to vehicle group that was treated with control bacteria. ^¶P<0.05; ^¶¶P<0.01 compared to chIL18 and ^†P<0.05; ^††P<0.01 compared to chIFN-α.

Fig. 3.

NDV-specific IgA levels in lung and duodenal lavages of chickens immunized via the systemic and mucosal routes following oral co-administration of live attenuated Salmonella Typhimurium expressing chIFN-α and chIL-18. (A and B) IgA levels in lung (A) and duodenal (B) lavages of chickens 14 days after booster vaccination via i.m. route. (C and D) IgA levels in lung (C) and duodenal (D) lavages of chickens 14 days after booster vaccination via i.n. route. Data represent the mean and standard error derived from six chickens per group. *P<0.05; **P<0.01; ***P<0.001 compared to vehicle group that was treated with control bacteria. ^¶P<0.05; ^¶¶P<0.01 compared to chIL18 and ^†P<0.05; ^††P<0.01 compared to chIFN-α.

Fig. 4.

Enhanced Th1-biased immunity in chickens immunized via the systemic route following oral co-administration of Salmonella Typhimurium expressing chIFN-α and chIL-18. (A and B) NDV antigen-specific proliferation of PBMCs. PBMCs were collected 14 days after booster vaccination and used for NDV Ag-specific stimulation. PBMCs that were not stimulated with Ag (Unst.) were also used for a negative control. (C and D) Expression of IFN-γ and IL-4 mRNA by PBMCs following stimulation with specific NDV (B1 strain) antigen. PBMCs were incubated in the presence (w/ Ag) or absence (w/o Ag) of NDV Ag, and RNAs extracted from PBMCs were used for real-time qRT-PCR to determine IFN-γ and IL-4 mRNA. Data represent the mean and standard error of IFN-γ and IL-4 expression obtained from six chickens per group, after normalization to GAPDH. ***P<0.001 compared to vehicle group that was treated with control bacteria. ^¶¶¶P<0.001 compared to chIL-18-treated chickens. ^†††P<0.001 compared to chIFN-α- treated chickens.

Fig. 5.

Enhanced Th1-biased immunity in chickens immunized via the mucosal route following oral co-administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18. (A and B) NDV antigen-specific proliferation of PBMCs. PBMCs were collected 14 days after booster vaccination and used for NDV Ag-specific stimulation. PBMCs that were not stimulated with Ag (Unst.) were also used for a negative control. (C and D) Expression of IFN-γ and IL-4 mRNA by PBMCs following stimulation with specific NDV (B1 strain) antigen. PBMCs were incubated in the presence (w/ Ag) or absence (w/o Ag) of NDV Ag, and RNAs extracted from PBMCs were used for real-time qRT-PCR to determine IFN-γ and IL-4 mRNA. Data represent the mean and standard error of IFN-γ and IL-4 expression levels obtained from six chickens per group, after normalization to GAPDH. ***P<0.001 compared to vehicle group that was treated with control bacteria. ^¶¶¶P<0.001 compared to chIL-18-treated chickens. ^†††P<0.001 compared to chIFN-α -treated chickens.