Bivalent Oral Vaccine Using Attenuated Salmonella Gallinarum Delivering HA and NA-M2e Confers Dual Protection Against H9N2 Avian Influenza and Fowl Typhoid in Chickens

Abstract

Background: Fowl typhoid (FT), a septicemic infection caused by Salmonella Gallinarum (SG), and H9N2 avian influenza are two economically important diseases that significantly affect the global poultry industry. Methods: We exploited the live attenuated Salmonella Gallinarum (SG) mutant JOL3062 (SG: ∆lon ∆pagL ∆asd) as a delivery system for H9N2 antigens to induce an immunoprotective response against both H9N2 and FT. To enhance immune protection against H9N2, a prokaryotic and eukaryotic dual expression plasmid, pJHL270, was employed. The hemagglutinin (HA) consensus sequence from South Korean avian influenza A virus (AIV) was cloned under the Ptrc promoter for prokaryotic expression, and the B cell epitope of neuraminidase (NA) linked with matrix protein 2 (M2e) was placed for eukaryotic expression. In vitro and in vivo expressions of the H9N2 antigens were validated by qRT-PCR and Western blot, respectively. Results: Oral immunization with JOL3121 induced a significant increase in SG and H9N2-specific serum IgY and cloacal swab IgA antibodies, confirming humoral and mucosal immune responses. Furthermore, FACS analysis showed increased CD4+ and CD8+ T cell populations. On day 28 post-immunization, there was a substantial rise in the hemagglutination inhibition titer in the immunized birds, demonstrating neutralization capabilities of immunization. Both IFN-γ and IL-4 demonstrated a significant increase, indicating a balance of Th1 and Th2 responses. Intranasal challenge with the H9N2 Y280 strain resulted in minimal to no clinical signs with significantly lower lung viral titer in the JOL3121 group. Upon SG wildtype challenge, the immunized birds in the JOL3121 group yielded 20% mortality, while 80% mortality was recorded in the PBS control group. Additionally, bacterial load in the spleen and liver was significantly lower in the immunized birds. Conclusions: The current vaccine model, designed with a host-specific pathogen, SG, delivers a robust immune boost that could enhance dual protection against FT and H9N2 infection, both being significant diseases in poultry, as well as ensure public health.

Keywords: H9N2; Salmonella; bivalent vaccine; dual expression system; fowl typhoid; immune response.

Figure 1

(A) A dual expression plasmid was developed to express H9N2 antigens in both Salmonella and eukaryotic cells. For Salmonella expression, HA gene sequences from the H9N2 influenza A virus strains of South Korea were utilized to prepare the HA consensus sequence. For eukaryotic expression, the B cell epitope of NA and the complete M2e sequence linked with furin protease cleavage were cloned for eukaryotic expression. (B) In vivo expression of recombinant H9N2 proteins assessed by Western Blot analysis. The expected immunoreactive bands at 63, 32, and 15 kDa indicated efficient expression of HA, NA, and M2e, respectively. No such bands were detected in the splenic tissues of chickens inoculated with JOL3122 (Vector control; VC). Mp: protein marker. Red arrows showing expected bands of antigens.

Figure 2

Humoral and mucosal immune responses. Chickens were vaccinated orally with PBS, JOL3122, or JOL3121. Blood and cloacal swab samples were collected on days 14 and 28 post-immunization. (A) SG-specific IgY and (B) H9N2-specific IgY were measured using serum samples via indirect ELISA at an absorbance of 492 nm. Similarly, (C) SG-specific secretory IgA and (D) H9N2-specific IgA were assessed using cloacal swab samples. The data were analyzed using two-way ANOVA, with significant differences presented as * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the PBS control.

Figure 3

Cellular immune response post-immunization. Flow cytometry was performed to measure the CD4+ and CD8+ T cells within PBMCs isolated from the immunized birds at day 28 post-immunization. (A) Bar diagram showing the percentages of CD4+ and CD8+ T cells in the PBMCs of immunized birds after stimulation with SG crude protein. (B) Bar diagram showing the CD4+ and CD8+ T cell percentages following stimulation of cells with formalin-inactivated H9N2. (C) Hemagglutination inhibition (HI) titer value against 0.75% chicken RBCs. (D) Changes in the cytokine expression profiles of IFN-γ and IL-4 in orally immunized chickens were determined by qRT-PCR after restimulating the cells with SG crude protein. (E) Cytokine expression profiles of IFN-γ and IL-4 after restimulation with H9N2 inactivated virus. The data were analyzed using one-way ANOVA with Tukey’s multiple comparisons or two-way ANOVA where applicable; with significant differences presented as * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the PBS control.

Figure 4

Evaluation of protection upon wild-type SG challenge. (A) Body weight changes. Changes in chicken body weight (n = 5 per group) were recorded post-challenge to assess the degree of protection against the wild-type challenge. (B) Post-challenge mortality. The survival of immunized birds challenged with the wild-type SG was compared with that of the non-immunized group. A Kaplan–Meier survival curve was developed using mortality records till 14 days post-challenge and immunized birds were compared with the PBS group. (C) Liver morphology. Morphological changes in the liver (n = 5 per group) were examined for hepatic lesions post-challenge. Th yellow colored arrows denote hepatic lesions. (D) Splenomegaly and spleen morphology. Post-challenge spleen weights (n = 5 per group) were measured and compared with those of naïve birds, and the spleen was examined for splenomegaly and other morphological changes post-challenge. (E) Microbial load of the challenge strain in the spleen. (F) The bacterial load of the challenge strain in the liver of the birds (n = 5 per group). The data were analyzed using one-way ANOVA with Tukey’s multiple comparisons or two-way ANOVA where applicable; with significant differences presented as * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the control group.

Figure 5

Histopathological changes and microscopic lesions in chickens orally infected with the wild-type SG strain. Histopathological analysis of the internal organs was performed using H&E staining. (A) Spleen: Disruption of normal cellular alignment and tissue architecture was evident in spleen sections. In the PBS group, degeneration and necrosis of the white pulp were observed, as indicated by arrows. (B) Liver: Liver sections displayed disrupted architecture and inflammatory lesions, along with degeneration and necrosis in the PBS group. Arrows indicate areas of inflammation and congestion. (C) Cecum: The PBS group exhibited tissue disruption in the cecum, including shortened and thickened villi, in contrast to the naïve and vaccinated groups. The arrows denote disturbed, thickened and shortened villi. Tissues from uninfected (naïve) chickens served as healthy controls. Data were visualized and analyzed using light microscopy. Scale bar: 200 µm.

Figure 6

Evaluation of protection against H9N2 challenge. (A) Gross pathology of lung samples collected on day 7 post-challenge with H9N2 Y280 influenza A virus. In the challenged PBS and vector control groups, edema, congestion, and hemorrhages were observed. The arrows indicate congestion and hemorrhages. (B) Photomicrographs of H&E-stained lung sections from chickens on day 7 post-H9N2 challenge. Samples from the PBS and vector control groups showed inflammatory cell infiltrates, edema, and thickened alveolar walls. The arrows indicate the inflammatory cells and thickening of alveolar walls. Scale bar: 200 µm. (C) Viral load in lungs after challenge. The viral load in chicken (n = 5 per group) lung samples was measured by EID₅₀/g using the Reed and Muench method. The data were analyzed using one-way ANOVA with Tukey’s multiple comparisons, with significant differences presented as *** p < 0.001, compared to the PBS and vector control.