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. 2025 Jan;104(1):104602.
doi: 10.1016/j.psj.2024.104602. Epub 2024 Nov 27.

Expression and delivery of HA1-M2e antigen using an innovative attenuated Salmonella-mediated delivery system confers promising protection against H9N2 avian influenza challenge

Affiliations

Expression and delivery of HA1-M2e antigen using an innovative attenuated Salmonella-mediated delivery system confers promising protection against H9N2 avian influenza challenge

Ram Prasad Aganja et al. Poult Sci. 2025 Jan.

Abstract

This study explores a dual expression vector system for delivering prokaryotic and eukaryotic antigens to improve conventional vaccination strategies. To enhance immune protection against H9N2 avian influenza virus (AIV), which threatens poultry and humans, we used the previously constructed pJHL270 and pJHL305 plasmids with the Ptrc and CMV promoters to stimulate MHC class II and I responses through exogenous and endogenous antigenic presentation. Salmonella Gallinarum (SG), a delivery vector, was engineered to have defective lipopolysaccharide structures through lon, pagL, and rfaL deletion. It demonstrated a safety profile with lower induction of inflammatory cytokines than the wild-type strain. Bioinformatics tools predicted that the HA1 and M2e sequences, which were designed as consensus sequences of South Korean strains (2000-2021), would have high antigenicity and favorable structures. In vitro expression of the vaccine constructs was validated by western blotting. Birds immunized with attenuated SG harboring pJHL270 (JOL3025) or pJHL305 (JOL3027) containing HA-M2e showed significant increases in serum IgY and mucosal IgA antibodies, indicating strong humoral and mucosal immune responses, comparable with inactivated commercial vaccine. Post-immunization, we found a substantial rise in the hemagglutination inhibition titer, suggesting effective prevention of viral attachment and robust cell-mediated immunity, with a 1.96-fold and 2.80-fold increase in CD4+ and CD8+ T cells, respectively, for JOL3025 and a 1.75-fold and 2.49-fold increase for JOL3027. Furthermore, MHC class I and II expression increased 1.35-fold and 1.63-fold, for JOL3025, and 1.61-fold and 1.68-fold, respectively, for JOL3027. The IL-4 and IFN-γ levels were elevated, indicating a balanced Th-1 and Th-2 response. Post-challenge, birds immunized with vaccine candidates or the commercial vaccine exhibited minimal to no clinical signs, reduced lesions, lower lung viral titers, and negligible impacts on egg production compared to controls. In conclusion, both plasmids successfully delivered HA1-M2e immunogens through the engineered SG strains, eliciting strong humoral, mucosal, and cell-mediated immune responses and co-stimulating MHC class I and II antigen presentation pathways to provide effective protection against H9N2 AIV with minimal adverse effects.

Keywords: Cell-Mediated response; Dual expression system; H9N2; Salmonella; Viral titer.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig 1
Fig. 1
Conceptual layout of plasmids and their expression. A. Expression and stimulation of immune response through the pJHL270 and pJHL305 vector plasmids for dual expression. B. The consensus sequence of hemagglutinase-P2A-matrix protein 2e (HA1-P2A-M2e) was introduced within the eukaryotic region of the plasmids. An optimized sequence of hemagglutinin-glycine linker-matrix protein 2e from the H9N2 strain was cloned in the prokaryotic expression system. The expression of immunogens via the prokaryotic expression system elicits T helper cells, especially CD4+ T cells, to activate B cells through antigen-presenting cells and induce a humoral immune response. The transcribed mRNA of HA1-P2A-M2e is translated to produce cleaved HA1 and M2e that can be presented through MHC class I molecules and stimulate CD8+ T cells. DC: Dendritic cells, ER: Endoplasmic reticulum, PRR: Pathogen recognition receptor, CSE: Conserved sequence elements, nsp1–4: Non-structural proteins 1–4.
Fig 2
Fig. 2
Structural prediction of the recombinant antigens. A. Linear B cell epitope for individual HA1 and M2e antigens and the combined HA1-M2e antigen predicted by the IEDB server. B. Structural validation of the selected antigens, shown using a Ramachandran plot. C. In silico 3D structures of the selected antigens, highlighting their conformational accuracy. D. The structural quality of the selected antigens predicted based on Z-scores.
Fig 3
Fig. 3
Expression of recombinant proteins and attenuated Salmonella Gallinarum strain endotoxicity. A. Assessment of the endotoxicity induced by the attenuated Salmonella Gallinarum strain was measured using the concentration of TNF-α, IL-1β, and IFN-γ in serum. Data were analyzed by Dunnett's multiple comparison test, with significant differences presented as *p < 0.05, **p < 0.01, and ****p < 0.0001, compared with the PBS control. B. Western blot analysis of LPS. C. Expression of mRNA for HA1-M2e in BMDM cells using bactofection. D. Western blot analysis of recombinant protein expression.
Fig 4
Fig. 4
Immunization schema and immune response. A. Details of the immunization schedule and the experimental plan for the study. The concentration of B. IgY and C. IgA in sera and cloacal swabs was determined by indirect ELISA to evaluate the systemic humoral and mucosal immune responses in immunized birds 28 days post-immunization.
Fig 5
Fig. 5
Cellular immune response post-immunization. A. Hemagglutination inhibition (HI) titer value against 0.75 % chicken RBC. B. Histogram representation of the percentages of CD4+ and CD8+ T cells in immunized birds. C. Representative flow cytometry scatterplots showing the gating of CD4+ and CD8+ cells post-immunization. Data were analyzed by multiple comparison tests, with significant differences presented as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with the PBS control.
Fig 6
Fig. 6
Post-immunization immune response. A. Representative flow cytometry scatterplots showing the gating of MHC class I and II cells post-immunization. B. Histogram representing the percentage of MHC class I and II cells in immunized birds. C. The expression of cytokines (IL-4 and IFN-γ). Relative fold changes in the expression of the cytokines were determined by quantitative real-time PCR after stimulating PBMCs with recombinant protein. The changes were measured at the mRNA transcript level using the 2-∆∆CT method, with GADPH as the internal control. Data were analyzed by multiple comparison tests, with significant differences presented as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with the PBS control.
Fig 7
Fig. 7
Morphological and histopathological changes in the lung. A. Gross pathological observation of lung samples collected on the 7th-day post-challenge with H9N2 influenza A virus. In both the control and mock control groups, edema, congestion, and hemorrhage were noted. The blue arrow indicates congestion and hemorrhage. B. Photomicrographs of H&E-stained lung sections of chickens on the 7th-day post-H9N2 challenge. Chickens (N = 15) were vaccinated with JOL3025, JOL3027, JOL3028, JOL3029, or a commercial vaccine (inactivated Y280 strain). Two weeks later chickens were booster immunized with the developed strains. Three weeks after the booster immunization, all the vaccinated chickens were challenged with 1 × 106 EID50 H9N2 virus. On the 7th-day post-challenge, chickens (n = 5) were sacrificed, and lung tissues were collected for histological analysis. Chickens that received PBS, JOL3028, or JOL3029 (vector controls) showed hyperemia with infiltration of mononuclear inflammatory cells in their bronchi and lung parenchyma, whereas birds vaccinated with JOL3025 or JOL3027 showed minimal inflammatory lesions. The black arrow indicates inflammatory cell infiltrates, edema, and thickening of the alveolar walls. The green arrow indicates the desquamation of epithelial cells. C. TUNEL staining of the oviduct in H9N2-infected hens. Oviducts were collected 7 days post-challenge, paraffin-embedded, sectioned, and stained for apoptotic nuclei by terminal deoxynucleotidyl transferase (TdT), which binds to exposed 3’-OH ends of DNA fragments generated in response to apoptotic signals. The brown spots indicated by open arrows represent apoptotic cells. Oviducts from the naïve group (virus-free hens) served as the negative control. Images at 200 × magnification.
Fig 8
Fig. 8
Post-infection changes. A. Post-challenge viral load in bird lung samples evaluated by EID50/g using the Reed and Muench method. B. The effects of H9N2 infection on the egg production rate.

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