Nanoparticle-Based mRNA Vaccine Induces Protective Neutralizing Antibodies Against Infectious Bronchitis Virus in In-Vivo Infection

Abstract

Background: Live attenuated and inactivated virus vaccines are commonly used against infectious bronchitis virus (IBV) in chickens, but they have limitations such as mutation risks and short efficacy. This study explores cationic bovine serum albumin (BSA) polyamine nanoparticles (NPs) for delivering IBV spike protein mRNA, aiming to develop a safer and more effective vaccine. Methods: A BSA-based nanoparticle system was designed with positive surface charges and characterized using dynamic light scattering (DLS), Zetasizer, and transmission electron microscopy (TEM). Its cytotoxicity, cellular uptake, and ability to deliver IBV spike protein mRNA were evaluated in macrophage-like chicken cell lines (HD11), followed by immunogenicity studies in SPF chickens to assess immune responses. Results: The study demonstrated successful binding and transfection efficiency of the in vitro transcription (IVT)-mRNA complexed with the NPs, which was enhanced with chloroquine. Immunogenicity studies in SPF chickens showed a significant increase in antibody titers in chickens vaccinated with the mRNA vaccine compared to the PBS control, indicating an effective immune response against the IBV S protein. Furthermore, the neutralization index doubled after a higher-dose mRNA booster with chloroquine, and PBMCs from immunized chickens exhibited a threefold higher stimulation index than the PBS control. Conclusions: BSA-based NPs effectively deliver IBV spike protein mRNA, enhancing immune responses and offering a promising strategy for a safer, more effective IBV vaccine.

Keywords: infectious bronchitis virus; mRNA vaccine; nanoparticles.

Figure 1

Characterization of BSA-PEHA NPs. (A) Dynamic Light Scattering of BSA-PEHA (10 mM phosphate buffer pH 7.0); (B) Zeta potential; (C) TEM image of BSA-PEHA (pH 4.0); (D) Circular Dichroism shows no loss in ellipticity for BSA-PEHA (blue) compared to BSA (red). All samples show a double minimum at 208 and 222 nm.

Figure 2

(A) Cell viability of BSA-PEHA NPs treated HD11 cells using MTT assay. Cells were treated with 100 to 0.097 µg/mL of BSA-PEHA NPs and incubated at 24, 48 and 72 h. [p values < 0.05 (*), <0.01 (**)]. (B) Cellular uptake study of BSA-PEHA NPs using confocal microscopy. Scale bar: 20 µm. (C) Electrophoretic Mobility Shift assay of BSA-PEHA and eGFP mRNA binding. Lane 1: mRNA, Lane 2: BSA-PEHA (25 µg/mL) and 1 µg mRNA, Lane 3: BSA-PEHA (25 µL, 25 µg/mL) and 2 µg mRNA, Lane 4: BSA-PEHA (25 µg/mL) NPs. (a) Gel image taken in a UV transilluminator (b) Gel after staining with Coomassie blue. pH of the phosphate buffer was maintained at 7.0.

Figure 3

(A) CD spectra of BSA-PEHA, mRNA and BSA. (B) Expression of eGFP encoding mRNA using Lipofectamine 2000 and BSA-PEHA NPs as the delivery vehicle in HD11 cells. (a) Expression of eGFP encoding mRNA with Lipofectamine 2000 (10×). (b) Expression of eGFP encoding mRNA with BSA-PEHA (25 µg/mL) (40×). (c) Expression of eGFP encoding mRNA BSA-PEHA (25 µg/mL) in presence of chloroquine (25 µM) (10×). (C) Denaturing gel electrophoresis of IBV S protein encoding mRNA. (D) Western blot image of IBV S protein encoding mRNA (128 kDA) in HD11 cells expressed in presence and absence of chloroquine.

Figure 4

(A) Titer of antibody against IBV S protein in different groups after first (p values < 0.0001 (****), and (B) second dose. (C) PBMC stimulation with killed IBV virus. (D) Virus neutralization study in embryonated chicken eggs. When the virus is neutralized, embryos exhibit normal development (left two). In contrast, if the virus remains active, the embryos display stunted growth (right three) (E) Serum neutralization assay result. Serum neutralization of chickens immunized with different combinations of BSA-PEHA/IVT IBV S protein encoding mRNA at two and three weeks after vaccination.