Preparation and immunogenicity studies of NvIBDV VP2-ferritin nanoparticles

Abstract

Background: Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is a highly contagious disease that is prevalent worldwide and poses a significant threat to the poultry industry. While commercially available vaccines are used for prevention, IBD outbreaks remain frequent.

Objective: The continuous mutation of virulent strains and their ability to evade traditional vaccine protection complicate IBD control, which necessitates the development of novel vaccines and a deeper understanding of viral mutation mechanisms.

Method: Utilizing the self-assembly capability of ferritin (Fe), the hypervariable region (HVR) protein of a novel variant IBDV (NvIBDV) VP2 was displayed on the ferritin shell, forming regular nanoparticles. The full-length NvIBDV VP2 protein and the NvIBDV VP2-HVR-Fe fusion protein were prokaryotically expressed in E. coli and purified to prepare a VP2 protein vaccine and a VP2-Fe nanoparticle vaccine. An inactivated NvIBDV vaccine served as a control for evaluating immunogenicity and protection.

Results: Recombinant prokaryotic expression vectors pET-VP2-Fe (encoding VP2-HVR-Fe) and pET-VP2 (encoding full-length VP2) were successfully constructed. Soluble VP2-Fe and VP2 proteins were expressed and purified. Electron microscopy confirmed the formation of a cage-like nanoparticle structure for VP2-Fe. Immunization of SPF chickens with NvIBDV VP2-Fe nanoparticles induced a robust immune response characterized by high antibody titers and a significantly high protection rate against viral challenge.

Conclusion: The successfully constructed recombinant subunit nanoparticle vaccine, which displays the NvIBDV VP2 HVR on ferritin, effectively increased the antibody titer and provided superior immune protection. This approach offers a feasible strategy for developing novel IBDV subunit vaccines.

Keywords: Ferritin; Infectious bursal disease virus; Nanoparticles; Novel mutant strain; Subunit vaccine.

Fig. 1

Construction of VP2-Fe, VP2 recombinant plasmid. (Figures A-C show the cropped images.) (A) Linearization of the target gene VP2 and the prokaryotic expression vector pET-32a M: DL 5000 DNA Marker; 1: indicates the full length of VP2. 2: VP2-pET-32a carrier; 3: VP2 high variable region; 4: VP2-Fe-pET-32a carrier. (B) pET - VP2, pET - VP2 - Fe bacteria liquid PCR M: DL 5000 DNA Marker; 1–5: pET-VP2 single colony; 6–10: pET-VP2-Fe single colony. (C) Recombinant plasmid pET - VP2 - Fe, pET - VP2 enzyme identification M: DL 5000 DNA Marker; 1: uncut pET-VP2-Fe; 2: pET-VP2-Fe after double digestion; 3: uncut pET-VP2; 4: pET-VP2 after double digestion

Fig. 2

Expression of VP2-Fe, VP2 recombinant protein. (Figures A-F show the cropped images.) (A)/(B) VP2-Fe and VP2 under different temperatures and precipitation on the expression of recombinant proteins A: VP2-Fe recombinant protein; B: VP2 recombinant proteinM: Protein molecular quality standard; 1: no induced supernatant; 2: no induced precipitation; 3:37 °C supernatant; 4:37 °C precipitation; 5:30 °C supernatant; 6:30 °C precipitation; 7:25 °C supernatant; 8:25 °C precipitation; 9:20 °C supernatant; 10:20 °C precipitation; 11:16 °C supernatant; 12:16 °C precipitation. (C) Different induction time of VP2-Fe and VP2 clear expression on the results M: Protein molecular quality standard; 1: VP2-Fe not induce supernatant; 2–6: VP2-Fe induced, respectively 6 h, 12 h, 18 h, 24 h, 30 h; 7: VP2 did not induce supernatant; 8–12: VP2, respectively induced 6 h, 12 h, 18 h, 24 h, 30 h. (D) Different IPTG concentration of VP2-Fe and VP2 clear expression on the results M: Protein molecular quality standard; 1: Supernatant induced by pET-32a empty carrier; 2–7: The final concentration of IPTG was 0.2, 0.4, 0.6, 0.8, 1, 1.2 mmol/L to induce 24 h VP2-Fe recombinant protein supernatant; 8–13: The final concentration of IPTG was 0.2, 0.4, 0.6, 0.8, 1, 1.2 mmol/L to induce 18 h of VP2 recombinant protein supernatant (E)/(F) Purification results of VP2-Fe and VP2 recombinant proteins E: VP2-Fe recombinant protein; F: VP2 recombinant protein M: Protein molecular quality standard; 1: flow through liquid; 2: The first wash; 3: The second wash; 4: First elution; 5: Second elution; 6: third elution; 7: Fourth elution; 8: Fifth elution; 9: Dialysis concentration (G) BCA protein standard curve

Fig. 3

Western blotting assay and electron microscopy of VP2-Fe, VP2 recombinant protein. (Figures A show the cropped images.). (A) VP2 - Fe, VP2 recombinant protein of Western blotting test results M: Protein molecular quality standard; 1: VP2-Fe recombinant protein; 2: VP2 recombinant protein. (B)The structure of VP2-Fe recombinant protein was observed under electron microscopy

Fig. 4

Vaccine quality test results. (A) Centrifuge after emulsification. (B) Coating plate after emulsification. (C) Emulsify and drop into the water

Fig. 5

Detection of IBDV antibody levels in serum and proliferation of peripheral blood lymphocytes. (A) The antibody titers of chickens in each group were detected by ELISA(*** P < 0.001, ns P > 0.05). (B) Peripheral blood lymphocyte proliferation results(*** P < 0.001,** P < 0.01,* P < 0.05)

Fig. 6

Attack Protection Experiment. (A) Comparison of bursa of Fabris and spleen in each group 7 days after challenge. (B) Groups of spleen index, the index of bursa results figure(** P < 0.01, ns P > 0.05). (C) The standard curve of real-time fluorescent quantitative q-PCR for PMD18-T-VP2