Immunogenicity Of Hemagglutinin Protein Of H9N2 Subtype Avian Influenza Virus Expressed In Rice Endosperm And Identification Of B Cell Epitopes

The H9N2 subtype avian influenza virus(AIV)is a low-pathogenicity type A influenza virus belonging to the Orthomyxoviridae family and the Influenza virus genus.H9N2 AIV is widely prevalent worldwide and causes significant economic losses.Due to its broad host range,tendency for avian-to-human transmission,and rapid antigenic variation,it has attracted widespread attention.Furthermore,there is evidence that H9N2 AIV can provide internal genes to produce new avian influenza viruses that infect humans,such as H7N9,H10N8,and H5N6,leading to the emergence and evolution of recombinant avian influenza viruses,posing a persistent threat to public health.Currently,the prevention of avian influenza mainly relies on vaccination.China launched a national poultry vaccine program in 1998,and commercial H9N2 AIV inactivated vaccines have been used in Chinese poultry farms for about 25 years.However,the virus is still widely prevalent in China and has become the main subtype of influenza virus in chicken and duck populations.Therefore,the development of a new generation of safe,efficient,and low-cost H9N2 AIV vaccines is urgent.Hemagglutinin(HA)is the main surface glycoprotein of influenza virus,which participates in binding to host cell receptors and membrane fusion during influenza virus infection.The HA protein induces the body to produce neutralizing antibodies that neutralize the virus,so it is the main target of immune protection and usually used as an immunogen for subunit vaccines.However,HA proteins produced by traditional expression systems have limitations such as low immunogenicity,high cost,and safety concerns.Currently,there is no commercialized subunit vaccine for avian influenza.Transgenic plants have been used as recombinant protein expression systems for nearly 30 years,with advantages such as safety,low cost,and easy scalability.In recent years,many plant-produced recombinant proteins have been shown to be comparable to those produced by traditional expression systems.Therefore,our laboratory previously conducted research on the expression of structural antigens in transgenic rice.Based on this,our study focused on the recombinant H9N2 AIV HA subunit vaccine expressed in rice and screening B cell epitopes.The detail content is as follows:Part 1: Design and N-glycosylation analysis of H9N2 AIV HA-dimer protein expressed in riceIn order to develop a safe and efficient H9N2 AIV subunit vaccine,we designed a "head-to-tail" dimer immunogen based on the spacing of B-cell receptors(BCRs)on B cells,which allows for crosslinking of multivalent antigens.We successfully obtained rice recombinant HA dimer protein(Oryza sativa recombinant HA dimer,Osr2HA)through the rice expression system.The purified Osr2 HA protein was identified,and the results showed that the molecular weight of the Osr2 HA protein was approximately 130 k Da,with an average particle size of 12.32 nm.Most of the sequences,including linking peptide(GGGGS)3,were detected by mass spectrometry,indicating that the expressed Osr2 HA conformed to the expected design.Biological activity tests showed good reactivity of Osr2 HA with H9N2 AIV chicken positive serum.The expression level of the recombinant protein was increased to 101 mg/kg by hybridization,which was 2.6 times higher than before hybridization.N-glycosylation profiling analysis of the Osr2 HA protein was performed,and a total of 68 complete N-glycopeptides were identified,located in 7 peptides and 3 glycosylation sites at positions 123,200,and 474 of the N-asparagine residues.The predominant sugar types in the glycans were mannose,α-1,3-fucose,and β-1,2-xylose.These results provide a foundation for the development and immunogenicity evaluation of H9N2 plant-derived recombinant subunit vaccines.Part 2: Immunogenicity study of rice-expressed H9N2 AIV hemagglutinin proteinThe immunogenicity of the "head-to-tail" Osr2 HA vaccine expressed in rice was evaluated using SPF chickens.To verify that the designed protein could elicit highaffinity antibodies and meet the requirements for low-dose,high-efficiency immunization,Osr2 HA vaccine was used for two immunization(Osr2HA protein levels of 1 μg,3 μg,6 μg,9 μg)and a single immunization(Osr2HA protein levels of 3 μg,6μg)to verify the immune effect.Immunization evaluation results showed that both single-dose and two-dose immunization in SPF chickens maintained hemagglutination inhibition(HI)antibody titers above 24 for 42 days,and the HI titer of the two immunized 6 μg group reached 28.The geometric mean titer of the neutralizing antibody antibodies reached 27.6 after two immunizations of 6 μg,and the neutralizing antibody titer reached 26.6 after a single immunization of 6 μg.After virus challenge,the negative control group appeared clinical symptoms and histopathological observations revealed primarily inflammatory changes,with a 15.2% decrease in body weight.Additionally,14 days after challenge,the weight gain of SPF chickens immunized twice with 6 μg of Osr2 HA protein was 10.3% higher than that of commercial inactivated vaccines,which could generate higher economic value in the poultry farming industry.Part 3: Vaccine immunization optimization of rice-expressed H9N2 AIV Osr2 HA vaccineIn order to improve the immunogenicity of Osr2 HA vaccine,the present study optimized the vaccine’s buffer system,adjuvants,storage temperature,and coimmunization with other antigens.The evaluation of immune response based on the level of HI antibodies induced by the vaccine.The results are summarized as follows:(1)Optimization of the rice-expressed Osr2 HA vaccine was performed by selecting a more suitable Tris-HCl buffer system.After storage at room temperature for 114 days,the antibody titers produced in SPF chickens immunized with the optimized vaccine showed no significant difference compared to the vaccine stored at 4°C.The HI titers remained above 26 during the 63-day testing period.This provides the possibility of developing an influenza vaccine that can be stored at room temperature.(2)Co-immunization with rice-expressed Osr2 HA and Newcastle disease virus Osr2HN(Oryza sativa recombinant HN dimer,Osr2HN)protein was performed.SPF chickens were immunized with a single dose of 10 μg.The antibody levels produced were not significantly different from those of individual immunizations,indicating that the Osr2 HA vaccine maintained good immunogenicity in the presence of other antigens,allowing for the preparation of multivalent vaccines tailored to specific needs,implementing a "one shot,multiple protections" vaccine strategy.(3)The HI antibody titers induced by a single dose of 10 μg Osr2 HA vaccine stored at room temperature remained at a high level for up to 105 days.The H9N2 AIV-specific HI antibodies remained above 28,with no significant difference compared to the commercial inactivated vaccine.Part 4: Study on the oral and intranasal immunization effects of rice-expressed H9N2 AIV hemagglutinin proteinTo investigate the mucosal immune effects of Osr2 HA,in this study,intranasal vaccines were prepared using SEPPIC MONTANIDE(?) GEL 02 adjuvant combined with Osr2 HA,and oral vaccines were prepared by mixing saponin crude extract with Osr2 HA protein.The intranasal and oral vaccines containing Osr2 HA doses of 1 μg,3 μg,9 μg,and 27 μg all induced effective immune responses.Among them,the Ig G and HI antibody levels induced by the 9 μg Osr2 HA intranasal vaccine and 27 μg Osr2 HA oral vaccine were not significantly different from those induced by the Osr2 HA subcutaneous injection vaccine.Besides,the levels of Ig A antibodies in mouse serum and bronchoalveolar lavage fluid induced by the 9 μg Osr2 HA intranasal vaccine and 27 μg Osr2 HA oral vaccine were significantly higher than those induced by the Osr2 HA subcutaneous injection vaccine.These results provide a basis for the development of intranasal and oral vaccines using Osr2 HA.Part 5: Identification of H9N2 AIV HA-specific monoclonal antibodies and B-cell epitope recognitionThe designed recombinant HA dimer antigen in this study aims to fully expose antigenic epitopes,facilitating the screening and detailed analysis of new epitopes.Monoclonal antibodies generated using rice-expressed Osr2 HA protein as the immunogen successfully identified a novel linear B-cell epitope,480HKCDDQCM487.Spatial localization revealed that this epitope is located at the C-terminus of HA2,near the disulfide bonds of HA1 and HA2.Sequence homology analysis showed that this epitope is conserved among H9N2 subtype avian influenza viruses,providing a basis for H9N2 AIV differentiation,diagnosis,and vaccine strategies,and laying the foundation for H9N2 AIV prevention and control.In conclusion,this study developed a novel rice-expressed recombinant HA subunit vaccine using the rice endosperm expression system.It lays the foundation for the development of novel influenza vaccines,providing candidate vaccines.The monoclonal antibodies generated using Osr2 HA protein identified a new linear B-cell epitope,providing a new epitope target for the differentiation and diagnosis of H9N2 AIV.