1. Introduction
Avian influenza, often referred to as AI, is a severe and rapidly spreading disease which is mainly caused by avian influenza virus (AIV), and the latter belongs to the Orthomyxoviridae family [
1,
2]. In 1966, H9N2 subtype AIV was reported to have been isolated from turkey flocks in Wisconsin for the first time [
3]. The low pathogenicity H9 subtype AIV was initially reported in China in 1994 [
4]. Notably, the 1997 AI outbreak in Hong Kong led to substantial poultry mortality and marked the first instances of avian-to-human transmission [
5], posing a grave threat both to the healthy development of the poultry industry and to public health and safety.
The AIV genome encodes various viral structural proteins, in which alkaline polymerase 1 (PB1), alkaline polymerase 2 (PB2), and acid polymerase (PA) together constitute the virus RNA-dependent RNA polymerase complex. This complex is responsible for catalyzing virus RNA synthesis and plays a vital function in virus replication and transcription [
6]. Among these components, the functional domains of the PB1 protein encompass the polymerase active region situated in the middle, the N-terminal nuclear localization (NLS), and interaction domains with PB2 and PA subunits [
7]. The NLS at the N-terminal of PB1 directs its nuclear localization, which is a crucial process during viral RNA replication and transcription. The RNA polymerase’s active region, consisting of finger and palm domains, is responsible for transcribing RNA templates into mRNA [
8]. Additionally, the interaction between PB1 and the cellular importin α/β complex mediates the translocation of viral RNA polymerase into the nucleus [
9].
Mutations of the PB1 protein significantly influence the adaptability and pathogenicity of the influenza virus [
10]. It has been reported that specific amino acid positions in PB1, such as 66S, 198K, and 701M, can enhance the pathogenicity of avian influenza viruses [
11,
12]. Furthermore, the PB1 protein interacts with host immune regulatory proteins, including interactions with RIG-I (Retinoic Acid-Inducible Gene I) receptors, which modulate the type I interferon (IFN-I) pathway within host cells [
13]. Additionally, viral PB1 protein can degrade the innate immunity of the crucial adapter protein MAVS through selective autophagy mediated by host NBR1, hindering innate immune pathway signaling and type I interferon production [
14], thereby promoting influenza virus replication. Therefore, the PB1 protein plays a significant role in the viral infection process.
Recently, H9N2 subtype AIV has increasingly become a predominant strain among clinical epidemics. It is essential to further examine the key determinants within the PB1 protein of the H9N2 subtype. The monoclonal antibodies targeting the PB1 protein and the identification of B cell epitopes hold significant implications for AIV infection diagnosis, vaccine development, antiviral drug discovery, and immunotherapy. To probe the novel determinants of the PB1 protein, in this study, a highly antigenic region of PB1 was selected and cloned, and expressed using a prokaryotic expression system. Following immunization of mice and cell fusion, four monoclonal antibodies targeting the PB1 protein were successfully generated, and two B cell epitopes were identified. Moreover, the analysis of the conservation and structural characteristics of these B cell epitopes plays a crucial role in understanding the function and antigenic structure of the PB1 protein in AIV, which facilitates a deeper comprehension of the viral pathogenic mechanism and offers guidance for prevention and novel therapeutic strategies against avian influenza.
2. Materials and Methods
2.1. Virus and Cells
H9N2AIV (strain A/chicken/Shandong/LY1/2017), examined in this research, was collected and maintained by our laboratory as referenced [
15]. It was amplified in the allantoic fluid of 9-day-old specific-pathogen-free (SPF) eggs.
For the cultivation of SP2/0 cells, we utilized RPMI-1640 medium (350-000-CL, Wisent Biotechnology, Nanjing, China) supplemented with 20% Fetal Bovine Serum (FBS, C04001-500, VivaCell, Shanghai, China), along with 1% penicillin and 100 µg/mL streptomycin. The cells were kept in an incubator at 37 °C and 5% CO2.
HeLa cells were grown in a mixture of RPMI-DMEM medium (same source as above) containing 10% FBS (FSP500, ExCell, Suzhou, China), and the same concentrations of antibiotics as used for the SP2/0 cells. These cells were also maintained at 37 °C and 5% CO2.
2.2. Gene Amplification and Recombinant Expression of PB1
This step was based on the PB1 amino acid sequence from GenBank (MH018675.1). The PB1 protein’s antigenic hydrophobicity was utilized to identify a target region spanning amino acids 1 to 245 with a high likelihood of harboring B cell epitopes. Specific primers designed using Snapgene are detailed in
Table 1.
According to the TRIZOL reagent procedure, allantoic fluid infected with H9N2 AIV was collected, total viral RNAs were extracted, and reverse transcription was employed to generate cDNA templates for PCR amplification. PCR conditions started with 94 °C initial denaturation, for 5 min, followed by 35 cycles of 94 °C denaturation for 30 s, 56 °C annealing for 30 s, and 72 °C extension for 2 min. PCR products were digested using BamH I and Hind III restriction enzymes and ligated into the pET-28a(+) vector, followed by transformation into E.coli BL21(DE3) strain to express the PB1 recombinant protein. The soluble recombinant protein was obtained and confirmed by SDS-PAGE and Western blotting.
Purification of recombinant PB1 proteins were conducted via Nickel Affinity Chromatography. Imidazole elution buffer containing 50–500 Mm of different concentrations of imidazole (imidazole, 50 mM Tris, 0.05 M NaCl) was used to elute the target protein, and the 80–150 Mm concentrations of imidazole eluate was collected as immune target protein.
2.3. Mouse Immunization
Five female BALB/c mice aged 6~8 weeks were immunized via intraperitoneal injection with 100 μg purified PB1 protein and Freund’s complete adjuvant by full oscillation emulsification. Then, on the 14th and 28th days after the initial immunization, the mice were immunized with PB1 protein and Freund’s incomplete adjuvant. Seven days after the last immunization, the antibody titers of the mouse serum were assessed using an enzyme-linked immunosorbent assay (ELISA), with purified PB1 recombinant protein employed as the coated antigen. Mice exhibiting high antibody titers were intraperitoneally injected with 200 μg PB1 protein without adjuvant on the third day prior to cell fusion to boost immunity.
2.4. Cell Fusion and Monoclonal Antibody (mAb) Preparation
Splenocytes were isolated from the boosted mice, and then fused with SP2/0 cells under the presence of polyethylene glycol (PEG4000) solution (P7171, SIGMA, St. Louis, MO, USA) on the third day following booster immunization. The fused cells were cultured with 20% FBS, 100 mg/mL streptomycin, 100 U/mL penicillin, and 1% HAT medium. The supernatant from the fused cells was screened by ELISA. The selected hybridoma cell line was then injected into the BALB/c mice to generate the ascites antibody. The type and isotype of the monoclonal antibody was detected with a mouse mAb isotype identification kit (PK20003, Proteintech, Rosemont, IL, USA). Subsequently, the specificities of the mAbs were assessed using Western blot and indirect immunofluorescence (IFA) techniques.
To enhance the titer of mouse ascites, each mouse was sensitized by intraperitoneal injection of 500 μL Freund’s incomplete adjuvant for one week, followed by injection of 200 μL containing 105 to 106 hybridoma cells. Ascites samples were collected one week after immunization.
2.5. ELISA Detection
To establish an indirect ELISA diagnosis, the purified recombinant PB1 protein served as the coating antigen. A total of 2 μg/mL purified recombinant PB1 protein was coated in CBS buffer (0.2 mol/L Na2CO3, 0.2 mol/L NaHCO3, pH = 9.6) at 4 °C overnight. After washing, the plate was blocked with PBST containing 5% skim milk and washed by PBST three times. Subsequently, the supernatant of hybridoma cells was incubated with the coated PB1 protein at 37 °C for 1 h. After washing, 100 μL with a dilution ratio of 1:5000 of HRP-labeled goat anti-mouse IgG antibody was incubated at 37 °C for 45 min. Next, 100 μL of TMB color solution was added into the plates, and color development proceeded at 37 °C for 15 min, and then was halted with 50 μL KPL termination solution. Absorbance was checked at 450 nm using an enzyme labeling instrument.
Furthermore, the titer of collected mouse ascites was determined by ELISA, and the subtypes of monoclonal antibodies were identified using a subtype identification kit (PK20002, Proteintech, Rosemont, IL, USA). The ascites antibody was diluted with 1× PBST at a ratio of 1:100,000 (50 μL/well). Sheep anti-mouse IgA + IgM + IgG-HRP was incubated with screened mAbs in plate wells for 1 h, and washed three times. Finally, the color developer and termination buffer were added into each well, and the data were analyzed following absorbance detection with a microplate reader.
2.6. IFA Experiment
HeLa cells were infected with H9N2 subtype AIV (MOI = 0.1) at 37℃, 5% CO2 for 24 h. Normal HeLa cells that were not infected with the virus were set up as a negative control. After washing, the cells were fixed with 4% methanol for 10 min at room temperature, and treated with 0.1% Triton for 10 min. Then, cells were incubated with the screened mAbs at 4 °C overnight. Then, the cells were incubated with CoraLite594-conjugated goat anti-mouse IgG (H + L) fluorescent antibody (SA00013-3, Proteintech, Rosemont, IL, USA) for 45 min, followed by incubation with DAPI. Subsequently, the cells were observed by fluorescence microscopy (Axiovert A1, Carl Zeiss AG, Jena, Germany).
2.7. Immunoblotting Analysis
The collected protein samples were separated through SDS-PAGE electrophoresis. The gel containing the target protein samples was then stained in Coomassie Brilliant Blue solution for 1 h and decolorized with decolorization solution.
For immunoblotting analysis, the gel containing the target protein was transferred onto a PVDF membrane pre-soaked in methanol following SDS-PAGE. Then, the transferred PVDF membrane was immersed in PBST solution containing 5% skim milk for 2 h, and was incubated with the corresponding primary antibody overnight at 4 °C on a shaking table. Subsequently, HRP-labeled goat anti-mouse IgG (H + L) diluted at 1:5000 was incubated with PVDF membrane at 37 °C for 45 min. After washing with PBST three times, the results of exposure and color development were observed under a chemiluminescence imager.
2.8. Design of Truncated PB1 Gene and Identification of B Cell Epitope
To ascertain the B cell epitope region recognized by the monoclonal antibody, a recombinant truncated plasmid was constructed for identification purposes. The truncated PB1 gene was designed according to the prediction results for the PB1 protein epitope by the Protean 7.1.0 software antigenicity binding website (
http://tools.iedb.org/bcell/ (accessed on 3 January 2024)). The designed truncated gene, PB1-1 and PB1-95, was cloned by PCR and subsequently inserted into the pET-28a(+) vector utilizing restriction endonuclease
EcoR I and
Sal I. The positive recombinant plasmid was then transformed into
E. coli BL21 (DE3 strain) for expression. Monoclonal antibodies and His-labeled antibodies were used as primary antibodies for Western blotting identification. To determine the final epitope, the truncated gene PB1-67, PB1-17, PB1-27, PB1-37, PB1-47, and PB1-57 was designed, and a prokaryotic expression plasmid was constructed using the cloned PB1 gene fragment and the pEGX-4T-1 plasmid. The recombinant plasmid was expressed and identified by Western blotting using a monoclonal antibody and a GST-tagged antibody. Primers for the aforementioned truncated genes are listed in
Table 1.
2.9. Bioinformatics
The homology of the epitopes of the four monoclonal antibodies was examined using MEGA-X 10.1.7 software to investigate the formation of B cell epitope homology of the PB1 protein among different subtypes of AIV. Additionally, the structure of the PB1 protein of H9N2 AIV was predicted using the Zhang Lab’s tool (
https://zhanggroup.org/I-TASSER/ (accessed on 26 February 2024)). Pymol 2.6 software was utilized to analyze the spatial characteristics and biological functions of the PB1 3D model and B cell epitopes based on the predicted modeling results.
4. Discussion
AIV infection poses a disastrous threat to the health of the poultry industry and human public safety. As a crucial component of core RNA polymerase in AIV, the PB1 protein plays a pivotal role during viral replication and regulation [
16,
17]. Screening viral protein epitopes with monoclonal antibodies is crucial for understanding the characteristics of viral proteins, identifying specific regions of the PB1 protein that elicit B cell immune responses, and revealing their importance in virus–host interactions.
In this study, we selected the region amino acids 1 to 245 in the PB1 protein of the H9N2 subtype AIV based on hydrophobicity and surface probability indices, which exhibited high antigenicity and B cell antigenic determinants. After recombinant truncated PB1 protein expression and mice immunization, four monoclonal antibodies (2H9, 3E6, 5B3, and 5F12) against the PB1 protein were screened based on cell fusion. Their reactivity with the viral PB1 protein was confirmed through various methods, including ELISA, Western blotting, and IFA. The results demonstrated that all four monoclonal antibodies could specifically recognize the PB1 protein.
Studies on B cell epitopes of the PB1 protein in AIV are limited. Some studies have identified potential B cell epitopes of the PB1 protein, providing insights into its immunogenicity and immune protection. The antibody epitopes in influenza internal proteins have been identified using serum from survivors of H5N1 infection, including the PB1 epitopes PB1
1348MISKCRTKEGRRKT
1361 and PB1
1420TNGTSKIKMKWGMEMRRC
1437 [
18]. Also, a bioinformatics tool has been utilized to design peptides with high immunogenicity for immunization and screen B cell epitopes for the PB1 protein, including PB1 202–221, PB1 180–199, and PB1 458–477 [
19]. In our study, two B cell epitopes,
67NPIDGPLPED
76 and
97TITYSSPMMW
106, were successfully identified by constructing truncated epitopes of screened monoclonal antibodies, which were the first reported PB1 protein determinant. Furthermore, through gradually truncating the PB1 protein, the recombinant plasmids containing PB1 17–66, 27–76, 37–86, 47–96, 57–106 were constructed and expressed, and analyzed with Western blotting. The results showed that the 2H9 monoclonal antibody could only recognize the sequence of 57–106. According to screening of the above amino acid sites, it was finally determined that the amino acid site that the 2H9 monoclonal antibody could recognize was
97ESHPGIFENS
106, which was another screened epitope. These epitopes provide ideas for key targets in vaccine design and could be used to design vaccines that can stimulate the host immune system to produce an effective immune response. In addition, they can be used to identify and quantify specific pathogens or their components in immunodiagnostic reagents, which provides research ideas for the prevention and control of avian influenza.
Furthermore, these epitopes, identified through homology, were highly conserved across different strains and located on the surface of the PB1 protein. It is reported that the PB1 protein interacts with host immunomodulatory proteins in innate immunity, thereby affecting host natural immune signaling pathways and type I interferon production. The PB1 protein also affects host inflammatory signaling pathways, such as the NF-κB (Nuclear Factor-κB) pathway, which regulates inflammatory factor production [
20]. These results might enhance the theoretical understanding of the PB1 protein antigen structure. However, the role of these epitopes in cellular immune responses and virus replication requires further investigation.
In summary, four monoclonal antibodies (2H9, 3E6, 5B3, and 5F12) against the AIV PB1 protein were screened in this study, and two B cell epitopes were identified. The key amino acids recognized by these antibodies were conserved among different AIV genotypes. The B cell epitopes identified are of great significance for vaccine research and immunodiagnostics. Localization of B cell epitopes is helpful for the design of new epitope vaccines with strong immunogenicity and high safety. Diagnostic combinations based on B cell epitopes can be established with multi-epitope peptides. The findings not only provide a tool for further exploration of the PB1 antigen structure and function, but also lay a necessary theoretical foundation for new AIV immunodiagnostic techniques.