Study on immunogenicity of recombinant ferritin hemagglutinin of canine distemper virus

Abstract

Background

Canine distemper virus (CDV) hemagglutinin protein (H), as one of the surface glycoproteins of the virus, helps attach the virus to the host cell through its interaction with cell receptors, makes hemagglutinin protein a key target for the development of neutralizing antibodies.

Methods

In this study, H protein sequences were analyzed by conservation analysis and prediction of multiple immune cell epitopes, three possible epitope sequences were identified. The epitopes were ligated to Ferritin vector with linkers to construct pET30a-Ferritin-Hemagglutinin-Canine distemper virus (FHCDV). pET30a-FHCDV was transfected into E.coli BL21, the expressed protein was extracted by nickel column affinity chromatography and dialysis concentration. Evaluate vaccine efficacy through nasal mucosal and muscular administration.

Results

The best expression conditions of FHCDV in E. coli transfected with pET30a-FHCDV were 25 °C and 1 mM Isopropyl β-D-1-Thiogalactopyranoside (IPTG). The results of Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE) and Non-denaturing Polyacrylamide Gel Electrophoresis (Native-PAGE)showed that the purity of purified FHCDV was about 95% and had capable of self-assembly. The results of Dynamic Light Scattering (DLS) and Transmission Electron Microscope (TEM) characterization showed the recombinant protein FHCDV self-assembly into tetrameric caged nanoparticles with a diameter of 16.56 nm. Significant secreted IgA (sIgA) was generated in BALB/C female mice with nasal mucosal immunization. The titers of sIgA antibodies against FHCDV in nasal wash and bronchoalveolar lavage fluid were about 1865 ng/ml and 3943 ng/ml. FHCDV was used as an intramuscular vaccination to inject BALB/C female mice with aluminium adjuvant (Al), Oligodeoxynucleotide (CpG), and polyethyleneimine (PEI) respectively. All immunization groups produced distinct antibodies against the antigenic epitopes of FHCDV. The antibody titer of FHCDV group was 1.28 × 10⁴, that of Ferritin-Hemagglutinin- Canine distemper virus + aluminium adjuvant (FHCDV + Al) group was 5.12 × 10⁴, Ferritin-Hemagglutinin- Canine distemper virus + Oligodeoxynucleotide (FHCDV + CpG) group was 1.28 × 10⁴, and Ferritin-Hemagglutinin- Canine distemper virus + polyethyleneimine (FHCDV + PEI) group was 2.56 × 10⁴. The results of cytokine assays showed that FHCDV generated a Th1 immune response. Immunoaffinity protein serum exhibited varying degrees of neutralizing activity against Canine distemper virus-11(CDV-11); ID₅₀ of FHCDV groups was 39.8, that of FHCDV + Al groups was 106.7, FHCDV + CpG groups was 58.7, and FHCDV + PEI groups was 55.4. FHCDV did not cause damage to mouse organs.

Conclusions

These results indicate that self-assembled multi-epitope FHCDV nano vaccines elicit effective immune responses with a favorable safety profile, providing a promising strategy for canine distemper virus vaccine development.

Introduction

Canine distemper virus is a highly contagious morbillivirus characterized by elevated morbidity and mortality rates [1, 2]. Outbreaks of canine distemper have caused significant mortality in domestic dogs (Canis lupus familiaris), foxes (Vulpes spp.), and minks (Neovison vison), with reported mortality rates ranging from 30 to 80%, reaching 100% in ferrets (Mustela putorius furo) [3]. This poses a substantial threat to both domestic canine populations and wildlife conservation efforts. Consequently, developing safer and more efficacious vaccines against canine distemper remains an urgent priority in veterinary medicine.

The host range of canine distemper virus (CDV) has expanded significantly, achieving global epidemic status. Current vaccine development efforts focus on multiple platforms: Traditional CDV vaccines primarily consist of live-attenuated formulations, which elicit protective immune responses against viral challenge in domestic canines. However, residual virulence in these attenuated strains precludes their application in wildlife immunization protocols. DNA-based vaccination strategies have been extensively investigated. Nielsen et al. engineered plasmid constructs encoding wild-type and attenuated CDV hemagglutinin (H) and nucleocapsid (N) genes. Experimental immunization of minks with these DNA vaccines demonstrated comparable protective efficacy against wild-type virus challenge [4]. Recombinant live-vector vaccines represent another approach, exemplified by the commercially available canarypox virus-vectored vaccine expressing H and fusion (F) glycoproteins from the Onderstepoort vaccine strain. Canary pox virus cannot replicate in mammalian cells, so this vaccine is considered very safe [5]. Nevertheless, this platform requires annual booster immunization due to transient humoral immunity persistence [6, 7]. Researchers used the probiotic strain casei 393 to express the CDV H protein, the recombinant vaccine showed good immune protection after nasal immunization [8]. Researchers constructed a nanoparticle vaccine based on CDV H gene. when they immunized mice with the vaccine, the mice produced high levels of neutralizing antibodies. The vaccine is a new type of vaccine [9]. It has been demonstrated that a strain isolated from Japan called Yanaka does not cause infection in dogs but still stimulates an effective immune response [10]. We designed for this strain and constructed a novel CDV epitope nanoparticle vaccine.

Canine distemper virus (CDV) is a member of the family Paramyxoviridae, genus Morbillivirus [11]. The enveloped virions exhibit pleomorphism, predominantly adopting spherical morphology with particle diameters of ~ 150 nm. The CDV genome comprises 15,690 base pairs (bp) encoding six structural proteins [12]. Viral entry is principally mediated by H glycoprotein surface spikes [13]. Through specific binding to signaling lymphocyte activation molecule (SLAM) receptors [14], the H protein governs viral cellular tropism and histopathological progression, thereby determining host specificity [15]. This interaction triggers conformational rearrangement of the F glycoprotein, an envelope-anchored type I transmembrane protein. Activation of the F protein’s membrane fusion machinery mediates viral envelope-host cell membrane integration [16, 17].

Epitope vaccines is a novel strategy in modern vaccinology.Epitopes has become a common strategy in vaccine development because they are easily processed and presented by antigen-presenting cells, leading to strong cell-mediated immune responses [18]. However, epitope vaccines often show weaker immune responses compared to traditional vaccines, likely due to the short length of the epitopes and the better delivery systems. Recent work on self-assembling nanoparticle vaccines has introduced new ideas, like using Ferritin to carry epitopes. Ferritin can self-assemble in vitro into small particles around 10–12 nm [19, 20]. These nanoparticles are safe for biological use, have consistent sizes, and can be modified on their surface to display antigens, which boosts their immune activity [21]. In 2020, Hatlem et al. [22] created a COVID-19 vaccine using Ferritin nanoparticles linked with SpyTag-SpyCatcher technology. Their goal was to make a Ferritin-based vaccine that keeps the antigen’s shape intact and makes the protein more stable, improving its ability to trigger immunity.In this experiment, the H protein epitopes were screened by bioinformatics. Then tandem Ferritin was used as antigen presentation vector. The E. coli expression system was selected for expression, and FHCDV was combined with aluminum adjuvant, CpG adjuvant and PEI adjuvant. The immunogenicity of the vaccine was verified by immunizing BALB/c mice via two different routes: intramuscular and nasal mucosal.

In this study, immunodominant epitopes of the H glycoprotein were predicted using immunoinformatic approaches. A tandem-repeat ferritin nanoparticle scaffold was engineered as an antigen display platform. The recombinant FHCDV construct was expressed in Escherichia coli BL21(DE3) and adjuvanted with aluminum hydroxide (Al), CpG oligodeoxynucleotide (CpG), and polyethyleneimine (PEI). The immunogenicity of the vaccine was verified by immunizing BALB/c mice via two different routes: intramuscular and nasal mucosal.

Materials and methods

Plasmid and cells

The pET30a-FHCDV plasmid was synthesized by Nanjing Kingsley Biotechnology Co. African green monkey kidney cells (Vero) were provided by Jilin Five-Star Animal Health Co., Ltd. E.coli BL21 (DE3) was purchased from Beijing All Style Gold Biotechnology Co.

Vaccines, animals and strains

Canine Distemper, Parainfluenza, Adenovirus and Parvovirus Vaccine (CDPAP) was Provided by Jilin Five-Star Animal Health Co, Ltd. CDV-11 strain was provided by Jilin Five-Star Animal Health Co, Ltd. BALB/C mice (female, 6 weeks old, body weight: 18–20 g) were obtained from Changchun Yise Laboratory Animal Technology Co.

The rearing conditions were as follows: clean-grade animal room (barrier system) of Jilin Five Star Animal Health Co. The temperature of the animal room was 19–23℃, relative humidity: 40–65% Light: 12 h bright and 12 h dark alternately. IVC. Cage Rearing All cages and bedding were sterilized by autoclaving and used, and they were free to eat and drink.

All animal experiments were approved by the Animal Ethics Committee of Changchun Longsheng Experimental Animal Technology Co., Ltd. (CCLSLL-2023112901). The protocols were performed in accordance with the guidelines for the Welfare and Ethics of Laboratory Animals of China.

Plasmid construction

The corresponding H protein sequences were downloaded from the NCBI database (National Center for Biotechnology Information (nih.gov)) and saved as Fasta files (GenBank accession number BBB35231.1). Highly conserved regions of H protein of Yanaka strain were obtained by conservativeness analysis of the collected sequences. The transmembrane region as well as the signal peptide region of the H protein of Yanaka strain was analyzed using the bioinformatics software TMHMM (http://www.cbs.dtu.dk/services/TMHMM/). Prediction of H protein glycosylation sites was performed using the Net NGlyc (https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0) tool Based on the information of the primary structure of the predicted amino acid sequence of the resulting H protein, the signal peptide region, transmembrane region peptides, intracellular peptides and possible potential glycosylation sites were excluded. Secondary structure prediction of the full sequence of H protein was performed using the bioinformatics software SOPMA (https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html). The highly conserved H protein regions were screened for B-cell epitope and T-cell epitope prediction by IEDB web site (https://www.iedb.org/). Two potential immune cell linear epitopes (366–377, 226–234) were finally selected in combination with the T and B cell epitopes of the highly conserved fragments of H protein and a potential neutralizing antibody epitope of H protein was screened from other literature searches (238–244). This epitope was recognized in the literature with the prepared neutralizing monoclonal antibody 2D 12 against CDV H protein and was the smallest linear epitope [23]. The three sequences were joined by Linker (AAY) and the Ferritin sequence (5-167) was linked to the three sequences by Linker (GGGGS), ending with a 6 × His tag. Two enzymatic cleavage sites, Nde I, BamH I, were selected. The sequences were cloned into the pET30a vector, synthesized by Nanjing Kingsley Bio-technology Co. Ltd, and the plasmid pET30a-FHCDV was constructed, and the plasmid was sequenced and identified.

FHCDV protein expression

The recombinant plasmid pET30a-FHCDV was transformed into the receptor cell E.coli BL21 (DE3), and the bacteria were cultured at 37 °C, 220 rpm until the OD₆₀₀ value was 0.6–0.8, and the expression was induced by the addition of 1 mM IPTG at 25 °C, 220 rpm for 14 h.

Purification and identification of FHCDV protein

The organisms after induced expression were collected, and the organisms were re-solubilized with PBS at pH 7.4, and ultrasonically crushed, and the ultrasonicated supernatant stock was collected, and then purified by nickel column affinity chromatography (NmTRAP 5 ml. UniNTA-80Ni. Nan Micr, China), and then the purified protein solution was put into a 100KD dialysis bag, which was placed into dialysis buffer, and then stirred at 4 °C. Then the purified protein solution was placed in a 100 KD dialysis bag, and the bag was placed in dialysis buffer and stirred at 4℃, and the solution was changed every 6 h for three times to obtain the target protein. The protein was then concentrated by passing it through 10 KD ultrafiltration tubing Amicon Ultra-15 centrifugal filters (Merck Millipore Ltd). The concentrated protein was passed through BCA protein concentration assay kit (Beyotime, China) to determine its final protein concentration, and the recombinant protein FHCDV was stored at -80 °C for reserve. The bacterial fluids before and after induction, the supernatant and precipitated samples after ultrasonic crushing were taken for polyacrylamide gel electrophoresis (SDS-PAGE). The purified proteins were subjected to 6% Native-PAGE and 15% SDS-PAGE. The effect of Ferritin self-assembly was tested. It also tested whether a single target band was obtained, and the purified FHCDV protein was identified by Western-blot, using His Tag Mouse Monoclonal Antibody for primary antibody and HRP-labelled goat anti-mouse IgG for secondary antibody.

Morphological detection of FHCDV protein

The assembly effect of Ferritin was detected using dynamic light scattering, with the objective of determining whether the size of FHCDV particles conformed to the theoretical order. Purified recombinant proteins were placed in a detection dish, and the diameters were detected according to the procedure for using the instrument. To further verify the assembly effect of Ferritin, FHCDV was examined by transmission electron microscopy. The sample was dropped on a copper carbonate grid and stained negatively with phosphotungstic acid. Detection and observation were performed at an accelerating voltage of 100 kV, and images with a magnification of 40 K were obtained using a charge-coupled device camera system.

Preparation of immunogens and immunization of mice

Immunization was initiated after a 5-day acclimatization period in each group, and mice in each immunization group were immunized once on day 0, once on day 7, and once on day 14 for intramuscular immunization and nasal mucosal immunization, for a total of three times. Proteins were mixed with aluminum adjuvant (Al) at 1:1 volume (20 µg, 100 µL/mouse), proteins with polydeoxyribonucleotide (CpG) adjuvant at 1:1 mass (20 µg, 100 µL/mouse), and proteins with polyethyleneimine (PEI) adjuvant at 1:1 mass (20 µg, 100 µL/mouse) for two hours prior to immunization. Mouse serum was collected by tail-vein means every other week in the muscle-immunized group. Blood was collected from mice by eyeball removal on day 42. The serum was inactivated by complementation in a water bath at 56 ℃ for 30 min, and the serum was labelled and stored at -80 ℃.

The nasal mucosal immunization group was required to be immunized with adjuvant-free immunogen at (20 µg, 40 µL/mouse) prior to immunization by mixing FHCDV with oligodeoxynucleotide (CpG) adjuvant at 1:1 mass (20 µg, 40 µL/mouse).The group performed euthanasia on the mice 14 days after triple immunization and collected bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF).The fractions were labelled and stored frozen at -80 °C.

Detection of sIgA in bronchoalveolar lavage and nasal washings in mice

Mouse BALF and NLF were diluted five times and used for sIgA assay, and the specific procedure was followed according to the instruction of sIgA assay kit. (JL11763 by Jiang Lai Bio)

ELISA, antibody isotyping assay and cytokines assay

We selected peptides chemically synthesized by Nanjing Jinsi Rui Biotechnology Co., LTD. (4 mg/mL). We use synthetic peptides for encapsulating antigens. We used ELISA to detect specific antibody levels in serum. The peptide was encapsulated in a 96-well plate,100 µL (10 µg/mL) was added to each well and incubated at 4 °C overnight. After 5 washes with Wash buffer (0.05% Tween20 in PBS), 250 µL of Block buffer (2% BSA in wash buffer) was added to each well and incubated at 37 °C for 2 h. After 5 washes with Wash buffer,100 µL of gradient diluted serum samples were added and incubated at 37 °C for 1 h. After 5 washes with Wash buffer,100 µL of Horseradish Peroxidase (HPR) was added. After 5 washes with Wash buffer, the samples were incubated with 100 µL of Horseradish peroxidase-labelled goat anti-mouse IgG (Beyotime, China) dilution for 45 min at 37 °C. After 5 washes with Wash buffer, the samples were incubated with 100 µL of TMB color development solution for 20 min at room temperature and protected from light. 50 µL of the stop solution (2M H₂SO₄) was added to the samples and the absorbance of OD₄₅₀ was measured by an ELISA system. A function curve was fitted to the serum dilution ratio and the OD value, and 2.0 times the OD₄₅₀ value of the serum-free group corresponded to the dilution of the experimental group, which was the potency of the antibody in the serum.

Antibody typing assay: The peptide was encapsulated in 96-well plates, 100 µL of peptide (10 µg/mL) was added to each well and incubated at 4℃ overnight; after washing with Wash buffer for 3 times, 250 µL of Block buffer was added to each well and incubated at 37℃ for 2 h; after washing with Wash buffer for 3 times, 100 µL of serum samples diluted by gradient was added; 100 µL of diluted samples was added to each well in the negative group, and 100 µL of diluted Mouse IgG isotype control mixture was added to each well in the positive group. Add 100 µL of diluted Mouse IgG isotype control mixture to each well of negative group and positive group, incubate at 37℃ for 2 h. Wash 3 times, add 100 µL of diluted IgG1, IgG2a, IgG2b, IgG3 and IgM antibodies to each well, incubate at 37℃ for 2 h. Wash 3 times with Wash buffer, add 100 µL of secondary antibody ( HRP conjugated Rabbit Anti-Mouse IgG) was added to each well and incubated at room temperature for 1 h. The wells were washed 5 times with Wash buffer and incubated with 100 µL of TMB color development solution for 20 min at room temperature and protected from light, and then 50 µL of stop solution was added to the wells to detect the absorbance of OD₄₅₀ in the zymography. Detect the cytokine mass concentration of IL-4, IFN-γ after five-fold dilution of mouse serum to be tested, the specific operation procedure is in accordance with the instructions of the cytokine assay kit. (S203358, S203579, Shanghai Dibao Biotechnology Co.)

Microneutralization assay

Make a 10-fold serial dilution of CDV with 2% MEM, take 100µL of each dilution and add it to a 96-well cell culture plate, and make 4-well replicates of each dilution. Add 100µL of Vero cell suspension (cell concentration 2 × 10⁵-3 × 10⁵ cells/mL) to each well, mix gently and put into 37℃ 5% carbon dioxide incubator, and determine the results after 5 days. Cells with lesions in each well were marked as positive “+”, and those without lesions were marked as negative “-“. TCID₅₀ was calculated according to the Reed-Muench method.

The mouse serum samples to be examined were diluted 2-fold in 96-well plates, and 4 duplicate wells were made for each dilution, and after dilution, 50µL of virus dilution with 100 TCID₅₀ was added to each well, and then placed in 37℃ 5% carbon dioxide constant temperature incubator for 1 h. 100µL of Vero cell suspension was added to each well (the concentration of the cells was 2 × 10⁵-3 × 10⁵ cells/mL), and then gently mixed, and then placed in 37℃ 5% carbon dioxide constant temperature incubator for 1 h.5% carbon dioxide incubator and set up a virus control group (100 TCID₅₀ viruses diluted in this assay were further diluted in a 10-fold gradient, and 100, 10, 1, 0.1, 0.01 TCID₅₀ viruses were added to 50µL per well, respectively). the results showed that 0.01, 0.1 TCID₅₀ should not cause lesions, 100, 10 TCID₅₀ should not cause lesions, 100, 10 TCID₅₀ must cause cell lesions) and normal cell controls (to avoid experimental errors caused by the plates themselves), and the results were determined after 5 days. Antibody neutralization potency of sera was expressed as the inhibitory dose in half (ID₅₀) and calculated according to the Reed-Muench method.

H&E stains of tissue sections

The tissue organs of mice were rinsed with saline and fixed in 4% paraformaldehyde for 48 h. The fixed tissues were sequentially dehydrated, transparent, permeable to wax, embedded, sectioned, extended and dried. The sections after melting wax were sequentially dewaxed and hydrated for H&E staining. The stained sections were dehydrated, transparent, sealed, and finally scanned and photographed.

Statistical analysis

GraphPad Prism 8.0 software was used for statistical analysis. Sample data were calculated using mean ± standard deviation, and t-test was used to analyze the significance of differences in the experimental results. (*, 0.01 < P < 0.05 indicates significant difference; **, P < 0.01, ***, P < 0.001 indicates extremely significant difference; ns, not statistically significant).

Results

Plasmid construction

In this experiment, 20 sequences of CDV H protein were found and screened through the NCBI website (https://www.ncbi.nlm.nih.gov/), and the sequences were compared and analyzed using Mage11.0 software (https://www.megasoftware.net/), and the compared sequences were analyzed for conserved sequences through the website Weblogo (https://weblogo.berkeley.edu/logo.cgi) (Fig. 1a).

Fig. 1.

Design of epitope nanoparticles. a. Analysis of CDV H protein conservation; b. Deep THMHH tool predicts protein transmembrane region results; c. NetNGlyc tool glycosylation site prediction results; d. Blue: Alpha helix, Green: Beta turn, Yellow: Random coil, Red: Extended strand. SOPMA software predicts CDV-H secondary structure, e. Schematic representation of constructed epitope nanoparticles

The CDV H protein was found to be relatively concentrated in the mutation region, although not very conserved, which is one of the potential advantages of developing an epitope vaccine against the CDV H protein. Subsequently, the mutations in the H protein of Yanaka strain were analyzed, and by comparing it with 19 others and using Clustal W software (https://www.genome.jp/tools-bin/clustalw) for conservativeness analysis, it was found that the conservativeness rate could be up to more than 93%, and the consecutive conserved fragments were collated and summarized. The transmembrane region is usually an α-helical structure with about 20–25 amino acid residues, most of which are hydrophobic, which can easily result in the inability to be expressed in a prokaryotic expression system, so it is necessary to predict the transmembrane region of the H protein of Yanaka strain. Based on the amino acid sequence of the H protein of Yanaka strain, the transmembrane domains and signal peptide of the protein were predicted using the Deep TMHMM tool (http://www.cbs.dtu.dk/services/TMHMM) (Fig. 1.b). In the amino acid 607aa coding for the H protein, the amino acid 59-607aa is the extracellular region, the amino acid 1-37aa, is the intracellular region, and the region 38-58aa contains transmembrane structural domains, which is presumed to be transmembrane region, and the protein does not have a signal peptide. In addition, the amino acid sequence of the H protein was predicted to have nine potential glycosylation sites using the Net NGlyctool (https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0), and the predicted amino acid residue positions are shown in the figure (Fig. 1.c). When screening the Yanaka strain H protein for dominant T- and B-cell antigenic epitopes, the signal peptide region, transmembrane region, intracellular region, and the highly glycosylated sites were removed. According to SOPMA (https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) prediction results H protein has the highest content of irregularly curled amino acid residues of 40.86%, α-helix is composed of 25.37% amino acid residues, and β-fold contains 6.59% amino acid residues (Fig. 1.d). The β-turns and irregular coils are usually located on the surface of the protein and are easier to recognize. This is also one of the evaluation criteria for selecting suitable sites of amino acid residues with irregular curls and β-folds as potential antigenic epitopes of CDV-H.

Subsequently, the T-cell and B-cell epitopes of the full sequence of the H protein were predicted and screened using the IEDB (https://www.iedb.org/) website. This entailed an assessment of the epitopes’ hydrophilicity, hydrophobicity, epitope sequence length, and major histocompatibility complex (MHC) source species. The H protein conserved fragments and secondary structure amino acid residues were screened using Mage11.0, SOPMA and Novopro software, in accordance with the predictions made regarding their location. Ultimately, two linear epitopes, H-B and H-T, were identified as the most promising candidates. Then one epitope H with neutralizing resistance was identified from the literature. This can be recognized in the literature with the prepared neutralizing monoclonal antibody 2D 12 against the anti-CDV H protein. Notably, this epitope is the smallest linear epitope [23].The three linear epitopes were linked by Linker AAY, and they were linked to the Ferritin epitope by Linker GGGGS. A 6 × His tag was added to the end of Ferritin. The enzymatic cleavage site was selected as Nde I at the 5’ end and Bam H I at the 3’ end. construct the epitope sequence. (Fig. 1e).

Nde I, Bam H I were selected as the two cleavage sites, the three epitope sequences were linked together by Linker AAY, and Ferritin was linked to the epitope sequences by Linker GGGGS, ending with a 6 × His tag.

Expression and identification of target proteins

The sequenced recombinant plasmid pET30a-FHCDV was transformed into E.coli BL21 (DE3) expression sensory state, and the monoclonal strain was picked for expansion culture, when the strain was in the logarithmic growth stage, 1mM IPTG was added to induce protein expression at 25℃, 220 rpm/min for 14 h. The bacterial liquid was broken by ultrasonication and subjected to SDS-PAGE. The results (Fig. 2a) showed that specific bands at about 25 KD could be seen in lanes 1,2,3 and 4, and the molecular mass size was consistent with that of the FHCDV protein, indicating that the expression of FHCDV was successful and most of the proteins were expressed in a soluble manner (lane 3).

Fig. 2.

Expression of target proteins. (a) Expression of target proteins analysis by SDS–PAGE showing the molecular weights. Lane1: FHCDV before induction; Lane2: FHCDV after induction; Lane3: FHCDV supernatant after sonication and fragmentation; Lane4: FHCDV precipitate after sonication and fragmentation. (b) purified proteins analysis by SDS–PAGE showing the molecular weights. Lane:1. FHCDV supernatant after sonication and fragmentation; Lane2: FHCDV purified protein. (c) purified proteins analysis by nonreducing SDS–PAGE showing the molecular weights. Lane1: Ferritin; Lane2: FHCDV. (d) Western blot analysis of the purified proteins effect, Lane1: FHCDV

FHCDV protein was purified by nickel column affinity chromatography with imidazole gradient elution. The purified protein was subjected to SDS-PAGE for identification. The results showed (Fig. 2b) that 25 KD was approximately the target protein, and the purity of lane 2 target protein could be more than 95%.

The purified protein was subjected to Native-PAGE, and the results were shown in the figure (Fig. 2c), the size of FHCDV protein was above 250 KD, which was in line with the theoretical size, indicating that the recombinant FHCDV protein underwent the process of self-assembly.

Western-Blot identification of the purified proteins showed (Fig. 2d) that the purified proteins specifically bound to His Tag Mouse Monoclonal Antibody, and a clear immunoblot appeared at 25 KD, indicating that the recombinant strains successfully expressed the FHCDV recombinant proteins.

In order to verify the assembly effect of recombinant proteins, we used TEM to show the profile of the recombinant protein and the shape of the particles. The results showed that the assembly effect of empty Ferritin was the best and formed a large number of hollow spherical particles. The results showed that empty Ferritin has a strong ability to form 24 - dimer. As the assembly effect of FHCDV protein carrying the epitope sequence was slightly lower than that of unloaded Ferritin, but still formed many uniformly shaped and densely distributed hollow spherical particles, indicating that recombinant nanoparticles that can be used for immunization and efficiently displaying the epitope polypeptide as expected have been successfully expressed. (Fig. 3a, b) The particle size of the recombinant protein was also analyzed using DLS on the obtained FHCDV samples (Fig. 3c). The results showed compliance with the expected results.

Fig. 3.

Characterization of target proteins. Observations of Ferritin nanoparticles by TEM (a), ­FHCDV (b) c. Dynamic light scattering (DLS) of target proteins. Red line represents FHCDV; Blue line represents Ferritin

Mucosal IgA antibody response

Nasal mucosal immunization of BALB/C mice was carried out in four groups, namely, PBS, Ferritin, FHCDV, and FHCDV + CpG, and all mice were immunized three times, once on day 0, once on day 7 and once on day 14. Bronchoalveolar lavage fluid and nasal lavage fluid were collected on day 28 (Fig. 4a), and heart, liver, spleen, lungs and kidneys were taken.

Fig. 4.

(a) Mouse study design. Schematic diagram of the immunological cycle of nasal mucosal immunity. Mice were immunized with nasal drops at 0, 7 and 14 days. Mice titrated with Ferritin and PBS were used as negative controls. (b) Detection of sIgA in nasal washings of mice after immunization with FHCDV vaccine nasal drops. (c) Detection of sIgA in the bronchoalveolar lavage fluid of mice immunized with FHCDV vaccine by nasal drops. (d) Mouse study design. Schematic diagram of the immune cycle for muscular immunity. Mice were immunized intramuscularly at 0, 7 and 14 days and blood were pre-collected. Mice injected with Ferritin and PBS were used as negative controls, (e) Using commercially synthesized peptides as antigens, the titer of specific IgG in the serum was detected by ELISA. Specific antibody titers to FHCDV in mice after 7 days of primary immunization (f) Using commercially synthesized peptides as antigens, the titer of specific IgG in the serum was detected by ELISA. Specific antibody titers to FHCDV in mice after 14 days of triple immunization (g) Using commercially synthesized peptides as antigens, the titer of specific IgG in the serum was detected by ELISA.FHCDV-specific antibody titers at different time points. CDPAP is an acronym for Canine Quadruple Inactivated Vaccine. (*,0.01 < P < 0.05 indicates significant difference; **, P < 0.01, ***, P < 0.001 indicates extremely significant difference; ns, not statistically significant)

In nasal mucosal immunization, mice were inoculated with FHCDV nanoparticles and the first to be stimulated and perform local specific immunity were the nasal associated lymphoid tissues and mediastinal lymph nodes of the lungs. Therefore, in this experiment, nasal wash and bronchoalveolar lavage fluid from mice 14 days after triple immunization were collected and their secretory mucosal specific antibodies (sIgA) were measured. The results showed (Fig.4b, c) that both the FHCDV group and the FHCDV + CpG group of the nasal mucosal immunization group could induce highly significant levels of sIgA in nasal wash and bronchoalveolar lavage fluid (P < 0.001), which were highly significant higher than those in the PBS group (P < 0.001).And it can also be seen from the figure that the sIgA antibody titer produced in bronchoalveolar lavage fluid by the FHCDV + CpG group was significantly lower than that of the FHCDV group (P < 0.01), suggesting that CpG adjuvant did not have an enhancement effect on the production of nasal mucosal immune response by FHCDV. Therefore, sIgA titers were measured in bronchoalveolar lavage and nasal washings of mice 14 days after three immunizations. The results showed that the antibody titers of FHCDV to sIgA in nasal wash and bronchoalveolar lavage fluid were about 1865 ng/ml and 3943 ng/ml. The ability of the FHCDV-immunized group to induce high titers of sIgA antibody levels suggests that intranasal inoculation with FHCDV nanoparticle vaccine induces a significant mucosal immune response. Meanwhile, the immunized group with added CpG adjuvant produced lower sIgA antibody titers in lung wash compared to the FHCDV-immunized group without adjuvant. This indicates that CpG adjuvant is less suitable for nasal mucosal immunization and does not promote efficient nasal mucosal immune response to the immunogen.

Specific antibody titer of immunized mice

Seven groups of BALB/C mice were immunized intramuscularly with PBS, Ferritin, canine quadruple inactivated vaccine (CDPAP), FHCDV and FHCDV supplemented with aluminum adjuvant, oligodeoxynucleotide (CpG) and polyethyleneimine (PEI) respectively. All mice were immunized three times on days 0, 7 and 14; blood was collected from the tail vein on days 0, 7, 14, 21, 28, 35 and 42 (Fig. 4d). The specific IgG titer of mouse antisera 7 days after the first immunization was determined by ELISA (Fig. 4e). The results showed that all protein immunogen groups produced high levels of specific antibody IgG titers compared to the negative control group. Moreover, the FHCDV + Al immunized group produced significantly higher specific antibody IgG titers than the FHCDV immunized group (P < 0.001). The specific antibody titers produced in the FHCDV + CpG group were not significantly different from those in the FHCDV + PEI group. Then the specific IgG titers of mouse antisera 14 days after the third immunization were determined by ELISA. The results showed (Fig. 4f) that the specific antibody IgG titer produced by the FHCDV-immunized group was higher than that of the CDPAP group (P < 0.01), and that the specific antibody IgG titer produced by the FHCDV + Al-immunized group was significantly higher than that of the FHCDV-immunized group (P < 0.001). The FHCDV + CpG group produced significantly higher specific antibody titers than the FHCDV + PEI group (P < 0.05). This indicates that Al promotes the specific antibody IgG titer produced by the immunogen. After three intramuscular immunizations, the induced serum IgG antibodies were significantly elevated in all experimental groups, and specific antibodies against FHCDV antigenic epitopes were produced in all immunized groups, with titers of 1.28 × 10⁴ for FHCDV, 5.12 × 10⁴ for FHCDV + Al, 1.28 × 10⁴ for FHCDV + CpG, and 2.56 × 10⁴ for FHCDV + PEI. indicating a good humoral immune response.

Then the trend of specific antibody IgG titers of mouse antisera from 7 days to 42 days was determined by ELISA (Fig.4 g). The results showed that all immunogen groups produced high levels of specific antibody IgG titers. The level of specific antibody IgG titer reached the highest level on day 21. At day 42, all the specific antibody IgG titers produced by each immunogen group decreased but were still higher than those of the positive group.

Classified detection of antibody isotypes and detection of cytokines

We selected sera from mice 14 days after three immunizations. The mouse antibody typing kit was used to detect the antibody typing of anti-epitopes in serum The results showed (Fig. 5a) that FHCDV, FHCDV + Al, FHCDV + CpG, FHCDV + PEI and CDPAP groups all induced high levels of IgG1, IgG2a, IgG2b, IgG3 and IgM antibodies. The IgG1/IgG2a ratios for each immunization group were 0.65, 0.637, 0.62, and 0.609. The IgG1/IgG2a ratios were all less than 1, indicating that all groups tended to activate the Th1 pathway to promote cellular immune responses.

Fig. 5.

Antibody isotyping assay and cytokine detection. (a) Determination of serum IgG1, IgG2a, IgG2b, IgG3 and IgM titers in muscle-immunized mice (b) ELISA for IL-4 cytokine secretion in serum of muscle-immunized mice (c) ELISA for IFN-γ cytokine secretion in serum of muscle-immunized mice. CDPAP is an acronym for Canine Quadruple Inactivated Vaccine. (*,0.01 < P < 0.05 indicates significant difference; **, P < 0.01, ***, P < 0.001 indicates extremely significant difference; ns, not statistically significant)

Meanwhile, the results showed (Fig. 5a) that the FHCDV group produced the other three antibodies IgG2b, IgG3, and IgM levels highly significantly higher than the negative PBS group (P < 0.001). IgM is the earliest antibody synthesized and secreted during individual development, and also the earliest antibody to appear in the initial humoral immune response and is the ‘vanguard’ of the body’s specific anti-infection.

In order to further evaluate the level of cytokines secreted by T cells in each immunization group, the levels of cytokines IL-4 and INF-γ were measured in the serum of mice at 14 days after the three intramuscular immunizations. The results showed (Fig. 5b and c) that the cytokine IL-4 levels in the FHCDV group were highly significantly higher than those in the PBS and Ferritin groups (P < 0.001) and significantly higher than those in the CDPAP group (P < 0.01). The FHCDV + Al group had the highest cytokine IL-4 level, which was significantly higher than that of the FHCDV group (P < 0.01), highly significantly higher than that of the FHCDV + PEI group (P < 0.001) and did not differ significantly from that of the FHCDV + CpG group.

The level of cytokine IFN-γ in the FHCDV group was highly significantly higher than that in the PBS and CDPAP groups (P < 0.001). The level of cytokine IFN-γ in the FHCDV + CpG group was extremely significantly higher than that in the FHCDV group (P < 0.001), and significantly higher than that in the FHCDV + Al group and the FHCDV + PEI group (P < 0.01).

The above results indicated that the FHCDV group could trigger both cellular and humoral immune responses.

Neutralization effect of antibody on CDV‑11 strain

In order to verify the effectiveness of the antibodies produced by immunized mice, a microtiter neutralization assay was carried out to test the sera of mice after 14 days of triple immunization. The results showed (Fig. 6) that PBS and Ferritin in the negative group before and after immunization did not cause a change in the neutralizing antibody titer against CDV in the serum, which could reach 112 in the CDPAP group, and 39.8 in the recombinant protein FHCDV, and it can be seen from the graphs that the addition of Al produced a stronger neutralizing antibody, which could reach 106.7, which was higher than that of the FHCDV + CpG adjuvant in the immunized group (P < 0.01) and higher than FHCDV + PEI (P < 0.01). And it can also be seen from Fig. 6 that the commercial canine quadruple vaccine CDPAP group was not significantly different from the Al adjuvant group. The canine commercial quadruple vaccine CDPAP group was significantly higher than the FHCDV + CpG group (P < 0.01). The canine commercial quadruple vaccine CDPAP group was extremely significantly higher than the FHCDV + PEI group (P < 0.001).

Fig. 6.

Detection of the titer of neutralizing antibody induced by self-assembled immunogen the neutralization titer of the antibody was detected by the microneutralization method. Under the condition of the 100TCID₅₀ CDV-11 strain, the half inhibitory dose of neutralizing antibody (ID₅₀) was calculated. (*,0.01 <  P  < 0.05 indicates significant difference; **, P  < 0.01, ***, P  < 0.001 indicates extremely significant difference; ns, not statistically significant)

This indicates that the immunogen induced the organism to produce neutralizing antibodies with a certain degree of protection against CDV.

Pathological histological analysis of the major organs of the mice

In order to preliminarily evaluate the biosafety of the multi-epitope no-vaccine, histological analyses of heart, liver, spleen, lungs and kidneys of mice in each immunization group were carried out using H&E staining, and the results were shown in the graphs (Fig.7a, b), which showed that the main organs in each immunization group did not have any obvious lesions, and this proved to some extent that multi-epitope no-vaccine based on the Ferritin nano-platform has a better biosafety in vivo, and it will not have effects on the composition of main organs of mice.

Fig. 7.

Protective efficacy of multiepitope nano vaccines against CDV challenge in mice. Pathological analysis of major organs taken 14 days after triple exemption. (a) H&E stains of various tissue sections of muscle immunized mice. (b) H&E stains of various tissue sections of nasal mucosa immunized mice

Discussion

In conjunction with the exponential growth of nanobiotechnology, there has been a notable increase in interest surrounding the development of nano vaccines [24, 25]. Nano vaccines have been shown to play an instrumental role in the advancement of novel vaccine development, exhibiting notable advantages and being regarded as a highly effective strategy for overcoming the low immunogenicity of antigen-based vaccines. Epitope vaccines have now become a general trend in the development of novel vaccines as they are easily processed and presented by antigen-presenting cells, leading to an effective cell-mediated immune response [18].

The process of screening epitopes involves the identification of potential antigenic epitope sequences within the amino acid sequences of pathogens. While experimental techniques and computer-based methods can both be employed for epitope screening, computer-based methods have become the predominant approach due to reduce the time and cost of vaccine development [26].

The initial and pivotal stage in the development of a multi-epitope vaccine is the selection of target proteins. It has been demonstrated that a strain isolated from Japan called Yanaka does not cause infection in dogs, but can still stimulate an effective immune response [10]. Accordingly, the objective of this study was to integrate the CDV infection mechanism, with a particular focus on the extramembrane region of the H protein as a target. Furthermore, bioinformatics software was employed to conduct a comprehensive analysis of the conserved regions of its protein, primary structure, secondary structure, and other parameters for screening purposes [27].

In addition, T lymphocytes represent a crucial element of the immune system, exhibiting a range of biological functions. These include the direct killing of target cells, the modulation of antibody production by B cells, the production of cytokines, etc. They are primarily responsible for the cellular immunity of the body [28, 29]. The principal physiological function of B cells is to mediate the humoral immune response through the proliferation and differentiation of plasma cells, which results in the production of antibodies. However, they also participate in the process of antigen presentation and cytokine secretion [30]. In order to ensure the protective effect of the multi-epitope vaccine, both T-cell antigenic epitopes and B-cell antigenic epitopes were predicted in this study.

Two potential linear epitopes of canine distemper haemagglutinin protein, H-B: 366–377 and H-T: 226–234, were finally screened. Subsequently, one epitope with neutralizing resistance, H: 238–244, was screened through a review of the literature, which was the smallest linear epitope that could be recognized in the literature by the preparation of a neutralizing monoclonal antibody, 2D 12, against the anti-CDV H protein [31].

The selection of appropriate Linkers between different epitopes is also important because the connection between epitopes using Linkers is always complicated and is often overlooked in the design of epitope-based vaccines. Without Linkers, direct fusion of antigenic epitopes may lead to many undesirable results, including misfolding of epitope-based vaccines, low vaccine yields, and impaired biological activity [32]. Therefore, when designing the epitopes, we chose two Likers, AAY and GGGGS, to avoid the above undesirable results.

Ferritin nanoparticles of Helicobacter pylori (H. pylori) are hollow, near-spherical nanocages self-assembled from 24 Ferritin subunits, which are surface-modifiable, thermally stable and pH stable [33], mimic the size and structure of pathogens [34], and facilitate antigen-immune cell. Masaru Kanekiyo et al. constructed Ferritin nanoparticles (HA-np) by fusing the HA of A/New Caledonia/20/1999 (H1N1) with Ferritin, and HA-np induced hemagglutination-inhibiting antibody titers more than 10-fold higher than those of inactivated vaccines [35]. The H1-HA2-Ferritin protein vaccine (Stabilized headless HA stem nanoparticles) based on this has entered phase I clinical trials (NCT03814720). This nano vaccine induced a significantly higher immune response when compared to subunit monoclonal vaccines that employ linkage systems such as SpyTag/SpyCatcher [36] or GvTagOpti/SdCatcher [37]. These linkage systems facilitate the in vitro linkage of prokaryotic-expressed Ferritin to eukaryotic-expressed antigens. The administration of Ferritin-based bovine viral diarrhea virus vaccines [38] and rotavirus vaccines [7] has been demonstrated to induce specific humoral and mucosal immunity.

Therefore, based on the above properties, in this work, to better present antigenic epitopes, we chose Ferritin nanocages as vaccine vectors and successfully demonstrated E. coli expression nanoparticles of FHCDV and the good homogeneity of the nanoparticles was clearly demonstrated by TEM and DLS analyses.

Adjuvants can enhance the innate immune response at the site of vaccination [39]. Adjuvants can promote the recognition and presentation of antigens by immune cells, which ultimately improves the overall immune efficacy of vaccines. In order to be able to enhance the immunogenicity of the vaccine, three different types of adjuvants were selected for this study. Aluminium(Al) adjuvant, which promotes the inflammatory response by directly activating the NLRP3/NALP3 inflammatory complex or activating NOD-like receptors (NLRs). Aluminium adjuvants mainly stimulate humoral immunity by producing higher titers of IgG, IgE, and activating Th2 cells, and these effects are associated with IL-4 production [40, 41].CpG adjuvant (Oligodeoxynucleotide) which is a DNA sequence with a core of non-methylated CpG, is used by organismal cells during bacterial infections by Toll-like receptor 9 (TLR9). TLR9 recognizes bacterial CpG-DNA during bacterial infection, triggering the body to generate a protective immune response against the bacteria and improving the ability of host cells to clear the bacteria. It has been shown that polyethyleneimine (PEI) acts as an adjuvant for HIV gp140 glycoprotein. The adjuvant could induce strong immune responses in mice by nasal mucosal immunization, subcutaneous immunization and intramuscular immunization. The adjuvant assists the antigen in inducing a high concentration of antibodies in mice. Among those, the branched-chain PEI with a molecular weight of 25KD was the most effective.

In this study, we chose two different modes of immunization because the route of infection of FHCDV includes the mucosal route, and the injection may lead to severe adverse reactions in some susceptible animals. BALB/C mice were immunized three times by intramuscular injection using prepared FHCDV with CpG, Al, and PEI adjuvants, and mouse sera were collected. Mice were immunized using prepared FHCDV, and FHCDV + CpG adjuvant through the nasal mucosa. Bronchoalveolar lavage and nasal washings were collected from mice.

The mucosal immune system is one of the important components of the body’s immune system, and its main function is to remove pathogenic microorganisms that invade the body through the mucosal surface. SIgA plays an important defensive role in mucosal immunity, neutralizing viruses, activating complement bypass, enhancing phagocytosis, and so on. The sIgA titers were measured in bronchoalveolar lavage and nasal washings of mice 14 days after three immunizations. The results showed that the FHCDV immunization group was able to induce high titer sIgA antibody levels. Thus, intranasal administration of FHCDV nanoparticle vaccine was able to induce a significant mucosal immune response. Meanwhile, the immunized group with added CpG adjuvant produced lower sIgA antibody titers in lung wash compared to the FHCDV-immunized group without adjuvant. These findings indicate that the CpG adjuvant is not an optimal choice for nasal mucosal immunization, as it does not effectively promote nasal mucosal immune responses to the immunogen.

After three intramuscular immunizations, the induced serum IgG antibodies were markedly elevated in all experimental groups, and specific antibodies against antigenic epitopes were produced in all immunized groups, indicating a robust humoral immune response. Notably, the FHCDV-immunized groups commenced production of specific antibodies (IgG) seven days after the initial immunization, demonstrating an expedient onset.

Interferon-gamma and IgG2a and interleukin IL-4 and IgG1 reflect Th1 and Th2 cell types, respectively. In general, activation of Th2 cells increases the production of IgG1 and inhibits the production of IgG2a activated by Th1 cells. IgG2a is a representative antibody of cellular immunity, while IgG1 levels reflect the state of humoral immune response. The results of the antibody typing test and cytokine assay indicated that high levels of IgG1 and IgG2a antibodies were induced in each immunization group. Additionally, the IgG1/IgG2a ratio was less than 1 in each immunization group, suggesting that all the immune responses in favor of Th1. In terms of antibody typing, the FHCDV, FHCDV + Al, FHCDV + CpG, FHCDV + PEI and CDPAP groups all induced high levels of IgG2b, IgG3 and IgM antibodies, and IgM, as the primary antibody that reacts when exposed to antigens, can play a complement-activating role and thus achieve the immune effects of bacterial melting, cell killing and virus neutralization.

The production of the cytokine IFN-γ was mediated by Th1 cells, which are involved in the antiviral effects of the cellular immune response. In contrast, IL-4 was mediated by Th2 cells, which are associated with the humoral immune response. The results showed that all experimental groups increased the secretion of both cytokine levels in serum. Meanwhile, the FHCDV + Al immunized group produced significantly higher levels of IL-4 cytokines than the other experimental groups, and the FHCDV + CpG immunized group produced significantly higher levels of IFN-γ cytokines than the other experimental groups. It indicates that several immunogens can induce humoral and cellular immune responses. Moreover, the immunized group with Al adjuvant could enhance the humoral immune response, and the immunized group with CpG adjuvant could promote the cellular immune response.

To verify the effectiveness of antibodies produced by immunized mice, serum neutralizing antibody titers are a more intuitive way of reflecting the effectiveness of immunization. Neutralizing antibody levels were assayed with the CDV-11 strain at 100 TCID 50. The results showed that PBS and Ferritin in the negative group did not cause changes in the neutralizing antibody titer against CDV in serum before and after immunization. As illustrated in the graph, all immunogen groups demonstrated the generation of more robust neutralizing antibodies. Among these, the neutralizing antibody titer produced by FHCDV + Al reached 256, a value that was markedly higher than that observed in the other immunogen groups. This indicates that each immunogen group has a certain degree of protection against CDV.

During the vaccination period, no abnormalities were observed in the mice of each immunization group. Additionally, the pathological tissue test results demonstrated that the FHCDV nanoparticle vaccine did not induce any damage to the major organs of the mice, indicating that the nanoparticles possess a certain degree of biosafety. Therefore, in conclusion, FHCDV has robust immunogenicity, and this multi-epitope sequence can be a key antigenic recognition site for CDV and more effective in preventing CDV.

In this study, an epitope nanoparticle vaccine was designed through immunoinformatic and based on the Ferritin nanoparticle platform. The relevant immune evaluations were performed, although it was demonstrated that the FHCDV nanoparticle vaccine exhibited some neutralizing activity in vitro. However, due to the limitations of the mouse model, there is a greater need to evaluate the protective power in canines, which will be the next step in our work.

Conclusions

In this experiment, we screened the dominant epitope of the CDV H protein and presented it on the nanocarrier ferritin to obtain an epitope nanoparticle vaccine. Then we evaluated its immunogenicity in different immunological modes in mouse animal experiments. The results obtained demonstrated that the antigenic epitope exhibited good antigenicity and immunogenicity. Consequently, it is expected to be a suitable antigen for use in a CDV vaccine. This provides a new idea and theoretical basis for the development of novel vaccines against canine distemper virus or even other viruses. In addition, the efficient self-assembly ability of Ferritin enables it to be used as a potential presentation vector for epitope targets.

Abbreviations

CDV

Canine distemper virus

FHCDV

Ferritin-Hemagglutinin-Canine distemper virus

HA

Haemagglutinin

E. coli

Escherichia coli

TEM

Transmission electron microscopy

DLS

Dynamic light scattering

SDS

Sodium dodecyl sulfate

PAGE

Polyacrylamide gel electrophoresis

IL-4

Interleukin-4

IFN

Interferon

TCID₅₀

Tissue culture infective dose

ID₅₀

Inhibitory dose

Al

Aluminium adjuvant

sIgA

secreted IgA

BSA

Bovine serum albumin

CDPAP

Canine Distemper, Parainfluenza, Adenovirus and Parvovirus Vaccine

CpG

Oligodeoxynucleotide

PEI

Polyethyleneimine

Author contributions

Conceptualization: L, W and Y; Methodology: L, W, L and Y. Investigation: L,Y, W,Z, Y,G, Z,L; Writing—original draft: L; Writing—Review & Editing: L, W and Y; Supervision—Z, Y,G, Z,L. All authors read and approved the final manuscript.

Funding

Jilin Scientific and Technological Development Program (Grant number:20200404195YY).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Animal Ethics Committee of Changchun Longsheng Experimental Animal Technology Co., Ltd. (CCLSLL-2023112901). The protocols were performed in accordance with the guidelines for the Welfare and Ethics of Laboratory Animals of China.

Consent for publication

All authors agreed to submit this manuscript.

Competing interests

The authors declare no competing interests.