Immunogenicity analyses and indirect ELISA application of a chimeric virus-like particle presenting a highly conserved peptide of Akabane virus Gc protein

Abstract

Background

Akabane virus (AKAV) is the causative agent of an economically significant disease in ruminants, manifested notably by outbreaks of abortion and congenital abnormalities. Vaccination stands as the primary defense against this disease. However, the development of safer, more stable, and efficient AKAV vaccines, including epitope-based designs, remains unexplored. Prior work by our group has pinpointed a neutralizing epitope, ¹¹³⁴SVQSFDGKL¹¹⁴², located in the Gc protein of AKAV. We further demonstrated its high degree of conservation across diverse AKAV genotypes.

Methods

 We produced and verified a novel virus-like particle (VLP) by incorporating the neutralizing epitope ¹¹³⁴SVQSFDGKL¹¹⁴² into a recombinant hepatitis B virus core antigen (HBcAg) scaffold. Then the immunogenicity of this VLP was evaluated by detecting the antibody titer targeting the AKAV Gc antigen and the neutralizing activity against AKAV in sera from the VLP-immunized mice. Furthermore, a preliminary indirect ELISA method was established based on this VLP for AKAV detection.

Results

The successful construction of VLP expressing the AKAV epitope was confirmed by using SDS-PAGE, followed by Western blot (WB) and transmission electron microscopy (TEM). Indirect ELISA results indicated that antisera from immunized mice contained antibodies specific to the AKAV Gc protein. Furthermore, neutralization assays demonstrated that the antisera could effectively neutralize AKAV in vitro and inhibit its replication in BHK-21 cells. The developed VLP-based indirect ELISA method successfully identified AKAV antibody-positive serum, with a detection sensitivity of up to a 1:1600 serum dilution.

Conclusions

In conclusion, we successfully constructed a VLP presenting the highly conserved neutralizing epitope of AKAV. This VLP is proved to be immunogenic and can serve as an effective coating antigen to establish an indirect ELISA method for AKAV detection. Collectively, our findings provide proof-of-concept for this epitope-presenting VLP as a promising candidate in the pursuit of a safe and effective epitope-based vaccine against AKAV and also highlight its utility as a diagnostic antigen for serological detection.

Introduction

Akabane virus (AKAV) is categorized within the Simbu serogroup and falls under the genus Orthobunyavirus, part of the Bunyaviridae family. As the pathogen responsible for Akabane disease, AKAV is spread among cattle, goats, and sheep through bites from infected Culicoides midges, and results in significant reproductive failure, manifesting as abortions, stillbirths, and offspring with congenital defects [1]. Since the initial isolation of the prototypical AKAV JaGAr39 strain in Japan in 1959 [2], subsequent infections have been reported.

in multiple continents such as Australia, Asia (Southeast and East), Africa, and the Middle East [3]. In addition to the severe economic impact that AKAV outbreaks have on the livestock industry [4, 5], they have also hindered international trade for endemic areas. Even with the application of both live attenuated and inactivated vaccines, complete protection is hindered, as evidenced by sporadic cases in vaccinated regions [6], which is potentially linked to the antigenic diversity of AKAV strains [7–9]. The progress in molecular vaccinology and genetic engineering has intensified the need for more effective and safer vaccines in various forms.

AKAV is an enveloped pathogen whose genome comprises three negative-sense, single-stranded RNA: the Large (L), Medium (M), and Small (S) segments. The classification of AKAV isolates relies on the genetic sequences of their S RNA segment, giving rise to five genogroups (I-V) [10]. AKAV gene expression yields four structural proteins and two non-structural proteins. The structural proteome comprises the nucleocapsid (N) protein, the RNA-dependent RNA polymerase (RdRp), and the Gn and Gc glycoproteins, while the non-structural ones are NSs and NSm [11]. As the primary target for neutralizing antibodies, the Gc protein is crucial for the induction of protective immunity in the host [12, 13]. Previous investigations have demonstrated that antibodies targeting the Gc protein exhibit potent neutralizing activity against the corresponding simbuvirus [12–14], making Gc protein a promising target for AKAV subunit vaccine design.

In our previous studies, we generated and characterized three neutralizing mAbs directed against the AKAV (TJ2016 strain) Gc protein. The virus strain is documented under GenBank accession numbers MT761689, MT761688, and MT755621. Epitope mapping revealed that all three mAbs recognize the same linear sequence (¹¹³⁴SVQSFDGKL¹¹⁴²). This epitope demonstrates a high degree of conservation across various AKAV genotypes [15]. Besides, one of these mAbs demonstrated the ability to protect Kunming mice against a lethal AKAV challenge (Wang et al., manuscript submitted to BMC Veterinary Research). However, whether the epitope can elicit neutralizing antibodies against AKAV infection is not well studied.

Hepatitis B core antigen (HBcAg) is a nucleocapsid protein of the Hepatitis B virus, comprising 183 amino acids (aa). It possesses the intrinsic capacity to self-assemble into nucleocapsid structures approximately 30 nm in diameter [16, 17]. HBcAg consists of two domains: an N-terminal assembly segment (aa 1 ~ 149, known as ΔHBcAg), which is the only region required for self-assembly, and a basic C- terminal tail (aa 150 ~ 183) [18, 19]. To display foreign epitopes, short peptide sequences from other viruses are commonly fused into the major immunodominant region (MIR, residues 76 ~ 81) of HBcAg [16, 20–22], which can realize successful self-assembly and presentation of the foreign antigen. The properties of HBcAg make it an attractive platform for a diverse range of biomedical applications [23–26].

In the present study, we designed a fusion protein based on the first 149 amino acids of HBcAg and a highly conserved epitope (¹¹³⁴SVQSFDGKL¹¹⁴²) of the AKAV Gc protein and confirmed its ability to elicit neutralizing antibodies against AKAV in a mouse model. Based on the generated fusion protein, we preliminarily established an indirect ELISA method for AKAV detection, which demonstrates favorable sensitivity. These findings hold promise for the rational design of epitope-based vaccines and diagnostics for AKAV.

Materials and methods

Cell lines, viral strain, antibodies, and animals

BHK-21 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, USA). The cell line was maintained under standard conditions at the Institute of Animal Inspection and Quarantine, Chinese Academy of Quality and Inspection & Testing (our lab).

• The bacterial strain E. coli BL21 (DE3) was procured from TransGen Biotech, located in Beijing, China.

The AKAV strain TJ2016 (GenBank accession nos: MT761689, MT761688, and MT755621) used in this study originated from our laboratory’s collection, where it was isolated and characterized [27].

• Two antibodies utilized for detection, mAb 4F12 4F12 (specific for AKAV Gc) and a rabbit-derived polyclonal antibody (pAb, specific for AKAV), were generated in-house [15, 28]. All secondary antibodies, including HRP-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG, along with the mouse anti-His mAb, were procured from Solarbio Life Sciences in Beijing, China.

Six-week-old, specific pathogen-free (SPF) female BALB/c mice (weighing 16 ~ 18 g) were sourced from Beijing Vital River Laboratory Animal Technology Co., Ltd.

Plasmid construction

According to the nucleotide sequence of the AKAV (TJ2016 strain) M gene segment (GenBank accession no: MT761688), the epitope (¹¹³⁴SVQSFDGKL¹¹⁴²) was inserted between the 80 A and 81S positions of the backbone sequence of ΔHBcAg (GenBank registration NC_003977.2) by linkers. To facilitate purification, a C-terminal 6 × His tag was incorporated into the construct. Following codon optimization for E. coli expression, the combined DNA sequence was subsequently synthesized and inserted into the pJET-28a (+) vector by Tsingke Biological Technology. A control vector expressing the C-terminal 6 × His-tagged ΔHBcAg was generated in parallel using the same backbone. The recombinant clones were verified by DNA sequencing and subsequently named HBc-AGc9 and HBc-C, respectively. The design of the plasmids is shown in Fig. 1.

Fig. 1.

Design strategy for the construction of the HBc-C control and the chimeric HBc-AGc9 VLP. The core assembly domain of hepatitis B core antigen (ΔHBcAg, aa 1 ~ 149) constitutes the backbone of each construct. A C-terminal His × 6 tag tag was incorporated into this backbone to facilitate protein purification. (A) The HBc-AGc9 VLP was engineered on theΔHBcAg backbone, with a 9-aa AKAV epitope inserted into its major immunodominant region (MIR) between residues 80 and 81. (B) The control construct HBc-C VLP

Protein expression and purification

Plasmids HBc-AGc9 and HBc-C were transformed into BL21 (DE3) competent bacteria, respectively. Selected positive clones were grown overnight at 37℃ in 10 mL of kanamycin-supplemented LB medium (50 mg/L) with shaking at 200 rpm. The main culture was initiated by inoculating 500 mL of fresh, kanamycin-supplemented LB medium (50 mg/L) with 5 mL of the overnight pre-culture, followed by incubation for about 3 h at 37℃ and 200 rpm. Upon reaching an OD600 of 0.6 ~ 0.8, the bacterial culture was induced by supplementing with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma-Aldrich, USA). Protein expression was carried out for 20 h at 16℃ with shaking at 160 rpm. To collect the soluble fraction, the cell pellets obtained from low-speed centrifugation (1000 rpm, 10 min) was resuspended in lysis buffer and disrupted by sonication. The target proteins were purified via gel filtration chromatography using a NI Sepharose 6FF (Cytiva, Sweden) resin according to the manufacturer’s instructions. The purity and identity of the eluted proteins were then confirmed by SDS-PAGE coupled with Western blotting (WB). Protein concentrations were measured with the Thermo Scientific Pierce BCA assay, followed by aliquoting and storage at −80 °C until further use.

SDS-PAGE and WB analyses

Electrophoresis of the protein samples was carried out on 12% SDS-PAGE gels. The run was initiated at 80 V for 30 min, after which the voltage was increased to 120 V and maintained for 1 h, with subsequent visualization using Coomassie brilliant blue staining.

Following transfer onto PVDF membranes (Millipore, USA), an overnight incubation was carried out at 4 °C in PBS buffer containing 4% skim milk for blocking. Mouse anti-His mAb at a dilution of 1:10,000 was used to detect the HBc149 antigen. For detection of the AKAV Gc epitope, the mAb 4F12 (1:10,000 dilution) that is specific to the AKAV Gc protein was used. The membranes were probed with the diluted primary antibodies with a one-hour incubation at at room temperature. After being washed three times with PBST (PBS containing 0.05% Tween 20), the membranes were subjected to a one-hour room temperature incubation with HRP-conjugated goat anti-mouse IgG diluted at 1:10,000. A final series of three PBST washes was then performed. Following development with the EasySee Western blot kit (TransGen Biotech, China), the immunoreactive bands were documented using a FluorChem E instrument (ProteinSimple, USA).

Electron microscopy

The purified recombinant proteins were examined using negative staining electron microscopy (EM). In brief, the protein samples were diluted to a concentration of 0.5 mg/ml and were absorbed to 200-mesh copper grids for 3 min to allow for adherence. Subsequently, a 60-s negative staining procedure was applied to the sample using 2% phosphotungstic acid. Protein samples were imaged using an HT7800 transmission electron microscope (HITACHI, Japan) operated at 80 kV, and micrographs were captured at 40,000 × magnification.

Mouse immunization

The immunogenicity of the HBc-AGc9 virus-like particle (VLP) was assessed in a mouse model using six-week-old female BALB/c mice. There were four mice in each treatment group. One group was administered intramuscularly (IM) with 25 μg of adjuvant-formulated HBc-AGc9 at weeks 0 and 3. The other group was immunized with 5 μg of adjuvant-formulated HBc-AGc9 at weeks 0 and 3 by the IM route. Two mice received an injection of an equivalent volume of adjuvant as a control, following the same immunization procedure. Sera were collected on weeks 0, 1, 2, 3, 4, and 5 by tail bleeding. For subsequent analysis, the collected serum samples were aliquoted and frozen at −80℃.

Evaluation of serum antibodies targeting the AKAV Gc protein

The antibody titers targeting the AKAV Gc protein in mouse serum were detected by an indirect ELISA assay. Briefly, AKAV recombinant Gc protein (aa991 ~ 1232) [15] was adsorbed to ELISA plates at 0.1 μg/well in a carbonate-buffered environment (pH 9.6) at 4 °C overnight. For blocking, the plates were subjected to a 2-h incubation at 37 °C with 4% BSA prepared in PBS. Following four PBST washes, the wells were treated with tenfold serially diluted mouse serum (10² to 10⁹) for 1 h at 37 °C. A final series of four PBST washes was then performed. Each well was supplied with 100 μL of HRP-labeled goat anti-mouse IgG (diluted 1:5000), and the plate was subsequently incubated for 60 min at 37℃. After four PBST washes, the TMB substrate (100 μL/well) was added to initiate the chromogenic reaction, which proceeded for 15 min in the dark at room temperature. The reaction was then terminated by adding of 2 M H₂SO₄ (50 μL/well). The absorbance at 450 nm was recorded with a Multiskan spectrum microplate reader (Thermo Fisher Scientific, USA).

In vitro neutralization assay

Prior to use, the serum samples were subjected to heat inactivation at 56 °C for 30 min and subjected to a two-fold serial dilution (from 2³ to 2¹²) in triplicate. Subsequently, each dilution was mixed with an equal volume of AKAV suspension (200 TCID₅₀) and subjected to a 1-h incubation at 37 °C. Confluent BHK-21 cell cultures prepared in 96-well plates were infected with the virus-serum mixtures and incubated at 37℃ with 5% CO₂. Virus-only preparations (without serum) and mock-infected BHK-21 cells were designated the negative and positive controls, respectively. After 48 h, cytopathic effect (CPE) development was evaluated using an immunofluorescence assay (IFA). Briefly, the cells were fixed for a duration of 30 min using ice-cold absolute ethanol following the removal of the supernatant. For immunofluorescence staining, the rabbit pAb specific to AKAV (1:1000 dilution) and the FITC-labeled goat anti-rabbit IgG (1:500 dilution) served as the primary and secondary antibodies, respectively. Image acquisition was performed with the Invitrogen EVOS FL fluorescence imaging system (Thermo Fisher Scientific, USA). In this assay, the neutralization titer corresponds to the reciprocal of the serum dilution that confers 50% protection against viral infection. For the serum collected at week 7, the viral inhibition at each serum dilution was carefully observed and recorded in detail.

Establishment of an indirect ELISA method with HBc-AGc9 VLP

ELISA plates were coated with 100 μL of HBc-AGc9 VLP (20 ng/well, 10 ng/well, 5 ng/well, 2.5 ng/well) in carbonate buffer and incubated at 4℃ overnight. Following a 1-h blocking step at 37℃ with 2% skim milk in PBS, the diluted AKAV positive and negative control serum samples (1:50 to 1:400 dilution) were dispensed into the wells (100 μL/well) and subsequently maintained at 37℃ for 1 h. The HRP-labeled goat anti-mouse IgG (1:5000 dilution), used as the detection antibody, was applied to the wells (100 μL/well) and maintained at 37℃ for 1 h. After each incubation step, the plates underwent four PBST washes. Thereafter, color development was initiated by adding TMB substrate (100 μL/well) and incubating for 15 min at room temperature in the dark, followed by reaction termination with 2 M H₂SO₄ (50 μL/well). Finally, the absorbance of each well was read at 450 nm.

Determination of cutoff value

A panel of 24 AKAV-negative sera was subjected to the indirect ELISA after a 50-fold PBS dilution, and the OD₄₅₀ was recorded (Table 1). The positive cutoff value was defined as the mean optical density of negative samples plus three times their standard deviation (Mean + 3SD) .

Table 1.

Indirect ELISA results of 24 AKAV-negative sera

No	OD450nm	No	OD450nm	No	OD450nm
1	0.268	9	0.21	17	0.156
2	0.199	10	0.14	18	0.252
3	0.23	11	0.162	19	0.179
4	0.216	12	0.148	20	0.207
5	0.207	13	0.149	21	0.175
6	0.201	14	0.126	22	0.185
7	0.246	15	0.158	23	0.17
8	0.119	16	0.187	24	0.192

Sensitivity analysis

The AKAV-positive serum was initially diluted 100-fold, followed by serial two-fold dilutions (from 1:100 to 1:12,800). All dilutions were tested using the established indirect ELISA method, with two replicates per dilution. The positivity or negativity of each serum dilution was determined based on the cutoff value.

Results

Production and characterization of recombinant proteins

As presented in Fig. 2, the HBc-AGc9 and HBc-C fusion proteins were observed to be expressed in the E. coli BL21(DE3) strain. According to the SDS-PAGE analysis, the apparent molecular masses of the fusion protein HBc-AGc9 and the HBc-C protein were approximately 20.6 kDa (Fig. 2A) and 17.3 (Fig. 2B), respectively. Following purification, both the HBc-AGc9 and HBc-C fusion proteins were obtained as pure products, as evidenced by the data in Fig. 3A. In addition, mouse anti-His mAb recognized the fusion proteins HBc-AGc9 and HBc-C (Fig. 3B), while the mAb 4F12 specific to AKAV Gc protein only recognized the fusion protein HBc-AGc9 (Fig. 3C).

Fig. 2.

Purification of the recombinant proteins. (A) Validation of HBc-AGc9 protein expression by SDS-PAGE, showing a band at the expected 20.6 kDa. (B) Validation of HBc-C protein expression by SDS-PAGE, showing a band at the expected 17.3 kDa. The recombinant proteins were purified from the sonication supernatant with nickel-affinity resin. The labels (0, 20, 50, 100, 250, and 500 mM) denote the imidazole concentration in the elution buffer. The base composition of all buffers was 20 mM Tris–HCl and 0.25 M NaCl, pH 7.5

Fig. 3.

Identification of the purified recombinant proteins. (A) The purified HBc-AGc9 and HBc-C proteins were analyzed by SDS-PAGE and visualized with Coomassie brilliant blue staining. (B) The purified proteins were detected by WB analysis using an anti-His mAb. (C) Identification of the purified recombinant proteins via WB with the 4F12 mAb specific to AKAV Gc protein. M, molecular mass marker; 1, purified HBc-AGc9; 2, purified HBc-C

The VLPs HBc-AGc9 and HBc-C were demonstrated by negative stain EM. As Fig. 4 shows, numerous particles with an average diameter of 30 nm were observed in both protein samples. VLP formation was observed for both the HBc-AGc9 (Fig. 4B) and HBc-C (Fig. 4A) proteins, with the resulting particles from both proteins exhibiting similar morphological characteristics.

Fig. 4.

Microstructural analysis of the purified VLPs by transmission electron microscopy (TEM). (A) HBc-C VLP. (B) HBc-AGc9 VLP. For TEM imaging, purified protein samples were negatively stained using 2% phosphotungstic acid (PTA) for 60 s. The presented micrographs were captured at a magnification of 40,000 ×, with a scale bar representing 200 nm

Immunogenicity of chimeric VLP antigens in BALB/c mice

To assess the immunogenicity of HBc-AGc9, BALB/c mice were immunized with two doses at 0 and 3 weeks. Serum was collected at various time points to monitor the kinetics and neutralizing efficacy of the anti-AKAV Gc antibody response (Fig. 5A). The results demonstrate that significant serum titers against the AKAV Gc protein were detected only in the mice immunized with HBc-Ag9, excluding the possibility of nonspecific binding and neutralization (Fig. 5B). Following booster immunization, serum antibody levels in HBc-Ag9-immunized mice continued to increase. Mice receiving the higher dose exhibited higher antibody titers after the booster compared to the low-dose group, but no statistically significant difference was found (Fig. 5B). The potential of the VLP HBc-AGc9, which incorporates a neutralizing epitope, to elicit neutralizing antibodies in mice was investigated. To this end, the neutralizing capacity of the antiserum against AKAV was quantified using an in vitro neutralization assay with BHK-21 cells. The data in Fig. 5C reveal that serum samples from the HBc-AGc9 immunization group was able to neutralize AKAV from 7 to 35 days post immunization (DPI). The analysis revealed no intergroup differences between mice immunized with 25 μg and 5 μg doses of HBc-AGc9 from week 1 to week 6, and both groups exhibited peak neutralizing antibody titers on 35 DPI (Fig. 5C). The inhibitory effects of each serum dilution from 35 DPI on AKAV infection were meticulously quantified. The results demonstrated that the neutralizing antibody efficacy decreased from 100 to 0% as the dilution factor increased. The serum neutralization titer in mice immunized with the 25 μg dose was approximately 1:2^9.5, whereas that in the 5 μg dose group was about 1:2^7.8 (Fig. 5D). At serum dilution factors of 1:512 and 1:1024, the high-dose vaccination group exhibited significantly higher inhibition rates against AKAV infection compared to the low-dose group (Fig. 5D).

Fig. 5.

Immunogenicity of the purified HBc-AGc9 VLP. (A) Schematic of the mouse immunization and serum collection schedule. The prime immunization, boost immunization, and blood collection time points (red drops) are shown. DPI, days post immunization. (B) Kinetics of anti-AKAV Gc antibody titers in mice following immunization. The mice immunized intramuscularly with 25, 5, and 0 μg doses of the HBc-AGc9 were bled at 0, 7, 14, 21, 28, and 35 DPI to measure the anti-Gc immunoglobulin titers by ELISA. (C) Measurement of neutralizing antibody titers in immune sera. The assay was performed by incubating two-fold serially diluted sera (2³ to 2¹²) with an equal volume of AKAV (200 TCID50). Following incubation, the virus-mAb mixtures were transferred to BHK-21 cell monolayers, and the neutralization titers were subsequently assessed. Neutralization titers were calculated as the maximum serum dilution achieving a 50% inhibitory effect. (D) Assessment of serum neutralization activity against AKAV. AKAV infection of BHK-21 cells was challenged with serial dilutions of sera obtained at 35 days post-immunization. Significance (** P ≤ 0.01; *** P ≤ 0.001) is indicated for the 25 μg challenge dose group compared to the 5 μg challenge dose group

Preliminary establishment of the indirect ELISA method

To investigate whether the HBc-Ag9 protein can be applied for AKAV antibody detection, an indirect ELISA method was preliminarily established using HBc-Ag9 as the coating agent. The optimal assay conditions, including peptide coating concentration and serum dilution, were established using a checkerboard titration (Fig. 6A). The concentration of the peptide was fixed at 20 ng/well, and the test serum dilution was 1:50, respectively. Under the optimal conditions, the signal-to-noise ratio reached (P/N) 22.762. The mean optical density was 0.187 with a standard deviation (SD) of 0.039. Accordingly, the cutoff value (mean + 3 × SD) was determined as 0.304. This value subsequently served as the criterion for distinguishing positive from negative samples. AKAV-positive sera were titrated starting from a 1:100 dilution, undergoing serial two-fold dilutions to determine the maximum positive dilution. A positive signal was detected at a dilution as high as 1:1600 (Fig. 6B), indicating the high sensitivity of the assay.

Fig. 6.

Preliminary establishment of indirect ELISA method (A) Results of checkerboard titration assays used to optimize HBc-AGc9 VLP concentrations and serum dilution. (B) Sensitivity analysis. Using the established assay, the AKAV antiserum exhibited a maximum sensitivity of 1:1600 (cutoff value: 0.304) when tested in two-fold serial dilutions ranging from 1:100 to 1:12,800

Discussion

As a key determinant of viral pathogenesis and the AKAV-neutralizing antigen, glycoprotein Gc orchestrates several critical processes including host cell attachment, membrane fusion, and immune evasion [29]. The conserved C-terminal domain of the Gc protein is indispensable for membrane fusion during viral entry. In contrast, the immunodominant and highly variable N-terminal region (Gc head) serves as the primary target for neutralizing antibodies [12, 13]. Yohsuke Ogawa et al. [30] successfully expressed the recombinant AKAV Gc protein and confirmed its immunogenicity and capacity to generate neutralizing antibodies in a mouse model. This work indicates that the two domains (Gc1 ~ 97 and Gc189 ~ 397) located in the Gc head encompass neutralizing epitopes, positioning them as viable subunit vaccine targets against AKAV. The Schmallenberg virus (SBV), which belongs to the Orthobunyavirus family along with AKAV and shares most of its characteristics, has seen its Gc protein, mainly the Gc head, widely applied in the development of new vaccines [31]. Nevertheless, the high genetic variability of the Gc head region poses a challenge for vaccines designed based on this domain, limiting their effectiveness against diverse AKAV genotypes and newly emerging mutant strains.

In our previous study, a neutralizing epitope, ¹¹³⁴SVQSFDGKL¹¹⁴², was identified within the highly conserved C-terminus of the Gc protein. Its conservation across diverse AKAV genotypes suggests its potential as a key component in a broad-spectrum, epitope-based vaccine. Owing to its small size, the target epitope can be readily modified to correspond with any natural variants present in wild populations or with emerging escape mutants. The limited immunogenicity of a single epitope peptide, which often results in inadequate protective immunity, can be significantly enhanced by presenting the epitope on specialized platforms including phages, lipids, recombinant viruses, or VLPs [32]. As the first reported VLP carrier, HBcAg represents the most adaptable and promising carrier system for the rational design of chimeric peptides to date [33]. HBcAg can self-assemble into highly immunogenic particles even in recombinant form. Furthermore, this carrier is highly effective at presenting foreign antigens to the immune system, eliciting a potent response [34]. Numerous studies have constructed epitope-presenting VLPs using HBcAg as a carrier platform, and these VLPs have consistently demonstrated robust immunogenicity [32, 33, 35].

In the present study, we use the HBcAg as a VLP carrier to present the conserved epitope peptide we identified previously. The VLP HBc-AGc9 was expressed and purified and was successfully constructed by SDS-PAGE, Western blot, and negative stain EM analysis and identification. In both the 25 μg/dose group and the 5 μg/dose group, the VLP HBc-AGc9 elicited high-titer antibodies targeting the AKAV Gc protein in mouse sera. Furthermore, the antiserum was able to neutralize the AKAV with a high neutralization titer in vitro. Additionally, the VLP HBc-AGc9 has the potential to be developed as a coating antigen for an indirect ELISA method to detect AKAV. The findings verified that HBcAg retains its ability to fold and spontaneously form VLPs in vitro, with the inserted peptide being correctly displayed on the particle surface.

In this study, the quality and yield of protein purification still require optimization and improvement, for example, by adjusting factors such as the IPTG concentration, induction temperature, and induction time. Additionally, the two VLP doses chosen (5 μg and 25 μg) were suboptimal. Including a wider range of doses would better elucidate the impact of dosage on immunogenicity. A further limitation of this study is that, due to the unavailability of other AKAV genotypes in our laboratory, we could not evaluate the neutralizing activity of the mouse antisera induced by the VLP against different strains in vitro. Consequently, we were unable to demonstrate its broad-spectrum neutralizing capability against diverse AKAV genotypes.

For subsequent investigations, we may attempt to find more neutralizing epitopes of AKAV and change the number of the epitopes inserted into the HBcAg to increase the immunogenicity of the chimeric VLP. In addition, further improvements could be made to parameters such as the immunization route, the choice of adjuvant, and the animal model.

In conclusion, this study successfully generated a chimeric VLP based on the HBc protein, into which a neutralizing epitope of AKAV was inserted. Mouse immunization experiments demonstrated that this VLP elicited strong immunogenicity, indicating its potential to serve as a foundation for new AKAV vaccines and detection methodologies.

Author contributions

JJ.W.: Conceptualization, methodology, formal analysis, funding acquisition, writing—original draft preparation. F.W. and RY.Y.: investigation, methodology. HY.W. and L.M.: funding acquisition, formal analysis. DJ.C. and SQ.W.: supervision, funding acquisition, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Fundamental Research Funds of the Chinese Academy of Quality and Inspection & Testing (2024JK002 and 2025JK024) and the Beijing Natural Science Foundation (6254044).

Data availability

All data associated with this study are included in the paper.

Declarations

Ethics approval and consent to participate

The animal study was carried out in strict accordance with the experimental animal care and use guidelines of the Beijing Animal Control Committee. The experimental protocol was reviewed and approved by the Animal Welfare Ethics Committee of Beijing MDKN Biotechnology Co., LTD., with approval No. MDKN-2025–045. All efforts were made to minimize animal suffering.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.