Fusion with immunoglobulin Fc fragment enhances the immunogenicity of the African swine fever virus p30/p54 fusion protein

Abstract

Abstract

African swine fever (ASF) is a significant threat to the global pig breeding industry, making the development of a vaccine extremely urgent. In this study, the key antigenic proteins p30 and p54 of African swine fever virus (ASFV) were fused with the Fc fragment of immunoglobulin (IgG) to construct recombinant fusion proteins with strong immunity. Here, we successfully expressed four recombinant fusion proteins comprising the ASFV proteins p30 and p54 (p30-54 and p54-30) alone and the porcine IgG Fc-fused p30 and p54 proteins (p30-54-Fc and p54-30-Fc) using an insect baculovirus expression system. All expressed proteins demonstrated excellent reactivity with specific antibodies, confirming their immunological activity. In vitro studies revealed that the Fc-fused proteins exhibited significantly increased binding affinity to Fcγ receptors (FcγRs) on antigen-presenting cells (APCs). Immunization studies in mice revealed that p30-54-Fc and p54-30-Fc elicited greater humoral and cellular immune responses than p30-54 and p54-30, respectively. Notably, sera from mice immunized with the Fc-fused proteins significantly inhibited ASFV-GFP infection in porcine alveolar macrophages. These results suggest that these ASFV p30-54-Fc and p54-30-Fc recombinant fusion proteins represent promising candidate antigens for the development of an effective ASF subunit vaccine.

Key points

• Fc-fused ASFV p30/p54 proteins elicit stronger humoral and cellular immune responses in mice.

• Sera from Fc-fused antigen-immunized mice show enhanced inhibition of ASFV infection in PAMs.

• Fc fusion strategy improves immunogenicity of ASFV antigens, supporting promising subunit vaccine candidates.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-026-13919-6.

Introduction

African swine fever (ASF), caused by the virus ASFV, is a highly contagious hemorrhagic disease, with highly pathogenic strains causing near-100% mortality in domestic swine (Pikalo et al. 2020; Wang et al. 2021a). Since its first report in Kenya in 1921, ASF has spread to numerous countries around the world (Eustace Montgomery 1921). Although several commercial ASF vaccines have been licensed in some regions (Wu et al. 2020; Zhang et al. 2023b), they still have considerable limitations in safety, efficacy, and broad applicability. Therefore, the development of safer, more effective, and optimized ASF vaccines remains urgently needed.

ASFV is a large, enveloped, double-stranded DNA virus with an icosahedral morphology and an average diameter of 200 nm that belongs to the Asfarviridae family (Gaudreault et al. 2020). Its genome, ranging from 170 to 194 kb, contains 150 to 167 open reading frames (ORFs) and encodes more than 50 structural and 100 nonstructural proteins (Wang et al. 2021b). Owing to the complexity and size of the ASFV genome, as well as the unclear mechanisms of viral infection and immune evasion, no commercial ASF vaccine has been developed to date. Recent research on ASF vaccines reveals a complex scenario: while inactivated ASF vaccines can trigger humoral immune responses under specific conditions, they fail to provide effective immune protection and often do not stimulate a robust cellular immune response (Blome et al. 2014; Zhang et al. 2023b). In contrast, live attenuated vaccines can induce immune protection against homologous or heterologous strains, but vaccinated animals often exhibit adverse reactions, and there are risks of virus shedding, recombination, and reversion of virulence (Chen et al. 2020; Borca et al. 2021). In this context, subunit vaccines using recombinant proteins as antigenic components have demonstrated significant advantages in terms of safety and immune protection. Recent studies suggest that proteins such as p30, p54, p72, and CD2v are key candidates for developing subunit vaccines against ASFV. Immunizing pigs with these proteins as antigens can provide a certain level of protection against ASFV (Li et al. 2022; Duan et al. 2022; Liu et al. 2023). Therefore, with ongoing research into ASFV and the discovery of immune-protective antigens, subunit vaccines have emerged as promising vaccine types.

Both p30 and p54 are structural proteins located in the inner membrane of the virus particle. p30 is encoded by the CP204L gene, and p54 is encoded by the E183L gene (Neilan et al. 2004). In vitro experiments have shown that specific antibodies targeting p54 and p30 can effectively inhibit virus binding and internalization, respectively (Oviedo et al. 1997; Gómez-Puertas et al. 1998). The use of recombinant p30 and p54, either alone or as a fusion protein (p54/30), has successfully elicited efficient neutralizing antibody responses in pigs, providing varying degrees of protection against ASFV (Indrabalan et al. 2021; Zhang et al. 2021; Imdhiyas et al. 2023). Additionally, published data indicate that both antibodies and T cells play crucial roles in controlling viral infections, with robust specific T cell responses being particularly important for protection (Oura et al. 2005; Argilaguet et al. 2012; Takamatsu et al. 2013; Lu et al. 2023). Therefore, in the development of recombinant subunit vaccines for ASF, it is essential to not only induce neutralizing antibodies but also promote cellular immune responses for comprehensive and effective protection.

While subunit vaccines show great potential, their protective effects need further enhancement. One innovative strategy is the fusion of antigens with Fc fragments. The Fc fragment, a critical component of the IgG constant region, has numerous biological functions and offers novel vaccine design possibilities. By binding to the neonatal Fc receptor (FcRn), Fc can significantly extend the circulating half-life of fusion proteins, ensuring stable antigen concentrations in the body (Mackness et al. 2019; Li et al. 2022). Additionally, Fc binding to FcγRs plays a vital role in antibody-mediated immune responses, facilitating mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP), thereby enhancing antiviral capabilities (Thulin and Wang 2018; Ren et al. 2021; Yang et al. 2023). Furthermore, Fc-fused proteins may have intrinsic adjuvant effects. For example, a plant-expressed tetravalent dengue vaccine with Fc-fused proteins has demonstrated significant adjuvant properties, highlighting the unique value of Fc fragments in vaccine design (Kim et al. 2018). Thus, the strategy of fusing antigens with Fc fragments is expected to enhance antigen transport across membrane barriers through FcRn- and FcγRs-mediated mechanisms, significantly boosting both humoral and cellular immune responses.

In this study, we expressed ASFV p30 and p54 fusion proteins (p30-54 and p54-30) and novel Fc-fused proteins (p30-54-Fc and p54-30-Fc) using a baculovirus expression system. We further investigated whether these Fc-fused proteins could elicit a stronger immune response in mice than the p30 and p54 fusion proteins. This study provides a foundation and strategy for future research on ASFV subunit vaccines.

Materials and methods

Plasmid construction

The p30 gene and p54 gene were derived from ASFV-SY18 (GenBank: MH766894.1). To generate a modified version of p54, the transmembrane region (His30-Phe52) was replaced with a long flexible linker “(GGGGS)3,” while p30 remained unchanged when expressed independently. The IgG Fc fragment, comprising the hinge, extended CH2, and CH3 domains, was sourced from porcine IgG (AK405774.1). To evaluate the impact of different fusion strategies on protein expression and the immune response, p30 was fused either to the C-terminus or N-terminus of modified p54, resulting in the constructs p30-54 and p54-30, respectively. Additionally, p30-54 and p54-30 were further fused to the N-terminus of the porcine IgG Fc fragment to produce p30-54-Fc and p54-30-Fc, respectively (Fig. 1A). To ensure proper folding of the fusion proteins, the long flexible linker “(GGGGS)3” was utilized to connect the protein domains. All the plasmids were synthesized by GenScript (Nanjing, China) and cloned and inserted into the pFastBac1 vector (Thermo Fisher Scientific, Waltham, MA, USA). The integrity and accuracy of the constructed expression plasmids were verified by DNA sequencing.

Fig. 1.

Design of antigens and expression identification of recombinant fusion proteins. A Schematic design of the recombinant expression plasmids. Four fusion proteins (p30-54, p54-30, p30-54-Fc, and p54-30-Fc) were constructed by fusing p30 and a modified p54 (with its transmembrane region replaced by a (GGGGS)₃ linker) in different orientations, and selected constructs were further fused to the porcine IgG Fc fragment via a flexible linker. The fusion genes were cloned into the pFastBac™1 vector under the control of the polyhedrin promoter, with a C-terminal His tag for detection. B IFA confirming the expression of recombinant proteins in Sf9 cells. The primary antibodies used were anti-His monoclonal antibody, anti-ASFV p30 antibody, and anti-ASFV p54 antibody. The secondary antibodies were Alexa Fluor 594 or 488-labeled goat anti-mouse and goat anti-pig antibodies, respectively

The preparation of recombinant baculovirus

The recombinant plasmids were transformed into Escherichia coli DH10Bac competent cells (Weidi Biotechnology, Shanghai, China) for transposition. The bacterial suspension was then spread onto LB agar plates supplemented with 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL Bluo-GAL (5-bromo-3-indolyl-β-D-galactopyranoside), and 40 µg/mL IPTG (isopropyl-β-D-1-thiogalactopyranoside). The plates were incubated at 37 °C for 48 h. Positive recombinant strains were identified through multiple rounds of blue-white screening. The extracted recombinant bacmids were designated as Bacmid-p30-54, Bacmid-p54-30, Bacmid-p30-54-Fc, and Bacmid-p54-30-Fc. These recombinant bacmids were subsequently transfected into Sf9 cells (Thermo Fisher Scientific, Waltham, MA, USA) using Expifectamine SF™ transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). The transfected cells were incubated at 27 °C with 5% CO₂ and constant shaking at 120 rpm/min for 72 to 96 h. After cytopathic effect occurred, the supernatant was collected to harvest the first-generation recombinant viruses (rBac-p30-54, rBac-p54-30, rBac-p30-54-Fc, and rBac-p54-30-Fc). The virus titers (50% tissue culture infectious dose, TCID₅₀) were determined.

Indirect immunofluorescence assay (IFA)

The expression of the recombinant fusion proteins p30-54, p54-30, p30-54-Fc, and p54-30-Fc in Sf9 cells was assessed using IFA. Briefly, Sf9 cells were infected with respective recombinant baculoviruses (rBac-p30-54, rBac-p54-30, rBac-p30-54-Fc, or rBac-p54-30-Fc) and cultured at 27 °C for 72 h. The cells were then fixed with 4% paraformaldehyde (PFA) at room temperature, permeabilized with 2% Triton X-100, and blocked with 5% bovine serum albumin (BSA) for 30 min. Subsequently, the cells were incubated with anti-His monoclonal antibody (Abcam, Cambridge, UK), anti-ASFV p30 antibody (prepared in our laboratory), and anti-ASFV p54 antibody (prepared in our laboratory) at room temperature for 2 h. After incubation, the cells were treated with Alexa Fluor 488-labeled goat anti-pig antibody (1:1000 dilution) or Alexa Fluor 594-labeled goat anti-mouse antibody (1:1000 dilution) in the dark for 1 h, followed by nuclear staining with DAPI (4′,6-diamidino-2-phenylindole) in the dark for 15 min. Images were captured using an inverted fluorescence microscope (Nikon, Tokyo, Japan).

Protein purification and identification

For protein purification, Sf9 cells were infected with the respective recombinant baculoviruses and cultured at 27 °C until cell viability reached 60–80%. The cells were harvested, solubilized in lysis buffer, and treated with nucleases and protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). Following sonication and centrifugation, the supernatant was collected, filtered, and subjected to protein purification using nickel affinity chromatography (GE, Boston, MA, USA) according to the manufacturer’s instructions. The purified proteins were analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and native-PAGE (native polyacrylamide gel electrophoresis). SDS-PAGE was performed under denaturing conditions. Briefly, protein samples were heated with SDS loading buffer and separated on polyacrylamide gels. Native-PAGE was carried out in the absence of SDS and sample heating to preserve the native protein structure, allowing separation based on the intrinsic characteristics of proteins. Following SDS-PAGE separation, the gel samples were subjected to western blotting. Proteins were transferred to a membrane after electrophoresis and blocked with 5% skimmed milk for 1 h at room temperature. The membrane was then incubated overnight at 4 °C with primary antibodies (anti-His monoclonal antibody, anti-ASFV p30 antibody, and anti-ASFV p54 antibody). This was followed by incubation with HRP-labeled goat anti-mouse antibody (1:10,000 dilution) or goat anti-pig antibody (1:10,000 dilution) as secondary antibodies at room temperature for 1 h. Finally, protein bands were visualized by treating the membrane with a chemiluminescence (ECL) reagent (Thermo Fisher Scientific, Waltham, MA, USA) and imaging with a high-resolution gel documentation system (Bio-Rad, Hercules, CA, USA).

Analysis for the binding of Fc-fused proteins to porcine FcγRI

The binding capacity of p30-54-Fc and p54-30-Fc to the FcγRI was examined by IFA. Porcine alveolar macrophages (PAMs) were cultured overnight in RPMI-1640 medium (10% FBS + 100 IU/mL penicillin + 100 µg/mL streptomycin) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA). Cells were then treated separately with p30-54, p54-30, p30-54-Fc, and p54-30-Fc recombinant proteins and incubated at 37 °C for 30 min. After treatment, cells were fixed, permeabilized, and blocked as described in previous methods. Subsequently, the cells were incubated with rabbit anti-CD64 (FcγRI) polyclonal antibody (1:1000, Absin, Shanghai, China) and anti-His monoclonal antibody (1:1000) at 37 °C for 2 h, followed by a 1-h incubation in the dark at room temperature with Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 594-conjugated goat anti-mouse IgG (both at 1:1,000). Finally, images were acquired using a laser scanning confocal microscope (Leica, Wetzlar, Germany).

Animal immunization

Thirty 6- to 8-week-old female BALB/c mice were randomly divided into five groups (n = 6 per group). For the primary immunization, antigens for Groups A to D (p30-54, p54-30, p30-54-Fc, and p54-30-Fc, respectively) were thoroughly emulsified with an equal volume of complete Freund’s adjuvant (TargetMol, Shanghai, China). Each mouse in the experimental groups received a 200 µL subcutaneous multi-point injection of the emulsified vaccine containing 40 µg of the corresponding protein, while the control group (Group E) was administered an equal volume of PBS emulsified in the same manner. A booster immunization was performed 21 days later using the same antigen dose emulsified with incomplete Freund’s adjuvant (TargetMol, Shanghai, China) at the same volume and ratio. Blood samples were collected from the orbital vein every 7 days starting from the primary immunization. After collection, the blood was allowed to stand at room temperature for 2 h for serum separation, followed by centrifugation at 3000 r/min for 20 min. The serum was aliquoted and stored at −80 °C for subsequent analysis. All animal procedures were conducted in strict accordance with the Guidelines for Animal Ethics and Welfare of the People’s Republic of China and were approved by the Animal Management and Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval No. LVRIAEC-2024–056).

Detection of specific antibodies by enzyme-linked immunosorbent assay (ELISA)

Serum levels of IgG specific to p30-54, p54-30, p30-54-Fc, and p54-30-Fc were measured by indirect ELISA. Briefly, each purified protein (1 µg/mL) was dissolved in Na₂CO₃ coating buffer and used to coat 96-well plates, which were then incubated at 4 °C overnight. After blocking with 5% BSA at 37 °C for 1 h, diluted mouse serum samples (1:1000) were added and incubated at 37 °C for 1 h. Plates were then incubated with HRP-conjugated goat anti-mouse IgG at 37 °C for 45 min, and OD₄₅₀ was measured using a microplate reader. Additionally, endpoint titers of specific antibodies were detected 42 days after immunization by indirect ELISA. Serum samples were serially diluted twofold from 1:1000 to 1:256,000, and the OD₄₅₀ values were detected.

Lymphocyte proliferation assay

On day 42 post-primary immunization, three mice per group were euthanized, and spleens were aseptically collected. Splenic lymphocytes were isolated using a commercial mouse lymphocyte isolation kit (Solebio, Beijing, China). The lymphocyte density was diluted to 2.5 × 10⁶ cells/mL and seeded into 96-well cell culture plates at 100 µL/well. To assess antigen-specific proliferation, lymphocytes were stimulated with 50 µL of the corresponding purified protein (p30-54, p54-30, p30-54-Fc, or p54-30-Fc) at a final concentration of 10 µg/mL. Concanavalin A (ConA; 10 µg/mL, Beyotime, Shanghai, China) was used as a positive control, while RPMI-1640 medium was added to blank control wells. Negative control wells received no stimulant. All conditions were performed in triplicate, and plates were incubated at 37 °C with 5% CO₂ for 48 h. Thereafter, 10 µL of CCK-8 solution (5 mg/mL, Cambridge, UK) was added to each well, followed by an additional 4 h of incubation. The OD₄₅₀ values were detected using a microplate reader. The stimulation index (SI) was calculated as follows: SI = (OD _experimental − OD _{blank control})/(OD _{negative control} − OD _{blank control}).

IL-4 and IFN-γ detection by enzyme-linked immunospot (ELISpot)

Lymphocytes were seeded into 96-well microplate pre-coated with anti-IL-4 and IFN-γ specific antibodies (Dakewe Biotech, Shenzhen, China) at a density of 2.5 × 10⁵ cells per well. For the experimental groups, 50 µL of specific antigen stimuli (p30-54, p54-30, p30-54-Fc, and p54-30-Fc proteins) was added to each well at a final concentration of 10 µg/mL. The positive control wells were treated with the positive stimulus at the working concentration as specified in the kit instructions. The negative control wells were supplemented with an equal volume of RPMI-1640 medium, while the blank control wells received no stimuli. All procedures were performed in accordance with the instructions provided with the IL-4 and IFN-γ detection kits (Dakewe Biotech, Shenzhen, China). The number of spots was quantified using an ELISpot analyzer.

Flow cytometry analysis

The lymphocyte density was adjusted to 1 × 10⁵ cells/mL. Next, 1 mL of lymphocyte suspension was centrifuged and resuspended in 200 µL of PBS. Subsequently, 1 µL each of fluorescein isothiocyanate (FITC)–conjugated anti-CD3, phycoerythrin (PE)–conjugated anti-CD4, and allophycocyanin (APC)–conjugated anti-CD8 antibodies were added, and the mixture was gently vortexed. All samples were prepared in triplicate. Staining was performed in the dark on ice for 30 min. The cells were suspended by 300 µL of PBS, and the flow cytometry analysis was performed to detect T lymphocyte subsets.

Serum inhibition assay

On day 42 post-primary immunization, serum samples from each group were inactivated by incubation in a 56 °C water bath for 30 min. The serum was then serially diluted at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 using RPMI-1640 medium. Recombinant ASFV virus expressing GFP was generated using ASFV CN/GS/2018 as the genome backbone in Biosafety Level 3 (BSL-3) facilities at the Lanzhou Veterinary Research Institute (LVRI) of the Chinese Academy of Agricultural Sciences (CAAS). PAMs were cultured overnight in 96-well plates at 37 °C with 5% CO₂. The diluted serum was mixed with ASFV-GFP at a 1:1 volume ratio and incubated at 37 °C for 2 h to allow complete interaction between the virus and serum. The virus-serum mixture was then inoculated into PAMs and cultured at 37 °C with 5% CO₂ for 48 h. Infected cells were observed under a fluorescence microscope. Subsequently, the infected cells were lysed, and the supernatants were collected for DNA extraction. The viral genome copy number in the serum was quantified using qPCR.

Statistical analysis

Data are presented as the mean ± SD. The significance of differences between groups was calculated by one-way analysis of variance and independent sample t-tests using GraphPad Prism 8.0 software (La Jolla, CA, USA). P > 0.05 indicates no significant difference (ns), *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences.

Results

Design of the recombinant fusion proteins and their expression in the insect baculovirus expression system

To analyze the immunogenicity of ASFV p30 and p54 fusion proteins, four recombinant plasmids encoding p30-54, p54-30, p30-54-Fc, and p54-30-Fc were designed and constructed. As shown in Fig. 1A, p30 was fused to the C-terminus or N-terminus of modified p54 to generate p30-54 and p54-30, respectively. These constructs were subsequently fused to the N-terminus of the porcine IgG Fc fragment to produce p30-54-Fc and p54-30-Fc. DNA sequencing confirmed that all expression plasmids were correctly constructed with intact sequences, ensuring their suitability for further experiments.

Protein expression of ASFV p30-54, p54-30, p30-54-Fc, and p54-30-Fc was detected IFA. The results revealed that Sf9 cells infected with the rBac-p30-54, rBac-p54-30, rBac-p30-54-Fc, and rBac-p54-30-Fc presented strong fluorescence signals when probed with anti-His, anti-ASFV p30, and anti-ASFV p54 antibodies, whereas uninfected cells presented no signal (Fig. 1B). These results demonstrated that all four fusion proteins were successfully expressed in the insect baculovirus expression system.

Purification and identification of the recombinant fusion proteins

The recombinant fusion proteins p30-54, p54-30, p30-54-Fc, and p54-30-Fc were expressed in Sf9 cells and purified using nickel affinity chromatography. SDS-PAGE analysis indicated that both the p30-54 and p54-30 migrated at their anticipated molecular weights (Fig. 2A). In contrast, the p30-54-Fc and p54-30-Fc proteins each exhibited a major band near 70 kDa, along with additional bands around 35 kDa, suggestive of possible degradation or impurities (Supplementary Fig. S1). To assess whether these lower molecular weight bands resulted from SDS-induced denaturation or fragmentation, we performed native-PAGE. This analysis revealed a single band at approximately 70 kDa for both Fc-fusion constructs (Fig. 2B), indicating the intact, non-denatured state of the proteins under native conditions. Western blotting was subsequently conducted to confirm protein identity and antigenicity. Western blot analysis using anti-His monoclonal (Fig. 2C), anti-ASFV p30 (Fig. 2D), and anti-ASFV p54 (Fig. 2E) antibodies revealed distinct bands at approximately 40 kDa for the p30-54 and p54-30 proteins. In contrast, the p30-54-Fc and p54-30-Fc proteins showed specific bands at around 70 kDa, along with additional impurity bands at 35 kDa. These observations align with the SDS-PAGE and native-PAGE results. Overall, the recombinant fusion proteins p30-54, p54-30, p30-54-Fc, and p54-30-Fc were successfully expressed and purified, and each demonstrated strong antigenic properties.

Fig. 2.

Identification of recombinant fusion proteins. A SDS-PAGE analysis of purified p30-54 and p54-30. B Native-PAGE analysis of purified p30-54-Fc and p54-30-Fc. C–E Western blot analysis of the purified proteins p30-54, p54-30, p30-54-Fc, and p54-30-Fc probed with C anti-His tag monoclonal antibody, D anti-ASFV p30 polyclonal antibody, and E anti-ASFV p54 polyclonal antibody

Functional characterization of the recombinant Fc-fused proteins

The functionality of the Fc-fused proteins was preliminarily assessed by western blot. Given the high homology between mouse and pig IgG Fc regions (Liu et al. 2023), the membranes were probed with HRP-conjugated goat anti-pig or goat anti-mouse secondary antibodies to evaluate Fc integrity and binding capacity. The results show that the proteins p30-54-Fc and p54-30-Fc could be directly recognized by HRP-conjugated goat anti-pig (Fig. 3A) and goat anti-mouse secondary antibodies (Fig. 3B). In contrast, the control proteins p30-54 and p54-30, which lack the Fc domain, showed no reactivity. The results demonstrate that the Fc portion of the recombinant fusion proteins is correctly folded and retains the ability to bind antibodies specific to both mouse and porcine IgG.

Fig. 3.

Functional validation of the Fc-fused proteins. A, B Western blot analysis of the recombinant proteins probed with HRP-conjugated A goat anti-pig or B goat anti-mouse secondary antibodies. C Confocal microscopy images demonstrating the binding of Fc-fused proteins to FcγRI on PAMs. FcγRI was stained with an Alexa Fluor 488-conjugated antibody (green). The recombinant proteins were detected using an anti-His tag antibody followed by an Alexa Fluor 594-conjugated secondary antibody (red). Nuclei were counterstained with DAPI (blue). Co-localization appears in yellow (merge). Scale bar, 25 µm

We next assessed whether the incorporated Fc domain could leverage the effector function by binding to FcγRI on APCs. Laser confocal analysis revealed that both p30-54-Fc and p54-30-Fc bound to FcγRI with significantly greater efficiency than the p30-54 and p54-30 proteins (Fig. 3C), confirming the functional role of the Fc segment.

Fc-fused proteins induce potent antigen-specific IgG responses in mice

To evaluate the potential of Fc fusion to enhance the humoral immune response, mice were immunized subcutaneously with purified p30-54, p54-30, p30-54-Fc, or p54-30-Fc proteins, according to the schedule in Fig. 4A. Serum levels of antigen-specific IgG were monitored by indirect ELISA. All immunized groups generated specific antibodies following the primary immunization, with the p30-54-Fc and p54-30-Fc groups exhibiting more rapid kinetics of antibody generation (Fig. 4B). After the booster immunization, antibody levels increased markedly in all groups, but were significantly higher in mice receiving the Fc-fused proteins compared to those immunized with p30-54 or p54-30 alone (Fig. 4B). At 42 days post-primary immunization, antibody titers in the Fc-fusion groups continued to rise, whereas titers in the non-Fc groups plateaued or began to decline, indicating a more sustained antibody response in the presence of the Fc domain. No specific antibodies were detected in the PBS group. No significant differences in antibody levels were observed between the p30-54 and p54-30 groups, or between the p30-54-Fc and p54-30-Fc groups.

Fig. 4.

Fc-fused proteins enhance humoral immune responses in mice. A Immunization scheme. B Kinetics of antigen-specific IgG levels in mouse serum measured by indirect ELISA at the indicated days post-primary immunization. C Endpoint titers of antigen-specific antibodies in serum collected on day 42 post-primary immunization

On day 42, endpoint titers of antigen-specific antibodies were determined. The mean serum antibody titer reached 1:16,000 in both the p30-54 and p54-30 groups, compared to 1:64,000 in the p30-54-Fc and p54-30-Fc groups (Fig. 4C). Together, these findings demonstrate that Fc fusion significantly enhances the immunogenicity of p30-54 and p54-30, leading to higher and more durable antigen-specific antibody responses in mice.

The Fc-fused proteins elicit enhanced cellular immune responses

To evaluate antigen-specific cellular immune responses, splenic lymphocytes isolated from immunized mice were stimulated with the corresponding recombinant proteins, and proliferation was measured by CCK-8 assay. The SIs of all protein-immunized groups were significantly higher than those of the PBS control group. Notably, the SIs of the p30-54-Fc and p54-30-Fc groups were significantly greater than those of the p30-54 and p54-30 groups (Fig. 5A), indicating that Fc fusion enhances antigen-induced lymphocyte proliferation.

Fig. 5.

The Fc-fused proteins induce more potent cellular immune responses in mice. A Lymphocyte proliferation was tested by the CCK-8 lymphocyte proliferation assay at 42 days after primary immunization. B, C Number of IL-4 (B) and IFN-γ (C) cytokine-secreting cells in splenocytes after antigen-specific stimulation, measured by ELISpot. D, E Flow cytometric analysis of CD8⁺ (D) and CD4⁺ (E) T cell populations in splenic lymphocytes

Cytokine secretion by activated splenocytes was further assessed by ELISpot. As shown in Fig. 5B and C, stimulation with p30-54-Fc or p54-30-Fc induced higher numbers of IFN-γ (induced by the Th1 cell response) and IL-4 (induced by the Th2 cell response) spot-forming cells compared to stimulation with p30-54 or p54-30, suggesting a more robust cytokine response elicited by the Fc-fused antigens.

Furthermore, flow cytometric analysis of splenic T lymphocytes revealed that immunization with all recombinant proteins significantly increased the proportions of both CD8⁺ (Fig. 5D) and CD4⁺ T cells (Fig. 5E) compared to the PBS control. Importantly, the p30-54-Fc and p54-30-Fc immunization groups exhibited significantly higher frequencies of CD8⁺ and CD4⁺ T cells than the non-Fc protein groups. No significant differences were observed between the p30-54 and p54-30 groups or between the two Fc-fusion groups. Collectively, these results demonstrate that the Fc-fused proteins p30-54-Fc and p54-30-Fc induce stronger cellular immune responses than their non-Fc counterparts.

Antibodies against p30-54, p54-30, p30-54-Fc, and p54-30-Fc inhibit ASFV replication

To further evaluate the immunogenicity of the recombinant fusion proteins, the ability of mouse antisera to inhibit ASFV replication was analyzed. PAMs were infected with ASFV-GFP pre-incubated with serial dilutions of antisera, and fluorescence intensity was examined at 48 h post-infection. The results demonstrated that antisera from all immunized groups significantly suppressed ASFV-GFP replication across multiple dilutions. Notably, antisera from the p30-54-Fc and p54-30-Fc groups exhibited stronger inhibitory effects at higher dilutions than those from the p30-54 and p54-30 groups (Fig. 6A).

Fig. 6.

Antisera from immunized mice inhibit ASFV replication in vitro. A Fluorescence images of PAMs infected with ASFV-GFP pre-incubated with serial dilutions of mouse antisera, taken at 48 h post-infection. B Viral genome copies in infected PAMs quantified by qPCR at 48 h post-infection. ASFV-GFP was pre-incubated with the indicated dilutions of antisera or control serum prior to infection

To further quantify viral inhibition, viral DNA was extracted from infected PAMs, and viral genome copies were measured by qPCR. Consistent with the fluorescence observations, all antisera significantly reduced viral DNA copy numbers in a dilution-dependent manner. Again, antisera from the Fc-fused proteins groups showed more potent inhibition of ASFV replication at higher dilutions (Fig. 6B).

Collectively, these results indicate that polyclonal antibodies generated against p30-54, p54-30, p30-54-Fc, and p54-30-Fc effectively restrict ASFV replication, with Fc-fused proteins eliciting antisera of superior neutralization potency.

Discussion

The development of a safe and effective ASF vaccine is crucial for ensuring sustainable pig production and remains an urgent challenge. While the ASFV proteins p30 and p54 have been extensively investigated as subunit vaccine candidates, previous studies have demonstrated their limited efficacy. Earlier studies showed that pigs immunized with a combination of recombinant p30 and p54 or the chimeric protein p54/30 were partially protected against ASFV challenge owing to neutralizing antibodies (Argilaguet et al. 2012). However, subsequent studies revealed that immunization with multiple ASFV proteins (p30, p54, p72, and p22) only marginally delayed clinical disease onset by 2 days post-challenge (Blander 2018). These findings underscore the need to develop more immunogenic antigen formulations capable of eliciting robust humoral and cellular immune responses, which are critical for effective ASFV prevention and control. Recent advances in vaccine development have highlighted the potential of IgG Fc-fused proteins to enhance antigen presentation and induce stronger immune responses (Li et al. 2019, 2020; Park et al. 2020; Chen et al. 2021; Richel et al. 2023; Zhang et al. 2024). On the basis of these findings, two of the most antigenic and immunogenic ASFV proteins were fused with porcine IgG Fc to further enhance protective immune responses. Using the insect baculovirus expression system, we successfully expressed four recombinant proteins, namely, the ASFV proteins p30 and p54 (p30-54 and p54-30) alone and the porcine IgG Fc-fused p30 and p54 proteins (p30-54-Fc and p54-30-Fc), and all four recombinant proteins could be recognized by anti-ASFV p30 and p54 serum, confirming their excellent reactivity. In mouse immunization studies, all four fusion proteins elicited robust humoral and cellular immune responses, confirming their excellent antigenicity. Notably, no significant differences in immunogenicity were observed between p30-54 and p54-30 or between their Fc-fused counterparts, indicating that the tandem sequence of p30 and modified p54 proteins does not affect the expression or immunogenicity of the fusion protein. These findings suggest that the structural configuration of these fusion proteins maintains the antigenic integrity of both components, while Fc fusion enhances their immunogenic potential, making them promising candidates for ASFV subunit vaccine development.

Extensive research has demonstrated that Fc-fused recombinant proteins can significantly promote antigen recognition and phagocytosis, thereby facilitating antigen presentation and increasing immunogenicity. This enhanced immune response is primarily mediated through the interaction of the Fc region with FcγRI and serum complement proteins, which play crucial roles in immune effector functions (Czajkowsky et al. 2012; Chun et al. 2019; Li et al. 2019). Specifically, porcine IgG Fc can bind to porcine FcγR, which are abundantly expressed on the surface of APCs, thereby potentiating immune responses (Zhang et al. 2006). Given that APCs are the primary target cells for ASFV infection, the fusion of porcine IgG Fc with the ASFV protective antigens p30 and p54 represents a strategic approach to enhance antigen recognition and presentation. In our study, western blotting analysis revealed distinct immunological properties of the Fc-fused proteins. The purified p30-54-Fc and p54-30-Fc fusion proteins were specifically recognized by both sheep anti-mouse and sheep anti-pig secondary antibodies, whereas the p30-54 and p54-30 proteins could not be recognized by these antibodies. Furthermore, the binding efficiencies of the Fc fusion recombinant proteins p30-54-Fc and p54-30-Fc to FcγRI on PAMs were greater. These findings suggest that the p30-54-Fc and p54-30-Fc proteins may promote the phagocytosis ability of APCs, resulting in enhanced antigen presentation or cross-presentation to T cells, thereby enhancing the adaptive immune response.

The humoral immune response is a critical defense mechanism against ASFV infection, as evidenced by previous studies (Orosco 2023; Zhang et al. 2023a). Our preliminary experiments confirmed that the porcine Fc fragments used in this study could specifically bind to mouse secondary antibodies, validating the use of mice as experimental models for evaluating immune responses. To assess the immunogenicity of our constructs, we measured antigen-specific antibody levels in sera from mice immunized with the ASFV fusion proteins. The immunization results revealed that all four fusion proteins induced robust immune responses in mice, and the antibody titers of the p30-54-Fc and p54-30-Fc protein groups were significantly greater than those of the p30-54 and p54-30 protein groups. We also observed a longer duration of antibody generation induced by p30-54-Fc and p54-30-Fc. In total, the p30-54-Fc and p54-30-Fc fusion proteins can increase the level of specific antibody production and the humoral immune response by targeting antigens on APCs through binding to the FcγRI of APCs.

Cellular immune responses play pivotal roles in eliminating intracellular infections caused by ASFV(Argilaguet et al. 2012; Takamatsu et al. 2013; Lu et al. 2023). Given the critical functions of IFN-γ (a Th1 cytokine) and IL-4 (a Th2 cytokine) in orchestrating immune responses against viral infections, we employed the ELISpot assay to quantify antigen-specific cytokine secretion by splenic lymphocytes. Our results demonstrated that immunization with p30-54-Fc and p54-30-Fc proteins elicited significantly higher levels of both IFN-γ and IL-4 than immunization with the p30-54 and p54-30 proteins. To comprehensively evaluate the cellular immune response, we further assessed lymphocyte proliferation and T cell subset differentiation. In terms of lymphocyte proliferation, the lymphocyte proliferation levels of the mice in the p30-54-Fc and p54-30-Fc immunized groups were significantly greater than those in the p30-54 and p54-30 immunized groups. Flow cytometric analysis of T lymphocyte subsets revealed increased proportions of both CD8⁺ and CD4⁺ T cells in the p30-54-Fc and p54-30-Fc immunized groups. These findings were consistent with the observed cytokine profiles, demonstrating a coordinated enhancement of cellular immunity. In summary, the above findings suggest that the fusion of the IgG Fc with the ASFV p30 and p54 proteins induces increased levels of cellular immune responses in mice.

Additionally, we systematically analyzed the capacity of sera from mice immunized with p30-54, p54-30, p30-54-Fc, and p54-30-Fc proteins to inhibit ASFV-GFP infection of PAMs. First, the fluorescence and qPCR results revealed that sera at various dilutions could effectively decrease the fluorescence intensity and copy number of the viral genome after viral infection, suggesting that the antibodies in these sera could bind to the viral particles, thereby preventing or reducing the replication and spread of the virus within PAMs. Notably, sera from the p30-54-Fc and p54-30-Fc immunized groups at high dilutions presented more pronounced virus inhibitory effects, which might imply that the antibodies induced by these two fusion proteins have greater affinity and/or neutralizing activity. Their specific mechanisms of action in antiviral immunity should be explored in future studies.

In conclusion, we successfully expressed the ASFV p30-54, p54-30, p30-54-Fc, and p54-30-Fc fusion proteins using a baculovirus expression system and demonstrated that incorporating porcine IgG Fc into the fusion proteins of p30 and p54 could enhance both humoral immunity and cellular immunity in mice, as well as their potential to inhibit ASFV infection. It should be noted that the present study was only conducted in a mouse model, and further evaluation in pigs is still necessary to confirm the immunogenicity and protective efficacy in the natural host. First, our study confirmed that Fc fusion effectively improved the immunogenicity of ASFV p30/p54 antigens in mice, including increased antibody titers, sustained humoral responses, and robust cellular immunity, all of which are well-recognized immune correlates of vaccine protection against viral pathogens and provide a solid basis for further evaluation in pig. Second, the porcine IgG Fc domain used in this study retained complete biological activity and was capable of binding to mouse Fc receptors in vitro, thereby achieving efficient antigen presentation through Fc-mediated pathways. This core mechanism is fully recapitulated in the mouse model, confirming that mice are suitable for evaluating the immune-enhancing effect of Fc-fused antigens. In addition, numerous previous ASF vaccine studies have routinely used mouse models for the initial screening and optimization of subunit vaccines, mRNA vaccines, and viral vector vaccines before subsequent testing in pigs (Liu et al. 2023; Sun et al. 2025; Hu et al. 2025). Accumulated evidence has shown that immunogenicity results obtained in mice are highly consistent with protective potential in pigs, strongly supporting the rational use of mouse models for early-stage ASF vaccine development. Collectively, these findings indicate that the ASFV p30-54-Fc and p54-30-Fc fusion proteins represent promising candidate antigens for future ASF subunit vaccine development. This study not only provides a feasible strategy for improving the immunogenicity of ASFV subunit vaccines but also offers a valuable reference for the design of next-generation vaccines against African swine fever.

Conclusions

This study demonstrates that fusion of porcine IgG Fc domain to ASFV p30 and p54 antigens significantly enhances their immunogenicity and functional antibody responses. Using a baculovirus expression system, we successfully produced four recombinant proteins (p30-54, p54-30, p30-54-Fc, and p54-30-Fc), with the Fc-fusion variants showing superior performance in multiple assays. The Fc-fused proteins induced stronger and more durable humoral immune responses, enhanced T cell proliferation, promoted both Th1 and Th2 cytokine production, and increased CD4⁺ and CD8⁺ T cell populations. Critically, antisera from Fc-fusion immunized mice exhibited more potent neutralization capacity against ASFV infection in vitro, even at high dilutions.

Our findings validate Fc fusion as an effective strategy to improve the vaccine potential of ASFV subunit antigens. The p30-54-Fc and p54-30-Fc constructs represent promising candidates for further development as subunit vaccines against ASFV. Future studies should focus on evaluating the protective efficacy of these Fc-fusion vaccines in swine models and elucidating the precise mechanisms underlying Fc-mediated enhancement of anti-ASFV immunity.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Y.Y. and H.Z. is the corresponding author, who plays a guiding role in the study design, laboratory supervision, and manuscript editing. H.D. contributed to the study design, experiments, data analysis, and manuscript drafting, editing, and writing. Y.R. contributed to the expression of the recombinant proteins, design experiments and assist in preparing and revising manuscripts. G.W., R.H. and J.X. contributed to the revision of the manuscripts. Y.L., C.J., L.Z. and B.L. contributed to laboratory quality control and data collection. X.H, S.H and D.Z. provided assistance for the experimental work.

Funding

This work was supported by the National Key R&D Program of China (2025YFD1800703), Major S&T Special Program of Chengguan District, Lanzhou, Gansu (2025KJZD00098), Major Project of Gansu Joint Research Fund (23JRRA1477), Gansu Industry-Research Integration Key Technology Empowerment Program (25FNNA002), the Science and Technology Department of Gansu Province (23ZDKA0002), the Central Public-interest Scientific Institution Basal Research Fund (CAAS-ZDRW202409), the Earmarked Fund for National Pig Industry Technology System (CARS-35), the Youth Science and Technology Talent Innovation Program of Lanzhou (2023-QN-3), and the Fundamental Research Funds for the Central Universities (lzujbky-2022-ct02).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. The datasets supporting the findings of this study, including uncropped Western blot images and raw numerical data for all graphs, are available from the corresponding author upon reasonable request.

Declarations

Ethics approval

All animal experiments were approved by the Animal Management and Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval Number: LVRIAEC-2024-056) and were conducted in accordance with the institutional guidelines for animal welfare.

Informed consent

All participants received information about this research and their participation and signed a digital informed consent form.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.