Development and immunological evaluation of a ferritin nanoparticle vaccine displaying chicken infectious anemia virus VP1-VP2 proteins

Abstract

Background

Chicken infectious anemia virus (CIAV) causes immunosuppression, aplastic anemia, and lymphoid atrophy in young chicks, causing major economic losses to the poultry industry. Vaccination is the primary strategy for disease control. This study aimed to develop a ferritin-based nanoparticle vaccine displaying CIAV VP1-VP2 proteins and evaluate its protective efficacy in chicks.

Results

Using the SpyTag003/SpyCatcher003 system, we expressed ferritin-SpyCatcher003 (SC-FN) in insect cells and SpyTag003-VP2-VP1Nd129 in E. coli, achieving in vitro assembly of FN-VP2-VP1Nd129 nanoparticles, which were confirmed by SDS-PAGE, Western blotting, and transmission electron microscopy. Two-week-old chicks immunized with the nanoparticle vaccine developed significantly higher CIAV-specific antibody levels compared to the control group. Following lethal challenge with CIAV Cux-1 strain, the vaccine group showed significantly higher hematocrit values and thymus indices (p < 0.001), and importantly, no viral nucleic acid was detected in their thymus tissues.

Conclusions

We successfully constructed a ferritin nanoparticle-based antigen display system for CIAV. The vaccine induced robust humoral immunity, effectively protected against viral replication and clinical disease, and offers a promising new strategy for developing safe and effective subunit vaccines against CIAV.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12917-026-05694-3.

Introduction

Chicken infectious anemia virus (CIAV) is a major pathogen causing immunosuppression and aplastic anemia in young chicks. Since its first report in Japan in 1979, it has become widely distributed globally [1–3]. CIAV infection leads to thymic atrophy, bone marrow adipose replacement, and severe anemia in chicks [4–6], with mortality rates of 5%-30%. More importantly, the immunosuppression it induces often co-occurs with infections by Marek’s disease virus (MDV), Newcastle disease virus (NDV), avian leukosis virus subgroup J (ALV-J), fowl adenovirus (FAdV), and infectious bursal disease virus (IBDV) [7–11], significantly exacerbating disease severity, compromising vaccine efficacy, and causing substantial economic losses to the poultry industry.

To develop an effective subunit vaccine, understanding the functions of CIAV-encoded proteins is essential. The CIAV genome encodes three proteins, including VP1, VP2, and VP3. VP1 is the sole structural protein forming the viral capsid and is a key antigen for inducing neutralizing antibodies [12]. VP2 acts as a scaffold protein; although not part of the mature virion, it is crucial for the correct folding of VP1 and the exposure of neutralizing epitopes [13]. Studies have shown that key neutralizing epitopes are fully exposed only when VP1 and VP2 are co-expressed, thereby inducing effective neutralizing antibodies [14]. VP3, also known as apoptin, is the main virulence factor of CIAV and specifically induces apoptosis in chicken lymphoblastic T cells and myeloid cells [15]. However, the N-terminal arginine-rich, highly positively charged region of full-length VP1 is toxic to host cells, resulting in very low or undetectable expression levels in prokaryotic systems [16]. Constructing a truncated VP1 lacking the N-terminal 129 amino acids (VP1Nd129) significantly increases its expression yield in prokaryotic systems, effectively overcoming the challenge of expressing full-length VP1 and laying the foundation for subunit antigen preparation [17, 18].

Ferritin (FN) is a hollow spherical nanocage formed by the self-assembly of 24 subunits, with a diameter of approximately 12 nm. It possesses good biocompatibility, stability, and modifiability, making it a promising antigen display platform for vaccine development [19, 20]. Displaying exogenous antigens on the ferritin surface in high-density repeating arrays mimics the native structure of pathogens, thereby effectively activating B cells and inducing potent immune responses [21]. The SpyTag/SpyCatcher system, a protein ligation technology based on covalent bond formation, enables rapid and stable coupling of antigens to carrier proteins and is widely used in bioconjugation, particularly in vaccine development [22, 23]. Currently, no ferritin nanoparticle-based vaccine against CIAV has been reported. Based on the highly efficient covalent ligation system SpyTag003/SpyCatcher003, this study constructed a novel CIAV subunit vaccine, in which ferritin fused with SpyCatcher003 (SC-FN) was expressed via the baculovirus system and the VP2-VP1Nd129 protein fused with SpyTag003 was expressed in a prokaryotic system [24]. FN-VP2-VP1Nd129 nanoparticles were assembled in vitro using this system, and their immunogenicity and protective efficacy against challenge in chicks were evaluated, aiming to provide a new vaccine candidate for CIAV control.

Materials and methods

Animals, cells, and virus

Fourteen-day-old specific pathogen-free (SPF) chickens were purchased from Guangdong Wens Dahuanong Biotechnology Co., Ltd. (Yunfu, China). Spodoptera frugiperda 9 (Sf9) cells (Pricell, Wuhan, China) were cultured in Sf-900™ II SFM medium (Gibco, Grand Island, NY, USA) supplemented with 1% penicillin-streptomycin (10,000 U/mL, Gibco, USA) in a shaking incubator at 27 °C and 120 rpm. CIAV strain Cux-1 (GenBank accession No.M55918.1) was propagated and titrated in MDCC-MSB-1 cells. The virus stock had a titer of 10^3.4 TCID₅₀/mL. For the challenge experiment, the virus stock was diluted and administered at a dose of 10⁵ TCID₅₀ per bird.

Plasmid design and construction

The SC-FN coding sequence consisted of the engineered SpyCatcher003 variant and Helicobacter pylori ferritin (GenBank ID: WP_000949190). To enhance its utility as a display platform, two modifications were introduced into the ferritin sequence: (i) deletion of the N-terminal four amino acids to improve solubility, and (ii) introduction of an N19Q point mutation to eliminate a potential N-linked glycosylation site, ensuring protein homogeneity. SpyCatcher003 was fused to the N-terminus of the modified ferritin via a short flexible linker (SGG). A 6× His tag was added to the C-terminus of ferritin for purification. An insect cell-preferred Kozak sequence (GCCACC) was introduced upstream of the start codon (ATG) to enhance translation initiation efficiency in the baculovirus expression system. The codon-optimized gene sequence was synthesized by Shanghai Sangon Biotech (Shanghai, China) and cloned downstream of the Ph promoter in the pFastBacDual vector. Subsequently, the SC-FN gene fragment was amplified by PCR using the forward primer SC-FN-F (5′-CCCTCGAGGCCACCATGGTGACTACTCTGTCTG-3′) and the reverse primer SC-FN-R (5′-GGGGTACCTTAGTGGTGGTGATGGTGGTGAGACTTTCTAGACTTAGC-3′), and cloned downstream of the P10 promoter in the same vector, yielding the dual-promoter recombinant transfer vector pFastBacDual-SC-FN.

The ST-VP2-VP1Nd129 fusion gene was constructed as follows: the C-terminus of SpyTag003 was linked to the N-terminus of CIAV VP2 via a GSGSG linker; concurrently, CIAV VP1 lacking the N-terminal 129 amino acids (VP1Nd129) was linked to the C-terminus of VP2 via another GSGSG linker. Based on the genomic sequence of CIAV strain Cux-1, specific primers were designed using SnapGene 5.2 software (San Diego, CA, USA). The fusion gene was amplified stepwise using overlap extension PCR. The first round of PCR used primers ST-L-VP2-F1 (5′-GCGTATAAACGTTACAAGGGGTCTGGAAGTGGCATGCACGGGAACGGCGGAC-3′) and VP2-L-VP1-R (5′-TCCGCAATCAACTCACCGGCGCCACTTCCAGACCCCACTATACGTACCGGGGC-3′) to generate the ST-VP2 fragment. The VP1Nd129 fragment was amplified using primers VP1Nd129-F (5′-GCCGGTGAGTTGATTGCGGA-3′) and VP1-R (5′-CCCTCGAGGGGCTGCGTCCCCCAGTACATGGTGCTGTTG-3′). The full-length ST-VP2-VP1Nd129 fusion gene was then assembled by overlap extension PCR using primers ST-L-VP2-F2 (5′-CGGAATTCATGAGGGGAGTTCCCCACATAGTAATGGTCGATGCGTATAAACGTTACAAG-3′) and VP1-R. The final full-length fragment was cloned into the pET28a vector, generating the recombinant expression plasmid pET28a-ST-VP2-VP1Nd129.

For challenge protection evaluation, a conserved 723 bp region of the CIAV genome was amplified by PCR using the forward primer CIAV-F (5′-GCTCTCCAAGAAGATACTCCA-3′) and the reverse primer CIAV-R (5′-tatgtcaggttcattgaggct-3′).

Expression and purification of SC-FN recombinant protein

The recombinant transfer plasmid pFastBacDual-SC-FN was transformed into MAX Efficiency™ DH10Bac competent cells (Thermo Fisher, Waltham, MA, USA). Recombinant bacmids were obtained through blue-white screening and purified using a bacmid isolation kit. Sf9 cells were transfected with the recombinant bacmid using LipoInsect™ transfection reagent (Beyotime, Shanghai, China). The supernatant was collected 96 h post-transfection as the P1 recombinant baculovirus stock, and transfected cell pellets were collected as P1 cell samples. P1 virus was used to infect Sf9 cells for amplification; the supernatant was harvested 96 h post-infection as the P2 virus stock, and infected cell pellets were collected as P2 cell samples. P1 and P2 cell samples were lysed and analyzed for recombinant protein expression by SDS-PAGE and Western blotting. For indirect immunofluorescence assay (IFA), Sf9 cells seeded on coverslips were infected with P2 virus. After 72 h of culture, IFA was performed to assess protein expression and subcellular localization. For large-scale expression, Sf9 cells were infected with P2 virus at a multiplicity of infection (MOI) of 0.5 and cultured with shaking at 27 °C for 96 h. Cells were harvested by centrifugation at 500 rpm for 5 min, resuspended in PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF; Beyotime, Shanghai, China), and lysed by three freeze-thaw cycles. The lysate was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm membrane. The filtrate was subjected to Ni-NTA affinity chromatography (Biodragon, Beijing, China) to purify the SC-FN protein. The eluate was dialyzed against PBS, concentrated with PEG 6000, and the protein concentration was determined using a BCA protein assay kit (Beyotime, Shanghai, China). Protein purity was analyzed by SDS-PAGE, and the purified protein was aliquoted and stored at − 80 °C until use.

Expression and purification of ST-VP2-VP1Nd129 recombinant protein

The recombinant plasmid pET28a-ST-VP2-VP1Nd129 was transformed into E. coli BL21(DE3) competent cells. A single colony was inoculated into LB medium containing 50 µg/mL kanamycin and cultured at 37 °C until the OD₆₀₀ reached 0.6–0.8. Protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM, followed by continued incubation at 37 °C for 6 h. Cells were harvested by centrifugation, sonicated on ice, and centrifuged at 12,000 rpm for 15 min. The inclusion body pellet was collected, washed once with PBS containing 2 M urea, and then dissolved in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0) supplemented with 8 M urea. The dissolved solution was purified by Ni-NTA affinity chromatography, and the target protein was eluted with elution buffer (containing 8 M urea) supplemented with 250 mM imidazole. The eluate was collected and sequentially dialyzed against refolding buffers (PBS containing 0.4 M L-arginine, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, and 5% glycerol) with decreasing urea concentrations (6 M, 4 M, and 2 M) at 4 °C, followed by a final dialysis against refolding buffer without urea overnight. The refolded protein was concentrated with PEG 6000, and the protein concentration was determined by BCA assay. The purified and refolded protein was analyzed by SDS-PAGE and Western blotting and stored at − 80 °C until use.

Assembly and purification of FN-VP2-VP1Nd129 nanoparticles

Purified SC-FN protein and ST-VP2-VP1Nd129 protein were mixed at a 1:8 molar ratio and incubated overnight at 4 °C to allow covalent coupling via the SpyTag003/SpyCatcher003 system. The coupling products were verified by SDS-PAGE analysis. Subsequently, the mixture was centrifuged at 4 °C using Amicon Ultra centrifugal filters (Merck Millipore, Burlington, MA, USA) with a 50 kDa molecular weight cutoff to remove unreacted free proteins. The retentate was collected as purified FN-VP2-VP1Nd129 nanoparticles. The purified nanoparticles were identified by Western blotting using a mouse anti-His tag monoclonal antibody (Proteintech, Rosemont, IL, USA) as the primary antibody and HRP-labeled goat anti-mouse IgG (Proteintech) as the secondary antibody. The purified nanoparticles were aliquoted and stored at 4 °C until use.

Transmission Electron Microscopy (TEM) observation

Purified SC-FN protein and FN-VP2-VP1Nd129 nanoparticle samples were separately applied to carbon-supported copper grids and allowed to stand at room temperature for 1 min. Excess liquid was removed with filter paper. The grids were negatively stained with 2% phosphotungstic acid (pH 7.0) for 1 min, excess stain was removed, and the grids were air-dried at room temperature. Particle morphology was observed under a transmission electron microscope (Hitachi HT-7800, Tokyo, Japan) at an accelerating voltage of 80 kV. Particle size distribution was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Animal immunization and challenge protection trial

Purified FN-VP2-VP1Nd129 nanoparticles (0.6 mg/mL) were emulsified with Freund’s complete adjuvant (for the primary immunization) or Freund’s incomplete adjuvant (for the booster immunization) at a 1:1 (v/v) ratio. A stable water-in-oil emulsion was formed by repeatedly passing the mixture through a double-syringe connector with a three-way valve. The final concentration of nanoparticle protein in the emulsified vaccine was 0.3 mg/mL.

Twenty-one 14-day-old specific pathogen-free (SPF) chicks were randomly divided into three groups (n = 7 per group). The nanoparticle vaccine immunization group received a subcutaneous injection of 200 µL of the vaccine (containing 60 µg of protein) in the neck at 14 and 28 days of age. The challenge control group and blank control group received 200 µL of sterile PBS via the same route. At 42 days of age, the immunization group and challenge control group were challenged with CIAV strain Cux-1 (10⁵ TCID₅₀/bird) by intramuscular injection in the thigh muscle. The blank control group was left unchallenged.

Blood samples were collected from the wing vein before immunization and at 14, 21, 28, and 42 days post-primary immunization (i.e., at 28, 35, 42, and 56 days of age). Sera were separated, and CIAV-specific antibody levels were measured using a chicken infectious anemia virus antibody ELISA kit (Shanghai Yuanju, Shanghai, China), with OD₄₅₀ values recorded. Anticoagulated blood was collected from the wing vein at 7 and 14 days post-challenge (i.e., at 49 and 56 days of age). Hematocrit (HCT) values were determined using a Mindray BC-30 Vet automated hematology analyzer (Mindray, Shenzhen, China). At 14 days post-challenge (56 days of age), each chicken was weighed and then euthanized by cervical dislocation. Thymus tissues were aseptically removed and weighed to calculate the thymus index (thymus weight in mg/body weight in g).

Approximately 0.1 g of thymus tissue collected at 14 days post-challenge was used for total RNA extraction with TRIzol reagent (Takara, Shiga, Japan). After reverse transcription to synthesize cDNA, PCR amplification of a conserved region (expected size: 723 bp) of the CIAV genome was performed using specific primers (CIAV-F and CIAV-R). PCR products were analyzed by 1.5% agarose gel electrophoresis.

Statistical analysis

All data were analyzed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to assess differences between groups. Differences were considered statistically significant at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Nanoparticle sizes were analyzed using ImageJ software, and size distribution histograms were plotted using Origin software (OriginLab, Northampton, MA, USA).

Results

Expression and purification of SC-FN recombinant protein

To evaluate SC-FN expression in the baculovirus system, protein samples from uninfected Sf9 cells, P1 virus-infected cells, and P2 virus-infected cells were analyzed by SDS-PAGE. A distinct protein band at approximately 32.1 kDa was observed in P2-infected cell samples, which was absent in the uninfected control. Notably, P1-infected cell samples did not exhibit a clear band at this position (Fig. 1A), suggesting that SC-FN expression levels were low during the initial infection stage and fell below the detection limit of SDS-PAGE.

Fig. 1.

Expression analysis of SC-FN recombinant protein. A SDS-PAGE analysis of SC-FN expression. M: protein molecular weight marker; Lane 1: uninfected Sf9 cells (negative control); Lane 2: P1 virus-infected cells; Lanes 3–4: P2 virus-infected cells. The arrow indicates the specific protein band at approximately 32.1 kDa. B Western blot analysis using an anti-His tag monoclonal antibody. The specific immunoreactive band at 32.1 kDa was detected in both P1- and P2-infected cell samples, with a stronger signal in P2-infected cells, while no band was observed in the uninfected control

To further confirm expression, Western blotting was performed using an anti-His tag antibody. Specific immunoreactive bands at 32.1 kDa were detected in both P1- and P2-infected cell samples, with a stronger signal in P2-infected cells, whereas no band was observed in the uninfected control (Fig. 1B). These results confirmed specific SC-FN expression and its good immunoreactivity.

Indirect immunofluorescence assay (IFA) further revealed specific green fluorescence in the cytoplasm of P2 virus-infected Sf9 cells, while no fluorescence was detected in the control group (Fig. 2), indicating successful SC-FN expression in the cytoplasm of Sf9 cells.

Fig. 2.

Subcellular localization of SC-FN protein in Sf9 cells detected by indirect immunofluorescence assay (IFA). Sf9 cells were infected with P2 recombinant baculovirus. Green fluorescence represents SC-FN protein labeled with anti-His tag antibody followed by FITC-conjugated secondary antibody. Blue fluorescence (DAPI) indicates the nucleus. No fluorescence was observed in the uninfected control group. Scale bar = 50 μm

Following successful expression verification, large-scale culture and purification were performed. The purified SC-FN protein appeared as a single clear band on SDS-PAGE (Fig. 3), and the protein concentration was determined to be 1.18 mg/mL by BCA assay.

Fig. 3.

SDS-PAGE analysis of purified SC-FN protein. M: protein molecular weight marker; Lane 1: purified SC-FN protein. The protein appeared as a single clear band at approximately 32.1 kDa. The protein concentration was determined to be 1.18 mg/mL by BCA assay

Expression and purification of ST-VP2-VP1Nd129 recombinant protein

Following transformation of E. coli BL21(DE3) with pET28a-ST-VP2-VP1Nd129 and induction with IPTG, SDS-PAGE analysis revealed a specific protein band at approximately 68 kDa, which was predominantly expressed in inclusion bodies. After denaturing purification and gradient dialysis refolding, highly pure soluble protein was obtained (Fig. 4A). Western blotting using an anti-His tag antibody confirmed the specific reactivity of the protein (Fig. 4B). The concentration of the refolded protein was 0.6 mg/mL.

Fig. 4.

Purification and identification of ST-VP2-VP1Nd129 recombinant protein. A SDS-PAGE analysis of purified and refolded ST-VP2-VP1Nd129 protein expressed in E. coli BL21(DE3). M: protein molecular weight marker (kDa); Lane 1: purified and refolded ST-VP2-VP1Nd129 protein, showing a specific band at approximately 68 kDa. B Western blot analysis using an anti-His tag monoclonal antibody (1:5,000) followed by HRP-conjugated goat anti-mouse IgG (1:10,000). The specific immunoreactive band at 68 kDa confirms the identity of the recombinant protein. The protein concentration was 0.6 mg/mL

Assembly and identification of FN-VP2-VP1Nd129 nanoparticles

After mixing purified SC-FN and ST-VP2-VP1Nd129 proteins at a 1:8 molar ratio and incubating overnight, SDS-PAGE analysis showed a new protein band at approximately 100 kDa (Fig. 5A), corresponding to the covalently linked FN-VP2-VP1Nd129 complex. Following ultrafiltration purification, Western blotting using an anti-His tag antibody detected a specific hybridization signal at 100 kDa (Fig. 5B), confirming successful coupling.

Fig. 5.

Assembly and characterization of FN-VP2-VP1Nd129 nanoparticles via the SpyTag003/SpyCatcher003 system. A SDS-PAGE analysis of coupling reaction. M: marker; Lane 1: SC-FN (32.1 kDa); Lane 2: ST-VP2-VP1Nd129 (68 kDa); Lane 3: coupling product (100 kDa, arrow). B Western blot of purified nanoparticles using anti-His tag antibody, showing a specific band at 100 kDa. C TEM images of SC-FN monomers (left) and FN-VP2-VP1Nd129 nanoparticles (right). Scale bar = 100 nm. D Particle size distribution. SC-FN monomers: peak ~ 15 nm (12–18 nm); FN-VP2-VP1Nd129: peak ~ 23 nm (18–28 nm)

TEM was used to characterize nanoparticle morphology before and after assembly. SC-FN monomers presented as typical spherical particles with good dispersion, smooth surfaces, and clear boundaries. In contrast, assembled FN-VP2-VP1Nd129 nanoparticles were also spherical but exhibited significantly increased particle sizes with rough, irregular protrusions on the surface (Fig. 5C). Statistical analysis of particle size distribution indicated that SC-FN monomers were mainly distributed in the 12–18 nm range, with a peak around 15 nm, whereas FN-VP2-VP1Nd129 nanoparticles were mainly distributed in the 18–28 nm range, with the peak shifting to approximately 23 nm (Fig. 5D). Compared with SC-FN monomers, the peak diameter of the assembled nanoparticles increased by approximately 8 nm, indicating successful display of the VP2-VP1Nd129 antigen on the ferritin surface.

Immune response induced by the nanoparticle vaccine

To evaluate the ability of the FN-VP2-VP1Nd129 nanoparticle vaccine to induce humoral immune responses, sera were collected at different time points post-immunization (14, 21, 28, and 42 days) and CIAV-specific antibody levels were measured by ELISA. Antibody levels in the nanoparticle vaccine immunization group began to rise 14 days post-prime, with an average OD₄₅₀ value reaching 0.45. Antibody levels increased significantly 7 days post-boost, peaked at 14 days post-boost, and remained at a relatively high level until 56 days of age (Fig. 6A). In contrast, OD₄₅₀ values in the PBS blank control group remained below the cut-off value (OD₄₅₀ = 0.23) throughout the detection period, all testing negative. These results indicate that the FN-VP2-VP1Nd129 nanoparticle vaccine effectively induces high levels of CIAV-specific humoral immune responses in chicks, with antibodies persisting for a considerable duration.

Fig. 6.

Immunoprotective efficacy of the FN-VP2-VP1Nd129 nanoparticle vaccine against CIAV challenge in chicks. A Serum CIAV-specific antibody levels (ELISA). Dashed line: cut-off (OD₄₅₀ = 0.23). Data are mean ± SD (n = 7). B Hematocrit (HCT) values on days 7 and 14 post-challenge. C Thymus indices on day 14 post-challenge. D PCR detection of CIAV nucleic acid in thymus tissues of challenge control group (100% positive, 7/7). E PCR detection of CIAV nucleic acid in vaccine immunization group (0% positive, 0/7). For (D) and (E): M: DL2000 marker; Lane 1: positive control; Lane 2: negative control; Lanes 3–9: individual samples (n = 7)

Changes in hematocrit post-challenge

Hematocrit (HCT) is an important clinical indicator for evaluating hematopoietic suppression caused by CIAV infection. HCT values were measured on days 7 and 14 post-challenge (Fig. 6B). On day 7 post-challenge, the HCT value of the nanoparticle vaccine immunization group was significantly higher than that of the challenge control group (p < 0.01) and showed no significant difference from the blank control group (p > 0.05). On day 14 post-challenge, the HCT value of the nanoparticle vaccine immunization group was highly significantly higher than that of the challenge control group (p < 0.001) and remained at the same level as the blank control group. These results demonstrate that the FN-VP2-VP1Nd129 nanoparticle vaccine effectively protects chicks against hematopoietic suppression caused by CIAV challenge.

Comparison of thymus index post-challenge

The thymus is a primary target organ for CIAV, and infection leads to severe thymic atrophy. The thymus index was measured on day 14 post-challenge (Fig. 6C). The PBS blank control group had the highest thymus index, while the CIAV challenge group had the lowest, with highly significant differences observed between all pairs of groups (p < 0.001). Specifically, the thymus index of the nanoparticle vaccine immunization group was highly significantly higher than that of the CIAV challenge control group (p < 0.001), indicating that immunization effectively protects against thymic atrophy. However, the thymus index of the immunized group remained significantly lower than that of the blank control group (p < 0.001), suggesting that the vaccine did not fully restore the thymus to normal physiological levels.

Detection of CIAV nucleic acid in thymus tissues

At 14 days post-challenge, CIAV nucleic acid in thymus tissues was detected by PCR. All seven thymus tissue samples from the challenge control group displayed specific amplification bands at 723 bp, with a positive rate of 100% (7/7) (Fig. 6D). In contrast, none of the seven samples from the nanoparticle vaccine immunization group showed specific bands, with a positive rate of 0% (0/7) (Fig. 6E). These results indicate that the FN-VP2-VP1Nd129 nanoparticle vaccine effectively inhibits CIAV replication in the target organ.

Discussion

CIAV poses a serious threat to the global poultry industry, significantly impacting chicken health and productivity. Due to the generally low proliferation titer of CIAV in chicken embryos or MDCC-MSB-1 cells, the production cost of inactivated vaccines is high, representing a major bottleneck for industrial production [25]. Although live attenuated vaccines are commercially available, they carry risks of reversion to virulence and potential interference with the chick’s immune system, limiting their use to chickens older than 8 weeks. Therefore, developing a safe, efficient, and early-immunization suitable novel CIAV vaccine is of significant practical importance. In this study, we successfully constructed a ferritin nanoparticle vaccine displaying the CIAV VP2-VP1Nd129 fusion protein using the SpyTag003/SpyCatcher003 covalent ligation system and confirmed its good immunogenicity and protective efficacy in a chick model.

Antigen design is a critical factor determining the immune efficacy of CIAV subunit vaccines. Studies have shown that the neutralizing epitopes of CIAV depend on the co-expression of VP1 and VP2 proteins; neither protein alone can induce effective protective antibodies. This is because VP2 acts as a scaffold protein, assisting VP1 in forming the correct conformation to expose its neutralizing epitopes [13, 14]. In this study, we fused VP2 with truncated VP1 (VP1Nd129) via a flexible GSGSG linker, mimicking the natural co-expression state of the two proteins and laying a structural foundation for inducing effective immune responses. The use of VP1Nd129 instead of full-length VP1 was based on previous findings that the arginine-rich N-terminus of full-length VP1 is toxic to prokaryotic host cells, severely limiting its expression efficiency [16, 17]. Lee et al. demonstrated that truncating the N-terminal 129 amino acids significantly increases VP1 expression levels in E. coli [17], and the successful high-level expression of the ST-VP2-VP1Nd129 fusion protein in this study further confirms this point.

After optimizing the antigen design, an efficient platform for antigen presentation was required to maximize its immunogenicity. Ferritin self-assembles into 24-subunit nanocages, displaying antigens in high-density repeating arrays that mimic the native structure of pathogens, thereby effectively activating B cells and inducing potent immune responses [19–21]. The SpyTag003/SpyCatcher003 system provided an efficient and stable protein coupling strategy for this study. Currently, two main strategies exist for linking antigens to nanoparticle carriers: direct genetic fusion expression to produce recombinant nanoparticle vaccines [26] or separate production of the antigen and nanocarrier followed by covalent or non-covalent linkage [27]. Owing to its high efficiency, the SpyTag/SpyCatcher system has emerged as a particularly popular covalent coupling method and has been successfully applied in constructing various nanoparticle vaccines [23, 28]. This study adopted the separate expression strategy, producing SC-FN nanocarriers via the baculovirus system and ST-VP2-VP1Nd129 antigen protein via the prokaryotic system, followed by in vitro covalent coupling using the SpyTag003/SpyCatcher003 system. SDS-PAGE and Western blot results showed a new band at approximately 100 kDa, corresponding to the assembled FN-VP2-VP1Nd129 complex, and high-purity nanoparticles were obtained after ultrafiltration purification. These results demonstrate that the SpyTag003-labeled ST-VP2-VP1Nd129 protein efficiently binds to SC-FN nanoparticles, further confirming that the SpyTag/SpyCatcher system is a reliable platform for efficient antigen coupling in nanovaccine construction.

Immune protection efficacy is the core indicator for evaluating vaccine potency. Characteristic lesions of CIAV infection include aplastic anemia and thymic atrophy, manifested by decreased hematocrit (HCT) and thymus index, respectively [29, 30]. In this study, the HCT values of chicks in the nanoparticle vaccine immunization group at 7 and 14 days post-challenge were significantly higher than those in the challenge control group and showed no significant difference from the blank control group, indicating that the vaccine effectively protected the hematopoietic system. The thymus index of the immunized group was highly significantly higher than that of the challenge control group, confirming significant protection against CIAV-induced thymic atrophy. Although the thymus index of the immunized group remained lower than that of the blank control group, suggesting that the vaccine did not fully restore the thymus to normal physiological levels, the viral nucleic acid detection results (no CIAV nucleic acid detected in the thymus of the immunized group) collectively indicate that the vaccine effectively inhibited viral replication in the target organ.

Compared with previous studies, Liu et al. reported that a DNA prime-protein boost strategy induced peak ELISA antibody titers at 4 weeks post-boost [18]; Chellappa et al. found that an NDV-vectored CIAV vaccine required booster immunization to induce specific antibodies [31]. In this study, specific antibodies were detectable 14 days after a single immunization, peaked 14 days post-boost, and remained at relatively high levels until 56 days of age, indicating that the ferritin nanoparticle display technology significantly enhanced the immunogenicity of the VP2-VP1Nd129 fusion protein.

Maternal antibodies have been shown to protect chicks from CIAV infection, maintaining normal thymic structure and HCT values [32]. Ge et al. demonstrated that vaccination with a recombinant MDV vaccine expressing VP1 and VP2 induced CIAV-specific ELISA antibodies up to 1 × 10⁵ and neutralizing antibody titers up to 1:2⁵, significantly inhibiting viral replication in the thymus [33]. In this study, the nanoparticle vaccine immunization group induced high levels of CIAV-specific antibodies, consistent with the negative viral nucleic acid results post-challenge, suggesting that antibody-mediated immune responses played a key role in viral clearance. Cellular immune responses are also important and cannot be ignored. Liu et al. further discovered that a DNA prime-protein boost strategy significantly promoted splenocyte proliferation and upregulated the expression of cytokines such as IL-2, IL-6, and IFN-γ, indicating effective stimulation of cellular immune responses [18]. Shen et al. found that an rVP1 subunit vaccine combined with IFN-γ significantly induced the expression of the Th1-type cytokine IFN-γ [34]. Although cytokine levels and lymphocyte proliferation were not directly measured in this study, the good protective effect observed in the vaccine group post-challenge indirectly suggests the involvement of cellular immunity, which warrants further investigation in future studies.

Compared with existing CIAV vaccine development strategies, the nanoparticle vaccine constructed in this study offers unique advantages. Moeini et al. used Lactobacillus acidophilus as a live vector to display VP1 protein on the bacterial surface, providing a new approach for oral CIAV vaccine development [35]; Fang et al. compared the effects of CpG-ODN and Freund’s adjuvant on the immune response to CIAV VP1, VP2, and VP3 subunit proteins, finding that CpG-ODN adjuvant induced higher antibody titers with less individual variation [36]. While these studies explored strategies to improve CIAV vaccines from different perspectives, they did not address the core issue of antigen presentation efficiency. In contrast, our study, through high-density multivalent display on ferritin nanoparticles, mimics the natural presentation mode of viral particles, significantly enhancing antigen immunogenicity and providing a novel strategy for the development of CIAV subunit vaccines.

Limitations and future directions

We acknowledge that the present study has certain limitations. First, the humoral immune response was followed only until 56 days of age (14 days post-challenge). Since the vaccine is intended for use in breeding chickens, long-term antibody persistence should be monitored until the onset of egg production (approximately 18–20 weeks of age). Such studies will help determine whether a second or third booster dose is required to maintain protective antibody levels throughout the laying period. Second, although the challenge was performed at 42 days of age rather than in younger chicks, successful infection was confirmed by the detection of CIAV nucleic acid in the thymus tissues of all challenged control birds. While this provided a valid basis for evaluating vaccine protection, the hematocrit reduction was less severe than the 6–20% drop typically reported in 1- to 2-week-old chicks, which may be attributed to the reduced susceptibility of older chickens to the clinical manifestations of CIAV. Third, while we demonstrated that the vaccine effectively protected chicks against clinical disease and inhibited CIAV replication in the thymus, we did not assess its ability to block vertical transmission. For a vaccine intended for breeders, it is crucial to evaluate whether vaccination can prevent CIAV from reaching the ovary, egg yolk, or progeny chicks. Future studies should therefore include longer-term monitoring of antibody persistence in breeding chickens, challenge of vaccinated breeders during the laying period with detection of viral nucleic acid in ovarian tissues, egg yolks, and hatched chicks, as well as challenge experiments in younger chicks (e.g., 1–7 days old) to fully evaluate the vaccine’s efficacy against the classical severe anemia induced by velogenic CIAV strains. These investigations, although requiring extended experimental timelines, are essential for a comprehensive evaluation of the vaccine’s full protective potential under field conditions.

In summary, this study successfully constructed a ferritin nanoparticle vaccine displaying the CIAV VP2-VP1Nd129 fusion protein using the SpyTag003/SpyCatcher003 system. This vaccine induced high levels of specific antibodies in chicks and effectively protected against hematopoietic suppression, thymic atrophy, and viral replication caused by CIAV challenge. It provides a new strategy for the development of safe and efficient CIAV subunit vaccines and offers a valuable technological platform for vaccine development against other avian viral diseases.

Abbreviations

CIAV

Chicken infectious anemia virus

FN

Ferritin

SC-FN

SpyCatcher003-ferritin

IFA

Indirect immunofluorescence assay

HCT

Hematocrit

TCID₅₀

50% tissue culture infectious dose

SPF

Specific pathogen-free

ELISA

Enzyme-linked immunosorbent assay

Authors’ contributions

L.Z. and Z.L. contributed equally to this work. L.Z. performed the experiments, analyzed the data, and drafted the manuscript. Z.L. assisted with the experiments and data analysis. Y.-M.C.Y. and H.S. contributed to the animal experiments. F.W. conceived and designed the study, supervised the research, and revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (NSFC) (32473004).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. The raw Western blot images are available as supplementary files.

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Experimental Animal Administration and Ethics Committee of the South China Agricultural University (protocol No. SCAUADL2020-009). All experiments were performed in accordance with the ARRIVE guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.