Clostridium perfringens epsilon-ferritin nanoparticles induced robust immune responses in mice and sheep

Clostridium perfringens epsilon-ferritin nanoparticles induced robust immune responses in mice and sheep | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clostridium perfringens epsilon-ferritin nanoparticles induced robust immune responses in mice and sheep Huiyu Zhao, Qianru Xing, Guangzhi Zhang, Yinuo Lei, Peng Li, Heleer Cha, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9380767/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Clostridium perfringens ( C. perfringens ) is an opportunistic pathogen, widely distributed in the environment. Epsilon toxin secreted by B and D type C. perfringens can cause enterotoxemia or necrotic enteritis in goats, sheep and cattle. Vaccination is one of the most effective measures to prevent such diseases. However, current toxoid vaccines still exhibit some problems, thus this study aims to engineer C. perfringens epsilon-ferritin nanoparticles antigen and evaluate its immunogenicity in mice and sheep. The r-epsilon successfully assembled with r-ferritin to form epsilon-ferritin nanoparticles via SDS-PAGE, transmission election microscopy and dynamic light scattering test. In the mouse experiment, epsilon-ferritin nanoparticles could significantly improve specific antibody levels including serum IgG, IgG subtypes antibodies, the ratio of IgG1/IgG2a and neutralizing antibodies. Moreover, epsilon-ferritin nanoparticles elicited more production of IFN-γ cytokines and more activation of memory CD8 + T cells in mice. In the sheep experiment, epsilon-ferritin nanoparticles could enhance the level of neutralizing antibodies and more production of IFN-γ cytokines. The present study revealed that epsilon-ferritin nanoparticles remarkably enhanced the immunogenicity of r-epsilon in mice and sheep, providing a new strategy for the development of C. perfringens vaccines. C. perfringens epsilon toxin ferritin nanoparticles immunogenicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction C. perfringens , also known as Clostridium welchii , is a Gram-positive, anaerobic, spore-forming bacterium with a large, non-flagellated body [ 1 ] and capsules. The pathogen has been identified as a significant etiological agent of various enteric disorders in livestock species [ 2 , 3 ] , frequently causing diseases such as gas gangrene, necrotizing enterocolitis [ 4 ] and enterotoxicosis in livestock, including cattle, sheep, and chickens [ 5 , 6 ] . These diseases typically manifest acutely [ 7 ] , progress rapidly with high mortality rates [ 8 ] , sometimes leading to sudden death within 1–2 days if treatment is delayed [ 9 , 10 ] . The bacterium has negatively impacted the farming industry in China [ 11 ] . The virulence of C. perfringens largely depends on the release of six main exotoxins: α (alpha), β (beta), ε (epsilon), ι (iota), enterotoxin (cpe), and NetB [ 12 , 13 ] . Based on its toxin production pattern, C. perfringens is classified into seven types labelled A through G [ 14 ] . Type C and D are particularly significant in veterinary clinical practices; type C produces alpha and beta toxins and type D produces alpha and epsilon toxins. When epsilon toxin is activated by proteolysis, with the consequent release of carboxy-terminal and amino-terminal peptides from the protein, the bacterial virulence will substantially increase [ 15 ] . It is the third most potent toxin after the botulinum and tetanus toxins, making it a significant potential bioterrorist agent recognized by the international community [ 16 , 17 ] . By an unidentified mechanism, this toxin passes through gut wall of affected animals and enters the blood stream in the intestine. Epsilon toxins can further disseminate to other organs via blood circulation, and brain and kidney are preferentially targeted by these toxins [ 18 ] . For cattle and sheep, vaccines against C. perfringens are primarily toxoid-based. Some toxoid components can also cause local inflammatory or allergic reactions [ 19 ] . Thus, these limitations have prompted endeavour to design novel vaccines [ 20 ] . The use of biomolecular methods to express recombinant toxins for novel vaccine technology offers an effective way, which can bypass the complexities and biosafety risks, preserving immunogenicity and enhancing potency [ 21 ] . However, most recombinantly expressed proteins pose challenges for vaccine development due to their low immunogenicity and rapid release rates [ 22 ] . Thus, it is necessary to take advantage of some adjuvants or delivery systems to stimulate the immune response. Nanoparticle vaccine is a new type of vaccine emerging in recent years, and has been widely used in the prevention of many diseases [ 23 , 24 ] . Various distinctive features of nanotechnology have been demonstrated to improve drug delivery, such as increasing drug solubility/stability, controlling drug release, prolonging systemic circulation, crossing tissue barriers, enhancing accumulation in target tissues and facilitating cellular uptake. Ferritin is one of nanoparticles with a hollow cage-like protein structure that occurs naturally in the host body [ 25 , 26 ] . The outer surface of ferritin can be chemically or genetically modified with targeted sequences to display multiple ligands, thereby improving binding affinity [ 27 , 28 ] , and it can prepare the multivalent nanovaccines. Additionally, ferritin nanoparticles rapidly circulate into lymph nodes and target dendritic cells [ 29 ] , particularly CD8α + cell populations, following subcutaneous immunization [ 30 ] . Ferritin has been examined in preclinical research as a vaccine carrier candidate for various diseases, including HIV-1 [ 31 ] , influenza (H1N1 strain) [ 32 ] , demonstrating its satisfactory efficacy and promising potentials in combating pathogenic infections. In this study, a non-toxic r-epsilon with mutation H106P and SpyTag, and soluble r-ferritin with SpyCatcher were obtained respectively. Subsequently, self-assembling ferritin nanoparticles were bound to exogenous r-epsilon onto their surface through SpyTag-SpyCatcher docking peptides. Then, using this technology, the immunological effect and immune response type of epsilon-ferritin nanoparticles were evaluated and analyzed in mice and sheep. Our findings demonstrated that epsilon-ferritin nanoparticles efficiently stimulated humoral and cellular immunity in mice and sheep and induced robust cellular immune responses. Results The r-epsilon can be successfully displayed on r-ferritin through SpyTag/SpyCatcher To conveniently and stably load specific antigens of C. perfringens on ferritin nanoparticles, we employed SpyTag/SpyCatcher docking technique [ 33 ] (Fig. 1A,B,C). Western blotting assay confirmed the successful expression of the r-epsilon and r-ferritin and the predominance of expression in the supernatant (Fig. 1D,E). The r-epsilon and r-ferritin were purified by Ni-NTA affinity chromatography. Imidazole was subsequently removed by dialysis against PBS (pH 7.4). The r-epsilon was demonstrated high quality, as evident from SDS-PAGE gels (Fig. 1F). Similarly, SDS-PAGE showed a clear single band, indicating that the purified r-ferritin was obtained (Fig. 1G). Ferritin and epsilon-ferritin nanoparticles were examined by TEM (Fig. 1H,I). The TEM image confirmed that ferritin and epsilon-ferritin nanoparticles were successfully self-assembled and had a uniform size distribution. The result of dynamic light scattering test showed that the particle size of ferritin protein ranged from 11.7 ~ 15.7 nm. The particle size of epsilon-ferritin nanoparticles ranged from 32.7 ~ 50.7 nm, and the main peak was 37.8 nm, which indicated that r-epsilon successfully docked with ferritin protein to form epsilon-ferritin nanoparticles(Fig. 1J). The SDS-PAGE figure (Fig. 1K) showed a uniform increase in molecular weight from 35 kDa to 110 kDa. It indicated that r-epsilon bound to r-ferritin. These results showed that r-epsilon successfully assembled with r-ferritin to form epsilon-ferritin nanoparticles. Muscular inoculation with epsilon-ferritin nanoparticles induced strong specific humoral immune response in mice In the standard prime-boost regimen, animals are administered different antigens via intramuscular immunization (Fig. 2 A). To evaluate the humoral antibody response, the antibody level of the mice from epsilon-ferritin nanoparticles groups and r-epsilon groups were measured using ELISA. As expected, there was no difference in the specific antibody levels between the PBS group and the r-ferritin group, indicating that r-ferritin does not stimulate a specific B-cell immune response. After vaccination, mice immunized with epsilon-ferritin nanoparticles produced significantly higher IgG antibody levels than those immunized with r-epsilon (Fig. 2 B). In addition, the epsilon-ferritin nanoparticles groups showed a plateau at 35 dpi, whereas the r-epsilon groups showed a plateau after 42 dpi. On the 7 dpi and 28 dpi, the IgM level was also significantly higher than that in the r-epsilon groups (Fig. 2 C, P < 0.0001). The results of the IgG subtypes showed that the epsilon-ferritin nanoparticles group produced notably higher levels of both IgG1 and IgG2a antibodies compared to the r-epsilon group (Fig. 2 D,E, P < 0.0001). Besides, the IgG1/IgG2a ratio was lower than that of the r-epsilon group at day 42 (Fig. 2 F, P < 0.01). In addition to antibody levels, neutralizing antibody titers were also one of an essential feature of vaccine with high efficacy. Firstly, minimum lethal dose (MLD) of the CV-81 natural toxin caused cytopathic effect in MDCK cells was dilution 2 13 . Then, serum collected at 21 dpi and 42 dpi were were used to determine neutralizing antibody titer in serum using the fixed virus-diluted serum method (Fig. 2 G). The results demonstrated that at 42 dpi, serum from the epsilon-ferritin nanoparticles group exhibited higher neutralizing antibody titer than r-epsilon group ( P < 0.05). Epsilon-ferritin nanoparticles induced an effective cellular immune response through muscular immunity T cells are the main components of lymphocytes, and their immune response from T cells is known as cellular immunity [ 34 ] . Splenocyte cells from experimental mice were isolated, then T-lymphocyte subpopulation and IFN-γ cytokine levels were assessed to determine whether the nanoparticles vaccine induced Th1-type cellular immunity. There were no statistically significant differences in T-lymphocyte subpopulation among the groups at weeks 7 (Fig. 3 A, P > 0.05). However, the epsilon-ferritin nanoparticles group elicited the highest IFN-γ levels, which were significantly higher than the PBS groups and the r-epsilon group (Fig. 3 B, P < 0.0001). Moreover, staining of splenocytes taken 15 weeks after immunization revealed that memory CD8 + T cells were significantly higher in the epsilon-ferritin nanoparticles group than that in the r-epsilon group (Fig. 3 C, P < 0.001). Detection of Antibody Level in the Immunized Sheep The immunogenicity of epsilon-ferritin nanoparticles in sheep was also assessed. Blood samples were collected at 21 dpi and 42 dpi. IgG levels in the serum of both groups were measured by ELISA. Unlike the results observed in mice, the epsilon-ferritin nanoparticles group did not induce significantly higher IgG antibody levels than the r-epsilon group in sheep (Fig. 4 A, P > 0.05). Next, the neutralizing capacity of the serum from both groups were evaluated using the same method as described previously. The results showed that there were no statistically significant differences in neutralizing antibody level among the vaccine group at 21 dpi (Fig. 4 B, P > 0.05). However, the epsilon-ferritin nanoparticles group elicited higher neutralizing antibody level at 42 dpi ( P < 0.05). Anticoagulated blood from both groups were stimulated by r-epsilon and the cytokine IFN-γ in supernatant was assayed (Fig. 4 C). The results showed that stimulation of the epsilon-ferritin nanoparticles group produced higher levels of IFN-γ than the r-epsilon group ( P < 0.05). Discussion C. perfringens often settles in the intestines of healthy animals and humans in an asymptomatic manner [ 35 , 36 ] , and can cause diseases such as gas gangrene, necrotizing enterocolitis and enterotoxaemia when the host's immune function is disrupted [ 37 ] . Epsilon toxin is a virulence factor produced by C. perfringens types B and D [ 38 ] . And it is characterized by a rapid onset, short disease course, and high mortality rate [ 39 , 40 ] . Treatment with medication alone is often ineffective due to the acute feature of the disease, resulting in significant losses to cattle and sheep. Currently, the vaccines on the market are mainly toxoid vaccines, which can also cause local inflammatory or allergic reactions [ 19 , 40 ] . Therefore, it is particularly important to develop a new vaccine to prevent this disease [ 41 ] . Epsilon toxin is a relatively inactive prototoxin consisting of 311 amino acids. Besides, the histidine residue (domain I) appeared to be integral to the active site of epsilon toxin. Of the two histidine residues in the mature epsilon toxin, the histidine at 106 was predicted to be located in the middle of the β-folding region in a weakly hydrophobic environment, whereas the one at 149 is predicted to be located close to a turn region in the hydrophilic environment [ 42 ] . H106P has been shown to be completely non-toxic to cultured MDCK cells, and is a safe, non-toxic, and immunoprotective candidate vaccine antigen for enterotoxaemia [ 38 , 43 ] . Therefore, this site was chosen for mutation and then r-epsilon was expressed in this study. The r-epsilon was confirmed to be non-lethal to MDCK cells by cellular assays. Recently, advances in nanotechnology have been widely used to enhance the immunogenicity of antigens, which has led to significant breakthroughs in the field of medicine because of its high safety, delivery capacity and modifiability [ 44 – 46 ] . Nanoparticle materials mainly include protein/peptides, lipid nanoparticles, polymer nanoparticles and inorganic nanoparticles [ 47 ] , among which ferritin nanoparticles are an attractive protein nanoplatform [ 48 , 49 ] . In the present study, prokaryotic expression system was utilized to express ferritin nanoparticles, which not only consumes low cost, but also has high soluble expression, purity and stability. SpyTag/SpyCatcher coupling technology is a versatile and efficient protein-linking method that enables covalent attachment of proteins by simply mixing components under various pH, temperature, and buffer conditions. This reaction yields high specificity and stability, unaffected by competing peptides [ 33 , 50 ] . In this study, the SpyTag/SpyCatcher coupling technique was introduced into ferritin nanoparticle vaccines construction. And r-epsilon with SpyTag tag and r-ferritin with SpyCatcher tag were prepared, respectively. The fusion of r-ferritin and r-epsilon occurred after mixing them in vitro. The fusion products could be observed as nanoparticles by TEM and a range of diameters for docking products were greater than the average FeSC diameter by DLS, indicating the successful docking of r-ferritin and r-epsilon. It is generally believed that inactivated vaccines and subunit vaccine antigens are processed by antigen-presenting cells and are primarily presented through the MHC-II pathway, mediating the recognition by CD4 + T lymphocytes. The main function of these CD4 + effector T cells is to secrete cytokines, which can activate phagocytes, neutrophils, and eosinophils, and stimulate B cells to produce antibodies, thereby triggering a specific humoral immune response that can effectively neutralize and eliminate extracellular pathogens. To evaluate the immunological effect of nanoparticles antigen, mice were subcutaneously immunized with epsilon-ferritin nanoparticles or r-epsilon. It was found that IgG antibody in the epsilon-ferritin nanoparticles group was significantly higher than that in the r-epsilon group, and its plateau was earlier than that in the r-epsilon group; The in vitro neutralization assay at 42 dpi showed that the epsilon-ferritin nanoparticles group also were significantly higher than in the r-epsilon group. These results suggested that nanoparticles can stimulate a stronger humoral immune response in mice. Second, in mice, it is widely accepted that the IgG1 response reflects the paracrine activity of Th2 CD4 + T cells and that IgG2a is generated by Th1 CD4 + T cell activity [ 51 ] . Therefore, the lower the IgG1/IgG2a ratio indicates a strong trend toward a Th1 immune response. Both IgG1 and IgG2a antibodies were significantly higher in the epsilon-ferritin nanoparticles group than in the r-epsilon group and the IgG1/IgG2a ratio was lower than that of the r-epsilon group at 42 dpi, which indirectly proved that the epsilon-ferritin nanoparticles group induced stronger Th1 and Th2 immune response in mice. Tang et al. [ 22 ] immunized mice with SARS- CoV-2 ORF8 protein after coupling it with 8-arm PEG and the results showed that the coupling group with 8-arm PEG stimulated the organism to produce higher IgG1 and IgG2a, inducing both humoral immune response and cellular immunity. This indicated that nanoparticles antigen has better immune activation capacity, and aligned with the conclusions that we obtained from our experimental results. Th1 cells enhance CTL functions through the secretion of cytokines, such as IL-2, and IFN-γ, which activate other players of the innate system, as NK cells and macrophages, thereby resulting in higher pathogen or tumor cells/apoptotic tumor bodies elimination [ 52 , 53 ] . Th2 cells stimulate the secretion of antibodies by B cells, which additionally activate other phagocytic cells [ 54 ] . In our experiments, the epsilon-ferritin nanoparticles group showed a preference for a stronger Th1 type response (Fig. 3 B), inducing a cellular immune response in mice. In addition, the nanoparticles group were found to have significantly more memory CD8 + cells than the r-epsilon group. It demonstrated that nanoparticles induced a durable level of cellular immunity in mice. Thus, these data suggested that nanoparticles can stimulate stronger and longer-lasting cellular immunity in mice. Sheep were booster immunized with epsilon-ferritin nanoparticles or r-epsilon. Neutralizing antibody titer and IFN-γ cytokine level were found to be significantly higher in the epsilon-ferritin nanoparticles group than in the r-epsilon group. Ma et al. [ 55 ] designed a nanoparticles vaccine using ferritin against the RBD and Heptad Repeat (HR) antigens of SARS-CoV-2 to immune in rhesus monkeys. The results showed the nanoparticles antigen induced the production of neutralizing antibodies as well as T and B cell responses in rhesus monkeys prior to booster immunization. Thus, these suggest that nanoparticles not only induce stronger humoral and cellular immunity in animal model, but also stimulate stronger immune response in another animal. Although this study achieved promising results, additional refinements are required. For instance, improving the docking efficiency between r-epsilon and r-ferritin, as well as optimizing immunization doses and adjuvant selection for different animal models, will be necessary. The immune effects of different immune doses in the same animal are different. For example, it has been reported in the literature that when mice were initially injected, the immune response (antibody titer) was enhanced with the increase of the antigen dose up to 50 µg, but after that the increase of the antigen dose did not have any effect on the antibody titer [ 56 ] ; The immune effects of identical adjuvants in different animals are also different. In our other tests, aluminium adjuvant was not the best adjuvant for immunity in mice, whereas aluminium adjuvant was better than aqueous adjuvant for immunity in sheep (Data not shown). Conclusion In this study, the epsilon-ferritin nanoparticles antigen was successfully prepared by in vitro docking. As verified by the mice model and sheep, the epsilon-ferritin nanoparticles antigen increased the immune response better than r-epsilon, which provided a new insight for the development of C. perfringens vaccines. Methods Cells and Strains MDCK cells were obtained from National Center for Veterinary Culture collection (CVCC code: CL22). All cells were confirmed to be free of mycoplasma contamination. Then cells were cultured in DMEM (Gibco, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Logan, UT, USA) and 1% penicillin-streptomycin (Solarbio, Beijing, China) at 37°C with 5% CO 2 . Cells were maintained in DMEM supplemented with 0.5% FBS and 1% penicillin- streptomycin. The C. perfringens type D strain CV-81(CVCC code: 811802), along with its naturally produced toxins, was supplied by our laboratory. Mice and Sheep Specific Pathogen Free (SPF) female Balb/c mice, aged 6–8 weeks, were obtained from Beijing Viton Lihua Laboratory Animal Technology Co. Ltd. Two-month old healthy Small Tailed Han sheep self-propagated were used in the experiment as well. Gene Synthesis and Plasmid Construction Previous studies have shown that toxicity of epsilon is greatly reduced at 106 site mutation compared to other sites [ 42 , 57 ] . Thus, the epsilon gene sequence with mutation H106P (GenBank accession No. 93001271) was downloaded from NCBI. A 6×His tag was added to the N-terminal end of epsilon gene sequence, three repeat GGGGS linker and a SpyTag were added to its C-terminal sequentially. This fusion fragment was constructed and named epsilon-ST. The ferritin sequence (GenBank accession No. NP_223316) was obtained from NCBI. A fusion gene named ferritin-SC was constructed. The structure, from the N-terminus to the C-terminus, was shown below: 6×His, ferritin gene sequence, three repeats GGGGS linker and a SpyCatcher. Gene with codon optimized preference to E. coli cell was synthesized by Sangon(Biotech,Shanghai,China). Then, epsilon-ST gene was cloned in the pCold II vector and named pCold-epsilon. The ferritin-SC gene was inserted into pET28a vector and named pET-ferritin. Expression and Identification of Protein The pCold-epsilon and pET-ferritin plasmids were amplified by E. coli JM101 (TransGen Biotech, Beijing, China) and extracted following the instructions of the PurePlasmid Mini Kit (Tiangen Biotech, Beijing,China). The plasmids were transformed into BL21 (DE3) competent E. coli cells (TransGen Biotech, Beijing, China), and single colonies were inoculated into 5 mL of LB culture containing ampicillin sodium and kanamycin (Solarbio, Beijing, China), respectively, then incubated at 37°C. When the optical density at 600 nm (OD 600 ) was approximately 0.6, IPTG (TransGen Biotech, Beijing, China) was added to a final concentration of 1 mM. pCold-epsilon cultures were shaken at 16°C for 20 h, and pET-ferritin cultures were shaken at 28°C for 6 h. Appropriate amounts of expression products samples were mixed with 5×loading buffer (YEASEN, Shanghai, China), heated at 95°C for 10 min, and separated on a 12.5% polyacrylamide Tris-glycine gel (Biotides, Beijing, China) at 180 V for 90 min. And then expression products were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA), and blocked with 5% (w/v) skim milk in Phosphate-Buffered Saline (PBS) containing 0.1% Tween (PBST) for 1 h at 37°C. PVDF membranes were then incubated overnight at 4°C with His-tag antibodies (Sigma-Aldrich, St. Louis, MO, USA, 1:10000). Following three washes with PBST, membranes were incubated with HRP-conjugated goat anti-mouse antibodies (Sigma-Aldrich, St. Louis, MO, USA, 1:2000) diluted in PBST for 1 h at 37°C. The chemiluminescent signal was developed using Immobilon Classico Western HRP substrate (Millipore, Burlington, MA, USA). Protein Purification The bacterial cultures were collected by centrifugation after induction. After centrifugation, the bacterial pellets were resuspended in lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0), lysed by ultrasonication, and cell debris was removed by centrifugation at 8,000 rpm for 40 min at 4°C. The supernatant was filtered through a 0.45 µm pore size microfilter, and the samples were loaded onto gravity-flow Ni-NTA chromatography columns(DiNing, Beijing, China). The column was incubated at 4°C for 3 h. Non-binding proteins were washed out as thoroughly as possible using a washing buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, and 20 mM imidazole), and finally, the target proteins were washed out using an elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, and 250 mM imidazole). SDS-PAGE was used to confirm the target protein in collection products. After determining the purity and yield, the proteins were dialyzed to replace the imidazole with 0.01 M PBS (pH 7.4). The purified products obtained were named r-epsilon and r-ferritin, respectively. Epsilon-Ferritin Nanoparticles Preparation and Identification To prepare epsilon-ferritin nanoparticles, r-epsilon and r-ferritin were mixed thoroughly at a molar ratio of 2:1 and incubated at 37°C for 2 h. SDS-PAGE was used to confirm the docking efficacy of the two proteins. The docking products were proceeded to be observed by transmission electron microscopy (HITACHI, HT7700, Japan). Briefly, 10 µL of each sample (0.1 mg/mL) were applied to a freshly discharged 300-mesh copper grid and incubated for 1 min. Excess liquid was blotted away with filter paper. The grid was then washed twice with double-distilled water, blotted, and negatively stained with freshly prepared 2% (w/v) hydrogen peroxide acetate for 45 s, and air-dried. The grids were imaged using a transmission electron microscope operated at 80 kV [ 58 ] . Images were recorded at a magnification of 60,000×. Triplicate measurements of 20 acquisitions each at 5 s per acquisition were taken on a DynaPro Nanostar instrument at 25°C in a 1 mL quartz cuvette (Wyatt Technology Corp.) and using auto-attenuation of the laser to analyze the size distribution of particles. Vaccination of Mice and Sheep For mice vaccination, thirty female Balb/c mice (6–8 weeks old) were randomly divided into four groups: PBS group (n = 5), r-ferritin group (n = 5), r-epsilon group (n = 10), epsilon-ferritin nanoparticles group (n = 10) [ 34 ] . Ten mice were immunized subcutaneously with r-epsilon/ quick antibody aqueous adjuvant(Biodragon, Suzhou, China), while another ten mice were immunized subcutaneously with epsilon-ferritin nanoparticles/aqueous adjuvant. The immune dose was 10 µg r-epsilon protein monomer or the epsilon-ferritin nanoparticles(in which contained 10 µg of r-epsilon protein). An empty r-ferritin-only control(containing an equivalent amount of r-ferritin in epsilon-ferritin nanoparticles group) were set up. Each sample was mixed 1:1(50 µL + 50 µL) with aqueous adjuvant. All the mice were vaccinated in a prime/boost manner which was vaccinating mice at week 0 and weeks 3. Serum was randomly collected through the venous sinus in each group every one week. Blood samples were incubated at 37°C for 2 h, then centrifuged at 5,000 rpm for 15 min to separate the serum. They were stored at -20°C. Mice of PBS group (n = 5), r-ferritin group (n = 5), r-epsilon group (n = 5) and epsilon-ferritin nanoparticles group (n = 5) were euthanized at weeks 7 to analyze the percentages of activated CD4 + and effector CD8 T cells subsets. Mice of r-epsilon group (n = 5) and epsilon-ferritin nanoparticles group (n = 5) were euthanized at weeks 15 to analyze the percentages of memory CD8 + T cell subsets. For sheep vaccination, six sheep were randomly divided into two groups. Three sheep were immunized subcutaneously with r-epsilon/aqueous adjuvant, while another three sheep were immunized subcutaneously with epsilon-ferritin nanoparticles. Immunization dose for all the groups was normalized based on the mass quantity of r-epsilon. Each sheep received 300 µg of r-epsilon in the first and third weeks. Blood samples were collected through jugular vein in the third and sixth weeks and treated in the same manner as described for mice. Detection of Specific Antibody Level by ELISA Antibody levels in the serum from each group of mice and sheep were measured using ELISA [ 59 , 60 ] . Briefly, the purified r-epsilon were coated on high-binding 96-well plates at 1 µg/mL with 100 µL per well overnight at 4°C. After washing the plates 4 times with PBS containing 0.05% Tween at 300 µL per well, plates were blocked with 3% fish gelatin (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37°C. After washing with PBST, immunized serum were diluted 1:100 with 3% fish gelatin (PBST) and added into each well in duplicate. Then the plates were incubated at 37°C for 1 h. After incubation, the plates were washed as described above. The detection of antigen-specific IgG antibody in serum of mice or sheep was conducted through adding 1:4000 dilution of horseradish peroxidase (HRP)-coupled goat anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO, USA) or a 1:10,000 dilution of HRP-coupled monoclonal anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO, USA), respectively. After a final wash to remove any nonspecifically bound antibodies, 3,5'-tetramethylbenzidine (TMB, Solarbio, Beijing, China) substrate was added to initiate the color development reaction. The reaction was allowed to proceed for 10 min at room temperature, protected from light, and then stopped by adding 100 µL of 1 M H 2 SO 4 . The absorbance at 450 nm (OD 450nm ) was immediately recorded using an ultraviolet spectrophotometer. For IgG subtype detection reaction of mice, the purified r-epsilon were coated on high-binding 96-well plates at 5 µg/mL with 100 µL per well overnight at 4°C. After washing according to the above conditions, plates were blocked with 3% fish gelatin (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37°C. After washing with PBST, immunized mice serum were diluted 1:100 with 3% fish gelatin (containing 0.05% Tween) and incubated at 37°C for 1 h. Then the plates were washed as described above. The goat anti-mouse IgG1 or goat anti-mouse IgG2a (Biodragon, Suzhou, China) was added. After washed, 3,5'-tetramethylbenzidine (TMB, Solarbio, Beijing, China) substrate was added to initiate the color development reaction. The reaction was allowed to proceed for 10 min at 37°C, protected from light, and then stopped by adding 100 µL of 1 M H 2 SO 4 . The absorbance at 450 nm (OD 450nm ) was immediately recorded using an ultraviolet spectrophotometer. Detection of Neutralizing Antibody Titers Serum was prepared from mice and sheep at 21 and 42 days post immunization (dpi) and inactivated at 56°C for 30 min in a water bath. Serial 2-fold diluted sera and 2 MLD/100 µL of the type D natural toxin were mixed in equal proportions in 96-well microtiter plates. Subsequently, 100 µL of well-grown MDCK cells at a concentration of 1×10 4 cells/mL were added and incubated for 24 h in a 37°C, 5% CO 2 incubator. Neutralizing antibodies titers were detected by observing CPE. Serum samples from the PBS group were used as the negative control, and toxin wells served as the positive control. Splenocytes Isolation of Mice Mice were euthanized at weeks 7 and 15. Then, they were soaked in 75% alcohol for 10 min. The spleen was surgically removed out and washed in PBS containing 1% penicillin, then homogenized through a 40 µm strainer until no visible tissue remained. After centrifugation at 1,500 rpm for 5 min, the supernatant was discarded. Erythrocyte lysis buffer (Solarbio, Beijing, China) was added, and the samples were incubated at room temperature for 10 min to remove red blood cells debris. Next, 5 mL of PBS with 0.5% FBS was added, and the suspension was centrifuged at 1,500 rpm for 5 min. This wash step was repeated once. The resulting cell pellet was resuspended in 1 mL of RPMI 1640 medium (Gibco, Logan, UT, USA) and followed by passing through a 40 µm strainer to obtain scattered splenocytes. A portion of the cells were used for IFN-γ induction analysis, and the other portion of the cells were subjected to T-lymphocyte subpopulation analysis by flow cytometry. Detection of T-lymphocyte Subpopulation by Flow Cytometry in Mice Mice splenocytes obtained from the isolation procedure above were resuspended to a concentration of 10 6 cells and placed into a 96-well plate. The cells were washed twice with a flow staining buffer and then stained with a panel of antibodies, including DAPI anti-mouse L/D, BV500 violetFluorTM 500 Anti-Mouse CD3, PerCP-Cyanine 5.5 anti-mouse CD4, ApC-Cy7 anti-mouse CD8a, ApC anti-mouse CD44, FITC anti-mouse CD62L (Tonbo Biosciences, USA). The plate was incubated with the antibodies at 4°C for 30 min. After incubation, the cells were centrifuged and the supernatant were removed. Then, 0.7% paraformaldehyde was used to fix the cells. Finally, the cells were washed with a flow staining buffer and were analyzed using a BD LSR Fortessa Flow Cytometer (Becton Dickinson, USA), The data were analyzed by FlowJo 10.8.0 software. Detection of IFN-γ Level in Mice by ELISA The splenocyte cell suspension at weeks 7 were diluted to 1×10 5 /mL and transferred to a 96-well plate with 100 µL per well (1×10 4 cells per well). Then r-epsilon was added into the 96-well plate with final concentration of 5 µg/mL. The plate was incubated at 37°C 5% CO 2 for 48 h. After stimulation, cells were centrifuged at 1,500 rpm for 15 min and then supernatant were collected. The supernatant was diluted 2-fold, and detected with Mouse IFN-gamma ELISA Kit (Solarbio, China) according to the manufacturer’s instructions. Detection of IFN-γ Level in Sheep by ELISA Anticoagulated blood was collected at 49 dpi. 900 µL of anticoagulated blood was added to a 24-well cell plate, and 100 µL of r-epsilon at a final concentration of 100 µg/mL was added. 100 µL of PBS was added as the negative control. The plates were put in the cell incubator at 37°C 5% CO 2 for 72 h. After stimulation, blood was centrifuged at 1,500 rpm for 15 min and supernatant were collected. The supernatant was detected with Sheep IFN-gamma ELISA Kit (Solarbio, China) according to the manufacturer’s instructions. Statistical analysis All data in this study were expressed as mean ± standard deviation (SD), and a t test or two-way ANOVA was performed using GraphPad Prism 8.0 software(San Diego,CA). Subsequently, multiple comparisons were used to compare between groups. A P value < 0.05 was considered to indicate a significant difference; Ns, no significant differences were observed (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Declarations Supplementary Information The percentages of memory CD8+ T cell subsets. Acknowledgements We would like to express our thanks to the staff of the Central Laboratory at the Institute of Animal Science, Chinese Academy of Agricultural Sciences, for their assistance in preparing and observing TEM samples. And thanks to Lin Jiang and Yabin Pu researchers, the scientific and technological innovation team of Livestock and Poultry Quality Resources Protection and Utilization, for their support in the sheep immune evaluation test. Author Contributions Conceptualization, X.F. and H.Z.; Methodology, Q.X. and Y.Z.; Validation, H.Z. and Y.L.; Formal analysis, H.Z. and Q.X.; Investigation, H.Z. and Y.L.; Resources, J.D.,H.J. and Q.S.; Data curation, H.Z.; Writing-original draft preparation, H.Z. and Q.X.; Writing-review and editing, H.C. and G.Z; Visualization, H.Z. and Q.X.; Supervision, X.F. and S.J.. All authors have read and agreed to the published version of the manuscript. Fundings This study was supported by the the Ningxia Hui Autonomous Region Key R&D Program (2024BBF02014), and the Agricultural Science and Technology Innovation Program (ASTIP‐IAS15). Data availability The data will be shared upon request by the readers. Ethics approval and consent to participate The permissions to conduct these experiments were granted by The Animal Welfare & Ethics Committee of Institute of Animal Science, Chinese Academy of Agricultural Sciences, China (IAS2024−177, IAS2024−84). Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Soncini SR, Hartman AH, Gallagher TM, Camper GJ, Jensen RV, Melville SB. Changes in the expression of genes encoding type IV pili-associated proteins are seen when Clostridium perfringens is grown in liquid or on surfaces. BMC Genomics. 2020; 21(1):45. Zhou B, Yuan Y, Zhang S, Guo C, Li X, Li G, et al. Intestinal Flora and Disease Mutually Shape the Regional Immune System in the Intestinal Tract. Front Immunol. 2020; 11:575. Posthaus H, Kittl S, Tarek B, Bruggisser J. Clostridium perfringens type C necrotic enteritis in pigs: diagnosis, pathogenesis, and prevention. J Vet Diagn Invest. 2020; 32(2):203-12. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9380767","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":635972213,"identity":"f6efb467-4858-4c6b-8f93-998e7f9bf6b0","order_by":0,"name":"Huiyu Zhao","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Huiyu","middleName":"","lastName":"Zhao","suffix":""},{"id":635972215,"identity":"31628bc7-95b1-4964-b0a4-ab537352b221","order_by":1,"name":"Qianru Xing","email":"","orcid":"","institution":"Chinese Academy of Agricultural 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r-epsilon protein docking schematic. \u003cstrong\u003e(D)\u003c/strong\u003e Identification diagram of r-epsilon. \u003cstrong\u003e(E)\u003c/strong\u003e Identification diagram of r-ferritin. M. Protein marker; 1. The bacterial lysates without induction; 2. The supernatant of bacterial lysates induced with IPTG; 3. The precipitation of bacterial lysates induced with IPTG. \u003cstrong\u003e(F)\u003c/strong\u003e Purification diagram of r-epsilon. \u003cstrong\u003e(G)\u003c/strong\u003e Purification diagram of r-ferritin. M. Protein marker; 1. The supernatant before purification; 2. The flow-through solution; 3. The eluent product (20 mmol·L\u003csup\u003e-1\u003c/sup\u003e Imidazole); 4-6. The eluent product (250 mmol·L\u003csup\u003e-1\u003c/sup\u003e Imidazole). \u003cstrong\u003e(H)\u003c/strong\u003e Transmission electron microscopy image of r-ferritin. \u003cstrong\u003e(I)\u003c/strong\u003e Transmission electron microscopy image of epsilon-ferritin nanoparticles. \u003cstrong\u003e(J)\u003c/strong\u003e Dynamic light scattering diagram of epsilon-ferritin nanoparticles and r-ferritin. \u003cstrong\u003e(K)\u003c/strong\u003e SDS-PAGE diagram of epsilon-ferritin nanoparticles. M. Protein marker; 1. r-epsilon and r-ferritin docking product; 2. r-ferritin; 3. r-epsilon.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9380767/v1/2dea981b4184105399fe36bf.jpg"},{"id":109116661,"identity":"5cd70dfe-2bb8-4e1f-9be0-003300d1b4a9","added_by":"auto","created_at":"2026-05-12 16:32:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73771,"visible":true,"origin":"","legend":"\u003cp\u003eHumoral Immune Responses in Nanoparticles Vaccinated Balb/c Mice. \u003cstrong\u003e(A) \u003c/strong\u003eSchematic of Balb/c vaccination. Mice from each group were prime or boost-vaccinated at week 0 or weeks 3. Serum was collected every week. All mice were euthanized at weeks 7 or 15.\u003cstrong\u003e (B-E)\u003c/strong\u003e The r-epsilon specific IgG,IgM, IgG1, and IgG2a antibody levels of immunized Balb/c mice at each time point were detected by ELISA. \u003cstrong\u003e(F)\u003c/strong\u003eThe ratio of IgG1/IgG2a. \u003cstrong\u003e(G)\u003c/strong\u003e Detection of neutralizing antibody titers in Balb/c mice serum. Statistical significance was calculated using two-way ANOVA with multiple comparisons corrected. Ns, \u003cem\u003eP\u003c/em\u003e\u0026gt; 0.05; *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9380767/v1/986be47515cff0095c96d474.jpg"},{"id":109116660,"identity":"13537ea1-5cad-4bd9-8f4a-218fd6dc6523","added_by":"auto","created_at":"2026-05-12 16:32:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44838,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of T-lymphocyte subpopulation and IFN-γ cytokine level. Splenocytes were stimulated with 5 µg r-epsilon. (A) The percentages of activated CD4+ and effector CD8 T cells were determined by flow cytometry at weeks 7. (B) ELISA assays were conducted for IFN-γ secretion in splenocytes at weeks 7. (C) The percentages of memory CD8+ T cell subsets at weeks 15. Statistical significance was calculated using two-way ANOVA with multiple comparisons corrected. Ns, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9380767/v1/9aed63a4545e3646909936c1.jpg"},{"id":109116659,"identity":"c9cbe960-118d-422c-b5be-1d80408f1e51","added_by":"auto","created_at":"2026-05-12 16:32:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":42531,"visible":true,"origin":"","legend":"\u003cp\u003eImmunological evaluation in Sheep. \u003cstrong\u003e(A)\u003c/strong\u003e Detection of IgG in sheep serum. \u003cstrong\u003e(B)\u003c/strong\u003e Detection of neutralizing antibody titers in sheep serum. \u003cstrong\u003e(C)\u003c/strong\u003e ELISA assays were conducted for IFN-γ secretion. Statistical significance was calculated using two-way ANOVA with multiple comparisons corrected or t test (and nonparametric tests). Ns,\u003cem\u003e P\u003c/em\u003e \u0026gt; 0.05; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9380767/v1/c830d7e19cd406c41ead14df.jpg"},{"id":109207324,"identity":"d57aff90-c377-4757-b34e-0b3eb0211547","added_by":"auto","created_at":"2026-05-13 15:19:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":514660,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9380767/v1/3d7952b5-b871-4d7b-a14c-768b5535b83b.pdf"},{"id":109116657,"identity":"83498d64-1d9e-4454-be00-1b68d6edf6e2","added_by":"auto","created_at":"2026-05-12 16:32:51","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":63208,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9380767/v1/d88285e4adfd11fda99f5e4b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clostridium perfringens epsilon-ferritin nanoparticles induced robust immune responses in mice and sheep","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eC. perfringens\u003c/em\u003e, also known as \u003cem\u003eClostridium welchii\u003c/em\u003e, is a Gram-positive, anaerobic, spore-forming bacterium with a large, non-flagellated body\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e and capsules. The pathogen has been identified as a significant etiological agent of various enteric disorders in livestock species\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, frequently causing diseases such as gas gangrene, necrotizing enterocolitis\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e and enterotoxicosis in livestock, including cattle, sheep, and chickens\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. These diseases typically manifest acutely\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, progress rapidly with high mortality rates\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, sometimes leading to sudden death within 1\u0026ndash;2 days if treatment is delayed\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The bacterium has negatively impacted the farming industry in China\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe virulence of \u003cem\u003eC. perfringens\u003c/em\u003e largely depends on the release of six main exotoxins: α (alpha), β (beta), ε (epsilon), ι (iota), enterotoxin (cpe), and NetB\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Based on its toxin production pattern, \u003cem\u003eC. perfringens\u003c/em\u003e is classified into seven types labelled A through G\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Type C and D are particularly significant in veterinary clinical practices; type C produces alpha and beta toxins and type D produces alpha and epsilon toxins. When epsilon toxin is activated by proteolysis, with the consequent release of carboxy-terminal and amino-terminal peptides from the protein, the bacterial virulence will substantially increase\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. It is the third most potent toxin after the botulinum and tetanus toxins, making it a significant potential bioterrorist agent recognized by the international community\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. By an unidentified mechanism, this toxin passes through gut wall of affected animals and enters the blood stream in the intestine. Epsilon toxins can further disseminate to other organs via blood circulation, and brain and kidney are preferentially targeted by these toxins\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor cattle and sheep, vaccines against \u003cem\u003eC. perfringens\u003c/em\u003e are primarily toxoid-based. Some toxoid components can also cause local inflammatory or allergic reactions\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Thus, these limitations have prompted endeavour to design novel vaccines\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. The use of biomolecular methods to express recombinant toxins for novel vaccine technology offers an effective way, which can bypass the complexities and biosafety risks, preserving immunogenicity and enhancing potency\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. However, most recombinantly expressed proteins pose challenges for vaccine development due to their low immunogenicity and rapid release rates\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Thus, it is necessary to take advantage of some adjuvants or delivery systems to stimulate the immune response.\u003c/p\u003e \u003cp\u003eNanoparticle vaccine is a new type of vaccine emerging in recent years, and has been widely used in the prevention of many diseases\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Various distinctive features of nanotechnology have been demonstrated to improve drug delivery, such as increasing drug solubility/stability, controlling drug release, prolonging systemic circulation, crossing tissue barriers, enhancing accumulation in target tissues and facilitating cellular uptake. Ferritin is one of nanoparticles with a hollow cage-like protein structure that occurs naturally in the host body\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The outer surface of ferritin can be chemically or genetically modified with targeted sequences to display multiple ligands, thereby improving binding affinity\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, and it can prepare the multivalent nanovaccines. Additionally, ferritin nanoparticles rapidly circulate into lymph nodes and target dendritic cells\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, particularly CD8α\u0026thinsp;+\u0026thinsp;cell populations, following subcutaneous immunization\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Ferritin has been examined in preclinical research as a vaccine carrier candidate for various diseases, including HIV-1\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, influenza (H1N1 strain)\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, demonstrating its satisfactory efficacy and promising potentials in combating pathogenic infections.\u003c/p\u003e \u003cp\u003eIn this study, a non-toxic r-epsilon with mutation H106P and SpyTag, and soluble r-ferritin with SpyCatcher were obtained respectively. Subsequently, self-assembling ferritin nanoparticles were bound to exogenous r-epsilon onto their surface through SpyTag-SpyCatcher docking peptides. Then, using this technology, the immunological effect and immune response type of epsilon-ferritin nanoparticles were evaluated and analyzed in mice and sheep. Our findings demonstrated that epsilon-ferritin nanoparticles efficiently stimulated humoral and cellular immunity in mice and sheep and induced robust cellular immune responses.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe r-epsilon can be successfully displayed on r-ferritin through SpyTag/SpyCatcher\u003c/h2\u003e \u003cp\u003eTo conveniently and stably load specific antigens of \u003cem\u003eC. perfringens\u003c/em\u003e on ferritin nanoparticles, we employed SpyTag/SpyCatcher docking technique\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e (Fig.\u0026nbsp;1A,B,C). Western blotting assay confirmed the successful expression of the r-epsilon and r-ferritin and the predominance of expression in the supernatant (Fig.\u0026nbsp;1D,E). The r-epsilon and r-ferritin were purified by Ni-NTA affinity chromatography. Imidazole was subsequently removed by dialysis against PBS (pH 7.4). The r-epsilon was demonstrated high quality, as evident from SDS-PAGE gels (Fig.\u0026nbsp;1F). Similarly, SDS-PAGE showed a clear single band, indicating that the purified r-ferritin was obtained (Fig.\u0026nbsp;1G).\u003c/p\u003e \u003cp\u003eFerritin and epsilon-ferritin nanoparticles were examined by TEM (Fig.\u0026nbsp;1H,I). The TEM image confirmed that ferritin and epsilon-ferritin nanoparticles were successfully self-assembled and had a uniform size distribution. The result of dynamic light scattering test showed that the particle size of ferritin protein ranged from 11.7\u0026thinsp;~\u0026thinsp;15.7 nm. The particle size of epsilon-ferritin nanoparticles ranged from 32.7\u0026thinsp;~\u0026thinsp;50.7 nm, and the main peak was 37.8 nm, which indicated that r-epsilon successfully docked with ferritin protein to form epsilon-ferritin nanoparticles(Fig.\u0026nbsp;1J). The SDS-PAGE figure (Fig.\u0026nbsp;1K) showed a uniform increase in molecular weight from 35 kDa to 110 kDa. It indicated that r-epsilon bound to r-ferritin. These results showed that r-epsilon successfully assembled with r-ferritin to form epsilon-ferritin nanoparticles.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMuscular inoculation with epsilon-ferritin nanoparticles induced strong specific humoral immune response in mice\u003c/h3\u003e\n\u003cp\u003eIn the standard prime-boost regimen, animals are administered different antigens via intramuscular immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To evaluate the humoral antibody response, the antibody level of the mice from epsilon-ferritin nanoparticles groups and r-epsilon groups were measured using ELISA. As expected, there was no difference in the specific antibody levels between the PBS group and the r-ferritin group, indicating that r-ferritin does not stimulate a specific B-cell immune response. After vaccination, mice immunized with epsilon-ferritin nanoparticles produced significantly higher IgG antibody levels than those immunized with r-epsilon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In addition, the epsilon-ferritin nanoparticles groups showed a plateau at 35 dpi, whereas the r-epsilon groups showed a plateau after 42 dpi. On the 7 dpi and 28 dpi, the IgM level was also significantly higher than that in the r-epsilon groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The results of the IgG subtypes showed that the epsilon-ferritin nanoparticles group produced notably higher levels of both IgG1 and IgG2a antibodies compared to the r-epsilon group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,E, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Besides, the IgG1/IgG2a ratio was lower than that of the r-epsilon group at day 42 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eIn addition to antibody levels, neutralizing antibody titers were also one of an essential feature of vaccine with high efficacy. Firstly, minimum lethal dose (MLD) of the CV-81 natural toxin caused cytopathic effect in MDCK cells was dilution 2\u003csup\u003e13\u003c/sup\u003e. Then, serum collected at 21 dpi and 42 dpi were were used to determine neutralizing antibody titer in serum using the fixed virus-diluted serum method (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The results demonstrated that at 42 dpi, serum from the epsilon-ferritin nanoparticles group exhibited higher neutralizing antibody titer than r-epsilon group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEpsilon-ferritin nanoparticles induced an effective cellular immune response through muscular immunity\u003c/h3\u003e\n\u003cp\u003eT cells are the main components of lymphocytes, and their immune response from T cells is known as cellular immunity\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Splenocyte cells from experimental mice were isolated, then T-lymphocyte subpopulation and IFN-γ cytokine levels were assessed to determine whether the nanoparticles vaccine induced Th1-type cellular immunity. There were no statistically significant differences in T-lymphocyte subpopulation among the groups at weeks 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the epsilon-ferritin nanoparticles group elicited the highest IFN-γ levels, which were significantly higher than the PBS groups and the r-epsilon group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Moreover, staining of splenocytes taken 15 weeks after immunization revealed that memory CD8\u0026thinsp;+\u0026thinsp;T cells were significantly higher in the epsilon-ferritin nanoparticles group than that in the r-epsilon group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDetection of Antibody Level in the Immunized Sheep\u003c/h3\u003e\n\u003cp\u003eThe immunogenicity of epsilon-ferritin nanoparticles in sheep was also assessed. Blood samples were collected at 21 dpi and 42 dpi. IgG levels in the serum of both groups were measured by ELISA. Unlike the results observed in mice, the epsilon-ferritin nanoparticles group did not induce significantly higher IgG antibody levels than the r-epsilon group in sheep (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Next, the neutralizing capacity of the serum from both groups were evaluated using the same method as described previously. The results showed that there were no statistically significant differences in neutralizing antibody level among the vaccine group at 21 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the epsilon-ferritin nanoparticles group elicited higher neutralizing antibody level at 42 dpi (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Anticoagulated blood from both groups were stimulated by r-epsilon and the cytokine IFN-γ in supernatant was assayed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The results showed that stimulation of the epsilon-ferritin nanoparticles group produced higher levels of IFN-γ than the r-epsilon group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eC. perfringens\u003c/em\u003e often settles in the intestines of healthy animals and humans in an asymptomatic manner\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, and can cause diseases such as gas gangrene, necrotizing enterocolitis and enterotoxaemia when the host's immune function is disrupted\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Epsilon toxin is a virulence factor produced by \u003cem\u003eC. perfringens\u003c/em\u003e types B and D\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. And it is characterized by a rapid onset, short disease course, and high mortality rate\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Treatment with medication alone is often ineffective due to the acute feature of the disease, resulting in significant losses to cattle and sheep. Currently, the vaccines on the market are mainly toxoid vaccines, which can also cause local inflammatory or allergic reactions\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is particularly important to develop a new vaccine to prevent this disease\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEpsilon toxin is a relatively inactive prototoxin consisting of 311 amino acids. Besides, the histidine residue (domain I) appeared to be integral to the active site of epsilon toxin. Of the two histidine residues in the mature epsilon toxin, the histidine at 106 was predicted to be located in the middle of the β-folding region in a weakly hydrophobic environment, whereas the one at 149 is predicted to be located close to a turn region in the hydrophilic environment\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. H106P has been shown to be completely non-toxic to cultured MDCK cells, and is a safe, non-toxic, and immunoprotective candidate vaccine antigen for enterotoxaemia\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Therefore, this site was chosen for mutation and then r-epsilon was expressed in this study. The r-epsilon was confirmed to be non-lethal to MDCK cells by cellular assays.\u003c/p\u003e \u003cp\u003eRecently, advances in nanotechnology have been widely used to enhance the immunogenicity of antigens, which has led to significant breakthroughs in the field of medicine because of its high safety, delivery capacity and modifiability\u003csup\u003e[\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Nanoparticle materials mainly include protein/peptides, lipid nanoparticles, polymer nanoparticles and inorganic nanoparticles\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e, among which ferritin nanoparticles are an attractive protein nanoplatform\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. In the present study, prokaryotic expression system was utilized to express ferritin nanoparticles, which not only consumes low cost, but also has high soluble expression, purity and stability.\u003c/p\u003e \u003cp\u003eSpyTag/SpyCatcher coupling technology is a versatile and efficient protein-linking method that enables covalent attachment of proteins by simply mixing components under various pH, temperature, and buffer conditions. This reaction yields high specificity and stability, unaffected by competing peptides\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. In this study, the SpyTag/SpyCatcher coupling technique was introduced into ferritin nanoparticle vaccines construction. And r-epsilon with SpyTag tag and r-ferritin with SpyCatcher tag were prepared, respectively. The fusion of r-ferritin and r-epsilon occurred after mixing them in vitro. The fusion products could be observed as nanoparticles by TEM and a range of diameters for docking products were greater than the average FeSC diameter by DLS, indicating the successful docking of r-ferritin and r-epsilon.\u003c/p\u003e \u003cp\u003eIt is generally believed that inactivated vaccines and subunit vaccine antigens are processed by antigen-presenting cells and are primarily presented through the MHC-II pathway, mediating the recognition by CD4\u0026thinsp;+\u0026thinsp;T lymphocytes. The main function of these CD4\u0026thinsp;+\u0026thinsp;effector T cells is to secrete cytokines, which can activate phagocytes, neutrophils, and eosinophils, and stimulate B cells to produce antibodies, thereby triggering a specific humoral immune response that can effectively neutralize and eliminate extracellular pathogens. To evaluate the immunological effect of nanoparticles antigen, mice were subcutaneously immunized with epsilon-ferritin nanoparticles or r-epsilon. It was found that IgG antibody in the epsilon-ferritin nanoparticles group was significantly higher than that in the r-epsilon group, and its plateau was earlier than that in the r-epsilon group; The in vitro neutralization assay at 42 dpi showed that the epsilon-ferritin nanoparticles group also were significantly higher than in the r-epsilon group. These results suggested that nanoparticles can stimulate a stronger humoral immune response in mice.\u003c/p\u003e \u003cp\u003eSecond, in mice, it is widely accepted that the IgG1 response reflects the paracrine activity of Th2 CD4\u0026thinsp;+\u0026thinsp;T cells and that IgG2a is generated by Th1 CD4\u0026thinsp;+\u0026thinsp;T cell activity\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Therefore, the lower the IgG1/IgG2a ratio indicates a strong trend toward a Th1 immune response. Both IgG1 and IgG2a antibodies were significantly higher in the epsilon-ferritin nanoparticles group than in the r-epsilon group and the IgG1/IgG2a ratio was lower than that of the r-epsilon group at 42 dpi, which indirectly proved that the epsilon-ferritin nanoparticles group induced stronger Th1 and Th2 immune response in mice. Tang et al.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e immunized mice with SARS- CoV-2 ORF8 protein after coupling it with 8-arm PEG and the results showed that the coupling group with 8-arm PEG stimulated the organism to produce higher IgG1 and IgG2a, inducing both humoral immune response and cellular immunity. This indicated that nanoparticles antigen has better immune activation capacity, and aligned with the conclusions that we obtained from our experimental results. Th1 cells enhance CTL functions through the secretion of cytokines, such as IL-2, and IFN-γ, which activate other players of the innate system, as NK cells and macrophages, thereby resulting in higher pathogen or tumor cells/apoptotic tumor bodies elimination\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Th2 cells stimulate the secretion of antibodies by B cells, which additionally activate other phagocytic cells\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. In our experiments, the epsilon-ferritin nanoparticles group showed a preference for a stronger Th1 type response (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), inducing a cellular immune response in mice. In addition, the nanoparticles group were found to have significantly more memory CD8\u0026thinsp;+\u0026thinsp;cells than the r-epsilon group. It demonstrated that nanoparticles induced a durable level of cellular immunity in mice. Thus, these data suggested that nanoparticles can stimulate stronger and longer-lasting cellular immunity in mice.\u003c/p\u003e \u003cp\u003eSheep were booster immunized with epsilon-ferritin nanoparticles or r-epsilon. Neutralizing antibody titer and IFN-γ cytokine level were found to be significantly higher in the epsilon-ferritin nanoparticles group than in the r-epsilon group. Ma et al.\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e designed a nanoparticles vaccine using ferritin against the RBD and Heptad Repeat (HR) antigens of SARS-CoV-2 to immune in rhesus monkeys. The results showed the nanoparticles antigen induced the production of neutralizing antibodies as well as T and B cell responses in rhesus monkeys prior to booster immunization. Thus, these suggest that nanoparticles not only induce stronger humoral and cellular immunity in animal model, but also stimulate stronger immune response in another animal.\u003c/p\u003e \u003cp\u003eAlthough this study achieved promising results, additional refinements are required. For instance, improving the docking efficiency between r-epsilon and r-ferritin, as well as optimizing immunization doses and adjuvant selection for different animal models, will be necessary. The immune effects of different immune doses in the same animal are different. For example, it has been reported in the literature that when mice were initially injected, the immune response (antibody titer) was enhanced with the increase of the antigen dose up to 50 \u0026micro;g, but after that the increase of the antigen dose did not have any effect on the antibody titer\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e; The immune effects of identical adjuvants in different animals are also different. In our other tests, aluminium adjuvant was not the best adjuvant for immunity in mice, whereas aluminium adjuvant was better than aqueous adjuvant for immunity in sheep (Data not shown).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the epsilon-ferritin nanoparticles antigen was successfully prepared by in vitro docking. As verified by the mice model and sheep, the epsilon-ferritin nanoparticles antigen increased the immune response better than r-epsilon, which provided a new insight for the development of \u003cem\u003eC. perfringens\u003c/em\u003e vaccines.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCells and Strains\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMDCK cells were obtained from National Center for Veterinary Culture collection (CVCC code: CL22). All cells were confirmed to be free of mycoplasma contamination. Then cells were cultured in DMEM (Gibco, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Logan, UT, USA) and 1% penicillin-streptomycin (Solarbio, Beijing, China) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were maintained in DMEM supplemented with 0.5% FBS and 1% penicillin- streptomycin. The \u003cem\u003eC. perfringens\u003c/em\u003e type D strain CV-81(CVCC code: 811802), along with its naturally produced toxins, was supplied by our laboratory.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMice and Sheep\u003c/h2\u003e \u003cp\u003eSpecific Pathogen Free (SPF) female Balb/c mice, aged 6\u0026ndash;8 weeks, were obtained from Beijing Viton Lihua Laboratory Animal Technology Co. Ltd. Two-month old healthy Small Tailed Han sheep self-propagated were used in the experiment as well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGene Synthesis and Plasmid Construction\u003c/h2\u003e \u003cp\u003ePrevious studies have shown that toxicity of epsilon is greatly reduced at 106 site mutation compared to other sites\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. Thus, the epsilon gene sequence with mutation H106P (GenBank accession No. 93001271) was downloaded from NCBI. A 6\u0026times;His tag was added to the N-terminal end of epsilon gene sequence, three repeat GGGGS linker and a SpyTag were added to its C-terminal sequentially. This fusion fragment was constructed and named epsilon-ST. The ferritin sequence (GenBank accession No. NP_223316) was obtained from NCBI. A fusion gene named ferritin-SC was constructed. The structure, from the N-terminus to the C-terminus, was shown below: 6\u0026times;His, ferritin gene sequence, three repeats GGGGS linker and a SpyCatcher. Gene with codon optimized preference to \u003cem\u003eE. coli\u003c/em\u003e cell was synthesized by Sangon(Biotech,Shanghai,China). Then, epsilon-ST gene was cloned in the pCold II vector and named pCold-epsilon. The ferritin-SC gene was inserted into pET28a vector and named pET-ferritin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExpression and Identification of Protein\u003c/h2\u003e \u003cp\u003eThe pCold-epsilon and pET-ferritin plasmids were amplified by \u003cem\u003eE. coli\u003c/em\u003e JM101 (TransGen Biotech, Beijing, China) and extracted following the instructions of the PurePlasmid Mini Kit (Tiangen Biotech, Beijing,China). The plasmids were transformed into BL21 (DE3) competent \u003cem\u003eE. coli\u003c/em\u003e cells (TransGen Biotech, Beijing, China), and single colonies were inoculated into 5 mL of LB culture containing ampicillin sodium and kanamycin (Solarbio, Beijing, China), respectively, then incubated at 37\u0026deg;C. When the optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) was approximately 0.6, IPTG (TransGen Biotech, Beijing, China) was added to a final concentration of 1 mM. pCold-epsilon cultures were shaken at 16\u0026deg;C for 20 h, and pET-ferritin cultures were shaken at 28\u0026deg;C for 6 h. Appropriate amounts of expression products samples were mixed with 5\u0026times;loading buffer (YEASEN, Shanghai, China), heated at 95\u0026deg;C for 10 min, and separated on a 12.5% polyacrylamide Tris-glycine gel (Biotides, Beijing, China) at 180 V for 90 min. And then expression products were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA), and blocked with 5% (w/v) skim milk in Phosphate-Buffered Saline (PBS) containing 0.1% Tween (PBST) for 1 h at 37\u0026deg;C. PVDF membranes were then incubated overnight at 4\u0026deg;C with His-tag antibodies (Sigma-Aldrich, St. Louis, MO, USA, 1:10000). Following three washes with PBST, membranes were incubated with HRP-conjugated goat anti-mouse antibodies (Sigma-Aldrich, St. Louis, MO, USA, 1:2000) diluted in PBST for 1 h at 37\u0026deg;C. The chemiluminescent signal was developed using Immobilon Classico Western HRP substrate (Millipore, Burlington, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProtein Purification\u003c/h2\u003e \u003cp\u003eThe bacterial cultures were collected by centrifugation after induction. After centrifugation, the bacterial pellets were resuspended in lysis buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 300 mM NaCl, 10 mM imidazole, pH 8.0), lysed by ultrasonication, and cell debris was removed by centrifugation at 8,000 rpm for 40 min at 4\u0026deg;C. The supernatant was filtered through a 0.45 \u0026micro;m pore size microfilter, and the samples were loaded onto gravity-flow Ni-NTA chromatography columns(DiNing, Beijing, China). The column was incubated at 4\u0026deg;C for 3 h. Non-binding proteins were washed out as thoroughly as possible using a washing buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 300 mM NaCl, and 20 mM imidazole), and finally, the target proteins were washed out using an elution buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 300 mM NaCl, and 250 mM imidazole). SDS-PAGE was used to confirm the target protein in collection products. After determining the purity and yield, the proteins were dialyzed to replace the imidazole with 0.01 M PBS (pH 7.4). The purified products obtained were named r-epsilon and r-ferritin, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEpsilon-Ferritin Nanoparticles Preparation and Identification\u003c/h2\u003e \u003cp\u003eTo prepare epsilon-ferritin nanoparticles, r-epsilon and r-ferritin were mixed thoroughly at a molar ratio of 2:1 and incubated at 37\u0026deg;C for 2 h.\u003c/p\u003e \u003cp\u003eSDS-PAGE was used to confirm the docking efficacy of the two proteins. The docking products were proceeded to be observed by transmission electron microscopy (HITACHI, HT7700, Japan). Briefly, 10 \u0026micro;L of each sample (0.1 mg/mL) were applied to a freshly discharged 300-mesh copper grid and incubated for 1 min. Excess liquid was blotted away with filter paper. The grid was then washed twice with double-distilled water, blotted, and negatively stained with freshly prepared 2% (w/v) hydrogen peroxide acetate for 45 s, and air-dried. The grids were imaged using a transmission electron microscope operated at 80 kV\u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. Images were recorded at a magnification of 60,000\u0026times;.\u003c/p\u003e \u003cp\u003eTriplicate measurements of 20 acquisitions each at 5 s per acquisition were taken on a DynaPro Nanostar instrument at 25\u0026deg;C in a 1 mL quartz cuvette (Wyatt Technology Corp.) and using auto-attenuation of the laser to analyze the size distribution of particles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eVaccination of Mice and Sheep\u003c/h2\u003e \u003cp\u003eFor mice vaccination, thirty female Balb/c mice (6\u0026ndash;8 weeks old) were randomly divided into four groups: PBS group (n\u0026thinsp;=\u0026thinsp;5), r-ferritin group (n\u0026thinsp;=\u0026thinsp;5), r-epsilon group (n\u0026thinsp;=\u0026thinsp;10), epsilon-ferritin nanoparticles group (n\u0026thinsp;=\u0026thinsp;10)\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Ten mice were immunized subcutaneously with r-epsilon/ quick antibody aqueous adjuvant(Biodragon, Suzhou, China), while another ten mice were immunized subcutaneously with epsilon-ferritin nanoparticles/aqueous adjuvant. The immune dose was 10 \u0026micro;g r-epsilon protein monomer or the epsilon-ferritin nanoparticles(in which contained 10 \u0026micro;g of r-epsilon protein). An empty r-ferritin-only control(containing an equivalent amount of r-ferritin in epsilon-ferritin nanoparticles group) were set up. Each sample was mixed 1:1(50 \u0026micro;L\u0026thinsp;+\u0026thinsp;50 \u0026micro;L) with aqueous adjuvant. All the mice were vaccinated in a prime/boost manner which was vaccinating mice at week 0 and weeks 3. Serum was randomly collected through the venous sinus in each group every one week. Blood samples were incubated at 37\u0026deg;C for 2 h, then centrifuged at 5,000 rpm for 15 min to separate the serum. They were stored at -20\u0026deg;C. Mice of PBS group (n\u0026thinsp;=\u0026thinsp;5), r-ferritin group (n\u0026thinsp;=\u0026thinsp;5), r-epsilon group (n\u0026thinsp;=\u0026thinsp;5) and epsilon-ferritin nanoparticles group (n\u0026thinsp;=\u0026thinsp;5) were euthanized at weeks 7 to analyze the percentages of activated CD4\u0026thinsp;+\u0026thinsp;and effector CD8 T cells subsets. Mice of r-epsilon group (n\u0026thinsp;=\u0026thinsp;5) and epsilon-ferritin nanoparticles group (n\u0026thinsp;=\u0026thinsp;5) were euthanized at weeks 15 to analyze the percentages of memory CD8\u0026thinsp;+\u0026thinsp;T cell subsets.\u003c/p\u003e \u003cp\u003eFor sheep vaccination, six sheep were randomly divided into two groups. Three sheep were immunized subcutaneously with r-epsilon/aqueous adjuvant, while another three sheep were immunized subcutaneously with epsilon-ferritin nanoparticles. Immunization dose for all the groups was normalized based on the mass quantity of r-epsilon. Each sheep received 300 \u0026micro;g of r-epsilon in the first and third weeks. Blood samples were collected through jugular vein in the third and sixth weeks and treated in the same manner as described for mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDetection of Specific Antibody Level by ELISA\u003c/h2\u003e \u003cp\u003eAntibody levels in the serum from each group of mice and sheep were measured using ELISA\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. Briefly, the purified r-epsilon were coated on high-binding 96-well plates at 1 \u0026micro;g/mL with 100 \u0026micro;L per well overnight at 4\u0026deg;C. After washing the plates 4 times with PBS containing 0.05% Tween at 300 \u0026micro;L per well, plates were blocked with 3% fish gelatin (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37\u0026deg;C. After washing with PBST, immunized serum were diluted 1:100 with 3% fish gelatin (PBST) and added into each well in duplicate. Then the plates were incubated at 37\u0026deg;C for 1 h. After incubation, the plates were washed as described above. The detection of antigen-specific IgG antibody in serum of mice or sheep was conducted through adding 1:4000 dilution of horseradish peroxidase (HRP)-coupled goat anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO, USA) or a 1:10,000 dilution of HRP-coupled monoclonal anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO, USA), respectively. After a final wash to remove any nonspecifically bound antibodies, 3,5'-tetramethylbenzidine (TMB, Solarbio, Beijing, China) substrate was added to initiate the color development reaction. The reaction was allowed to proceed for 10 min at room temperature, protected from light, and then stopped by adding 100 \u0026micro;L of 1 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The absorbance at 450 nm (OD\u003csub\u003e450nm\u003c/sub\u003e) was immediately recorded using an ultraviolet spectrophotometer.\u003c/p\u003e \u003cp\u003eFor IgG subtype detection reaction of mice, the purified r-epsilon were coated on high-binding 96-well plates at 5 \u0026micro;g/mL with 100 \u0026micro;L per well overnight at 4\u0026deg;C. After washing according to the above conditions, plates were blocked with 3% fish gelatin (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37\u0026deg;C. After washing with PBST, immunized mice serum were diluted 1:100 with 3% fish gelatin (containing 0.05% Tween) and incubated at 37\u0026deg;C for 1 h. Then the plates were washed as described above. The goat anti-mouse IgG1 or goat anti-mouse IgG2a (Biodragon, Suzhou, China) was added. After washed, 3,5'-tetramethylbenzidine (TMB, Solarbio, Beijing, China) substrate was added to initiate the color development reaction. The reaction was allowed to proceed for 10 min at 37\u0026deg;C, protected from light, and then stopped by adding 100 \u0026micro;L of 1 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The absorbance at 450 nm (OD\u003csub\u003e450nm\u003c/sub\u003e) was immediately recorded using an ultraviolet spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDetection of Neutralizing Antibody Titers\u003c/h2\u003e \u003cp\u003eSerum was prepared from mice and sheep at 21 and 42 days post immunization (dpi) and inactivated at 56\u0026deg;C for 30 min in a water bath. Serial 2-fold diluted sera and 2 MLD/100 \u0026micro;L of the type D natural toxin were mixed in equal proportions in 96-well microtiter plates. Subsequently, 100 \u0026micro;L of well-grown MDCK cells at a concentration of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL were added and incubated for 24 h in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Neutralizing antibodies titers were detected by observing CPE. Serum samples from the PBS group were used as the negative control, and toxin wells served as the positive control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSplenocytes Isolation of Mice\u003c/h2\u003e \u003cp\u003eMice were euthanized at weeks 7 and 15. Then, they were soaked in 75% alcohol for 10 min. The spleen was surgically removed out and washed in PBS containing 1% penicillin, then homogenized through a 40 \u0026micro;m strainer until no visible tissue remained. After centrifugation at 1,500 rpm for 5 min, the supernatant was discarded. Erythrocyte lysis buffer (Solarbio, Beijing, China) was added, and the samples were incubated at room temperature for 10 min to remove red blood cells debris. Next, 5 mL of PBS with 0.5% FBS was added, and the suspension was centrifuged at 1,500 rpm for 5 min. This wash step was repeated once. The resulting cell pellet was resuspended in 1 mL of RPMI 1640 medium (Gibco, Logan, UT, USA) and followed by passing through a 40 \u0026micro;m strainer to obtain scattered splenocytes. A portion of the cells were used for IFN-γ induction analysis, and the other portion of the cells were subjected to T-lymphocyte subpopulation analysis by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDetection of T-lymphocyte Subpopulation by Flow Cytometry in Mice\u003c/h2\u003e \u003cp\u003eMice splenocytes obtained from the isolation procedure above were resuspended to a concentration of 10\u003csup\u003e6\u003c/sup\u003e cells and placed into a 96-well plate. The cells were washed twice with a flow staining buffer and then stained with a panel of antibodies, including DAPI anti-mouse L/D, BV500 violetFluorTM 500 Anti-Mouse CD3, PerCP-Cyanine 5.5 anti-mouse CD4, ApC-Cy7 anti-mouse CD8a, ApC anti-mouse CD44, FITC anti-mouse CD62L (Tonbo Biosciences, USA). The plate was incubated with the antibodies at 4\u0026deg;C for 30 min. After incubation, the cells were centrifuged and the supernatant were removed. Then, 0.7% paraformaldehyde was used to fix the cells. Finally, the cells were washed with a flow staining buffer and were analyzed using a BD LSR Fortessa Flow Cytometer (Becton Dickinson, USA), The data were analyzed by FlowJo 10.8.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDetection of IFN-γ Level in Mice by ELISA\u003c/h2\u003e \u003cp\u003eThe splenocyte cell suspension at weeks 7 were diluted to 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e/mL and transferred to a 96-well plate with 100 \u0026micro;L per well (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well). Then r-epsilon was added into the 96-well plate with final concentration of 5 \u0026micro;g/mL. The plate was incubated at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e for 48 h. After stimulation, cells were centrifuged at 1,500 rpm for 15 min and then supernatant were collected. The supernatant was diluted 2-fold, and detected with Mouse IFN-gamma ELISA Kit (Solarbio, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDetection of IFN-γ Level in Sheep by ELISA\u003c/h2\u003e \u003cp\u003eAnticoagulated blood was collected at 49 dpi. 900 \u0026micro;L of anticoagulated blood was added to a 24-well cell plate, and 100 \u0026micro;L of r-epsilon at a final concentration of 100 \u0026micro;g/mL was added. 100 \u0026micro;L of PBS was added as the negative control. The plates were put in the cell incubator at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e for 72 h. After stimulation, blood was centrifuged at 1,500 rpm for 15 min and supernatant were collected. The supernatant was detected with Sheep IFN-gamma ELISA Kit (Solarbio, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data in this study were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), and a t test or two-way ANOVA was performed using GraphPad Prism 8.0 software(San Diego,CA). Subsequently, multiple comparisons were used to compare between groups. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a significant difference; Ns, no significant differences were observed (*, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe percentages of memory CD8+ T cell subsets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our thanks to the staff of the Central Laboratory at the Institute of Animal Science, Chinese Academy of Agricultural Sciences, for their assistance in preparing and observing TEM samples. And thanks to Lin Jiang and Yabin Pu researchers, the scientific and technological innovation team of Livestock and Poultry Quality Resources Protection and Utilization, for their support in the sheep immune evaluation test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, X.F. and H.Z.; Methodology, Q.X. and Y.Z.; Validation, H.Z. and Y.L.; Formal analysis, H.Z. and Q.X.; Investigation, H.Z. and Y.L.; Resources, J.D.,H.J. and Q.S.; Data curation, H.Z.; Writing-original draft preparation, H.Z. and Q.X.; Writing-review and editing, H.C. and G.Z; Visualization, H.Z. and Q.X.; Supervision, X.F. and S.J.. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the the Ningxia Hui Autonomous Region Key R\u0026amp;D Program (2024BBF02014), and the Agricultural Science and Technology Innovation Program (ASTIP‐IAS15).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data will be shared upon request by the readers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe permissions to conduct these experiments were granted by The Animal Welfare \u0026amp; Ethics Committee of Institute of Animal Science, Chinese Academy of Agricultural Sciences, China (IAS2024\u0026minus;177, IAS2024\u0026minus;84).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSoncini SR, Hartman AH, Gallagher TM, Camper GJ, Jensen RV, Melville SB. 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Microorganisms. 2020; 8(3):436.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"one-health-advances","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [One Health Advances](https://onehealthadv.biomedcentral.com/)","snPcode":"44280","submissionUrl":"https://submission.springernature.com/new-submission/44280/3","title":"One Health Advances","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"C. perfringens, epsilon toxin, ferritin, nanoparticles, immunogenicity","lastPublishedDoi":"10.21203/rs.3.rs-9380767/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9380767/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eClostridium perfringens\u003c/em\u003e (\u003cem\u003eC. perfringens\u003c/em\u003e) is an opportunistic pathogen, widely distributed in the environment. Epsilon toxin secreted by B and D type \u003cem\u003eC. perfringens\u003c/em\u003e can cause enterotoxemia or necrotic enteritis in goats, sheep and cattle. Vaccination is one of the most effective measures to prevent such diseases. However, current toxoid vaccines still exhibit some problems, thus this study aims to engineer \u003cem\u003eC. perfringens\u003c/em\u003e epsilon-ferritin nanoparticles antigen and evaluate its immunogenicity in mice and sheep. The r-epsilon successfully assembled with r-ferritin to form epsilon-ferritin nanoparticles via SDS-PAGE, transmission election microscopy and dynamic light scattering test. In the mouse experiment, epsilon-ferritin nanoparticles could significantly improve specific antibody levels including serum IgG, IgG subtypes antibodies, the ratio of IgG1/IgG2a and neutralizing antibodies. Moreover, epsilon-ferritin nanoparticles elicited more production of IFN-γ cytokines and more activation of memory CD8\u0026thinsp;+\u0026thinsp;T cells in mice. In the sheep experiment, epsilon-ferritin nanoparticles could enhance the level of neutralizing antibodies and more production of IFN-γ cytokines. The present study revealed that epsilon-ferritin nanoparticles remarkably enhanced the immunogenicity of r-epsilon in mice and sheep, providing a new strategy for the development of \u003cem\u003eC. perfringens\u003c/em\u003e vaccines.\u003c/p\u003e","manuscriptTitle":"Clostridium perfringens epsilon-ferritin nanoparticles induced robust immune responses in mice and sheep","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-12 16:32:19","doi":"10.21203/rs.3.rs-9380767/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-04T23:14:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-15T14:06:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T14:05:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"One Health Advances","date":"2026-04-10T14:32:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"one-health-advances","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [One Health Advances](https://onehealthadv.biomedcentral.com/)","snPcode":"44280","submissionUrl":"https://submission.springernature.com/new-submission/44280/3","title":"One Health Advances","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b07005a4-7fda-4cab-be46-46802779a3ae","owner":[],"postedDate":"May 12th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"42","date":"2026-05-04T23:14:19+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T16:32:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-12 16:32:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9380767","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9380767","identity":"rs-9380767","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}