Evaluation of vaccine efficacy with 2B/T epitope conjugated porcine IgG-Fc recombinants against foot-and-mouth disease virus

Abstract

The inactivated vaccine is effective in controlling foot-and-mouth disease (FMD), but it has drawbacks such as the need for a biosafety level 3 laboratory facility to handle live foot-and-mouth disease virus (FMDV), high production costs, and biological safety risks. In response to these challenges, we developed a new recombinant protein vaccine (2BT-pIgG-Fc) containing porcine IgG-Fc to enhance protein stability in the body. This vaccine incorporates two-repeat B-cell and one-single T-cell epitope derived from O/Jincheon/SKR/2014. Our study confirmed that 2BT-pIgG-Fc and a commercial FMDV vaccine induced FMDV-specific antibodies in guinea pigs at 28 days post-vaccination. The percentage inhibition (PI) value of 2BT-pIgG-Fc was 90.43%, and the commercial FMDV vaccine was 81.75%. The PI value of 2BT-pIgG-Fc was 8.68% higher than that of commercial FMDV vaccine. In pigs, the primary target animals for FMDV, all five individuals produced FMDV-specific antibodies 42 days after vaccination with 2BT-pIgG-Fc. Furthermore, serum from 2BT-pIgG-Fc-vaccinated pigs exhibited neutralizing ability against FMDV infection. Intriguingly, the 2BT-pIgG-Fc recombinant demonstrated FMDV-specific antibody production rates and neutralization efficiency similar to commercial inactivated vaccines. This study illustrates the potential to enhance vaccine efficacy by strategically combining well-known antigenic domains in the development of recombinant protein-based vaccines.

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease that primarily affects cloven-hoofed animals. More than 70 wildlife species, including domesticated ruminants such as cattle, cows, pigs, sheep, goats, camels, and buffalos, are susceptible to this disease. FMD is characterized by fever, lameness, inappetence, and the development of vesicular lesions in various areas, including the tongue, mouth, snout, nipple, hoof, and other hairless skin regions. During the acute phase, animals experience severe discomfort, leading to significant decreases in growth, milk yield, and mobility, thereby impacting overall productivity [1, 11, 51]. The causative agent of FMD is the foot-and-mouth disease virus (FMDV), a member of the Picornaviridae family, genus Aphthovirus, with an approximately 8.4 kb nonenveloped single-stranded positive-sense RNA genome. This virus encodes a single open reading frame (ORF) flanked by a long 5′-untranslated region (5′-UTR) and a short 3′-UTR. The ORF is translated into a polypeptide chain and processed into four structural proteins (SP; VP1, VP2, VP3, VP4) and 10 non-structural proteins (NSP; L, 2A, 2B, 2C, 3A, 3B_1–3, 3C, 3D). FMDV comprises seven immunologically distinct serotypes: O, A, C, Asia 1, Southern African Territories (SAT) 1, SAT 2, and SAT 3, with various subtypes due to the virus’s high mutation rate [23, 26, 54]. Serotype O is the most prevalent globally, including in Korea [1].

Vaccination is the best way to prevent and control FMD [49]. In Korea, commercially used FMDV vaccines are inactivated vaccines that originate from inactivated sources, exhibiting robust immunogenicity and providing protection [29]. However, these vaccines are associated with drawbacks such as high production costs, the requirement for biosafety level 3 facilities in their production, and potential biological safety risks during vaccine manufacture [11, 51]. These challenges underscore the need for alternative vaccine development. Ongoing vaccine development efforts, including recombinant protein, DNA, virus-like particle (VLP), and subunit vaccines, are gaining attention due to their absence of infectious ingredients [2, 9, 13, 40, 43, 44, 47, 50, 52, 56, 57]. Escherichia coli (E. coli)-derived recombinant protein vaccines, in particular, offer advantages such as low manufacturing costs, high production yields, ease of mass production, and short production periods [7, 22, 31].

Previous studies identified the amino acid residues 135–160 of VP1, located on the viral surface, as the dominant B-cell antigenic epitope triggering the production of neutralizing antibodies [4, 6, 16, 19, 21, 28]. However, vaccines composed solely of B-cell epitopes have limited efficacy in inducing neutralizing antibodies and protection against FMD. To address this, T-cell epitopes that stimulate cellular immunity, present in 3A (21–35 a.a.) and 3D (341–370 a.a.) of NSP, are incorporated when designing recombinant protein vaccine constructs [5, 15, 24, 35, 60]. Guinea pig studies revealed that a B-cell epitope peptide vaccine containing 3D residue elicited a higher IgG titer and neutralizing antibodies compared to one containing 3A residue. FMDV-specific neutralizing antibodies were also produced more effectively in the former than in an inactivated vaccine. In FMDV challenges, the 3D residue-containing recombinant peptide vaccine and inactivated vaccine provided complete protection, while the 3A residue-containing recombinant peptide vaccine showed 66% protection in guinea pigs. In cattle, a recombinant peptide vaccine consisting of VP1 and 3D yielded higher neutralizing antibody titers and protection rates than a recombinant peptide vaccine containing 3A residue. However, the protection rate of 3D residue-containing recombinant peptide vaccines was lower than that of inactivated vaccines, with only 3 out of 5 animals avoiding FMDV infection. Other studies suggested that incorporating the immunoglobulin G (IgG) Fc region could enhance the immunogenicity of antigenic epitopes as a vaccine carrier. Utilizing the porcine IgG-Fc region as a vaccine carrier stimulates B-cell and cell-mediated T cells, thereby increasing antibody production [32, 36, 48, 58].

This study constructed and expressed a recombinant FMDV protein vaccine include the incorporation of T cell epitopes from 3D protein rather than 3A, along with two B cell epitopes, and conjugation with porcine IgG. FMDV-specific antibodies were compared between the recombinant FMDV vaccine and commercial inactivated vaccine in guinea pigs and pigs. Based on the results, we speculate that this recombinant protein vaccine platform can serve as an alternative to commercialized FMDV vaccines.

MATERIALS AND METHODS

Virus

FMDV/O/Boeun/SKR/2017 (KVCC-VR1700004) was provided by the Animal and Plant Quarantine Agency (APQA) and propagated in fetal porcine kidney (LFBK, supplied by APQA) cells with DMEM supplemented with 2% FBS at 37°C for 2 days. Virus titer was determined by the 50% tissue culture infectious dose (TCID₅₀) method, and viral stocks were stored at −80°C. All FMDV experiments were performed at a biosafety level 3 laboratory (KCDC-15-3-02) using personal protection equipment following the biosafety manual instructions issued by the Korea Zoonosis Research Institute of Jeonbuk National University (JBNU 2022-01-00).

Construction of plasmids

Based on the VP1 and 3D sequences of the FMDV type O Jincheon/SKR/2014 isolate (GenBank accession number KX162590.1), two immunogens corresponding to amino acid (a.a.) 135–178 of VP1 and 341–370 of 3D of FMDV type O were selected for the antigenic epitopes and named B and T, respectively. The two-repeat B cell epitopes were linked to the N-terminus of the porcine IgG-Fc region, and the T-cell epitope was linked to the C-terminus of the porcine IgG-Fc region. A hinge region was added between the two-repeat B epitopes and before the porcine IgG-Fc region to enhance structural flexibility. A linker sequence (GGGGSGGGGSGGGGS) was also included between porcine IgG-Fc and the T epitope to prevent antigenic interference. The 2BT-pIgG-Fc was synthesized by GenScript (GenScript Biotech, Piscataway, NJ, USA). To compare vaccine efficacy according to the number of B cell epitopes, BT-pIgG-Fc was synthesized by GenScript, consisting of one single B epitope, one T epitope, and porcine IgG-Fc region. The control gene (pIgG-Fc), consisting of the hinge, porcine IgG-Fc, and linker sequence, was also synthesized GenScript. Both synthesized genes were inserted into the pET30a (+) vector (Sigma-Aldrich, St. Louis, MO, USA) for protein expression in E. coli.

Expression of recombinant FMDV protein in E. coli

The expression plasmids, pET30a-2BT-pIgG-Fc, pET30a-BT-pIgG-Fc, and pET30a-pIgG-Fc, were separately introduced into E. coli BL21 (DE3) competent cells (Thermo Fisher Scientific, Waltham, MA, USA). These transformed bacteria were cultured in a shaking incubator in 500 mL of Luria-Bertani broth medium containing 50 μg/mL kanamycin at 37°C overnight. The cells were incubated until the optical density (OD₆₀₀) reached 0.5–0.6. Subsequently, isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma-Aldrich) was added at a final concentration of 300 μM, and the culture was incubated at 20°C for 16 hr. Cells were harvested by centrifugation at 7,000 × g for 10 min at 4°C, lysed with bugbuster master mix (Sigma-Aldrich). Cell lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant proteins were determined by Coomassie brilliant blue staining and western blotting using anti-6xHis tag antibody (Bethyl, Montgomery, TX, USA) and anti-FMDV antibody (Lab-made anti-rabbit polyclonal).

Fractionation of recombinant FMDV protein in E. coli

The IPTG-treated E. coli were suspended with the bugbuster master mix, and the lysed by using sonicator (KBT, KUS-650, South Korea) with 10 cycles of 30 sec on/30 sec off, amplitude 60% on ice. After centrifugation at 15,000 × g for 30 min at 4°C, the target proteins were purified using NI-nitrilotriacetic acid (NTA) resin (Thermo Fisher Scientific) following the manufacturer’s manual. Briefly, protein samples were mixed with NI-NTA resin and rotated at 4°C for 16 hr to bind the resin to the protein. The protein-resin mixture was loaded into a column. Subsequently, protein-resin was washed with 10 resin-bed volumes of wash buffer containing phosphate-buffered saline (PBS) and 50 μM imidazole (Sigma-Aldrich), and then target proteins were eluted with 2 resin-bed volumes of elution buffer containing PBS and 250 μM imidazole. The target proteins were eluted 20 times during the protein elution step and each purified recombinant protein was labeled from fraction 1 to fraction 20. The purified proteins were concentrated and dialyzed with PBS using an Amicon Ultra-15 centrifugal filter unit (10 kDa molecular weight cutoff, Sigma-Aldrich). The concentration of the purified protein was determined using a Micro BCA protein assay kit (Thermo Fisher Scientific). Absorbance was measured at 562 nm on a GloMax Discovers System (Promega Corp., Madison, WI, USA) to determine the protein concentration.

Vaccination and sampling

Recombinant proteins were diluted in PBS and emulsified with oil-adjuvant Montanide ISA 201 (Seppic, Quai d’Orsay, Paris, France) in a 50:50 (w/w) ratio for animal vaccination. All animal experiment procedures were approved by the Institutional Animal Care and Use Committee (IACUC, JBNU 2020-0152) of Jeonbuk National University. The commercial vaccine utilized was PRO-VAC FMD (Swine 313) by Komipharm lnc. Twelve specific pathogen-free grade guinea pigs (250–300 g) were randomly assigned to four groups (n=3; PBS, pIgG-Fc, 2BT-pIgG-Fc, and commercial inactivated vaccine). Guinea pigs were immunized by intramuscular injection of 1 mL/1 dose of vaccine candidates (200 μg) or commercial FMDV vaccine or PBS into the left hind limb. The second vaccination was performed at the same dose 14 days after the first vaccination. Serum samples were collected from each animal on 28 days post-vaccination (dpv) and analyzed to confirm FMDV-specific antibody production. Twenty-five white, cross-bred, Landrace female pigs (8 weeks old) were assigned to five groups with five animals each. The pigs were immunized by intramuscular injection in the hind neck with 2 mL/dose of vaccine candidates (1 mg) or commercial FMDV vaccine. At 28 dpv, the second vaccination was performed at the same dose of the antigen mixture. Serum samples were collected from pigs at 0, 28, and 42 dpv for verification of FMDV-specific antibody production.

Evaluation of antibody productivity after vaccination of 2BT-pIgG-Fc

FMDV-specific antibodies were detected using a PrioCHECK™ FMDV Type O Antibody ELISA Kit (Thermo Fisher Scientific) [34]. All procedures were done following the manufacturer’s instructions. In brief, 90 μL of ELISA buffer was dispensed into all wells of a pre-coated 96-well plate with inactivated FMDV type O. Then, 10 μL of serum samples or reference sera was added to each well of a 96-well plate and incubated for 1 hr at 25°C. The wells were washed 6 times with 200 μL of wash buffer included in the ELISA kit. Subsequently, 100 μL of the diluted conjugate was dispensed into all wells of a 96-well plate and incubated for 1 hr at room temperature. The wells were washed six times with 200 μL of wash buffer. Next, 100 μL of chromogen (TMB) substrate was dispensed into the 96-well plate and incubated for 15 min at room temperature. Sequentially, 100 μL of the stop solution was added to each well of a 96-well plate, and absorbance at OD₄₅₀ was measured for the 96-well plate using GloMax Discover Microplate. Percentage inhibition (PI) was calculated according to the formula provided by the manufacturer.

$\mathrm{PI}=100-\frac{\left({\mathrm{OD}}_{450}\mathrm{sample}-{\mathrm{OD}}_{450}\mathrm{blank}\right)}{\left({\mathrm{OD}}_{450}\max -{\mathrm{OD}}_{450}\mathrm{blank}\right)}\times 100$

PI values above 50 indicate the presence of FMDV-specific antibodies in the test serum, and values below 50 were considered to indicate their absence.

Determination of virus neutralizing antibody titer

Virus neutralization test was conducted following the World Organization for Animal Health (WOAH) manual [55]. Sera were inactivated at 56°C for 30 min. Sera were serially twofold diluted with serum-free DMEM in 96-well plates and incubated with 100 TCID₅₀ of FMDV/O/Boeun/SKR/2017 at 37°C for 60 min. After incubation, sera-virus mixture was adsorbed to LFBK cells for 60 min and the titer of the serum-neutralizing antibodies were calculated after culturing for 2–3 days. The titers of the serum neutralizing antibodies were calculated as the highest dilutions that neutralized 100 TCID₅₀ of FMDV in 50% of the wells, calculated using the Reed–Muench method [45].

Statistical analysis

Statistical analyses were performed by one-way ANOVA according to the Dunnett’s multiple comparison tests using GraphPad Prism 8.0.1 (GraphPad, San Diego, CA, USA). Statistical significance is indicated with asterisks. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

RESULTS

Design of recombinant FMDV protein vaccine

The B-cell epitope of VP1 and T-cell epitope of 3D of FMDV genome sequences were obtained from NCBI Reference Sequence KX162590.1, and porcine IgG-Fc region sequences were obtained from other studies [8, 30]. 2BT-pIgG-Fc consists of a two-repeat B-cell and one-single T-cell epitope and porcine IgG-Fc (Fig. 1A). And, BT-pIgG-Fc is composed of one B cell and T cell epitope and a porcine IgG-Fc region (Supplementary Fig. 1A). We also designed a control recombinant protein comprising the porcine IgG-Fc region without the B-cell and T-cell epitope of FMDV (Fig. 1B). B-cell and T-cell epitopes were selected based on FMDV serotype O/Jincheon/SKR/2014.

Fig. 1.

Design and bacterial expression of foot-and-mouth disease virus (FMDV) recombinant protein. (A) Schematic diagram of 2BT-pIgG-Fc consisting two B epitopes (135–178 a.a., VP1), two hinges, one porcine IgG, one linker, and one T epitope (341-370 a.a., 3D). (B) Schematic diagram of pIgG-Fc consisting one hinge, one porcine IgG, and one linker. (C) To express the recombinant proteins, E. coli was cultured with 300 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 20°C for 16 hr. The recombinant proteins were determined by Coomassie brilliant blue staining. Expression of the recombinant protein was detected by western blot using 6xHis tag antibody (D) and anti-FMDV polyclonal antibody (E).

Expression, purification and quantification of recombinant FMDV protein

The pET30a-2BT-pIgG-Fc, pET30a-BT-pIgG-Fc, and pET30a-pIgG-Fc plasmids were transformed into E. coli BL21 (DE3) competent cells. The transformed bacteria were induced for recombinant protein expression by treating IPTG. The optimal protein expression and purification conditions were 300 μM IPTG at 25°C for 16 hr (data not shown). Coomassie brilliant blue staining on SDS-PAGE revealed that 2BT-pIgG-Fc, BT-pIgG-Fc, and pIgG-Fc proteins were expressed with molecular weights of 59, 40, and 32 kDa, respectively (Fig. 1C, Supplementary Fig. 1B). The expression of the three recombinant proteins was also confirmed by western blotting using anti-6x-His-tag and anti-FMDV antibodies. The 2BT-pIgG-Fc, BT-pIgG-Fc, and pIgG-Fc proteins were detected by the anti-6x-His antibody (Fig. 1D, Supplementary Fig. 1C), while the pIgG-Fc protein was not detected using the FMDV antibody (Fig. 1E). As the pIgG-Fc protein lacked the FMDV epitopes, only the 2BT-pIgG-Fc, BT-pIgG-Fc proteins have FMDV epitopes. Next, the three recombinant proteins were purified by affinity-based chromatography with Ni-NTA resin. Confirming the purification yield after fractionation, 2BT-pIgG-Fc and pIgG-Fc showed high purity from the 5th fraction, and BT-pIgG-Fc from the 3rd fraction (Fig. 2A, 2B, and Supplementary Fig. 1E). 2BT-pIgG-Fc and pIgG-Fc were pooled from the 5th to the 20th fraction, and BT-pIgG-Fc was pooled from the 3rd to the 20th fraction. The concentration of the purified recombinants was calculated using the BCA protein assay. The results showed that the concentrations of 2BT-pIgG-Fc, BT-pIgG-Fc, and pIgG-Fc proteins were 2 mg/mL, 1.8 mg/mL, and 15 mg/mL, respectively (Supplementary Fig. 2).

Fig. 2.

Purification of recombinant proteins using affinity chromatography. The whole-cell lysates that isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced recombinant protein were purified using NI-nitrilotriacetic acid (NTA) resins. The protein-resin was eluted 20 times during the elution step of purification. Each purified recombinant protein was labeled from fraction 1 to fraction 20. Purified 2BT-pIgG-Fc (A) and pIgG-Fc (B) were confirmed by Coomassie brilliant blue staining.

Vaccination of recombinant FMDV vaccine in guinea pigs

To determine the immunogenic effect of the recombinant proteins, each protein was immunized in guinea pigs twice at a 14-day interval. Commercial FMDV vaccine and PBS were used as positive and negative controls. Sera were collected from guinea pigs 28 days after the primary vaccination. The PI value of 2BT-pIgG-Fc was as high as 90.43 (± 9.38), while that of commercial FMDV vaccine was 81.75 (± 1.28). In contrast, the PI value of pIgG-Fc was only 2.96 (± 2.80), while that of PBS, a negative control, was 0.45 (± 0.52) (Fig. 3). The PI value of 2BT-pIgG-Fc was 8.68% higher than that of the commercial FMDV vaccine. However, there was no difference in significance of PI value between 2BT-pIgG-Fc and commercial FMDV vaccine. pIgG-Fc and PBS, which does not contain the FMDV immunogenic epitope, failed to induce FMDV-specific antibodies in guinea pigs. These results indicated that recombinant 2BT-pIgG-Fc induced FMDV-specific antibodies equivalently to the inactivated FMDV vaccine in guinea pigs.

Fig. 3.

Evaluation of foot-and-mouth disease virus (FMDV)-specific antibody production by recombinant protein vaccine in guinea pigs. The percentage of inhibition (PI) against FMDV type O was analyzed by PrioCheck FMDV ELISA Kit in serum samples collected at day 28 after vaccination. The dotted horizontal line represents the cut-off value for the absence or presence of FMDV type O antibody. Data represent the mean ± SD. Statistical significance was analyzed by one-way ANOVA according to the Dunnett’s multiple comparison tests using GraphPad Prism version 8.0.1 (n=3, ****P<0.0001).

Vaccination of recombinant FMDV vaccine in pigs

To confirm the vaccine efficacy of recombinant 2BT-pIgG-Fc protein in pigs, the main target animals of FMDV vaccines, and to verify the difference in immunogenicity according to the number of B epitopes, a recombinant protein (BT-pIgG-Fc) consisting of a single-B epitope and T epitope was designed and expressed (Supplementary Fig 1). Five immunization groups, 2BT-pIgG-Fc, BT-pIgG-Fc, pIgG-Fc, inactivated commercial vaccine, and PBS were injected into five pigs each at day 0 and day 28. Pig sera were collected at 14, 28, and 42 dpv, and the antibody production efficacy was evaluated using ELISA. As shown in Fig. 4, the commercial FMDV vaccine produced FMDV-specific antibodies in 3 of 5 animals at 14 dpv and in all 5 animals at 28 dpv after the first vaccination. The 2BT-pIgG-Fc steadily increased with vaccination, but no individuals had FMDV-specific antibodies until 28 dpv. However, after the second vaccination, all five pigs immunized with 2BT-pIgG-Fc induced FMDV-specific antibodies. The pigs immunized with BT-pIgG-Fc induced FMDV-specific antibodies in 2 of 5 animals at 42 dpv. The pIgG-Fc and PBS did not induce FMDV antibodies. These results confirm that 2BT-pIgG-Fc also strongly produces FMDV-specific antibodies in pigs. Furthermore, we found that the two-repeat B-cell epitope recombination enhances the vaccine efficacy against FMDV.

Fig. 4.

Evaluation of foot-and-mouth disease virus (FMDV)-specific antibody production in pigs using ELISA. Sera were collected at a 14-day interval. FMDV-specific antibody was analyzed by PrioCheck FMDV ELISA Kit. The dotted horizontal line represents the cut-off value for the absence or presence of FMDV type O antibody. The final percentage of inhibition (PI) values of 2BT-pIgG-Fc, commercial FMDV vaccine, BT-pIgG-Fc, pIgG-Fc, and phosphate-buffered saline (PBS) group were 62.44 (± 9.64), 79.49 (± 3.31), 41.80 (± 29.86), 5.42 (± 3.42), and 2.74 (± 2.43), respectively. Data represent the mean ± SD. Statistical significance was analyzed by one-way ANOVA according to the Dunnett’s multiple comparison tests using GraphPad Prism version 8.0.1 (n=5, ****P<0.0001).

Neutralization efficacy of recombinant FMDV vaccine

To determine whether the antibodies induced by the recombinant FMDV vaccine have neutralization activity against FMDV infection, we mixed infectious live FMDV with pig sera collected at 42 dpv. The virus-serum mixture was then transferred into cells. As shown in Fig. 5, 42 days post-vaccination, the neutralizing antibody titers of 2BT-pIgG-Fc and commercial vaccines were increased by 4.1-fold and 4.7-fold higher than PBS, respectively. Furthermore, vaccination with 2BT-pIgG-Fc induced a higher neutralization antibody level than BT-pIgG-Fc. The neutralizing antibody titers of PBS and pIgG-Fc showed no significant difference from before vaccination. From this result, we found that the combination of the two-repeat B epitope can increase neutralizing antibody productivity. Additionally, we have shown that 2BT-pIgG-Fc can produce neutralizing antibodies similar to inactivated vaccines after a second vaccination.

Fig. 5.

Verification of the effectiveness of recombinant protein vaccines through foot-and-mouth disease virus (FMDV) neutralization test. The sera were collected at 42 days after vaccination. The neutralizing antibody titers were calculated as the reciprocal of the last serum dilution to neutralize 100 50% tissue culture infectious dose (TCID₅₀) of FMDV in 50% of the wells and expressed as a relative fold change compared to phosphate-buffered saline (PBS)-injected pigs. The dotted horizontal line represents the possible protection for FMDV according to the World Organization for Animal Health (WOAH) terrestrial manual (55).

DISCUSSION

FMDV, especially serotype O, is prevalent in many countries, including Korea. Upon a farm’s FMDV infection, the only recourse is the culling of all cloven-hoofed animals to halt transmission, leading to substantial economic losses for livestock farmers. Vaccination stands as a viable approach to FMD control. Available FMDV vaccines primarily rely on inactivated virus preparations, commercially accessible for their high immunogenicity and protection rate. Despite drawbacks like the need for high virus titers, elevated production costs, and safety concerns, inactivated vaccines remain the preferred choice. While various researchers explore alternative vaccine types (recombinant protein, antigenic protein expressing DNA, viral vectors, live attenuated viruses, VLPs) to overcome inactivated vaccine limitations, the latter remains the favored option due to its robust immunogenicity and protection efficacy. Methods enhancing immunogenicity and protection efficacy, such as inducing cellular immunity, altering adjuvants, changing epitopes inducing immunity, or modifying vaccine composition, are under investigation [17, 18, 20, 27, 33, 37,38,39, 46, 53, 57, 59]. Developing a recombinant protein vaccine necessitates establishing a major virus immunogen [3, 10, 12, 14, 25, 41, 42]. This study produces a recombinant protein vaccine comprising three major subunits: B and T-cell epitopes from FMDV and the porcine IgG-Fc region. The B epitope used in this paper is 135−178 a.a. of VP1, which has 18 additional amino acids compared to other papers. Because the diversity of the antibody was expected by increasing the antigenic determinant by adding 18 amino acids, VP1 of 135–178 a.a. was used. We constructed and expressed a recombinant protein, including a two-repeat B-cell epitope, one-single T-cell epitope, and pIgG-Fc, to investigate the association between increased immune epitopes and immunogenicity. Based on the gene sequence, the size of the recombinant protein was predicted the 2BT-pIgG-Fc was 59 kDa, BT-pIgG-Fc was 40 kDa, and pIgG-Fc was 32 kDa. As a result of expressing the recombinant protein in E. coli and performing Coomassie brilliant blue staining and western blot on an SDS-PAGE, BT-pIgG-Fc was confirmed to be a protein similar to the predicted size. However, 2BT-pIgG-Fc and pIgG-Fc were confirmed to be larger proteins than predicted, with sizes of approximately 63 kDa and 37 kDa. To find the cause, the entire sequence of the cloned plasmid was analyzed, but all genes were identical to the existing information. Additionally, in Fig. 1D and 1E, we validated specificity by using antibodies for detecting His-tagged domain and FMDV antigenic domain simultaneously. Administering the 2BT-pIgG-Fc protein to guinea pigs twice at 2-week intervals resulted in an antibody production rate equivalent to that of the commercial vaccine. Subsequently, we constructed BT-pIgG-Fc, expressing a single-B cell epitope, to assess the immunogenicity of a two-repeat B-cell epitope. Despite delayed antibody production, 2BT-pIgG-Fc produced FMDV-specific antibodies at levels similar to the commercial inactivated vaccine in both guinea pigs and pigs. The production of neutralizing antibodies correlated with the number of B-cell epitopes. BT-pIgG-Fc showed no significant change in FMDV-specific antibodies until 28 dpv, producing antibodies in 2 out of 5 pigs after the second vaccination. In contrast, 2BT-pIgG-Fc induced FMDV-specific antibodies in all 5 pigs at 42 dpv, with a PI value of 62.44% compared to BT-pIgG-Fc’s 41.80%. Additionally, the neutralizing antibody titers of 2BT-pIgG-Fc after the second vaccination were significantly increased and comparable to the commercial vaccine, highlighting the essential role of the second vaccination in inducing strong immunogenicity. The mechanism underlying the potent immunogenicity induced by two B-cell epitopes remains unclear. We believed that having two B-cell epitopes may increase the antigenic determinants structurally. Based on these findings, the next studies aim to observe changes in the vaccine efficacy after increasing the number of B cell epitopes to three or more, and the analyze the structural change in the protein according to the number of B cell epitopes. Furthermore, we needed to confirm the function of pIgG-Fc in the 2BT-pIgG-Fc recombinant vaccine and its applicability to a wide range of genetic properties in type O FMDVs; these results underscore the involvement of the number of B-cell epitopes in immunogenicity.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest