Abstract
Vaccination prevents and controls foot-and-mouth disease (FMD). However, the current FMD vaccine remains disadvantageous since it cannot overcome maternally-derived antibody (MDA) interference in weeks-old animals, which suppress active immunity via vaccination. To address this, we developed the immune-enhancing O PA2-C3d and A22-C3d FMD vaccine strains that can stimulate receptors on the surface of B cells by inserting C3d (a B cell epitope) into the VP1 region of O PA2 (FMDV type O) and A22 (FMDV type A). We purified inactivated viral antigens from these vaccine strains and evaluated their immunogenicity and host defense against FMDV infection in mice. We also verified its efficacy in inducing an adaptive immune response and overcome MDA interference in MDA-positive (MDA(+), FMD-seropositive) and -negative (MDA(−), FMD-seronegative) pigs. These results suggest a key strategy for establishing novel FMD vaccine platform to overcome MDA interference and induce a robust adaptive immune response.
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Introduction
Foot-and-mouth disease (FMD), an acute infectious disease in cloven-hooved animals, especially pigs and cattle, causes significant economic loss to the livestock industry as it rapidly spreads, thereby causing high mortality in young individuals and reducing productivity1,2. The current commercial FMD vaccine requires periodic and repeated vaccination in both cattle and pigs. Following vaccination, the maternally-derived antibodies (MDA) are transferred to the offspring through the placenta or ingestion of colostrum to form passive immunity. Upon initial infection with the FMD virus (FMDV), the MDA have a short-term protective effect in calves and piglets. Early vaccination of an FMD vaccine in young-week-old animals causes interference via passive immunity by inhibiting antigen-specific antibody production in plasma cells and memory B cells, resulting in immunological tolerance, which reduces the efficacy of the vaccine and inhibits the formation of active immunity3. Therefore, the current FMD vaccination program in Korea recommends that calves and piglets be vaccinated 2–3 months after birth, when the MDA levels decrease. Since the level, titer, and half-life of MDA vary between individuals, it is difficult to determine the appropriate timing for FMD vaccination in practice. Moreover, the commercially available FMD vaccine cannot overcome the interference by MDA.
Various studies have reported the relationship between MDA interference and reduced efficacy of FMD vaccines4,5,6, and the optimal timing for vaccination in young animals7,8. However, few studies have suggested strategies for inducing a strong immune response by effectively overcoming MDA. Vaccines are also being developed against other viruses, such as NDV9,10, AIV11, PRRSV12, PCV-213, IAV12, and CSFV The recombinant plasmid was prepared as follows17,65. The whole FMD-O1 Manisa virus genome (GenBank Accession No. AY593823.1) was PCR-amplified and inserted into the pBluescript SK II (Agilent, Santa Clara, CA, USA) plasmid to produce the pO-Manisa plasmid. In the pO-Manisa plasmid, the gene encoding the structural protein was substituted with the gene encoding structural proteins from O-serotype FMDV O PA2 (GenBank Accession No. AY593829.1) or A-serotype FMDV A22/Iraq/24/64 (GenBank Accession No. AY593764.1) to prepare two types of plasmids: pOm-O PA2-P1 or pOm-A22-P1. For type O, O PA2 was determined as the strongest candidate vaccine strain. This was based on the results of a previous study which confirmed the vaccine matching rate in floating cells—especially the antigen-mediated immunogenicity in experimental animals (mice) and target animals (pigs). For type A, the vaccine matching rate tends to be low in terms of global incidence; however, A22 was suitable. The C3d (B cell epitope) sequence (5′-GGTAAGCAGCTCTACAACGTGGAGGCCACATCCTATGCC-3′, corresponding to the amino acid sequence, GKQLYNVEATSYA) was inserted in the VP1 protein coding sequence between the 456th and 457th base pairs (amino acid positions, 152 and 153) in PA2-C3d, and between the 453rd and 454th base pairs (amino acid positions, 151 and 152) in A22-C3d. Next, 300 ng/μL of pOm-A22-P1 (PCR template), 1 μL (10 pmol/μL) of the C3d F primer (5′-GGAGGCCACATCCTATGCCCGCGAGAGGCCCTAGGTCGC-3′), and 1 μL (10 pmol/μL) of the C3d R primer (5′-ACGTTGTAGAGCTGCTTACCGCGAGGGTCGCCGCTCAGCT-3′) were used to prepare the target plasmid using the same self-ligating method used in the previous study17,65. Figure 1a and b illustrates the schematic of the final plasmid for O PA2-C3d and A22-C3d, respectively. The PCR conditions were as follows: 10 μL of the 5X Phusion HF buffer (Thermo Scientific, Waltham, MA, USA), 1 μL of 10 mM dNTP (Invitrogen, Carlsbad, CA, USA), 1 μL of 2 U/μL Phusion DNA polymerase (Thermo Scientific), and 35 μL of sterile distilled water were subjected to 98 °C (30 s), followed by PCR amplification for 25 cycles at 98 °C (10 s), 65 °C (20 s), and 72 °C (2 min and 30 s), followed by a final cycle at 72 °C (10 min). Next, 1 μL of DpnІ (Enzynomics, Daejeon, Korea) was added to the 25 μL of PCR product and allowed to incubate at 37 °C for 1 h. Next, 35 μL of sterile distilled water, 5 μL of Ligation High (TOYOBO, Osaka, Japan), and 1 μL of 5 U/μL T4 polynucelotide kinase (TOYOBO) were added to 4 μL of the DpnI-treated product. The mixture was ligated in a 16 °C water bath for 1 h, following which the plasmid was transformed into 100 μL of DH5α cells (Yeastern Biotech, Taipei, Taiwan) according to the manufacturer’s protocol. The transformed cells were smeared onto an agar plate containing ampicillin and incubated overnight at 37 °C. A colony was picked from the plate with a pipette tip and mixed with 18 μL of sterile distilled water, 1 μL (10 pmol/μL) of a universal forward primer for VP1 (5′-AGNGCNGGNAARTTTGA-3′), and 1 μL (10 pmol/μL) of a universal reverse primer for VP1 (5′-CATGTCNTCCATCTGGTT-3′) in a colony PCR tube. This mixture was subjected to 94 °C (5 min), followed by PCR amplification for 25 cycles at 94 °C (30 s), 55 °C (30 s), and 72 °C (1 min), followed by a final cycle at 72 °C (5 min). In the aforementioned universal primer, N can represent any nucleotide. Next, 5 μL of the PCR sample was mixed with 1 μL of 6X loading buffer (DYNE BIO, Gyeonggi, Korea) before being loaded onto an agarose gel alongside 5 μL of 100 bp marker (DYNE BIO). After electrophoresis at 100 V (30 min), the bands were assessed on a Gel Doc (Bio-Rad, Hercules, CA, USA) system. Next, 5 μL of PCR product was mixed with 2 μL of ExoSAP (Thermo Scientific) and PCR amplified at 37 °C (15 min) and 85 °C (15 min). The insertion of the epitopes into the VP1 sequence was confirmed via full DNA sequencing. Next, the colony was placed in 200 mL of LB media containing ampicillin and incubated overnight at 37 °C with shaking. The midi-prep method (Macherey-Nagel, Duren, Germany) was used to prepare the plasmid17. The recombinant FMD virus was recovered by transfecting BHKT7-9 (a cell line that expresses T7 RNA polymerase) with the recombinant plasmid prepared above using the Lipofectamine 3000 reagent (Invitrogen), followed by incubation for 2–3 days. The prepared virus was passaged in fetal goat tongue (ZZ-R) cells or baby hamster kidney-21 (BHK-21) cells for viral proliferation17. The purified antigen (inactivated virus) was prepared in BHK-21 cells infected with the recombinant immunostimulatory FMDV O PA2-C3d and A22-C3d constructed for the swift phenotype of VP1 (referred sequence) by reverse genetics according to the method described by Lee et al., with modifications17. For viral infection, the culture medium was replaced with serum-free Dulbecco’s modified Eagle’s medium (HyClone, Logan, UT, USA), and the cells were inoculated with the virus by incubating for 1 h at 37 °C in a 5% CO2 atmosphere. The extracellular viruses were then removed. Twenty-four hours post-infection, the viruses were inactivated by two treatments of 0.003 N binary ethylenimine for 24 h in a shaking incubator, followed by concentration with polyethylene glycol (PEG) 6000 (Sigma-Aldrich, St. Louis, MO, USA)66. The virus concentrate was layered on 15–45% sucrose density gradients and centrifuged. After ultracentrifugation, the bottom of the centrifuge tube was punctured and 1 mL fractions were collected. The presence of FMDV particles was confirmed in a sample of each fraction by performing optical density measurements using a lateral flow device (BioSign FMDV Ag; Princeton BioMeditech, Princeton, NJ, USA). Prior to use in field experiments, the pre-PEG treated supernatant was passage through ZZ-R and BHK-21 cells at least twice to ensure that no cytopathic effects (CPE) occurred, thereby confirming the absence of any live virus in the supernatant. The SPs of purified antigen expression were confirmed in cells infected with immunopotent recombinant FMDV O PA2-C3d, A22-C3d, O PA2 and A22 using rapid antigen kits (PBM kit, PBM Co Ltd., Princeton, NJ, USA). The results showed band formation for the SPs and no band formation for the NSPs of FMDV. The virus particle (146 S) was characterized by TEM imaging17. The animal protocol was conducted according to the method described by Lee et al. and Jo et al.17,67. Age- and sex-matched wild-type C57BL/6 mice (females, 6–7 weeks old) were purchased from KOSA BIO Inc. (Gyeonggi, Korea). All mice were housed in microisolator cages with ad libitum access to food and water in a specific pathogen-free biosafety level 3 animal facility at the Animal and Plant Quarantine Agency. All animals were allowed to adapt for at least one week before use in experiments. The housing room was set to a 12 h:12 h light/dark cycle, a temperature of approximately 22 °C, and relative humidity of approximately 50%. The studies were performed according to institutional guidelines and approved by the Ethics Committee of the Animal and Plant Quarantine Agency (accreditation number: IACUC-2021-584). Naïve mice were anesthetized using CO2 and sacrificed. The peritoneal cavities were lavage with 5 mL of chilled Hank’s balanced salt solution (HBSS, Gibco, Waltham, MA, USA) buffer without Ca2+/Mg2+/phenol-red. The peritoneal lavage fluid was centrifuged at 300 × g for 10 min at 4 °C. The pelleted PECs were resuspended and counted using a Bio-Rad TC20 Automated Cell Counter (Bio-Rad). All cells were freshly isolated before use. No cryopreserved cells were used in any experiment. Purified PECs were then cultured in a complete medium consisting of Roswell Park Memorial Institute (RPMI) 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (HyClone), 3 mM L-glutamine (Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich), 100 U/mL penicillin/streptomycin (Sigma-Aldrich), and 0.05 mM 2-beta-mercaptoethanol (Sigma-Aldrich). Incubations were carried out at 37 °C and 5% CO2. Porcine PBMCs were isolated from whole blood of FMD antibody-seronegative pigs as donors (8-9 weeks old animals, n = 3/group) according to the method described by Lee et al. and Jo et al.17,67. Whole blood (20 mL/donor) was independently collected in BD Vacutainer heparin tubes (BD, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). PBMCs were isolated using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) gradient centrifugation. Residual red blood cells were lysed with ammonium–chloride–potassium (ACK) lysing buffer (Gibco). The PBMCs were suspended in Ca2+/Mg2+-free DPBS (Gibco) and counted using a Bio-Rad TC20 Automated Cell Counter (Bio-Rad). All cells were freshly isolated before use. No cryopreserved cells were used in any experiment. Purified PBMCs were then resuspended in RPMI-1640 (Gibco) medium supplemented with 10% FBS (Gibco), 3 mM L-glutamine (Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich), and 100 U/mL penicillin–streptomycin (Sigma-Aldrich). Incubations were carried out at 37 °C and 5% CO2. O PA2-C3d and A22-C3d antigen-mediated IFNγ secretion was analyzed using commercial ELISpot assay kits (catalog no. EL485 and EL985 for mouse and porcine, respectively; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, isolated murine PECs or porcine PBMCs (5 × 105 cells/well) were cultured in a 96-well PVDF-backed microplates containing a monoclonal capture antibody specific for mouse or porcine IFNγ and stimulated with 4 μg/mL (Final concentration) of inactivated FMDV (O PA2, O PA2-C3d, A22, A22-C3d) antigen at each concentration for 18 h in a humidified incubator at 37 °C with 5% CO2. As negative and positive control, PBS and 5 μg/mL of phorbol myristate acetate (PMA, Sigma-Aldrich) were used, respectively. The plates were washed with wash buffer and incubated with biotinylated anti-mouse IFNγ antibodies (1:119) or anti-porcine antibodies (1:119) overnight at 4 °C, followed by AP-conjugated streptavidin (1:119) at RT for 2 h. The plates were washed, developed with 5-Bromo-4-Chloro-3’ Indolyphosphate p-Toluidine Salt (BCIP)/Nitro Blue Tetrazolium Chloride (NBT), and counted using an ImmunoSpot ELISpot reader (AID iSpot Reader System; Autoimmune Diagnostika GmbH, Strassberg, Germany). The results were presented as spot forming unit (SFU). The animal protocol was conducted according to the method described in Lee et al. and Jo et al.17,67 as mentioned in the Mice of the method section. To validate the immunogenicity and short-term immunity of purified antigens isolated from immunopotent FMDV O PA2-C3d and A22-C3d, and to verify their potential as a master seed virus for the development of an FMD vaccine, we conducted animal experiments as follows. The vaccine compositions used in the experiments were as follows: purified antigens isolated from O PA2-C3d and A22-C3d (15 μg/dose/mL; 1/10–1/640 of the dose for pigs), ISA 206 (Seppic, Paris, France; 50% w/w), 10% Al(OH)3, and 15 μg/mouse Quil-A (InvivoGen, San Diego, CA, USA). Mice were vaccinated by I.M. injection in the thigh muscle (0 dpv) and challenged with FMDV (100 LD50 of O/VET/2013, ME-SA topotype or 100 LD50 A/Malay/97, SEA topotype) by I.P. injection at 7 dpv. Mice in the NC group received an equal volume of PBS (pH 7.0) administered via the same route. Survival rates and changes in body weight were monitored for up to 7 dpc to assess short-term immunogenicity (Fig. 2a). The PD50 test was conducted as a preliminary experiment to verify the immunogenicity of the bivalent study vaccine (containing the O PA2-C3d + A22-C3d antigens) in pigs (Fig. 3a). The results were compared to those of the group that received the study vaccine (containing the O PA2 + A22 antigens) used as the backbone of the immune-enhancing vaccine strain. The vaccine compositions used in the experiment were as follows; O PA2-C3d + A22-C3d antigens (15 μg + 15 μg/dose/mL, 1/10–1/640 dose) or O PA2 + A22 antigens (15 μg + 15 μg/dose/mL, 1/10–1/640 dose), ISA 206 (50%, w/w), 10% Al(OH)3, and 15 μg Quil-A/mouse. Animals in the NC group were administered the same volume of PBS by the same route. In mice, vaccination was administered I.M. on 0 dpv, and FMDV (100 LD50 of O/VET/2013, ME-SA topotype or 100 LD50 of A/Malay/97, SEA topotype) was administered I.P. at 7 dpv. Survival and changes in body weight were monitored until 7 dpc. To evaluate the potential of O PA2-C3d and A22-C3d as an FMDV vaccine strain and to investigate its ability to induce cellular and humoral immune responses and long-term immunity, preliminary experiments were conducted using pigs according to the method described by Lee et al. and Jo et al.17,67. The pigs (8–9 weeks old; n = 32) were screened based on antibody titers (PI value: 50%) using the ELISA tests for SP O and SP A, and VN titers (1.65 log10), and were classified as MDA(+) and MDA(−) (n = 16 per group). In each group, the pigs were further divided into 3 groups: NC (negative control), O PA2 + A22-treated (positive control, PC), and O PA2-C3d + A22-C3d-treated. The animals were randomly divided into three groups (n = 5/group) (Fig. 4a). The animals were isolated in closed ABSL3 containments during the study, provided with ad libitum access to food and water, and used for the experiment after at least one week of adaptation. The housing room was set to a 12 h:12 h light/dark cycle, a temperature of approximately 22 °C, and a relative humidity of approximately 50%. These studies were performed according to institutional guidelines and approved by the Ethics Committee of the Animal and Plant Quarantine Agency (accreditation number: IACUC-2021-584). We used MDA(+) (FMD-seropositive) and MDA(−) (FMD-seronegative) wild pigs in the experiments to evaluate the immunogenicity of the antigens isolated and purified from the immune-enhancing FMD vaccine strains, O PA2-C3d and A22-C3d, and assessed their ability to induce an adaptive immune response and overcome MDA interference. The compositions of the vaccines were as follows: a total of 1 mL of vaccine was considered 1 dose, and contained O PA2 + A22 antigens (15 μg + 15 μg; PC group, n = 6/group) or O PA2-C3d + A22-C3d antigens (15 μg + 15 μg; experimental group, n = 6/group), ISA 206 (50% w/w), 10% Al(OH)3, and 150 μg Quil-A. Animals in the NC group received the same volume of PBS via the same route. During the experiment, 1 mL of vaccine was administered I.M. twice at 28-day intervals (0 and 28 dpv). Blood samples were collected from the vaccinated pigs at 0, 7, 14, 28, 42, 56, 70, and 84 dpv for use in serological assays such as ELISAs (SP O and SP A), VN titer confirmation and isotype specific antibody immunoassay. To detect SP antibodies in the sera, we used the PrioCheckTM FMDV type O or FMDV type A (catalog no. 7610420 and 7610850 for FMDV type O and FMDV type A, respectively; Prionics AG, Switzerland) kits and the VDPro® FMDV type O or FMDV type A (catalog no. EM-FMD-05 and EM-FMD-03 for FMDV type O and FMDV type A, respectively; Median Diagnostics, Gangwon, Korea) kits. Absorbance in the ELISA plate was converted to a PI value. When the PI value was ≥50% for the PrioCheckTM FMDV kit or ≥40% for the VDPro® FMDV kit, the animals were considered antibody positive. A virus neutralization test (VNT) was performed according to the OIE manual68. The sera were heat-inactivated at 56 °C for 30 min in a water bath. Cell density was adjusted to form a 70% monolayer, and 2X serial dilutions of sera samples (1:8–1:1024) were prepared. The diluted sera samples were then incubated with a 100-tissue culture infectious dose (TCID)50/0.5 mL homologous virus for 1 h at 37 °C. After 1 h, an LF-BK (bovine kidney) cell suspension was added to all wells. After 2–3 days, CPE was evaluated to determine the titers, which were calculated as log10 of the reciprocal antibody dilution required to neutralize 100 TCID50 of the virus69,70. FMDV O/PA2 and FMDV A22/IRAQ were used for the VNT. To detect isotype specific antibody, ELISA for porcine IgG, IgA, and IgM (catalog no. E101-104, E101-102 and E101-117 for IgG, IgA and IgM, respectively; Bethyl Laboratories. Inc., Montgomery, Texas, USA) were performed on sera according to the manufacturer’s instructions. Briefly, one hundred microliters per well of serially diluted sera and standards were added to the appropriate wells, and the plates were incubated at RT for 1 h. After another washing and drying step, 100 μL/well of the 1X biotinylated detection antibodies were added to all wells, and the plates were incubated at RT for 1 h. The wells were washed and patted dry, 100 μL/well of 1X streptavidin-horseradish peroxidase conjugate was added, and the plates were incubated at RT for 30 min. Subsequently, the plates were washed again and dried. The peroxidase was developed with 100 μL/well of 1X TMB solution for 30 min at RT, and the reaction was stopped with 100 μL 2 N H2PO4. Absorbance was measured within 30 min using a Hidex 300SL spectrophotometer (Hidex, Turku, Finland) set at 450 nm17,67. To evaluate the O PA2-C3d and A22-C3d mediated cellular immune response and related gene expression, porcine PBMCs were isolated from the whole blood of vaccinated pigs (n = 5/group) at the time points described in Fig. 4a according to the method described by Lee et al. and Jo et al.17,67. PBMC isolation was performed as described in the PBMCs isolation of the method section. All cells were freshly isolated before use, and no cryopreserved cells were used in any experiment. Total RNA was extracted from the purified porcine PBMCs using TRIzol reagent (Invitrogen) and RNeasy Mini Kits (QIAGEN, Valencia, CA, USA). The cDNA was prepared by reverse transcription using a GoScript Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The synthesized cDNAs were amplified using quantitative-real-time PCR (qRT-PCR) on a Bio-Rad iCycler using the iQ SYBR Green Supermix (Bio-Rad)17,67. Gene expression levels were normalized to hprt levels and presented as a relative ratio compared to the control values. The primers used in this study are listed in Table S1. All quantitative data were expressed as the mean ± standard error (SEM) unless otherwise stated. Between-group statistical differences was assessed using two-way ANOVA followed by Tukey’s post hoc test or one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was denoted as follows: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ****p < 0.0001. Parametric tests were used to compare different groups. Survival curves were built using the Kaplan-Meier method, and differences were analyzed using the log-rank sum test. The GraphPad Prism 9.1.2 (GraphPad, San Diego, CA, USA) and IBM SPSS (IBM Corp., Armonk, NY, USA) software were used for all statistical analyses. Further information on the experimental design is available in the Nature Research Reporting Summary linked to this article.Methods
Preparation of the recombinant plasmid
Preparation of the immunostimulating recombinant FMD vaccine strain
Purification of the antigen from recombinant FMDV type O and type A presenting C3d-epitopes
Confirmation of structural and non-structural proteins using purified antigens and examination of 146 S particles using TEM
Mice
PECs isolation and cell culture
PBMCs isolation and cell culture
Antigen-induced IFNγ ELISpot assay on PECs and PBMCs in vitro
Evaluation of immunogenicity in experimental animals (mice) vaccinated with the immune-enhancing FMD vaccine strains, O PA2-C3d and A22-C3d
Evaluation of immunogenicity in pigs vaccinated with the immune-enhancing FMD vaccine strains, O PA2-C3d and A22-C3d
RNA isolation, cDNA synthesis, and quantitative real-time PCR
Statistical analysis
Reporting summary
Data availability
All data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
This work was supported by grants from the Animal and Plant Quarantine Agency (APQA) (B-1543386-2021-24). We would like to thank the staff and researchers of the Center for Foot-and-Mouth Disease Vaccine Research at the APQA for hel** us with this study.
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Conceptualization, M.J.L.; methodology, M.J.L., and J.-H.P.; software, M.J.L.; validation, M.J.L.; formal analysis, M.J.L.; investigation, M.J.L., H.M.K., S.S., S.H.P., and H.J.; resources, M.J.L., S.-M.K., and J.-H.P.; writing—original draft preparation, M.J.L.; writing—review and editing, M.J.L.; visualization, M.J.L.; supervision, M.J.L.; project administration, M.J.L.; funding acquisition, M.J.L. All authors have read and agreed to the published version of the manuscript.
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Lee, M.J., Kim, H.M., Shin, S. et al. The C3d-fused foot-and-mouth disease vaccine platform overcomes maternally-derived antibody interference by inducing a potent adaptive immunity. npj Vaccines 7, 70 (2022). https://doi.org/10.1038/s41541-022-00496-8
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DOI: https://doi.org/10.1038/s41541-022-00496-8
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