ABSTRACT
Vaccination of domestic animals with chemically inactivated foot-and-mouth disease virus (FMDV) is widely practiced to control FMD. Currently, FMD vaccine manufacturing requires the growth of large volumes of virulent FMDV in biocontainment-level facilities. Here, two marker FMDV vaccine candidates (A24LL3DYR and A24LL3BPVKV3DYR) featuring the deletion of the leader coding region (Lpro) and one of the 3B proteins were constructed and evaluated. These vaccine candidates also contain either one or two sets of mutations to create negative antigenic markers in the 3D polymerase (3Dpol) and 3B nonstructural proteins. Two mutations in 3Dpol, H27Y and N31R, as well as RQKP9-12→PVKV substitutions, in 3B2 abolish reactivity with monoclonal antibodies targeting the respective sequences in 3Dpol and 3B. Infectious cDNA clones encoding the marker viruses also contain unique restriction endonuclease sites flanking the capsid-coding region that allow for easy derivation of custom designed vaccine candidates. In contrast to the parental A24WT virus, single A24LL3DYR and double A24LL3BPVKV3DYR mutant viruses were markedly attenuated upon inoculation of cattle using the natural aerosol or direct tongue inoculation. Likewise, pigs inoculated with live A24LL3DYR virus in the heel bulbs showed no clinical signs of disease, no fever, and no FMD transmission to in-contact animals. Immunization of cattle with chemically inactivated A24LL3DYR and A24LL3BPVKV3DYR vaccines provided 100% protection from challenge with parental wild-type virus. These attenuated, antigenically marked viruses provide a safe alternative to virulent strains for FMD vaccine manufacturing. In addition, a competitive enzyme-linked immunosorbent assay targeted to the negative markers provides a suitable companion test for differentiating infected from vaccinated animals.
INTRODUCTION
Foot-and-mouth disease (FMD) is an extremely contagious viral disease of cloven-hoofed ungulates, including a variety of wild and domestic (cattle, pigs, and sheep, among others) animals. The disease is distributed worldwide and has great negative economic impact not only on livestock health and production but also on international trade. Disease outbreaks occur frequently on almost every continent; outbreaks in previously FMD-free countries often have devastating economic consequences. Regular prophylactic vaccination using vaccine antigens that antigenically match circulating viruses is practiced in many countries as a primary control measure. However, the emergence or introduction of new strains renders these vaccines ineffective and thus require the development of new viruses.
FMD virus (FMDV) is a member of the genus Aphthovirus in the family Picornaviridae and exists as an antigenically variable virus of 7 serotypes, including A, O, C, Asia-1, and South African Territories (SATs) 1 to 3, as well as multiple subtypes. The viral genome consists of 8,500 nucleotides of a single-stranded positive-sense RNA protected by an icosahedral capsid containing 60 copies of each of the four structural proteins (18). FMDV is translated as a single polyprotein that is posttranslationally cleaved to produce partial and full cleavage products resulting in four structural proteins (VP1, VP2, VP3, and VP4) and 10 nonstructural proteins (Lpro, 2A, 2B, 2C, 3A, 3B1-3, 3Cpro, and 3Dpol).
FMD control is largely based on the FMD status of a geographical region. In endemic countries, it is based on regular (twice a year) vaccinations to reduce disease and transmission. On the other hand, the control policy for FMD-free countries usually includes the slaughter of animals in affected regions, as well as in neighboring regions, regardless of the disease status. However, large outbreaks in the United Kingdom and the Netherlands in 2001, as well as more recent outbreaks in Japan and Korea, where millions of animals that were mostly noninfected were sacrificed and burned or buried, resulted in public outcry and questioning of these control measures. As a result, there is a need for emergency vaccination programs accompanied by “vaccinate to live” policies as an alternative to mass culling of infected animals (35).
Current FMD vaccines consist of FMD virus antigen that has been chemically inactivated and formulated with adjuvants. Although this vaccine has been very successful in reducing disease outbreaks and virus transmission in countries where FMD is endemic, there are numerous risks and limitations associated with the current product (18, 42). First, vaccine production requires large cultures of live virulent virus prior to inactivation, which poses the risk of virus escape from the manufacturing facilities. Due to the lack of cross protection between serotypes and subtypes, vaccine strains must be selected to provide adequate protection and control of the virus circulating in particular regions. Also, vaccine production requires additional antigen purification process to remove cellular contaminants, as well as nonstructural viral proteins (NSPs), in order to support DIVA (differentiating infected from vaccinated animals) diagnostic testing, which is critical in outbreak control and serosurveillance. Since vaccine antigens consisting of killed virus do not replicate or induce antibodies against NSPs, anti-NSP antibodies have often been used as markers of infection. The highly conserved FMDV 3D polymerase (3Dpol) has been long identified as the main determinant of infection and has been called the FMDV infection-associated antigen (3, 4, 12, 27, 46). Interestingly, studies by Newman and Brown (30, 31) suggested that FMDV purified 140S particle preparations contain small quantities of 3Dpol and therefore could account for seroconversion to 3Dpol in animals that have received multiple doses of inactivated FMD vaccines. At present, antibodies against NSP 3ABC are considered the most reliable indicator of infection in both bovine and porcine sera (23), and numerous 3AB/3ABC-based enzyme-linked immunosorbent assay (ELISA) diagnostic tests are available to differentiate between infected and vaccinated animals (4, 8, 13, 24, 41, 43, 44, 46).
Past strategies to generate attenuated vaccines for FMDV have failed due to unstable phenotypes, variable pathogenic profiles in different species, and failure to induce adequate protection (25, 28, 29, 50). However, stably attenuated FMDV could provide a safe platform to produce inactivated antigen vaccines. Recent advances in infectious cDNA technology have led to opportunities to study and produce genetically engineered FMDV with altered virulence. FMDV virulence factor, leader protease (Lpro), is a papain-like proteinase (22, 40, 45) and is produced in two forms, Lab and Lb, through translation initiation codon at the first or the second AUG site, respectively. The presence of this inter-AUG region (the 84 nucleotides [nt] between the two AUG codons), as well as second AUG initiation codon of the Lpro, has been shown to be essential for viral virulence and replication of the FMDV type A24 virus (6, 36, 37). Both forms of Lpro can autocatalytically remove itself from the N terminus of the nascent polyprotein as well as cleave the eukaryotic initiation factor 4G, resulting in the shutoff cap-dependent host cell translation machinery. However, studies have demonstrated that polyprotein translation from the first AUG codon results in low translational efficiency with no viable virus production in vitro, and polyprotein translation is favored from the second AUG site through an unknown mechanism (6, 37). Lpro has also been shown to induce degradation of nuclear factor κB (NF-κB) in FMDV-infected cells, leading to inhibition of the host innate immune response (14, 15). Numerous studies with genetically engineered serotype A FMDVs lacking Lpro have been shown to be infectious but grow more slowly in cell culture (37) and is attenuated in both cattle and swine (5, 7, 26).
In the present study, we developed experimental marker FMD vaccine candidates that can aid in FMD control while providing DIVA capabilities. The negative antigenic marker virus candidates, A24LL3DYR and A24LL3BPVKV3DYR derived by infectious cDNA technology, lack the functional Lpro but contain the 84-nt inter-AUG sequence, as well as the second initiation codon of Lpro. These vaccine candidates also lack one of three 3B copies (3B1) and also contain replacements of immunodominant epitopes in 3B and 3Dpol with sequences corresponding to the closely related bovine rhinitis virus 2 (BRV-2). Aerosol inoculation of cattle with the live leaderless marker viruses results in very limited virus replication and the absence of FMD signs. Moreover, none of the infected animals shed significant amounts of virus into the environment. Likewise, swine inoculated in the heel bulbs with live A24LL3DYR virus showed no clinical signs of FMD, nor did the inoculated animals transmit the disease to contact animals. Both A24LL3DYR and A24LL3BPVKV3DYR vaccines produced by chemical inactivation with binary ethylenimine (BEI) proved to be as effective as a commercially available FMD vaccine in protecting cattle from challenge with the parental virus. Finally, serum from animals inoculated with these marker viruses can be readily distinguished from parental FMDV-infected animals utilizing companion DIVA serological ELISAs based on either one or two built-in antigenic markers.
MATERIALS AND METHODS
Viruses and cells.FMDV type A24 Cruzeiro was derived from the infectious cDNA clone, pA24Cru (referred to here as A24WT for simplicity [39]). A plasmid containing the BRV-2 (pBRV2, accession number EU236594) sequence from poly(C) to poly(A) described previously (20) was used as a source of bovine rhinitis virus 2 genetic material. The baby hamster kidney strain 21, clone 13, cell line (BHK-21) was maintained in Eagle basal medium (BME; Life Technologies, Gaithersburg, MD) supplemented with 10% calf serum (HyClone, South Logan, UT), 10% tryptose phosphate broth, and antibiotic/antimycotic. Cells were grown at 37°C with 5% CO2 in a humidified atmosphere.
Derivation of A24LL negative marker FMDVs.The 3Dpol region of pA24Cru (A24WT) was modified by PCR utilizing the mutagenic oligonucleotides P1266 (5′-ACCGTTGCGTACGGTGTGTTCCGTCCTGAGTTCGGG) and P1267 (5′-CCCGAACTCAGGACGGAACACACCGTACGCAACGGT) engineered to introduce mutations at codons 27 and 31 of 3Dpol protein (see Fig. 1B). The deletion of Lpro and the introduction of FseI at the beginning of the coding region for the capsid viral protein VP4 and NheI site in 2A were generated by overlap PCR fusion, created by mixing PCR amplified fragments, and reamplifying through the product of the fusion of these two fragments. This was accomplished by using the oligonucleotides P819 (5′-CGAGCCACAGGAAGGATGGGGGCCGGCCAATCCAG) and P820 (5′-CTGGATTGGCCGGCCCCCATCCTTCCTGTGGCTCG) containing an FseI site added by silent mutation in VP4 and sense (5′-GACCTGCTTAAGCTAGCCGGAGACGTTGA) and antisense (5′-TCAACGTCTCCGGCTAGCTTAAGCAGGTC) oligonucleotides containing a silent mutation that introduces NheI in 2A. To introduce these unique restriction sites and the deletion of the functional Lpro into pA24Cru, two mutant PCR products (PCR-LLfseI and PCR-NheI) were generated. PCR-LLfseI (a 1,315-bp product) lacking the Lpro and containing the unique restriction sites XbaI, FseI, and SpeI was digested with XbaI and SpeI and cloned into pA24Cru digested with the same enzymes to generate pA24-LLNheI. The PCR-SacII/MfeI fragment (2,333 bp) containing unique restriction sites SacII, NheI and MfeI was digested with SacII and MfeI, and this mutant product was cloned into pA24-LLNheI plasmid digested with the same restriction enzymes. Although all leaderless plasmids generated in the present study lack the functional leader protein, they do contain the 84-nt region between the first and the second AUG codons of the leader-coding region. The generated plasmids pA24Cru, pA24WT3DYR, pA24LL, and pA24LL3DYR all contain a T7 promoter sequence in front of a hammerhead ribozyme at the 5′ terminus of the S fragment of the FMDV genome, terminate with a poly(A) tract of 15 residues, and possess a unique restriction site (SwaI) that can be used for linearization. The double-negative epitope mutants, A24WT3BPVKV3DYR and A24LL3BPVKV3DYR, were derived from plasmids pA24WT3DYR and pA24LL3DYR, respectively, and lack one of the 3B (3B1; also known as VPg1) proteins but contain a substitution in 3B2 that abolishes reactivity with monoclonal antibody (MAb) F83B (a gift from Alfonso Clavijo, National Centre for Foreign Animal Disease, Winnipeg, Manitoba, Canada). The cDNA template corresponding to the 3B1 deletion mutant virus, A24WT-5853, arose from transfection of a RNA that contained a transposon insertion in 3B1 at position 5853 (33). This virus has been shown to grow in vitro and produce signs of FMD in cattle similar to A24WT. Therefore, to derive these double mutant plasmids, a PCR product spanning sequences between the unique restriction sites SalI and AgeI was produced that lacked 3B1 and harbored a substitution in 3B2 RQKP9-12 with PVKV found at a similar position in BRV-2. The sequence encoding 3B2 was modified by utilizing mutagenic sense (5′-GCCCGATGGAGAGACCAGTTAAAGTTAAAGTGAAAGCAAAAGCC) and antisense (5′-GGCTTTTGCTTTCACTTTAACTTTAACTGGTCTCTCCATCGGGC) oligonucleotides to introduce mutations in 3B2 (also known as VPg2; see Fig. 1B). Full-length genomic clones were linearized with SwaI and were in vitro transcribed using the T7 Megascript system (Ambion, Austin, TX). Transcript RNAs were transfected into BHK-21 cells by electroporation as previously described (38). The transfected cells were seeded in six-well plates and incubated for 24 to 48 h at 37°C. All six viruses were passaged up to four times in BHK-21 cells, and complete viral genomes were verified by sequence analysis. Virus stocks at passage 4 were stored at −70°C. Passage 4 virus stocks were used in animal experiments and for the production of inactivated vaccines. Virus titers were determined by plaque assays as described below.
Rescue of parental and mutant viruses, viral growth, and plaque assays.For virus growth curves, BHK-21 monolayers were infected with A24WT, A24WT3DYR, A24LL, A24LL3DYR, A24WT3BPVKV3DYR, and A24LL3BPVKV3DYR at a multiplicity of infection (MOI) of 5 PFU/cell. After 1 h of adsorption at 37°C, the monolayers were rinsed once with MES buffer (morpholine ethanesulfonic acid [25 mM], 145 mM NaCl [pH 5.5]) and then twice with phosphate-buffered saline (PBS), followed by the addition of fresh BME containing no serum. At various times postinfection, virus titers were determined by plaque assays (38) using 0.6% gum tragacanth overlay and incubated for 48 to 72 h at 37°C. Plates were fixed and then stained with crystal violet (0.3% in Histochoice; Amresco, Solon, OH), and the plaques were counted. Titers were expressed as PFU/ml and determined in duplicate.
Western blotting.BHK-21 cells were either mock infected or infected with A24WT, A24WT3DYR, A24LL, A24LL3DYR, A24WT3BPVKV3DYR, or A24LL3BPVKV3DYR at an MOI of 5. The following day, cells were lysed with radioimmunoprecipitation assay buffer (PBS supplemented with 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], and protease inhibitors). Next, 10 μl of each cell lysate was run under denaturing conditions in a SDS–12% PAGE gel (Invitrogen) and transferred onto nitrocellulose membranes using an XCell II transfer system (Invitrogen). The blots were blocked with 5% skim milk in PBS-Tween (PBS-T) for 1 h at room temperature, followed by an additional incubation of 1 h with 1:200 FMDV-specific MAbs (MAb F32-44, MAb F19-2, and MAb F83B) in PBS-T and 1% skim milk. Blots were then washed twice with PBS-T for 5 min each and incubated for an additional hour with goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP; Bethyl Laboratories) at 1:20,000 diluted in PBS-T and 1% skim milk. The blots were subsequently washed three times with PBS-T and developed with SuperSignal West Dura extended duration substrate (Thermo Scientific). Mouse anti-tubulin IgG conjugated to HRP (Abcam) was used at a dilution of 1:2,000 for a loading control.
Immunohistochemistry assay.The MAbs used in the present study were the MAbs F19-2 and F32-44 directed against the FMDV 3Dpol (49) and MAb F83B directed against the 3B nonstructural protein (19). Briefly, cell monolayers grown in 24-well plates were infected with A24WT, A24WT3DYR, A24LL, A24LL3DYR, A24WT3BPVKV3DYR, or A24LL3BPVKV3DYR at an MOI of 5. The following day, the infected cells were fixed with cold acetone-methanol (50/50) mix for 20 min, followed by two washes with PBS. Fixed cells were stained using FMDV-specific MAbs according to the manufacturer's instructions in a Vectastain ABC alkaline phosphatase kit (Vector Labs).
Antigen production and vaccine formulation.The A24LL3DYR and A24LL3BPVKV3DYR vaccine antigens were harvested from infected BHK-21 monolayers (a total of 3 × 108 cells) and inactivated with 5 mM BEI for 24 h at 25°C. The inactivated antigens were then concentrated and partially purified with 8% polyethylene glycol 8000. The yield of the inactivated antigens ranged between 18 and 23 μg. The vaccines were prepared as water-in-oil-in-water (WOW) emulsion with Montadine ISA 206 (Seppic, Paris, France) according to the manufacturer's instructions. Briefly, the oil adjuvant was mixed into the aqueous antigen phase (50:50) at 30°C for 15 min and stored at 4°C for 24 h, followed by another brief mixing cycle for 10 min. The integrity of 146S particles and antigen concentration present in the experimental vaccines (15 μg/dose of chemically inactivated A24LL3DYR or A24LL3BPVKV3DYR antigen) were determined by 10 to 30% sucrose density gradient and 260 nm densitometry. The commercial vaccine used for comparison was a polyvalent vaccine (Biogenesis-Bagó, Bioaftogen series 565 composed of O1 Campos, A24 Cruzeiro, A Arg 2001, and C3 Indaial) with an antigen load of 4 to 6 μg/dose.
Virulence study in cattle.Groups of Holstein steers—two animals each for A24WT, A24WT3BPVKV3DYR, and A24LL3BPVKV3DYR viruses and three animals each for A24WT3DYR and A24LL3DYR viruses—were marked and housed in a single room for a week of acclimation. Prior to infection, the animals were moved to separate rooms, and each of them was inoculated by aerosol with either 1 × 107 50% tissue culture infective dose(s) (TCID50) (for live A24WT) or 1 × 106 to 3 × 106 TCID50 (for live A24WT3DYR, A24LL3DYR, and A24WT3BPVKV3DYR mutants) using a method previously described (32). A dose of 7 × 106 TCID50 of live A24LL3BPVKV3DYR virus was inoculated intradermolingually into cattle. Sera and oral secretions were collected daily for up to 9 days for A24WT and for up to 21 days for A24WT3DYR, A24LL3DYR, A24WT3BPVKV3DYR, and A24LL3BPVKV3DYR, and temperature and clinical evaluation were recorded for the same period of time. Shedding of virus in the air was also monitored using a dry filter unit model 1000 air pump developed by the Program Executive Office for Chemical Biological Defense (PEO-CBD), as previously published (34). Clinical signs were scored as one credit for each affected foot and one credit for the affected head (vesicles in mouth, nostrils, tongue, or lips). FMDV RNA was measured in sera, swabs, and air samples by real-time reverse transcription-PCR (rRT-PCR) as described below.
Swine study.Mutant (A24LL3DYR) virus was tested for its virulence in swine. Briefly, two 20-kg pigs were inoculated intradermally in the heel bulb of the foot with 105 TCID50 of live virus, and 24 h later two naive pigs were added in direct contact. Sera and nasal and oral secretions were collected daily for up to 9 days postinoculation (dpi) and twice a week thereafter. Infected animals underwent clinical evaluation, and rectal temperature was determined daily throughout the experiment. Shedding of virus in the air was also monitored as described above. FMDV RNA was measured in sera, swabs, and air samples by rRT-PCR as described below.
Vaccination and challenge of cattle.Sixteen Holstein steers, between 250 and 300 kg, were allowed to acclimatize from shipping for 1 week before testing was initiated. Groups of four steers were vaccinated intramuscularly in the neck with the commercial vaccine (cattle 863, 864, 865, and 866), with chemically inactivated A24LL3DYR/water-in-oil-in-water (WOW, cattle 867, 868, 869, and 870) vaccine or A24LL3BPVKV3DYR/water-in-oil-in-water (WOW, cattle 1018, 1019, 1020, and 1021) vaccine. Cattle 871, 872, 1022, and 1023 were vaccinated with sterile PBS to be used as unvaccinated controls. On day 21 postvaccination (dpv), all 16 cattle were challenged intradermolingually with 104 BTID50 (50% bovine tongue infectious doses [10]) of parental A24WT. The animals were then monitored at 0, 4, 7, and 10 days postchallenge (dpc) for the appearance of localized and generalized lesions. Sera, nasal swabs (cotton tip, immersed in 2 ml of minimum essential medium with 25 mM HEPES and 1% fetal bovine sera), and temperature data were collected daily. Clinical signs were scored as one credit for each affected foot, and the presence of vesicles in the head was not considered due to lingual inoculation of challenge. FMDV RNA was measured in sera, swabs, and air samples by rRT-PCR as described below.
Foot-and-mouth disease virus RNA detection and DNA sequence analysis.Sera and swabs were processed for RNA extraction and rRT-PCR as previously described (2, 32). Briefly, 50 μl of each sample (sera, nasal, or oral swab suspension) for each cow was transferred to 96-well plates (King Fisher no. 97002540) containing 150 μl of lysis/binding solution. RNA was then extracted using MagMax-96 viral RNA isolation kit (Ambion, catalog no. 1836) on a King Fisher-96 magnetic particle processor (Thermo Electron Corp.). After an initial 5-min lysis/binding step, the RNA samples underwent a series of four washing steps, a drying step, and a final elution step. RNA was eluted in a final volume of 25 μl. At each of the above steps, RNA was magnetically bound to the beads contained in the lysis/binding solution and was transferred to the different extraction solutions. For filters containing the air samples, 1/4 of the filters were processed with 600 μl of RLT/β-mercaptoethanol and 106-μm acid-washed glass beads (Sigma). The sample was then disrupted using a Retsch tissue Mixer Mill (model MM400) at 30 beats/s for 3 min, and the liquid suspension was used for RNA extraction with the standard RNeasy RNA extraction. RNA extracted from all of the previous described samples was analyzed by rRT-PCR using 2.5 μl of RNA on the ABI 7000 as previously described (36). The cutoff to consider a positive value for preclinical samples (sera and swabs) was 102.69 RNA copies/ml, while the cutoff for air samples was 100.8 RNA copies number/1,000 liters of air. When necessary, PCR amplicons were sequenced using gene-specific primers, BigDye termination cycle sequencing kits (Applied Biosystems, Foster City, CA) and a Prism 3700 automated sequencer (Applied Biosystems). Primers and probes were designed using Primer Express software (Applied Biosystems).
Expression of recombinant FMDV 3Dpol protein.Expression clone for 3Dpol was prepared by using standard recombinant DNA methods. Briefly, PCR was used to amplify the 3Dpol-coding sequence of a type A FMDV. Forward primer P727 (5′-GCGGAATTCCCGCGGTGGAGGGTTAATCGTTGATAC) containing a SacII restriction site was designed to fuse the three amino acids of C terminus of ubiquitin (48) to the coding sequence for 3Dpol. The antisense primer, P728 (5′- GCGGAATTCGGATCCTGCGTCACCGCACACGGCGTTCACCC), encoding the C-terminal residues of 3Dpol also contains a BamHI restriction site for cloning purposes. The PCR product was cloned into pET26cHis, and the protein was expressed and purified from E. coli.
Serology and antigen differentiation assays.Serum samples from all animals were tested for the presence of neutralizing antibodies against FMDV in a serum neutralization assay. Neutralizing titers were reported as the reciprocal of the last serum dilution to neutralize 100 TCID50 of homologous FMDV in 50% of the wells (17). To study the anti-3Dpol response in the animals, we utilized sera collected from cattle following aerosol inoculation with live A24WT (bovines 7109 and 7110), A24WT3DYR (bovines 7199, 1, and 2), and A24WT3BPVKV3DYR (bovines 9143 and 9144) viruses and four animals introdermolingually inoculated with live A24WT virus (bovines 871, 872, 1022, and 1023). A competitive ELISA (cELISA) was performed according to the protocol of Yang et al. (49) with minor modifications. Briefly, recombinant 3Dpol was diluted in buffer carbonate-bicarbonate (pH 9.6) to obtain 0.25 μg/ml, and 100 μl/well was used to coat Nunc Maxisorp plates (Fisher Scientific). After 2 h of incubation at 37°C on a rotary shaker, the plates were washed four times with 0.01 M PBS and 0.05% Tween 20 (PBS-T). Triplicates of 50 μl of test sera/well (1/15 in PBS-T) and 50 μl of F32-44 hybridoma culture supernatant/well (1/5 in PBS-T) were applied to the coated plates, followed by incubation at 37°C for 1 h on a rotary shaker. After four washes, 100 μl of peroxidase labeled goat antibody to mouse-IgG (H+L) (KPL)/well diluted 1:2,000 in 5% skim milk in PBS-T was added, followed by incubation for 1 h at 37°C. After four additional washes, the antigen-antibody complexes were detected by the addition of 100 μl/well of SureBlue Reserve (KPL) and stopped in 25 min with 50 μl of TMB BlueSTOP solution (KPL)/well. The optical density (OD) was determined at 630 nm on an automated ELISA plate reader. For cELISA based on MAb F83B, a similar protocol was utilized with minor modifications. In particular, the antigen consisted of a peptide encoding the 3B sequence GPYAGPLETQKPLK and was applied at a concentration of 0.5 μg/well. Test sera were assayed at a 1/15 dilution in PBS-T, and MAb F83B was used at a 1/125 dilution. The results were expressed as the percentage of inhibition using mean OD values of test sera as well as known FMDV-positive and -negative bovine sera. The percent inhibition (PI) of samples was derived according to the following formula: PI = [(negative reference serum OD − test sample OD)/(negative reference serum OD − positive reference serum OD)] × 100.
RESULTS
Generation of negative marker FMDVs.Two mutant plasmids designated pA24WT3DYR and pA24LL3DYR were derived by site-directed mutagenesis using either FMDV pA24Cru (39) or the backbone of functional leader-deleted pA24LL infectious cDNA clones (Fig. 1A). Plasmids pA24WT3DYR, and pA24LL3DYR were engineered with a substitution in 3Dpol for residues found in BRV-2 at the respective locations that would eliminate an important antigenic epitope in FMDV 3Dpol; His27 was replaced by Tyr (H27Y), and Asn31 was changed to the basic amino acid, Arg (N31R) (Fig. 1B). Likewise, the two double-negative marker plasmids, pA24WT3BPVKV3DYR and pA24LL3BPVKV3DYR, were derived by deleting one of the 3B copies (3B1) and introducing a substitution in 3B2. The substitution in 3B2 was at amino acid positions 9 to 12 (RQKP), which were replaced by PVKV based on the sequence found at similar position in BRV-2 (Fig. 1B). The integrity of each construct for the genetic alterations was verified by DNA sequencing. Infectious virus was derived from each of the six infectious cDNA plasmids by electroporation of transcript RNA into BHK-21 cells. All mutant viruses were sequenced to confirm the presence of marker mutations in 3B and/or 3Dpol. The genetic stability of 3B and 3Dpol epitope mutations were confirmed by nucleotide sequence analysis of virus recovered after up to 15 serial passages in BHK-21 cells (data not shown).
Schematic representation of the WT and mutant FMDV genomes and the modifications introduced in the present study. (A) A24WT3DYR and A24LL3DYR viruses were generated by site-directed mutagenesis of a full-length clone pA24Cru of the FMDV outbreak strain A24 Cruzeiro. In addition, A24WT3BPVKV3DYR and A24LL3BPVKV3DYR viruses lack one of the 3B (3B1) proteins and contain a substitution in 3B2. Additional modifications present in the mutant plasmids are indicated: Lab and Lb, deletion of the functional leader-coding region but including the two AUG codons as well as the 84-nt inter-AUG region between Lab and Lb; 3B23, contains only two copies of 3B; and two unique restriction endonuclease enzyme cloning sites 1 and 2 (◆; RE1 and RE2). The plaque phenotypes of A24WT and mutant FMDVs on BHK-21 monolayers are also shown. (B) 3B2 contains a substitution at RQKP9-12 with PVKV, while 3Dpol contains two amino acid substitutions at positions H27Y and N31R.
In vitro characterization of negative marker FMDVs.In vitro characterization studies of negative marker viruses were performed to compare these viruses to their respective parental A24WT and A24LL viruses. The plaque phenotypes of A24WT3DYR and A24WT3BPVKV3DYR viruses were a mix of various sizes after 48 h, similar to the parental A24WT virus (Fig. 1A). However, the plaques of A24LL, A24LL3DYR and A24LL3BPVKV3DYR viruses were significantly smaller after 48 h of incubation and therefore required a 72-h overlay incubation.
In vitro growth kinetics of A24WT and A24LL viruses with their respective negative marker mutants were determined by using a high MOI of 5 on BHK-21 monolayers (Fig. 2A). Although the growth of the A24WT, A24WT3DYR, and A24WT3BPVKV3DYR viruses reached similar titers by 24 h postinfection (hpi), A24LL and the two marker viruses, A24LL3DYR and A24LL3BPVKV3DYR, were slightly delayed in their initial proliferation. By 24 hpi, virus production in A24LL-, A24LL3DYR-, and A24LL3BPVKV3DYR-infected cells yielded titers that were lower by 0.5 to 1 log compared to their respective parenteral WT strains.
In vitro characterization of marker vaccine candidates. (A) FMDVs. Monolayers were mock infected or infected with A24WT, A24WT3DYR, A24WT3BPVKV3DYR, A24LL, A24LL3DYR, or A24LL3BPVKV3DYR at an MOI of 5 PFU/cell. (B) Analysis of the marker epitope expression of mutant FMD viruses by Western blotting. Cells were mock infected or infected with A24WT, A24WT3DYR, A24LL, A24LL3DYR, A24WT3BPVKV3DYR, or A24LL3BPVKV3DYR at an MOI of 5. The following day, the cell lysates were collected and run under denaturing conditions using a SDS–12% PAGE. The nitrocellulose blots were probed with MAbs F83B for FMDV 3B protein and F19-6 and F32-44 for FMDV 3Dpol protein. (C) Analysis of the marker epitope expression of mutant FMD viruses by immunohistochemistry. Cells were mock infected or infected with A24WT, A24WT3DYR, A24LL, A24LL3DYR, A24WT3BPVKV3DYR, or A24LL3BPVKV3DYR at an MOI of 5. The following day, the cells were fixed and processed for immunohistochemistry using MAbs specific for 3B (F83B) and 3Dpol proteins (F19-2 and F32-44).
In order to assay the antigenic profile of mutant versus parental viruses, virus-infected cells were examined by Western blot analysis and immunohistochemistry (Fig. 2B and C, respectively). As shown in Fig. 2, BHK cells infected with each of the six viruses were immunoreactive with MAb F19-2, which detects an epitope in 3Dpol protein that was not altered. In contrast, only A24WT and A24LL viruses were immunoreactive with MAb F32-44. Failure of MAb F32-44 to react with A24WT3DYR, A24WT3BPVKV3DYR, A24LL3DYR, and A24LL3BPVKV3DYR mutant viruses indicated that the mutation of the 3Dpol epitope (a two-amino-acid replacement) affected the ability of mutant FMDVs to be recognized by MAb F32-44 but not by MAb F19-2. Likewise, both A24WT3BPVKV3DYR and A24LL3BPVKV3DYR viruses failed to react against MAb F83B antibody, which recognizes an epitope that was altered in the 3B2 region of the double-negative marker viruses.
Assessment of attenuation of live recombinant A24LL3DYR virus in pigs.An experiment protocol for swine challenge model was developed by Pacheco et al. (34). In that and subsequent experiments, naive pigs were inoculated intradermally in the heel bulbs with a range of 105 to 107 TCID50 of live A24WT. These animals developed disease with fever and lesions starting at 2 dpi (16, 34; unpublished data). In addition, these directly inoculated pigs were able to transmit the virus to naive pigs by direct contact, all of which succumbed to disease. Therefore, we wanted to determine the virulence of the recombinant A24LL3DYR in pigs. For this, 105 TCID50 (similar to the experiment used above) of live A24LL3DYR virus was used to inoculate the heel bulbs of two animals (Table 1, animals 40 and 41), and two naive animals (animals 43 and 44) were moved to the room at 24 hpi and housed together for 20 days. Among the directly inoculated pigs, only one (animal 41) showed detectable viral RNA in serum (105.44 viral RNA copies/ml) at 1 dpi and 105.45 viral RNA copies/ml in oral swabs at 2 dpi. This animal also developed low serum neutralizing antibodies titers starting at 5 dpi, but in the absence of any clinical manifestation of FMD. Interestingly, no virus transmission occurred from this pig to the second directly inoculated pig or to the two in-contact animals (animals 43 and 44). None of the pigs were ever pirexic (temperatures remained below 40°C) during the course of this experiment. No virus was detected in the air samples collected in this room during 21 days (results not shown).
Response of swine inoculation with live FMDV A24LL3DYR
Assessment of attenuation of live single- and double-negative marker viruses in cattle.To examine the influence of mutations on pathogenicity and the serological response to live recombinant A24WT3DYR, A24WT3BPVKV3DYR, A24LL3DYR, and A24LL3BPVKV3DYR compared to the parental A24WT virus in cattle, we performed aerosol or intradermolingual inoculation of these viruses, followed by measurement of clinical disease and virus shedding to the environment. After inoculation, several parameters, including fever, clinical score, viremia, neutralizing antibodies, and the presence of virus in air and oral swabs samples were recorded and analyzed (see Materials and Methods and Table 2).
Responses of cattle directly inoculated with live A24WT, A24WT3DYR, A24LL3DYR, A24WT3BPVKV3DYR, or A24LL3BPVKV3DYR virus
Two animals (bovines 7109 and 7110) that received aerosol inoculation with 107 TCID50 of live A24WT showed viremia, virus in saliva, and fever by 2 dpi. Clinical signs appeared by 2 to 4 dpi and reached a high clinical score by 5 to 7 dpi when neutralizing antibodies were first detectable. Virus was also detected in air samples when collected, but only from animal 7109. For the negative single marker viruses, six steers housed in separate rooms were aerosol inoculated with approximately 1 × 106 to 3 × 106 TCID50 of either live A24LL3DYR or A24WT3DYR virus. Bovines 7199, 1, and 2 inoculated with live A24WT3DYR virus showed viremia by 2 dpi and reached a peak at 3 dpi. Virus was detectable in saliva starting at 2 to 3 dpi and peaking at 4 to 6 dpi. Fever appeared at 2 or 6 dpi and lasted up to 3 or 8 dpi. Clinical signs appeared by 4 to 6 dpi, and virus was detected in air samples when collected (only from bovines 1 and 2). Virus was shed in the air starting by 3 dpi and peaked at 4 to 7 dpi. Serum neutralizing antibodies were first detectable by 5 to 6 dpi in all three cows (Table 2). Vesicular fluid was also collected from lesions on 4 and 6 dpi (bovines 1, 2, and 7199), and each sample was separately processed for rRT-PCR and sequencing. These fluids contained viruses that were indistinguishable from the inoculated virus in their genome sequences, further indicating that the A24WT3DYR virus had not changed during growth in bovines. In clear contrast to the pathogenic profiles of A24WT and A24WT3DYR viruses, the three animals that received aerosol inoculation with live A24LL3DYR (bovines 7201, 3, and 4; Table 2) showed absence of fever, viremia, clinical manifestation, or shedding of virus in saliva or air samples. The level of attenuation was such that these animals did not develop significant levels of neutralizing antibodies during the course of the experiment (Table 2), even though antibodies against viral structural proteins were demonstrated by 21 dpi by radioimmunoprecipitation assays (data not shown).
In order to determine the virulence profiles of double-negative marker viruses, groups of two steers were inoculated with either live A24WT3BPVKV3DYR or the A24LL3BPVKV3DYR virus. Bovines 9143 and 9144 that received aerosol inoculation with 7 × 106 TCID50 of live A24WT3BPVKV3DYR showed a pathogenic profile similar to A24WT and A24WT3DYR viruses. These animals showed viremia by 2 dpi and reached a peak at 4 dpi. Likewise, virus was detected in saliva starting at 2 to 3 dpi and peaking at 5 dpi. Fever was detected by 3 dpi and lasted up to 6 dpi, while clinical signs appeared by 4 to 5 dpi. Virus shedding in the air was detected by 6 to 7 dpi, and serum neutralizing antibodies appeared by 6 dpi in both cows. On the other hand, the lack of virulence of A24LL3BPVKV3DYR was similar to that of A24LL3DYR. Two cows, animals 9145 and 9146, were inoculated intradermolingually with 106 TCID50 of the live virus, and the results shown in Table 2 demonstrate that this virus is attenuated with lack of fever, disease, viremia, and viral shedding in air and saliva. However, unlike bovines infected with A24LL3DYR, these animals were able to develop neutralizing antibodies starting at 9 dpi.
Efficacy of chemically inactivated antigen vaccines prepared with single- or double-negative marker viruses in cattle.To determine the efficacy of inactivated antigen vaccines prepared using attenuated single or double marker viruses in providing immunological protection against challenge with parental A24WT virus, BEI-inactivated A24LL3DYR or A24LL3BPVKV3DYR vaccines were tested in cattle and compared to a commercial FMDV vaccine. As shown in Table 3, four cattle (animals 863 to 866) were each inoculated intramuscularly with one dose of a commercial polyvalent vaccine, while groups of four steers, animals 867 to 870 and animals 1018 to 1021, received the A24LL3DYR and A24LL3BPVKV3DYR BEI-inactivated vaccine, respectively. Steers 871, 872, 1022, and 1023 were mock vaccinated with PBS. All A24LL3DYR- and A24LL3BPVKV3DYR-vaccinated animals except one (animal 1020) developed a detectable FMDV-specific neutralizing antibody response by 7 days postvaccination (dpv). Bovine 1020 had no detectable neutralizing antibodies at 7 dpi, but they appeared by 14 dpi. Vaccination with A24LL3DYR or A24LL3BPVKV3DYR yielded slightly higher neutralizing titers compared to vaccination with the commercial vaccine, which is more likely due to a high content of antigen mass (approximately 15 μg/dose of A24LL3DYR or A24LL3BPVKV3DYR). By day 21, all animals but one (bovine 864) had increased titers of serum neutralizing antibodies. In contrast, the unvaccinated animals (bovines 871, 872, 1022, and 1023) that received PBS had no detectable FMDV-specific antibody response (Table 3). Furthermore, the 12 immunized cattle, regardless of the vaccine, did not show any FMD clinical signs (data not shown).
Specific neutralizing antibody response and protection against challenge with the FMDV A24WT strain after vaccination with commercial polyvalent, A24LL3DYR, or A24LL3BPVKV3DYR chemically inactivated antigen vaccine
At 21 dpv, all animals were challenged by intradermal inoculation at four sites in the tongue with 104 bovine infectious doses (BTID50) of parental FMDV A24WT. All control animals developed fever within 1 to 3 dpc, followed by typical FMD lesions on all four feet. In contrast, none of the vaccinated animals showed clinical signs of FMD during the course of the experiment and were fully protected (up to 10 dpc, Table 3). Only one animal vaccinated with the commercial vaccine showed fever at 3 dpc, but it did not succumb to FMDV. Unfortunately, cattle 866 and 1020 were euthanized due to illnesses unrelated to FMDV. All unvaccinated animals developed viremia from 1 to 5 dpc. In contrast, no virus was detected in the sera of any of the vaccinated animals (data not shown).
Antibody responses against 3B and 3Dpol in animals inoculated with negative marker viruses determined using competitive ELISA.Preimmune (0 dpv) or convalescent-phase (9 to 21 dpv) sera collected from animals inoculated with live A24WT by the intradermolingual route or infected by aerosol exposure to live A24WT or A24WT3DYR were assayed for reactivity to FMDV 3Dpol protein in cELISA (see Materials and Methods). The assay (Fig. 3A) allows the distinction of the serological responses of cattle inoculated with either parental (A24WT) from those inoculated with mutant (A24WT3DYR) virus. Although seroconversion after inoculation with A24WT resulted in significant inhibition of the anti-3Dpol response in cELISA, sera from animals inoculated with A24WT3DYR showed little inhibition, demonstrating that the YR mutation effectively removes reactivity with MAb F32-44. Likewise, sera from animals inoculated with A24WT3BPVKV3DYR via an aerosol route also showed a decreased antibody inhibition compared to sera collected from A24WT-infected animals (Fig. 3B). Therefore, cELISA using MAb F32-44 allows for differentiation of animals infected with A24WT from those inoculated with the negative marker viruses. In addition, antibody responses to FMDV 3B epitopes were measured by cELISA developed using MAb F83B and a 3B peptide (see Materials and Methods). The results demonstrate that convalescent-phase sera from animals infected with A24WT3BPVKV3DYR (bovines 9143 and 9144) were unable to compete with MAb F83B, unlike sera obtained from A24WT-infected animals (Fig. 3C). Finally, sera collected from convalescent animals inoculated with the marker A24LL3BPVKV3DYR virus by the intradermolingual route were analyzed against both FMDV 3Dpol protein and 3B peptide in cELISAs, but no antibodies against nonstructural 3Dpol and 3B were detected (data not shown).
cELISA. (A) Differential antibody response in animals infected with live A24WT and A24WT3DYR using MAb F32-44 raised against 3Dpol. (B) Differential antibody response in animals infected with live A24WT and A24WT3BPVKV3DYR using MAb F32-44 raised against 3Dpol. (C) Differential antibody response in animals infected with live A24WT and A24WT3BPVKV3DYR using MAb F83B raised against 3B. For each group, the average ± the standard deviation of two to three cows infected with either virus is shown. Samples were collected before inoculation and at day 21. DPI, days postinoculation; IDL, intradermolingual. Neutralization titers against A24Cru for all samples used in the cELISA were determined to be about ≥2.1 (see Tables 2 and 3). No significant difference in the day 9 and day 21 neutralizing titers against A24Cru were observed.
DISCUSSION
Currently, FMD vaccines are produced in expensive biological containment facilities where cell culture-adapted, live virulent FMDVs are grown in large volumes. This process has resulted in the escape of virulent virus from manufacturing facilities, causing costly outbreaks in livestock (11, 21). After growth, the live virus is then inactivated using chemicals, and antigen concentrates are prepared by purification steps required to remove contaminant proteins that make it difficult to differentiate infected from vaccinated animals (DIVA) through serological diagnostic tests. Since there is little to no cross-protection across serotypes and subtypes, appropriate matching between vaccine and circulating field strains is required to achieve protection. Despite these limitations, billions of vaccine doses are manufactured every year around the world. Their use has been the basis for eradicating FMDV from Europe and controlling the associated disease in many parts of the world through mass vaccination campaigns. Thus, there is an urgent need for the development of effective marker FMD vaccine candidates with improved DIVA capabilities.
We present here a rational approach to design novel negative marker FMD vaccine viruses for use in conjunction with a companion serological test. The vaccine candidate viruses, A24LL3DYR and A24LL3BPVKV3DYR, harbor negative antigenic markers for potential DIVA capabilities which are encoded in either the 3Dpol alone or both in 3B and 3Dpol, respectively. An additional modification of the vaccine candidates included the deletion of the nonessential Lpro and one of three 3B coding sequence that rendered these leaderless viruses markedly attenuated in both cattle and swine. Our data also showed that the leaderless marker viruses were not transmissible between cattle or swine. These results are consistent with previous studies on A12LLV2 FMD virus showing that deletion of the FMDV Lpro coding sequence produces viruses that maintained the ability to infect BHK-21 cells but displayed low virulence for cattle or pigs (1, 5, 7, 26). The vaccine platform also includes strategically located restriction enzyme sites that allow easy exchange of the capsid region of any virus serotype and subtype into the vaccine backbone. This feature allows rapid development of new vaccines in response to emerging FMDV antigenic variants.
Marker mutations introduced on the vaccine viruses were based on similar sequences found in BRV-2, the closest relative of FMDV. Comparison of the 3Dpol sequences of FMDV and BRV-2 viruses revealed 64% identity (20) at the amino acid level. Amino acids 16 to 32 comprise an important antigenic site in the FMDV 3Dpol protein (49), and this peptide is 76% identical between these two closely related viruses. MAbs F32-44 and F83B targeted against native FMDV 3Dpol and 3B protein, respectively, showed high reactivity against A24WT protein but did not react with BRV-2. These MAbs also failed to react with the mutants, A24LL3DYR and A24LL3BPVKV3DYR, suggesting that these antigenic markers and MAbs are of significant value for the development of companion DIVA diagnostic tests for the vaccine platform.
The two mutant viruses, A24WT3DYR and A24WT3BPVKV3DYR, carrying the leader coding sequence but lacking the 3Dpol epitope did not react with MAb F32-44. Moreover, the additional deletion of 3B epitope in the double-mutant A24WT3BPVKV3DYR failed to react with MAb F83B. Regardless of these mutations in the NSP, these marker viruses containing the leader proteinase showed similar tissue culture phenotypes as the parental A24WT virus. Furthermore, mutant viruses containing leader were virulent in the natural hosts, demonstrating that the mutations introduced in 3B and 3Dpol do not significantly attenuate the virus and that deletion of leader is critical to abrogate viral virulence. It is notable that the 3B and 3Dpol mutations were stably maintained in tissue culture (unchanged even after 15 serial passages in BHK-21) and also during replication in animals.
Attenuation of FMDV virulence is a critical aspect to be addressed in developing a safe vaccine production platform particularly for regions that are FMD free. Given that cattle infected with live A24LL3DYR and A24LL3BPVKV3DYR by the aerosol or the intradermolingual route, and pigs inoculated intradermally in the heel bulb of the foot with live A24LL3DYR showed no signs of FMD and no transmission to naive animals in direct contact, an outbreak in animals due to escape or through incomplete inactivation of these marker vaccine viruses from the manufacturing is highly unlikely. Also notable is that infection with the live leaderless marker viruses resulted in low or no antibody responses to FMDVs, indicating that very little or no replication of these viruses occurred. Therefore, in order to test the serological profile induced by the markers in 3B and 3Dpol, we used live single- and double-marker FMDVs with the leader containing backbone (A24WT3DYR and A24WT3BPVKV3DYR, respectively). Convalescent-phase sera from animals infected with these WT marker viruses had a serological profile that was easily distinguished by cELISA from that observed in WT-infected animals. The presence of a 3Dpol marker in the vaccine candidate is particularly important since the FMDV 3Dpol protein is known to stimulate a strong humoral response that is detectable early after infection and therefore is valuable in assessing infectious status during control of an outbreak (9, 12). In the present study, we utilized a modified cELISA (49) to capture the 3Dpol antigen to the solid phase, and the ability of test sera to inhibit the binding of the MAb F32-44 to the antigen was evaluated. Likewise, a similar in-house cELISA based on MAb F83B (targeted against 3B protein) was used to detect serological responses to the epitope contained in the 3B viral protein. Therefore, the presence of these 3B and 3Dpol epitopes in the parental virus but the lack of them in the mutant A24LL3DYR and A24LL3BPVKV3DYR viruses provides a potential DIVA companion test to be used in conjunction with these marker FMD vaccine candidates.
Killed FMD vaccines are currently commercially available and have been shown to be safe and effective for the control of FMD. The killed-virus vaccine is prepared from virus grown in BHK-21 cells, chemically inactivated using BEI and formulated with adjuvants. In this report, we have demonstrated that both BEI-inactivated vaccine candidates, A24LL3DYR and A24LL3BPVKV3DYR, as well as a commercial polyvalent FMD vaccine (with a standard antigen payload), protected cattle from direct challenge after only one vaccine dose. The analysis of the humoral responses against FMDV revealed that the three vaccine formulations were able to induce levels of neutralizing antibodies predictive of protection before challenge. However, the higher performance by the experimental vaccines in the induction of neutralizing antibodies was most likely due to higher amounts of antigens present in the experimental vaccines. We did not detect a significant antibody response against NSPs after a single-dose vaccination with either A24LL3DYR or A24LL3BPVKV3DYR (data not shown). On the other hand, animals infected with the live A24WT3DYR or A24WT3BPVKV3DYR virus developed antibody responses to NSPs that were unable to compete with the 3B and 3Dpol MAbs targeted against the marker epitopes. This demonstrates the robustness of the DIVA markers built into the vaccine platform (Fig. 3).
In control programs, particularly in countries where FMD is endemic, multiple doses of inactivated FMDV vaccines are applied for the control and prevention of FMD, and antibodies against 3Dpol are commonly detected in animals vaccinated multiple times (44). Our results suggest that vaccines prepared using our platform would consistently allow for easy differentiation of vaccinated from infected animals, even after multiple vaccinations. Furthermore, the absence of the two epitopes in 3B and 3Dpol would simplify the downstream processing during vaccine production, making it unnecessary to remove NSPs from the vaccine antigens. This will not only decrease production cost but could also potentially increase the potency of the vaccine since it is often hypothesized that presence of NSPs is desirable for the induction of a stronger and potentially cross-reactive immune response to FMD. Nonstructural proteins of FMDV are much more conserved across all clades (see the introduction). Some additional desirable characteristics of A24LL3DYR and A24LL3BPVKV3DYR marker vaccine candidates include the fact that they can be differentiated from field FMDV strains by genetic (e.g., PCR targeting the leader region) and immunological methods targeting the two antigenic markers.
Considering the high economic damage that FMD can elicit on livestock (47), current vaccination programs are now supporting the “vaccinate-to-live” policy for FMD outbreaks. In this scenario, the A24LL3DYR, and A24LL3BPVKV3DYR vaccine candidates have built-in capabilities (negative markers) to differentiate between vaccinated and WT-infected animals. As a result, these vaccines can be used in conjunction with a companion DIVA test (cELISA) that could assist in FMD control measurements and support the differentiation of infected versus vaccinated animals.
Taken together, the marker viruses developed here provide a viable alternative to using antigen produced from virulent virus for vaccine, allowing for safe production of FMD vaccines using existing production facilities with little or no modification. This innovative vaccine platform allows for the easy exchange sequences encoding the capsid proteins that can be utilized to rapidly derive contemporary outbreak strains of other subtypes and serotypes. The safety features of these marker vaccine viruses open the door for the interesting possibility of vaccine production in regions that do not currently allow the use of live FMDV for vaccine production.
ACKNOWLEDGMENTS
This research was supported in part by the Plum Island Animal Disease Research Participation Program administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Department of Agriculture (appointment of Sabena Uddowla), by CRIS projects 1940-32000-053-00D and 1940-32000-057-00D (Agricultural Research Service, U.S. Department of Agriculture) (E.R.), and by reimbursable agreement 60-1940-9-028, Task 2a, with the Department of Homeland Security (E.R.).
We thank Ian Olesen for determination of the 140S particles in BEI-inactivated viruses, Betty Bishop and Ethan Hartwig for technical support, and Marvin Grubman, Paul Lawrence, and William Golde for fruitful discussions. We are also thankful to the Plum Island Animal Disease Center caretakers for their professional support and assistance with animal experiments.
FOOTNOTES
- Received 18 May 2012.
- Accepted 9 August 2012.
- Accepted manuscript posted online 22 August 2012.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.