Introduction

Foot-and-mouth disease (FMD) is an extremely contagious viral disease of cattle, pigs, sheep, goats and many cloven-hoofed wild animals. The causative agent, FMD virus (FMDV), is a member of the genus Aphthovirus in the family Picornaviridae. There are seven serotypes of FMDV found throughout the world, which are designated: A, O, C, Asia 1 and South African Territories (SAT) 1–3 (Forss et al. 1984). These distinct serotypes are determined by the structural proteins (SPs) expressed by the viruses. Both SP (VP4, VP2, VP3 and VP1) and non-structural proteins (NSPs) (L, 2A, 2B, 2C, 3A, 3B, 3C, 3D; or some complex, such as 3AB or 3ABC) are produced during the process of FMDV replication (Belsham 1993). Accordingly, two types of antibodies arise in animals infected with FMDV.

The strategy currently used to characterize FMDV isolated from vaccinated animals is to detect NSP antibodies. NSP antibodies exist in infected animals. However, in vaccinated animals there are no live viruses and the majority of NSPs are eliminated in the process of vaccine production, so it is not easy to detect NSP antibodies. The reliability of detection of 3AB or 3ABC antibodies is higher than that of other NSPs (Sorensen et al. 1998). Hence, ELISA methods to detect 3ABC, 3AB or 3A antibodies have been established in many laboratories around the world (Clavijo et al. 2004).

For FMD, an inactivated vaccine, generated from virus propagation in the cell line BHK-21, is commonly used. Although SPs and NSPs are expressed at the same time, some of the NSPs are eliminated during the process of vaccine production. However, dissociated NSPs and NSP complexes are difficult to eliminate and can induce antibodies after repeated vaccination, making it difficult to differentiate infected animals from vaccinated animals (Bergmann et al. 2000). FAO/OIE provides vaccine evaluation criteria to analyze NSP antibodies after three rounds of vaccination (http:\\www.oie.int, Manual of diagnostic tests and vaccines for terrestrial animals).

A genetically engineered FMD vaccine is currently being developed and its biosafety is under investigation. However, in this case some NSP genes were altered to increase their expression (Abrams et al. 1995) and consequently NSP antibodies appeared after several rounds of vaccination. This also led to difficulties in differentiating infected animals from vaccinated animals.

Therefore, it appears that regardless of the type of vaccine used, 3ABC antibodies can be induced after repeated vaccination, interfering with the diagnosis of infection or vaccination. In this study, the NSP mAb -sepharose conjugant (3BmAb-6BFF) was mixed with inactivated FMDV to reduce NSPs by affinity chromatography, with the aim of developing a FMD vaccine which contains less NSPs.

Materials and methods

Animals

Chinese yellow cattle, 1–2 years old, seronegative for FMDV NSP 3ABC, type O and Asia 1 antibodies, and PCR detection negative for O/P fluid, were used in this study. Also used in this study were 30–60-day-old long white pigs, seronegative for FMDV NSP 3ABC, type O and Asia 1 antibodies, and guinea pigs, weighing 300–400 g that were seronegative for FMDV NSP 3ABC antibodies.

Cell lines

BHK-21 passed with DMEM containing 10% fetus calf serum.

FMDV strains

OH/99 strain was isolated from pigs (Liu et al. 2004), ID50 = 10−5.0; Asia1/JS/05/S strain (GenBank No. EF149009) was isolated from cattle and adapted in pigs, ID50 = 10−4.5.

FMDV NSP monoclonal antibody

A FMDV NSP 3B monoclonal antibody, named 3BIgG, against the prokaryotic-expressed FMDV O/China/99 strain NSP 3B (GenBank No. AY539138) was obtained by screening. Its relative activity was ≥1:100000.

FMDV replication

Strain OH/99 or Asia1/JS/05/S was used to inoculate BHK-21 cells. When the cytopathogenic effect (CPE) reached 90%, the virus culture was stored at between −20 and −40°C. Meanwhile, the TCID50 was detected.

FMDV inactivation and safety test

The virus cultures were treated by addition of 0.035 M/L binary ethylenimine at 30°Cfor 24 h for virus inactivation. Inactivation was stopped by addition of 0.04 M sodium thiosulphate (Bahnemann 1973).

Eight 2–3-day-old suckling mice were subcutaneously injected with 0.2 ml of inactivated samples. Two mice were treated as negative controls. These two groups of mice were observed for clinical signs of FMD for seven days. The inactivated viral cultures were stored at 2–8°C.

Preparation of the NSP mAb-sepharose conjugant

This procedure was carried out according to the manufacturer’s guidelines. 5–10 mg/ml of purified 3B monoclonal antibody (3BIgG) was added to 1 ml of epoxy-activated sepharose 6BFF(Pharmacia, Sweden) mixed and incubated for 4 h, then centrifuged at 3,000 g . The resulting 3BmAb-6BFF conjugate antibody was stored at 4°C.

NSP elimination from inactivated FMDV cultures

3BMcA-6BFF was added to inactivated FMDV cultures (ratio=1:20), mixed and incubated for 3–4 h, then centrifuged at 3,000 g to remove the sepharose 6BFF. The supernatant was stored at 2–8°C and then used as the antigen to manufacture vaccines.

NSP detection in inactivated virus cultures treated with or without 3BmAb-6BFF

The content of 3B protein in inactivated virus cultures was tested by liquid phase-blocking-ELISA. A 96-well ELISA plate was coated with 3B protein (0.5 g/ml). The optimal concentrations of 3BmAb (1:80,000) and goat anti-mouse HRP IgG (1:40,000) were determined by checkerboard titration. To test samples, the purified 3B protein was serially diluted as the standard control, and the WLOGIT software (ED Nieuwenhuys, Netherlands) was used to assess the 3B protein concentration in samples.

Vaccine preparation

Four kinds of inactivated FMDV vaccines, designated “Improved OH/99 FMD vaccine” or “Improved Asia1/JS/05/S FMD vaccine” (the antigens treated with 3BmAb-6BFF) and “OH/99 FMD vaccine” or “Asia1/JS/05/S FMD vaccine” (the antigens not treated with 3BmAb-6BFF), were prepared by emulsifying the antigens with ISA206 (SEPPIC, France), according to the manufacturer’s guidelines.

Vaccine safety tests

Three of the 1-month-old piglets without FMDV antibody were intramuscularly inoculated at the ear-root-neck area with 5 ml of each inactivated vaccine respectively. The piglets were observed over 14 days.

Vaccination animals

Vaccination guinea pigs

The 3ABC antibody titers were detected when guinea pigs were inoculated with antigen or antigen with oil-adjuvant vaccine in four groups of guinea pigs (10 in each group), weighing 300–400 g, were vaccinated by intramuscular injection, the other three animals were included in a control group. The inactivated Asia1/JS/05/S antigen (0.5 ml/animal) was used in the first group. The Asia1/JS/05/S FMD vaccine (0.5 ml/animal) was used in the second group. The inactivated Asia1/JS/05/S antigen (0.5 ml/animal) treated with 3BmAb-6BFF was used in the third group. The improved Asia1/JS/05/S FMD vaccine (0.5 ml/animal) treated with 3BmAb-6BFF was used in the fourth group. BHK-21 cell cultures (0.5 ml/animal) were used in the control group. The second vaccination was carried out after two weeks, and then eight subsequent vaccinations were carried out on a weekly basis. The sera were collected to test for 3ABC antibody from the third vaccination.

Vaccination pigs and cattle

Two groups of pigs (10 in each group) were vaccinated by neck intramuscular injection, and other two pigs were included in a control group. The OH/99 FMD vaccine (2 ml/animal) was used in the first group. The improved OH/99 FMD vaccine (2 ml/animal) was used in the second group. The sera were collected to test SP and NSP (3ABC) antibodies after 28 days.

The other two groups (13 pigs and six cattle in each group) were vaccinated by neck intramuscular injection, and two pigs and two cattle were included in a control group. The Asia1/JS/05/S FMD vaccine (2 ml/animal) was used in the first group. The improved Asia1/JS/05/S FMD vaccine (2 ml/animal) was used in the second group. The second vaccination was carried out after two weeks, and then nine subsequent vaccinations were carried out on a weekly basis. The sera were collected to test SP and NSP (3ABC) antibodies.

Challenge test of vaccinated animals

Two groups of pigs (10 in each group) vaccinated with OH/99 FMD vaccine and the improved OH/99 FMD vaccine and the control group were challenged with OH/99 strain (1000ID50/ animal) by neck intramuscular inoculation. The pigs were observed for 10 days.

Sera test of vaccinated animals

Previously reported methods for LPB-ELISA and 3ABC-ELISA (Lu et al. 2007, 2008) were adopted to detect antibodies.

Results

Preparation of FMDV cultures

200 ml of OH/99 (TCID50 = 10−8.33) and 400 ml of Asia1/JS/05/S (TCID50 = 10−8.5) were used to inoculate BHK-21 cells.

FMDV inactivation test

The 16 suckling mice inoculated with inactivated OH/99 and Asia1/JS/05/S vaccines survived and remained healthy throughout the observation period.

Vaccine safety test

The 12 piglets inoculated with four kinds of vaccines remained healthy throughout the observation period and did not show any clinical symptoms of FMDV infection.

NSP concentration in inactivated FMDV cultures

The 3B concentration in the OH/99 vaccine was 256 ng/ml (untreated) and 59 ng/ml (treated with 3BmAb-6BFF), indicating that 76.95% of the 3B protein had been eliminated. The 3B concentration in the Asia1/JS/05/S vaccine preparation was 207 ng/ml (untreated) and 70 ng/ml (treated with 3BmAb-6BFF), indicating that 66.18% of the 3B protein had been eliminated.

Challenge to vaccinated animals

On the fourth day of the challenge, one of the 10 pigs vaccinated with OH/99 vaccine showed clinical symptoms of FMD (No.139). On the third day of the challenge, one of the 10 pigs vaccinated with improved OH/99 vaccine showed clinical symptoms of FMD (No.167). The percentage of protected animals in both groups was therefore 9/10 (see Table 1). Two control pigs also showed clinical symptoms of FMD.

Table 1 Challenge result of pigs vaccinated with OH/99 or improved OH/99 vaccine
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Sera antibody titers of vaccinated animals

NSP (3ABC) and SP antibody titers of mono-vaccinated pigs

Ten pigs were vaccinated with OH/99 vaccine or improved OH/99 vaccine respectively. Thirty days later, all of the animals were negative for 3ABC antibodies. The highest SP antibody titer was 1:360, and the lowest was less than 1:4 (see Table 2). In the challenge experiment, one pig showed an antibody titer lower than 1:4 in each group, and these animals were not protected (No.139 and No.167) (Table 1).

Table 2 Sera titers of pigs vaccinated with OH/99 or improved OH/99 vaccine
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Antibody titer of 3ABC in guinea pigs vaccinated 10 times

The 3ABC antibodies were detected after the third vaccination in animals not treated with 3BmAb-6BFF, and the herd positive rate (highest value: 63%) increased gradually during repeated vaccination. The 3ABC antibody titers were different when guinea pigs were inoculated with antigen or antigen with oil-adjuvant vaccine. The appearance time of 3ABC positive sera inoculated with antigen emulsified by oil-adjuvant was earlier and the positive rate was higher than only inoculated with antigen. However, 3ABC antibody production was delayed in animals treated with 3BmAb-6BFF, with antibodies only being detected after the seventh vaccination. The herd positive rate (highest value: 43%) was lower than in the untreated group (see Table 3). The control group remained negative for 3ABC antibodies.

Table 3 Antibody titer of 3ABC in guinea pigs vaccinated 10 times
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The NSP 3ABC and SP antibody titers of pigs and cattle vaccinated nine times

Sera from pigs and cattle were tested and one of the 3ABC titers was neutral (1/13 pigs, No.9) after the sixth vaccination with Asia1/JS/05/S vaccine, and all of the 3ABC titers were negative after the sixth vaccination with improved Asia1/JS/05/S vaccine. Three 3ABC sera were positive (3/13 pigs, No.9, No.13 and No.16) and one was questionable (1/13 pigs, No.12) after the seventh vaccination with Asia1/JS/05/S vaccine, and only one was questionable (1/13 pigs, No.7) after the seventh vaccination and it became positive after the eighth vaccination with improved Asia1/JS/05/S vaccine (see Table 4). Therefore, the percentage of sera positive for 3ABC antibody was 30.77% (4/13) for the Asia1/JS/05/S vaccine (including one questionable serum) and 7.69% (1/13) for the improved Asia1/JS/05/S vaccine, indicating that the improved Asia1/JS/05/S vaccine more effectively reduced the rate of detection of NSP 3ABC in pigs.

Table 4 The NSP 3ABC and SP antibody titers of pigs vaccinated nine times
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Of the sera tested from cattle, one 3ABC titer was positive (1/6 cattle, No.46) after the second vaccination with Asia1/JS/05/S vaccine, but the 3ABC antibodies were not detected until the fourth vaccination with improved Asia1/JS/05/S vaccine. Both of theses vaccines gave positive rates of 2/6 after the fifth vaccination. The positive rate was 4/6 (66.67%, No.43, No.46, No.48 and No.53) after the eighth vaccination with Asia1/JS/05/S vaccine, but remained at 2/6 (33.33% No.59 and No.61) after the eighth vaccination with improved Asia1/JS/05/S vaccine. One questionable serum was added to each of these two respective groups (No.49 and No.47) after the ninth vaccination. Therefore, the 3ABC positive percentage was 83.33% (5/6) for the Asia1/JS/05/S vaccine (including one questionable serum) and 50% (3/6) for the improved Asia1/JS/05/S vaccine (see Table 5), indicating that the improved Asia1/JS/05/S vaccine reduced the NSP 3ABC positive rate in cattle.

Table 5 The NSP 3ABC and SP antibody titers of cattle vaccinated nine times
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Compared with pigs, the production of 3ABC antibody in cattle occurred earlier and the positive rate was higher. The FMDV SP sera titers in both animals increased with repeated vaccination, but the whole antibody levels in cattle were higher than those detected in pigs (Tables 4 and 5).

Discussion

The most important and effective methods of prevention and control of FMD are vaccination and elimination of infected animals in developing countries. However, one challenge that is faced is how to differentiate FMDV infected animals from vaccinated animals. Inactivated FMD vaccines are still widely used (Parida 2009), although new types of vaccines have been developed. Techniques for detecting NSP antibodies have shown that a small amount of NSP proteins present within vaccine preparations can stimulate antibodies after repeated vaccination. If a 5% NSP positive rate appeared in a billion animals post vaccination, 50 million animals would be slaughtered according to the current policy. Therefore, the development of FMD vaccines without NSPs could avoid the misdiagnosis of vaccinated animals without infection, meaning that only infected animals would be slaughtered.

Although most inactivated vaccines are prepared from FMDV infected BHK-21 cell culture supernatants via concentration and purification methods, the residual NSPs cause difficulties in differentiating infected animals from vaccinated animals. Here, we used a NSP mAb-sepharose conjugant (3BmAb-6BFF) to reduce NSPs in order to explore the possibility of FMD vaccine manufacture without NSPs.

All the guinea pigs, pigs and cattle inoculated with FMD antigens or vaccines treated with 3BmAb-6BFF, showed no adverse side-effects, such as shock, body damage or aberrance. The vaccine potency data for pigs showed that the protection rates of the improved and untreated vaccines were the same, indicating that the treatment of antigens with 3BmAb-6BFF did not affect vaccine potency.

The reason that 3ABC antibody production was delayed and the positive rates were lower when guinea pigs, pigs and cattle were inoculated repeatedly with FMD antigens or vaccines treated with 3BMcA-6BFF, is that some NSPs related with 3B such as 3B, 3AB and 3ABC antigens (Xiang et al. 1998) were eliminated. Although improved FMD vaccines were shown here to reduce 3ABC antibodies, positive samples were found. There are two possible reasons for this. Either the 3A and 3C proteins were not eliminated because the 3B mAb did not react with them, or the concentration of the 3B mAb was not enough to combine 3B, 3AB and 3ABC completely. Future work should involve adding mAbs of 3A and 3C proteins, to confirm their suitable working concentrations.

The 3ABC antibody values were different when guinea pigs were inoculated with antigen or antigen with oil-adjuvant vaccine. The appearance time of 3ABC positive sera was earlier and the positive rate was higher, which means that oil-adjuvant can boost the immunoreaction.

When pigs and cattle were vaccinated with the same vaccine at the same dose, the time of 3ABC antibody appearance and the positive rates differed. 3ABC antibody in vaccinated cattle appeared earlier and the positive rate was higher compared with pigs. Similarly, the SP antibody levels in cattle were higher than in pigs. Therefore, reducing NSPs in FMD vaccines using mAbs is a promising strategy for future vaccine development.