A Vaccine Strain of the A/ASIA/Sea-97 Lineage of Foot-and-Mouth Disease Virus with a Single Amino Acid Substitution in the P1 Region That Is Adapted to Suspension Culture Provides High Immunogenicity

Abstract

There are seven viral serotypes of foot-and-mouth disease virus (FMDV): A, O, C, Asia 1, and Southern African Territories 1, 2, and 3 (SAT 1-3). Unlike serotype O FMDV vaccine strains, vaccine strains of serotype A FMDV do not provide broad-range cross-reactivity in serological matching tests with field isolates. Therefore, the topotype/lineage vaccine strain circulating in many countries and a highly immunogenic strain might be advantageous to control serotype A FMDV. We developed a new vaccine strain, A/SKR/Yeoncheon/2017 (A-1), which belongs to the A/ASIA/Sea-97 lineage that frequently occurs in Asian countries. Using virus plaque purification, we selected a vaccine virus with high antigen productivity and the lowest numbers of P1 mutations among cell-adapted virus populations. The A/SKR/Yeoncheon/2017 (A-1) vaccine strain has a single amino acid mutation, VP2 E82K, in the P1 region, and it is perfectly adapted to suspension culture. The A/SKR/Yeoncheon/2017 (A-1) experimental vaccine conferred high immunogenicity in pigs. The vaccine strain was serologically matched with various field isolates in two-dimensional virus neutralization tests using bovine serum. Vaccinated mice were protected against an A/MAY/97 virus that was serologically mismatched with the vaccine strain. Thus, A/SKR/Yeoncheon/2017 (A-1) might be a promising vaccine candidate for protection against the emerging FMDV serotype A in Asia.

Keywords: A/ASIA/Sea-97; foot-and-mouth disease virus; immunogenicity; vaccine.

Figure 1

Cell adaptation of A/SKR/Yeoncheon/2017 over serial passages. Foot-and-mouth disease virus (FMDV) A/SKR/Yeoncheon/2017 was serially passaged as follows for adaptation to the cells: five times in porcine kidney (LFBK) cells and three times in baby hamster kidney (BHK)-21 adherent cells (L5B3); five times in LFBK cells, four times in BHK-21 adherent cells, and one time in BHK-21 suspension cells (L5B4S1); five times in LFBK cells, four times in BHK-21 adherent cells, and four times in BHK-21 suspension cells (L5B4S4); five times in LFBK cells, four times in BHK-21 adherent cells, and six times in BHK-21 suspension cells (L5B4S6); five times in LFBK cells, four times in BHK-21 adherent cells, and seven times in BHK-21 suspension cells (L5B4S7). The 50% tissue culture infective dose (TCID₅₀) of the virus during passaging was measured in BHK-21 adherent cells expressing the integrin receptor and in CHO-K1 cells expressing the heparan sulfate (HS) receptor. Error bars indicate standard deviations (SDs) from the mean.

Figure 2

Growth of cell-adapted A/SKR/Yeoncheon/2017 (A-1) and the production of 146S antigen in BHK-21 suspension cells. (A) BHK-21 suspension cells were infected with cell-adapted A/SKR/Yeoncheon/2017 (A-1) at a multiplicity of infection of 0.001 and cultured at 37 °C in 5% CO₂. Supernatants were collected at 0, 8, 12, 16, and 24 h post-infection (hpi) and the TCID₅₀ was determined (line, left Y-axis). Inactivated antigen (146S) was quantified in the supernatants collected at 8, 12, and 16 hpi by sucrose density gradient centrifugation (bar, right Y-axis). Error bars indicate SDs from the mean. (B) Inactivated antigen (146S) from the supernatant collected at 16 hpi was confirmed by transmission electron microscopy.

Figure 3

Induction of virus-neutralizing (VN) antibody in pigs vaccinated with the A/SKR/Yeoncheon/2017 (A-1) experimental vaccine. The virus neutralization test (VNT) was carried out using sera collected from pigs vaccinated with A/SKR/Yeoncheon/2017 (A-1) at 0, 1, 2, 3, 4, 5, and 6 weeks post-vaccination. The dotted line indicates the 1:45 (log₁₀ 1.65) VN antibody titer cut-off level. Statistical analysis was conducted using an unpaired t-test (* p < 0.05, *** p < 0.005, ns: not significant). “Boosting” indicates the second vaccination.

Figure 4

Comparisons of neutralizing titers against heterologous field viruses in pigs. The virus neutralization test (VNT) was carried out using sera from pigs vaccinated with the A/SKR/Yeoncheon/2017 (A-1) experimental vaccine and A/SKR/Yeoncheon/2017(homologous virus), A/VIT/2013, A/SKR/Pocheon/2010, and A22/IRQ/24/64, and VN antibody titers were compared. Error bars indicate SDs from the mean. Statistical analysis was conducted using an unpaired t-test (* p < 0.05, ** p < 0.01, ns: not significant).

Figure 5

Serological cross-reactivity against heterologous viruses. A two-dimensional (2D) virus neutralization test (VNT) was performed using sera from cows vaccinated with the A/SKR/Yeoncheon/2017 (A-1) experimental vaccine and field viruses including A/SKR/Yeoncheon/2017 (A/ASIA/Sea-97-G2, homologous field virus), A/VIT/2013 (A/ASIA/Sea-97), A22/IRQ/24/64 (A/ASIA), A/SKR/Gimpo/2017 (A/ASIA/Sea-97), A/MAY/97 (A/ASIA/Sea-97), and A/SKR/Pocheon/2010 (A/ASIA/Sea-97-G1). The dotted line indicates the cut-off value of 0.3, above which the vaccine is considered to antigenically match with the field virus. Error bars indicate SDs from the mean. Experiments were performed twice independently.

Figure 6

Heterologous virus challenge in mice vaccinated with the A/SKR/Yeoncheon/2017 (A-1) experimental vaccine. C57BL/6 mice were intramuscularly injected with the A/SKR/Yeoncheon/2017 experimental vaccine or phosphate-buffered saline (PBS). Each animal was inoculated with vaccine containing serially 1/4 diluted antigen (1, 0.25, 0.063, or 0.0016 μg) per animal (A) or 1 or 0.1 μg of antigen per animal (B). The animals were challenged with 200 LD₅₀ of mouse-adapted FMDV A/MAY/97 via intraperitoneal injection at 10 (A) or 21 (B) days post-vaccination (DPV). Survival was monitored for 7 days.