Evaluation of a 3A-truncated foot-and-mouth disease virus in pigs for its potential as a marker vaccine

Abstract

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals in the world. The disease can be effectively controlled by vaccination of susceptible animals with the conventional inactivated vaccine. However, one major concern of the inactivated FMD virus (FMDV) vaccine is that it does not allow serological discrimination between infected and vaccinated animals, and therefore interferes with serologic surveillance and the epidemiology of disease. A marker vaccine has proven to be of great value in disease eradication and control programs. In this study, we constructed a marker FMDV containing a deletion of residues 93 to 143 in the nonstructural protein 3A using a recently developed FMDV infectious cDNA clone. The marker virus, r-HN/3A93-143, had similar growth kinetics as the wild type virus in culture cell and caused a symptomatic infection in pigs. Pigs immunized with chemically inactivated marker vaccine were fully protected from the wild type virus challenge, and the potency of this marker vaccine was 10 PD50 (50% pig protective dose) per dose, indicating it could be an efficacious vaccine against FMDV. In addition, we developed a blocking ELISA targeted to the deleted epitope that could clearly differentiate animals infected with the marker virus from those infected with the wild type virus. These results indicate that a marker FMDV vaccine can be potentially developed by deleting an immunodominant epitope in NSP 3A.

Figure 1

Growth curves of r-HN and r-HN/3A_93–143 in BHK-21 cells. BHK-21 cells were infected either with r-HN or r-HN/3A_93–143 at an MOI of 1. At 0, 4, 8, 12 and 24 h post-infection, cells and supernatants were harvested and the virus titers were determined by TCID₅₀/mL on BHK-21 cells. The values of the viral titers represent the average obtained from triplicate experiments.

Figure 2

Analysis of the marker epitope expression of the recombinant FMDV by immunofluorescence. Confluent BHK-21 cells were either mock infected or infected with r-HN or r-HN/3A_93–143 at an MOI of 1, incubated for 6 h, fixed and probed with anti-3A or anti-3B MAb, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated secondary antibody. The cells were visualized under an Olympus BX40 fluorescence microscope. Magnification, ×10.

Figure 3

Analysis of the marker epitope expression of the recombinant FMDV by western blotting. BHK-21 cells were either mock infected or infected with r-HN or r-HN/3A_93–143 at an MOI of 1, and incubated at 37 °C. Infected cell extracts were prepared at 12 h post-infection. Proteins were separated on a 12% SDS-PAGE, blotted, and probed with anti-3A or anti-3B MAb. Protein marker is indicated on the right.

Figure 4

bELISA. (A) Differential antibody response in animals infected with WT virus using MAb 3A24 raised against 3A. (B) Differential antibody response in animals infected with the marker virus using MAb 3A24 raised against 3A. Samples were collected before inoculation and at 28 dpi.