A safe foot-and-mouth disease vaccine platform with two negative markers for differentiating infected from vaccinated animals

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 (A(24)LL3D(YR) and A(24)LL3B(PVKV)3D(YR)) featuring the deletion of the leader coding region (L(pro)) 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 (3D(pol)) and 3B nonstructural proteins. Two mutations in 3D(pol), H(27)Y and N(31)R, as well as RQKP(9-12)→PVKV substitutions, in 3B(2) abolish reactivity with monoclonal antibodies targeting the respective sequences in 3D(pol) 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 A(24)WT virus, single A(24)LL3D(YR) and double A(24)LL3B(PVKV)3D(YR) mutant viruses were markedly attenuated upon inoculation of cattle using the natural aerosol or direct tongue inoculation. Likewise, pigs inoculated with live A(24)LL3D(YR) 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 A(24)LL3D(YR) and A(24)LL3B(PVKV)3D(YR) 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.

Fig 1

Schematic representation of the WT and mutant FMDV genomes and the modifications introduced in the present study. (A) A₂₄WT3D_YR and A₂₄LL3D_YR viruses were generated by site-directed mutagenesis of a full-length clone pA₂₄Cru of the FMDV outbreak strain A₂₄ Cruzeiro. In addition, A₂₄WT3B_PVKV3D_YR and A₂₄LL3B_PVKV3D_YR 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 RQKP_9-12 with PVKV, while 3D^pol contains two amino acid substitutions at positions H₂₇Y and N₃₁R.

Fig 2

In vitro characterization of marker vaccine candidates. (A) FMDVs. Monolayers were mock infected or infected with A₂₄WT, A₂₄WT3D_YR, A₂₄WT3B_PVKV3D_YR, A₂₄LL, A₂₄LL3D_YR, or A₂₄LL3B_PVKV3D_YR 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, A₂₄WT3D_YR, A₂₄LL, A₂₄LL3D_YR, A₂₄WT3B_PVKV3D_YR, or A₂₄LL3B_PVKV3D_YR 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 3D^pol protein. (C) Analysis of the marker epitope expression of mutant FMD viruses by immunohistochemistry. Cells were mock infected or infected with A₂₄WT, A₂₄WT3D_YR, A₂₄LL, A₂₄LL3D_YR, A₂₄WT3B_PVKV3D_YR, or A₂₄LL3B_PVKV3D_YR at an MOI of 5. The following day, the cells were fixed and processed for immunohistochemistry using MAbs specific for 3B (F83B) and 3D^pol proteins (F19-2 and F32-44).

Fig 3

cELISA. (A) Differential antibody response in animals infected with live A₂₄WT and A₂₄WT3D_YR using MAb F32-44 raised against 3D^pol. (B) Differential antibody response in animals infected with live A₂₄WT and A₂₄WT3B_PVKV3D_YR using MAb F32-44 raised against 3D^pol. (C) Differential antibody response in animals infected with live A₂₄WT and A₂₄WT3B_PVKV3D_YR 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.