SAT2 Foot-and-Mouth Disease Virus Structurally Modified for Increased Thermostability

Abstract

Foot-and-mouth disease virus (FMDV), particularly strains of the O and SAT serotypes, is notoriously unstable. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of immunity with a poor duration and hence require more frequent vaccinations to ensure protection. In silico calculations predicted residue substitutions that would increase interactions at the interpentamer interface, supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses in regard to their growth kinetics, antigenicity, plaque morphology, genetic stability, and temperature, ionic, and pH stability by using Thermofluor and inactivation assays in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutant for temperature and pH stability was the S2093Y single mutant, while other promising mutants were the E3198A, L2094V, and S2093H single mutants and the F2062Y-H2087M-H3143V triple mutant. Although the S2093Y mutant had the greatest stability, it exhibited smaller plaques, a reduced growth rate, a change in monoclonal antibody footprint, and poor genetic stability properties compared to those of the wild-type virus. However, these factors affecting production can be overcome. The addition of 1 M NaCl was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilizing SAT2 vaccines.IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of some serotypes, such as SAT2, affects the quality of vaccines and therefore the duration of immunity. We have shown that we can improve the stability of SAT2 viruses by mutating residues at the capsid interface through predictive modeling. This is an important finding for the potential use of such mutants in improving the stability of SAT2 vaccines in countries where FMD is endemic, which rely heavily on the maintenance of the cold chain, with potential improvement to the duration of immune responses.

Keywords: FMDV; SAT2; stability.

FIG 1

Stabilization of the SAT2 interpentamer interface and design of the models used for molecular dynamics simulations. (A) Cartoon representation of atomic structure of a virus capsid, with two pentameric subunits delineated and enlarged to show the interpentamer interface. The external proteins forming an icosahedral protomeric unit are labeled as follows: blue, VP1; green, VP2; and red, VP3. A truncated model was generated by trimming the protomers to include VP2 and VP3 atoms within a 13-Å radius from the interface. Yellow spheres represent amino acids interacting at the interface that were replaced in this study. Wild-type (B) and substituted (C) residues capping the 2-fold symmetry-related helix for the SAT serotype are highlighted and labeled in green for VP2 and red for VP3.

FIG 2

Schematic representation of the mutagenesis strategy used to introduce stabilizing mutations into SAT2/ZIM/7/83. The predicted mutations of the VP2 and VP3 capsid proteins are indicated in single, triple, or quadruple cassettes. The amino acid residues are numbered independently for each protein. In the mutant names, the first digit indicates the protein (VP1, VP2, or VP3) and the last three digits the amino acid position. Following overlap extension mutagenesis, the stabilizing mutated P1 regions were cloned into the SspI and XmaI sites of pSAT2, a genome-length cDNA clone of SAT2/ZIM/7/83. IRES, internal ribosome entry sequence; UTR, untranslated region.

FIG 3

One-step growth kinetics study performed on BHK-21 cells. The average log₁₀ titers for duplicate wells are shown for different time points (0, 1, 2, 4, 6, 8, 12, and 24 h postabsorption), as indicated on the graph, after infection over a 24-h period with the wild-type, S2093H, S2093Y, and F2062Y-H2087M-H3143V mutant viruses. The standard deviations of the titers determined for quadruplicate wells are indicated on the graph.

FIG 4

Heat map of the five SAT2-specific MAb (DA10, GG1, GE11, 1D5, and GD12) reactivity ratios for the mutant and wild-type viruses. Absorbance values (averages for three repeats) are shown as the ratio of each MAb's reactivity for the indicated virus. Mock-infected cell supernatants were used as a background control. The ratio depicts a specific profile for each virus. Green shaded areas represent ratio values closest to 1, indicative of the highest reactivity of a given virus to the MAb. Values of <0.5 (orange to red) are indicative of poor reactivity to that MAb and of a result most disparate from the wild-type profile. The binding pattern ratio for the wild-type virus (0.8:0.7:0.9:0.7:1) is used as a control against which mutant virus reactivities can be compared.

FIG 5

Thermal and pH inactivation kinetics of wild-type and mutant SAT2 viruses tested in duplicate. Inactivation of SDG-purified wild-type and mutant SAT2 particles was determined following heat treatment at 49°C for 1 h (A) or 42°C for 4 h (C) or treatment with TNE buffer at pH 6.0 for 2 h (E). The average log₁₀ virus titers determined for two different inactivation experiments are shown. The respective logarithmic values of the virus titers at the different time points (0, 15, 30, 45, 60, 90, 120, 180, and 240 min postinfection) were linearly fitted and the slopes determined. The average virus titers following heat inactivation at 49°C (B) or 42°C (D) or following pH treatment (F) were used to determine the percentages of residual infectious particles remaining over time.

FIG 6

Fluorescence thermal shift assay (41) to determine the dissociation temperatures of the wild-type and mutant SAT2 viruses. The A24 virus was used as a representative control of an A-serotype stable virus. (A) Dissociation temperatures of mutants showing improved temperature stability in comparison to that of controls. (B and C) Heat maps showing average dissociation temperatures of duplicate repeats, with error values. Green shading is indicative of a more stable virus, while red shading shows a more unstable virus. (B) pH stability tested with a range of pH buffers (pH 6.1 to 9.1). (C) Ionic stability of 15 different buffers and their effects as additional improvements to stability. The control was 0.8× PBS. ds*, viruses were dissociated under the experimental conditions, and no reading was obtained.