Engineering viable foot-and-mouth disease viruses with increased thermostability as a step in the development of improved vaccines

Abstract

We have rationally engineered foot-and-mouth disease virus to increase its stability against thermal dissociation into subunits without disrupting the many biological functions needed for its infectivity. Amino acid side chains located near the capsid intersubunit interfaces and either predicted or found to be dispensable for infectivity were replaced by others that could establish new disulfide bonds or electrostatic interactions between subunits. Two engineered viruses were normally infectious, genetically stable, and antigenically indistinguishable from the natural virus but showed substantially increased stability against irreversible dissociation. Electrostatic interactions mediated this stabilizing effect. For foot-and-mouth disease virus and other viruses, some evidence had suggested that an increase in virion stability could be linked to an impairment of infectivity. The results of the present study show, in fact, that virion thermostability against dissociation into subunits may not be selectively constrained by functional requirements for infectivity. The thermostable viruses obtained, and others similarly engineered, could be used for the production, using current procedures, of foot-and-mouth disease vaccines that are less dependent on a faultless cold chain. In addition, introduction of those stabilizing mutations in empty (nucleic acid-free) capsids could facilitate the production of infection-risk-free vaccines against the disease, one of the economically most important animal diseases worldwide.

FIG. 1.

(A) Schematic quaternary structure of the FMDV capsid. Numbers 1, 2, and 3 denote proteins VP1, VP2, and VP3, respectively, in one protomeric subunit. A pentameric subunit that includes the labeled protomer is delimited by thick lines. A black pentagon, triangle, or ellipse indicates the position of a capsid fivefold, threefold, or twofold symmetry axis, respectively. (B) A cartoon view that includes parts of three neighboring VP2 subunits (colored red, blue, or green) belonging to different pentamers in the FMDV C-S8c1 capsid. Their N-terminal segments (Nt) (colored magenta, cyan, or yellow) form a β annulus around a threefold axis (white line).

FIG. 2.

Some targeted residues to engineer new interpentamer interactions in the FMDV capsid. (a) Thr2023 and Ala3145 were mutated to Cys to allow the formation of 60 interpentamer disulfide bonds. (b) Gln2057 and Thr2053 were mutated to Lys and Asp, respectively, to allow the formation of 60 new interpentamer ion pairs. (c) Asp3069 was mutated to Glu to allow the formation of 60 interpentamer salt bridges with nonmutated Lys2198. (d) Ala2065 was mutated to either His or Lys to allow the formation of 60 interpentamer ion pairs with nonmutated Glu3137. In panels a to d, other residues from either of two interacting subunits belonging to different pentamers are colored magenta or yellow. (e) Localization on an FMDV capsid protomer of residues 3069 (red), where a lethal mutation from Asp to Glu was introduced (compare panel c), and 2188 (green), where a second-site mutation from Thr to Ala that restored infectivity occurred during virus replication.

FIG. 3.

Thermal-inactivation kinetics at 42°C of some engineered FMD virions. The remaining percent infectivity is represented in a logarithmic scale as a function of the incubation time. Circles, nonmutated virus; triangles, mutant A2065H; inverted triangles, mutant A2065K. The values and error bars (standard deviations) correspond to the averages from three or two independent experiments (nonmutated virus or A2065H, respectively). All data were fitted to single-order exponential processes.

FIG. 4.

Sucrose gradient profiles of purified radiolabeled FMDV virions subjected to thermal-dissociation conditions. In this example, nonmutated virions were incubated at 50°C for 0 min (circles), 90 min (triangles), or 180 min (squares); centrifuged; and processed as described in Materials and Methods. The peak on the right corresponds to intact (nondissociated) virions (sedimentation coefficient, 140S), and the peak on the left corresponds to dissociated pentameric subunits (sedimentation coefficient, 12S).

FIG. 5.

Comparison of the biological-inactivation kinetics and virus dissociation kinetics of nonmutated FMDV C-S8c1 at 42°C (left) or 4°C (right). The values correspond to four inactivation experiments (open symbols; left ordinate axis) using independent nonpurified virus (diamonds, triangles, and inverted triangles) or purified virus (circles) preparations and two dissociation experiments (solid symbols; right ordinate axis) using independent purified virus preparations (circles and squares). The same purified virus preparation was used to obtain the inactivation and dissociation curves represented by open or solid circles, respectively. All data were fitted to single-order exponential processes.

FIG. 6.

Thermal-dissociation kinetics of purified FMDV mutant D3069E/T2188A (a and b) or A2065H (c and d) at 42°C (a and c) or 4°C (b and d). The remaining percent of intact (nondissociated) 140S virions is represented in a logarithmic scale as a function of the incubation time. All data were fitted to single-order exponential processes. (a and b) Mutant D3069E/T2188A (triangles) and the corresponding nonmutated control (circles) were purified and assayed in parallel. The average values and error bars (standard deviations) corresponding to two independent measurements at 42°C (a) or 4°C (b) are shown. The average dissociation rate constants for D3069E/T2188A (k_mut) versus the nonmutated control virus (k_wt) were as follows: k_mut = 0.033 h⁻¹ versus k_wt = 0.106 h⁻¹ at 42°C and k_mut = 0.022 days⁻¹ versus k_wt = 0.075 days⁻¹ at 4°C. Further, independent preparations of D3069E/T2188A and its nonmutated control were also analyzed at 4°C and yielded similar results (not shown). (c and d) Mutant A2065H (triangles) and the corresponding nonmutated control (circles) were purified and assayed in parallel. The average values and error bars corresponding to two independent measurements at 42°C (c) or three measurements at 4°C (d) are shown. The average dissociation rate constants for A2065H versus the nonmutated control virus were as follows: k_mut = 0.041 h⁻¹ versus k_wt = 0.094 h⁻¹ at 42°C and k_mut = 0.004 days⁻¹ versus k_wt = 0.055 days⁻¹ at 4°C. The analysis of an independently engineered A2065H mutant and its nonmutated control at either temperature yielded similar results (not shown).

FIG. 7.

Reactivity in immunodot assays of nonmutated (C-S8c1; wt) FMDV and mutants A2065H and D3069E/T2188A with neutralizing monoclonal antibodies (MAbs) directed against each of the identified antigenic sites (A, C, D1, D2, and D3) in serotype C FMDV. In each strip, each of the three viruses was applied in duplicate. As a negative control, no virus was added to the bottom (seventh) well in each strip (no signal was obtained). The first strip is a negative control (no MAb was added). For the remaining strips, the numbers 1, 2, and 3 correspond to 10-fold serial dilutions of the indicated MAb. The results obtained with MAbs that recognize four other epitopes within antigenic site A (not shown) were similar to those obtained with MAb SD6.