Determining the Epitope Dominance on the Capsid of a Serotype SAT2 Foot-and-Mouth Disease Virus by Mutational Analyses

Abstract

Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible ␤G-␤H loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/ 7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the ␤G-␤H loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV. IMPORTANCEMonoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been identified. We have followed a unique approach using an infectious SAT2 cDNA genome-length clone. Ten structurally surface-exposed, highly varied loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of the SAT2/ZIM/7/83 virus. These regions were replaced with the corresponding regions of an antigenically disparate virus, SAT2/KNP/19/89. Antigenic profiling of the epitope-replaced and parental viruses with SAT2-specific MAbs led to the identification of two unique antibody-binding footprints on the SAT2 capsid. In this report, evidence for the structural engineering of antigenic sites of a SAT2 capsid to broaden cross-reactivity with antisera is provided. Genetically modified viruses provide a valuable tool for the manipulation of the biological properties of field and laboratory strains and present a promising avenue for the design of safe and effective vaccines. The modification of antigenic regions of human immunodeficiency virus (HIV) by amino acid (aa) substitutions in a recombinant virus has been used to confirm m...

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

“…The readings were expressed as ratios of the mutant readings to the readings for the wild-type SAT2 virus. The five MAbs reacted as expected to wild-type vSAT2, as described previously (40). All mutants reacted to DA10, GG1, GE11, and 1D5 similarly to wild-type virus, as shown by similar reactivity profiles ( Fig.…”

“…The antigenic reactivity of the mutants was assessed to ascertain if the amino acid changes had affected the typical binding of five SAT2 MAbs to the epitopes of the ZIM/7/83 virus as characterized by Opperman et al (40). The reactivity of the S2093Y mutant to MAb GD12 was lowered to 37% compared to that of the wild type, which is indicative of changes to the GD12 epitope footprint by the S2093Y mutation in VP2.…”

“…Although we do not have structural data to confirm this, we hypothesize that increased hydrophobic interactions may possibly cause reduced flexibility or local distortion in surface-exposed loops, locking the exposed side chains into a position not optimally aligned to GD12's complementarity-determining regions (CDRs). The exact footprint of GD12 has not yet been mapped, but previous evidence (40) suggests that amino acids at the 2-fold interface form part of the GD12 footprint. The S2093Y mutation is not expected to affect the critical domain for GD12 binding but perhaps to cause imperfect alignment (50-52) of the functional binding region of the capsid and the GD12 CDRs.…”

“…Sandwich antibody ELISA with SAT2-specific MAbs. Five nonneutralizing, SAT2-specific MAbs (mouse IgG1 isotype MAbs 1D5 [14 g/ml], DA10 [8 g/ml], GE11 [19 g/ml], GD12 [15 g/ml], and GG1 [22 g/ml]) have been described elsewhere (40). The MAbs, prepared by inoculating mice with purified 146S antigens (40), were found by mutational analysis of the virus to interact with either (i) two sites on the VP2 protein, i.e., residues 71 and 72 and residues 133 and 134, or (ii) a site that spans the 2-fold axis and involves residues on VP2 and VP3 (40).…”

Establishment of persistent foot-and-mouth disease virus (FMDV) infection in MDBK cells

“…In our in vitro MDBKbased model of FMDV type O persistence, sequencing the FMDV Op23 region encoding structural protein VP1 revealed only one point mutation (V50A) located in the bB-bC loop (residues 42-51), the second hypervariable region of FMDV after the bG-bH loop, which contains antigenic site 3 [46]. Interestingly, mutations in this loop can perturb the stability of the immunodominant VP1 GH loop [18], and monoclonal antibodies can bind to residues 48 to 50 in VP1 of the SAT2 serotype of FMDV [42]. Moreover, an amino acid substitution from valine (V) to alanine (A) at position 50 may disrupt a predicted SUMO binding site.…”