Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O

Abstract

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV "O" 100 GPID challenge virus.

Optimized Adenoviral Vector That Enhances the Assembly of FMDV O1 Virus-Like Particles in situ Increases Its Potential as Vaccine for Serotype O Viruses

“…Thus, the introduced double-phenylalanine substitution at positions S93 and Y98 increased the yields of assembly of FMDV O1/Campos empty capsids. The phenotype exhibited by this mutated VP2 confirmed previously reported results indicating that a 10-residue region spanning α-helix A (amino acids 88–98 for the O serotype) of VP2 protein is relevant for the infectivity and stability of FMDV ( Ellard et al, 1999 ; Porta et al, 2013 ; Kotecha et al, 2015 ; Ganji et al, 2018 ). Furthermore, these studies have shown that residues within this region are responsible for non-covalent interactions between adjacent pentamers at the icosahedral two-fold axis, where neighboring VP2–VP2 proteins interact.…”

“…Secondly, to favor the self-assembly of the FMDV O1/Campos capsid proteins into VLPs, we introduced substitutions in amino acids located in the N-terminal of the VP2 capsid protein, whose side chains are engaged in inter-pentamer interactions. In particular, the replacements S93F and Y98F were described as essential to increase the pH and thermal stability of the less stable FMD O1/Manisa and SAT2 virus particles and recombinant empty capsids ( Kotecha et al, 2015 ; Rincon et al, 2015 ; Ganji et al, 2018 ). We surmised that introducing these mutations in both residues would favor O1/Campos empty capsid formation in situ .…”

Cell culture propagation of foot-and-mouth disease virus: adaptive amino acid substitutions in structural proteins and their functional implications

“…Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1126 2-019-01714 -7) contains supplementary material, which is available to authorized users. the case of FMDV, this often leads to an attenuated clinical phenotype in the natural host [13] and can change important physical properties of the viral capsid such as resistance to acids [16][17][18] and temperature [19]. For vaccine production, the acquisition of mutations that confer an extended receptor tropism is a welcome event that facilitates adaptation of the virus to cell culture.…”

Molecular Mechanisms of Immune Escape for Foot-and-Mouth Disease Virus

“…The tyrosine at position 98 of VP2 mutated to phenylalanine (Y98F) enhanced the thermal stability of the virus. This mutant presented optimal immunogenicity, and neutralizing antibodies could be induced by immunizing guinea pigs [55]. Thus, identifying these specific sites of VP2 protein in FMDV provides an idea for the preparation of a heat-resistant and immunogenetically superior FMD antigen.…”