Mutation of FMDV Lpro H138 reside outside substrate binding domain drives viral attenuation in vitro and in swine

Authors: Azzinaro, Paul; Medina, Gisselle; RAI, DEVENDRA - Pfizer, Inc; Ramirez-Medina, Elizabeth; Spinard Iii, Edward; Zhu, James; Rieder, Aida - Elizabeth; De Los Santos, Teresa; Diaz San Segundo, Fayna

Submitted to: Frontiers in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/3/2022
Publication Date: 10/31/2022
Citation: Azzinaro, P.A., Medina, G.N., Rai, D., Ramirez Medina, E., Spinard Iii, E.J., Zhu, J.J., Rieder, A.E., De Los Santos, T.B., Diaz San Segundo, F.C. 2022. Mutation of FMDV Lpro H138 reside outside substrate binding domain drives viral attenuation in vitro and in swine. Frontiers in Veterinary Science. https://www.frontiersin.org/articles/10.3389/fvets.2022.1028077/full.
DOI: https://doi.org/10.1016/j.vetmic.2016.12.018

Interpretive Summary: Adequate control of viral infections has proven to be a universal challenge for preserving human and animal health. Foot-and-mouth disease virus (FMDV) is one of the most contagious agents that can affect livestock and wild cloven hoofed species. In developing countries, where FMD is enzootic, control of the disease involves surveillance, slaughter of infected and in contact animals and use of an inactivated whole virus vaccine that can induce protection in 7 days. However, recent outbreaks in FMD-free countries have proved that these measures fall short of restricting the occurrence and spread of FMD worldwide, seriously affecting, economically and socially, under-developed as well as developed nations. Live attenuated viral vaccines can induce both, humoral and cell-mediated immune responses, potentially affording long-term protection. In an effort to develop improved FMD control strategies, including attenuated vaccines, we have previously shown that the viral nonstructural protein leader protein (Lpro) is a virulence factor involved in controlling the innate immune response. In this work we characterize a new domain outside the catalytic domain that tolerated mutations and mutant virus derived showed attenuated phenotype in vitro and in vivo, confirming that Lpro is a reliable target to derive numerous viable FMDV strains that alone or in combination could be exploited for the development of novel FMD vaccine platforms.

Technical Abstract: The foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) is a papain like protease that cleaves the viral polyprotein and several host factors affecting host cell translation and induction of innate immunity. Introduction of Lpro mutations ablating catalytic activity is not tolerated by the virus; however, complete coding sequence deletion or introduction of targeted amino acid substitutions can render viable progeny. In proof-of-concept studies, we have previously identified and characterized FMDV Lpro mutants that are attenuated in cell culture and in animals, while retaining their capacity for inducing a strong adaptive immunity. By using molecular modeling, we have now identified a new mutation outside of the substrate binding and catalytic domain in a highly conserved across serotype His residue (H138), which rendered viable FMDV variants of reduced virulence in vitro and in vivo. Kinetics studies showed that FMDV A12-LH138L mutant replicates similarly to wild type (WT) virus in cells that do not offer immune selective pressure, but attenuation is observed upon infection of primary or low passage porcine epithelial cells. Western blot analysis on protein extracts from these cells revealed that while processing of translation factor eIF-4G was slightly delayed, no degradation of innate sensors or effector molecules such as NF-'B or G3BP2 was observed, and higher levels of interferon (IFN) and IFN-stimulated genes (ISGs) were induced after infection with A12-LH138L as compared to WT FMDV. Consistently with the results in porcine cells, inoculation of swine with this mutant only resulted in mild, or in some cases no clinical disease, but induction of a strong serological adaptive immune response. These results confirm that Lpro is a reliable target to derive numerous viable FMDV strains that alone or in combination could be exploited for the development of novel FMD vaccine platforms.