Use of Synonymous Deoptimization to Derive Modified Live Attenuated Strains of Foot and Mouth Disease Virus

Abstract

Foot-and-mouth disease (FMD) is one of the most economically important viral diseases that can affect livestock. In the last 70 years, use of an inactivated whole antigen vaccine has contributed to the eradication of disease from many developed nations. However, recent outbreaks in Europe and Eastern Asia demonstrated that infection can spread as wildfire causing economic and social devastation. Therefore, it is essential to develop new control strategies that could confer early protection and rapidly stop disease spread. Live attenuated vaccines (LAV) are one of the best choices to obtain a strong early and long-lasting protection against viral diseases. In proof of concept studies, we previously demonstrated that “synonymous codon deoptimization” could be applied to the P1 capsid coding region of the viral genome to derive attenuated FMDV serotype A12 strains. Here, we demonstrate that a similar approach can be extended to the highly conserved non-structural P2 and P3 coding regions, providing a backbone for multiple serotype FMDV LAV development. Engineered codon deoptimized P2, P3 or P2, and P3 combined regions were included into the A24Cruzeiro infectious clone optimized for vaccine production, resulting in viable progeny that exhibited different degrees of attenuation in cell culture, in mice, and in the natural host (swine). Derived strains were thoroughly characterized in vitro and in vivo. Our work demonstrates that overall, the entire FMDV genome tolerates codon deoptimization, highlighting the potential of using this technology to derive novel improved LAV candidates.

Impact of Synonymous Genome Recoding on the HIV Life Cycle

“…A similar approach involving synonymous deoptimization of the poliovirus capsid coding region generated a virus that exhibited a neuro-attenuated phenotype in transgenic mice (Mueller et al, 2006). Largescale synonymous codon usage recoding has been used to generate prototypes of live attenuated vaccines for several RNA viruses, including poliovirus (Burns et al, 2006;Mueller et al, 2006), influenza virus (Mueller et al, 2010;Yang et al, 2013), respiratory syncytial virus (RSV) (Nouën et al, 2014), vesicular stomatitis virus (Wang et al, 2015), porcine reproductive and respiratory syndrome virus (Ni et al, 2014), dengue virus (Shen et al, 2015), zika virus (Li et al, 2018), echovirus 7 (Fros et al, 2017), foot and mouth disease virus (Diaz-San Segundo et al, 2021), and the plant cucumber mosaic virus (Mochizuki et al, 2018); arboviruses, such as Chikungunya virus (Nougairede et al, 2013) and tick-borne encephalitis virus (de Fabritus et al, 2015); and DNA viruses, such as Marek's disease herpesvirus (Conrad et al, 2018;Eschke et al, 2018). Clinical trials have been performed using codon-deoptimized type 2 poliovirus.…”

Evaluation of Potential In Vitro Recombination Events in Codon Deoptimized FMDV Strains

“…Plasmid Construction and RNA Synthesis. pA 24 Cru and pA 24 Cru- Nhe I P2P3Deopt have been previously described [ 14 ]. pA24-ΔP1 was constructed by replacing the P1 coding region of pA 24 Cru-(containing unique Nhe I and Fse I) with a 60 bp de novo synthesized fragment (GenScript, Piscataway, NJ, United States) that contained 15 bp of VP4 directly followed by 27 bp of VP1 via subcloning ( Figure S1 ) [ 47 ].…”

“…Recently, the creation of attenuated FMDV strains has shifted from the direct targeting of virulence factors to altering the codon bias (CB) or codon-pair bias (CPB) of the genome [ 14 , 15 ]. In short, all organisms exhibit a preference toward the use of certain codons or separately, codon pairs.…”

“…Complete or partial genome codon deoptimization (CD) or codon-pair deoptimization (CPD) has been leveraged to create a multitude of attenuated viruses [ 16 , 17 , 18 , 19 , 20 , 21 , 22 ]. Indeed, CPD of the FMDV P1 capsid coding region of strain A12 and CD of the P2 and/or P3 non-structural (NS) coding region of FMDV A24 has led to attenuated strains both in vitro and in vivo [ 14 , 15 ].…”

“…After verifying the validity and reproducibility of the assay, the effects of deoptimization on recombination within the conserved P2P3 region were tested by replacing the donor template ΔP1 with A24-ΔP1-P2P3Deopt (ΔP1-P2P3Deopt). A24-ΔP1-P2P3Deopt is based on the previously described LAV DIVA candidate strain A24-P2P3Deopt [ 14 ]. Following transfection of the two genetically engineered non-infectious RNAs, viable recombinant viruses from various time points were plaque purified and analyzed by NGS to identify the general sites of recombination and the stability of the P2P3 deoptimized regions post-recombination.…”