Codon-deoptimized single-round infectious virus for therapeutic and vaccine applications

Fig. 1. Codon deoptimization of the 3CD region of CVB3 resulted in reduced viral replication in correlation with the number of deoptimized codons (a) Schematic representations of codon-deoptimized Coxsackievirus B3 (CVB3) genomes. The gray bars represent the deoptimized regions. (b) The CpG ratio and the GC content in the polyprotein open reading frame sequence of wild-type (WT) or codon-deoptimized CVB3 genomes were analyzed using SSE V1.4. WT sequences were obtained from the National Center for Biotechnology Information website. (c) Plaque morphologies of WT and codon-deoptimized CVB3s in Vero cells at 3 days post-infection. (d) Comparison of the growth kinetics of the WT and codon-deoptimized CVB3s. Vero cells were infected with each virus at a multiplicity of infection of 0.02, and the viral titers at the indicated time points were evaluated in Vero cells using a plaque assay (n = 3). Red, blue, orange and green lines represent WT, 3C37cd, 3CD50cd, and 3CD65cd, respectively. (e-h) Mice were infected with three different doses (103, 104 or 105 plaque-forming units [PFU]) of WT virus or 105 PFU of codon- deoptimized viruses (3C37cd, 3CD65cd) via the intraperitoneal (i.p.) route. Body…

Fig. 2. A single-dose infection with 3CD65cd via the intramuscular route induced protective immunity in mice. (a, b) Mice were infected with 103 PFU of WT or 3CD65cd via the intranasal (i.n.) or the intramuscular (i.m.) route. Body weight changes (a) and survival (b) were monitored daily for 2 weeks (n = 5). Solid lines with filled circles and open triangles represent i.n. and i.m. infection, respectively (red: WT; green: 3CD65cd). Asterisks in the weight change graph indicate a statistical comparison between the mock and 3CD65cd (i.n.) groups with corresponding P-values. (c) Experimental design of the challenge study. Mice were infected with two different doses (102 PFU or 103 PFU) of WT or 3CD65cd virus via the i.m. route. (d) Body weight changes were monitored daily for 2 weeks (n = 5). (e) Neutralization titers of sera from mice infected with CVB3WT or 3CD65cd. Sera were collected 3 weeks after infection. (f) Mice were challenged with 104 PFU of CVB3WT and body weight changes were monitored daily for 2 weeks (n = 4 or 5). Solid red and green lines represent WT and 3CD65cd, respectively. Red and green indicate 103 PFU, while pink and light green indicate 102 PFU. Lines in the…

Fig. 3. Simultaneous infection of CVB3s with DVPs via the intramuscular route reduced the pathogenicity of CVB3 without compromising immunogenicity in mice. (a) Schematic representation of WT- or 3CD65cd-based defective viral genomes (DVGWT and DVG65cd, respectively). The gray box indicates the deoptimized region. (b) HeLa cells were infected with defective viral particles (DVPs) containing DVGWT or DVG65cd. Establishment of infection with the DVPs was confirmed based on green fluorescent protein (GFP) expression and double-stranded (ds) RNA production. Green, red, and blue indicate GFP, dsRNA, and DNA, respectively. Scale bar = 50 µm. (c) Experimental design for co-infections. Mice were infected with WT or 3CD65cd (103 PFU/mouse) together with DVPWT or DVP65cd (2 × 104 IU/mouse) via the i.m. route. Body weight change and survival were monitored daily for 2 weeks. Sera were collected 3 weeks post-infection, and the induction of neutralizing antibodies was evaluated (n = 8). The results are shown separately for the WT (d–f) and 3CD65cd (g–i) viruses. In panels d, e, g, and h, different line and marker styles indicate experimental groups. Solid red lines with filled red circles represent…