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

Abstract

Coxsackievirus B3 (CVB3) is a major cause of myocarditis and acute pancreatitis, particularly in neonates, in whom infections result in severe symptoms and high mortality rates. Despite the urgent need for effective preventive strategies, no vaccines or therapeutic agents have been developed. Live-attenuated vaccines hold promise for combating viral infections; however, their pathogenicity must be carefully regulated without compromising immunogenicity. Here, we investigated codon deoptimization and defective viral genomes (DVGs) as strategies to modulate CVB3 pathogenicity, while preserving its immune-activating capacity. Codon-deoptimized CVB3s with increased CpG dinucleotide content in their 3CD region were engineered, leveraging the innate immunostimulatory properties of CpG. These modified CVB3s exhibited attenuated pathogenicity proportional to the level of codon deoptimization and induced protective immunity against wild-type CVB3 (CVB3_WT), making them viable live-attenuated vaccine candidates. Additionally, DVGs derived from codon-deoptimized CVB3 demonstrated superior viral interference and enhanced stimulation of neutralizing antibody production compared to DVGs derived from CVB3_WT. These findings highlight that CpG-enriched genomes and DVGs are promising tools for regulating viral pathogenicity, enhancing vaccine safety, and developing therapeutic strategies against viral infections.

Keywords: Codon deoptimization; Coxsackievirus B3; CpG dinucleotide; Defective viral genomes; Live-attenuated vaccines.

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, 3C_37cd, 3CD_50cd, and 3CD_65cd, 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 (3C_37cd, 3CD_65cd) via the intraperitoneal (i.p.) route. Body weight change (e, g) and survival (f, h) were monitored daily for 2 weeks (n = 4). The solid red line with filled circles, the dashed dark pink line with crosses, and the dashed light pink line with open circles represent different doses of WT virus: 10³, 10⁴, and 10⁵ PFU, respectively. The solid gray line with open circles, the solid blue line with filled circles, and the solid green line with filled circles represent the mock control, 10⁵ PFU of 3C_37cd, and 10⁵ PFU of 3CD_65cd, respectively. All data are presented as the mean ± standard deviation (SD). Two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test was used for statistical analysis of viral growth. Survival rates were compared using the log-rank test. **P < 0.01, ****P < 0.0001.

Fig. 2

A single-dose infection with 3CD_65cd via the intramuscular route induced protective immunity in mice. (a, b) Mice were infected with 10³ PFU of WT or 3CD_65cd 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: 3CD_65cd). Asterisks in the weight change graph indicate a statistical comparison between the mock and 3CD_65cd (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 3CD_65cd 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 CVB3_WT or 3CD_65cd. Sera were collected 3 weeks after infection. (f) Mice were challenged with 10⁴ PFU of CVB3_WT and body weight changes were monitored daily for 2 weeks (n = 4 or 5). Solid red and green lines represent WT and 3CD_65cd, respectively. Red and green indicate 10³ PFU, while pink and light green indicate 10² PFU. Lines in the neutralization assay graph represent the median, while all other data are presented as the mean ± SD. Two-way ANOVA with Sidak’s multiple comparisons test was performed for statistical analysis of body weight changes or neutralizing antibody titers. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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 3CD_65cd-based defective viral genomes (DVG_WT and DVG_65cd, respectively). The gray box indicates the deoptimized region. (b) HeLa cells were infected with defective viral particles (DVPs) containing DVG_WT or DVG_65cd. 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 3CD_65cd (10³ PFU/mouse) together with DVP_WT or DVP_65cd (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 3CD_65cd (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 the WT virus only. Solid pink lines with open light red circles and light green open circles represent WT with DVP_WT and WT with DVP_65cd, respectively. Solid green lines with filled green circles represent the 3CD_65cd virus only. Solid light green lines with open pink circles and open light green circles represent 3CD_65cd with DVP_WT and 3CD_65cd with DVP_65cd, respectively. In panels f and i, filled red and green circles represent WT and 3CD_65cd alone, respectively. Filled pink and filled light green circles indicate WT and 3CD_65cd co-infected with DVP_WT, respectively, while open pink and open light green circles represent co-infection with DVP_65cd in WT and 3CD_65cd groups, respectively. Lines in the neutralization assay graph represent the median, while all other data are presented as the mean ± SD. For statistical analysis of weight changes and neutralizing antibody titers, two-way ANOVA with Sidak’s multiple comparisons test was performed. For the comparison of survival rates, the log-rank test was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.