A rotavirus vaccine candidate attenuated by codon deoptimization protects neonatal mice against wild-type virus infection

Figures

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  Fig 1. Design, generation, and in vitro characteristics of codon deoptimized rotaviruses. (A) Illustration of a rotavirus particle and codon deoptimized regions of rotavirus gene segments. (B) Electrophoresis of rotavirus double-stranded RNA genomes (top to bottom, VP1, VP2, VP3, VP4, NSP1, VP6, NSP2, NSP3, VP7, NSP4, and NSP5 gene segments). Viral segments with codon deoptimization migrate slower on the polyacrylamide gel electrophoresis (indicated with orange arrowheads). (C) Replication of single-segment codon deoptimized rotaviruses in MA104 and HT-29 cells. Cells were infected with viruses at a multiplicity of infection (MOI) of 0.01 and harvested at 24 hours post-infection (hpi). Data show the means of three biological replicates. Error bars, SD. P values calculated by one-way ANOVA (*p < 0.05, ***p < 0.001). (D, E and F) Multi-step viral growth kinetics of multi-segment codon deoptimized rotaviruses in (D) MA104 cells, (E) HT-29 cells, and (F) human intestinal epithelial cells. Cells were infected with viruses at an MOI of 0.01 and harvested at the indicated timepoints. Data show the means of three biological replicates. Error bars, SD. P values comparing rSA11 and MS8cd…

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  Fig 2. Codon deoptimization interferes with rotavirus protein production, independent of mRNA stability. (A) Western blotting of rSA11 and MS8cd protein production in MA104 and HT-29 cells. Cells were infected with viruses at an MOI of 1 and harvested at 8 hpi. WT: rSA11, CD: MS8cd. (B) Immunofluorescence images of cells infected with rSA11 and MS8cd stained with anti-NSP4 or anti-VP7 antibody at 16 hpi. (C) Violin plots showing the quantification of NSP4 and VP7 fluorescence intensity of single cells. All infected cells in each well of 96-well plate were counted for this analysis. The number of infected cells in each well of a 96-well plate was counted. The specific cell numbers were as follows: 77 rSA11-infected cells and 70 MS8cd-infected cells were counted for the NSP4 immunofluorecence intensity analysis; and 43 rSA11-infected cells and 42-MS8cd-infected cells were counted for the VP7 immunofluorescence intensity analysis. Violin plots showing the quantification of fluorescence intensity of single cells in (B). P values calculated by one-way ANOVA (**p < 0.01, P > 0.05 was considered statistically non-significant). (D) Violin plots showing the quantification of NSP4 and VP7…

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  Fig 3. In vivo replication and pathogenicity of codon deoptimized rotavirus. (A) In vivo rotavirus replication in BALB/c mice. Each 3-week-old female mouse was orally inoculated with rSA11 or MS8cd at 1.0 × 107 FFU, and samll intestine, cecum and codon samples were collected 2 days later. Created in BioRender. Chen, Z. (2026) https://BioRender.com/fr8p7v5. (B) Viral RNA copies in intestine, cecum and colon samples detected by qRT-PCR (n = 6 each). *p < 0.05, **p < 0.01, by Mann-Whitney U-tests, with p > 0.05 considered statistically non-significant. (C) Infectious viral titers in intestine, cecum and colon samples detected by immunofluorescence assays (n = 6 each). P values calculated by t-tests (*p < 0.05, **p < 0.01; p > 0.05 was considered statistically non-significant). (D) Timeline of the mouse challenge experiment. Each 5-day-old mouse was orally inoculated with rSA11 or MS8cd at 1.0 × 106 FFU or PBS as a control, and their body weight and diarrhea symptoms were continuously monitored for one week. (E) Percentage of body weight change (b) relative to baseline 0 days post-inoculation (n = 6 each). P values calculated by two-way ANOVA (*p < 0.05, ***p < 0.001; p > 0.05 was…

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  Fig 4. Antibody responses elicited by codon deoptimized rotavirus. (A) Timeline of mouse immunization. Each 4-week-old female BALB/c mice were orally inoculated with rSA11, MS8cd at 1.0 × 107 FFU three times at regular intervals followed by serum collection. Created in BioRender. Chen, Z. (2026) https://BioRender.com/fr8p7v5. (B) Rotavirus-specific IgG in serum samples after the first, second and third inoculations, as measured by ELISA. (C and D) Rotavirus-specific IgA in serum (C) and fecal (D) samples following tertiary immunization, as measured by ELISA. (E) Rotavirus neutralizing antibody titers in serum samples following primary, secondary and tertiary immunization. P values calculated by Kruskal-Wallis tests, with p > 0.05 considered statistically non-significant.

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  Fig 5. Codon deoptimized rotavirus elicits protective immunity in a maternal immunization model. (A) Timeline of the maternal challenge experiment. Created in BioRender. Chen, Z. (2026) https://BioRender.com/fr8p7v5. (B) Percentage of body weight change relative to baseline. In the graph legend, the first term indicates the inoculum used for the maternal immunization, and the second term indicates the inoculum used for the neonatal mice challenge. P values calculated by two-way ANOVA (***p < 0.001), with p > 0.05 considered statistically non-significant. (C) Diarrheal symptoms recorded for 7 days after inoculation. In the graph legend, the first term indicates the inoculum used for the maternal immunization, and the second term indicates the inoculum used for the neonatal mice challenge. The vertical axis shows the percentage of mice showing diarrheal symptoms.

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  Fig 6. Generation of a panel of codon deoptimization attenuated rotaviruses carrying different VP4 and VP7 genes of human rotaviruses as vaccine candidates. (A) Electrophoresis of rotavirus double-stranded RNA of MS8cd monoreassortants carrying VP4 and/or VP7 genes of human rotaviruses. The reassorted gene segments are indicated with orange arrowheads. (B) Multi-step viral growth kinetics of MS8cd monoreassortants in MA104 cells. Cells were infected at an MOI of 0.01 and harvested at each indicated timepoint. (C) Neutralization activity of MS8cd monoreassortants carrying different VP4 or VP7 genotypes in SA11-immunized mouse serum samples. P values were calculated by one-way ANOVA (***p < 0.001), with p > 0.05 considered statistically non-significant. (D) Neutralizing titers of serum samples from MS8cd-immunized mice against MS8cd (G3P[2]) and MS8cd-G9 (G9P[2]). P values calculated using Wilcoxon tests (*p < 0.05). (E) Neutralizing titers of serum samples from MS8cd-G9-immunized mice against MS8cd (G3P[2]) and MS8cd-G9 (G9P[2]). P values calculated using Wilcoxon tests (*p < 0.05).