Construction of a Vero cell line expression human KREMEN1 for the development of CVA6 vaccines

Figures

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  Fig. 1. Transient expression of KRM1 renders vero cells susceptible to CVA6 infection. (A) Representative cytopathic effect images of CVA6 and EV71 infections in wide-type Vero cells and transfected Vero cells, and control cells without infection. Scale bar, 100 μm. (B) Representative fluorescence confocal images of CVA6 and EV71 infections in wide-type Vero cells and transfected Vero cells, and control cells without infection. The primary antibodies were CVA6-specific mAb 4D6 or EV71-specific mAb CT11F9 and the second antibody (green) was Alexa Fluor 488-conjugated goat anti-mouse. The nuclei were stained with DAPI (blue). Scale bar, 100 μm

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  Fig. 2. Construction of a transfected Vero cell line with stable expressing of KRM1. (A) Schematic of the experimental workflow for constructing a transfected Vero cell line with stable expressing of KRM1. (B) Western blot analysis of RD, Vero, Vero-KRM1_#11 and Vero-KRM1_#26 cells with anti-KRM1 polyclonal antibody. GAPDH was used as the internal reference. (C) The levels of KRM1 mRNA as measured by quantitative real-time RT-PCR. The experiments were performed independently in triplicate. The values are expressed as the mean ± SD. Statistical significance was determined using unpaired Student’s t test. ****p < 0.0001, n.s., not significant

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  Fig. 3. Vero-KRM1_#11 cells susceptible to replication of CVA6. (A) Representative cytopathic effect images of CVA6-infected Vero-KRM1_#11 cells or uninfected cells. Scale bar, 100 μm. (B) Representative fluorescence confocal images of CVA6-infected Vero-KRM1_#11 cells or uninfected cells. The primary antibody was CVA6-specific mAb 4D6 and the second antibody (green) was Alexa Fluor 488-conjugated goat anti-mouse IgG. The nuclei were stained with DAPI (blue). Scale bar, 50 μm. (C) Growth kinetics of CVA6-TW-00141 stain infection on RD, Vero and Vero-KRM1_#11 cells after infection at an MOI of 0.01. The virus samples were collected at the indicated time points and then titrated. The experiments were independently performed in triplicate. The values are expressed as the mean ± SD. Statistical significance was determined using unpaired Student’s t test. n.s., not significant. (D) Virus titers of CVA6-TW-00141 strain grown on Vero-KRM1_#11 cells from P3 to P20 generation of serial passages. The experiments were performed independently in triplicate. The values are expressed as the mean ± SD

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  Fig. 4. Characterization of the purified CVA6-TW-00141 particles. (A) Representative negative-staining TEM images of the purified CVA6-TW-00141 particles prepared on either Vero-KRM1_#11 or RD cells. Scale bars, 200 nm in left pannels and 100 nm in right pannels. (B) SDS-PAGE analysis of the purified CVA6-TW-00141 particles prepared on either Vero-KRM1_#11 or RD cells

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  Fig. 5. Immunogenicity andin vivo protection of the purified CVA6-TW-00141 particles. (A) The animal experiment was performed following the procedures explained in the schematic diagram. Groups of 6–8 weeks old BALB/c mice were immunized i.p. with the inactivated CVA6 particles prepared on either Vero-KRM1_#11 or RD cells. A control group was inoculated with adjuvant alone. Each mice was immunized at weeks 0 and 2 with a dosage of 1.5 μg (n = 5 per group), and subsequently bled at weeks 0, 4, 6, 8 and 10. The green arrows represent immunization, and the red arrows represent blood collection. (B and C) Antibody response induced by CVA6 particles in mice was measured. Serum anti-CVA6 IgG titer (B) and neutralizing titer (C) were presented as the geometric mean titre (GMT) and converted to the logarithmic scale. The values are expressed as the mean ± SD. Significance was determined using unpaired Student’s t test. n.s., not significant. (D-F)In vivo animal protective efficacy of antisera collected from mice immunized with the CVA6 particles prepared on Vero-KRM1_#1. One-day-old BALB/c mice were firstly administered with diluted antisera 6 h before challenged with CVA6-TW-00141 strain. Mice…