Effective in vivo therapeutic IgG antibody against VP3 of enterovirus 71 with receptor-competing activity

Figures

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  Figure 1. Mab 5H7 recognizes all 11 EV71 subgenogroups and does not cross-react with coxsackievirus A16. IFA of Vero cells infected with heterologous EV71 virus strains and coxsackievirus A16 (GenBank U05876). Cytopathic effects were visible at 2 days post-infection of cells fixed and labeled with supernatant of Mab 5H7-secreting hybridoma cells. FITC-conjugated anti-mouse Mab was used to detect viral signals. While all 11 EV71 subgenogroups were recognized by Mab 5H7, no signal was observed for CVA16. Magnification: 100×.

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  Figure 2. S74L mutation of VP3 confers evasion from neutralization with 5H7. Virus infection was indicated by CPE observed in inoculated Vero cells. No CPE was observed when neutralization occurred. RG: virus generated by reverse genetics; WT: wild-type virus; EM: escape mutant. 4B12: a non-neutralizing antibody against EV71 3CD; 51: a neutralizing antibody against VP1. Magnification: 100×.

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  Figure 3. S74L escape mutation of VP3 protein abolishes 5H7 binding. Escape mutants of 5H7 were created by incubating EV71 B4 strain with an excess of Mab. Vero cells were then fixed and labeled with 5H7, followed by FITC-coupled secondary antibody. Cells infected with wild-type viruses served as positive controls. Mab 5H7 staining was abolished in the escape mutant carrying the mutation S74L (second row). 4B12, an antibody against EV71 3CD, was used as positive control for infection. RG: virus generated by reverse genetics; WT: wild-type virus; EM: escape mutant. Magnification: 100×.

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  Figure 4. Stereographic images of 5H7 and 10D3 epitopes on VP3. The 3D crystal structure of EV71 C4 virus (MMDB ID: 97658) was downloaded from NCBI and viewed in the Cn3D program. (A) VP3 protein. EV71 VP3 protein is shown as a side-on view with the outside of the virion located at the top of the image and the inside on the bottom. 5H7 epitope is indicated as amino acid 74. 10D3 epitope is indicated in the “knob” structure as amino acids 55 to 69 of VP3. The escape mutation sites are indicated in yellow. (B) An EV71 protomer of the virus capsid is shown, containing one of each of the viral capsid proteins VP1 (pink), VP2 (blue), VP3 (brown), and VP4 (green). The mapped epitopes of EV71-specific Mabs 10D3 (VP3 59P, 62A and 67E), 7C7 (VP2 142-EDSHP-146) and 51 (VP1 215-KQEKD-219) as well as the escape mutation site of 5H7 at VP3 74S are indicated in yellow (arrows).

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  Figure 5. 5H7 inhibits EV71 virus attachment. Virus-binding assays were performed in Vero cells with B4 EV71 virus. Cells were washed with PBS and lysed 1 hour after virus inoculation. Cell lysate was analyzed by Western blot with Mab 57 against VP2. (A) Binding assays detected in Western blot were performed with different EV71 antibodies individually (500 μg of each antibody). Cropped blots were presented. No viral protein was detected in the sample incubated with 5H7, indicating its inhibitory effect against EV71 attachment. 4B12: a non-neutralizing antibody against EV71 3CD; VP2: purified VP2 protein as the control for Western blot. (B) Binding assays detected in Western blot were performed with different amounts of 5H7. 250 μg of 5H7 was sufficient to neutralize 2 × 108 TCID50 of B4 viruses. (C) Binding assays detected in antigen-capture ELISA were performed with different amounts of 5H7. No viral protein was detected in the samples incubated with at least 250 μg of 5H7.

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  Figure 6. 10D3 neutralizes EV71 post-attachment. Post-attachment neutralization assays were performed in Vero cells with varying amounts of 5H7, 10D3 and anti-EV71 polyserum (positive control). Virus infection was detected by IFA with 4B12, an antibody against 3CD. PBS indicated a control for B4 infection. Magnification: 100×.

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  Figure 7. 5H7 prevents EV71 from binding to SCARB2 receptor. (A): A pull-down assay was performed with SCARB2, B4 virus, and different antibodies. Virus binding to beads was detected by Western blot with Mab 57 against VP2. Cropped blots were presented. No virus band was detected in the samples incubated with 5H7. IgG: a non-EV71 IgG antibody served as a negative control. B4 only: a control for Western blot. (B) Competitive ELISA was also performed with SCARB2, B4 virus, and different antibodies. B4 virus was incubated with different antibodies, and added to wells coated with SCARB2. Virus binding to the coated wells was detected by guinea pig polyserum against B4. No virus was detected in the samples incubated with 5H7. IgG: a non-EV71 IgG antibody acted as a negative control.

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  Figure 8. Prophylactic and therapeutic efficacy of Mab 5H7 and 10D3 against EV71 B4 infection of AG129 mice (A): Survival rate of mice in a prophylactic study. (B): Survival rate of mice in a therapeutic study. Survival and clinical signs of mice were monitored daily until death or until 20 dpi. PBS: represents infected group of mice without any treatment. n = 10.

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  Figure 9. Immunohistochemistry and histopathology of EV71-infected AG129 mice without and with therapeutic protection with 5H7. Brain (A–D) and spinal cord (E,F) tissues were harvested and sectioned. Mice infected with a lethal dose of EV71, and treated with control antibody (A,B,E,F) displayed viral antigen and neuronal vacuolation. However, neither viral antigen nor neuronal lesion was observed in infected mice subjected to therapeutic protection with 5H7 (C,D,G,H). (B,D,F,H) Are views at higher magnification (200×) of (A,C,E and G) (40×).

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  Figure 10. Therapeutic efficacy of chimeric 5H7 against EV71 B4 infection of AG129 mice. Clinical disease scores and survival rates were recorded and evaluated daily until death or until 20 dpi. PBS: represents infected group of mice without any treatment. n = 5.