An auto-antibody identified from phenotypic directed screening platform shows host immunity against EV-A71 infection

Figures

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  Fig. 1. Construction of human scFv library into pDisplay-STOP vector, expression of scFv in RD cells, and panning for candidates against EV-A71 infection. A Modified pDisplay-STOP vector with stop codon before PDGFR transmembrane domain sequence in the original pDisplay vector. Expression of scFv in cells was validated by using HA tag antibody on Western blotting and flow cytometric assay B Left figure: flowchart of biopanning. Right figure: Total RNA was extracted and reverse transcribed from surviving cells of different panning rounds, and amplified scFv genes were cloned into pDisplay-STOP vector. RD cells were transfected with the libraries from different rounds and challenged with EV-A71 (M.O.I. = 0.01). After 48 h of infection, WST-8 reduction assay was performed to measure the cell viability. C Cell viability after EV-A71 infection of cells expressed with 10 scFv clones derived from 4th biopanning. Total RNA was extracted and reverse transcribed from surviving cells after 4th panning round, and amplified scFv genes were cloned into the pDisplay-STOP vector. RD cells were transfected with 10 of the different clones from the 4th panning rounds and challenged with EV-A71 (M.O.I. =…

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  Fig. 2. Inhibition of viral growth by scFv07 in cells. A RD cells were infected with different titers (0, 1, and 10 TCID50) of EV-A71 at 2 days post-transfection with scFv07 expressing plasmid (pTT5-scFv07) or pTT5 vector control. After 24 h of infection, the cells were fixed, and ELISA was performed by using anti-EV-A71 antibody to determine the expression of viral antigen. The absorbance of 450 nm was determined. B Post-transfection with scFv07 expressing pTT5-scFv07 plasmid or pTT5 vector cells were infected with EV-A71 (M.O.I. = 0.1) for 24 h followed by staining with anti-HA tag and EV-A71 antibody (MAB979). Magnification: 10x. C Viral supernatants from above EV-A71-infected cells as indicated were used to calculate virus titer by plaque assay. D Viral growth (M.O.I. = 0.01) of pTT5 or pTT5-scFv07 expressed RD cell were examined at 6, 12, 24 and 36 h post infection by plaque assay. *p < 0.05; **p < 0.001

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  Fig. 3. Intracellular scFv07 and 07-IgG1 do not recognize EV-A71 capsid proteins. A Cells were infected with EV-A71 (MOI 0.1) for 24 h followed by staining with rabbit anti-HA tag and mouse anti-EV-A71 (Mab979) antibody. Goat anti-mouse IgG-488 and goat anti-rabbit IgG-594 were used for further detection. B EV-A71 infected cell lysate (500 μg) was used for immunoprecipitation assays. MAB979 (2 μg) was captured by protein G magnetic beads (20 μL) and incubated with lysate for 24 h at 4 °C. After several washes, eluents were analyzed by WB using MAB979 and anti-HA tag antibody. C PEG precipitated EV-A71 were coated onto ELISA wells. Serial dilution of 07-IgG1 and MAB979 were added as primary antibody and incubated at 37 °C for 1 h. HRP labeled goat anti-mouse and human IgG were used for detecting bound antibody

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  Fig. 4. Characterization of 07-IgG1 using mass spectrum identification. A Mass spectrum identification of 07-IgG1 pull-down proteins could be HSA, β-actin, or ENO1. B ENO1, HSA, or β-actin recombinant protein was used in indirect ELISA with 07-IgG1. C EV-A71 patients’ sera were used to detect IgG against hENO1 protein. The levels of anti-ENO1 antibody were quantified by ELISA using 07-IgG1 as the standard antibody. **p < 0.001

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  Fig. 5. ENO1 is upregulated in EV-A71 infection and enhances viral replication. A ENO1 protein expression of EV-A71 infected RD cells were examined by WB. The fold change of ENO1 was normalized with the intensity of α-tubulin. B RD cells were infected EV-A71 (M.O.I. = 0.01) and ENO1 expression was analyzed at different hours post infection (h.p.i.). C ENO1 overexpressed RD cells were infected with EV-A71 for 24 h at the indicated M.O.I. of 0.001 and 0.01. Virus titers in the supernatant were determined by plaque assay. D ENO1 knock-down or supplement with ENO1 RD cells were infected with EV-A71, and viral titers were determined by plaque assay. E ENO1 overexpressed or knock-down RD cells were infected with EV-A71 at M.O.I. of 0.01 and virus growth was analyzed by plaque assay. *p < 0.05; **p < 0.001, ***p < 0.0001

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  Fig. 6. Effects of 07-IgG1 upon EV-A71 infection in animal model. A Scheme of the experimental design using 6-days-old ICR sucking mice. B Survival rate, C Percentage of initial body weight, and D clinical score of each suckling mice was recorded for 5 days. ***p < 0.0001

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  Fig. 7. 07-IgG1 inhibits EV-A71 replication in mice. A Brain tissues from sacrificed mice were used for ENO1 and EV-A71 qPCR. B H&E stain was performed to analyze ventral striatum immune cells infiltration. Upper panel: 4 × objective; Lower panel: 40 × objective. Black arrows indicate the cluster of immune cell infiltration. C H&E and IHC stain of brainstem from sacrificed mice. Red arrows indicate gliosis and green arrows indicate necrotic and partially lytic forms of basophilic neuronal necrosis. *p < 0.05; **p < 0.001, ***p < 0.0001