Antigenic characterization and mapping of a conserved neutralizing epitope on VP1 of coxsackievirus A5

Figure 1. Purification and characterization of distinct CV-A5 particle populations. CV-A5 particles were purified by CsCl gradient ultracentrifugation. A total of 23 fractions were collected from the CsCl gradient. (A) The positions of empty particles (EP-1, EP-2), full particles (FP), and A-particles (AP) are indicated. (B) Absorbance ratios at 260 nm/280 nm (blue line) and CsCl density values (orange line) of the 23 fractions. Red arrows indicate the CsCl densities corresponding to the four particle types. Fractions within the red box were pooled for further analysis. (C) Western blotting analysis of the 23 fractions and mock-infected Vero cells, using antibodies against CV-A5 virion, VP2, and VP3. The molecular weight markers (M) are shown on the left, and the detected proteins are indicated on the right. (D) Western blotting analysis of the four pooled fractions and mock-infected Vero cells, using an anti-VP4 antibody.

Figure 2. Purification and characterization of the heat-treated full particles (FPs) and other particles. CV-A5 FPs were heat-treated at 56 °C for 10 minutes, followed by RNase treatment and CsCl gradient ultracentrifugation. A total of 21 fractions were collected. (A) The positions of unheated and heat-treated full particles are indicated. (B) Absorbance ratios at 260 nm/280 nm (blue line) and CsCl density values (orange line) of the 21 fractions of heated FPs. Red arrows indicate the CsCl density corresponding to the heated FPs. Fractions within the red box were pooled and named heated FPs (BPs). (C) Western blotting analysis of unheated and heat-treated FPs, using an anti-CV-A5 virion antibody. (D) SDS-PAGE (4-20%) analysis of proteins from mock-infected Vero cells, EP-1, EP-2, FP, AP and heated FPs, stained with Coomassie brilliant blue. The detected proteins (VP0, VP1, VP2, VP3, and VP4) are indicated. (E) Transmission electron microscopy (TEM) images of the purified EP-1, EP-2, FP, AP and heated FPs (BP). Scale bar = 100 nm. (F) Humoral immune responses of the five purified particles. Six- to eight-week-old BALB/c mice were inoculated intraperitoneally (i.p.) on day 0 and 14 with…

Figure 3. Screening and identification of neutralizing monoclonal antibodies (mAbs) against CV-A5. (A) Representative neutralization assays using CV-A5-r611-iLOV to identify neutralizing mAbs. Hybridoma cell supernatants were diluted 1:8 or 1:64 and inoculated with CV-A5-r611-iLOV for 36 hours. Autofluorescence was observed; wells without fluorescence at 1:8 dilution were considered neutralizing-positive (+), and wells with fluorescence were considered negative (−). Scale bar = 100 μm. (B) Representative Western blotting analysis of hybridoma supernatants to identify proteins recognized by the mAbs. Cell supernatants were diluted 1:10 and used as primary antibodies, with goat anti-mouse IgA, IgM, and IgG (1:10,000) as the secondary antibody. VP1 is indicated by an arrow. (C) Isotype determination of ten neutralizing mAbs. (D) ELISA analysis to assess the specificity of ten neutralizing mAbs against CV-A5. Plates were coated with purified particles from 10 enterovirus serotypes, with pre-immune mouse serum and rabbit antiserum used as negative and positive control, respectively. (E) Western blotting analysis of ten neutralizing mAbs. Protein of purified CV-A5 FPs (1 ng per lane) were…

Figure 4. Specificity and binding activity of monoclonal antibody (mAb) 4D6. (A) Western blotting analysis to identify the specificity of mAb 4D6. Purified particles from nine enterovirus serotypes were separated by SDS-PAGE (4-20%), and mAb 4D6 (50 μg/mL) was used as the primary antibody. Goat anti-mouse IgM was used as the secondary antibody. VP1 is indicated by an arrow. (B) ELISA analysis to assess the binding activity of mAb 4D6. Plates were coated with purified particles from 9 enterovirus serotypes, and mAb 4D6 (50 μg/mL) was used to detect binding. (C) ELISA analysis of mAb 4D6’s binding activity to CV-A5 VP1.

Figure 5. Epitope mapping of mAb 4D6 on CV-A5 VP1. (A) Peptide ELISA to map the binding site of mAb 4D6 on CV-A5 VP1. A set of 39 overlapping peptides covering the entire VP1 region (10 amino acids overlap) were tested for reactivity with mAb 4D6. Overlapping residues from peptides 20 to 22 are shown in bold. (B) Truncated peptides from the epitope region were tested in ELISA for binding reactivity to mAb 4D6. The inferred minimal epitope, from G158 to R165, is underlined. (C) Alanine scanning peptides spanning the epitope region were synthesized and tested in ELISA for reactivity with mAb 4D6. Mutated residues are indicated in bold. A control mAb (1H4) that recognizes a conserved, linear epitope on VP1 of enterovirus A species without neutralizing activity was included. (D) Sequence alignment of the identified epitope region (G158-R165) across different CV-A5 serotypes. Variant residues are indicated. (E) ELISA analysis to assess the binding activity of mAb 4D6 to peptides containing mutations from panel (D) Mutated residues are indicated in bold.

Figure 6. Protective efficacy of mAb 4D6 against lethal CV-A5 challenge. (A) Determination of LD50 for CV-A5–611 in 14-day-old Kunming mice. Mice were inoculated intraperitoneally (i.p.) with serial virus doses of CV-A5-611 (3.16 × 107 to 3.16 × 104 CCID50 per mouse), and the control group received MEM medium. (B) Passive protection of mice by mAb 4D6 following CV-A5–611 lethal challenge. Mice were i.p. inoculated with 3.16 × 107 CCID50 of CV-A5-611 (44 LD50). Two hours later, mice were treated with 4D6 at doses ranging from 2.22 μg to 60 μg per mouse. The control group received CV-A5–611 challenge followed by PBS injection.