Identification of a Conserved, Linear Epitope on VP3 of Enterovirus A Species Recognized by a Broad-Spectrum Monoclonal Antibody
<p>Western blotting analysis of mAb 1A11 binding. Proteins of purified CV-A5 full particles (FP) and empty particles (EP) were separated by 4–20% SDS-PAGE. The proteins were blotted to the membrane and were incubated with rabbit anti-CV-A5 serum or mAb 1A11. The lysate of mock-infected Vero cells was used as control. Molecular weight markers in KDa and viral proteins are indicated on the left and right.</p> "> Figure 2
<p>Cross reactivity of mAb 1A11 to 9 serotypes of different enterovirus species. (<b>a</b>) Binding specificity by IFA of the 1A11 mAbs to the <span class="html-italic">Enterovirus</span> species <span class="html-italic">A</span>, <span class="html-italic">B</span> and <span class="html-italic">C</span> was performed. RD cells were infected with 7 serotypes (CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16 and EV-A71) of <span class="html-italic">Enterovirus A</span>, 1 serotype (echovirus 11) of <span class="html-italic">Enterovirus B</span> and 1 serotype (poliovirus Sabin 3) of <span class="html-italic">Enterovirus C</span> and were stained with 1A11 in IFA. Mock-infected cells were incubated with mAb as a negative control. (<b>b</b>) Western blotting of proteins of purified viruses of the same 9 serotypes used in (<b>a</b>) and rotavirus G8 was performed using mAb 1A11. Lysate of mock-infected cells was used as a negative control.</p> "> Figure 2 Cont.
<p>Cross reactivity of mAb 1A11 to 9 serotypes of different enterovirus species. (<b>a</b>) Binding specificity by IFA of the 1A11 mAbs to the <span class="html-italic">Enterovirus</span> species <span class="html-italic">A</span>, <span class="html-italic">B</span> and <span class="html-italic">C</span> was performed. RD cells were infected with 7 serotypes (CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16 and EV-A71) of <span class="html-italic">Enterovirus A</span>, 1 serotype (echovirus 11) of <span class="html-italic">Enterovirus B</span> and 1 serotype (poliovirus Sabin 3) of <span class="html-italic">Enterovirus C</span> and were stained with 1A11 in IFA. Mock-infected cells were incubated with mAb as a negative control. (<b>b</b>) Western blotting of proteins of purified viruses of the same 9 serotypes used in (<b>a</b>) and rotavirus G8 was performed using mAb 1A11. Lysate of mock-infected cells was used as a negative control.</p> "> Figure 3
<p>Indirect ELISA of the binding specificity and affinity against 9 serotypes of different enterovirus species and a non-enterovirus control rotavirus-G8 (Rota-G8). Data are means ± SDs of the OD<sub>450</sub> reading from triplicate wells. ELISA plates were coated with purified viruses including CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, EV-A71, Echo11, Sabin 3, and Rota-G8, at 1 μg/mL. The 10-fold serial dilution of mAb 1A11 was used as detection antibody.</p> "> Figure 4
<p>Epitope mapping of 1A11 mAb binding epitope to CV-A5 VP3. (<b>a</b>) CV-A5 VP3 peptides ELISAs were performed using a panel of 31 peptides spanning the entire VP3 region with 10 overlapped residues to detect reactivity to 1A11. Overlapped residues of peptides 2 to 4 are underlined. Data are means ± SDs of the OD<sub>450</sub> reading from triplicate wells. (<b>b</b>) Amino acid sequences of the epitope region among multiple serotypes of different enterovirus species are aligned. Conserved residues are shadowed, and a core conserved sequence is indicated in the box. (<b>c</b>) A panel of 10 truncated peptides in the epitope region, as indicated, was used to detect reactivity to 1A11 in ELISA. The inferred minimal epitope is underlined. Dashes indicate deleted amino acids of peptides b–j from the original sequence of a. Data are means ± SDs of the OD<sub>450</sub> reading from triplicate wells.</p> "> Figure 5
<p>Binding ability of 1A11 mAb to single mutation peptides of enterovirus. A panel of 9 single-mutation peptides (mutated residues highlighted) on the epitope region were used to detect reactivity to 1A11 in ELISA. Two amino acids were retained upstream of the N-termini of the mutant epitopes to reduce the effect of the N-terminal amino on the binding reactivity. Data are means ± SDs of the OD<sub>450</sub> reading from triplicate wells.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Cells, Viruses and Anti-Serum
2.3. Generation of Monoclonal Antibody (mAb)
2.4. Indirect Immunofluorescence Assay (IFA)
2.5. Western Blotting
2.6. Indirect ELISA
2.7. Epitope Mapping of mAb
2.8. BLAST of mAb Epitope
3. Results
3.1. Generation and Characterization of mAb 1A11
3.2. Binding Specificity of the Antibody to the Enterovirus A Species
3.3. Epitope Mapping of mAb 1A11
3.4. BLAST Analysis of 1A11 Epitope
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Enterovirus Species | Serotypes | Total Sequence No. | Identical Sequence No. | Sequence with a Single Mutation (No.) 1 | Percentage of Identical Sequence (%) |
---|---|---|---|---|---|
Enterovirus A (taxid: 138948) | CV-A2 | 63 | 63 | 0 | 100 |
CV-A4 | 107 | 107 | 0 | 100 | |
CV-A5 | 62 | 62 | 0 | 100 | |
CV-A6 | 977 | 977 | 0 | 100 | |
CV-A10 | 247 | 243 | PILHGF (2) PILPRF (2) | 98.4 | |
CV-A16 | 523 | 522 | PILPNF (1) | 99.8 | |
EV-A71 | 1491 | 3 | PILPNF (1487) | 0.2 | |
EV-A91 | 2 | 0 | PILPGY (2) | 0 | |
EV-A121 | 1 | 0 | PILPGY (1) | 0 | |
EV-A125 | 1 | 0 | PVLPGF (1) | 0 | |
SV19 | 9 | 8 | PILPGY (1) | 88.9 | |
Other serotypes | 171 | 171 | 0 | 100 | |
Enterovirus D (taxid: 138951) | EV-D68 | 1052 | 0 | PILPCF (3) | 0 |
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Fu, L.; Zhang, X.-Y.; Jin, W.-P.; Wang, C.; Qian, S.-S.; Wang, M.-J.; Wang, W.-H.; Meng, S.-L.; Guo, J.; Wang, Z.-J.; et al. Identification of a Conserved, Linear Epitope on VP3 of Enterovirus A Species Recognized by a Broad-Spectrum Monoclonal Antibody. Viruses 2023, 15, 1028. https://doi.org/10.3390/v15041028
Fu L, Zhang X-Y, Jin W-P, Wang C, Qian S-S, Wang M-J, Wang W-H, Meng S-L, Guo J, Wang Z-J, et al. Identification of a Conserved, Linear Epitope on VP3 of Enterovirus A Species Recognized by a Broad-Spectrum Monoclonal Antibody. Viruses. 2023; 15(4):1028. https://doi.org/10.3390/v15041028
Chicago/Turabian StyleFu, Lie, Xiao-Yu Zhang, Wei-Ping Jin, Chen Wang, Sha-Sha Qian, Meng-Jun Wang, Wen-Hui Wang, Sheng-Li Meng, Jing Guo, Ze-Jun Wang, and et al. 2023. "Identification of a Conserved, Linear Epitope on VP3 of Enterovirus A Species Recognized by a Broad-Spectrum Monoclonal Antibody" Viruses 15, no. 4: 1028. https://doi.org/10.3390/v15041028