Characterization of an In Vivo Neutralizing Anti-Vaccinia Virus D8 Single-Chain Fragment Variable (scFv) from a Human Anti-Vaccinia Virus-Specific Recombinant Library
Abstract
:1. Introduction
2. Material and Methods
2.1. Immunization and Lymphocyte Preparation
2.2. Library Construction
2.3. Cells and Viruses
2.4. Gradient Purification of Vaccinia Viruses Elstree and Munich 1
2.5. ScFv Selection Using Purified Vaccinia Virus Elstree
2.6. Screening of Randomly Selected scFv-Producing HB2151 Clones in Indirect ELISAs
2.7. Purification of Selected scFvs
2.8. Competitive ELISA for Epitope Detection
2.9. Engineering of Specific Binding scFv to Human scFv-Fc and IgG1 Molecules
2.10. SDS-PAGE and Western Blotting for the Detection of the Target Virus Protein
2.11. Enzyme-Linked Immunosorbent Assay (ELISA)
2.12. In Vitro Plaque Reduction Neutralization Test (PRNT)
2.13. In Vivo Neutralization
3. Results
3.1. Immunization, Library Construction, and Characterization
3.2. Selection of Vaccinia-Virus-Specific scFv
3.3. Binding Characteristics of scFv-1.2.2.H9, scFv-Fc-1.2.2.H9, and IgG1-1.2.2.H9 in ELISA
3.4. Epitope Mapping
3.5. In Vitro Neutralization
3.6. In Vivo Passive Protection
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Epitope ID | MAb | Isotype | Virus Strain Used for mAb Production | Isotype |
---|---|---|---|---|
2A | 1B3/1A11 | IgG2a | VACV M1 | IgG2a |
2B | 1F7/2F9 | IgG2b | ECTV M1 | IgG2b |
2D | 3D11/2G7 | IgG2a | CPXV KR2 Brighton | IgG1 |
2G | 4C4/2B6 | IgG2a | CPXV KR2 Brighton | IgG1 |
Equality Test over Levels | ||||
---|---|---|---|---|
Test | Chi-Quadrat | DF | Pr > Chi-Quadrat | |
Log-Rank | 20.8477 | 5 | 0.0009 | |
Adjustment for Multiple Comparisons for the Log-Rank Test | ||||
Strata Comparison | Chi-Quadrat | p-Values | ||
Group | Group | Raw | Sidak Adjustment | |
1 | 2 | 1.2919 | 0.2557 | 0.9881 |
1 | 3 | 0.00968 | 0.9216 | 1.0000 |
1 | 4 | 0.8085 | 0.3686 | 0.9990 |
1 | 5 | 2.1580 | 0.1418 | 0.8992 |
1 | 6 | 6.8927 | 0.0087 | 0.1222 |
2 | 3 | 1.4918 | 0.2219 | 0.9768 |
2 | 4 | 4.2725 | 0.0387 | 0.4471 |
2 | 5 | 0.1233 | 0.7255 | 1.0000 |
2 | 6 | 1.9182 | 0.1661 | 0.9344 |
3 | 4 | 0.6326 | 0.4264 | 0.9998 |
3 | 5 | 2.3846 | 0.1225 | 0.8593 |
3 | 6 | 7.3013 | 0.0069 | 0.0985 |
4 | 5 | 5.7236 | 0.0167 | 0.2237 |
4 | 6 | 13.4020 | 0.0003 | 0.0038 |
5 | 6 | 0.9339 | 0.3339 | 0.9977 |
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Diesterbeck, U.S.; Ahsendorf, H.P.; Frenzel, A.; Sharifi, A.R.; Schirrmann, T.; Czerny, C.-P. Characterization of an In Vivo Neutralizing Anti-Vaccinia Virus D8 Single-Chain Fragment Variable (scFv) from a Human Anti-Vaccinia Virus-Specific Recombinant Library. Vaccines 2021, 9, 1308. https://doi.org/10.3390/vaccines9111308
Diesterbeck US, Ahsendorf HP, Frenzel A, Sharifi AR, Schirrmann T, Czerny C-P. Characterization of an In Vivo Neutralizing Anti-Vaccinia Virus D8 Single-Chain Fragment Variable (scFv) from a Human Anti-Vaccinia Virus-Specific Recombinant Library. Vaccines. 2021; 9(11):1308. https://doi.org/10.3390/vaccines9111308
Chicago/Turabian StyleDiesterbeck, Ulrike S., Henrike P. Ahsendorf, André Frenzel, Ahmad Reza Sharifi, Thomas Schirrmann, and Claus-Peter Czerny. 2021. "Characterization of an In Vivo Neutralizing Anti-Vaccinia Virus D8 Single-Chain Fragment Variable (scFv) from a Human Anti-Vaccinia Virus-Specific Recombinant Library" Vaccines 9, no. 11: 1308. https://doi.org/10.3390/vaccines9111308
APA StyleDiesterbeck, U. S., Ahsendorf, H. P., Frenzel, A., Sharifi, A. R., Schirrmann, T., & Czerny, C.-P. (2021). Characterization of an In Vivo Neutralizing Anti-Vaccinia Virus D8 Single-Chain Fragment Variable (scFv) from a Human Anti-Vaccinia Virus-Specific Recombinant Library. Vaccines, 9(11), 1308. https://doi.org/10.3390/vaccines9111308