{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T17:02:41Z","timestamp":1773680561099,"version":"3.50.1"},"reference-count":31,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T00:00:00Z","timestamp":1758585600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2023YFC3404000"],"award-info":[{"award-number":["2023YFC3404000"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Adeno-associated virus (AAV) vectors are the preferred gene delivery tool in gene therapy owing to their safety, long-term gene expression, broad tissue tropism, and low immunogenicity. Affinity ligands that can bind multiple AAV serotypes endure harsh clean-in-place (CIP) conditions and are critical for industrial-scale purification. However, current ligands lack broad serotype recognition and adequate alkaline stability, which limits their reusability in large-scale manufacturing. In this study, we employed a competitive biopanning strategy to isolate a single-domain antibody (VHH) that simultaneously binds AAV2, AAV8, and AAV9. The VHH retained structural integrity and binding activity after exposure to 0.1 M NaOH, demonstrating robust alkaline stability. Structural modeling revealed that the VHH primarily recognizes the DE loop region of the VP3 capsid protein across the three serotypes, explaining its cross-serotype reactivity. Affinity chromatography using the VHH yielded infectious AAV particles, confirming its potential for downstream processing. This strategy provides a versatile platform for developing high-performance AAV affinity ligands and may be extended to other viral vector systems.<\/jats:p>","DOI":"10.3390\/v17101289","type":"journal-article","created":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T13:48:37Z","timestamp":1758635317000},"page":"1289","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Screening of Single-Domain Antibodies to Adeno-Associated Viruses with Cross-Serotype Specificity and a Wide pH Tolerance"],"prefix":"10.3390","volume":"17","author":[{"given":"Hailing","family":"Guo","sequence":"first","affiliation":[{"name":"Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6275-9675","authenticated-orcid":false,"given":"Shuo","family":"Wang","sequence":"additional","affiliation":[{"name":"Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lujin","family":"Feng","sequence":"additional","affiliation":[{"name":"Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weiwei","family":"Xu","sequence":"additional","affiliation":[{"name":"YSK Bio Co., Ltd., Zhuji 311899, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiandong","family":"Zhang","sequence":"additional","affiliation":[{"name":"YSK Bio Co., Ltd., Zhuji 311899, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaoju","family":"Zhou","sequence":"additional","affiliation":[{"name":"School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ningning","family":"Ma","sequence":"additional","affiliation":[{"name":"Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3515","DOI":"10.1016\/j.ymthe.2022.09.015","article-title":"AAV Vectors: The Rubik\u2019s Cube of Human Gene Therapy","volume":"30","author":"Pupo","year":"2022","journal-title":"Mol. Ther."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1007\/s40259-017-0234-5","article-title":"Adeno-Associated Virus (AAV) as a Vector for Gene Therapy","volume":"31","author":"Naso","year":"2017","journal-title":"Biodrugs"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1903","DOI":"10.1016\/j.ymthe.2025.03.064","article-title":"AAV Vector Development, Back to the Future","volume":"33","author":"Song","year":"2025","journal-title":"Mol. Ther. J. Am. Soc. Gene Ther."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1038\/gt.2008.170","article-title":"Tailoring the AAV Vector Capsid for Gene Therapy","volume":"16","author":"Vandenberghe","year":"2009","journal-title":"Gene Ther."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"849","DOI":"10.1016\/S0140-6736(17)31868-8","article-title":"Efficacy and Safety of Voretigene Neparvovec (AAV2-hRPE65v2) in Patients with RPE65-Mediated Inherited Retinal Dystrophy: A Randomised, Controlled, Open-Label, Phase 3 Trial","volume":"390","author":"Russell","year":"2017","journal-title":"Lancet"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1713","DOI":"10.1056\/NEJMoa1706198","article-title":"Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy","volume":"377","author":"Mendell","year":"2017","journal-title":"N. Engl. J. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1038\/s41392-024-01780-w","article-title":"Adeno-Associated Virus as a Delivery Vector for Gene Therapy of Human Diseases","volume":"9","author":"Wang","year":"2024","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1013","DOI":"10.1056\/NEJMoa2113708","article-title":"Valoctocogene Roxaparvovec Gene Therapy for Hemophilia A","volume":"386","author":"Ozelo","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1038\/s41576-019-0205-4","article-title":"Engineering Adeno-Associated Virus Vectors for Gene Therapy","volume":"21","author":"Li","year":"2020","journal-title":"Nat. Rev. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"706","DOI":"10.1056\/NEJMoa2211644","article-title":"Gene Therapy with Etranacogene Dezaparvovec for Hemophilia B","volume":"388","author":"Pipe","year":"2023","journal-title":"N. Engl. J. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"5887","DOI":"10.1128\/JVI.00254-08","article-title":"In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses","volume":"82","author":"Grimm","year":"2008","journal-title":"J. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"824","DOI":"10.1186\/s12967-024-05599-5","article-title":"AAV-DJ Is Superior to AAV9 for Targeting Brain and Spinal Cord, and de-Targeting Liver across Multiple Delivery Routes in Mice","volume":"22","author":"Chauhan","year":"2024","journal-title":"J. Transl. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"679483","DOI":"10.3389\/fbioe.2021.679483","article-title":"PhP.B Enhanced Adeno-Associated Virus Mediated-Expression Following Systemic Delivery or Direct Brain Administration","volume":"9","author":"Pietersz","year":"2021","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Kim, J.J., Kurial, S.N., Choksi, P.K., Nunez, M., Lunow-Luke, T., Bartel, J., Driscoll, J., Her, C.L., Dhillon, S., and Yue, W. (2025). AAV Capsid Prioritization in Normal and Steatotic Human Livers Maintained by Machine Perfusion. Nat. Biotechnol.","DOI":"10.1038\/s41587-024-02523-6"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1016\/j.omtm.2022.12.014","article-title":"Characterization of the Humanized FRG Mouse Model and Development of an AAV-LK03 Variant with Improved Liver Lobular Biodistribution","volume":"28","author":"Navarro","year":"2023","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Zhao, Y., Zhang, L., Wang, D., Chen, B., and Shu, Y. (2022). Approaches and Vectors for Efficient Cochlear Gene Transfer in Adult Mouse Models. Biomolecules, 13.","DOI":"10.3390\/biom13010038"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.omtm.2023.05.016","article-title":"In-Depth Comparison of Anc80L65 and AAV9 Retinal Targeting and Characterization of Cross-Reactivity to Multiple AAV Serotypes in Humans","volume":"30","author":"Schwartz","year":"2023","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1186\/s12985-025-02662-5","article-title":"Long-Term Inhibition of Hepatitis B Virus Gene Expression by a Primary Microrna Expressing Ancestral Adeno-Associated Viral Vector","volume":"22","author":"Mnyandu","year":"2025","journal-title":"Virol. J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2712","DOI":"10.1002\/bit.26031","article-title":"Insight into the Mechanisms of Enhanced Retinal Transduction by the Engineered AAV2 Capsid Variant -7m8","volume":"113","author":"Khabou","year":"2016","journal-title":"Biotechnol. Bioeng."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1016\/j.omtm.2020.10.001","article-title":"Characterization of AAV-Specific Affinity Ligands: Consequences for Vector Purification and Development Strategies","volume":"19","author":"Mietzsch","year":"2020","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1016\/j.omtm.2022.12.009","article-title":"High-Efficiency Purification of Divergent AAV Serotypes Using AAVX Affinity Chromatography","volume":"28","author":"Florea","year":"2023","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"15040","DOI":"10.1038\/mtm.2015.40","article-title":"Identification of an Adeno-Associated Virus Binding Epitope for AVB Sepharose Affinity Resin","volume":"2","author":"Wang","year":"2015","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2283","DOI":"10.1002\/bit.28495","article-title":"Peptide Ligands for the Affinity Purification of Adeno-Associated Viruses from HEK 293 Cell Lysates","volume":"120","author":"Chu","year":"2023","journal-title":"Biotechnol. Bioeng."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e2300230","DOI":"10.1002\/biot.202300230","article-title":"Rational Design and Experimental Evaluation of Peptide Ligands for the Purification of Adeno-Associated Viruses via Affinity Chromatography","volume":"19","author":"Shastry","year":"2024","journal-title":"Biotechnol. J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1038\/s41434-020-00198-7","article-title":"Engineered AAV8 Capsid Acquires Heparin and AVB Sepharose Binding Capacity but Has Altered in Vivo Transduction Efficiency","volume":"30","author":"Stegelmeier","year":"2023","journal-title":"Gene Ther."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Jin, B., Odongo, S., Radwanska, M., and Magez, S. (2023). NANOBODIES\u00ae: A Review of Generation, Diagnostics and Therapeutics. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24065994"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jim.2007.04.005","article-title":"Improved Anti-IgG and HSA Affinity Ligands: Clinical Application of VHH Antibody Technology","volume":"324","author":"Klooster","year":"2007","journal-title":"J. Immunol. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Zhang, L., Zang, B., Huang, C., Ren, J., and Jia, L. (2019). One-Step Preparation of a VHH-Based Immunoadsorbent for the Extracorporeal Removal of \u03922-Microglobulin. Molecules, 24.","DOI":"10.3390\/molecules24112119"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Issa, S.S., Shaimardanova, A.A., Solovyeva, V.V., and Rizvanov, A.A. (2023). Various AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12.","DOI":"10.3390\/cells12050785"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3760","DOI":"10.1038\/s41467-019-11668-x","article-title":"Divergent Engagements between Adeno-Associated Viruses with Their Cellular Receptor AAVR","volume":"10","author":"Zhang","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"192","DOI":"10.4161\/mabs.3.2.14874","article-title":"A Tool for Increasing the Lifetime of Chromatography Resins","volume":"3","author":"Eriksson","year":"2011","journal-title":"MAbs"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/10\/1289\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:47:54Z","timestamp":1760035674000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/10\/1289"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,23]]},"references-count":31,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["v17101289"],"URL":"https:\/\/doi.org\/10.3390\/v17101289","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,23]]}}}