{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T10:18:24Z","timestamp":1787566704249,"version":"build-2736575974"},"reference-count":39,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T00:00:00Z","timestamp":1787443200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project","award":["2025ZD01902100"],"award-info":[{"award-number":["2025ZD01902100"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Population immune pressure from vaccination and prior infection continues to drive the evolution of SARS-CoV-2. Systematic characterization of RBD mutations under complex immune backgrounds is essential for understanding viral adaptation and evolutionary trajectories. Here, we applied a deep mutational scanning (DMS) to comprehensively map the neutralization escape landscape of the Omicron variant JN.1 and its descendant lineage XEC, under immune pressure from individuals who experienced Omicron breakthrough infections following three doses of inactivated vaccines. A neutralization escape map for the single amino acid substitutions in the RBD of JN.1 or XEC was generated, and the escape efficiency of each mutation was determined. The results show that RBD escape mutations are hierarchically organized: low-intensity signals are widespread, whereas high-intensity escape is confined to a few key sites. These escape mutations are not confined solely to the receptor-binding motif (RBM) but are broadly distributed across the entire RBD. Many escape sites could accommodate multiple amino acid substitutions. Integration of DMS data with genomic surveillance of circulating variants from 2024 to 2025 revealed significant overlap between experimentally identified escape sites and mutations observed in natural isolates. This overlap increased substantially in 2025, with site concordance rising from 27.17% and 26.81% to 45.09% and 47.10% for JN.1 and XEC, respectively. The natural prevalence of these escape mutations is further shaped by factors such as receptor-binding affinity, protein stability, and epistatic interactions. Overall, our findings suggest that SARS-CoV-2 antigenic evolution follows the pattern of multiple pathways within a constrained space, providing new insights into the adaptive mechanisms of Omicron-derived variants under hybrid immune pressure.<\/jats:p>","DOI":"10.3390\/microorganisms14091872","type":"journal-article","created":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T08:03:41Z","timestamp":1787558621000},"page":"1872","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Serum Escape Landscape of SARS-CoV-2 Omicron JN.1 and XEC RBD Under COVID-19 Vaccine Breakthrough Immunity in China"],"prefix":"10.3390","volume":"14","author":[{"given":"Chengwei","family":"Shao","sequence":"first","affiliation":[{"name":"School of Public Health, Southeast University, Nanjing 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0424-7492","authenticated-orcid":false,"given":"Jianguang","family":"Fu","sequence":"additional","affiliation":[{"name":"Jiangsu Provincial Medical Innovation Center, National Health Commission Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Academy of Preventive Medicine), Nanjing 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Deng","sequence":"additional","affiliation":[{"name":"Jiangsu Provincial Medical Innovation Center, National Health Commission Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Academy of Preventive Medicine), Nanjing 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huiyan","family":"Yu","sequence":"additional","affiliation":[{"name":"Jiangsu Provincial Medical Innovation Center, National Health Commission Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Academy of Preventive Medicine), Nanjing 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huan","family":"Fan","sequence":"additional","affiliation":[{"name":"Jiangsu Provincial Medical Innovation Center, National Health Commission Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Academy of Preventive Medicine), Nanjing 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanjun","family":"Chen","sequence":"additional","affiliation":[{"name":"Jiangsu Provincial Medical Innovation Center, National Health Commission Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Academy of Preventive Medicine), Nanjing 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ke","family":"Xu","sequence":"additional","affiliation":[{"name":"Jiangsu Provincial Medical Innovation Center, National Health Commission Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Academy of Preventive Medicine), Nanjing 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingwei","family":"Wei","sequence":"additional","affiliation":[{"name":"Jiangsu Provincial Medical Innovation Center, National Health Commission Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Academy of Preventive Medicine), Nanjing 210009, China"},{"name":"School of Public Health, National Vaccine Innovation Platform, Nanjing Medical University, Nanjing 211166, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siyue","family":"Jia","sequence":"additional","affiliation":[{"name":"Jiangsu Provincial Medical Innovation Center, National Health Commission Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Academy of Preventive Medicine), Nanjing 210009, China"},{"name":"School of Public Health, National Vaccine Innovation Platform, Nanjing Medical University, Nanjing 211166, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoyan","family":"Jia","sequence":"additional","affiliation":[{"name":"Institute of Pediatrics, Guangzhou Women and Children\u2019s Medical Center, Guangzhou Medical University, Guangzhou 511300, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liguo","family":"Zhu","sequence":"additional","affiliation":[{"name":"Jiangsu Provincial Medical Innovation Center, National Health Commission Key Laboratory of Enteric Pathogenic Microbiology, Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Academy of Preventive Medicine), Nanjing 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7033-5151","authenticated-orcid":false,"given":"Jingxin","family":"Li","sequence":"additional","affiliation":[{"name":"School of Public Health, Southeast University, Nanjing 210009, China"},{"name":"School of Public Health, National Vaccine Innovation Platform, Nanjing Medical University, Nanjing 211166, China"},{"name":"Taizhou Institute of Pharmaceutical Industry, Nanjing Medical University, Taizhou 225300, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"eabq1841","DOI":"10.1126\/science.abq1841","article-title":"Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure","volume":"377","author":"Reynolds","year":"2022","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1038\/s41586-022-05644-7","article-title":"Imprinted SARS-CoV-2 humoral immunity induces convergent Omicron RBD evolution","volume":"614","author":"Cao","year":"2023","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1038\/s41579-021-00573-0","article-title":"SARS-CoV-2 variants, spike mutations and immune escape","volume":"19","author":"Harvey","year":"2021","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1038\/s41586-021-04385-3","article-title":"Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies","volume":"602","author":"Cao","year":"2022","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/s41586-021-04388-0","article-title":"Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2","volume":"602","author":"Liu","year":"2022","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.chom.2022.11.012","article-title":"Enhanced neutralization resistance of SARS-CoV-2 Omicron subvariants BQ.1, BQ.1.1, BA.4.6, BF.7, and BA.2.75.2","volume":"31","author":"Qu","year":"2023","journal-title":"Cell Host Microbe"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1038\/s41586-024-07636-1","article-title":"Spike deep mutational scanning helps predict success of SARS-CoV-2 clades","volume":"631","author":"Dadonaite","year":"2024","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"850","DOI":"10.1126\/science.abf9302","article-title":"Prospective mapping of viral mutations that escape antibodies used to treat COVID-19","volume":"371","author":"Starr","year":"2021","journal-title":"Science"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Crawford, K.H.D., Eguia, R., Dingens, A.S., Loes, A.N., Malone, K.D., Wolf, C.R., Chu, H.Y., Tortorici, M.A., Veesler, D., and Murphy, M. (2020). Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses, 12.","DOI":"10.1101\/2020.04.20.051219"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1038\/s41591-021-01678-y","article-title":"An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes neutralization by therapeutic monoclonal antibodies","volume":"28","author":"VanBlargan","year":"2022","journal-title":"Nat. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1038\/s41586-021-04389-z","article-title":"Considerable escape of SARS-CoV-2 Omicron to antibody neutralization","volume":"602","author":"Planas","year":"2022","journal-title":"Nature"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1038\/nmeth.3027","article-title":"Deep mutational scanning: A new style of protein science","volume":"11","author":"Fowler","year":"2014","journal-title":"Nat. Methods"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4008","DOI":"10.1016\/j.cell.2022.08.024","article-title":"Deep mutational learning predicts ACE2 binding and antibody escape to combinatorial mutations in the SARS-CoV-2 receptor-binding domain","volume":"185","author":"Taft","year":"2022","journal-title":"Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1038\/s41392-021-00863-2","article-title":"SARS-CoV-2 Omicron RBD shows weaker binding affinity than the currently dominant Delta variant to human ACE2","volume":"7","author":"Wu","year":"2022","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2003","DOI":"10.1038\/s41564-025-02030-7","article-title":"Viral evolution prediction identifies broadly neutralizing antibodies to existing and prospective SARS-CoV-2 variants","volume":"10","author":"Jian","year":"2025","journal-title":"Nat. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1038\/s41586-020-2456-9","article-title":"Convergent antibody responses to SARS-CoV-2 in convalescent individuals","volume":"584","author":"Robbiani","year":"2020","journal-title":"Nature"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"abm0829","DOI":"10.1126\/science.abm0829","article-title":"mRNA vaccines induce durable immune memory to SARS-CoV-2 and variants of concern","volume":"374","author":"Goel","year":"2021","journal-title":"Science"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1038\/s41586-023-06753-7","article-title":"Repeated Omicron exposures override ancestral SARS-CoV-2 immune imprinting","volume":"625","author":"Yisimayi","year":"2024","journal-title":"Nature"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1263","DOI":"10.1016\/j.cell.2023.02.001","article-title":"A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike","volume":"186","author":"Dadonaite","year":"2023","journal-title":"Cell"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2686472","DOI":"10.1080\/22221751.2026.2686472","article-title":"Deep mutational scanning reveals the antibody escape and infectivity landscape of SARS-CoV-2 Omicron JN.1 and XEC receptor-binding domains","volume":"15","author":"Shao","year":"2026","journal-title":"Emerg. Microbes Infect."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2404","DOI":"10.1093\/bioinformatics\/btac137","article-title":"BWA-MEME: BWA-MEM emulated with a machine learning approach","volume":"38","author":"Jung","year":"2022","journal-title":"Bioinformatics"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1093\/bib\/bbs017","article-title":"Integrative Genomics Viewer (IGV): High-performance genomics data visualization and exploration","volume":"14","author":"Robinson","year":"2013","journal-title":"Brief. Bioinform."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Bushnell, B., Rood, J., and Singer, E. (2017). BBMerge\u2014Accurate paired shotgun read merging via overlap. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0185056"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1138","DOI":"10.1089\/cmb.2017.0096","article-title":"cutPrimers: A New Tool for Accurate Cutting of Primers from Reads of Targeted Next Generation Sequencing","volume":"24","author":"Kechin","year":"2017","journal-title":"J. Comput. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1913","DOI":"10.1093\/bioinformatics\/btv053","article-title":"Starcode: Sequence clustering based on all-pairs search","volume":"31","author":"Zorita","year":"2015","journal-title":"Bioinformatics"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"463","DOI":"10.1016\/j.chom.2021.02.003","article-title":"Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies","volume":"29","author":"Greaney","year":"2021","journal-title":"Cell Host Microbe"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4196","DOI":"10.1038\/s41467-021-24435-8","article-title":"Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies","volume":"12","author":"Greaney","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e0142325","DOI":"10.1128\/jvi.01423-25","article-title":"Spike mutations that affect the function and antigenicity of recent KP.3.1.1-like SARS-CoV-2 variants","volume":"99","author":"Dadonaite","year":"2025","journal-title":"J. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Ramasamy, R. (2025). Perspective Overview of Changing Population Immunity to COVID-19 in the Context of Infection, Vaccination, and Emerging SARS-CoV-2 Variants. Pathogens, 14.","DOI":"10.3390\/pathogens14121197"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.chom.2020.11.007","article-title":"Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition","volume":"29","author":"Greaney","year":"2021","journal-title":"Cell Host Microbe"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1126\/science.abb9983","article-title":"Site-specific glycan analysis of the SARS-CoV-2 spike","volume":"369","author":"Watanabe","year":"2020","journal-title":"Science"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"evag049","DOI":"10.1093\/gbe\/evag049","article-title":"Structural Constraints Acting on the SARS-CoV-2 Spike Protein Reveal Limited Space for Viral Adaptation","volume":"18","author":"Herzig","year":"2026","journal-title":"Genome Biol. Evol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"101452","DOI":"10.1016\/j.cels.2025.101452","article-title":"One thousand SARS-CoV-2 antibody structures reveal convergent binding and near-universal immune escape","volume":"17","author":"Feng","year":"2026","journal-title":"Cell Syst."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"vead055","DOI":"10.1093\/ve\/vead055","article-title":"Fitness effects of mutations to SARS-CoV-2 proteins","volume":"9","author":"Bloom","year":"2023","journal-title":"Virus Evol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1457","DOI":"10.1038\/s41559-023-02123-8","article-title":"Immune evasion and ACE2 binding affinity contribute to SARS-CoV-2 evolution","volume":"7","author":"Ma","year":"2023","journal-title":"Nat. Ecol. Evol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"899","DOI":"10.1038\/s41564-021-00908-w","article-title":"The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets","volume":"6","author":"Peacock","year":"2021","journal-title":"Nat. Microbiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1126\/science.abo7896","article-title":"Shifting mutational constraints in the SARS-CoV-2 receptor-binding domain during viral evolution","volume":"377","author":"Starr","year":"2022","journal-title":"Science"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"145240","DOI":"10.1016\/j.molstruc.2026.145240","article-title":"Design, Synthesis and Computational Insights of 7-Hydroxystilbene-Coumarin Hybrid Scaffolds as SARS-CoV-2 3CLpro Inhibitors","volume":"1357","author":"Khamto","year":"2026","journal-title":"J. Mol. Struct."}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/9\/1872\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T08:19:37Z","timestamp":1787559577000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/9\/1872"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,23]]},"references-count":39,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["microorganisms14091872"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14091872","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,23]]}}}