{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T04:14:25Z","timestamp":1783743265042,"version":"3.55.0"},"reference-count":88,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T00:00:00Z","timestamp":1783555200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"CTL"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background: Despite the circulation of evolutionarily related cold-causing coronaviruses (CCCs) in the pre-COVID era, most individuals lacked pre-existing serum IgG and\/or class-switched memory B cell (Bmem) reactivity for the SARS-CoV-2 Spike (S) glycoprotein expressed by the ancestral Wuhan-Hu-1 (WH1) strain. Subsequent priming of the immune system through natural infection or prophylactic COVID-19 mRNA vaccination successfully generated robust Bmem responses against the WH1-S antigen, along with eliciting cross-reactivity for the future Omicron (BA.1) variant responsible for breakthrough infections (BTIs). However, to what extent immunological imprinting of Bmem towards the WH1-S antigen detrimentally constrains the elicitation of variant-specific antibody responses following subsequent booster vaccinations or BTIs\u2014a phenomena referred to as \u201coriginal antigenic sin\u201d\u2014remains an unresolved and open question. Methods: Using ImmunoSpot\u00ae, we evaluated peripheral blood mononuclear cells (PBMCs) from defined human cohorts for IgG+ ASC reactivity against Spike proteins representing CCCs and SARS-CoV-2. Additionally, we developed a novel dual-label inverted FluoroSpot assay to distinguish between strain-specific and cross-reactive IgG+ ASCs recognizing epitopes in the receptor binding domain (RBD) of SARS-CoV-2 Omicron variants. Results: Our data demonstrate a lack of appreciable back-boosting of IgG+ Bmem recognizing structurally conserved epitopes shared between CCCs and SARS-CoV-2. Moreover, we found evidence for immunological imprinting and the preferential expansion of Bmem recognizing cross-reactive epitopes in the RBD following BTI. Nevertheless, Omicron strain-specific Bmem were detected in PBMC donors collected in 2025. Conclusions: Our novel inverted dual-label FluoroSpot methodology evidenced preferential expansion of cross-reactive Bmem following breakthrough SARS-CoV-2 infection and supports the influence of original antigenic sin shaping the recall response. Moreover, the inverted dual-label assay provides a highly flexible and easily implementable technique for distinguishing between strain-specific and cross-reactive B cell responses and has broad applications in translational vaccine research against pathogens that undergo antigenic drift.<\/jats:p>","DOI":"10.3390\/vaccines14070604","type":"journal-article","created":{"date-parts":[[2026,7,9]],"date-time":"2026-07-09T10:16:22Z","timestamp":1783592182000},"page":"604","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluating Memory B Cell Cross-Reactivity Between Ancestral and Future SARS-CoV-2 Variants\u2014Evidence for Original Antigenic Sin"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-3134-8630","authenticated-orcid":false,"given":"Lingling","family":"Yao","sequence":"first","affiliation":[{"name":"Research and Development, Cellular Technology Ltd. (CTL), Shaker Heights, OH 44122, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zolt\u00e1n","family":"Megyesi","sequence":"additional","affiliation":[{"name":"CTL Software Ltd., V\u00e1g\u00f3 str. 2, H-6000 Kecskem\u00e9t, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul V.","family":"Lehmann","sequence":"additional","affiliation":[{"name":"Research and Development, Cellular Technology Ltd. (CTL), Shaker Heights, OH 44122, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6463-7623","authenticated-orcid":false,"given":"Greg A.","family":"Kirchenbaum","sequence":"additional","affiliation":[{"name":"Research and Development, Cellular Technology Ltd. (CTL), Shaker Heights, OH 44122, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1146\/annurev-virology-093022-013037","article-title":"The Emergence and Evolution of SARS-CoV-2","volume":"11","author":"Holmes","year":"2024","journal-title":"Annu. Rev. Virol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"3181","DOI":"10.1111\/tbed.14732","article-title":"The origins of COVID-19 pandemic: A brief overview","volume":"69","author":"Hao","year":"2022","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"929","DOI":"10.1056\/NEJMoa2001191","article-title":"First Case of 2019 Novel Coronavirus in the United States","volume":"382","author":"Holshue","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"190","DOI":"10.3181\/00379727-121-30734","article-title":"A New Virus Isolated from the Human Respiratory Tract","volume":"121","author":"Hamre","year":"1966","journal-title":"Proc. Soc. Exp. Biol. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1086\/374222","article-title":"An Outbreak of Coronavirus OC43 Respiratory Infection in Normandy, France","volume":"36","author":"Vabret","year":"2003","journal-title":"Clin. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1038\/nm1024","article-title":"Identification of a new human coronavirus","volume":"10","author":"Pyrc","year":"2004","journal-title":"Nat. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"884","DOI":"10.1128\/JVI.79.2.884-895.2005","article-title":"Characterization and Complete Genome Sequence of a Novel Coronavirus, Coronavirus HKU1, from Patients with Pneumonia","volume":"79","author":"Woo","year":"2005","journal-title":"J. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2940","DOI":"10.1128\/JCM.00636-10","article-title":"Epidemiology and Clinical Presentations of the Four Human Coronaviruses 229E, HKU1, NL63, and OC43 Detected over 3 Years Using a Novel Multiplex Real-Time PCR Method","volume":"48","author":"Gaunt","year":"2010","journal-title":"J. Clin. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/j.jcv.2011.11.011","article-title":"The dominance of human coronavirus OC43 and NL63 infections in infants","volume":"53","author":"Dijkman","year":"2012","journal-title":"J. Clin. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.jcv.2018.01.019","article-title":"Human coronavirus circulation in the United States 2014\u20132017","volume":"101","author":"Killerby","year":"2018","journal-title":"J. Clin. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1033","DOI":"10.1038\/s41591-020-0913-5","article-title":"A serological assay to detect SARS-CoV-2 seroconversion in humans","volume":"26","author":"Amanat","year":"2020","journal-title":"Nat. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1858","DOI":"10.1016\/j.cell.2021.02.010","article-title":"Seasonal human coronavirus antibodies are boosted upon SARS-CoV-2 infection but not associated with protection","volume":"184","author":"Anderson","year":"2021","journal-title":"Cell"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1093\/ajcp\/aqaa123","article-title":"SARS-CoV-2 Antibody Responses Do Not Predict COVID-19 Disease Severity","volume":"154","author":"Phipps","year":"2020","journal-title":"Am. J. Clin. Pathol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"19893","DOI":"10.1038\/s41598-020-76914-5","article-title":"Detection of SARS-CoV-2 antibodies is insufficient for the diagnosis of active or cured COVID-19","volume":"10","author":"Escribano","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1016\/j.cell.2020.11.029","article-title":"Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19","volume":"184","author":"Rodda","year":"2021","journal-title":"Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1489","DOI":"10.1016\/j.cell.2020.05.015","article-title":"Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals","volume":"181","author":"Grifoni","year":"2020","journal-title":"Cell"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"eabi6950","DOI":"10.1126\/sciimmunol.abi6950","article-title":"Distinct antibody and memory B cell responses in SARS-CoV-2 na\u00efve and recovered individuals after mRNA vaccination","volume":"6","author":"Goel","year":"2021","journal-title":"Sci. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"408","DOI":"10.1038\/s41586-021-04188-6","article-title":"The emergence, genomic diversity and global spread of SARS-CoV-2","volume":"600","author":"Li","year":"2021","journal-title":"Nature"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1038\/s41392-022-00996-y","article-title":"COVID-19 vaccine development: Milestones, lessons and prospects","volume":"7","author":"Li","year":"2022","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2603","DOI":"10.1056\/NEJMoa2034577","article-title":"Safety and efficacy of the BNT162b2 mRNA COVID-19 vaccine","volume":"383","author":"Polack","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"572","DOI":"10.1038\/s41586-021-03653-6","article-title":"BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans","volume":"595","author":"Sahin","year":"2021","journal-title":"Nature"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2893","DOI":"10.1016\/j.immuni.2021.09.011","article-title":"mRNA vaccination of naive and COVID-19-recovered individuals elicits potent memory B cells that recognize SARS-CoV-2 variants","volume":"54","author":"Sokal","year":"2021","journal-title":"Immunity"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2427","DOI":"10.1056\/NEJMoa2028436","article-title":"Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults","volume":"383","author":"Anderson","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1038\/s41586-021-03738-2","article-title":"SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses","volume":"596","author":"Turner","year":"2021","journal-title":"Nature"},{"key":"ref_25","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_26","doi-asserted-by":"crossref","unstructured":"Ciabattini, A., Pastore, G., Fiorino, F., Polvere, J., Lucchesi, S., Pettini, E., Auddino, S., Rancan, I., Durante, M., and Miscia, M. (2021). Evidence of SARS-CoV-2-Specific Memory B Cells Six Months After Vaccination With the BNT162b2 mRNA Vaccine. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.740708"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Burns, M.D., Boribong, B.P., Bartsch, Y.C., Loiselle, M., Denis, K.J.S., Sheehan, M.L., Chen, J.W., Davis, J.P., Lima, R., and Edlow, A.G. (2022). Durability and Cross-Reactivity of SARS-CoV-2 mRNA Vaccine in Adolescent Children. Vaccines, 10.","DOI":"10.1101\/2022.01.05.22268617"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Yao, L., Becza, N., Stylianou, G., Tary-Lehmann, M., Todryk, S.M., Kirchenbaum, G.A., and Lehmann, P.V. (2025). SARS-CoV-2 Infection or COVID-19 mRNA Vaccination Elicits Partially Different Spike-Reactive Memory B Cell Responses in Na\u00efve Individuals. Vaccines, 13.","DOI":"10.20944\/preprints202507.1994.v1"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"20628","DOI":"10.1038\/s41598-022-24464-3","article-title":"Early-stage antibody kinetics after the third dose of BNT162b2 mRNA COVID-19 vaccination measured by a point-of-care fingertip whole blood testing","volume":"12","author":"Hagiya","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Jo, D.-H., Minn, D., Lim, J., Lee, K.-D., Kang, Y.-M., Choe, K.-W., and Kim, K.-N. (2021). Rapidly Declining SARS-CoV-2 Antibody Titers within 4 Months after BNT162b2 Vaccination. Vaccines, 9.","DOI":"10.3390\/vaccines9101145"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1093\/infdis\/jiac035","article-title":"SARS-CoV-2 Infectivity and Antibody Titer Reduction for 6 Months After Second Dose of BNT162b2 mRNA Vaccine in Health Care Workers: A Prospective Cohort Study","volume":"226","author":"Kim","year":"2022","journal-title":"J. Infect. Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1088","DOI":"10.1056\/NEJMc2119912","article-title":"SARS-CoV-2 Omicron Variant Neutralization after mRNA-1273 Booster Vaccination","volume":"386","author":"Pajon","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1016\/j.cell.2021.12.033","article-title":"MRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant","volume":"185","author":"Denis","year":"2022","journal-title":"Cell"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e2210780","DOI":"10.1001\/jamanetworkopen.2022.10780","article-title":"Assessment of Neutralizing Antibody Response Against SARS-CoV-2 Variants After 2 to 3 Doses of the BNT162b2 mRNA COVID-19 Vaccine","volume":"5","author":"Furukawa","year":"2022","journal-title":"JAMA Netw. Open"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1875","DOI":"10.1016\/j.cell.2022.04.009","article-title":"Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine","volume":"185","author":"Goel","year":"2022","journal-title":"Cell"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1038\/s41586-022-04778-y","article-title":"Increased memory B cell potency and breadth after a SARS-CoV-2 mRNA boost","volume":"607","author":"Muecksch","year":"2022","journal-title":"Nature"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1038\/s41586-022-04411-y","article-title":"Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa","volume":"603","author":"Viana","year":"2022","journal-title":"Nature"},{"key":"ref_38","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_39","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1016\/S0140-6736(21)02844-0","article-title":"Reduced neutralisation of SARS-CoV-2 omicron B.1.1.529 variant by post-immunisation serum","volume":"399","author":"Dejnirattisai","year":"2021","journal-title":"Lancet"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"100529","DOI":"10.1016\/j.xcrm.2022.100529","article-title":"MRNA-1273 and BNT162b2 mRNA vaccines have reduced neutralizing activity against the SARS-CoV-2 omicron variant","volume":"3","author":"Edara","year":"2022","journal-title":"Cell Rep. Med."},{"key":"ref_41","unstructured":"U.S. Food and Drug Administration (2026, May 16). Updated COVID-19 Vaccines for Use in the United States Beginning in Fall 2024, Available online: https:\/\/www.fda.gov\/vaccines-blood-biologics\/updated-covid-19-vaccines-use-united-states-beginning-fall-2024."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"912","DOI":"10.1016\/j.immuni.2024.02.017","article-title":"Immunological imprinting shapes the specificity of human antibody responses against SARS-CoV-2 variants","volume":"57","author":"Johnston","year":"2024","journal-title":"Immunity"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"904","DOI":"10.1016\/j.immuni.2024.02.016","article-title":"Persistent immune imprinting occurs after vaccination with the COVID-19 XBB.1.5 mRNA booster in humans","volume":"57","author":"Tortorici","year":"2024","journal-title":"Immunity"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"eadu8067","DOI":"10.1126\/scitranslmed.adu8067","article-title":"The XBB.1.5 COVID-19 vaccine elicits a durable antibody response to ancestral and XBB.1.5 SARS-CoV-2 spike proteins","volume":"17","author":"Kumar","year":"2025","journal-title":"Sci. Transl. Med."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"e162192","DOI":"10.1172\/JCI162192","article-title":"Impact of antigenic evolution and original antigenic sin on SARS-CoV-2 immunity","volume":"133","author":"Fouchier","year":"2023","journal-title":"J. Clin. Investig."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Boonyaratanakornkit, J., and Taylor, J.J. (2019). Techniques to Study Antigen-Specific B Cell Responses. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.01694"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1038\/s41596-023-00930-8","article-title":"Production and use of antigen tetramers to study antigen-specific B cells","volume":"19","author":"Phelps","year":"2024","journal-title":"Nat. Protoc."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Kirchenbaum, G.A., Pawelec, G., and Lehmann, P.V. (2025). The Importance of Monitoring Antigen-Specific Memory B Cells, and How ImmunoSpot Assays Are Suitable for This Task. Cells, 14.","DOI":"10.3390\/cells14030223"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"206","DOI":"10.15585\/mmwr.mm7106a4","article-title":"Genomic Surveillance for SARS-CoV-2 Variants: Predominance of the Delta (B.1.617.2) and Omicron (B.1.1.529) Variants\u2014United States, June 2021\u2013January 2022","volume":"71","author":"Lambrou","year":"2022","journal-title":"MMWR-Morb. Mortal. Wkly. Rep."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Becza, N., Yao, L., Lehmann, P.V., and Kirchenbaum, G.A. (2025). Optimizing PBMC Cryopreservation and Utilization for ImmunoSpot\u00ae Analysis of Antigen-Specific Memory B Cells. Vaccines, 13.","DOI":"10.20944\/preprints202506.1606.v1"},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Fecher, P., Caspell, R., Naeem, V., Karulin, A.Y., Kuerten, S., and Lehmann, P.V. (2018). B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody. Cells, 7.","DOI":"10.3390\/cells7060050"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"313","DOI":"10.3390\/cells1030313","article-title":"Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies","volume":"1","author":"Ramachandran","year":"2012","journal-title":"Cells"},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Franke, F., Kirchenbaum, G.A., Kuerten, S., and Lehmann, P.V. (2020). IL-21 in Conjunction with Anti-CD40 and IL-4 Constitutes a Potent Polyclonal B Cell Stimulator for Monitoring Antigen-Specific Memory B Cells. Cells, 9.","DOI":"10.3390\/cells9020433"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1260","DOI":"10.1002\/eji.200839129","article-title":"Clonal dissection of the human memory B-cell repertoire following infection and vaccination","volume":"39","author":"Pinna","year":"2009","journal-title":"Eur. J. Immunol."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"1501","DOI":"10.1126\/science.abd0826","article-title":"Structure-based design of prefusion-stabilized SARS-CoV-2 spikes","volume":"369","author":"Hsieh","year":"2020","journal-title":"Science"},{"key":"ref_56","unstructured":"BEI Resources (2020). Vector pCAGGS Containing the SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike Glycoprotein Receptor Binding Domain (RBD), NR-52309, BEI Resources."},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Ecker, J.W., Kirchenbaum, G.A., Pierce, S.R., Skarlupka, A.L., Abreu, R.B., Cooper, R.E., Taylor-Mulneix, D., Ross, T.M., and Sautto, G.A. (2020). High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines. Vaccines, 8.","DOI":"10.3390\/vaccines8030462"},{"key":"ref_58","doi-asserted-by":"crossref","unstructured":"K\u00f6ppert, S., Wolf, C., Becza, N., Sautto, G.A., Franke, F., Kuerten, S., Ross, T.M., Lehmann, P.V., and Kirchenbaum, G.A. (2021). Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays. Cells, 10.","DOI":"10.1101\/2021.06.02.21258073"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1007\/978-1-0716-3690-9_5","article-title":"Artificial Intelligence-Based Counting Algorithm Enables Accurate and Detailed Analysis of the Broad Spectrum of Spot Morphologies Observed in Antigen-Specific B-Cell ELISPOT and FluoroSpot Assays","volume":"Volume 2768","author":"Karulin","year":"2024","journal-title":"Handbook of ELISPOT: Methods of Protocols"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1007\/978-1-4939-8567-8_10","article-title":"Multi-Color FLUOROSPOT Counting Using ImmunoSpot(R) Fluoro-X Suite","volume":"Volume 1808","author":"Megyesi","year":"2018","journal-title":"Handbook of ELISPOT: Methods and Protocols"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"906","DOI":"10.1007\/s10875-021-00997-6","article-title":"Serologic Cross-Reactivity of SARS-CoV-2 with Endemic and Seasonal Betacoronaviruses","volume":"41","author":"Hicks","year":"2021","journal-title":"J. Clin. Immunol."},{"key":"ref_62","doi-asserted-by":"crossref","unstructured":"Kirchenbaum, G.A., Becza, N., Yao, L., Karulin, A.Y., and Lehmann, P.V. (2026). Extending ImmunoSpot\u00ae Assays\u2019 Sensitivity for Detecting Rare Antigen-Specific B Cells to One in a Million\u2014And Possibly Lower. Vaccines, 14.","DOI":"10.3390\/vaccines14010088"},{"key":"ref_63","doi-asserted-by":"crossref","unstructured":"(2010). Writing Committee of the WHO Consultation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza. Clinical aspects of pandemic 2009 influenza A (H1N1) virus infection. N. Engl. J. Med., 362, 1708\u20131719.","DOI":"10.1056\/NEJMra1000449"},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"100040","DOI":"10.1016\/j.xcrm.2020.100040","article-title":"Rapid Generation of Neutralizing Antibody Responses in COVID-19 Patients","volume":"1","author":"Suthar","year":"2020","journal-title":"Cell Rep. Med."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"e2204607119","DOI":"10.1073\/pnas.2204607119","article-title":"Early human B cell signatures of the primary antibody response to mRNA vaccination","volume":"119","author":"Kardava","year":"2022","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"109823","DOI":"10.1016\/j.celrep.2021.109823","article-title":"High-affinity memory B cells induced by SARS-CoV-2 infection produce more plasmablasts and atypical memory B cells than those primed by mRNA vaccines","volume":"37","author":"Pape","year":"2021","journal-title":"Cell Rep."},{"key":"ref_67","doi-asserted-by":"crossref","unstructured":"Hoormann, M.J., Becza, N., Yao, L., Kuerten, S., Tary-Lehmann, M., Sautto, G.A., Lehmann, P.V., and Kirchenbaum, G.A. (2026). Assessing the affinity spectrum of an antigen-specific memory B cell repertoire by inverted ImmunoSpot. bioRxiv.","DOI":"10.64898\/2026.04.20.719720"},{"key":"ref_68","doi-asserted-by":"crossref","unstructured":"Lee, K., Demirev, A.V., Lee, S., Cho, S., Kim, H., Cho, J., Yang, J.-S., Kim, K.-C., Lee, J.-Y., and Shin, W. (2025). Forecasting framework for dominant SARS-CoV-2 strains before clade replacement using phylogeny-informed genetic distances. Front. Microbiol., 16.","DOI":"10.3389\/fmicb.2025.1619546"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1007\/s44197-025-00510-x","article-title":"The Emergence and Characterization of SARS-CoV-2 Variant XFG (\u201cStratus\u201d): Comparative Virological, Epidemiological, and Public-Health Perspectives","volume":"16","author":"Azhar","year":"2026","journal-title":"J. Epidemiol. Glob. Health"},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1016\/j.immuni.2023.04.012","article-title":"Immunological imprinting: Understanding COVID-19","volume":"56","author":"Koutsakos","year":"2023","journal-title":"Immunity"},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1038\/s41579-023-00878-2","article-title":"The evolution of SARS-CoV-2","volume":"21","author":"Markov","year":"2023","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1146\/annurev-immunol-083122-043054","article-title":"SARS-CoV-2: The Interplay Between Evolution and Host Immunity","volume":"43","author":"Case","year":"2025","journal-title":"Annu. Rev. Immunol."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"1579","DOI":"10.1056\/NEJMc2201849","article-title":"Neutralization of the SARS-CoV-2 Omicron BA.1 and BA.2 Variants","volume":"386","author":"Yu","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"1297","DOI":"10.1038\/s41591-022-01792-5","article-title":"Serum neutralization of SARS-CoV-2 Omicron sublineages BA.1 and BA.2 in patients receiving monoclonal antibodies","volume":"28","author":"Bruel","year":"2022","journal-title":"Nat. Med."},{"key":"ref_75","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_76","doi-asserted-by":"crossref","first-page":"eabn8543","DOI":"10.1126\/scitranslmed.abn8543","article-title":"SARS-CoV-2 BA.1 variant is neutralized by vaccine booster\u2013elicited serum but evades most convalescent serum and therapeutic antibodies","volume":"14","author":"Lusvarghi","year":"2022","journal-title":"Sci. Transl. Med."},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1038\/s41586-021-03720-y","article-title":"In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains","volume":"596","author":"Chen","year":"2021","journal-title":"Nature"},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"824","DOI":"10.1038\/s41467-023-36561-6","article-title":"Resistance of Omicron subvariants BA.2.75.2, BA.4.6, and BQ.1.1 to neutralizing antibodies","volume":"14","author":"Planas","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"e20251146","DOI":"10.1084\/jem.20251146","article-title":"Common cold embecovirus imprinting primes broadly neutralizing antibody responses to SARS-CoV-2 S2","volume":"222","author":"Changrob","year":"2025","journal-title":"J. Exp. Med."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"1782","DOI":"10.1093\/infdis\/jix173","article-title":"The Doctrine of Original Antigenic Sin: Separating Good from Evil","volume":"215","author":"Monto","year":"2017","journal-title":"J. Infect. Dis."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"eabq3511","DOI":"10.1126\/sciimmunol.abq3511","article-title":"Recall of preexisting cross-reactive B cell memory after Omicron BA.1 breakthrough infection","volume":"7","author":"Kaku","year":"2022","journal-title":"Sci. Immunol."},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"112278","DOI":"10.1016\/j.isci.2025.112278","article-title":"Efficient boosting of Omicron-reactive memory B cells after breakthrough infection protects from repeated exposure","volume":"28","author":"Chu","year":"2025","journal-title":"iScience"},{"key":"ref_83","first-page":"100157","article-title":"Breakthrough infections with SARS-CoV-2 omicron efficiently boost antibodies from previous BNT162b2 vaccinations","volume":"3","author":"Perkmann","year":"2023","journal-title":"J. Clin. Virol."},{"key":"ref_84","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":"2023","journal-title":"Nature"},{"key":"ref_85","doi-asserted-by":"crossref","first-page":"e20221006","DOI":"10.1084\/jem.20221006","article-title":"Memory B cell responses to Omicron subvariants after SARS-CoV-2 mRNA breakthrough infection in humans","volume":"219","author":"Wang","year":"2022","journal-title":"J. Exp. Med."},{"key":"ref_86","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1038\/s44298-026-00185-6","article-title":"SARS-CoV-2 crossreactive B-cells outnumber seasonal coronavirus spike-specific clones at the end of the COVID-19 pandemic","volume":"4","author":"Reinders","year":"2026","journal-title":"NPJ Viruses"},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"4047","DOI":"10.1128\/JVI.03422-13","article-title":"Flow Cytometry Reveals that H5N1 Vaccination Elicits Cross-Reactive Stem-Directed Antibodies from Multiple Ig Heavy-Chain Lineages","volume":"88","author":"Whittle","year":"2014","journal-title":"J. Virol."},{"key":"ref_88","doi-asserted-by":"crossref","first-page":"4720","DOI":"10.1128\/JVI.03152-15","article-title":"Design and Characterization of a Computationally Optimized Broadly Reactive Hemagglutinin Vaccine for H1N1 Influenza Viruses","volume":"90","author":"Carter","year":"2016","journal-title":"J. Virol."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/7\/604\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T04:11:16Z","timestamp":1783743076000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/7\/604"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,9]]},"references-count":88,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["vaccines14070604"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14070604","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,9]]}}}