Low pre-infection levels of neutralizing antibody in breakthrough infections after bivalent BA.4-5 vaccine and practical application of dried blood spots

The level of neutralizing antibodies required to confer protection against COVID-19 breakthrough infections (BIs) is unclear, and the ability to know the immune status of individuals against the rapidly changing endemic variants is limited. We assessed longitudinal serum anti-RBD antibody levels and neutralizing activities (NTs) against Omicron BA.5 and XBB.1.5 in healthcare workers following the fourth monovalent and fifth bivalent BA.4-5 vaccines. The occurrence of BIs was also followed, and pre-infection antibody levels were compared between patients who developed BI and those who did not. In addition, we collected whole blood samples on the same day as the sera and stored them on filter papers (nos. 545, 590, and 424) for up to two months, then measured their NTs using dried blood spots (DBS) eluates, and compared them with the NTs in paired sera. Pre-infection levels of NTs were lower in patients who developed BI than those who did not, but the anti-RBD antibody levels were not different between them. The NTs below 50% using 200-fold diluted sera might be one of the indicators of high risk for COVID-19 BI. However, the NTs against XBB.1.5 at 6 months after the fifth dose of bivalent BA.4-5 vaccine were lower than this threshold in almost half of infection-naive participants. NTs measured using DBS eluates were strongly correlated with those measured using paired sera, but the time and temperature stability varied with the type of filter paper; no. 545 filter paper was found to most suitable for NT evaluation. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by the Research Program on Emerging and Re-emerging Infectious Diseases from the AMED (grant no. JP20he0622035) (Y. Morinaga, H.T., and Y. Yamamoto) and (grant no. JP21fk0108588) (Y. Morinaga and H.T.), a research funding grant from the president of the University of Toyama (Y. Morinaga, H.N., and Y. Yamamoto), Toyama Pharmaceutical Valley Development Consortium (Y. Morinaga, H.N., and Y. Yamamoto), Japan Antibiotics Research Association (H.K.), and Japan Society for the Promotion of Science (JSPS) KAKENHI (grant nos. JP22K20768 and JP23K15364) (H.K.). The funding bodies played no role in the study design, collection, analysis, or interpretation of data, nor in writing the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was performed in accordance with the Declaration of Helsinki and approved by the ethical review board of the University of Toyama (approval no. R2019167). Written informed consent was obtained from all participants. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript