Correlates of nucleocapsid antibodies and a combination of spike and nucleocapsid antibodies against protection of SARS-CoV-2 infection during Omicron XBB.1.16 and EG.5 predominant wave

Background The role of nucleocapsid (N) antibodies and their combination with spike (S) antibodies against SARS-CoV-2 reinfection remains unclear. We aimed to examine the association between N antibodies, a combination of N and S antibodies, and protection against SARS-CoV-2 reinfection. Methods We conducted a prospective cohort study among staff at a national medical research center in Tokyo and followed them for the incidence of SARS-CoV-2 infection between June and September 2023 (Omicron XBB.1.16/EG.5 predominant wave). At baseline, participants donated blood samples to measure N-and S-specific antibodies in assays from three companies (Roche, Abbott, and Sysmex). Cox regression was used to estimate the hazard ratio (HR) and protection (1-HR*100) against subsequent SARS-CoV-2 infection across these antibody levels. Findings Of the 2549 staff included in the analysis, 237 SARS-CoV-2 infections were identified during follow-up. Among participants with previous infection, higher pre-reinfection N antibodies were associated with a lower risk of reinfection even after adjusting S antibody levels (P for trend<0.01). Estimation of the protection matrix for N and S antibodies yielded that high levels in both N and S antibodies conferred robust protection (>90%) against subsequent infection. In addition, a pattern of low pre-reinfection N antibodies but high vaccine-enhanced S antibodies showed high protection (>80%). Interpretation Pre-reinfection N antibody levels correlated with protection against reinfection, independent of S antibodies. If the N antibodies were low, vaccine-boosted S antibodies could enhance the reinfection protection. Funding National Center for Global Health and Medicine and Japan Health Research Promotion Bureau Research Fund. Evidence before this study We searched published and preprinted literature with the following keywords: “COVID-19,” “SARS-CoV-2,” “nucleocapsid,” “spike,” “antibody,” “protection,” and “reinfection.” We found few prospective or case-control studies examining the association between pre-reinfection anti-SARS-CoV-2 nucleocapsid (N) antibody levels and risk of SARS-CoV-2 reinfection; in particular, no studies were conducted for adults among Omicron-dominant phases. We also found no studies that examined the role of a combination of anti-spike (S) and anti-N antibodies in protection against SARS-CoV-2 infection. Added value of this study This study first revealed that pre-reinfection anti-N antibody levels correlated with protection against reinfection during the Omicron XBB.1.16 and EG.5 predominant waves even after adjusting S antibody levels. Further, we first estimated the protection matrix by combining anti-N and S antibody levels and showed that both high levels in N and S conferred robust protection (>90%). Vaccine-induced higher S antibody levels were associated with higher protection among previously infected individuals with low levels of N antibodies. Implications of all the available evidence The prolonged COVID-19 pandemic has resulted in diverse immune characteristics across individuals due to varying timing of infection and doses and timing of vaccination, making it challenging to decide the timing of additional vaccination. Our results suggest the utility of assessing both N and S antibody levels for considering the timing of additional vaccination for those with a history of COVID-19. If the N antibody level was low due to waning over time, additional vaccination enhances S antibodies and might improve the protection against reinfection. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the NCGM COVID-19 Gift Fund (grant number 19K059), Japan Health Research Promotion Bureau Research Fund (grant number 2020-B-09), and National Center for Global Health and Medicine (grant number 21A2013D). Abbott Japan and Roche Diagnostics provided reagents for the anti-SARS-CoV-2 antibody assays. ### 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 approved by the National Center for Global Health and Medicine, Japan (NCGM) ethics committee (approval number: NCGM-G-003598). 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 study are available upon reasonable request to the authors.