Omicron Breakthrough Infection Elicits Superior Humoral and Mucosal Immune Responses to SARS-CoV-2 Variants than an Intramuscular Booster Dose

Our understanding of the quality of cellular and humoral immunity conferred by COVID-19 vaccination alone versus vaccination plus SARS-CoV-2 breakthrough (BT) infection remains incomplete. While the current (2023) SARS-CoV-2 immune landscape of Canadians is complex, in late 2021 most Canadians had either just received a third dose of COVID-19 vaccine, or had received their two dose primary series and then experienced an Omicron BT. Herein we took advantage of this coincident timing to contrast cellular and humoral immunity conferred by three doses of vaccine versus two doses plus BT. Our results show that mild BT infection induces cell-mediated immune responses to variants comparable to an intramuscular vaccine booster dose. In contrast, BT subjects had higher salivary IgG and IgA levels against the Omicron Spike and enhanced reactivity to the ancestral Spike for the IgA isotype, which also reacted with SARS-CoV-1. Serum neutralizing antibody levels against the ancestral strain and the variants were also higher after BT infection. Our results support the need for mucosal vaccines to emulate the enhanced mucosal and humoral immunity induced by Omicron without exposing individuals to the risks associated with SARS-CoV-2 infection. ### Competing Interest Statement ACG has received research funds from a research contract with Providence Therapeutics Holdings, Inc. for other projects. ### Funding Statement This study was supported by funding from a CoVaRR-Net Rapid Response Research Grant (MB, CP, CQ, JR, ACG, HD, JG) under CIHR Rapid Response Network to SARS-CoV-2 Variants (Funding Reference Number : FRN 175622) as well as the following grants : - Canadian Institutes of Health Research GA1-177703 (ACG), GA2-177710 (JG) and VR2172712 (CQ, HD) - Coalition for Epidemic Preparedness Innovations and Canadian Institutes of Health Research 468231 (HD) - Ontario Together COVID-19 Rapid Response 20013418 (JG) - Public Health Agency of Canada 2122-HQ-000225 (CQ, HD) and 2223-HQ-000261 (JG). The robotics equipment used is housed in the Network Biology Collaborative Centre at the Lunenfeld-Tanenbaum Research Institute, a facility supported by the Canada Foundation for Innovation, the Ontario Government, Genome Canada and Ontario Genomics (OGI-139). ### 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: The Research Ethics Board of the University of Toronto granted approval for recruitment of participants from across the Greater Toronto Area for blood and saliva collection, as well as analysis of saliva via ELISA and sharing of samples under protocol number 23901. The Reserach Ethics Boards of the Unity Health Network and Mount Sinai Hospital granted approval for collection of saliva and blood as well as analysis via ELISA, pseudotyped lentiviral neutralization assays, T-cell and humoral immunity assays under protocol numbers 20-044 UHN, 20-0078-E and 22-0069-E Mount Sinai. The Research Ethics Board of Health Canada and the Public Health Agency of Canada - National Microbiology Laboratory approved use of human plasma and serum for Plaque Reduction Neutralization Test (PRNT) under study number REB 2020-004P. The Research Ethics Board of the Sainte-Justine University Hospital and Research Center approved RECOVER protocols under study numbers MP-21-2021-3035 and MP-21-2021-3046 which granted approval for recruitment of health care workers (HCWs) from the Greater Montreal area for blood collection, analysis of DNA, RNA, serum and peripheral blood mononuclear cells (PBMCs), as well as sharing serum samples for humoral studies and receiving samples for T cell assays. The Research Ethics Board of the Toronto Invasive Bacterial Diseases Network approved the use of serum and saliva samples from COVID-19 infected individuals under protocol numbers 20-044 UHN, 02-0118-U and 05-0016-C Mount Sinai Hospital. 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 The datasets generated and analysed in the current study are available from the corresponding author upon reasonable request.