IL-2 and IFN-γ are biomarkers of SARS-CoV-2 specific cellular response in whole blood stimulation assays

A proper description of the immune response to SARS-CoV-2 will be critical for the assessment of protection elicited after both infection and vaccination. Uncoupled T and B cell responses have been described in acute and convalescent patients and exposed individuals. We assessed the potential usefulness of whole blood stimulation assays to identify functional cellular immune responses to SARS-CoV-2. Blood from COVID-19 recovered individuals (5 months after infection) and negative subjects was stimulated for 24 hours with HLA predicted peptide “megapools” of the Spike and Nucleoprotein, or the mixture of them. After stimulation, cytokines were quantified using a beads-based multiplex assay. Interleukin-2 and IFN-γ were found to be specific biomarkers of SARS-CoV-2 cellular response. Using the Spike and Nucleoprotein mixture, 91.3% of COVID-19 recovered individuals presented an IL-2 stimulation index over the cut-off, while 82.6% showed IFN-γ. All the negative individuals presented an IL-2 response under the cut-off, while 5.3% of these subjects presented positive IFN-γ stimulation indexes. Moreover, IL-2 production correlated with IgG levels for Spike 1, RBD, and Nucleocapsid. In conclusion, we demonstrate the potential of whole blood stimulation assays and the quantification of IL-2 and IFN-γ for the analysis of SARS-CoV-2 functional cellular responses. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was financed by the Portuguese Foundation for Science and Technology (FCT) in the framework of the project RESEARCH4COVID 19 reference number 460. This work also received funds through the Research Unit 4293. Individual funding from FCT through CEECIND/02362/2017 (to JT). NS and BPC are funded respectively through the FCT projects PTDC/SAU-PAR/31013/2017 and PTDC/SAU‐PAR/31340/2017. FCT funded PhD scholarships SFRH/BD/140177/2018 (to IC) SFRH/BD/140119/2018 (to SE) and SFRH/BD/133276/2017 (AT). ### 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 Ethics Committees of Centro Hospitalar e Universitario de Coimbra (CHUC-084-20). Participants signed an informed consent form, according to the Helsinki principles, the Oviedo Convention and according to the General Data Protection Regulation Regulation (EU) 2016/679. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. 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 and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data referred to in the manuscript is available upon request to corresponding authors. * CHUC : Centro Hospitalar Universitário de Coimbra N : Nucleoprotein PBMC : Peripheral Blood Mononuclear Cells ROC : Receiving Operating Curves S : SARS-CoV-2 Spike S1 subunit SEB : Staphylococcus enterotoxin B SI : Stimulation Index S1 : Spike PepMix peptide pool S1 S2 : Spike PepMix peptide pool S2 WBS : Whole Blood Stimulation