Persistent Spike antigenemia is not associated with Post-COVID condition

A bstract Objectives Spike antigenemia has been proposed as one of the potential mechanisms underlying post-COVID condition (PCC). Several studies addressing Spike antigenemia have measured both free and antibody-bound Spike employing 10 mM DTT to dissociate Spike–IgG complexes. Methods Within the ORCHESTRA project, we analysed Spike and Nucleocapsid proteins in 188 participants, comprising PCC patients (n=112), non-PCC individuals (n=58) and pre-pandemic controls (n=18). For the post-pandemic cohort, 546 samples were studied from acute infection (n=99), and at 3, 6, 12, and 18 months follow-up (n=547). Impact of 10 mM DTT treatment on Spike detectability was evaluated. Correlations between Spike and proinflammatory markers were examined in both PCC and non-PCC groups. Results DTT treatment of recombinant Spike reduced its detectability in a dose-dependent manner, with a 91.3% reduction observed at 10 mM concentration (p<0.001). In plasma, DTT treatment increased detection signals in acute COVID-19 and PCC patients (n=13) by 2.6-fold, except in an acutely infected individual with exceptionally high acute Spike levels (p<0.001). Notably, pre-pandemic plasma (n=18) also exhibited a fourfold increase in measurable signal following DTT exposure (p<0.001). Analysis of free Spike and Nucleocapsid revealed that Nucleocapsid was detectable in nearly all acute cases (n=98/99), whereas Spike was observed less frequently (15/99). Neither Spike nor Nucleocapsid antigenemia differed between PCC and controls, and no association was measured between Spike levels and PCC. Interestingly, Spike levels at 3 and 6 months correlated with inflammatory cytokines IL-6, IL-2, and IL-1β, with stronger associations observed within the PCC cohort (all p<0.05). Conclusions Our findings indicate that DTT treatment induces Spike denaturation and artefactual neoepitope generation rather than genuine release of immune-complexed antigen. Furthermore, we demonstrate that PCC pathogenesis is less consistent with persistent Spike antigenemia. Together, these results call for harmonized biomarker methodologies for Spike antigenemia and more in-depth mechanistic studies of PCC.