Longitudinal assessment of SARS-CoV-2 spike and nucleocapsid antigenemia in the ORCHESTRA post-COVID cohort

Spike antigenemia has been proposed as a mechanism underlying post-COVID condition (PCC). We determined whether persistent free SARS-CoV-2 spike or nucleocapsid antigenemia is associated with PCC or systemic inflammation. As a secondary methodological aim, we evaluated whether dithiothreitol (DTT) treatment reliably unmasks antibody-bound spike.

Within the ORCHESTRA project, we quantified free spike and nucleocapsid in 200 participants (112 PCC, 58 non-PCC, and 30 prepandemic controls), analysing 576 samples collected during acute infection (n = 99) and at 3, 6, 12, and 18 months postinfection (n = 447). Correlations between free spike and proinflammatory markers were assessed. The effect of 10 mM DTT on spike detectability was validated before analysis.

During acute infection, nucleocapsid was detectable in nearly all participants (n = 98/99) and spike in 15/99 (15.2%). Low-level antigen persisted in a subset of participants up to 18 months; however, neither free spike nor nucleocapsid differed between PCC and controls or were independently associated with PCC (spike: OR, 0.99; 95% CI, 0.55-1.78; nucleocapsid: OR, 0.52; 95% CI, 0.14-2.03). At 3-6 months postinfection, free spike correlated with proinflammatory cytokines, with stronger associations in PCC than controls (all p < 0.05). Methodologically, DTT reduced recombinant spike detectability by 90.9% ± 0.7% at 10 mM (p < 0.001) yet increased measured plasma signal 2.6 ± 2.7-fold in pandemic samples and 5.5 ± 2.4-fold in prepandemic controls (p < 0.001), indicating nonspecific assay artefact rather than recovery of antibody-bound antigen.

Low-level systemic antigen persistence occurs in a subset of individuals but does not differentiate PCC from recovered controls. Early convalescent spike-cytokine correlations likely reflect transient, host-specific immune activation rather than a persistent viral driver. Furthermore, DTT does not recover true immune-complexed antigen but generates nonspecific assay artefacts. These data argue against persistent circulating antigenemia as a primary, generalisable driver of PCC, highlighting the need for cautious biomarker interpretation.