Objectives: 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 post-COVID condition (PCC) or systemic inflammation. As a secondary methodological aim, we evaluated whether dithiothreitol (DTT) treatment reliably unmasks antibody-bound Spike. Methods: Within the ORCHESTRA project, we quantified free Spike and Nucleocapsid in 200 participants (112 PCC, 58 non-PCC and 30 pre-pandemic controls), analysing 576 samples collected during acute infection (n=99) and at 3, 6, 12, and 18 months post-infection (n=447). Correlations between free Spike and proinflammatory markers were assessed. The effect of 10 mM DTT on Spike detectability was validated prior to analysis. Results: 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 post-infection, 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 pre-pandemic controls (p<0.001), indicating non-specific assay artifact rather than recovery of antibody-bound antigen. Conclusions: 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 non-specific assay artifacts. These data argue against persistent circulating antigenemia as a primary, generalizable driver of PCC, highlighting the need for cautious biomarker interpretation.
Longitudinal assessment of SARS-CoV-2 Spike and Nucleocapsid antigenemia in the ORCHESTRA Post-COVID cohort
Canziani, Lorenzo Maria;Gentilotti, Elisa;Tacconelli, Evelina;
In corso di stampa
Abstract
Objectives: 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 post-COVID condition (PCC) or systemic inflammation. As a secondary methodological aim, we evaluated whether dithiothreitol (DTT) treatment reliably unmasks antibody-bound Spike. Methods: Within the ORCHESTRA project, we quantified free Spike and Nucleocapsid in 200 participants (112 PCC, 58 non-PCC and 30 pre-pandemic controls), analysing 576 samples collected during acute infection (n=99) and at 3, 6, 12, and 18 months post-infection (n=447). Correlations between free Spike and proinflammatory markers were assessed. The effect of 10 mM DTT on Spike detectability was validated prior to analysis. Results: 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 post-infection, 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 pre-pandemic controls (p<0.001), indicating non-specific assay artifact rather than recovery of antibody-bound antigen. Conclusions: 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 non-specific assay artifacts. These data argue against persistent circulating antigenemia as a primary, generalizable driver of PCC, highlighting the need for cautious biomarker interpretation.
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