Multi-layered deep immune profiling, SARS-CoV-2 RNAemia and inflammation in unvaccinated COVID-19 individuals with persistent symptoms

Background: Long-COVID immunopathogenesis involves diverse factors. We longitudinally characterize hospitalized COVID-19 patients, examining the role of SARS-CoV-2 RNAemia and inflammation in immune dysregulation. Methods: Hospitalized patients are evaluated during acute infection (T0), 3 months post-symptom onset (T1), and 3 years if symptoms persisted (T2). Immune profile includes characterization of SARS-CoV-2-specific/non-specific T/B cells (flow cytometry) and antibodies (ELISA, neutralization, ADCC). RNAemia and cytokines are quantified (RT-PCR, cytometric beads array) and correlated. Statistics: non-parametric cross-sectional, longitudinal and correlation analyses. Results: Here we show 48 hospitalized individuals during acute COVID-19, 38 exhibit early persistent symptoms (EPS+) 3 months post-symptoms onset, 10 do not (EPS-). Groups are comparable for age, sex, co-morbidities. The EPS+ shows fatigue, dyspnoea, anosmia/dysgeusia, diarrhea, chronic pain, mnestic disorders. Over time, they show a reduction of neutralization ability and total SARS-CoV-2-specific CD4 T cells, with increased total CD4 TEMRA, and failure to increase RBD-specific B cells and IgA+ MBCs. EPS+ patients show higher levels of T0-IFN-γ + CD4 TEMRA, T1-IL-2 + CD4 TEM and T1-TNF-α + CD4 cTfh. In EPS+, baseline SARS-CoV-2 RNAemia positively correlates with CD4 TEMRA, follow-up SARS-CoV-2 RNAemia with ADCC. Among 38 EPS+ individuals at T1, 33 are evaluated 3 years after infection, 5 are lost at follow-up. 10/33 EPS+ show long-term symptoms (late persistent symptoms, EPS + LPS+), whereas 23/33 fully recover (EPS + LPS-). Antibodies, RNAemia, and cytokines show no differences between/within groups at any time point. Conclusions: Early persistent symptoms are associated with multi-layered SARS-CoV-2-specific/non-SARS-CoV-2-specific immune dysregulation. The shift towards non-Ag-specific TEMRA and ADCC trigger in EPS+ may relate to SARS-CoV-2 RNAemia. Early immune dysregulation does not associate with long-term persistent symptoms. Further research on SARS-CoV-2 RNAemia and early immune dysregulation is needed.