Understanding long-term SARS-CoV-2 immune and inflammatory responses and proteases interplay in individuals recovering from COVID-19: a longitudinal study

Marcin Poreba^1*Natalia Ćwilichowska-Puślecka^1*Aleksandra Makowiecka¹Małgorzata Kalinka¹Katarzyna Groborz²Tobiasz Puślecki¹Marcin Drag¹Krzysztof Simon^3,4Krystyna Dąbrowska¹Monika Pazgan-Simon^4*

• ¹Wrocław University of Science and Technology, Wrocław, Poland
• ²Genentech Inc., San Francisco, California, United States
• ³Wroclaw Medical University, Wrocław, Silesian, Poland
• ⁴Regional Specialist Hospital, Wroclaw, Poland

The immune and inflammatory responses following SARS-CoV-2 infection, particularly in the context of long COVID, remain critical areas of study. We conducted a longitudinal analysis of 72 convalescent COVID-19 patients, assessing their recovery at three key time points: immediately post-discharge, one month later, and at three months post-infection. Additionally, a subset of 15 patients was followed up two years post-COVID-19, providing insights into the prolonged recovery process. Clinical parameters, including demographics, comorbidities, treatment modalities, and COVID-19 severity, were analyzed to evaluate the long-term health impacts of the virus. Using CyTOF technology, we characterized over 30 immune cell subsets, including granulocytes, T cells, B cells, NK cells, and monocytes. In parallel, we performed multiplexed analyses of blood samples, focusing on cytokines, chemokines, growth factors, proteases, and COVID-19-related proteins. This comprehensive approach revealed significant immune system changes over time, highlighting the role of specific immune cells and proteases in the recovery process. Key findings include decreasing deregulatory effect on immune responses exerted by subsequent SARS-CoV-2 variants Alpha, Delta, and Omicron. Our study provides an in-depth understanding of the molecular dynamics of immune recovery, integrating clinical profiling, serum multiplex analysis, antibody profiling, mass cytometry immunophenotyping, and in vitro PBMC stimulation.