Deciphering Long-COVID: Uncovering the Significance of Immunological & Virological Dysregulation Underpinning Long-COVID Pathophysiology (DECI-LOCO Study)

After recovery of the acute phase of COVID-19, a significant proportion of patients suffer from persistent symptoms, known as long-COVID. This leads to chronic disability and reduced quality of life. Because the underlying causes of long-COVID are unclear, the disease course cannot be reliably predicted, and there are currently no optimal treatments available. It is found that several abnormalities, including a dysregulated immune system, an abnormal anti-viral response and viral persistence, and reactivation of latent human herpes viruses (HHVs) may contribute to the development of long-COVID. However, how these abnormalities lead to disease pathogenesis is unknown. Furthermore, the disease course and the factors influencing recovery or deterioration remain unclear. Therefore, in this project we will follow patients and convalescent COVID-19 patients for two years to investigate how the immune system and virus-specific responses are connected. We will link these findings to clinical symptoms in patients, including fatigue, shortness of breath, muscle aches, headaches, and loss of concentration, and disease course, and to findings in convalescent COVID-19 patients.   . Together, this will provide essential information to unravel the causes of long-COVID and how they contribute to disease course in individual patients. This allows us to develop objective diagnostic criteria and the best possible treatment for every patient.  

Our primary objective is to determine the relationship between immunological profiles and disease course in long-COVID, by characterizing phenotypic and functional changes in (virus-specific) immune cells, , as well as epigenetic and transcriptomic analyses, of several B cell, T cell, and monocyte subsets, which relate to immune activation/dysfunction in patients with long-COVID, and to link these to clinical parameters. In the lymphoid compartment, B and T cell subsets, including virus specific T cells, will be analyzed for shifts in subsets, as well as alterations in activation and/or exhaustion phenotype. B and T cell receptor signaling will be assessed, as well as cytokine production by monocytes and lymphocytes. Observed alterations in specific circulating lymphocyte or monocyte subsets can be further analyzed on transcriptomic and epigenetic level, using RNA-sequencing (seq) and ATAC-seq, respectively. All observed alterations will be combined to form unique immune profiles to discover possible disease underlying mechanisms, as well as  to serve as a diagnostic and prognostic tool for disease course to aid patient stratification for personalized treatment.

Our secondary objective is to generate in-depth virological profiles, to link aberrations in viral responses or reactivation to clinical parameters and disease course. To explore the possibility that chronic immune activation and dysregulation are caused or supported by viral persistence, we will measure presence of SARS-CoV-2 spike protein in serum and specific monocyte populations [22]. Also, we will determine the profile of SARS-CoV-2-specific B and T cells, and antibodies present in the circulation of included subjects. We will determine the breadth of the antibody response by measuring total antibody concentrations and virus-neutralizing capacity against SARS-CoV-2. To explore whether chronic immune activation may be caused by a reactivation of latent viruses, we will determine virus titers of EBV, CMV, and HHV6 in serum/plasma. To link virus reactivation to the presence of circulating virus-specific antibodies, we will also determine antibody titers against these latent viruses.   

Tertiary objective:   
Our tertiary objective is to  gain new knowledge on critical mechanisms responsible for disease persistence or recovery, and to identify novel therapeutic targets, based on our in-depth immunological and virological characterization of post-COVID profiles.

This study will be a longitudinal continuation of the DECI-LOCO study, supported by a ZonMw grant thereby acknowledging the urgent need for comprehensively following up long-COVID patients over an extended period. The DECI-LOCO is a comparative, non-randomized, observational study that included 40 recently diagnosed (WHO post-COVID definition) long-COVID patients (<12 0 months) and long-term ill patients (>24 months) with considerable disability (Fatigue Assessment Scale (FAS) >22, overall functioning <70% 15 27 40 and presence of post exertional malaise (pem)), convalescent controls. we will follow-up these participants for two years after baseline inclusion obtain both clinical (patient reported outcome measures [proms]) data additional blood samples at months inclusion. peripheral mononuclear cells (pbmcs), plasma, serum be isolated. all frozen stored until further use. different circulating lymphoid myeloid cell populations form pbmcs characterized quantified using flow cytometry. differences in specific subsets between long-covid patients ccs analyzed by assessing the genetic epigenetic landscape rna- atac-sequencing, respectively. plasma used quantification viral factors, load, cytokines, antiviral antibodies. We will use an innovative systems biology approach to integrate the obtained data with clinical parameters and disease course to extract a diagnostic and predictive immune signature. These integrated data will be used to identify potential pathogenic pathways, which we will further investigate using functional assays. Longitudinal analyses of clinical and biological follow-up data within all groups but also between long-COVID patients and CCs will be performed to identify biomarkers that predict disease course and recovery, and to identify mechanistic pathways that drive disease course and are associated with either recovery or ongoing disease.

Blood sampling, hair sample, and questionnaires.

Peripheral blood collection by venous phlebotomy can, in some cases, lead to hematoma formation. This leads to minor discomfort on the short-term and has no known long-term risks for the subjects. In patients with severe complaints of orthostatic tachycardia it will be offered to replace the drawn blood volume with 250ml NaCl 0.9% to reduce symptom burden afterwards.   The subjects participating in this research will not benefit directly from the results of our research. However, our study could lead to new insights, that are highly relevant in understanding the pathophysiology of long-COVID in general, finding a potential diagnostic and or prognostic tool, and potential treatment options in the future.