Persistence of dysfunctional immune response 12 months after SARS-CoV-2 infection and their relationship with pulmonary sequelae and long covid

doi:10.21203/rs.3.rs-5019121/v1

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Persistence of dysfunctional immune response 12 months after SARS-CoV-2 infection and their relationship with pulmonary sequelae and long covid

https://doi.org/10.21203/rs.3.rs-5019121/v1

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Introduction

Most patients recover fully after an acute infection by SARS-CoV-2. Some, however, may develop pulmonary sequelae (PS) and/or long COVID (LC), However, whether these two clinical conditions have similar or different pathogenic mechanisms is unknown.

Methods.

The levels of autoantibodies and 184 inflammatory and organ damage associated proteins in plasma were determined (by immunofluorescence and Olink panels, respectively) 1 year after an acute infection by SARS-CoV-2 in 51 patients with PS (DLCO < 80% ref), 31 patients with LC and 31 patients fully recovered (Rec). PS was defined by the presence of reduced carbon monoxide diffusing capacity (DLCO) lower than 80% ref. LC was defined by the presence of chronic symptoms in the absence of an alternative diagnosis.

Results.

We found that patients with PS or LC both showed increased levels than Rec of anti-microbial, immune cell activation and recruitment related proteins. Patients with PS showed higher levels of anti-nuclear autoantibodies, whereas LC patients had increased levels of organ-damage associated proteins. In patients with PS most of the elevated proteins correlate with the impairment of lung function (DLCO). Finally, in PS we additionally performed the determinations at an earlier time point (6 months) and showed that the expression of CCL20 and IFN-ɣ was already higher at 6 months, while CCL3 and CCL19 increase from 6 to 12 months, suggesting a pathogenic role in PS persistence.

Conclusions.

Patients with PS or LC have abnormal but different persistent circulatory immune and organ damage biomarkers, suggesting different underlying biology of both post-COVID conditions.

COVID-19

Pulmonary Sequelae

Long covid

persistence of viral reservoirs

autoantibodies

organ-damage markers

1 year of convalescence

biomarkers

After an acute infection of SARS-CoV-2 most patients recover fully. Yet, some patients develop structural and/or functional pulmonary sequelae (PS)¹ whereas others experience long-term symptoms, including fatigue, chest pain, cough, neurocognitive alterations and/or muscle and joint pain without a discernible diagnosis (long-COVID - LC)²,³. Whether the biologic mechanisms underlying PS and LC are similar or different is unclear. Previoius studies have shown that PS is associated with systemic endothelial dysfunction markers (VEGF-A, sITGaM and sITGb2) ⁴, mitochondrial dysfunction ⁵, inflammation (IL-1α and TGF-β)⁶, neutrophil activation (LCN2)⁷, and fibrotic markers (MMP-7 and HGF)⁸, but the role of these biologic mechanisms in LC is unclear. On the other hand, LC has been shown to be associated with autoimmunity abnormalities, including cross-reactive T-cells, antigen-specific tissue resident memory cells, and autoantibodies⁹. Higher titles of autoantibodies during the acute COVID-19 infection had been associated with the development of PS at 3 months follow up¹⁰, but the persistence of autoantibodies has not been explored in PS. Recent findings in LC patients 1 year after the acute episode point towards a persistent inflammation, (i.e complement and coagulation alterations) and tissue injury markers ¹¹. Finally, both PS and LC have been associated with an increased SARS-CoV-2 specific T cell response with an exhausted phenotype (CD3 + CD8 + CD28-), suggesting non-resolved, persistent, adaptive response potentially driven by persistence of viral reservoirs in some organs ^12,13. In this study, we sought to contrast the similarities, and differences in the inflammatory immune response, autoimmunity and host damage response in patients with PS and LC vs. patients who recovered fully (Rec) one year after the acute SARS-CoV2 infective episode.

Study Design, Patients and Ethics

This is a prospective, observational, controlled study that included 113 adults who were hospitalized in our institution between May and November 2020 because of PCR-confirmed COVID-19 pneumonia. The severity of the acute disease was determined according to the seven-category severity scale recommended by WHO¹⁴. During hospitalization patients were treated according to international recommendations¹⁵.

The participants included in this study, were followed-up in the outpatient clinic at 3, 6, 9 and 12 months (± 2 months) after hospital discharge because of the presence of respiratory symptoms, according to the Spanish Society of Pulmonology and Thoracic Surgery (SEPAR) consensus for follow-up of post-COVID-19 patients¹⁶; and/or in the post-COVID period, complained of fatigue, chest pain, arthralgia, myalgia, headache, neurocognitive dysfunction or autonomic dysfunction. For the current analysis we used data collected at 6 and 12 months. Patients were categorized in three groups by their health status at 12 months after hospital discharge: PS (defined by DLCO < 80% ref, with or without other non-pulmonary symptoms), LC (defined by non-pulmonary symptoms and DLCO > 80% ref.) and Rec (asymptomatic with DLCO > 80% ref).

Measurements

Clinical and lung function parameters

Symptoms were evaluated at each clinical visit using SF-36 and Fatigue structured questionnaires^2,17. Lung function (spirometry, DLCO) was measured also at each clinical visit following ERS/ATS standards using the Roca equations for reference values¹⁸.

Blood sampling

Blood was collected by peripheral venipuncture in EDTA and Vacutainer SST advance tubes (Fisher Scientific, US) at 6 and 12 months after discharge. Blood tubes were centrifuged at 600g during 10 min or 1000g during 15 mins (at 4ºC) to obtain plasma and serum, respectively. Samples were stored at -80ºC until analysis.

Detection of complement, immunoglobulins and autoantibodies

Complement (C3, C4) serum levels and activity (CH50) were measured by turbidimetric immunoassay (Atellica CH C3 and Atellica CH C4; Siemens, New York, NY, USA and Autokit CH50; Fujifilm Wako Chemicals, Neuss, Germany, all are tested in Atellica CH Solution, Siemens, New York, NY, USA). Reference values were: 0.870–1.700 g/L for C3, 0.110–0.540 g/L for C4 and 28–60 U/mL for CH50. Hypocomplementemia was considered when either CH50 activity or C3 or C4 levels were lower than reference values. Immunoglobulin subtypes were measured also by turbidimetric immunoassay in the core facility of our hospital. Reference values were 6.8–15.3 g/L, 0.66–3.65 g/L and 0.36–2.61 g/L for IgG, IgA and IgM, respectively.

The presence of IgG autoantibodies was determined by indirect immunofluorescence assays (IFA) on HEp-2 cells and on triple rat tissue (Werfen, US). Only positive results with a serum dilution higher than 1/80 were considered positive. Anti-nuclear and anti-cytoplasmic patterns of autoantibodies were reported, according to International Consensus on ANA Patterns (ICAP) nomenclature¹⁹. Additionally, IgG antigen-specific autoantibodies associated with systemic autoimmune diseases (autoantibodies anti-U1-RNP, Sm, Ro52, Ro60, SS-B, Scl-70, Jo-1, Cenp-B, and Ribosomal-P) were determined using the particle-based multi-analyte technology (PMAT) on the Aptiva instrument (CTD Essential Reagent Nº 723100, Werfen, US) and autoantibodies anti-IFNa and IFNw were quantify by Luminex as described before²⁰. The number of total anti-nuclear auto-reactivities (ANAs) per patient was calculated by adding the number of patients positive for the ANA patterns AC1, AC4, AC5, AC6, AC8-9-10 and AC10, and for the anti-cytoplasmic patterns AC16, AC19, AC20 and AC21 was considered.

Inflammation and organ damage proteins

The relative abundance in plasma of 184 proteins was quantified with two Olink multiplex proximity extension assays (Inflammation and Organ Damage panels) ²¹. A limit of detection (LOD) was established based on negative controls (included in each run). Biomarkers that did not pass quality control or the LOD were excluded from the analysis. ELISAS of Growth/differentiation factor 15 (GDF-15) and WAP Four-Disulfide Core Domain 2 (WFDC2) (R&D, US) were performed following manufacturing conditions.

Protein interaction and functional enrichment

The biological processes associated to differentially expressed proteins were computed using the Gene Ontology (GO) biological function and the KEGG pathways with Cluster profiler²². To explore the interactions between proteins, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) was used²³.

Statistical analysis

Results are presented as n, frequency, mean ± standard deviation (SD) or median [interquartile range -IQR]. Groups were compared using Kruskal-Wallis with Wilcox pairwise post-hoc comparison and FDR adjustment, and Chi-square, as appropriate. Bivariate correlations were analyzed using the Spearman rank test. A p value < 0.05 was considered significant. Analyses were performed using R version 4.3.

Patient characteristics

Table 1 presents the main characteristics of participants at the visit 6 and 12 months after hospital discharge. Groups were defined by the DLCO levels and extra-pulmonary symptoms at 12 months. At 12 months there were 51 with PS, 31 with LC and 31 Rec. Interestingly, up to 56.9% of patients with PS also reported non-pulmonary symptoms. Age and sex were similar between PS and Rec groups, but LC patients were slightly younger. The COVID-19 severity score during the acute episode was lower in patients with LC. Between 6 to 12 months after infection, the proportion of patients with non-pulmonary symptoms decrease from 64.9–53.1% (p = 0.22) while there was a minor increased in patients with PS, from 46.5–52% (p = 0.25).

Table 1

Main clinical characteristics of the enrolled patients. Results are expressed as mean and standard deviation. The comparison was made using a Mann-Whitney test between the PS and the LC groups with the recovered group.
	All	Rec	PS	p.val	LC		p.val	p.val
	N = 113	N = 31	N = 51	PS vs Rec	N = 31		LC vs Rec	PS vs LC
Age	57.2 (13.3)	57.4 (13.3)	59.6 (12.6)	0.464	51.7 (13.6)		0.127	0.023
Gender (m)	50 (47.6%)	11 (35.5%)	24 (47.1%)	0.425	15 (65.2%)		0.059	0.232
DLCO 12m	78.9 (14.5)	91.1 (10.1)	68.4 (9.53)	< 0.001	89.1 (7.04)		0.637	< 0.001
PS 12m, n(%)	51 (52.0%)	0 (0.00%)	51 (100%)	< 0.001	0 (0.00%)		.	< 0.001
DLCO_6m	80.4 (17.4)	92.8 (15.8)	71.7 (12.5)	< 0.001		82.2 (19.5)	0.164	0.152
PS_6m, n(%)	40 (46.5%)	4 (12.9%)	33 (71.7%)	< 0.001		3 (33.3%)	0.316	0.051
Non-pulmonary symptoms 12m	60 (53.1%)	0 (0.00%)	29 (56.9%)	< 0.001	31 (100%)		< 0.001	<0.001
Non-pulmonary symptoms 6m	74 (64.9%)	14 (44.0%)	52 (71.4%)	0.033	31 (100%)		0.001	0.03
ICU admission, n(%)	42 (39.3%)	13 (41.9%)	25 (49.0%)	0.693	4 (16.0%)		0.071	0.011
Corticoids during acute episode	37 (57.8%)	12 (54.5%)	23 (63.9%)	0.668	2 (33.3%)		0.648	0.202
Severity (WHO)				0.386			< 0.001	<0.001
1	27 (23.9%)	1 (3.23%)	6 (11.8%)		20 (64.5%)
2	1 (0.88%)	0 (0.00%)	1 (1.96%)		0 (0.00%)
3	20 (17.7%)	8 (25.8%)	9 (17.6%)		3 (9.68%)
4	17 (15.0%)	5 (16.1%)	8 (15.7%)		4 (12.9%)
5	21 (18.6%)	8 (25.8%)	10 (19.6%)		3 (9.68%)
6	8 (7.08%)	1 (3.23%)	7 (13.7%)		0 (0.00%)
7	11 (9.73%)	3 (9.68%)	7 (13.7%)		1 (3.23%)

Total Immunoglobulin levels and presence of autoantibodies

Total dose of IgM and the IgG/IgA and IgM/IgA ratios were increased in LC compared to Rec (Table 2). However, we did not find any statistically significant difference across the 3 study groups in the levels of specific autoantibodies and/or patterns, but total ANA reactivities were increased in PS and tended to be increased in LC patients (Supplementary figure S1).

Table 2

Immunoglobulin dosage, and presence of autoantibodies.
	Rec (n = 31)	PS (n = 51)	PS vs Rec p-val	LC (n = 31)	LC vs Rec p-val	PS vs LC p-val
Immunoglobulin subtypes
IgA (g/l)	2.38 [1.77;2.92]	2.11 [1.63;2.89]	0.586	1.85 [1.46;2.45]	0.092	0.229
IgG (g/l)	10.0 [8.30;11.5]	10.3 [9.00;12.7]	0.317	11.2 [9.25;12.6]	0.084	0.397
IgM (g/l)	0.96 [0.54;1.14]	0.89 [0.68;1.39]	0.456	1.08 [0.92;1.57]	0.032	0.182
Ratio IgG/IgM	11.0 [8.91;16.2]	11.6 [7.15;17.8]	0.800	10.4 [6.72;13.1]	0.257	0.393
Ratio IgG/IgA	4.68 [3.30;5.95]	4.71 [3.64;6.72]	0.371	6.51 [4.53;7.32]	0.013	0.062
Ratio IgM/IgA	0.48 [0.26;0.63]	0.40 [0.27;0.88]	0.506	0.63 [0.43;0.76]	0.012	0.196
ANAs in IF patterns (n and % of positive patients)
Homogeneous (AC-1)	0	6 (11.8%)	0.122	3 (9.7%)	0.126	1
Fine speckled (AC-4)	1 (3.2%)	3 (5.8%)	0.989	3 (9.7%)	0.317	0.84
Large speckled (AC-5)	0	2 (3.9%)	0.705	3 (9.7%)	0.106	0.56
Multiple (AC-6)	0	3 (5.8%)	0.442	0	NA	0.44
Nucleolar (AC-8-9-10)	0	3 (5.8%)	0.442	2 (6.4%)	0.294	1
Punctate (AC-10)	0	0	NA	2 (6.4%)	0.272	0.27
Total Anti-nuclear	1 (3.2%)	12	0.033	7 (22.6%)	0.058	0.91
ANCAs in IF patterns (n and % of positive patients)
Filamentous (AC-16)	0	0	NA	1 (3.2%)	0.8	0.8
Fine speckled (AC-20)	0	1 (2%)	1	0	NA	NA
AMA (AC-21)	0	1 (2%)	1	0	NA	1
Fine Nailing	0	0	NA	1 (3.2%)	0.8	1
Total Anti-cytoplasmic	0	2 (4%)	0.705	1 (3.2%)	1	1
Autoantibodies related with autoimmmune diseases (n and % of positive patients)
Anti-CPG antibodies	1 (3.2%)	5 (9.8%)		1 (3.2%)	1	0.2
Anti-DFS70	6 (19.4%)	14 (27.5%)	0.574	4 (12.9%)	0.730	1
Anti-Ro60	2 (6.45%)	1 (1.96%)	0.657	1 (3.23%)	1.000	1
Anti-SSB	0	1 (1.96%)	1.000	0	NA	1
Anti-U1-RNP	5 (16.1%)	2 (3.92%)	0.131	2 (6.45%)	0.422	0.7
Anti-Scl70	0	2 (3.92%)	0.705	0	NA	1
Anti-CENP-B	0	2 (3.92%)	0.705	2 (6.45%)	0.472	0.66
Total Anti-protein	10 (32.2%)	19 (31.4%)	0.825	10 (32.2%)	1.000	1
Total autoreactivities	12 (38.7%)	28 (55%)	0.232	14 (45.2%)	0.797	0.55
For immunoglobulin subtypes, results are expressed as median, 95% interquartile range, and compared to Rec with Mann-Whitney. For autoantibodies, the n and percentage of positive patients is presented, and were compared to Rec with a Chi-square. AC means Anti-Cell and is used by the International Consensus on ANA Patterns (ICAP) coding of HEp-2 IF patterns.

Inflammation proteins

The levels of the complement protein C3 were elevated in patients with PS (vs. LC) suggesting an active immune process in this group (Supplementary table S1). From the 96 inflammatory proteins of the Olink panel, 21 were different across the study groups (Fig. 1A, Table S1). IFN-γ, IL8, MCP-4, SIRT2 and TNFSF14 were elevated in both PS and LC vs. Rec, suggesting an ongoing INF-γ and TNF-α response in both groups. On the other hand, IL-6 was higher in PS vs. LC. CCL20 was exclusively elevated in PS and CCL3 and CCL19 were increased in PS vs. Rec, suggesting more leucocyte chemotaxis in PS. CXCL5, AXIN1, CXCL1, STAMBP, CXCL6 and IL7 were higher in LC than in both PS and Rec. The interaction network of the altered proteins in each comparison were represented in Supplementary figure S2.

Correlation with DLCO and its evolution from 6 to 12 months in patients with PS

Figure 2A shows that 6 out of the 8 cytokines with increased levels in PS (CCL19, CCL20, CCL3, INF-γ, IL-8, MCP-4) negatively correlate with the level of DLCO at 12 months. Thanks to the design of our cohort, with repeated visits, we could evaluate the levels of these mediators at an earlier time point (6 months). Interestingly, at 6 months only CCL20 and IFN-γ were increased in PS (Fig. 2B), suggesting a persistent elevation in PS, starting early post SARS-CoV2 infection. Finally, we performed a pair t-test to study their change from 6 to 12 months. Figure 2C shows that CCL3 and CCL19 increased their levels from 6 to 12 months. All these results are summarized on Supplementary table 2.

Functional enrichment

Proteins increased (vs. Rec) in both PS and LC patients were involved in immune cell migration, activation, and anti-microbial immune response (Supplementary Table 3). Specifically, the altered pathways were IL-17, viral response, NF-KB signaling, and Toll-like receptors (TLRs) pathways (Fig. 3). In addition, PS patients showed functional enrichment related to T cells. Compared with LC, PS showed acute inflammation and tissue repair ontologies, whereas antimicrobial responses were increased in LC (Supplementary Table 4).

Organ damage markers

Circulating organ damage markers were similar in PS and Rec, but LC patients had 22 proteins with altered plasma levels (vs. PS) and 17 vs. Rec (Fig. 1B and Table S5). All of them were increased in the LC group, except for TNNI3 and CALCA which were reduced. Some of the altered proteins had been associated with the acute COVID episode, cardiac injury or stress-response (Supplementary table 6). Finally, the plasma levels of GDF15 and WFDC2 were measure due to their previous association with damage in several chronic lung diseases^24,25. Figure 4 show that both proteins were elevated in patients with PS and that their levels correlated with the DLCO at 12 months.

The main and novel observation of this study is that, one year after the acute COVID-19 episodes, patients with both PS and LC have evidence of systemic inflammation but the former show increased levels of C3 protein and autoantibodies whereas the latter have increased circulating levels of organ damage markers.

Previous studies

In contrast to our study, most post-COVID studies did not discriminate between PS and LC and were performed during the first months after the acute episode. A few ones studied PS 1 year after hospital discharge and found that MMP1, MMP7, periostin and VEGF levels were not increased in plasma²⁶ but, in keeping with our findings here, they also found increased C3 levels too²⁷. Another study had described NK cells with markers of impaired activation or exhaustion and reduced proportion of B cells ²⁸. In the BAL of patients with PS a previous study has shown antigen-specific memory B and T cells with dysregulated CD8 T cell responses³⁰. All these alterations may contribute to the persistent systemic inflammation we found in our study in PS patients.

Studies in patients with LC studied at 1 year of convalescence have shown increased levels of inflammatory markers²⁹, cytotoxic features³⁰ and autoantibodies³¹. The latter is at variance with our results here, but it is of note that controls in these studies were healthy, age-matched controls who never suffered COVID-19 and that higher autoantibody levels were observed at 3 months with progressive temporal reduction. A recent publication has described alteration in the complement cascade as the main driver of inflammation¹¹. In this work the LC group had a severe acute episode of the disease, in line with our findings that we observed elevated levels of the C3 complement in the group with a more severe acute disease, the PS. Another previous study proposed the stratification of LC patients into an inflammatory and a non-inflammatory type based on neutrophil activity, B cell memory alterations, and autoreactivity, but this study failed to identify an association of such a biological classification with any clinical characteristic of the patients³². A recent review described the effect of COVID-19 vaccination reducing the risk to develop LC³³. The patients of our cohort were recruited during the first year of the pandemic and thus were not vaccinated when they suffered the acute episode of the disease. Finally, the study with the longest convalescent period so far has shown persistence of plasma metabolic alterations 2 years after the acute episode in patients with LC ³⁴.

Interpretation of novel findings

Both PS and LC patients presented elevated plasma levels of proteins implicated in anti-microbial immune response. This suggest that persistent viral reservoirs can be inducing a persistent inflammation both in PS and LC patients. Yet, both type of patients exhibited some noticeable differences.

On the one hand, several of the proteins increased in PS patients correlate negatively with DLCO level at 12 months, indicating that increased levels are associated with the severity of the lung impairment. Specifically, CCL3 and CCL19 were elevated at 12 months, but not at 6 months, suggesting that are biomarkers of the mechanism driving the perpetuation of PS. Both CCL3 and CCL19 are chemokines, one responsible of the polymorphonuclear cells and the latter of T and B cells migration to secondary lymphoid organs, suggesting that the adaptive immune response might have a role in the development of long term PS. In turn, CCL20 and IFN-γ were persistently elevated after SARS-CoV2 infection making them promising early biomarkers of PS development. PS patients also showed increased levels of GDF15 and WFDC2, both mediators of epithelial damage³⁵, associated with lung alterations and chronic lung diseases. GDF15 was associated with fibrotic interstitial lung diseases^35,36 so it may be indicating a fibrotic remodeling of the lung impairment. WFDC2 was elevated in PS and in the Rec, the groups with a more severe acute episode of COVID-19 and it had been recently described increased with the severity of the disease³⁷.

On the other, LC patients showed abnormal plasma levels of a range of cardiac and cellular stress markers (Table S5). Further, TNNI3 (Troponin I) myocardial protein and CALCA (calcitonin) were the only two proteins with reduced plasma levels in LC. Of note, both proteins have been associated with the severity and mortality of the COVID-19 acute episode³⁸. These observations are consistent with the results in our cohort where their levels continue to be increased after 12 months of convalescence in Rec. and in PS, the groups with a more severe disease. Several of the proteins elevated in patients with LC have been previously associated with different SARS-CoV2 infective mechanisms, suggesting again the existence of a viral reservoir. Specifically, FKBP1B³⁹, PRKAB1⁴⁰ and CSNK1D⁴¹ has been associated with SARS-CoV2 viral replication, and KIM1 was described as a viral entry factor. SMAD1⁴² increases endothelial permeabilization and its inhibition in mice models of SARS-CoV2 infection showed a reduce mortality. FOXO1⁴³ promotes airway inflammation and a knock-out mice model protected from severe COVID19 inflammation disease. Interestingly, three proteins (TNNI3, FKBP1B and FOXO1)⁴⁴ are related to cardiac function, suggesting potential cardiac dysfunction in these patients. Remarkably up to 13 of these proteins have immune-related functions. STX8 and YES1 are implicated in T-cell activation, MAP4K5, RRM2B, FOXO1, ERBB2IP and INPPL1 can promote inflammation and KIM1, MVK and BANK1 had been associated with autoimmune diseases. Collectively, these observations suggest a persistent antiviral response in both PS and LC, while PS also presents lymphocyte homing chemokines while in LC the biomarkers are related to cardiac and cellular organ damage.

Strengths and potential limitations

The strengths of our study are the wide range of immune mediators and antibodies measured, the long-term follow up period (12 months), the comparison of patients with PS, LC and Rec, and the exploration of the changes between 6 to 12 months of mediators associated to DLCO. We acknowledge, however, that our sample size is limited, and that we studied only plasma samples, so our observations may not reflect the lung pathobiology in PS. Likewise, lung function was not determined before the acute COVID-19 episode but to address this unavoidable limitation we only include in our analysis patients without any previously known pulmonary disease.

Patients with PS or LC show persistent systemic inflammation 12 months after the acute COVID-19 episode, but CCL3 and CCL19 are specifically associated with PS, and the deterioration of DLCO in these patients, whereas LC patients present increased circulating levels of organ-damage markers. Collectively, these observations suggest different underlying biology of both post-COVID conditions.

COVID Coronavirus disease

PS Pulmonary sequelae

LC Long COVID

Rec Recovered

DLCO Carbon monoxide diffusing capacity

IFN Interferon

VEGF-A Vascular endothelial growth factor A

sITGaM Integrin alpha M

sITGb2 Integrin beta 2

IL-1α Interleukin 1 alpha

TGF-β Transforming Growth Factor beta

LCN2 Lipocalin-2

MMP-7 Metalloproteinase 7

HGF Hepatocyte Growth Factor

WHO World Health Organization

SEPAR Spanish Society of Pulmonology and Thoracic Surgery

ERS/ATS European Respiratory Society / American Thoracic Society

Ig Immunoglobulin

IFA Immunofluorescence assays

ANA Anti-Nuclear Autoreactivities

PMAT Particle-based multi-analyte technology

LOD Limit of detection

GDF-15 Growth/differentiation factor 15

WFDC2 WAP Four-Disulfide Core Domain 2

GO Gene Ontology

STRING Search Tool for the Retrieval of Interacting Genes/Proteins

TLRs Toll-like receptors

TNNI3 Troponin I

CALCA Calcitonin

Ethics approval and consent to participate

The Ethical Review Board of Clinic Barcelona approved the study (HCB/2020/0422), and all patients signed their informed consent to participate. We followed the STROBE guidelines to report the results of this observational study.

Consent for publication

Not applicable

Availability of data and materials

All raw data generated during this study are available from the corresponding author on reasonable request. Tables with the full results of the analysis performed to support the conclusions are available in the main manuscript and the online supplement.

Competing interests

All authors have declared their conflict of interests.

Funding

The present study has been founded by a Research grant from Menarini, Fons Mecenatge HCB-IDIBAPS, SEPAR, and AGAUR (PANDEMIES 2020). Rosa Faner is a Serra Húnter Professor, and Tamara Cruz is a La Caixa Young Leadership Fellow (ID 100010434, LCF/BQ/PI22/11910042).

Authors' contributions

Study conception and design: (RF, TC, AG, OS, JS), biological sample collection (TC, NA, GL, LP, NM, PM), data acquisition: (TC, ER, AP, AT, OV), clinical data collection and interpretation (TC, NA, GL, LP, NM, PM), data analysis: (TC, RF, AP, ER, OV), manuscript preparation: (TC, RF, AA, OS, JS), manuscript revision: All.

Acknowledgements

Authors thank all participants in the study for their willingness to contribute to medical research, and all field workers for their dedication and quality of their daily work. We are indebted to the HCB-IDIBAPS Biobank for the biological human samples and data procurement and to the Fundació Glòria Soler for their support to the COVIDBANK collection.

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Competing interest reported. AA has grants with the following companies GSK, AZ, Menarini, Chiesi, Sanofi and has received payments as consulting fees or lecture honoraria from GSK, AZ, Menarini, Chiesi, Sanofi, Zambon. JS has grants with the following companies Boehringer, Roche and has received payments as consulting fees or lecture honoraria from Aflorfam and Boehringer RF has grants with the following companies AZ, GSK, Menarini and has received payments as consulting fees or lecture honoraria from AZ, Chiesi

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Persistence of dysfunctional immune response 12 months after SARS-CoV-2 infection and their relationship with pulmonary sequelae and long covid

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Version 1

Abstract

Figures

INTRODUCTION

METHODS

Study Design, Patients and Ethics

Measurements

Clinical and lung function parameters

Blood sampling

Detection of complement, immunoglobulins and autoantibodies

Inflammation and organ damage proteins

Protein interaction and functional enrichment

Statistical analysis

RESULTS

Patient characteristics

Total Immunoglobulin levels and presence of autoantibodies

Inflammation proteins

Correlation with DLCO and its evolution from 6 to 12 months in patients with PS

Functional enrichment

Organ damage markers

DISCUSSION

Previous studies

Interpretation of novel findings

Strengths and potential limitations

CONCLUSIONS

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1