key: cord-0858109-0iy8clcm
authors: Zollner, Andreas; Koch, Robert; Jukic, Almina; Pfister, Alexandra; Meyer, Moritz; Rössler, Annika; Kimpel, Janine; Adolph, Timon E.; Tilg, Herbert
title: Post-acute COVID-19 is characterized by gut viral antigen persistence in inflammatory bowel diseases
date: 2022-05-01
journal: Gastroenterology
DOI: 10.1053/j.gastro.2022.04.037
sha: b9e84989bf753c42d3d79717a1f4f3bb4c5e4665
doc_id: 858109
cord_uid: 0iy8clcm

Background and aims The coronavirus disease 2019 (COVID-19) pandemic affects populations, societies and lives for more than two years. Long-term sequelae of COVID-19, collectively termed the post-acute COVID-19 syndrome, are rapidly emerging across the globe. Here, we investigated whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen persistence underlies the post-acute COVID-19 syndrome. Methods We performed an endoscopy study with 46 inflammatory bowel disease (IBD) patients 219 days (range: 94-257) after a confirmed COVID-19 infection. SARS-CoV-2 antigen persistence was assessed in the small and large intestine by qPCR of four viral transcripts, immunofluorescence of viral nucleocapsid and virus cultivation from biopsy tissue. Post-acute COVID-19 was assessed by a standardized questionnaire, and a systemic SARS-CoV-2 immune response was evaluated by flow-cytometry and ELISA at endoscopy. IBD activity was evaluated by clinical, biochemical and endoscopic means. Results We report expression of SARS-CoV-2 RNA in the gut mucosa ∼7 months after mild acute COVID-19 in 32 of 46 patients with IBD. Viral nucleocapsid protein persisted in 24 of 46 patients in gut epithelium and CD8+ T cells. Expression of SARS-CoV-2 antigens was not detectable in stool and viral antigen persistence was unrelated to severity of acute COVID-19, immunosuppressive therapy and gut inflammation. We were unable to culture SARS-CoV-2 from gut tissue of patients with viral antigen persistence. Post-acute sequelae of COVID-19 were reported from the majority of patients with viral antigen persistence, but not from patients without viral antigen persistence. Conclusion Our results indicate that SARS-CoV-2 antigen persistence in infected tissues serves as a basis for post-acute COVID-19. The concept that viral antigen persistence instigates immune perturbation and post-acute COVID-19 requires validation in controlled clinical trials.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a single-stranded positivesense RNA virus causing respiratory, gastrointestinal, and central nervous system infections in humans collectively referred to as coronavirus disease 2019 . 1 Cellular SARS-CoV-2 infection is mediated by an interaction of membrane-bound angiotensin-converting enzyme 2 (ACE2) with the viral spike and is facilitated by the host proteases TMPRSS2, TMPRSS4 and CatB/L. 2 ACE2 is expressed in the brush border of enterocytes in the gut, 3 a site of SARS-CoV-2 associated inflammation. 4 Studies with intestinal epithelial organoids confirmed that SARS-CoV-2 infects human epithelium, triggering an interferon (IFN)signature. 5 Consequently, SARS-CoV-2 can be detected in anal swabs, a signal which remains positive long after nasopharyngeal swabs are negative. 6 The post-acute COVID-19 syndrome is characterised by persistent or prolonged symptoms for more than four weeks after acute COVID- 19 . Considerable disagreement about definition, and thus prevalence, of post-acute COVID-19 exists, ranging from 10% to 87% of patients with COVID-19. 7, 8 The syndrome was initially dismissed by many but is now recognized as a multiorgan disease, which reflects a significantly growing health care challenge. 9, 10 Post-acute COVID-19 typically involves symptoms including severe fatigue, cognitive dysfunction or pain. 11 Poor baseline health status and severe acute COVID-19 convey risk for the development of post-acute COVID-19, however, also mild COVID-19 (e.g. in non-hospitalised patients) may culminate in post-acute sequelae. 12 Although post-acute COVID-19 symptoms usually do not require hospitalisation, 13 disease burden weighs heavily on affected individuals. 12 While the pathophysiologic mechanisms of acute COVID-19 are well defined (e.g. viral toxicity, microvascular injury, immune dysregulation and inflammation), 1 post-acute COVID-19 sequelae are poorly understood. It appears plausible that viral immune perturbation and/or inflammatory tissue injury during the acute infection account for the post-acute COVID-19 syndrome. 9 For example, neural accumulation of memory T cells in COVID-19 was observed in post-acute COVID-19 neuropsychiatric sequelae (e.g. malaise and depression), and reflects a hallmark of immune senescence during aging and tissue injury. 9 Furthermore it was shown that post-acute COVID-19 was associated with persistently activated innate immune cells and hyperactivated T and B cells, along with increased proinflammatory cytokine expression, 14 while the cause of such prolonged immune perturbation is unknown.

Here, we took advantage of upper and lower gastrointestinal endoscopy (and related mucosal tissue availability) in patients with or without post-acute COVID-19 symptoms who were evaluated in our inflammatory bowel disease outpatient unit, to explore whether gut antigen persistence 15 

To decipher SARS-CoV-2 RNA persistence in the gastrointestinal tract we isolated RNA as described previously. 16 In brief, RNAlater (Qiagen, Hilden, Germany) preserved biopsy specimens were homogenised in lysing buffer using a Precellys® 24 Homogenisator and a duodenum, ileum or colon) ( Figure 1B) . We detected viral RNA in 31% of biopsies, with expression of the RNA dependent RNA polymerase (RdRP) in 13.6% of biopsies, the surface glycoprotein (Spike) in 11.4% of biopsies, the nucleocapsid phosphoprotein (Nucleocapsid) in 10 .6% of biopsies, and the envelope protein in 6.1% of biopsies ( Figure 1C ). Detection by qPCR was unrelated to the intestinal location, the time from COVID-19 diagnosis to endoscopy and IBD activity ( Figure 1B and Table 2 ). SARS-CoV-2 expression was not detectable in stool from patients in this cohort (Supplementary Figure 1) .

To confirm long-term viral antigen persistence in the mucosa and to confine the cellular Figure 2B ). In addition, 11 patients who were RdRP, spike, or envelope positive in qPCR (but without a nucleocapsid qPCR signal) displayed nucleocapsid immunoreactivity ( Figure 2B and Table 2 ), indicating a patchy expression pattern as previously reported. 15 Nucleocapsid immunoreactivity specifically localized to epithelial cells ( Collectively, these data demonstrated that SARS-CoV-2 viral antigen persistence frequently occurs in the gut mucosa even months after acute COVID-19, but was not detectable in stool and appeared unrelated to viral replication.

Intestinal_SARS-CoV-2 8

In a next step, we hypothesised that gut mucosal SARS-CoV-2 antigen persistence links to post-acute sequelae of COVID-19. We stratified patients into two groups based on a positive SARS-CoV-2 qPCR result along the gut. Patients with a positive qPCR result were compared with patients that displayed no evidence for viral antigen persistence by qPCR. Time from and disease characteristics of acute COVID-19 and IBD were comparable between both groups as shown in Table 1 and 2 and Supplementary Table 2 . Notably, only patients who displayed viral RNA expression in the gut reported symptoms compatible with post-acute COVID-19 sequelae ( Figure 3A ). Patients without evidence for viral antigen persistence in our cohort (n=14) did not display post-acute COVID-19 symptoms ( Figure 3A ). We confirmed this observation by stratifying patients according to nucleocapsid immunoreactivity assessed by immunofluorescence (rather than qPCR positivity) ( Figure 3B ). Viral antigen persistence occurred in patients with and without immunosuppressive therapy (i.e. azathioprine, anti-TNF therapy or vedolizumab; Figure 3C ), and was unrelated to gut inflammation indicated by faecal calprotectin ( Figure 3D and Supplementary Table 2 ).

Finally, we sought to define potential mechanisms of viral antigen persistence in the intestine. 15 We analysed SARS-CoV-2-associated humoral and cellular immune responses with enzymelinked immunosorbent assays, interferon gamma release assay (IGRA) and by surface and intracellular flow cytometry (ICFC) of peripheral blood cells (using peptide pools mapping the spike and the nucleocapsid proteins). Blood-derived immune cells from patients with gut antigen persistence exhibited a comparable IFN-γ release upon SARS-CoV-2 nucleocapsid exposure as patients without antigen persistence ( Figure 3E ). We rather noted that patients with gut viral antigen persistence more frequently lacked evidence of anti-nucleocapsid IgG antibodies ( Figure 3F ) and that anti-TNF immunosuppressive therapy was associated with impaired inflammatory T cell responses upon nucleocapsid peptide stimulation ( Figure 3G and Supplementary Figure 6 ).

, ranging from asymptomatic to severe cases, partly depending on immunocompetency of the host. 21 We rationed that viral antigen persistence was observed in patients with and without immunosuppressive therapy and was unrelated to severity of acute COVID-19 or gut inflammation in IBD at time of endoscopy. We argue that viral antigen persistence reflects incomplete clearance of SARS-CoV-2 rather than subclinical (latent or persistent) infection, as we were unable to replicate virus from biopsyderived tissue. In line with this, we usually detected only some (but not all) viral transcripts in biopsies from the same patient. Our experimental data rather suggest that immunosuppressive therapy with or without genetic predisposition (affecting the immune system) may promote incomplete viral clearance. 22 Indeed, we find that some patients exhibit a lack of humoral nucleocapsid IgG antibodies, which is pronounced in those with gut antigen persistence. In Interferon gamma release assay (IGRA)

The presence of SARS-CoV-2 specific T cells directed against the spike (S) and nucleocapsid (N) proteins were assessed by interferon gamma release assays (IGRA) and validated with intracellular flow cytometry. 19 To specifically stimulate SARS-CoV-2 specific T cells, lithium heparin whole blood or isolated peripheral blood mononuclear cells (PBMC), were coincubated with peptide pools (Miltenyi Biotech, Bergisch-Gladbach, Germany) consisting of 15-mer peptides with 11 amino acids overlap covering the entire sequence of the spike glycoprotein (pepS) and the complete sequence of the nucleocapsid phosphoprotein (pepN).

For the IGRA 600 µl lithium heparin aliquots whole blood were co-incubated with and without SARS-CoV-2 specific peptides. Four sample preparations were used to determine interferongamma (IFN-γ). One sample was mock treated to determine the steady state IFN-γ expression, 

Background and context

Long-term sequelae of coronavirus disease 2019 (COVID- 19) , collectively termed the postacute COVID-19 syndrome, reflects a significantly growing health care challenge. To date, the pathophysiology of this debilitating multi-organ disease is poorly understood. We investigated whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen persistence underlies the post-acute COVID-19 syndrome.

We report that SARS-CoV-2 antigens persist in the gut mucosa for months after acute COVID-19 in the majority of patients with IBD irrespective of immunosuppressive therapy or gut inflammation. Viral antigen persistence associates with post-acute COVID-19 symptoms.

The concept of viral antigen persistence as driver of immune perturbation and post-acute COVID-19 syndrome should be corroborated in controlled clinical trials beyond IBD.

Collectively, our findings suggest that viral antigen persistence is a basis for post-acute sequelae of COVID-19, a rapidly emerging disorder across the globe.

Post-acute COVID-19 is a rapidly growing health care challenge across the globe. This study identifies that post-acute COVID-19 is accompanied by viral antigen persistence in the gut from patients with inflammatory bowel diseases.

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