The researchers from the United States used a whole-body positron emission tomography (PET) with a novel radiopharmaceutical [18F]F-AraG (fluorine-18-labeled-arabino-furanosyl-guanine) to localize activated T lymphocytes in the post-acute COVID participants with or without symptoms of long COVID. They also used quantitative PCR assays and in situ hybridization of SARS-CoV-2 spike RNA to investigate the presence of virus-specific RNA in rectosigmoid cells from participants who had symptoms of long COVID and who had colorectal biopsy. The post-acute COVID group, which included those with and without long COVID symptoms, had higher uptake of [18F]F-AraG in numerous anatomical regions (increased T cell activation in these tissues) compared with pre-pandemic controls up to 2.5 years after initial infection. SARS-CoV-2 spike RNA was detected in the rectosigmoid lamina propria of nearly all participants who underwent biopsy.

The pathophysiology of post-acute sequelae of COVID-19 (or long COVID) remains unclear. The evidence suggests that viral persistence, inflammation, and immune dysregulation play a significant role.  https://discovermednews.com/a-pathophysiological-model-of-long-covid-based-on-the-persistence-of-the-sars-cov-2-virus/

As the significance of T cell responses in non-blood tissues during post-acute COVID remains uncertain, the authors of this study employed a novel radiopharmaceutical [18F]F-AraG (fluorine-18-labeled-arabino-furanosyl-guanine) to perform whole-body PET/CT imaging. The [18F]F-AraG is a highly selective and sensitive tracer that enables the anatomical localization of activated CD8+ and CD4+ T lymphocytes. In vivo murine models have confirmed that activated T cells selective uptake [18F]F-AraG.

About the study

The study included 24 participants who were divided into two groups: those who were in the early post-acute phase (less than 90 days after the onset of COVID-19 symptoms, with full recovery, n=3 or without full recovery, n=6), and those who were in the later post-acute phase (more than 90 days after the onset of COVID-19 symptoms, with complete recovery, n=3 and with long COVID symptoms, n=15). The median age of the participants was 39.5 years (range 26 to 65), 11 were women and 13 were men. During the first visit, participants completed a questionnaire that assessed demographics, medical history, SARS-CoV-2 infection, treatment history, and vaccinations.

The majority of participants were infected with SARS-CoV-2 prior to the emergence of Omicron variants, and only two were hospitalized during the acute phase of infection. One participant was infected during the ancestral wave but experienced two documented re-infections with presumed Omicron variants. There were no other participants with acute symptoms suggesting infection with another virus or reinfection with SARS-CoV-2 between the initial COVID-19 episode and PET imaging. Over the study period, none of the subjects had subsequent positive COVID-19 PCR or antigen test after the initial confirmatory test.

All participants, except one, had received at least one anti-SARS-CoV-2 vaccine prior to the PET imaging. The median number of days from the last dose of vaccine to the tracer injection was 183 days. The authors stated that they performed PET imaging more than 60 days after any vaccine dose to minimize the effect of vaccination on T cell activation. The study team was not informed that one participant received a booster vaccine dose 6 days before imaging.

T cell activation in tissues was determined by whole-body [18F]F-AraG PET/CT imaging at time points ranging from 27 to 910 days after the onset of COVID-19 symptoms. The [18F]F-AraG was given intravenously. Pre-pandemic controls were 6 participants who underwent [18F]F-AraG PET prior to 2020.

The median number of long COVID symptoms was 5.5, with a range of 0 to 15. The most common symptoms were fatigue (n=16) and neurocognitive disorders (n=14).

Results

The uptake of [18F]F-AraG was higher in many anatomical regions in the post-acute COVID group, which included those with and without long COVID symptoms, compared to the pre-pandemic group. It is noteworthy that elevated T cell activation in these tissues was also observed in numerous individuals who did not exhibit symptoms of long COVID. The uptake of [18F]F-AraG was higher in the thoracic spinal cord, cauda equina, brainstem (pons), aortic arch, pulmonary artery and lower lung lobes compared to pre-pandemic controls. An increase in [18F]F-AraG uptake was also observed in the nasopharyngeal and hilar lymphoid tissue, proximal colon wall, rectal wall, lumbar and iliac crest, bone marrow and pharyngeal tonsils in the post-acute COVID group compared with pre-pandemic controls. Interestingly, after dividing participants by time since the first onset of COVID-19 symptoms, [18F]F-AraG uptake in the right ventricle wall was found to be higher in post-acute COVID participants than in the pre-pandemic controls.

The uptake of [18F]F-AraG in certain tissues was associated with a greater number of long COVID symptoms. Long COVID symptoms were associated with the higher [18F]F-AraG uptake in spinal cord, hilar lymph nodes and colon/rectal wall. In addition, participants who reported more than five symptoms of long COVID at the time of imaging had higher levels of circulating inflammatory markers, such as proteins involved in immune responses, chemokine signaling, inflammatory responses, and nervous system development. There was an increase in the expression of proteins such as TGFb1, TANKIL7, TANK, IL20RA, CCL13, SPRY2, PRKAB1, BCR, and TAF2.

Participants with pulmonary symptoms of long COVID, such as cough, shortness of breath or dyspnea, had higher uptake of [18F]F-AraG in their lower lung and hilar regions of interest than participants who did not have these symptoms. Also, participants with higher uptake of [18F]F-AraG in the lower lung had upregulated clusters of gene products, including those involved in inflammatory response, cell signaling fibroblast transformation, and response to mitogenic stimulation. There was an increase in expression of IL-7, CXCL3, CD40, EGF, TRNSF14, TIMP3, CRKl, CXCL3, BKAP2, and PDGFB.

Interestingly, men had higher uptake of [18F]F-AraG in hilar regions of interest than women did.

Computerized tomography (CT) scan of the chest demonstrated mild apical scarring and/or reticulation in four participants, indicating a mild pulmonary fibrosis. Other participants had normal CT scans, except for incidental findings that were not attributable to prior COVID-19 infection (e.g., calcified granulomas).

Analysis of the correlation between the biodistribution of [18F]F-AraG and the timing of immunization revealed that the timing between the last immunization and imaging appeared to have minimal effect on the uptake of [18F]F-AraG in most tissues. The only exception was a modestly higher uptake in the lower gut wall in those who had received the last SARS-CoV-2 vaccine less than 180 days before PET imaging.

Assessment of CD4+ and CD8+ T-cell, NK-cell, and B-cell phenotypes, including markers of activation, naive/memory phenotypes, regulatory function and immune checkpoint/exhaustion was performed by flow cytometric analysis in peripheral blood mononuclear cells from 16 participants and in gut tissue from five participants who had colorectal biopsy. Compared to peripheral blood, higher frequencies of effector memory CD8+ and CD4+ T cells, and similar frequencies of CD8+ and CD4+ lymphocytes expressing the activation markers CD38/HLA-DR and immune checkpoint (PD-1) were found in gut tissue.

Since many post-acute COVID participants had higher uptake of [18F]F-AraG in the proximal colon and rectal wall compared to pre-pandemic controls, the authors hypothesized that viral persistence may be responsible, at least in part, for migration of activated T cells to the gastrointestinal tissues. Therefore, they investigated the persistence of the virus in rectosigmoid tissue collected by flexible sigmoidoscopy in five participants who underwent PET imaging 158 to 676 days after initial SARS-CoV-2 infection. At the time of biopsy, all five participants reported at least one symptom of long COVID. None of them had received the anti-SARS-CoV-2 vaccine in the previous month. Three out of five participants did not have detectable SARS-CoV-2 nucleocapsid IgG in a short time frame from tissue collection, which suggests that they did not have recent SARS-CoV-2 reinfection.

The quantitative PCR assays and in situ hybridization of SARS-CoV-2 spike RNA were conducted. In all participants with long COVID symptoms who had biopsy, virus-specific RNA was detected in rectosigmoid cells. Almost all the SARS-CoV-2 spike RNA was found in cells located in the lamina propria, without any epithelial signal. PCR was also performed on RNA isolated from bulk rectal tissue lysates (separate biopsy) that targets the N1, N2, envelope and RNA-dependent RNA polymerase regions of SARS-CoV-2. No RNA was detected in any participant.

A small number of spike RNA+ cells expressed CD68, a macrophage monocyte lineage marker, but many RNA+ cells did not express CD68 and none expressed CD3.

In four participants, virus-specific RNA was detected in all three tissue regions surveyed. Furthermore, the [18F]F-AraG SUVmax values in proximal colon and rectal tissue in post-acute COVID participants who had biopsy were at least three standard deviation higher than mean SUVmax in pre-pandemic controls.

The authors concluded that these results provide additional evidence for the role of tissue-based immune activation that contributes to the post-acute sequelae of SARS-CoV-2 infection.

The results of the study have been published on a preprint server and are currently being peer-reviewed.

Journal Reference

Peluso MJ et al. Multimodal Molecular Imaging Reveals Tissue-Based T Cell Activation and Viral RNA Persistence for Up to 2 Years Following COVID-19. MedRxiv preprint. (Open Access)  https://www.medrxiv.org/content/10.1101/2023.07.27.23293177v1.full