Australian Study Shows That Long COVID Could Be The Result Of Persistent Perturbation Of The Peripheral Immune System

Long-COVID: A new study by researchers from the South Australian Health and Medical Research Institute-Adelaide, University of Adelaide, Royal Adelaide Hospital and Flinders University-South Australia has revealed that long COVID could be the result of persistent immune disturbances.

According to the study team, increasing evidence suggests immune dysregulation in individuals recovering from SARS- CoV-2 infection.
 
The team had undertaken an integrated analysis of immune responses at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 individuals recovering from mild, moderate, severe, or critical COVID-19. Anti-Spike and anti-RBD IgG responses were largely stable up to 24wpi and correlated with disease severity.
 
Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper and regulatory T cells) in COVID-19 convalescents compared to healthy controls, which were most strongly evident at 12 and 16wpi. RNA sequencing suggested ongoing immune and metabolic dysregulation in convalescent months after infection.
 
The study findings show that variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.medrxiv.org/content/10.1101/2021.07.30.21261234v1
 
The COVID-19 disease, which is caused by the SARS-CoV-2coronavirus, has devastated the world’s healthcare and economic systems.
 
As of August 9, 2021, SARS-CoV-2 has caused over 4.35 million deaths and infected over 203 million people worldwide.
 
Although most patients ‘recover’, persistent symptoms have been repeatedly described.
 
The study findings discuss observable abnormalities in the immune response following COVID-19 in samples collected from patients who have recovered from COVID-19. While these alterations do not appear to affect the antibody response, they appear to affect immune cells and the immune transcriptional profile.
 
It has now known that In most cases, COVID-19 presents as asymptomatic or with mild symptoms. Aside from these cases, many people infected with COVID-19 have developed moderately severe symptoms like fever, cough, headache, tiredness, diarrhea, and disturbances of smell and taste.
 
However, a significant minority of individuals with COVID-19 will proceed to develop severe or critical disease. This is particularly true for individuals with underlying comorbidities such as obesity, diabetes, or cardiovascular disease, or who are older in age.
 
It was found was found that among those who recover from COVID-19, which comprises the vast majority of cases, some report persistent symptoms for months, independent of the severity of their symptoms.
 

This condition is often referred to as ‘long COVID span>,’ and has been reported to affect up to two-thirds of discharged patients, in some studies. While most of the COVID-19 long-haulers describe continuing tiredness, muscle weakness, sleep disruption, anxiety, and depression, those who survived severe acute lung injury often have residual impairment of respiratory function and abnormal findings on subsequent chest X-rays.
 
Significantly the commonality of post-infectious syndromes after COVID-19, Ebola virus, and COVID-19 indicates that these are underlaid by a persistent abnormality in immune regulation in these individuals.
 
In past research, flow cytometry of peripheral blood samples from COVID-19 patients showed that both innate and adaptive immune cells change in their frequency.
 
For example, recovered patients had altered CD4+ and CD8+ T cell activation and exhaustion markers. Classical CD14+ monocytes, which belong to the innate immune system, were increased and showed inflammatory profiles. In those with a history of severe COVID-19, plasmacytoid Dendritic Cells (pDCs) were decreased, while effector CD8+ T cells were higher than in healthy controls.
 
The research was carried out in Australia, which was under strict containment measures in force at the time at which the study was conducted. These measures largely prevented the re-infection of recovered COVID-19 patients.
 
Importantly three aspects of the immune responses to COVID-19 were evaluated in this study. These include the production of antibodies to the viral spike (S) and receptor-binding domain (RBD), multiple immunological phenotypic parameters, as well as transcriptomics. The tested cohorts included individuals who had recovered from both mild/moderate or severe/critical COVID-19, as well as healthy controls.
 
In all, a total of 69 cases were tested at 12, 16, and 24 weeks from the first positive SARS-CoV-2 test. Of the cases, 50 had a history of mild COVID-19, while 6, 7, and 6 had moderate, severe, or critical disease, respectively. These results were compared with 14 seronegative healthy controls.
 
The findings showed that antibodies were robustly induced by COVID-19 in samples collected six months after recovering from the infection. More specifically, the immunoglobulin G (IgG) antibody titers to the S and RBD antigens covered a wide range, but remained stable over time, with a tendency to go down as time increased.
 
It must be noted however that IgM and IgA titers decreased over time. Anti-RBD IgG3 and IgM titers showed a more rapid decline over time as compared to antibodies against the S protein.
 
Interestingly the anti-S protein IgG titers were higher in those who had recovered from severe or critical disease as compared to milder cases, either at all-time points or at 24 weeks only. Similar differences were observed with anti-RBD antibodies, but only at 24 weeks. The titers of total anti-S protein and anti-RBD IgG antibodies remained correlated throughout.
 
The study team explored ten types of immune cells that were separated in each category on functional markers. These immune cells showed marked alterations, particularly at 12 and 16 weeks after the infection. While the differences in these cellular phenotypes were most prominent at 12 weeks, only some persisted to 24 weeks.
 
For example, lymphocyte counts remained depressed for up to 16 weeks following COVID-19 infection, even as CD3+ T cells and CD19+ B cells increased. Additionally, CD38+CD27+ memory B cells showed a significant rise.
 
It was also noted conversely that both the CD4+ and CD8+ T cells were reduced at 12 and 16 weeks. This was accompanied by a reduction in CD4+ effector memory (EM) pools, as well as migratory central memory (CM) CD4+ T cells throughout the study period.
 
NK or Natural killer cells were also increased following the COVID-19 diagnosis, both total and tissue migratory phenotypes. Total granulocytes, including low density (LD) neutrophils, were increased for up to 16 weeks. Among the latter, those expressing CXCR3+, which indicates their active migration into injured tissues, were higher at 12 weeks and returned to normal by 16 weeks. Notably, CD14+CD16+ neutrophils remained low at both time points.
 
It was observed that at 12 weeks, monocytes levels remained consistent. However, tissue-homing monocytes bearing the CXCR3+ marker, both activated and regulatory monocytes, were elevated.
 
Importantly antibody titers were correlated with granulocytes, NK CD16+ cells, and CD4+ T cells, thereby suggesting that they both mirror disease severity and that each of these cells may help boost the T cell response.
 
The T helper cells type 9 (Th9) declined throughout the study period; however, Th2/22 cells rose at 16 weeks. Since all of these subsets are epithelial-homing cells, these alterations reflect a dysregulated lung mucosal repair process. This observation could also indicate that these cells left the peripheral circulation on a large scale in order to enter damaged sites.
 
Also the Th17 and Th22 cells both showed higher proportions of mature T cells. The frequencies of these cells were consistently reduced at all-time points, while their relative proportions remained constant. These may therefore be implicated in antiviral immune responses.
 
The memory Th12/Th22 cells were also increased at 12 weeks, thus indicating that these cells were establishing immune memory focused on tissue healing.
It was also observed that some subsets of T follicular helper (Tfh) cells were elevated, such as Tfh1 and Tfh2/22.
 
The T regulatory cells (Tregs) that modulate these T cell lineages were increased in their naïve form throughout the course of the study; however, mature cells were consistently reduced up to 16 weeks. The changes in various Treg subsets indicate that the follicular regulatory T cell lineages (TfhR), which regulate both Tfh and B cell help, were reduced during the same period; however, the levels of these cells returned to normal by 24 weeks. The signal recruiting Th cells into inflamed or damaged tissue may therefore continue to operate for months after the initial infection.
 
The study findings concluded that this immune subset apparently responded to the early infection. As the virus migrated into the tissue, the TfhR cells increased, thereby indicating their role in B cell help within the lymphoid germinal centers. These two phases are required for a robust antiviral B cell response.
 
Importantly both ThR2/22 and TfhR2/22 subsets were positively associated with anti-SARS-CoV-2 S protein and RBD IgG levels. This suggests an antibody-regulatory function for this epithelial homing lineage.
 
Just as important, RNA sequencing showed significant changes in the way genes were expressed following infection for up to 24 weeks following infection, irrespective of disease severity. Genes involving ribosome biosynthesis, oxidative phosphorylation, as well as platelet activation and signaling, were downregulated for up to 16 weeks. At 24 weeks, only the complement activation pathway was downregulated.
 
Numerous inflammatory pathways were upregulated in COVID-19 convalescents. Further analysis showed that these long-term disruptions were not explained by changes in any given immune cell population.
 
It should also be noted that another approach using blood transcriptional modules (BTMs) showed that while more recovering patients showed increasing BTM activity shifting towards the healthy control baseline profile, a set of patients continued to show persistent dysregulation, even at six months.
 
Co-corresponding author, Dr Simon C. Barry from the Molecular Immunology, Robinson Research Institute at University of Adelaide, Adelaide told Thailand Medical News, “These study data suggest ongoing inflammatory responses and immune dysregulation in COVID-19 convalescents weeks-to-months after infection.”
 
The study team said that in summary, their  integrated network analysis reveals a complex interplay of relationships between circulating immune cell populations, transcriptional dysregulation, and humoral immune responses in COVID-19 convalescent patients and provides a resource for further exploration and investigation of these relationships.
 
On the whole, many convalescents remained antibody-positive, reacting to both the S and RBD antigens for up to six months after infection. Antibody titers were higher in those recovering from severe disease, which mirrors recent studies investigating the neutralizing activity of serum in these patients.
 
The IgM and IgG1 target the S1 domain of the S protein, thus impacting virus binding to the host cell. This is the focus of activity for most neutralizing antibodies and monoclonal antibodies.
 
However IgG3 antibodies bind to the S2 domain with higher affinity, which may prevent virus entry and syncytia formation. Thus, further research may focus on the relative roles of these antibodies in virus neutralization, as well as inhibiting virus infection by preventing the membrane fusion triggered by viral binding.
 
Also some immune cell subsets continued to show marked alterations relative to the baseline, even at six months. Lymphopenia persisted until 16 weeks, which was accompanied by changes in other T cell subsets. Memory B cells also appeared to remain activated and exhausted at three months.
 
Also it was observed that Naïve Tregs appeared to expand, perhaps to restore their numbers that were previously depleted by inflammation and tissue damage. This supports current views that Tregs are required both to modulate the immune response and enhance tissue repair processes.
 
Importantly increased TEMRA Tregs are considered to be a sign of T cell exhaustion, but could instead represent cytotoxic, migratory, and tissue repair activity in a subset of polyfunctional effector Tregs. In fact, the study team comments, their presence may “suggest a competition between classical immune suppression and tissue repair by these cells in response to tissue damage in COVID-19 convalescents.”
 
Significantly the differences in how and when such cellular and transcriptional changes revert to normal may explain long COVID development; however, this requires further study. Moreover, persistent dysregulation is perhaps an accompaniment of impaired humoral responses, and therefore of shorter protective immunity.
 
The study team concluded, “These changes to the peripheral immune system could have implications for how individuals recovering from infection respond to other challenges encountered in this period and persistent immune activation may also exacerbate other chronic conditions.”
 
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