SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC).

Millions of people are suffering from Long COVID or post-acute sequelae of COVID-19 (PASC). Several biological factors have emerged as potential drivers of PASC pathology. Some individuals with PASC may not fully clear the coronavirus SARS-CoV-2 after acute infection. Instead, replicating virus and/or viral RNA-potentially capable of being translated to produce viral proteins-persist in tissue as a 'reservoir'. This reservoir could modulate host immune responses or release viral proteins into the circulation. Here we review studies that have identified SARS-CoV-2 RNA/protein or immune responses indicative of a SARS-CoV-2 reservoir in PASC samples. Mechanisms by which a SARS-CoV-2 reservoir may contribute to PASC pathology, including coagulation, microbiome and neuroimmune abnormalities, are delineated. We identify research priorities to guide the further study of a SARS-CoV-2 reservoir in PASC, with the goal that clinical trials of antivirals or other therapeutics with potential to clear a SARS-CoV-2 reservoir are accelerated.

Long COVID or Post-acute Sequelae of COVID-19 (PASC): An Overview of Biological Factors That May Contribute to Persistent Symptoms.

The novel virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic of coronavirus disease 2019 (COVID-19). Across the globe, a subset of patients who sustain an acute SARS-CoV-2 infection are developing a wide range of persistent symptoms that do not resolve over the course of many months. These patients are being given the diagnosis Long COVID or Post-acute sequelae of COVID-19 (PASC). It is likely that individual patients with a PASC diagnosis have different underlying biological factors driving their symptoms, none of which are mutually exclusive. This paper details mechanisms by which RNA viruses beyond just SARS-CoV-2 have be connected to long-term health consequences. It also reviews literature on acute COVID-19 and other virus-initiated chronic syndromes such as post-Ebola syndrome or myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) to discuss different scenarios for PASC symptom development. Potential contributors to PASC symptoms include consequences from acute SARS-CoV-2 injury to one or multiple organs, persistent reservoirs of SARS-CoV-2 in certain tissues, re-activation of neurotrophic pathogens such as herpesviruses under conditions of COVID-19 immune dysregulation, SARS-CoV-2 interactions with host microbiome/virome communities, clotting/coagulation issues, dysfunctional brainstem/vagus nerve signaling, ongoing activity of primed immune cells, and autoimmunity due to molecular mimicry between pathogen and host proteins. The individualized nature of PASC symptoms suggests that different therapeutic approaches may be required to best manage care for specific patients with the diagnosis.

Metagenomic compendium of 189,680 DNA viruses from the human gut microbiome.

Bacteriophages have important roles in the ecology of the human gut microbiome but are under-represented in reference databases. To address this problem, we assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Over 75% of genomes represent double-stranded DNA phages that infect members of the Bacteroidia and Clostridia classes. Based on sequence clustering we identified 54,118 candidate viral species, 92% of which were not found in existing databases. The Metagenomic Gut Virus catalogue improves detection of viruses in stool metagenomes and accounts for nearly 40% of CRISPR spacers found in human gut Bacteria and Archaea. We also produced a catalogue of 459,375 viral protein clusters to explore the functional potential of the gut virome. This revealed tens of thousands of diversity-generating retroelements, which use error-prone reverse transcription to mutate target genes and may be involved in the molecular arms race between phages and their bacterial hosts.

Re-framing the theory of autoimmunity in the era of the microbiome: persistent pathogens, autoantibodies, and molecular mimicry.

The theory of autoimmunity was developed at a time when the human body was regarded as largely sterile. Antibodies in patients with chronic inflammatory disease could consequently not be tied to persistent human pathogens. The concept of the "autoantibody" was created to reconcile this phenomenon. Today, however, the discovery of the human microbiome has revolutionized our understanding of human biology. Humans are superorganisms that harbor trillions of persistent microbial cells. Indeed, vast human microbiomes have been detected in human tissue and blood. These microbial ecosystems harbor thousands of newly identified bacteria, viruses, and other microorganisms -- most of which can act as pathogens under conditions of immunosuppression. The theory of autoimmunity must be revised to account for the human microbiome. Here, we propose a model in which "autoantibodies" are created in response to chronic, persistent microbiome pathogens. The structural homology (molecular mimicry) between pathogen and host proteins can result in "collateral damage" to surrounding human tissue. This calls for a paradigm shift in autoimmune disease treatment. Immunosuppressive medications palliate inflammatory symptoms at the expense of microbiome health and balance. In contrast, treatments that support the immune system in autoimmune disease could allow patients to target pathogens at the root of the disease process.

Microbe-microbe and host-microbe interactions drive microbiome dysbiosis and inflammatory processes.

An extensive microbiome comprised of bacteria, viruses, bacteriophages, and fungi is now understood to persist in nearly every human body site, including tissue and blood. The genomes of these microbes continually interact with the human genome in order to regulate host metabolism. Many components of this microbiome are capable of both commensal and pathogenic activity. They are additionally able to persist in both 'acute' and chronic forms. Inflammatory conditions historically studied separately (autoimmune, neurological and malignant) are now repeatedly tied to a common trend: imbalance or dysbiosis of these microbial ecosystems. Population-based studies of the microbiome can shed light on this dysbiosis. However, it is the collective activity of the microbiome that drives inflammatory processes via complex microbe-microbe and host-microbe interactions. Many microbes survive as polymicrobial entities in order to evade the immune response. Pathogens in these communities alter their gene expression in ways that promote community-wide virulence. Other microbes persist inside the cells of the immune system, where they directly interfere with host transcription, translation, and DNA repair mechanisms. The numerous proteins and metabolites expressed by these pathogens further dysregulate human gene expression in a manner that promotes imbalance and immunosuppression. Molecular mimicry, or homology between host and microbial proteins, complicates the nature of this interference. When taken together, these microbe-microbe and host-microbe interactions are capable of driving the large-scale failure of human metabolism characteristic of many different inflammatory conditions.

Inflammatory disease and the human microbiome.

The human body is a superorganism in which thousands of microbial genomes continually interact with the human genome. A range of physical and neurological inflammatory diseases are now associated with shifts in microbiome composition. Seemingly disparate inflammatory conditions may arise from similar disruption of microbiome homeostasis. Intracellular pathogens long associated with inflammatory disease are able to slow the innate immune response by dysregulating activity of the VDR nuclear receptor. This facilitates the ability of other species to gradually accumulate in tissue and blood, where they generate proteins and metabolites that significantly interfere with the body's metabolic processes. The microbes that contribute to this dysfunction are often inherited from family members. Immunosuppressive therapies for inflammatory disease allow pathogens driving these processes to spread with greater ease. In contrast to immunosuppression, treatments that stimulate the immune system seem to allow for reversal of this pathogen-induced genomic dysregulation.

Immunostimulation in the treatment for chronic fatigue syndrome/myalgic encephalomyelitis.

Chronic fatigue syndrome (CFS)/myalgic encephalomyelitis (ME) has long been associated with the presence of infectious agents, but no single pathogen has been reliably identified in all patients with the disease. Recent studies using metagenomic techniques have demonstrated the presence of thousands of microbes in the human body that were previously undetected and unknown to science. More importantly, such species interact together by sharing genes and genetic function within communities. It follows that searching for a singular pathogen may greatly underestimate the microbial complexity potentially driving a complex disease like CFS/ME. Intracellular microbes alter the expression of human genes in order to facilitate their survival. We have put forth a model describing how multiple species-bacterial, viral, and fungal-can cumulatively dysregulate expression by the VDR nuclear receptor in order to survive and thus drive a disease process. Based on this model, we have developed an immunostimulatory therapy that is showing promise inducing both subjective and objective improvement in patients suffering from CFS/ME.

The human microbiome and autoimmunity.

PURPOSE OF REVIEW: To demonstrate how dysbiosis of the human microbiome can drive autoimmune disease. RECENT FINDINGS: Humans are superorganisms. The human body harbors an extensive microbiome, which has been shown to differ in patients with autoimmune diagnoses. Intracellular microbes slow innate immune defenses by dysregulating the vitamin D nuclear receptor, allowing pathogens to accumulate in tissue and blood. Molecular mimicry between pathogen and host causes further dysfunction by interfering with human protein interactions. Autoantibodies may well be created in response to pathogens. SUMMARY: The catastrophic failure of human metabolism observed in autoimmune disease results from a common underlying pathogenesis - the successive accumulation of pathogens into the microbiome over time, and the ability of such pathogens to dysregulate gene transcription, translation, and human metabolic processes. Autoimmune diseases are more likely passed in families because of the inheritance of a familial microbiome, rather than Mendelian inheritance of genetic abnormalities. We can stimulate innate immune defenses and allow patients to target pathogens, but cell death results in immunopathology.

Immunostimulation in the era of the metagenome.

Microbes are increasingly being implicated in autoimmune disease. This calls for a re-evaluation of how these chronic inflammatory illnesses are routinely treated. The standard of care for autoimmune disease remains the use of medications that slow the immune response, while treatments aimed at eradicating microbes seek the exact opposite-stimulation of the innate immune response. Immunostimulation is complicated by a cascade of sequelae, including exacerbated inflammation, which occurs in response to microbial death. Over the past 8 years, we have collaborated with American and international clinical professionals to research a model-based treatment for inflammatory disease. This intervention, designed to stimulate the innate immune response, has required a reevaluation of disease progression and amelioration. Paramount is the inherent conflict between palliation and microbicidal efficacy. Increased microbicidal activity was experienced as immunopathology-a temporary worsening of symptoms. Further studies are needed, but they will require careful planning to manage this immunopathology.

Dysregulation of the vitamin D nuclear receptor may contribute to the higher prevalence of some autoimmune diseases in women.

Researchers have noted that the incidence of autoimmune diseases, such as Hashimoto's thyroiditis, is markedly higher in women than in men, but to date the reason for this disparity has been unclear. The vitamin D nuclear receptor (VDR) is expressed in the human cycling endometrium. Because the VDR controls expression of the cathelicidin and beta-defensin antimicrobial peptides (AmPs), dysregulation of the receptor greatly compromises the innate immune response. Increasing evidence indicates the presence of a chronic, intraphagocytic, metagenomic microbiota in patients with autoimmune disease that may survive by dysregulating the VDR. VDR dysregulation, in turn, prevents the breakdown of the active vitamin D metabolite 1,25-hydroxyvitamin D (1,25-D) by CYP24. In silico data suggest that when 1,25-D rises above its normal range, it binds the alpha/beta thyroid receptors, the glucocorticoid receptor (GCR), and the androgen receptor (AR), displacing their native ligands and causing an array of hormonal imbalances. If T3 is displaced from alpha-thyroid, thyroiditis may result. Because the VDR, GCR, and AR also express multiple families of AmPs, expression of these natural antibiotics further wanes in response to dysregulation by 1,25-D. The end result is a system-wide drop in AmP expression that may allow pathogens to spread with greater ease. Because women have an extra site of VDR expression in the endometrium, the drop in AmP expression associated with nuclear receptor dysregulation may disproportionately affect them. This would cause women to accumulate higher bacterial loads than their male counterparts, particularly during early pregnancy when 1,25-D levels rise by 40%.

Vitamin D metabolites as clinical markers in autoimmune and chronic disease.

Recent research has implicated vitamin D deficiency (serum levels of 25-hydroxyvitamin D <50 nmol/L) with a number of chronic conditions, including autoimmune conditions such as multiple sclerosis, lupus, and psoriasis, and chronic conditions such as osteoporosis, osteoarthritis, metabolic syndrome, fibromyalgia and chronic fatigue syndrome. It has been assumed that low levels of 25-hydroxyvitamin D (25-D) accurately indicate vitamin D storage and vitamin D receptor (VDR)-mediated control of calcium metabolism and innate immunity. To evaluate this assumption, 25-D and 1,25-dihydroxyvitamin D3 (1,25-D) levels were measured in 100 Canadian patients with these conditions. Additionally, other inflammatory markers (CK, CRP) were measured. Results showed a strong positive association between these autoimmune conditions and levels of 1,25-D >110 pmol/L. However, there was little association with vitamin D deficiency or the other inflammatory markers, meaning that the results challenge the assumption that serum levels of 25-D are a sensitive measure of the autoimmune disease state. Rather, these findings support the use of 1,25-D as a clinical marker in autoimmune conditions. High levels of 1,25-D may result when dysregulation of the VDR by bacterial ligands prevents the receptor from expressing enzymes necessary to keep 1,25-D in a normal range.

Autoimmune disease in the era of the metagenome.

Studies of autoimmune disease have focused on the characteristics of the identifiable antibodies. But as our knowledge of the genes associated with the disease states expands, we understand that humans must be viewed as superorganisms in which a plethora of bacterial genomes - a metagenome - work in tandem with our own. The NIH has estimated that 90% of the cells in Homo sapiens are microbial and not human in origin. Some of these microbes create metabolites that interfere with the expression of genes associated with autoimmune disease. Thus, we must re-examine how human gene transcription is affected by the plethora of microbial metabolites. We can no longer assume that antibodies generated in autoimmune disease are created solely as autoantibodies to human DNA. Evidence is now emerging that the human microbiota accumulates during a lifetime, and a variety of persistence mechanisms are coming to light. In one model, obstruction of VDR nuclear-receptor-transcription prevents the innate immune system from making key antimicrobials, allowing the microbes to persist. Genes from these microbes must necessarily impact disease progression. Recent efforts to decrease this VDR-perverting microbiota in patients with autoimmune disease have resulted in reversal of autoimmune processes. As the NIH Human Microbiome Project continues to better characterize the human metagenome, new insights into autoimmune pathogenesis are beginning to emerge.

Vitamin D: the alternative hypothesis.

Early studies on vitamin D showed promise that various forms of the "vitamin" may be protective against chronic disease, yet systematic reviews and longer-term studies have failed to confirm these findings. A number of studies have suggested that patients with autoimmune diagnoses are deficient in 25-hydroxyvitamin D (25-D) and that consuming greater quantities of vitamin D, which further elevates 25 D levels, alleviates autoimmune disease symptoms. Some years ago, molecular biology identified 25 D as a secosteroid. Secosteroids would typically be expected to depress inflammation, which is in line with the reports of symptomatic improvement. The simplistic first-order mass-action model used to guide the early vitamin studies is now giving way to a more complex description of action. When active, the Vitamin D nuclear receptor (VDR) affects transcription of at least 913 genes and impacts processes ranging from calcium metabolism to expression of key antimicrobial peptides. Additionally, recent research on the Human Microbiome shows that bacteria are far more pervasive than previously thought, increasing the possibility that autoimmune disease is bacterial in origin. Emerging molecular evidence suggests that symptomatic improvements among those administered vitamin D is the result of 25-D's ability to temper bacterial-induced inflammation by slowing VDR activity. While this results in short-term palliation, persistent pathogens that may influence disease progression, proliferate over the long-term.