While coronavirus disease 2019 (COVID-19) in children is relatively rare and usually mild, some have been known to return with a rare but serious, or even life-threatening, complication called Multisystem Inflammatory Syndrome in Children (MIS-C). These children present with gut symptoms followed by inflammatory symptoms in several organs and often cardiac damage.
A new paper in the Journal of Clinical Investigation reports that this condition is due to damage to the gut, mediated by a decrease in the protein called zonulin, which is responsible for the integrity of the gut epithelial barrier. Reduced zonulin levels are found in several inflammatory and autoimmune conditions.
Background
MIS-C presents with persistent fever, abdominal pain, nausea and/or vomiting, high cytokine levels, and poor heart function, sometimes leading to cardiogenic shock. In fact, 80% of patients have symptoms of heart problems. The cause remains unknown, adding powerful interest to the findings of the current study.
Earlier researchers discovered that the virus has a superantigen-like domain on the spike protein, near the S1/S2 cleavage site, triggering broad and intense inflammation. This explains why monocytes and phagocytes are increased in number, along with dysregulated cytokine levels and excessive T cell activation, as well as abnormally elevated inflammatory markers, all associated with immunoglobulin expansion.
Adult studies indicate that the gut is a fertile site for SARs-CoV-2 infection, which, when severe, leads to abnormal gut microbiome composition and the breakdown of the gut epithelial barrier to activate systemic inflammation.
Study details
The study included 100 children, of which about a quarter had acute COVID-19, and a fifth had MIS-C. Over half were healthy controls. The MIS-C patients reported three days (median duration) of acute MIS-C symptoms following a previous infection with the virus.
The median gap between infection and MIS-C was 26 days. Almost 90% of these patients had gut symptoms, but only just over a quarter of those with acute COVID-19.
All participants had their stool examined by reverse transcriptase-polymerase chain reaction (PCR) for the presence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition, zonulin levels were assessed in plasma.
Antigenemia and gut permeability
Even at this late stage, the gut samples from MIS-C patients showed the presence of viral RNA at 1.5x102 - 2.5x107 RNA copies/mL. Zonulin levels were high, as were lipopolysaccharide-binding protein (LBP) and soluble CD14concentrations. The latter are markers that show that microbes were crossing the gut barrier.
The fact that microbial translocation was detected in these patients tends to support the leakage of SARS-CoV-2 antigens as the basis for the cytokine storm that triggers MIS-C. Other conditions associated with microbial leakage into the blood show distinct symptoms not seen in these patients.
None of these markers were high in acute COVID-19, supporting the view that the presence of the virus affected tight junction integrity within the membrane.
Plasma levels of SARS-CoV-2 antigens were also measured, along with immunologic markers. This showed that, indeed, viral components cross the intestinal barrier to cause inflammation.
Superantigens are capable of avoiding specific T cell receptors to cause the over-reaction of these receptors in a broad-based way.
Leaky gut allows antigenemia leading to profound inflammation
The gut is the likely source of the viral antigens and the reason for the immune activation in MIS-C. Most of these patients had very severe gut symptoms. These findings warrant tissue-based studies to confirm the presence of SARS-CoV-2 infection in the gut for weeks after infection.
Chronic infection of the gut with this virus led to the release of zonulin, indicating the loosening of tight junctions and increased gut permeability, or ‘leaky gut’. This led to the escape of excessive levels of SARS-CoV-2 antigens into the bloodstream, inducing intense systemic inflammation manifesting as MIS-C.
The patients showed antibodies to the spike, namely, Immunoglobulin IgM, IgG, and IgA subsets, showing that they were still undergoing mucosal exposure to the virus, within the gut, especially against the spike and its S1 domain proteins. This sustained but surprising rise in IgA and IgG antibodies to the spike antigen supports the theory that MIS-C is due to ongoing antigenic exposure and inflammation.
The high titers of IgG antibodies, however, did not clear the virus and appear to have poor neutralizing capacity. This explains why high antigen levels were detectable in children with MIS-C, indicating ineffective neutralizing responses.
In adults, conversely, acute COVID-19 is accompanied by rapid seroconversion, which results in viral clearance.
Treatment with zonulin antagonist
Finally, one patient with MIS-C was treated with a zonulin antagonist called larazotide to visualize the effects on SARS-CoV-2 antigens in the blood and the overall clinical response.
The child treated with larazotide had multiple medical problems and had a history of severe COVID-19 with respiratory failure and cardiac arrest. One month later, the patient developed signs of MIS-C, failing to show improvement with steroids nor intravenous immunoglobulins (IVIG).
High viral RNA titers were present in the stool, as well as spike antigens in plasma even two weeks after these agents were started.
Treatment with larazotide led to a dramatic reduction in viral antigens, inflammatory markers and clinical parameters, compared to the ineffectiveness of either steroids or IVIG.
What are the implications?
“In MIS-C, zonulin-dependent loss of gastrointestinal tight junctions results in SARS-CoV-2 antigenemia.” Increased levels of inflammatory antibodies appear to be induced by the high antigen levels in plasma, which are due to the abnormal gut permeability.
This study is the first time viral antigens have been reported in MIS-C, mostly spike and S1 protein. In adults with COVID-19-related respiratory failure, nucleocapsid and S1 antigens are typically prominent in plasma. The different antigens identified in these two severe inflammatory conditions following SARS-CoV-2 infection may highlight distinctive underlying features in these complications.
It is noteworthy that the superantigen-like motif is on the S1 domain, which is raised in severe adult COVID-19 and in MIS-C. This explains why the T cell receptor beta variable gene (TRBV)11-2 is expanded, along with viral antigenemia.
The study not only describes a possible and plausible mechanism for MIS-C, but indicates the potential utility of zonulin measurement in SARS-CoV-2-infected children as a monitoring tool to identify those at risk of MIS-C.
It also serves as a “proof of concept that zonulin antagonism directly reduces SARS-CoV-2 antigenemia with abatement of the cytokine storm and subsequent clinical improvement in a single patient.”
Larazotide has a high safety margin and is undergoing phase 3 trials for the treatment of resistant celiac disease, an inflammatory bowel condition. Its use led to a 90% fall in spike antigen levels in the blood, with significant improvement in inflammation, fever and general illness.
Further therapies that reduce gut permeability could be useful in preventing or treating this condition.
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