Markers of COVID-19 severity and therapeutic targets possibly unveiled in patients’ blood plasma

An analysis conducted in the Brazilian state of São Paulo detected seven proteins in plasma from hospitalized patients that could be used in novel treatments and methods of identifying potentially severe or critical cases.

By Maria Fernanda Ziegler  |  Agência FAPESP – Researchers at the University of São Paulo (USP) in Bauru (Brazil) have detected seven proteins in blood plasma samples from hospitalized COVID-19 patients that could serve as indicators of severity of the disease and point to therapeutic targets.

The proteins are associated with immune response and lung protection. They may also be markers of the vascular complications and exacerbated inflammation (cytokine storm) frequently seen in COVID-19 patients. Their action and role in the progression of the disease will now be studied in depth by the group.

“Alterations in plasma protein levels are good indicators of how viral infections and other diseases develop. We analyzed hospitalized patients for variations in protein expression [proteomics] and selected seven molecules that seemed most worth investigating, given the physiopathology of COVID-19,” Marília Rabelo Buzalaf, a researcher in the Biological Science Department of USP’s Bauru Dental School (FOB-USP), told Agência FAPESP.

The study was part of the PhD research of Daniele Castro di Flora. Carlos Ferreira dos Santos, Director of FOB-USP, Deborah Maciel Cavalcanti Rosa, Director of Bauru Hospital (HEB), and Virginia Bodelão Richini Pereira at Adolfo Lutz Institute in Bauru, collaborated on the project.

Preliminary results are published on the preprint platform medRxiv in a paper that has yet to be peer-reviewed. The research group is supported by FAPESP via a Thematic Project and a Regular Research Grant, both of which have been redirected to the study of COVID-19.

“This is a disease with a significant variation in symptoms and severity. Knowledge of the proteins associated with different complications and infection stages can help identify therapeutic targets and biomarkers that support decision-making by health professionals,” Buzalaf said.

Between May 4 and July 4, 2020, the study evaluated 163 patients admitted to HEB with COVID-19 diagnosed by RT-PCR and divided into three groups: 76 mild cases discharged without admission to the ICU, 56 severe cases discharged after treatment in the ICU, and 31 critical patients who died after treatment in the ICU.

A proteomics-based comparison of the three groups showed high levels of the proteins IREB2, GELS, POLR3D, PON1, SFTPD, and ULBP6 in mild cases only.

Blood samples were taken at hospital admission for the purposes of this analysis. Additional weekly samples were taken to monitor their progression, and the data from longer-term analysis will be processed in the forthcoming stages of the project. 

“The proteomics analysis highlighted a protein exclusive to severe and critical cases [Gal-10], and once this has been validated, it can serve as a prognostic marker. Six others were found only in patients with mild cases of the disease. These could provide important clues to possible therapeutic targets,” Buzalaf said.

Gal-10 was only detected in plasma samples taken on admission to the hospital from patients who required ICU treatment. This protein is a known marker of the death of white blood cells called eosinophils, suggesting some degree of immune system impairment.

“When eosinophils die, they release Gal-10, which forms highly immunogenic crystals known as Charcot-Leyden crystals or CLCs,” Buzalaf explained. “CLCs promote type 2 immunity [which involves other lymphocyte subpopulations] but have a significant pro-inflammatory effect on the lungs, leading to an influx of other defense cells, such as neutrophils and monocytes. The increase in Gal-10 in critical and severe patients may be associated with the cytokine storm and exacerbated inflammation typical of the poorest COVID-19 outcomes.”

In a recent study, she added, antibodies against Gal-10 completely dissolved CLCs in mucus within two hours. “We’re currently awaiting the arrival of reagents to validate the increase in plasma levels of this protein in severe and critical cases. If the phenomenon is confirmed, antibodies against Gal-10 can be tested in the treatment of patients with high levels of the protein or CLCs at hospitalization,” she said.

Other studies supported by FAPESP have found prognostic markers. TREM proteins, especially sTREM-1, as well as SAA1 and SAA2, high levels of which are also present in severe cases, are considered important biomarkers that can help medical teams make clinical decisions (read more at: agencia.fapesp.br/34528 and agencia.fapesp.br/34668). In this latest study, SAA1 and SAA2 were augmented in severe and critical cases, confirming earlier findings.

Potential therapeutic targets

IREB2, another cytokine storm-related protein, prevents the formation of ferritin, a mediator of immune system regulation. In the FOB-USP study, IREB2 was found only in mild patients and presumably prevented progression of the cascade effect that leads to exacerbated inflammation.

“Previous research had shown that ferritin levels rise in severe patients, underscoring the importance of this rise in progression to a severe condition, so we weren’t surprised to find IREB2 only in mild patients,” Buzalaf said. The goal now, she added, is to develop drugs that increase IREB2 expression and hence reduce ferritin.

Another protein selected for further investigation is ULBP6, associated with adaptive immunity (the immune response specific to each pathogen). This molecule binds to and activates the receptor NKG2D, which is located on the surface of immune cells and plays a key role in immune control. “This receptor mediates the toxicity of immune cells of the natural killer type [lymphocytes that kill infected cells]. The protein also influences the clinical outcomes of a number of pathologies linked to the immune system, such as diabetic nephropathy and alopecia areata. But what interested us most was its polymorphism [having more than one form],” Buzalaf said.

There is an alternative form of ULBPC, she explained, in which an arginine is replaced by a leucine in the protein’s amino acid chain. The substitution increases its affinity for NKG2D, reducing natural killer cell activation and impairing the innate immune response.

“It’s possible that critical patients have this polymorphism. We need to investigate this possibility, which could entail impairment of the immune response,” Buzalaf said. 

If further research does indeed find that patients with this alternative form of ULBP6 tend to progress to the severe form of COVID-19, it will be possible to develop a method to identify this genetic susceptibility before they do so.

Gelsolin, another protein selected by the researchers, was found only in patients who did not require ICU treatment. “It has anti-inflammatory properties because it binds to calcium and removes actin filaments from the bloodstream,” Buzalaf said, explaining that actin filaments are involved in lung inflammation. “Clinical trials are now being conducted in other countries to investigate supplementation with recombinant gelsolin as a possible therapy. Our finding corroborates this hypothesis.”

The protein POLR3D was also found only in hospitalized patients with mild symptoms. “This enzyme is involved in the innate immune response and in production of type 1 interferon [an important cytokine in the response to viruses]. It plays a key role in limiting infection by intracellular bacteria and viruses. Increasing its expression in the early stages of the disease could be a therapeutic possibility,” she said.

Another protein found only in mild patients was SFTPD, which contributes to defense of the lungs against inhaled microorganisms by forming a protective layer on the lung’s surface. “It participates in the renewal of pulmonary surfactant, which lowers surface tension and keeps the alveoli from collapsing after exhalation,” Buzalaf said. “In our study, this protein was missing in severe and critical patients, who can’t have had much lung protection capacity as their condition worsened.”

A recent study, she added, showed that melatonin increases the formation of pulmonary surfactant (read more at: agencia.fapesp.br/35057). 

The last protein selected was PON-1, an enzyme that breaks down organic phosphate and could point to the development of new treatments. “It’s involved in the protection of low-density lipoproteins against oxidative damage and the formation of atheroma, so it prevents lipid peroxidation and blood vessel wall damage. But it’s also important to the immune response,” she said.

According to Buzalaf, a study by another group involving the analysis of candidate drugs for COVID-19 found that genes correlating with ACE2 (the receptor used by SARS-COV-2 to enter cells) are especially good at breaking down organic phosphate. “That’s another indication that PON-1 may play a role in the progression of the disease,” she said.

The article “Quantitative plasma proteomics of survivor and non-survivor COVID-19 patients admitted to hospital unravels potential prognostic biomarkers and therapeutic targets” can be read at: www.medrxiv.org/content/10.1101/2020.12.26.20248855v1.