Researchers from the University of São Paulo (USP) identified seven proteins present in the blood plasma of patients hospitalized for covid-19 that can serve as indicators of severity and even indicate therapeutic targets.
They are molecules associated with immune response, lung protection, vascular complications and inflammatory lack of control (cytokine storm), common in some patients with covid-19.
The action of proteins and their effects on the worsening of the disease will now be studied in greater depth by the group.
“Changes in blood plasma protein levels are good indicators of how disease develops, including viral infections. With the analysis of variations in protein expression [proteômica] of hospitalized patients, we selected seven molecules that we found most interesting to be investigated given the pathophysiology of covid-19 “, says Marília Rabelo Buzalaf, a researcher in the Department of Biological Sciences at the Faculty of Dentistry of Bauru (FOB-USP).
The study is part of the doctoral thesis of Daniele Castro di Flora and also has the collaboration of Carlos Ferreira dos Santos, director of FOB-USP, Deborah Maciel Cavalcanti Rosa, director of the State Hospital of Bauru, and Virginia Bodelão Richini Pereira, from Instituto Adolfo Lutz de Bauru.
Preliminary data were published on the medRxiv platform, still without peer review.
The group of researchers is supported by FAPESP through a Thematic Project and Regular Research Grants, whose resources were redirected to the study of covid-19.
“It is a disease with a wide range of symptoms and severity, and proteins related to different complications and stages of infection can pave the way for the identification of therapeutic targets and biomarkers that help decision making by health professionals” , says to FAPESP Agency.
The study evaluated, between May 4 and July 4, 2020, 163 patients admitted to the State Hospital of Bauru with a confirmed diagnosis of covid-19 by RT-PCR test.
The patients were divided into three groups: 76 individuals who were discharged from the hospital without intensive care unit (ICU) or mild cases, 56 who were discharged after remaining in the ICU (severe cases) and 31 who died even under follow-up at the ICU. ICU (critical).
When comparing the proteome of the three groups, the researchers observed high levels of the IREB2, GELS, POLR3D, PON1, SFTPD and ULBP6 proteins only in the mildest cases.
The blood samples analyzed were collected at the time the patients were admitted to the hospital. Weekly samples were also collected to follow the evolution of each participant. These data will be analyzed in the next stages of the research.
“Proteomic analysis showed us an exclusive protein for serious and critical cases [Gal-10] which, once validated, may serve as a prognostic marker. Another six were found only in mild patients and can give us important clues about possible therapeutic targets “, he says.
In the study, only patients who needed intensive treatment had Gal-10 protein in their blood plasma when they were admitted to the hospital. It is a well-known marker of defense cell death (eosinophils), which suggests, according to the researchers, some degree of impairment of the immune system.
“When eosinophils die, they release Gal-10 molecules, which bind to form highly immunogenic crystals – Charcot-Leyden crystals (CLCs) -, responsible for promoting type 2 immunity. [que envolve outras subpopulações de linfócitos]. These crystals have an important pro-inflammatory effect in the lung, leading to an influx of other defense cells, such as neutrophils and monocytes.
The increase in Gal-10 in critically ill and severe patients may be associated with the cytokine storm that leads to uncontrolled inflammation “, he explains.
According to the researcher, a recent study showed that antibodies against Gal-10 can completely dissolve the crystals present in the mucus in just two hours. “At the moment, we are waiting for the arrival of reagents to validate the increase of this protein in the plasma of severe and critical cases. Once the phenomenon is confirmed, antibodies against Gal-10 can be tested in the treatment of patients who present a high level of this protein or of CLCs at the time of hospital admission “, he says.
Other studies supported by FAPESP have identified prognostic markers.
The S-TREM proteins and SAA1 and SAA2, also increased according to the worsening of the disease, can serve as a “biological thermometer” capable of guiding the decision making by health teams (read more at: agencia.fapesp.br/34265/ and agencia.fapesp.br/34456/). In the present study, the SAA1 and SAA2 proteins were also increased in severe and critical cases, confirming the previous findings.
Potential therapeutic targets
Also associated with the cytokine storm, the IREB2 protein has the role of preventing the formation of ferritin, a mediator of the dysregulation of the immune system. According to the FOB-USP study, because it prevents the advance of the cascade effect that leads to unregulated inflammation, this protein was found only in mild patients.
“Previous research had already demonstrated the increase in ferritin levels in patients with severe cases of the disease and the importance that this phenomenon has in worsening. Therefore, it was not surprising to find IREB2 exclusively in patients who had a good prognosis. [casos suaves]”he says.
According to Buzalaf, the goal now is to identify drugs capable of increasing IREB2 expression and, consequently, reducing ferritin.
The ULBP6 protein, associated with the adaptive immune response (specific for each pathogen), was also selected for study. This molecule binds and activates the NKG2D receptor, located on the surface of immune cells, which plays an important role in immune control. “
The receptor mediates the toxicity of immune cells of the type natural killer [um tipo de linfócito que mata células infectadas]. In addition, these proteins have an influence on the clinical outcome of a variety of pathologies linked to the immune system, such as diabetic nephropathy and alopecia areata. But what interested us most was its polymorphism [forma alternativa da molécula]”, says the researcher.
Buzalaf explains that there is an alternative form of ULBPC in which, in the chain of amino acids that make up the protein, an arginine is exchanged for a leucine, which further increases the affinity for the NKG2D receptor. According to the researcher, this greater affinity reduces the activation of cells natural killer, impairing the response of the innate immune system.
“It is possible, and we still need to investigate, that critical patients have this polymorphism, which could lead to an impaired immune response”, he says.
According to the researcher, if it is proven that individuals with this alternative form of ULBP6 are more affected by the severe form of covid-19, it will be possible to develop a method that identifies this genetic susceptibility early.
Another protein selected by the researchers and found exclusively in patients who were not admitted to the ICU was geosoline. “This molecule has anti-inflammatory properties, as it binds to calcium and thus removes actin filaments that are circulating in the bloodstream, with an inflammatory action. Clinical studies conducted in other countries are already investigating the recombinant geosoline supplementation as a potential therapy Our finding corroborates this hypothesis “, he says.
The same will be investigated with the POLR3D protein, also found only among those hospitalized with a good prognosis. “This enzyme is involved in the innate immune response, in the production of type 1 interferon [citocina importante na resposta antiviral] and its function is to limit infection by intracellular bacteria and viruses. Perhaps a possible therapy is to increase the expression of this enzyme in the initial stages of the disease “, he states.
Another protein found only in mild patients is SFTPD, which is directly related to the defense of the lung against inhaled microorganisms, as it forms a kind of protective layer on the organ’s surface. “It is related to pulmonary surfactant, a liquid that significantly reduces the surface tension inside the pulmonary alveoli, preventing collapse during expiration. In our study, more severe patients did not have this protein. As the disease worsens, the patient will have little capacity to protect the lung “, he says.
Buzalaf comments that a recent study showed that melatonin increases the formation of surfactant in the lung (read more at agencia.fapesp.br/34959/).
The last protein selected, and which may indicate paths for the development of new treatments, is PON-1, an enzyme that degrades organic phosphates. “It is a molecule involved in the protection of low density lipoproteins against oxidative damage and the formation of atheroma. Therefore, it prevents lipid peroxidation with damage to the blood vessel wall. But it is also important for the immune response. “he says.
According to the researcher, a study carried out by another group, involving the analysis of candidate drugs against covid-19, found that genes correlated with ACE-2 (cell receptor for the entry of the SARS-COV-2 virus) are enriched in the degradation activity of organic phosphates. “This is another indication that PON-1 may have something to do with disease progression,” he says.
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.
This text was originally published by Agência FAPESP under the Creative Commons CC-BY-NC-ND license.