A study coordinated by researchers from the State University of Campinas (Unicamp) and published on the platform medRxiv brings new evidence that the virus SARS-CoV-2 it is capable of infecting and replicating within lymphocytes, which can cause these defense cells to die and at least temporarily compromise the immune system.
According to the authors, the new coronavirus acts by a mechanism similar to that of HIV, which causes AIDS. Both affect a type of lymphocyte known as T CD4, which is responsible for coordinating the so-called adaptive immune response – helping both B lymphocytes to produce antibodies and CD8 T lymphocytes – responsible for recognizing and killing infected cells – to proliferate. This coordination occurs through the release of signaling molecules known as cytokines.
In the image on the left it is possible to observe the RdRp enzyme of SARS-CoV-2 (in red), which regulates viral replication, inside the CD4 T lymphocyte. In the image on the right, the green spots represent the SARS-spike proteins. CoV-2 (spike proteins) close to the human defense cell membrane
“Our results suggest that, in some patients, the new coronavirus can cause acute immunodeficiency not only because it kills part of the CD4 T lymphocytes, but also because it impairs the function of these cells. This causes the CD8 T lymphocytes to proliferate less. and B lymphocytes produce antibodies with less affinity and duration. It would be an effect similar to that of HIV, only acute “, he explains to FAPESP Agency Alessandro Farias, head of the Department of Genetics, Evolution, Microbiology and Immunology at Unicamp’s Institute of Biology (IB), who coordinated the investigation alongside Professor Marcelo Mori.
The conclusions of the article, still in the process of peer review, are based mainly on experiments with primary cultures of lymphocytes (isolated from the blood of uninfected volunteers and patients with COVID-19) conducted at the Emerging Virus Studies Laboratory (Leve) IB-Unicamp, with support from FAPESP.
In the first stage of the research, the researchers incubated cells from healthy donors with SARS-CoV-2 and followed what happened in the next 24 hours using different techniques, such as hybridization in situ, transmission electron microscopy and RT-PCR (the same used to diagnose infection in the acute phase).
“We performed this assay only with T CD4 and T CD8 lymphocytes as they are the most reduced types in patients with severe COVID-19. The analyzes confirmed the presence of the new coronavirus within approximately 40% of the CD4 T, with 10% of these cells died at the end of the observation period. CD8 T lymphocytes were not affected “, says Farias.
The researchers further noted that the viral load more than doubled between measurements taken two and 24 hours after the start of the test – a sign that the virus was replicating in cultured cells.
The next step was to analyze the CD4 T lymphocytes isolated from patients diagnosed with COVID-19 with the same tools for signs of SARS-CoV-2. Few infected lymphocytes were found in people with moderate disease and they were producing, as expected, the interferon-gamma (IFN-?) Cytokine – important for the antiviral response. In critically ill patients, in addition to having a much larger number of lymphocytes with the virus, were the cells producing instead of IFN-? interleukin-10 (IL-10), a cytokine with anti-inflammatory action. That is, in these patients with severe COVID-19, CD4 T lymphocytes were signaling to the immune system the need to stop the fight against the virus.
According to Farias, this would explain why many people in this condition have changes in the adaptive immune response (the one that is specific to each pathogen), such as lymphopenia (reduction in the general concentration of lymphocytes in the blood), exhaustion of T cells and compromised production of antibodies.
“The production of IL-10 shuts down the immune system and allows the virus to stay in the body longer. For the time being it is not yet possible to know what is the cause and what is the consequence, that is, whether these patients have evolved to the serious form because had more infected CD4 T lymphocytes or the other way around. But there is a clear association between these two factors “, says Farias.
Several studies already published point to the molecule ACE2 (angiotensin-converting enzyme 2, in its acronym in English) as the main gateway for SARS-CoV-2 on the surface of human cells. However, CD4 T lymphocytes are known to express a very small amount of this enzyme on the surface of its plasma membrane, which is covered by the protein that gives the cell its name: CD4.
To unveil the strategy used by the new coronavirus to enter these lymphocytes – which are normally refractory to infection by viruses and bacteria – the Unicamp group carried out two new tests with samples from healthy donors. In the first, before placing the virus, antibodies capable of neutralizing the CD4 protein were added to the cell culture. In the second experiment, antibodies against ACE2 were placed.
“Our hypothesis was that SARS-CoV-2 would be able to enter the cell using only CD4, but when we also neutralized ACE2 the infection was completely blocked. This shows that, even in small quantities, ACE2 is necessary for the invasion of the lymphocyte “, says Farias.
Interaction tests between molecules in vitro revealed that the SARS-CoV-2 spike protein, the one that forms the crown present on the viral surface, is able to bind to the lymphocyte CD4.
“We believe that the virus has a trick to enter this cell. It uses the CD4 protein only to stay close to the cell membrane and to be able to locate ACE2, which then gives it passage to the intracellular environment”, explains the researcher.
In the third and final stage of the research, carried out in partnership with the professor at the University of São Paulo (USP) Helder Nakaya, the researchers used bioinformatics techniques to reanalyze data from a study published by Chinese scientists in May, in the journal Nature Medicine, in which the leukocytes isolated from the pulmonary secretion of patients with severe COVID-19 were sequenced by a technique known as single-cell sequencing.
“The algorithm developed by the Nakaya group made it possible to identify the viral genome also in the lymphocytes that were in the patients’ lungs, bringing a new level of evidence and even more reliability to the findings”, says Farias.
The research also had the collaboration of researchers from the Universidade Estadual Paulista (Unesp), the National Center for Research in Energy and Materials (CNPEM), the Oswaldo Cruz Foundation (Fiocruz) and the D’Or Institute for Research and Education (Idor) . FAPESP’s support was provided through various grants and grants (19 / 16116-4, 19 / 06372-3, 20 / 04583-4, 13 / 08293-7, 20 / 04579-7, 15 / 15626-8, 18 / 14933-2, 20 / 04746-0, 19 / 00098-7, 20 / 04919-2, 17 / 01184-9, 19 / 17007-4, 19 / 22398-2, 19 / 05155-9, 19 / 06459-1, 19 / 04726-2, 17 / 23920-9, 16 / 24163-4 and 16 / 23328-0).
At the moment, the group is trying to further detail the effects caused by the entry of SARS-CoV-2 into the CD4 T lymphocyte. The goal is to find ways to intervene in this process, which could theoretically help to fight infection.
“We already have lymphocytes isolated from more than 350 patients that we intend to use in the laboratory experiments and we are also going to do tests with mice genetically modified to express human ACE2. One of the ideas is to evaluate the effect of molecules capable of inhibiting the interaction between the protein of virus spicule and CD4 “, says the researcher.
The article SARS-CoV-2 Uses CD4 to Infect T Helper Lymphocytes can be read at www.medrxiv.org/content/10.1101/2020.09.25.20200329v1.full.pdf.