Current observations suggest that the coronavirus SARS-CoV-2 causes severe symptoms mainly in elderly patients with chronic disease.
However when two pairs of previously healthy young brothers from two families required mechanical ventilation at the intensive care unit in rapid succession, doctors and researchers at Radboud University Medical Center were inclined to consider that genetic factors had a key role in compromising their immune system.
Their research identified the gene TLR7 as an essential player in the immune response against SARS-CoV-2. A finding with potentially major consequences for understanding and possibly treatment of COVID-19.
During the wave of COVID-19 patients that flooded Dutch hospitals in the first half of 2020, two young brothers became seriously ill with the SARS-CoV-2 virus and had to be mechanically ventilated in the ICU.
One of them died from the consequences of the infection, the other recovered. The severe course of disease in otherwise healthy young brothers was a relatively rare occurrence, especially because the virus mainly affects the elderly.
This observation triggered the curiosity of an attentive physician from the MUMC+ department of clinical genetics. She contacted her colleagues in Nijmegen who then investigated why these two young brothers were so severely affected.
“In such a case, you immediately wonder whether genetic factors could play a role,” says geneticist Alexander Hoischen.
“Getting sick from an infection is always an interplay between – in this case – the virus and the human immune system. It may be a mere coincidence that two brothers from the same family become so severely ill.
But it is also possible that an inborn error of the immune system has played an important role. We investigated this possibility, together with our multidisciplinary team at Radboudumc.”
All genes (collectively called the exome) of both brothers were sequenced, after which the investigators combed through the data searching for a possible shared cause.
Cas van der Made, Ph.D. student and resident at the department of Internal Medicine: “We mainly looked at genes that play a role in the immune system.
We know that several of these genes are located on the X-chromosome, and with two brother pairs affected X-chromosomal genes were most suspicious. Women carry two X-chromosomes, while men possess a Y-chromosome apart from the X.
Therefore, men have only one copy of the X-chromosomal genes. In case men have a defect in such a gene, there is no second gene that can take over that role, as in women.”
That search quickly revealed mutations in the gene encoding for the Toll-like receptor 7, TLR7 for short. There are multiple TLR-genes, which belong to a family of receptors with an important role in the recognition of pathogens (such as bacteria and viruses) and the activation of the immune system.
Hoischen: “A few letters were missing in the genetic code of the TLR7 gene. As a result, the code cannot be read properly and hardly any TLR7 protein is produced.
TLR7 function has so far never been associated with an inborn error of immunity. But unexpectedly we now have an indication that TLR7 is essential for protection from this coronavirus.
So it seems that the virus can replicate undisturbed because the immune system does not get a message that the virus has invaded.
Because TLR7, which must identify the intruder and subsequently activate the defense, is hardly present. That could be the reason for the severity of the disease in these brothers.”
Then, quite unexpectedly, the doctors and researchers at Radboudumc come across another pair of brothers who have fallen seriously ill with COVID-19. Again, they are both under 35 years of age. Both of them were also in the ICU for mechanical ventilation.
“Then the question of the role of genetics became even more obvious.” says Hoischen. “We also investigated the genetic code of these two brothers, again via the ‘rapid-clinical exome’ method.
This time we saw no deletion, no loss of letters, but a single spelling mistake of one DNA-letter of the TRL7 gene. The effect on the gene is the same, however, because these brothers also do not make sufficient functional TLR7 protein.
Suddenly we had four young people with a defect in the same gene, all of whom had fallen seriously ill from the SARS-CoV-2 virus.”
Essential role in the defense
Van der Made and colleagues have investigated the consequences of improper functioning of the TLR7 receptor. “Once activated, TLR7 triggers the production of so-called interferons, signaling proteins that are essential in the defense against virus infections,” says van der Made.
“This immune response is perhaps all the more important in the fight against the SARS-CoV-2 virus, because we know from the literature that the virus has tricks to reduce the production of interferons by immune cells.
When we mimic an infection with the coronavirus, we see that immune cells of the patients without properly functioning TLR7 hardly respond, and that minimal amounts of interferons are produced.
These tests make it clear that the virus appears to have free rein in people without properly functioning TLR7 because it [the virus] is not recognized by the immune system.”
“Due to the serious illness of four brothers in two families, so serious that it cost one of the young men his life, we have discovered this condition,” says Hoischen.
“It seems to be a very specific abnormality, an immunodeficiency, which is mainly related to this coronavirus. None of the four men have previously suffered from immune-related diseases.
It is the first time that we can connect a clinical phenomenon so strongly with TLR7.”
“This discovery not only provides us with more insight into the fundamental workings of the immune system, but it may also have important consequences for the treatment of severely ill COVID-19 patients,” says Frank van de Veerdonk, immunologist and infectiologist.
“The substance interferon can be given as a therapy. It is currently being investigated whether administering interferon in COVID-19 can indeed help.”
What We Know about Immune Response to Coronaviruses
Knowledge about host immune response to SARS-CoV-2 is mostly derived from previous studies of other coronaviruses of the same family (betacoronavirus), namely SARS-CoV and MERS-CoV. Betacoronavirus are positive-sense, single-stranded RNA viruses of zoonotic origin .
SARS-CoV-2 genes share approximately 80% homology with SARS-CoV, suggesting that the two viruses belong to the same species. However, it is more likely that SARS-CoV-2 has developed from the bat coronavirus RaTG13, with which it has an even higher degree of homology, close to 96% .
Figure 1a illustrates the scenario of a typical immune response to viruses, discussing how it may be relevant to the case of SARS-CoV-2.
Even though the proposed sequence may not represent what happens specifically during Covid-19, it describes how distinct immune mechanisms may affect the fate of SARS-CoV-2 infection.
A Possible Scenario of Covid-19 Immune Response with Eective Recovery
Viral Entry via ACE2 Binding (Figure 1a I)
Both SARS-CoV and SARS-CoV-2 enter human cells exploiting the link with membrane-bound angiotensin converting enzyme II (ACE2) protein, which is widely expressed in many cells across the body, including type II alveolar cells (AT2), upper airways cells, endothelial cells, myocardial cells, proximal tubule cells of the kidney, ileum and esophagus epithelial cells, and bladder urothelial cells [13–17].
Coronavirus spike (S) glycoprotein binds to ACE2 initiating viral entry into host cells. Of note, the affinity of SARS-CoV-2 S protein for ACE2 is higher than that of SARS-CoV [18–20]. Whether higher density of ACE2 may facilitate or protect alveolar cells from the infection is still debated .
Binding of the S1 subunit of the S protein to ACE2 is followed by the fusion of the viral and cellular membranes mediated by the S2 subunit of S protein and requires S protein priming by cellular proteases.
For this purpose, SARS-CoV-2 mainly uses a serine protease, transmembrane serine protease 2 (TMPRSS2), and the endosomal cysteine proteases cathepsin B and L (CatB/L) .
Notably, TMPRSS2 is androgen-regulated and this may be related to higher prevalence of both infection and severe disease in males [22,23].
After viral entry into cells, expression of membrane ACE2 is downregulated because of endocytosis of the receptor together with the virus. This event results in reduced metabolization and increased levels of angiotensin II in lung tissues and increased stimulation of the Type 1 Angiotensin II Receptor (ATR1), which mediates angiotensin II-induced vascular permeability and severe acute lung injury [24,25].
Notably, injection of SARS-CoV S protein in mice is sufficient to worsen acute lung failure, and this effect is reduced by renin-angiotensin pathway blockage with the AT1R inhibitor losartan .
All these observations may explain why SARS-CoV-2 primarily causes pneumonia with vascular injury, differently from influenzavirus, and suggest that higher availability of membrane ACE2 may be protective.
Furthermore, activation of ATR1 receptor directly upregulates NF-kB, as well as a disintegrin and metalloprotease 17 (ADAM17) on cell surface; ADAM17 in turn cleaves membrane TNF to soluble TNF and processes membrane IL-6R to the soluble sIL-6R,
which allows IL6 responsiveness by many tissues through IL6 trans-signaling, leading to Signal Transducer and Activator of Transcription 3 (STAT3) activation and the amplification of Nuclear Factor kappa B (NF-kB) activation .
Once infected, cells are directly affected by SARS-CoV-2 replicative cycle, which is therefore a cytopathic virus causing direct cell death and resulting in increased inflammatory response .
Notably, SARS-CoV has been shown to cause caspase-1-mediated cell death (pyroptosis, a highly inflammatory form of cell death) via the activation of the Nod-like receptor family, pyrin domain-containing 3 (NLRP3) mediated by the viral 3a protein which acts as a potassium ion channel (viroporin) resulting in inflammasome activation .
Besides alveolar cells, ACE2 is widely expressed also on endothelial cells, macrophages, heart, intestine, and kidney, and this may explain the involvement of these cells in severe cases of Covid-19 [19,30,31].
The ability of SARS-CoV-2 to infect enterocytes has been demonstrated in human small intestinal organoids, and this may explain the relative frequency of gastrointestinal symptoms in COVID-19 .
Furthermore, it has been suggested that SARS-CoV2 may persist longer in the digestive system, and this may be a cause of protracted form of disease requiring readmission to hospital in some patients due to gastroenteritis symptoms with persistence of viral RNA in stools after resolution of respiratory symptoms .
Recent findings support a primary role of endothelial cell infection and resulting endotheliitis in Covid-19 pathogenesis, which may lead to vasculopathy, coagulopathy, and multiple organ injury .
Endothelial injury may be due to direct viral infection of endothelial cells as well as to endothelial activation and apoptosis from inflammatory cytokines, especially tumor necrosis factor alpha (TNFα) .
According to recent unpublished data from autopsies, it has been argued that vascular damage with peripheral lung microthrombi may be an early phenomenon directly linked to viral infection rather than to the inflammatory reaction.
A significant incidence of disseminated intravascular coagulation and thromboembolism has been also reported. This led to the suggestion to introduce prophylactic heparin in hospitalized patients with Covid-19 [35,36].
Overall, the occurrence of pulmonary vascular (micro)thrombosis and of vascular dysregulation due to angiotensin system abnormalities may account for the observation of severe hypoxemia despite high compliance of the lung, differently to what occurs in most other types of interstitial pneumonia.
These findings are likely associated with an increase of dead space ventilation (ventilation of poorly perfused lung), a defective hypoxic pulmonary vasoconstriction with areas not ventilated but perfused (shunt), and may also partly explain the efficacy of proning in patients with respiratory failure .
Recent findings demonstrated that SARS-CoV-2 also infect immune cells, causing activation and secretion of inflammatory cytokines . Direct infection of lymphocytes via ACE2 binding had been previously demonstrated for SARS-CoV .
Following this observation, it has been said that SARS-CoV could be considered halfway between a common respiratory virus and a lymphotropic virus such as HIV . SARS-CoV-2 seems unable to replicate in lymphocytes, and it is uncertain whether direct lymphocyte infection can contribute to the lymphopenia associated with severe Covid-19.
Indeed, lymphopenia may just be due to apoptosis as a part of a multilinear cytopenia induced by hypercytokinemia or by overt hemophagocytic lymphohistiocytosis [7,41].
Innate Immune Response (Figure 1a II)
The initial response to coronaviruses infection by the innate immune system plays a pivotal role in determining the outcome of the infection. The sensing of foreign nucleic acids is the first step in the pathway to an effective immune response leading to viral clearance.
Eukaryotic cells have several sensors, so-called pattern-recognition receptors (PRRs) that are activated by foreign pathogen-derived material.
In the endosomal compartment, Toll-like receptor 3 (TLR3) recognizes double-stranded RNA derived by viral replication, while TLR7, TLR8, and TLR9 recognize respectively single-stranded RNA (TLR7/8) and DNA (TLR9) sequences typical of virus and containing uCpG motifs.
In the cytoplasm the two RNA sensors Retinoic Acid-Inducible Gene I (RIG-I) and Melanoma Differentiation-Associated protein 5 (MDA5) recognize double-stranded RNA intermediates produced during viral replication.
Activation of cellular sensors elicits the production of type I IFNs and other inflammatory cytokines, which act on the infected cells and on the neighboring cells making them more resistant to the entry of other virus particles and act on resident dendritic cells (DCs) and macrophages to promote the activation and organization of the antiviral response .
The redundancy of cell sensors is not surprising given that viruses may develop evasion strategies. For example, SARS-CoV can modify the features of its immunostimulatory RNA, lowering the recognition by MDA5 [43,44].
Moreover, SARS-CoV can also dampen IFN-I production by distinct mechanisms, including degradation of interferon (IFN) pathway components by a papain-like protease [45,46], as well as IFN response by inhibition of STAT1 transport into the nucleus in response to interferon signaling .
Similarly, MERS CoV suppresses RIG-I-induced type I and type III IFN production interfering with Tripartite Motif Containing 25 (TRIM25)-mediated RIG-I ubiquitination .