The clinical utility of CD163 in viral diseases.

Macrophage activation and hypercytokinemia are notable presentations in certain viral infections leading to severe disease and poor prognosis. Viral infections can cause macrophage polarization into the pro-inflammatory M1 or anti-inflammatory M2 phenotype. Activated M1 macrophages usually restrict viral replication whereas activated M2 macrophages suppress inflammation and promote tissue repair. In response to inflammatory stimuli, macrophages polarize to the M2 phenotype expressing hemoglobin scavenger CD163 surface receptor. The CD163 receptor is shed as the soluble form, sCD163, into plasma or tissue fluids. sCD163 causes detoxification of pro-oxidative hemoglobin which produces anti-inflammatory metabolites that promote the resolution of inflammation. Hence, increased CD163 expression in tissues and elevated circulatory levels of sCD163 have been associated with acute and chronic inflammatory diseases. CD163 and other macrophage activation markers have been commonly included in the investigation of disease pathogenesis and progression. This review provides an overview of the involvement of CD163 in viral diseases. The clinical utility of CD163 in viral disease diagnosis, progression, prognosis and treatment evaluation is discussed.

CLEC5A expression can be triggered by spike glycoprotein and may be a potential target for COVID-19 therapy.

The immune response is crucial for coronavirus disease 19 (COVID-19) progression, with the participation of proinflammatory cells and cytokines, inducing lung injury and loss of respiratory function. CLEC5A expression on monocytes can be triggered by viral and bacterial infections, leading to poor outcomes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is able to induce neutrophil activation by CLEC5A and Toll-like receptor 2, leading to an aggressive inflammatory cascade, but little is known about the molecular interactions between CLEC5A and SARS-CoV-2 proteins. Here, we aimed to explore how CLEC5A expression could be affected by SARS-CoV-2 infection using immunological tools with in vitro, in vivo, and in silico assays. The findings revealed that high levels of CLEC5A expression were found in monocytes from severe COVID-19 patients in comparison with mild COVID-19 and unexposed subjects, but not in vaccinated subjects who developed mild COVID-19. In hamsters, we detected CLEC5A gene expression during 3-15 days of Omicron strain viral challenge. Our results also showed that CLEC5A can interact with SARS-CoV-2, promoting inflammatory cytokine production, probably through an interaction with the receptor-binding domain in the N-acetylglucosamine binding site (NAG-601). The high expression of CLEC5A and high levels of proinflammatory cytokine production were reduced in vitro by a human CLEC5A monoclonal antibody. Finally, CLEC5A was triggered by spike glycoprotein, suggesting its involvement in COVID-19 progression; therapy with a monoclonal antibody could be a good strategy for COVID-19 treatment, but vaccines are still the best option to avoid hospitalization/deaths.

ACE2 protein expression in lung tissues of severe COVID-19 infection.

Angiotensin-converting enzyme 2 (ACE2) is a key host protein by which severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enters and multiplies within cells. The level of ACE2 expression in the lung is hypothesised to correlate with an increased risk of severe infection and complications in COrona VIrus Disease 2019 (COVID-19). To test this hypothesis, we compared the protein expression status of ACE2 by immunohistochemistry (IHC) in post-mortem lung samples of patients who died of severe COVID-19 and lung samples obtained from non-COVID-19 patients for other indications. IHC for CD61 and CD163 was performed for the assessment of platelet-rich microthrombi and macrophages, respectively. IHC for SARS-CoV-2 viral antigen was also performed. In a total of 55, 44 COVID-19 post-mortem lung samples were tested for ACE2, 36 for CD163, and 26 for CD61, compared to 15 non-covid 19 control lung sections. Quantification of immunostaining, random sampling, and correlation analysis were used to substantiate the morphologic findings. Our results show that ACE2 protein expression was significantly higher in COVID-19 post-mortem lung tissues than in controls, regardless of sample size. Histomorphology in COVID-19 lungs showed diffuse alveolar damage (DAD), acute bronchopneumonia, and acute lung injury with SARS-CoV-2 viral protein detected in a subset of cases. ACE2 expression levels were positively correlated with increased expression levels of CD61 and CD163. In conclusion, our results show significantly higher ACE2 protein expression in severe COVID-19 disease, correlating with increased macrophage infiltration and microthrombi, suggesting a pathobiological role in disease severity.

Human-type sialic acid receptors contribute to avian influenza A virus binding and entry by hetero-multivalent interactions.

Establishment of zoonotic viruses, causing pandemics like the Spanish flu and Covid-19, requires adaptation to human receptors. Pandemic influenza A viruses (IAV) that crossed the avian-human species barrier switched from binding avian-type α2-3-linked sialic acid (2-3Sia) to human-type 2-6Sia receptors. Here, we show that this specificity switch is however less dichotomous as generally assumed. Binding and entry specificity were compared using mixed synthetic glycan gradients of 2-3Sia and 2-6Sia and by employing a genetically remodeled Sia repertoire on the surface of a Sia-free cell line and on a sialoglycoprotein secreted from these cells. Expression of a range of (mixed) 2-3Sia and 2-6Sia densities shows that non-binding human-type receptors efficiently enhanced avian IAV binding and entry provided the presence of a low density of high affinity avian-type receptors, and vice versa. Considering the heterogeneity of sialoglycan receptors encountered in vivo, hetero-multivalent binding is physiologically relevant and will impact evolutionary pathways leading to host adaptation.

The Human Liver-Expressed Lectin CD302 Restricts Hepatitis C Virus Infection.

C-type lectin domain-containing proteins (CTLDcps) shape host responses to pathogens and infectious disease outcomes. Previously, we identified the murine CTLDcp Cd302 as restriction factor, limiting hepatitis C virus (HCV) infection of murine hepatocytes. In this study, we investigated in detail the human orthologue's ability to restrict HCV infection in human liver cells. CD302 overexpression in Huh-7.5 cells potently inhibited infection of diverse HCV chimeras representing seven genotypes. Transcriptional profiling revealed abundant CD302 mRNA expression in human hepatocytes, the natural cellular target of HCV. Knockdown of endogenously expressed CD302 modestly enhanced HCV infection of Huh-7.5 cells and primary human hepatocytes. Functional analysis of naturally occurring CD302 transcript variants and engineered CD302 mutants showed that the C-type lectin-like domain (CTLD) is essential for HCV restriction, whereas the cytoplasmic domain (CPD) is dispensable. Coding single nucleotide polymorphisms occurring in human populations and mapping to different domains of CD302 did not influence the capacity of CD302 to restrict HCV. Assessment of the anti-HCV phenotype at different life cycle stages indicated that CD302 preferentially targets the viral entry step. In contrast to the murine orthologue, overexpression of human CD302 did not modulate downstream expression of nuclear receptor-controlled genes. Ectopic CD302 expression restricted infection of liver tropic hepatitis E virus (HEV), while it did not affect infection rates of two respiratory viruses, including respiratory syncytial virus (RSV) and the alpha coronavirus HVCoV-229E. Together, these findings suggest that CD302 contributes to liver cell-intrinsic defense against HCV and might mediate broader antiviral defenses against additional hepatotropic viruses. IMPORTANCE The liver represents an immunoprivileged organ characterized by enhanced resistance to immune responses. However, the importance of liver cell-endogenous, noncytolytic innate immune responses in pathogen control is not well defined. Although the role of myeloid cell-expressed CTLDcps in host responses to viruses has been characterized in detail, we have little information about their potential functions in the liver and their relevance for immune responses in this organ. Human hepatocytes endogenously express the CTLDcp CD302. Here, we provide evidence that CD302 limits HCV infection of human liver cells, likely by inhibiting a viral cell entry step. We confirm that the dominant liver-expressed transcript variant, as well as naturally occurring coding variants of CD302, maintain the capacity to restrict HCV. We further show that the CTLD of the protein is critical for the anti-HCV activity and that overexpressed CD302 limits HEV infection. Thus, CD302 likely contributes to human liver-intrinsic antiviral defenses.

Phage-Displayed Mimotopes of SARS-CoV-2 Spike Protein Targeted to Authentic and Alternative Cellular Receptors.

The evolution of the SARS-CoV-2 virus during the COVID-19 pandemic was accompanied by the emergence of new heavily mutated viral variants with increased infectivity and/or resistance to detection by the human immune system. To respond to the urgent need for advanced methods and materials to empower a better understanding of the mechanisms of virus's adaptation to human host cells and to the immuno-resistant human population, we suggested using recombinant filamentous bacteriophages, displaying on their surface foreign peptides termed "mimotopes", which mimic the structure of viral receptor-binding sites on the viral spike protein and can serve as molecular probes in the evaluation of molecular mechanisms of virus infectivity. In opposition to spike-binding antibodies that are commonly used in studying the interaction of the ACE2 receptor with SARS-CoV-2 variants in vitro, phage spike mimotopes targeted to other cellular receptors would allow discovery of their role in viral infection in vivo using cell culture, tissue, organs, or the whole organism. Phage mimotopes of the SARS-CoV-2 Spike S1 protein have been developed using a combination of phage display and molecular mimicry concepts, termed here "phage mimicry", supported by bioinformatics methods. The key elements of the phage mimicry concept include: (1) preparation of a collection of p8-type (landscape) phages, which interact with authentic active receptors of live human cells, presumably mimicking the binding interactions of human coronaviruses such as SARS-CoV-2 and its variants; (2) discovery of closely related amino acid clusters with similar 3D structural motifs on the surface of natural ligands (FGF1 and NRP1), of the model receptor of interest FGFR and the S1 spike protein; and (3) an ELISA analysis of the interaction between candidate phage mimotopes with FGFR3 (a potential alternative receptor) in comparison with ACE2 (the authentic receptor).

Escherichia coli recombinant expression of SARS-CoV-2 protein fragments.

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540-588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

SARS-CoV-2 Impairs Dendritic Cells and Regulates DC-SIGN Gene Expression in Tissues.

The current spreading coronavirus SARS-CoV-2 is highly infectious and pathogenic. In this study, we screened the gene expression of three host receptors (ACE2, DC-SIGN and L-SIGN) of SARS coronaviruses and dendritic cells (DCs) status in bulk and single cell transcriptomic datasets of upper airway, lung or blood of COVID-19 patients and healthy controls. In COVID-19 patients, DC-SIGN gene expression was interestingly decreased in lung DCs but increased in blood DCs. Within DCs, conventional DCs (cDCs) were depleted while plasmacytoid DCs (pDCs) were augmented in the lungs of mild COVID-19. In severe cases, we identified augmented types of immature DCs (CD22+ or ANXA1+ DCs) with MHCII downregulation. In this study, our observation indicates that DCs in severe cases stimulate innate immune responses but fail to specifically present SARS-CoV-2. It provides insights into the profound modulation of DC function in severe COVID-19.

An artificial neural network approach integrating plasma proteomics and genetic data identifies PLXNA4 as a new susceptibility locus for pulmonary embolism.

Venous thromboembolism is the third common cardiovascular disease and is composed of two entities, deep vein thrombosis (DVT) and its potential fatal form, pulmonary embolism (PE). While PE is observed in ~ 40% of patients with documented DVT, there is limited biomarkers that can help identifying patients at high PE risk. To fill this need, we implemented a two hidden-layers artificial neural networks (ANN) on 376 antibodies and 19 biological traits measured in the plasma of 1388 DVT patients, with or without PE, of the MARTHA study. We used the LIME algorithm to obtain a linear approximate of the resulting ANN prediction model. As MARTHA patients were typed for genotyping DNA arrays, a genome wide association study (GWAS) was conducted on the LIME estimate. Detected single nucleotide polymorphisms (SNPs) were tested for association with PE risk in MARTHA. Main findings were replicated in the EOVT study composed of 143 PE patients and 196 DVT only patients. The derived ANN model for PE achieved an accuracy of 0.89 and 0.79 in our training and testing sets, respectively. A GWAS on the LIME approximate identified a strong statistical association peak (rs1424597: p = 5.3 × 10-7) at the PLXNA4 locus. Homozygote carriers for the rs1424597-A allele were then more frequently observed in PE than in DVT patients from the MARTHA (2% vs. 0.4%, p = 0.005) and the EOVT (3% vs. 0%, p = 0.013) studies. In a sample of 112 COVID-19 patients known to have endotheliopathy leading to acute lung injury and an increased risk of PE, decreased PLXNA4 levels were associated (p = 0.025) with worsened respiratory function. Using an original integrated proteomics and genetics strategy, we identified PLXNA4 as a new susceptibility gene for PE whose exact role now needs to be further elucidated.

Intra-species sialic acid polymorphism in humans: a common niche for influenza and coronavirus pandemics?

The ongoing COVID-19 pandemic has led to more than 159 million confirmed cases with over 3.3 million deaths worldwide, but it remains mystery why most infected individuals (∼98%) were asymptomatic or only experienced mild illness. The same mystery applies to the deadly 1918 H1N1 influenza pandemic, which has puzzled the field for a century. Here we discuss dual potential properties of the 1918 H1N1 pandemic viruses that led to the high fatality rate in the small portion of severe cases, while about 98% infected persons in the United States were self-limited with mild symptoms, or even asymptomatic. These variations now have been postulated to be impacted by polymorphisms of the sialic acid receptors in the general population. Since coronaviruses (CoVs) also recognize sialic acid receptors and cause severe acute respiratory syndrome epidemics and pandemics, similar principles of influenza virus evolution and pandemicity may also apply to CoVs. A potential common principle of pathogen/host co-evolution of influenza and CoVs under selection of host sialic acids in parallel with different epidemic and pandemic influenza and coronaviruses is discussed.

ACE2, TMPRSS2, and L-SIGN Expression in Placentae From HIV-Positive Pregnancies Exposed to Antiretroviral Therapy-Implications for SARS-CoV-2 Placental Infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding receptor ACE2 and the spike protein priming protease TMPRSS2 are coexpressed in human placentae. It is unknown whether their expression is altered in the context of HIV infection and antiretroviral therapy (ART). METHODS: We compared mRNA levels of SARS-CoV-2 cell-entry mediators ACE2, TMPRSS2, and L-SIGN by quantitative polymerase chain reaction in 105 placentae: 45 from pregnant women with HIV (WHIV) on protease inhibitor (PI)-based ART, 17 from WHIV on non-PI-based ART, and 43 from HIV-uninfected women. RESULTS: ACE2 levels were lower, while L-SIGN levels were higher, in placentae from WHIV on PI-based ART compared to those on non-PI-based ART and to HIV-uninfected women. TMPRSS2 levels were similar between groups. Black race was significantly associated with lower expression of ACE2 and higher expression of L-SIGN. ACE2 levels were significantly higher in placentae of female fetuses. CONCLUSIONS: We identified pregnant women of black race and WHIV on PI-based ART to have relatively lower expression of placental ACE2 than those of white race and HIV-uninfected women. This may potentially contribute to altered susceptibility to COVID-19 in these women, favorably by reduced viral entry or detrimentally by loss of ACE2 protection against hyperinflammation.

An investigation into the molecular basis of cancer comorbidities in coronavirus infection.

Comorbidities in COVID-19 patients often worsen clinical conditions and may represent death predictors. Here, the expression of five genes, known to encode coronavirus receptors/interactors (ACE2, TMPRSS2, CLEC4M, DPP4 and TMPRSS11D), was investigated in normal and cancer tissues, and their molecular relationships with clinical comorbidities were investigated. Using expression data from GENT2 databases, we evaluated gene expression in all anatomical districts from 32 normal tissues in 3902 individuals. Functional relationships with body districts were analyzed by chilibot. We performed DisGeNet, genemania and DAVID analyses to identify human diseases associated with these genes. Transcriptomic expression levels were then analyzed in 31 cancer types and healthy controls from approximately 43 000 individuals, using GEPIA2 and GENT2 databases. By performing receiver operating characteristic analysis, the area under the curve (AUC) was used to discriminate healthy from cancer patients. Coronavirus receptors were found to be expressed in several body districts. Moreover, the five genes were found to associate with acute respiratory syndrome, diabetes, cardiovascular diseases and cancer (i.e. the most frequent COVID-19 comorbidities). Their expression levels were found to be significantly altered in cancer types, including colon, kidney, liver, testis, thyroid and skin cancers (P < 0.0001); AUC > 0.80 suggests that TMPRSS2, CLEC4M and DPP4 are relevant markers of kidney, liver, and thyroid cancer, respectively. The five coronavirus receptors are related to all main COVID-19 comorbidities and three show significantly different expression in cancer versus control tissues. Further investigation into their role may help in monitoring other comorbidities, as well as for follow-up of patients who have recovered from SARS-CoV-2 infection.

CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance.

Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.

Pig epidemics are the biggest threat to the pork industry. In 2019 alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. The porcine reproductive and respiratory virus (PRRS virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. Two coronaviruses ­ the transmissible gastroenteritis virus (or TGEV) and the porcine delta coronavirus ­ cause deadly diarrhea and could potentially cross over into humans. Unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. Traditionally, breeding pigs to have a particular trait is a slow process that can take many years. But with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. When viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the PRRS virus relies a protein called CD163, and TGEV uses pAPN. Xu, Zhou, Mu et al. used gene editing technology to delete the genes that encode the CD163 and pAPN proteins in pigs. When the animals were infected with PRRS virus or TGEV, the non-edited pigs got sick but the gene-edited animals remained healthy. Unexpectedly, pigs without CD163 and pAPN also coped better with porcine delta coronavirus infections, suggesting that CD163 and pAPN may also help this coronavirus infect cells. Finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. These experiments show that gene editing can be a powerful technology for producing animals with desirable traits. The gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale.

Existing bitter medicines for fighting 2019-nCoV-associated infectious diseases.

The sudden outbreak of COVID-19 has led to more than seven thousand deaths. Unfortunately, there are no specific drugs available to cure this disease. Type 2 taste receptors (TAS2Rs) may play an important role in host defense mechanisms. Based on the idea of host-directed therapy (HDT), we performed a negative co-expression analysis using big data of 60 000 Affymetrix expression arrays and 5000 TCGA data sets to determine the functions of TAS2R10, which can be activated by numerous bitter substances. Excitingly, we found that the main functions of TAS2R10 involved controlling infectious diseases caused by bacteria, viruses, and parasites, suggesting that TAS2R10 is a key trigger of host defense pathways. To quickly guide the clinical treatment of 2019-nCoV, we searched currently available drugs that are agonists of TAS2Rs. We identified many cheap, available, and safe medicines, such as diphenidol, quinine, chloroquine, artemisinin, chlorpheniramine, yohimbine, and dextromethorphan, which may target the most common symptoms caused by 2019-nCoV. We suggest that a cocktail-like recipe of existing bitter drugs may help doctors to fight this catastrophic disease and that the general public may drink or eat bitter substances, such as coffee, tea, or bitter vegetables, to reduce the risk of infection.