Complement Activation-Independent Attenuation of SARS-CoV-2 Infection by C1q and C4b-Binding Protein.

The complement system is a key component of the innate immune response to viruses and proinflammatory events. Exaggerated complement activation has been attributed to the induction of a cytokine storm in severe SARS-CoV-2 infection. However, there is also an argument for the protective role of complement proteins, given their local synthesis or activation at the site of viral infection. This study investigated the complement activation-independent role of C1q and C4b-binding protein (C4BP) against SARS-CoV-2 infection. The interactions of C1q, its recombinant globular heads, and C4BP with the SARS-CoV-2 spike and receptor binding domain (RBD) were examined using direct ELISA. In addition, RT-qPCR was used to evaluate the modulatory effect of these complement proteins on the SARS-CoV-2-mediated immune response. Cell binding and luciferase-based viral entry assays were utilised to assess the effects of C1q, its recombinant globular heads, and C4BP on SARS-CoV-2 cell entry. C1q and C4BP bound directly to SARS-CoV-2 pseudotype particles via the RBD domain of the spike protein. C1q via its globular heads and C4BP were found to reduce binding as well as viral transduction of SARS-CoV-2 spike protein expressing lentiviral pseudotypes into transfected A549 cells expressing human ACE2 and TMPRSS2. Furthermore, the treatment of the SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes with C1q, its recombinant globular heads, or C4BP triggered a reduction in mRNA levels of proinflammatory cytokines and chemokines such as IL-1ß, IL-8, IL-6, TNF-α, IFN-α, and RANTES (as well as NF-κB) in A549 cells expressing human ACE2 and TMPRSS2. In addition, C1q and C4BP treatment also reduced SARS-CoV-2 pseudotype infection-mediated NF-κB activation in A549 cells expressing human ACE2 and TMPRSS2. C1q and C4BP are synthesised primarily by hepatocytes; however, they are also produced by macrophages, and alveolar type II cells, respectively, locally at the pulmonary site. These findings support the notion that the locally produced C1q and C4BP can be protective against SARS-CoV-2 infection in a complement activation-independent manner, offering immune resistance by inhibiting virus binding to target host cells and attenuating the infection-associated inflammatory response.

Human surfactant protein D facilitates SARS-CoV-2 pseudotype binding and entry in DC-SIGN expressing cells, and downregulates spike protein induced inflammation.

Lung surfactant protein D (SP-D) and Dendritic cell-specific intercellular adhesion molecules-3 grabbing non-integrin (DC-SIGN) are pathogen recognising C-type lectin receptors. SP-D has a crucial immune function in detecting and clearing pulmonary pathogens; DC-SIGN is involved in facilitating dendritic cell interaction with naïve T cells to mount an anti-viral immune response. SP-D and DC-SIGN have been shown to interact with various viruses, including SARS-CoV-2, an enveloped RNA virus that causes COVID-19. A recombinant fragment of human SP-D (rfhSP-D) comprising of α-helical neck region, carbohydrate recognition domain, and eight N-terminal Gly-X-Y repeats has been shown to bind SARS-CoV-2 Spike protein and inhibit SARS-CoV-2 replication by preventing viral entry in Vero cells and HEK293T cells expressing ACE2. DC-SIGN has also been shown to act as a cell surface receptor for SARS-CoV-2 independent of ACE2. Since rfhSP-D is known to interact with SARS-CoV-2 Spike protein and DC-SIGN, this study was aimed at investigating the potential of rfhSP-D in modulating SARS-CoV-2 infection. Coincubation of rfhSP-D with Spike protein improved the Spike Protein: DC-SIGN interaction. Molecular dynamic studies revealed that rfhSP-D stabilised the interaction between DC-SIGN and Spike protein. Cell binding analysis with DC-SIGN expressing HEK 293T and THP- 1 cells and rfhSP-D treated SARS-CoV-2 Spike pseudotypes confirmed the increased binding. Furthermore, infection assays using the pseudotypes revealed their increased uptake by DC-SIGN expressing cells. The immunomodulatory effect of rfhSP-D on the DC-SIGN: Spike protein interaction on DC-SIGN expressing epithelial and macrophage-like cell lines was also assessed by measuring the mRNA expression of cytokines and chemokines. RT-qPCR analysis showed that rfhSP-D treatment downregulated the mRNA expression levels of pro-inflammatory cytokines and chemokines such as TNF-α, IFN-α, IL-1ß, IL- 6, IL-8, and RANTES (as well as NF-κB) in DC-SIGN expressing cells challenged by Spike protein. Furthermore, rfhSP-D treatment was found to downregulate the mRNA levels of MHC class II in DC expressing THP-1 when compared to the untreated controls. We conclude that rfhSP-D helps stabilise the interaction between SARS- CoV-2 Spike protein and DC-SIGN and increases viral uptake by macrophages via DC-SIGN, suggesting an additional role for rfhSP-D in SARS-CoV-2 infection.

Identification of functional tumor necrosis factor-alpha promoter variants associated with Helicobacter pylori infection in the Sudanese population: Computational approach.

BACKGROUND: Helicobacter pylori (H. pylori) is a ubiquitous bacterium that affects nearly half of the world's population with a high morbidity and mortality rate. Polymorphisms within the tumor necrosis factor-alpha (TNF-A) promoter region are considered a possible genetic basis for this disease. AIM: To functionally characterize the genetic variations in the TNF-A 5'-region (-584 to +107) of Sudanese patients infected with H. pylori using in silico tools. METHODS: An observational study was carried out in major public and private hospitals in Khartoum state. A total of 122 gastric biopsies were taken from patients who had been referred for endoscopy. Genomic DNA was extracted. Genotyping of the TNF-A-1030 polymorphism was performed using PCR with confronting two-pair primer to investigate its association with the susceptibility to H. pylori infection in the Sudanese population. Furthermore, Sanger sequencing was applied to detect single nucleotide polymorphisms in the 5'-region (-584 to +107) of TNF-A in H. pylori-infected patients. Bioinformatics analyses were used to predict whether these mutations would alter transcription factor binding sites or composite regulatory elements in this region. A comparative profiling analysis was conducted in 11 species using the ECR browser and multiple-sequence local alignment and visualization search engine to investigate the possible conservation. Also, a multivariate logistic regression model was constructed to estimate odds ratios and their 95% confidence intervals for the association between TNF-A-1030, sociodemographic characteristics and H. pylori infection. Differences were statistically significant if P < 0.05. Statistical analyses were performed using Stata version 11 software. RESULTS: A total of seven single nucleotide polymorphisms were observed in the TNF-A 5'-region of Sudanese patients infected with H. pylori. Only one of them (T > A, -76) was located at the in silico-predicted promoter region (-146 to +10), and it was predicted to alter transcription factor binding sites and composite regulatory elements. A novel mutation (A > T, +27) was detected in the 5' untranslated region, and it could affect the post-transcriptional regulatory pathways. Genotyping of TNF-A-1030 showed a lack of significant association between -1030T and susceptibility to H. pylori and gastric cancer in the studied population (P = 0.1756) and (P = 0.8116), respectively. However, a significant association was detected between T/C genotype and H. pylori infection (39.34% vs 19.67%, odds ratio = 2.69, 95% confidence interval: 1.17-6.17, P = 0.020). Mammalian conservation was observed for the (-146 to +10) region in chimpanzee (99.4%), rhesus monkey (95.6%), cow (91.8%), domesticated dog (89.3%), mouse (84.3%), rat (82.4%) and opossum (78%). CONCLUSION: Computational analysis was a valuable method for understanding TNF-A gene expression patterns and guiding further in vitro and in vivo experimental validation.

Human Properdin Released By Infiltrating Neutrophils Can Modulate Influenza A Virus Infection.

The complement system is designed to recognise and eliminate invading pathogens via activation of classical, alternative and lectin pathways. Human properdin stabilises the alternative pathway C3 convertase, resulting in an amplification loop that leads to the formation of C5 convertase, thereby acting as a positive regulator of the alternative pathway. It has been noted that human properdin on its own can operate as a pattern recognition receptor and exert immune functions outside its involvement in complement activation. Properdin can bind directly to microbial targets via DNA, sulfatides and glycosaminoglycans, apoptotic cells, nanoparticles, and well-known viral virulence factors. This study was aimed at investigating the complement-independent role of properdin against Influenza A virus infection. As one of the first immune cells to arrive at the site of IAV infection, we show here that IAV challenged neutrophils released properdin in a time-dependent manner. Properdin was found to directly interact with haemagglutinin, neuraminidase and matrix 1 protein Influenza A virus proteins in ELISA and western blot. Furthermore, modelling studies revealed that properdin could bind HA and NA of the H1N1 subtype with higher affinity compared to that of H3N2 due to the presence of an HA cleavage site in H1N1. In an infection assay using A549 cells, properdin suppressed viral replication in pH1N1 subtype while promoting replication of H3N2 subtype, as revealed by qPCR analysis of M1 transcripts. Properdin treatment triggered an anti-inflammatory response in H1N1-challenged A549 cells and a pro-inflammatory response in H3N2-infected cells, as evident from differential mRNA expression of TNF-α, NF-κB, IFN-α, IFN-ß, IL-6, IL-12 and RANTES. Properdin treatment also reduced luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles; however, it was increased in the case of pseudotyped H3N2 particles. Collectively, we conclude that infiltrating neutrophils at the site of IAV infection can release properdin, which then acts as an entry inhibitor for pandemic H1N1 subtype while suppressing viral replication and inducing an anti-inflammatory response. H3N2 subtype can escape this immune restriction due to altered haemagglutinin and neuraminindase, leading to enhanced viral entry, replication and pro-inflammatory response. Thus, depending on the subtype, properdin can either limit or aggravate IAV infection in the host.

Complement Proteins as Soluble Pattern Recognition Receptors for Pathogenic Viruses.

The complement system represents a crucial part of innate immunity. It contains a diverse range of soluble activators, membrane-bound receptors, and regulators. Its principal function is to eliminate pathogens via activation of three distinct pathways: classical, alternative, and lectin. In the case of viruses, the complement activation results in effector functions such as virion opsonisation by complement components, phagocytosis induction, virolysis by the membrane attack complex, and promotion of immune responses through anaphylatoxins and chemotactic factors. Recent studies have shown that the addition of individual complement components can neutralise viruses without requiring the activation of the complement cascade. While the complement-mediated effector functions can neutralise a diverse range of viruses, numerous viruses have evolved mechanisms to subvert complement recognition/activation by encoding several proteins that inhibit the complement system, contributing to viral survival and pathogenesis. This review focuses on these complement-dependent and -independent interactions of complement components (especially C1q, C4b-binding protein, properdin, factor H, Mannose-binding lectin, and Ficolins) with several viruses and their consequences.

Human Surfactant Protein D Binds Spike Protein and Acts as an Entry Inhibitor of SARS-CoV-2 Pseudotyped Viral Particles.

Human SP-D is a potent innate immune molecule whose presence at pulmonary mucosal surfaces allows its role in immune surveillance against pathogens. Higher levels of serum SP-D have been reported in the patients with severe acute respiratory syndrome coronavirus (SARS-CoV). Studies have suggested the ability of human SP-D to recognise spike glycoprotein of SARS-CoV; its interaction with HCoV-229E strain leads to viral inhibition in human bronchial epithelial (16HBE) cells. Previous studies have reported that a recombinant fragment of human SP-D (rfhSP-D) composed of 8 Gly-X-Y repeats, neck and CRD region, can act against a range of viral pathogens including influenza A Virus and Respiratory Syncytial Virus in vitro, in vivo and ex vivo. In this context, this study was aimed at examining the likely protective role of rfhSP-D against SARS-CoV-2 infection. rfhSP-D showed a dose-responsive binding to S1 spike protein of SARS-CoV-2 and its receptor binding domain. Importantly, rfhSP-D inhibited interaction of S1 protein with the HEK293T cells overexpressing human angiotensin converting enzyme 2 (hACE2). The protective role of rfhSP-D against SARS-CoV-2 infection as an entry inhibitor was further validated by the use of pseudotyped lentiviral particles expressing SARS-CoV-2 S1 protein; ~0.5 RLU fold reduction in viral entry was seen following treatment with rfhSP-D (10 µg/ml). These results highlight the therapeutic potential of rfhSP-D in SARS-CoV-2 infection and merit pre-clinical studies in animal models.

C4b Binding Protein Acts as an Innate Immune Effector Against Influenza A Virus.

C4b Binding Protein (C4BP) is a major fluid phase inhibitor of the classical and lectin pathways of the complement system. Complement inhibition is achieved by binding to and restricting the role of activated complement component C4b. C4BP functions as a co-factor for factor I in proteolytic inactivation of both soluble and cell surface-bound C4b, thus restricting the formation of the C3-convertase, C4b2a. C4BP also accelerates the natural decay/dissociation of the C3 convertase. This makes C4BP a prime target for exploitation by pathogens to escape complement attack, as seen in Streptococcus pyogenes or Flavivirus. Here, we examined whether C4BP can act on its own in a complement independent manner, against pathogens. C4BP bound H1N1 and H3N2 subtypes of Influenza A Virus (IAV) most likely via multiple sites in Complement Control Protein (CCP) 1-2, 4-5, and 7-8 domains of its α-chain. In addition, C4BP CCP1-2 bound H3N2 better than H1N1. C4BP bound three IAV envelope proteins: Haemagglutinin (~70 kDa), Neuraminidase (~55 kDa), and Matrix protein 1 (~25kDa). C4BP suppressed H1N1 subtype infection into the lung epithelial cell line, A549, while it promoted infection by H3N2 subtype. C4BP restricted viral entry for H1N1 but had the opposite effect on H3N2, as evident from experiments using pseudo-typed viral particles. C4BP downregulated mRNA levels of pro-inflammatory IFN-α, IL-12, and NFκB in the case of H1N1, while it promoted a pro-inflammatory immune response by upregulating IFN- α, TNF-α, RANTES, and IL-6 in the case of H3N2. We conclude that C4BP differentially modulates the efficacy of IAV entry, and hence, replication in a target cell in a strain-dependent manner, and acts as an entry inhibitor for H1N1. Thus, CCP containing complement proteins such as factor H and C4BP may have additional defense roles against IAV that do not rely on the regulation of complement activation.