Recognition and inhibition of SARS-CoV-2 by humoral innate immunity pattern recognition molecules.

The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

C1q/MASP Complexes-Hybrid Complexes of Classical and Lectin Pathway Proteins Are Found in the Circulation.

Complement pathways, traditionally regarded as separate entities in vitro, are increasingly noted for cross-communication and bypass mechanisms. Among these, the MBL/ficolin/CL-associated serine protease (MASP)-3, a component of lectin pathway pattern recognition molecules, has shown the ability to process critical substrates such as pro-factor D and insulin growth factor binding protein-5. Given shared features between lectin pathway pattern recognition molecules and C1q from the classical pathway, we hypothesized that C1q might be a viable in vivo binding partner for the MASPs. We used microscale thermophoresis, ELISA, and immunoprecipitation assays to detect C1q/MASP complexes and functionally assessed the complexes through enzymatic cleavage assays. C1q/MASP-3 complexes were detected in human serum and correlated well with MASP-3 serum levels in healthy individuals. The binding affinity between MASP-3 and C1q in vitro was in the nanomolar range, and the interaction was calcium-dependent, as demonstrated by their dissociation in the presence of EDTA. Furthermore, most of the circulating C1q-bound MASP-3 was activated. Based on immunoprecipitation, also C1q/MASP-2 complexes appeared to be present in serum. Finally, C1q/MASP-2 and C1q/MASP-3 in vitro complexes were able to cleave C4 and pro-factor D, respectively. Our study reveals the existence of C1q/MASP complexes in the circulation of healthy individuals, and both C1q/MASP-2 and C1q/MASP-3 complexes display proteolytic activity. Hence, this study uncovers a crosstalk route between complement pathways not previously described.

CL-11 circulates in serum as functionally distinct isoforms.

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway capable of interacting with collectin-10 (CL-10) and the MASPs to activate the complement cascade. Alternative splicing of the COLEC11 gene gives rise to two different isoforms found in serum (A and D). These isoforms vary in the length of their collagen-like region, which is involved in the stabilization of the trimeric subunit and the interaction with the MASPs. Here we aim at elucidating the biological differences of naturally occurring CL-11 isoforms A and D. We produced recombinant CL-11 as independent isoforms (CL-11A and CL-11D) and together with CL-10 (CL-10/11A, CL-10/11D). Both CL-11 isoforms associated with CL-10, but CL-11D did so to a lesser extent. CL-10/11 heterocomplexes were composed of trimeric subunits of CL-10 and CL-11, as opposed to CL-10 and CL-11 homotrimers. Heterocomplexes were more stable and migrated with higher apparent molecular weights. Immunoprecipitation of serum CL-11 and subsequent mass spectrometry analysis confirmed that native CL-11 circulates in the form of CL-10/11 heterocomplexes that associate with MASP-1, and MASP-3, but not necessarily MASP-2. Despite a shorter collagen region, CL-11D was capable to bind to the MASPs, suggesting that the missing exon 4 is not required for MASP association CL-11D had a reduced ligand binding compared to full-length CL-11A. Based on its reduced ability to oligomerize, form CL-10/11 heterocomplexes, and bind to ligands, we hypothesize that CL-11D may have a limited complement activation potential compared to full-length CL-11A.

MAP-2:CD55 chimeric construct effectively modulates complement activation.

The complement system is a complex, tightly regulated protein cascade involved in pathogen defense and the pathogenesis of several diseases. Thus, the development of complement modulators has risen as a potential treatment for complement-driven inflammatory pathologies. The enzymatically inactive MAP-2 has been reported to inhibit the lectin pathway by competing with its homologous serine protease MASP-2. The membrane-bound complement inhibitor CD55 acts on the C3/C5 convertase level. Here, we fused MAP-2 to the four N-terminal domains of CD55 generating a targeted chimeric inhibitor to modulate complement activation at two different levels of the complement cascade. Its biological properties were compared in vitro with the parent molecules. While MAP-2 and CD55 alone showed a minor inhibition of the three complement pathways when co-incubated with serum (IC50MAP-2+CD55 1-4 = 60.98, 36.10, and 97.01 nM on the classical, lectin, and alternative pathways, respectively), MAP-2:CD551-4 demonstrated a potent inhibitory activity (IC50MAP-2:CD55 1-4 = 2.94, 1.76, and 12.86 nM, respectively). This inhibitory activity was substantially enhanced when pre-complexes were formed with the lectin pathway recognition molecule mannose-binding lectin (IC50MAP-2:CD55 1-4 = 0.14 nM). MAP-2:CD551-4 was also effective at protecting sensitized sheep erythrocytes in a classical hemolytic assay (CH50 = 13.35 nM). Finally, the chimeric inhibitor reduced neutrophil activation in full blood after stimulation with Aspergillus fumigatus conidia, as well as phagocytosis of conidia by isolated activated neutrophils. Our results demonstrate that MAP-2:CD551-4 is a potent complement inhibitor reinforcing the idea that engineered fusion proteins are a promising design strategy for identifying and developing drug candidates to treat complement-mediated diseases.

Revaccination of patients with systemic lupus erythematosus or rheumatoid arthritis without an initial COVID-19 vaccine response elicits seroconversion in half of the patients.

OBJECTIVES: To investigate the effect of COVID-19 mRNA revaccination (two doses) on the antibody response in patients with rheumatic diseases (RD) who were initial vaccine non-responders. Further, to examine if B-cell levels or T-cell responses before revaccination predicted seroconversion. METHODS: From a RD cohort vaccinated with the standard two-dose COVID-19 vaccinations, we enrolled cases without detectable antibody responses (n=17) and controls with detectable antibody response (n=29). Blood donors (n=32) were included as additional controls. Samples were collected before and six weeks after completed revaccination. Total antibodies and specific IgG, IgA, and IgM against SARS-CoV-2 spike protein, SARS-CoV-2 neutralising antibodies, and SARS-CoV-2 reacting CD4+ and CD8+ T-cells were measured before and after revaccination. B-cells (CD19+CD45+) were quantified before revaccination. RESULTS: Forty-seven percent of cases had detectable neutralising antibodies after revaccination. However, antibody levels were significantly lower than in controls and blood donors. Revaccination induced an antibody class switch in cases with a decrease in IgM and increase in IgG. No significant difference was observed in T-cell responses before and after revaccination between the three groups. Only 29% of cases had measurable B-cells compared to 100% of controls and blood donors. Fifty percent of revaccinated cases who seroconverted had measurable B-cells before revaccination. CONCLUSIONS: Forty-seven percent of initial non-responders seroconverted after two-dose revaccination but still had lower levels of SARS-CoV-2 antibodies compared with controls and blood donors. RD patients without a detectable serological response after the initial COVID-19 mRNA vaccine had a T-cell response similar to immunocompetent controls and blood donors.

SARS-CoV-2 Antibody Responses Are Correlated to Disease Severity in COVID-19 Convalescent Individuals.

Globally, the COVID-19 pandemic has had extreme consequences for the healthcare system and has led to calls for diagnostic tools to monitor and understand the transmission, pathogenesis, and epidemiology, as well as to evaluate future vaccination strategies. In this study, we have developed novel, to our knowledge, flexible ELISA-based assays for specific detection of human SARS-CoV-2 Abs against the receptor-binding domain, including an Ag sandwich ELISA relevant for large population screening and three isotype-specific assays for in-depth diagnostics. Their performance was evaluated in a cohort of 350 convalescent participants with previous COVID-19 infection, ranging from asymptomatic to critical cases. We mapped the Ab responses to different areas on protein N and S and showed that the IgM, A, and G Ab responses against receptor-binding domain are significantly correlated to the disease severity. These assays and the data generated from them are highly relevant for diagnostics and prognostics and contribute to the understanding of long-term COVID-19 immunity.

SARS-CoV-2 Neutralizing Antibody Responses towards Full-Length Spike Protein and the Receptor-Binding Domain.

Tools to monitor SARS-CoV-2 transmission and immune responses are needed. We present a neutralization ELISA to determine the levels of Ab-mediated virus neutralization and a preclinical model of focused immunization strategy. The ELISA is strongly correlated with the elaborate plaque reduction neutralization test (ρ = 0.9231, p < 0.0001). The neutralization potency of convalescent sera strongly correlates to IgG titers against SARS-CoV-2 receptor-binding domain (RBD) and spike (ρ = 0.8291 and 0.8297, respectively; p < 0.0001) and to a lesser extent with the IgG titers against protein N (ρ = 0.6471, p < 0.0001). The preclinical vaccine NMRI mice models using RBD and full-length spike Ag as immunogens show a profound Ab neutralization capacity (IC50 = 1.9 × 104 to 2.6 × 104 and 3.9 × 103 to 5.2 × 103, respectively). Using a panel of novel high-affinity murine mAbs, we also show that a majority of the RBD-raised mAbs have inhibitory properties, whereas only a few of the spike-raised mAbs do. The ELISA-based viral neutralization test offers a time- and cost-effective alternative to the plaque reduction neutralization test. The immunization results indicate that vaccine strategies focused only on the RBD region may have advantages compared with the full spike.

The SARS-CoV-2 Y453F mink variant displays a pronounced increase in ACE-2 affinity but does not challenge antibody neutralization.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 from humans to animals has been reported for many domesticated species, including farmed minks. The identification of novel spike gene mutations appearing in minks has raised major concerns about potential immune evasion and challenges for the global vaccine strategy. One genetic variant, known as "cluster five," arose among farmed minks in Denmark and resulted in a complete shutdown of the world's largest mink production. However, the functional properties of this new variant are not established. Here we present functional data on the cluster-five variant, which contains a mutation resulting in a Y453F residue change in the receptor-binding domain (RBD) of the spike protein. Using an ELISA-based angiotensin-converting enzyme-2/RBD inhibition assay, we show that the Y453F variant does not decrease established humoral immunity from previously infected individuals or affect the neutralizing antibody response in a vaccine mouse model based on the original Wuhan strain RBD or spike as antigens. However, biolayer interferometry analysis demonstrates that it binds the human angiotensin-converting enzyme-2 receptor with a 4-fold higher affinity than the original strain, suggesting an enhanced transmission capacity and a possible challenge for viral control. These results also indicate that the rise in the frequency of the cluster-five variant in mink farms might be a result of the fitness advantage conferred by the receptor adaptation rather than evading immune responses.

C1q/TNF-Related Protein 6 Is a Pattern Recognition Molecule That Recruits Collectin-11 from the Complement System to Ligands.

C1q/TNF-related protein (CTRP) 6 is a member of the CTRP protein family associated with the regulation of cellular and endocrine processes. CTRP6 contains collagen and globular structures, resembling the pattern recognition molecules (PRMs) of the classical and lectin complement pathways. We expressed human CTRP6 in Chinese hamster ovary cells and investigated the binding to different putative ligands (acetylated BSA [AcBSA], zymosan, mannan, and LPS from Escherichia coli and Salmonella as well as to the monosaccharides l-fucose, d-mannose, N-acetylglucosamine, N-acetylgalactosamine, and galactose). Furthermore, we investigated the binding of CTRP6 to various Gram-negative bacteria as well as PRMs and enzymes of the lectin complement pathway. We found that CTRP6 bound to AcBSA and to a lesser extent to zymosan. Using EDTA as chelating agent, we observed an increased binding to AcBSA, zymosan and the two strains of LPS. We detected no binding to mannan and BSA. We identified l-fucose as a ligand for CTRP6 and that it bound to certain enteroaggregative Escherichia coli and Pseudomonas aeruginosa isolates, whereas to other bacterial isolates, no binding was observed. CTRP6 did not appear to interact directly with the activating enzymes of the lectin pathway; however, we could show the specific recruitment of collectin-11 and subsequent initiation of the complement cascade through deposition of C4. In conclusion, our results demonstrate the binding of CTRP6 to a variety of microbial and endogenous ligands identifying CTRP6 as a novel human lectin and PRM of importance for complement recognition and innate immunity.