Engineering Recombinantly Expressed Antiviral Lectins


Cyanovirin-N, or CV-N for short, is a broadly binding molecule to glycopeptides and glycan structures on viral envelope spikes, which allows for screening of these structures by protein-interaction based sensor technologies.

Dr. Irene Maier, Lecturer at the Medical University of Vienna, Austria, examines the molecular understanding of immune diseases. Maier has designed new antiviral CV-N variants and measured their binding constants to glycoproteins exposed at the surface of immune-stimulating cells, which has the potential to revolutionize treatments against viral infections.

Find out more about Irene’s work on ResearchGate

Read the original research:


Image source: Adobe Image Stock / Tatiana Shepeleva





Hello, and welcome to ResearchPod. Thanks for listening and joining us today.


In this episode, we look at Cyanovirin-N, or CV-N for short, which has recently been brought to the attention of the scientific community by Dr. Irene Maier, a researcher from the University of California, Los Angeles, USA, and current Lecturer at the Medical University of Vienna, Austria. CV-N is a broadly binding molecule to glycopeptides and glycan structures on viral envelope spikes, which allows for screening of these structures by protein-interaction based sensor technologies. Maier is interested in the molecular understanding of immune diseases, which has the potential to revolutionize treatments against viral infections, and designed new antiviral CV-N variants and measured their binding constants to glycoproteins exposed at the surface of immune-stimulating cells.


CV-N is a small cyanobacterial lectin which specifically binds high-mannose oligosaccharide modifications on virus envelope spike proteins, with antiviral activity. This means that the lectin is binding viruses near the receptor-binding site and interacts with virus internalization and dissemination. Among other topical microbicide candidates consisting of molecules or formulations that modify the genital environment, nonspecific microbicides were developed in the context of cervicovaginal defense. In particular, non-proteinogenic microbicides were less efficient treatments due to either inducing inflammatory responses or lacking adherence. CV-N, however, is an antiviral lectin known for its broad neutralizing activity against life-threatening viruses, including human immunodeficiency virus, and good adherence in vaginal mucosa as well as efficacy when delivered with a genetically engineered strain of Lactobacillus jensenii.


CV-N was found specifically active against HIV-1, influenza, herpes virus, hepatitis C, severe acute respiratory syndrome coronavirus 2, and bound glycoprotein from Ebola virus. CV-N does this by binding viral envelope N-glycans with nanomolar affinity, leading to the neutralization of, for example, HIV-1 and for this virus, blocked transfer to CD4+ cells. Binding of N-linked high-mannose glycan moieties was resolved in CV-N with two carbohydrate-binding sites of differing affinities located on opposite protein protomers. So, what is the binding mechanism of CV-N to viral envelope spikes characteristic of, and is it determined by dimerization of the molecule itself?


Historically, CV-N was characterized by biophysical and biochemical methods by Dr. Angela Gronenborn, University of Pittsburgh, USA. The carbohydrate-binding agent was developed as virus-neutralizing lectin targeting various viral envelope spikes as a monomer or solution-stabilized dimer. For several decades, an amino-terminally modified CV-N protein (P51G) has been tested for its antiviral potency against HIV at the National Institute of Health, and later against deadly infectious Ebola viruses.


With the potential application of this carbohydrate-binding agent and conjugated protein for antibody-dependent cell-mediated cytotoxicity, researchers around Dr. Stephen Mayo and Dr. Pamela Bjorkman from Caltech explored the hydrogen-bond network in CV-N to dimannose, hexamannoside and nonamannoside, tested dimers for its virus neutralization capacity, but further changed the glycosylation pattern of the antibody region, fragment crystallizable, which was shown to modulate cytotoxicity.


In Maier’s work, specific binding of N-glycans to CV-N’s high-affinity carbohydrate-binding site (H) was examined on domains B. Domain A formed the low-affinity carbohydrate-binding site (L), which bound chemically synthesized dimannosylated peptide and viral spikes with a minimum of two sites. A tandem-repeat of the engineered domain-swapped CVN2 dimer bound specific sites at hemagglutinin, Ebola and HIV spike glycoproteins, N-acetyl-D-glucosamine (or GlcNAc for short) and high-mannose oligosaccharides. This engineered domain-swapped CVN2L0 molecule showed binding to influenza hemagglutinin head domain and the whole molecule via H. Site-specific N-linked glycans on spikes were investigated to mediate both the infection with influenza viruses and coronaviruses.


Next, CV-N binding to spike glycoprotein is presented by testing CV-N binding to native spike protein, showing that CV-N is capable of restoring the binding at two carbohydrate-binding sites independently from each other by using agglutination assay and isothermal titration calorimetry. The spike of the coronavirus, responsible for its entry into the cell, is divided into two portions: the S1 subunit, which has binding sites to the SARS-CoV cell receptor, angiotensin-converting enzyme 2 (or ACE2 for short), and the S2 subunit, responsible for the conformational changes that result in the fusion of the membrane and viral envelope.


During the recent SARS-CoV-2 pandemic, C-type lectin receptors on dendritic cells have been linked to severe cases of COVID-19, possibly competing with virus-neutralizing antibodies to bind the receptor-binding domain, or RBD.


Various types of antibody-mediated agglutination assays have been reported that cross-linked antigen-coated particles by anti-spike antisera in a rapid point-of-care testing setup. Mixing mammalian-derived spike RBD variants, consisting of two novel fusion proteins engineered to present RBD on the surface of red blood cells, led to visible hemagglutination in the presence of antibodies against COVID-19 antigen. The assay was described by colleagues from the Johns Hopkins University, School of Medicine.


Other data showed more than 92% of patients had detectable antibodies on the day of a positive viral RNA test, thus making the agglutination antibody test a complement, rather than alternate, test to RNA testing for the diagnosis of SARS-CoV-2 infection in convalescent COVID-19 patients. Findings from Japan suggested that systemic therapies, including chemotherapy and immune checkpoint inhibitors, lowered anti-nucleocapsid-IgG or anti-spike-IgG levels against SARS-CoV-2 in cancer patients, with immune checkpoint inhibitor treatment showing less impact on the infection immune response.


Lentil lectin also blocked binding to ACE2 receptor and bound mutant spike, epidemic variants B.1.1.7, B.1.351, and P.1., and natural and artificial asparagine-linked glycosylation, called N-linked glycosylation. By comparison, N-acetylmannosamine, also well recognized by dimeric CVN2, is the first committed biological precursor of N-acetylneuraminic acid. Sialic acids are the negatively charged, terminal monosaccharides of carbohydrate chains that are attached to glycoproteins and glycolipids.


The targeting lectins, often multi-specific in their binding, are addressing antiviral glycosylated peptides from computational, natural and biological sources, while most antimicrobial peptides inhibit viral entry by interfering with virus and cell membrane fusion.


To determine CV-N’s binding specificity to high-mannose glycans, bivalent binding with glycopeptides and binding of CVN2 to mannose-derivatives and N-glycans were measured by surface plasmon resonance spectroscopy and isothermal titration calorimetry, respectively. Two disulfide bonds in CVN2 were replaced by ion-pairing residues that retrained potential ionic interactions between mutated E58 and R73 residues for binding a dimannosylated peptide, a 12mer peptide from hemagglutinin. The unmodified dimer CVN2L0 bound this peptide with an N-terminal cysteine with an equilibrium dissociation constant of 4 µM, but N-acetyl-glucosamine, the glycan attached to asparagine of N-glycans, with a dissociation constant of 327 nM.


We further expressed CV-N with a single-point mutation at site E41A, because of this position having been shown to reveal low effects on dimannose binding energies of the binding site in a mutation-stabilized fold of the monomeric CV-N. With two carbohydrate-binding sites involved, CVN-E41A showed lower molecular strength in binding N-acetyl-glucosamine than the four-site CVN2L0, but still bound with nanomolar affinity. Moreover, CVN-E41A showed higher agglutination to spike protein than spike-binding CV-N, whereas an interaction of CVN2L0 revealed active binding to viral spikes. CV-N variants and CV-N wildtype were expressed in Escherichia coli and cost-efficiently purified from the extracellular non-reducing periplasm using immobilized metal affinity chromatography.


N-glycosylation sites on spike protein, which were most interesting because of their selective interaction with known monoclonal virus-neutralizing antibodies, were N165, the high-mannose N-glycosylation site N234, and N343, a highly conserved site outside the RBD. Although the binding mechanism of CVN-E41A in terms of H versus L binding to viral spikes and sugars is currently unclear, we hypothesize different affinities to the various glycan structures, thereby introducing binding specificity and induced fit with the competitively bound poorly neutralizing antibodies from SARS-CoV-2 infected and ACE2-overexpressing cells.


The lectins that are low-affinity carbohydrate binders and a scaffold for computational protein design, will be utilized to evaluate the production of broadly neutralizing antibodies. The potentially multisite and unlocalized binding of CVN2 counteracts with high-affinity binding of neutralizing antibodies in response to escape mutations, as well as glycan masking.


Taken together, CV-N, a lectin isolated from Nostoc ellipsosporum, was first found an infusion inhibitory protein for HIV-1/AIDS. CV-N secreted from an engineered Lactobacillus strain reduced vaginal transmission of a chimeric simian/HIV in macaques for 69%. While also neutralizing influenza A and B virus strains, it may have the potential to sterically block broadly neutralizing antibodies which address either influenza viruses or coronavirus spikes, thereby influencing the glycan shield of these enveloped viruses.


In the words of Dr. Irene Maier:


‘we are searching for CV-N variants with enhanced binding capacity to retroviruses that can initiate immune responses such as antibody-dependent cell-mediated cytotoxicity, reduce virus spike interactions with C-type lectin receptors, or reduce secondary bacterial infections in high-risk and COVID-19 vaccinated cancer patients.’


That’s all for this episode – thanks for listening. Links to the original research can be found in the notes for this episode. And, as always, stay subscribed to ResearchPod for more of the latest science.


See you again soon.



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