Antiviral activity of marine sulfated glycans against pathogenic human coronaviruses.

Great interest exists towards the discovery and development of broad-spectrum antivirals. This occurs due to the frequent emergence of new viruses which can also eventually lead to pandemics. A reasonable and efficient strategy to develop new broad-spectrum antivirals relies on targeting a common molecular player of various viruses. Heparan sulfate is a sulfated glycosaminoglycan present on the surface of cells which plays a key role as co-receptor in many virus infections. In previous work, marine sulfated glycans (MSGs) were identified as having antiviral activities. Their mechanism of action relies primarily on competitive inhibition of virion binding to heparan sulfate, preventing virus attachment to the cell surface prior to entry. In the current work we used pseudotyped lentivirus particles to investigate in a comparative fashion the inhibitory properties of five structurally defined MSGs against SARS-CoV-1, SARS-CoV-2, MERS-CoV, and influenza A virus (IAV). MSGs include the disaccharide-repeating sulfated galactan from the red alga Botryocladia occidentalis, the tetrasaccharide-repeating sulfated fucans from the sea urchin Lytechinus variegatus and from the sea cucumber Isostichopus badionotus, and the two marine fucosylated chondroitin sulfates from the sea cucumbers I. badionotus and Pentacta pygmaea. Results indicate specificity of action against SARS-CoV-1 and SARS-CoV-2. Curiously, the MSGs showed decreased inhibitory potencies against MERS-CoV and negligible action against IAV. Among the five MSGs, the two sulfated fucans here studied deserve further attention since they have the lowest anticoagulant effects but still present potent and selective antiviral properties.

Fractionation of sulfated galactan from the red alga Botryocladia occidentalis separates its anticoagulant and anti-SARS-CoV-2 properties.

Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified cofactors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK-293T-hACE2 cells monitored by GFP. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation cofactors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including WT, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action.

Fractionation of sulfated galactan from the red alga Botryocladia occidentalis separates its anticoagulant and anti-SARS-CoV-2 properties

Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified co-factors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK293T cells monitored by green fluorescent protein. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation co-factors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including wild type, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action.

Immunization with Human Cytomegalovirus Core Fusion Machinery and Accessory Envelope Proteins Elicit Strong Synergistic Neutralizing Activities.

Human cytomegalovirus (HCMV) core fusion machinery proteins gB and gH/gL, and accessory proteins UL128/UL130/UL131A, are the key envelope proteins that mediate HCMV entry into and infection of host cells. To determine whether these HCMV envelope proteins could elicit neutralizing activities synergistically, we immunized rabbits with individual or various combinations of these proteins adsorbed to aluminum hydroxide mixed with CpG-ODN. We then analyzed serum neutralizing activities with multiple HCMV laboratory strains and clinical isolates. HCMV trimeric gB and gH/gL elicited high and moderate titers of HCMV neutralizing activity, respectively. HCMV gB in combination with gH/gL elicited up to 17-fold higher HCMV neutralizing activities compared to the sum of neutralizing activity elicited by the individual proteins analyzed with both fibroblasts and epithelial cells. HCMV gB+gH/gL+UL128/UL130/UL131A in combination increased the neutralizing activity up to 32-fold compared to the sum of neutralizing activities elicited by the individual proteins analyzed with epithelial cells. Adding UL128/UL130/UL131A to gB and gH/gL combination did not increase further the HCMV neutralizing activity analyzed with fibroblasts. These data suggest that the combination of HCMV core fusion machinery envelope proteins gB+gH/gL or the combination of gB and pentameric complex could be ideal vaccine candidates that would induce optimal immune responses against HCMV infection.