Barry R. O'Keefe

Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component

To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals (ARVs) that directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, we report a manufacturing breakthrough for griffithsin (GRFT), one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing GRFT. Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, we also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes.

Probing protein-carbohydrate interactions with microarrays of synthetic oligosaccharides.

Formerly a TMneglected dimension∫ of biochemistry, recent years have seen growing interest in studying the biological function of carbohydrates and glycoconjugates. An emerging understanding of the physiological role of these biomolecules has uncovered their vital participation in a host of fundamental cellular processes. In the form of glycopeptides, glycolipids, glycosaminoglycans, and proteoglyans, glycoconjugates are known to be involved in inflammation, cell ± cell interactions, signal transduction, fertility, and development. 5] Unfortunately, current methods for elucidating the biochemical roles of glycoconjugates are often cumbersome. This demonstrates the need to develop techniques that will satisfy this growing field of study by enabling rapid and facile exploration of biochemical events involving carbohydrates. Inspired by the success of DNA and protein microarrays, 7] the chip-based approach has been put forward as a useful tool in the emerging field of glycomics. Nitrocellulose-coated slides have been employed for the noncovalent immobilization of microbial polysaccharides and neoglycolipid-modified oligosaccharides. 12] Hydrophobic interactions have been utilized to anchor lipid-bearing carbohydrates on polystyrene microtiter plates. Self-assembled monolayers presenting benzoquinone groups enabled the Diels ±Alder-mediated immobilization of cyclopentadiene-derivatized monosaccharides on a gold surface. Another covalent immobilization chemistry involved treating maleimide-functionalized monoand disaccharide glycosylamines with a thiol-derivatized glass slide, or, alternatively, thiol-functionalized carbohydrates with a self-assembled monolayer presenting maleimide groups. Our motivation for developing a system for arraying carbohydrates is based on the need to have microarrays that are fully phosphate buffer (0.1M) and Perfect Hyb hybridization buffer (Sigma, 1:1 v/v) for 5 h to give double-stranded DNA assembly on the surface. The resulting surfaces were rinsed with the hybridization buffer and immersed in a solution of hemin (1.2 M) in buffer (25 mM HEPES, 20 mM KCl, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO; pH 7.4) for 12 h at room temperature. The resulting system was further treated with doxorubicin (5, 5 M) in phosphate buffer (0.1M, pH 7.4) for 1 h at room temperature.

Potent anti-influenza activity of cyanovirin-N and interactions with viral hemagglutinin.

ABSTRACT The novel antiviral protein cyanovirin-N (CV-N) was initially discovered based on its potent activity against the human immunodeficiency virus (HIV). Subsequent studies identified the HIV envelope glycoproteins gp120 and gp41 as molecular targets of CV-N. More recently, mechanistic studies have shown that certain high-mannose oligosaccharides (oligomannose-8 and oligomannose-9) found on the HIV envelope glycoproteins comprise the specific sites to which CV-N binds. Such selective, carbohydrate-dependent interactions may account, at least in part, for the unusual and unexpected spectrum of antiviral activity of CV-N described herein. We screened CV-N against a broad range of respiratory and enteric viruses, as well as flaviviruses and herpesviruses. CV-N was inactive against rhinoviruses, human parainfluenza virus, respiratory syncytial virus, and enteric viruses but was moderately active against some herpesvirus and hepatitis virus (bovine viral diarrhea virus) strains (50% effective concentration [EC50] = ∼1 μg/ml) while inactive against others. Remarkably, however, CV-N and related homologs showed highly potent antiviral activity against almost all strains of influenza A and B virus, including clinical isolates and a neuraminidase inhibitor-resistant strain (EC50 = 0.004 to 0.04 μg/ml). When influenza virus particles were pretreated with CV-N, viral titers were lowered significantly (>1,000-fold). Further studies identified influenza virus hemagglutinin as a target for CV-N, showed that antiviral activity and hemagglutinin binding were correlated, and indicated that CV-N′s interactions with hemagglutinin involved oligosaccharides. These results further reveal new potential avenues for antiviral therapeutics and prophylaxis targeting specific oligosaccharide-comprised sites on certain enveloped viruses, including HIV, influenza virus, and possibly others.

Domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding.

Summary The crystal structure of griffithsin, an antiviral lectin from the red alga Griffithsia sp., was solved and refined at 1.3 Å resolution for the free protein and 0.94 Å for a complex with mannose. Griffithsin molecules form a domain-swapped dimer, in which two β strands of one molecule complete a β prism consisting of three four-stranded sheets, with an approximate 3-fold axis, of another molecule. The structure of each monomer bears close resemblance to jacalin-related lectins, but its dimeric structure is unique. The structures of complexes of griffithsin with mannose and N-acetylglucosamine defined the locations of three almost identical carbohydrate binding sites on each monomer. We have also shown that griffithsin is a potent inhibitor of the coronavirus responsible for severe acute respiratory syndrome (SARS). Antiviral potency of griffithsin is likely due to the presence of multiple, similar sugar binding sites that provide redundant attachment points for complex carbohydrate molecules present on viral envelopes.

The Domain-Swapped Dimer of Cyanovirin-N Is in a Metastable Folded State: Reconciliation of X-Ray and NMR Structures

The structure of the potent HIV-inactivating protein cyanovirin-N was previously found by NMR to be a monomer in solution and a domain-swapped dimer by X-ray crystallography. Here we demonstrate that, in solution, CV-N can exist both in monomeric and in domain-swapped dimeric form. The dimer is a metastable, kinetically trapped structure at neutral pH and room temperature. Based on orientational NMR constraints, we show that the domain-swapped solution dimer is similar to structures in two different crystal forms, exhibiting solely a small reorientation around the hinge region. Mutation of the single proline residue in the hinge to glycine significantly stabilizes the protein in both its monomeric and dimeric forms. By contrast, mutation of the neighboring serine to proline results in an exclusively dimeric protein, caused by a drastic destabilization of the monomer.

Multisite and Multivalent Binding between Cyanovirin-N and Branched Oligomannosides: Calorimetric and NMR Characterization

Binding of the protein cyanovirin-N to oligomannose-8 and oligomannose-9 of gp120 is crucially involved in its potent virucidal activity against the human immunodeficiency virus (HIV). The interaction between cyanovirin-N and these oligosaccharides has not been thoroughly characterized due to aggregation of the oligosaccharide-protein complexes. Here, cyanovirin-Ns interaction with a nonamannoside, a structural analog of oligomannose-9, has been studied by nuclear magnetic resonance and isothermal titration calorimetry. The nonamannoside interacts with cyanovirin-N in a multivalent fashion, resulting in tight complexes with an average 1:1 stoichiometry. Like the nonamannoside, an alpha1-->2-linked trimannoside substructure interacts with cyanovirin-N at two distinct protein subsites. The chitobiose and internal core trimannoside substructures of oligomannose-9 are not recognized by cyanovirin-N, and binding of the core hexamannoside occurs at only one of the sites on the protein. This is the first detailed analysis of a biologically relevant interaction between cyanovirin-N and high-mannose oligosaccharides of HIV-1 gp120.

Resistance of human immunodeficiency virus type 1 to the high-mannose binding agents cyanovirin N and concanavalin A

ABSTRACT Due to the biological significance of the carbohydrate component of the human immunodeficiency virus type 1 (HIV-1) glycoproteins in viral pathogenesis, the glycosylation step constitutes an attractive target for anti-HIV therapy. Cyanovirin N (CV-N), which specifically targets the high-mannose (HM) glycans on gp120, has been identified as a potent HIV-1 entry inhibitor. Concanavalin A (ConA) represents another mannose-binding lectin, although it has a lower specificity for HM glycans than that of CV-N. For the present study, we selected CV-N- and ConA-resistant HIV-1 strains in the presence of CV-N and ConA, respectively. Both resistant strains exhibited a variety of mutations eliminating N-linked glycans within gp120. Strains resistant to CV-N or ConA displayed high levels of cross-resistance towards one another. The N-glycan at position 302 was eliminated in both of the lectin-resistant strains. However, the elimination of this glycan alone by site-directed mutagenesis was not sufficient to render HIV-1 resistant to CV-N or ConA, suggesting that HIV-1 needs to mutate several N-glycans to become resistant to these lectins. Both strains also demonstrated clear cross-resistance towards the carbohydrate-dependent monoclonal antibody 2G12. In contrast, the selected strains did not show a reduced susceptibility towards the nonlectin entry inhibitors AMD3100 and enfuvirtide or towards reverse transcriptase or protease inhibitors. Recombination of the mutated gp160 genes of the strains resistant to CV-N or ConA into a wild-type background fully reproduced the (cross-)resistance profiles of the originally selected strains, pointing to the impact of the N-glycan mutations on the phenotypic resistance profiles of both selected strains.

Broad-Spectrum In Vitro Activity and In Vivo Efficacy of the Antiviral Protein Griffithsin against Emerging Viruses of the Family Coronaviridae

ABSTRACT Viruses of the family Coronaviridae have recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection both in vitro and in vivo. We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals.

Treatment of influenza A (H1N1) virus infections in mice and ferrets with cyanovirin-N.

Cyanovirin-N (CV-N), a protein derived from Nostoc ellipsosporum, neutralizes influenza virus infectivity by binding to specific high-mannose oligosaccharides (oligomannose-8 and -9) at glycosylation sites on the viral hemagglutinin HA1 subunit. Mouse-adapted viruses lose sensitivity to CV-N due to HA1 mutations that eliminate these glycosylation sites. Recently we created a hybrid (reassortant) influenza A/WSN/33 (H1N1) virus containing the HA gene of A/New Caledonia/20/99 (H1N1) with an Asp225Gly mutation in the HA1, that was lethal to mice yet retained sensitivity to CV-N. We then utilized this model system to test the efficacy of CV-N against influenza. CV-N efficacy was dose-responsive from 0.0625 to 1 mg/kg/day when administered intranasally (i.n.) twice daily for 4 days starting 4h prior to virus exposure. In a second study, survival benefit was seen with CV-N treatments (0.5 mg/kg/day for 4 days) beginning at -4 or +6 h, but was significantly reduced at +12h. The early treatment resulted in up to 100% survival and 1000-fold reduction in lung virus titer on day 3 of the infection. In contrast, ribavirin (a positive control-75 mg/kg/day) treatment resulted in 30% survival and 30-fold decrease in lung virus titers. Lung consolidation scores and lung weights were significantly reduced by CV-N and ribavirin treatment on day 6 of the infection. Ferrets infected with a non-animal adapted influenza A/Charlottesville/31/95 (H1N1) virus were treated intranasally with CV-N (50 microg twice daily for 5 days starting 24 h before virus challenge). They exhibited 100-fold lower viral titers in nasal washes than placebos 1 day after treatment, but virus titers were equivalent on days 2-7. CV-N has the potential for prophylaxis and early initiation of treatment of influenza virus infections.

Investigation of Griffithsin's Interactions with Human Cells Confirms Its Outstanding Safety and Efficacy Profile as a Microbicide Candidate

Many natural product-derived lectins such as the red algal lectin griffithsin (GRFT) have potent in vitro activity against viruses that display dense clusters of oligomannose N-linked glycans (NLG) on their surface envelope glycoproteins. However, since oligomannose NLG are also found on some host proteins it is possible that treatment with antiviral lectins may trigger undesirable side effects. For other antiviral lectins such as concanavalin A, banana lectin and cyanovirin-N (CV-N), interactions between the lectin and as yet undescribed cellular moieties have been reported to induce undesirable side effects including secretion of inflammatory cytokines and activation of host T-cells. We show that GRFT, unlike CV-N, binds the surface of human epithelial and peripheral blood mononuclear cells (PBMC) through an exclusively oligosaccharide-dependent interaction. In contrast to several other antiviral lectins however, GRFT treatment induces only minimal changes in secretion of inflammatory cytokines and chemokines by epithelial cells or human PBMC, has no measureable effect on cell viability and does not significantly upregulate markers of T-cell activation. In addition, GRFT appears to retain antiviral activity once bound to the surface of PBMC. Finally, RNA microarray studies show that, while CV-N and ConA regulate expression of a multitude of cellular genes, GRFT treatment effects only minimal alterations in the gene expression profile of a human ectocervical cell line. These studies indicate that GRFT has an outstanding safety profile with little evidence of induced toxicity, T-cell activation or deleterious immunological consequence, unique attributes for a natural product-derived lectin.