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Abstract Background Sexual acquisition of the human immunodeficiency virus (HIV) through mucosal transmission may be prevented by using topically applied agents that block HIV transmission from one individual to another. Therefore, virucidal agents that inactivate HIV virions may be used as a component in topical microbicides. Results Here, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a low-molecular-weight molecule that inactivates HIV particles. Both HIV-1 and HIV-2 virions pretreated with this compound were unable to infect permissive cells. Moreover, WDO-217 was able to inhibit infections of a wide spectrum of wild-type and drug-resistant HIV-1, including clinical isolates, HIV-2 and SIV strains. Whereas the capture of virus by DC-SIGN was unaffected by the compound, it efficiently prevented the transmission of DC-SIGN-captured virus to CD4+ T-lymphocytes. Interestingly, exposure of virions to WDO-217 reduced the amount of virion-associated genomic RNA as measured by real-time RT-qPCR. Further mechanism-of-action studies demonstrated that WDO-217 efficiently ejects zinc from the zinc fingers of the retroviral nucleocapsid protein NCp7 and inhibits the cTAR destabilization properties of this protein. Importantly, WDO-217 was able to eject zinc from both zinc fingers, even when NCp7 was bound to oligonucleotides, while no covalent interaction between NCp7 and WDO-217 could be observed. Conclusion This compound is a new lead structure that can be used for the development of a new series of NCp7 zinc ejectors as candidate topical microbicide agents.

Abstract Background In a recent report, the carbohydrate-binding specificities of the plant lectins Galanthus nivalis (GNA) and the closely related lectin from Zea mays (GNAmaize) were determined by glycan array analysis and indicated that GNAmaize recognizes complex-type N-glycans whereas GNA has specificity towards high-mannose-type glycans. Both lectins are tetrameric proteins sharing 64% sequence similarity. Results GNAmaize appeared to be ~20- to 100-fold less inhibitory than GNA against HIV infection, syncytia formation between persistently HIV-1-infected HuT-78 cells and uninfected CD4+ T-lymphocyte SupT1 cells, HIV-1 capture by DC-SIGN and subsequent transmission of DC-SIGN-captured virions to uninfected CD4+ T-lymphocyte cells. In contrast to GNA, which preferentially selects for virus strains with deleted high-mannose-type glycans on gp120, prolonged exposure of HIV-1 to dose-escalating concentrations of GNAmaize selected for mutant virus strains in which one complex-type glycan of gp120 was deleted. Surface Plasmon Resonance (SPR) analysis revealed that GNA and GNAmaize interact with HIV IIIB gp120 with affinity constants (KD) of 0.33 nM and 34 nM, respectively. Whereas immobilized GNA specifically binds mannose oligomers, GNAmaize selectively binds complex-type GlcNAcβ1,2Man oligomers. Also, epitope mapping experiments revealed that GNA and the mannose-specific mAb 2G12 can independently bind from GNAmaize to gp120, whereas GNAmaize cannot efficiently bind to gp120 that contained prebound PHA-E (GlcNAcβ1,2man specific) or SNA (NeuAcα2,6X specific). Conclusion The markedly reduced anti-HIV activity of GNAmaize compared to GNA can be explained by the profound shift in glycan recognition and the disappearance of carbohydrate-binding sites in GNAmaize that have high affinity for mannose oligomers. These findings underscore the need for mannose oligomer recognition of therapeutics to be endowed with anti-HIV activity and that mannose, but not complex-type glycan binding of chemotherapeutics to gp120, may result in a pronounced neutralizing activity against the virus.

Abstract Background HIV-1 in genital secretions may be opsonized by several molecules including complement components. Opsonized HIV-1 by complement enhances the infection of various mucosal target cells, such as dendritic cells (DC) and epithelial cells. Results We herein evaluated the effect of HIV-1 complement opsonization on microbicide candidates' activity, by using three in vitro mucosal models: CCR5-tropic HIV-1JR-CSF transcytosis through epithelial cells, HIV-1JR-CSF attachment on immature monocyte-derived dendritic cells (iMDDC), and infectivity of iMDDC by CCR5-tropic HIV-1BaL and CXCR4-tropic HIV-1NDK. A panel of 10 microbicide candidates [T20, CADA, lectines HHA & GNA, PVAS, human lactoferrin, and monoclonal antibodies IgG1B12, 12G5, 2G12 and 2F5], were investigated using cell-free unopsonized or opsonized HIV-1 by complements. Only HHA and PVAS were able to inhibit HIV trancytosis. Upon opsonization, transcytosis was affected only by HHA, HIV-1 adsorption on iMDDC by four molecules (lactoferrin, IgG1B12, IgG2G5, IgG2G12), and replication in iMDDC of HIV-1BaL by five molecules (lactoferrin, CADA, T20, IgG1B12, IgG2F5) and of HIV-1NDK by two molecules (lactoferrin, IgG12G5). Conclusion These observations demonstrate that HIV-1 opsonization by complements may modulate in vitro the efficiency of candidate microbicides to inhibit HIV-1 infection of mucosal target cells, as well as its crossing through mucosa.

Abstract Background Formation of an HIV-1 particle with a conical core structure is a prerequisite for the subsequent infectivity of the virus particle. We have previously described that glycineamide (G-NH2) when added to the culture medium of infected cells induces non-infectious HIV-1 particles with aberrant core structures. Results Here we demonstrate that it is not G-NH2 itself but a metabolite thereof that displays antiviral activity. We show that conversion of G-NH2 to its antiviral metabolite is catalyzed by an enzyme present in bovine and porcine but surprisingly not in human serum. Structure determination by NMR suggested that the active G-NH2 metabolite was α-hydroxy-glycineamide (α-HGA). Chemically synthesized α-HGA inhibited HIV-1 replication to the same degree as G-NH2, unlike a number of other synthesized analogues of G-NH2 which had no effect on HIV-1 replication. Comparisons by capillary electrophoresis and HPLC of the metabolite with the chemically synthesized α-HGA further confirmed that the antiviral G-NH2-metabolite indeed was α-HGA. Conclusion α-HGA has an unusually simple structure and a novel mechanism of antiviral action. Thus, α-HGA could be a lead for new antiviral substances belonging to a new class of anti-HIV drugs, i.e. capsid assembly inhibitors.

Abstract Background Plant lectins such as Galanthus nivalis agglutinin (GNA) and Hippeastrum hybrid agglutinin (HHA) are natural proteins able to link mannose residues, and therefore inhibit HIV-target cell interactions. Plant lectins are candidate for microbicide development. Objective To evaluate the activity against HIV of the mannose-specific plant lectins HHA and GNA at the cellular membrane level of epithelial cells and monocyte-derived dendritic cells (MDDC), two potential target cells of HIV at the genital mucosal level. Methods The inhibitory effects of HHA and GNA were evaluated on HIV adsorption to genital epithelial HEC-1A cell line, on HIV transcytosis throughout a monolayer of polarized epithelial HEC-1A cells, on HIV adsorption to MDDC and on transfer of HIV from MDDC to autologous T lymphocytes. Results HHA faintly inhibited attachment to HEC-1A cells of the R5-tropic HIV-1Ba-L strain, in a dose-dependent manner, whereas GNA moderately inhibited HIV adsorption in the same context, but only at high drug doses. Only HHA, but not GNA, inhibited HIV-1JR-CSF transcytosis in a dose-dependent manner. By confocal microscopy, HHA, but not GNA, was adsorbed at the epithelial cell surface, suggesting that HHA interacts specifically with receptors mediating HIV-1 transcytosis. Both plant lectins partially inhibited HIV attachment to MDDC. HHA inhibited more efficiently the transfer of HIV from MDDC to T cell, than GNA. Both HHA and GNA lacked toxicity below 200 μg/ml irrespective the cellular system used and do not disturb the monolayer integrity of epithelial cells. Conclusion These observations demonstrate higher inhibitory activities of the lectin plant HHA by comparison to GNA, on HIV adsorption to HEC-1A cell line, HIV transcytosis through HEC-1A cell line monolayer, HIV adsorption to MDDC and HIV transfer from MDDC to T cells, highlighting the potential interest of HHA as effective microbicide against HIV.