Multivalent Manno‐Glyconanoparticles Inhibit DC‐SIGN‐Mediated HIV‐1 Trans‐Infection of Human T Cells

Viral entry is a critical step in human immunodeficiency virus (HIV) infection that takes place through a series of sequential interactions between the envelope glycoprotein gp120, cellular receptor CD4, and coreceptors CCR5 or CXCR4 on T cells. Through a mechanism named trans-infection, dendritic cells (DCs) efficiently transfer the virus to T lymphocytes where viral replication occurs. Interference in HIV transmission in the DC–lymphocyte synapse is a preferential but complex target in the development of new compounds displaying anti-HIV activity. HIV–DC interaction is mediated by the glycans of gp120 and the C-type lectin DC-SIGN (dendritic cell-specific ICAM 3grabbing nonintegrin) expressed on DCs. DC-SIGN is tetrameric and specifically recognizes N-linked high-mannose oligosaccharides (Man9GlcNAc2) through multivalent and Ca -dependent protein–carbohydrate interactions. Mimicking the cluster presentation of the oligomannosides on the surface of the virus is a strategy for designing carbohydrate-based antiviral agents. Mannose multivalent systems based on proteins, peptides, liposomes, and dendrimers as scaffolds have recently been prepared for targeting DCs and tested as inhibitors of DC-SIGN binding to gp120 in ELISA and SPR experiments. Nevertheless, none of these systems has so far been tested in cell-based trans-infection models. Glyconanoparticles (GNPs) are polyvalent, biocompatible sugar-functionalized gold nanoclusters. 10] Glyconanoparticle platforms offer the unique potential for simultaneous incorporation of different ligands in variable densities on a single gold cluster. We have prepared a small library of GNPs (mannoGNPs) coated with sets of different structural motifs of the Nlinked high-mannose undecasaccharide Man9ACHTUNGTRENNUNG(GlcNAc)2 of gp120 (or with an unnatural heptasaccharide) and have observed that GNPs that display multiple copies of the disacchaACHTUNGTRENNUNGride Mana1-2Mana block DC-SIGN/gp120 binding at 120 nm in surface plasmon resonance (SPR) experiments. The mannoGNPs were designed to target DC-SIGN receptors present on DCs by mimicking the clustered carbohydrate display of gp120. In this work we present the results obtained with manno-GNPs (Scheme 1) as inhibitors of DC-SIGN-mediated HIV trans-infection of human activated peripheral blood mononuclear cells (PBMCs). We show that manno-GNPs coated with the linear tetrasaccharide Mana1-2Mana1-2Mana1-3Mana inhibit HIV trans-infection of human T cells similarly to GNPs coated with more complex branched pentaand heptaoligomannosides. Hybrid gold manno-GNPs displaying different densities of linear and branched mannose oligosaccharides were prepared (Scheme 1). The manno-conjugates 6–10 were synthesized by conjugation of ethylamino mannosides 1–5, obtained by the protocol of Wong et al. , with isothiocyanate linker 11 and subsequent removal of the acetyl group (Scheme 1 A). A shorter five-carbon atom aliphatic linker was used for the preparation of 5’-mercaptopentyl b-d-glucopyranoside (GlcC5S), and was hidden internally in order to allow correct presentation of the oligomannosides. Manno-GNPs with 100, 50, or 10 % densities of dimannoside (D-100, D-50, and D-10, respectively), trimannoside (T-50 and T-10), tetramannoside (Te-50 and Te-10), pentamannoside (P-50 and P-10), and heptamannoside (H-50) were prepared from mixtures of manno-conjugates 6–10 and gluco-conjugate GlcC5S by using a previously described methodology (Scheme 1 B). GNPs were characterized by transmission electron microscopy (TEM), H NMR, IR, UV/Visible, and elemental analysis as previously reported. The average number of (oligo)mannosides per GNP and their average molecular weights (Table 1) were calculated from elemental analysis and gold cluster size (1–2 nm as determined by TEM; see the Supporting Information). For trans-infection studies, we used Raji DC-SIGN transfected lymphoblastoid B cells, which can capture and transmit HIV with an efficiency similar to that of monocyte-derived DCs. Manno-GNPs are nontoxic to Raji DC-SIGN and to human activated PBMCs at concentrations of 100 mg mL , as determined by the CellTiter cell viability assay (Figure S9 in the Supporting Information). The activities of manno-GNPs against R5 or X4 HIV-1 were evaluated through an original DC-SIGN transfer assay in which inhibition of HIV-1 infection by GNPs was assessed by use of recombinant viruses carrying the Renilla reporter genes in their genomes. In this assay, inhibition of viral replication is proportional to Renilla-luciferase activity in cell lysates. Briefly, Raji DC-SIGN cells were preincubated with GNPs for 1 h and were then pulsed with JR-Renilla (R5) or NL4.3-Renilla (X4) recombinant viruses for 2 h. Afterwards, the cell cultures were extensively washed and co-cultured with activated PBMCs that would be infected through transfer of the virus bound to DC-SIGN in Raji cells. Viral replication was as[a] Dr. O. Mart nezvila, Dr. M. Marradi, Dr. C. Clavel, Prof. Dr. S. Penad s Laboratory of GlycoNanotechnology, CIC biomaGUNE/CIBER-BBN P8 Miram n 182, 20009 San Sebasti n (Spain) Fax: (+ 34) 943-00-53-01 E-mail : spenades@cicbiomagune.es [b] Dr. L. M. Bedoya, Dr. J. Alcam AIDS Immunopathology Unit, National Center of Microbiology Instituto de Salud Carlos III, Ctra.Majadahonda-Pozuelo Km. 2,200 Madrid (Spain) E-mail : ppalcami@isciii.es [c] Dr. C. Clavel Current address : IBMM, UMR 5247CNRS-Universit s Montpellier 2 et 1 ENSCM, Rue de l’Ecole Normale 8, 34296 Montpellier Cedex 5 (France) Supporting information for this article is available on the WWW under http ://dx.doi. org/10.1002/cbic.200900294.