Dissecting the Carbohydrate Specificity of the Anti-HIV‑1 2G12 Antibody by Single-Molecule Force Spectroscopy

Broadly neutralizing anti-HIV-1 monoclonal antibody 2G12 exclusively targets a conserved cluster of high-mannose oligosaccharides present on outer viral envelope glycoprotein gp120. This characteristic makes the otherwise immunogenically “silent” glycan shield of gp120 a tempting target for drug and vaccine design. However, immune responses against carbohydrate-based mimics of gp120 have failed to provide immunization against HIV-1 infection, highlighting the need to understand the molecular events that determine immunogenicity better. In this work, the unbinding kinetics of the gp120–2G12 (<i>k</i>₀ = 0.002 ± 0.09 s^–1, <i>x</i>^⧧ = 1.5 ± 1.2 nm), Man₄–2G12 (<i>k</i>₀ = 0.35 ± 0.32 s^–1, <i>x</i>^⧧ = 0.6 ± 0.2 nm), and Man₅–2G12 interactions were measured by single-molecule force spectroscopy. To our knowledge, this is the first single-molecule study aimed at dissecting the carbohydrate–antibody recognition of the gp120–2G12 interaction. We were able to confirm crystallographic models that show both the binding of the linear Man₄ arm to 2G12 and also the multivalent gp120 glycan binding to 2G12. These results demonstrate that single-molecule force spectroscopy can be successfully used to dissect the molecular mechanisms underlying immunity

Cellular Uptake of Gold Nanoparticles Bearing HIV gp120 Oligomannosides

Dendritic cells are the most potent of the professional antigen-presenting cells which display a pivotal role in the generation and regulation of adaptive immune responses against HIV-1. The migratory nature of dendritic cells is subverted by HIV-1 to gain access to lymph nodes where viral replication occurs. Dendritic cells express several calcium-dependent C-type lectin receptors including dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN), which constitute a major receptor for HIV-1. DC-SIGN recognizes <i>N</i>-linked high-mannose glycan clusters on HIV gp120 through multivalent and Ca²⁺-dependent protein–carbohydrate interactions. Therefore, mimicking the cluster presentation of oligomannosides from the virus surface is a strategic approach for carbohydrate-based microbicides. We have shown that gold nanoparticles (<i>manno</i>GNPs) displaying multiple copies of structural motifs (di-, tri-, tetra-, penta-, or heptaoligomanosides) of the <i>N</i>-linked high-mannose glycan of viral gp120 are efficient inhibitors of DC-SIGN-mediated <i>trans-</i>infection of human T cells. We have now prepared the corresponding fluorescent-labeled glyconanoparticles (FITC-<i>manno</i>GNPs) and studied their uptake by DC-SIGN expressing Burkitt lymphoma cells (Raji DC-SIGN cell line) and monocyte-derived immature dendritic cells (iDCs) by flow cytometry and confocal laser scanning microscopy. We demonstrate that the 1.8 nm oligomannoside coated nanoparticles are endocytosed following both DC-SIGN-dependent and -independent pathways and part of them colocalize with DC-SIGN in early endosomes. The blocking and sequestration of DC-SIGN receptors by <i>manno</i>GNPs could explain their ability to inhibit HIV-1 <i>trans</i>-infection of human T cells <i>in vitro</i>

⁶⁸Ga-Labeled Gold Glyconanoparticles for Exploring Blood–Brain Barrier Permeability: Preparation, Biodistribution Studies, and Improved Brain Uptake via Neuropeptide Conjugation

New tools and techniques to improve brain visualization and assess drug permeability across the blood–brain barrier (BBB) are critically needed. Positron emission tomography (PET) is a highly sensitive, noninvasive technique that allows the evaluation of the BBB permeability under normal and disease-state conditions. In this work, we have developed the synthesis of novel water-soluble and biocompatible glucose-coated gold nanoparticles (GNPs) carrying BBB-permeable neuropeptides and a chelator of the positron emitter ⁶⁸Ga as a PET reporter for in vivo tracking biodistribution. The small GNPs (2 nm) are stabilized and solubilized by a glucose conjugate. A NOTA ligand is the chelating agent for the ⁶⁸Ga, and two related opioid peptides are used as targeting ligands for improving BBB crossing. The radioactive labeling of the GNPs is completed in 30 min at 70 °C followed by purification via centrifugal filtration. As a proof of principle, a biodistribution study in rats is performed for the different ⁶⁸Ga-GNPs. The accumulation of radioactivity in different organs after intravenous administration is measured by whole body PET imaging and gamma counter measurements of selected organs. The biodistribution of the ⁶⁸Ga-GNPs varies depending on the ligands, as GNPs with the same gold core size show different distribution profiles. One of the targeted ⁶⁸Ga-GNPs improves BBB crossing near 3-fold (0.020 ± 0.0050% ID/g) compared to nontargeted GNPs (0.0073 ± 0.0024% ID/g) as measured by dissection and tissue counting

Galactofuranose-Coated Gold Nanoparticles Elicit a Pro-inflammatory Response in Human Monocyte-Derived Dendritic Cells and Are Recognized by DC-SIGN

Galactofuranose (Gal<i>f</i>) is the five-membered ring form of galactose exclusively found in nonmammalian species, among which several are pathogens. To determine the putative role of this carbohydrate in host–pathogen interactions, we synthesized multivalent gold nanoparticles carrying Gal<i>f</i> (Gal<i>f</i>-GNPs) and show that they are recognized by the EB-A2 antibody, which is widely used to detect Gal<i>f</i>-containing galactomannan in the serum of Aspergillosis patients. We demonstrated that human monocyte-derived dendritic cells bound Gal<i>f</i>-GNPs via interaction with the lectin DC-SIGN. Moreover, interaction of dendritic cells with Gal<i>f</i>-GNPs resulted in increased expression of several maturation markers on these cells and induced secretion of the pro-inflammatory cytokines IL-6 and TNF-α. These data indicate that Gal<i>f</i> is able to modulate the innate immune response via dendritic cells. In conclusion, Gal<i>f</i>-GNPs are a versatile tool that can be applied in multiple functional studies to gain a better understanding of the role of Gal<i>f</i> in host–pathogen interaction

Residual CTAB Ligands as Mass Spectrometry Labels to Monitor Cellular Uptake of Au Nanorods

Gold nanorods have numerous applications in biomedical research, including diagnostics, bioimaging, and photothermal therapy. Even though surfactant removal and surface conjugation with antifouling molecules such as polyethylene glycol (PEG) are required to minimize nonspecific protein binding and cell uptake, the reliable characterization of these processes remains challenging. We propose here the use of laser desorption/ionization mass spectrometry (LDI-MS) to study the ligand exchange efficiency of cetyltrimethylammonium bromide (CTAB)-coated nanorods with different PEG grafting densities and to characterize nanorod internalization in cells. Application of LDI-MS analysis shows that residual CTAB consistently remains adsorbed on PEG-capped Au nanorods. Interestingly, such residual CTAB can be exploited as a mass barcode to discern the presence of nanorods in complex fluids and in vitro cellular systems, even at very low concentrations

Negatively Charged Glyconanoparticles Modulate and Stabilize the Secondary Structures of a gp120 V3 Loop Peptide: Toward Fully Synthetic HIV Vaccine Candidates

The third variable region (V3 peptide) of the HIV-1 gp120 is a major immunogenic domain of HIV-1. Controlling the formation of the immunologically active conformation is a crucial step to the rational design of fully synthetic candidate vaccines. Herein, we present the modulation and stabilization of either the α-helix or β-strand conformation of the V3 peptide by conjugation to negatively charged gold glyconanoparticles (GNPs). The formation of the secondary structure can be triggered by the variation of the buffer concentration and/or pH as indicated by circular dichoism. The peptide on the GNPs shows increased stability toward peptidase degradation as compared to the free peptide. Moreover, only the V3β-GNPs bind to the anti-V3 human broadly neutralizing mAb 447-52D as demonstrated by surface plasmon resonance (SPR). The strong binding of V3β-GNPs to the 447-52D mAb was the starting point to address its study as immunogen. V3β-GNPs elicit antibodies in rabbits that recognize a recombinant gp120 and the serum displayed low but consistent neutralizing activity. These results open up the way for the design of new fully synthetic HIV vaccine candidates

Effective Targeting of DC-SIGN by α‑Fucosylamide Functionalized Gold Nanoparticles

Dendritic Cells (DCs), the most potent antigen-presenting cells, play a critical role in the detection of invading pathogens, which are recognized also by multiple carbohydrate-specific receptors. Among them, DC-SIGN is one of the best characterized, with high-mannose and Lewis-type glycan specificity. In this study, we present a potent DC-SIGN targeting device developed using gold nanoparticles functionalized with α-fucosyl-β-alanyl amide. The nanoparticles bound to cellular DC-SIGN and induced internalization as effectively as similar particles coated with comparable amounts of Lewis^X oligosaccharide. They were found to be neutral toward dendritic cell maturation and IL-10 production, thus envisaging a possible use as targeted imaging tools and antigen delivery devices