Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (V) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization.

Cooperation between somatic mutation and germline-encoded residues enables antibody recognition of HIV-1 envelope glycans.

Viral glycoproteins are a primary target for host antibody responses. However, glycans on viral glycoproteins can hinder antibody recognition since they are self glycans derived from the host biosynthesis pathway. During natural HIV-1 infection, neutralizing antibodies are made against glycans on HIV-1 envelope glycoprotein (Env).

Star nanoparticles delivering HIV-1 peptide minimal immunogens elicit near-native envelope antibody responses in nonhuman primates.

Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites on the HIV envelope protein (Env) trimer or on other pathogens. However, the physical characteristics of peptide immunogens can limit their pharmacokinetic and immunological properties. Here, we have designed synthetic "star" nanoparticles based on biocompatible N-[(2-hydroxypropyl)methacrylamide] (HPMA)-based polymer arms extending from a poly(amidoamine) (PAMAM) dendrimer core.

HIV envelope V3 region mimic embodies key features of a broadly neutralizing antibody lineage epitope.

HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide ("Man-V3") for structural studies of an HIV Env third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage ("DH270"), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man-V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs-the conserved GDIR motif and the N332 glycan.

Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide.

A goal for an HIV-1 vaccine is to overcome virus variability by inducing broadly neutralizing antibodies (bnAbs). One key target of bnAbs is the glycan-polypeptide at the base of the envelope (Env) third variable loop (V3). We have designed and synthesized a homogeneous minimal immunogen with high-mannose glycans reflective of a native Env V3-glycan bnAb epitope (Man-V3).

Staged induction of HIV-1 glycan-dependent broadly neutralizing antibodies.

A preventive HIV-1 vaccine should induce HIV-1-specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target.

Vaccine Elicitation of High Mannose-Dependent Neutralizing Antibodies against the V3-Glycan Broadly Neutralizing Epitope in Nonhuman Primates.

Induction of broadly neutralizing antibodies (bnAbs) that target HIV-1 envelope (Env) is a goal of HIV-1 vaccine development. A bnAb target is the Env third variable loop (V3)-glycan site. To determine whether immunization could induce antibodies to the V3-glycan bnAb binding site, we repetitively immunized macaques over a 4-year period with an Env expressing V3-high mannose glycans.

Recognition of synthetic glycopeptides by HIV-1 broadly neutralizing antibodies and their unmutated ancestors.

Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. Broadly neutralizing antibodies (BnAbs) are not induced by current vaccines, but are found in plasma in ∼20% of HIV-1-infected individuals after several years of infection. One strategy for induction of unfavored antibody responses is to produce homogeneous immunogens that selectively express BnAb epitopes but minimally express dominant strain-specific epitopes.

Chemical synthesis of highly congested gp120 V1V2 N-glycopeptide antigens for potential HIV-1-directed vaccines.

Critical to the search for an effective HIV-1 vaccine is the development of immunogens capable of inducing broadly neutralizing antibodies (BnAbs). A key first step in this process is to design immunogens that can be recognized by known BnAbs. The monoclonal antibody PG9 is a BnAb that neutralizes diverse strains of HIV-1 by targeting a conserved carbohydrate-protein epitope in the variable 1 and 2 (V1V2) region of the viral envelope.

An advance in the chemical synthesis of homogeneous N-linked glycopolypeptides by convergent aspartylation.

We describe a useful advance in glycopeptide synthesis. We have developed a one-flask aspartylation/deprotection method, wherein long peptide fragments, bearing proximal pseudoproline functionality are merged with complex glycan domains. Following aspartylation, acidmediated global deprotection reveals the elaborated glycopeptide.

Total synthesis of the α-subunit of human glycoprotein hormones: toward fully synthetic homogeneous human follicle-stimulating hormone.

Described herein is the first total chemical synthesis of the unique α-subunit of the human glycoprotein hormone (α-hGPH). Unlike the biologically derived glycoprotein hormones, which are isolated as highly complex mixtures of glycoforms, α-hGPH obtained by chemical synthesis contains discrete homogeneous glycoforms. Two such systems have been prepared.

A Diels-Alder Route to Angularly Functionalized Bicyclic Structures.

A Diels-Alder based route to trans-fused angularly functionalized bicyclic structures has been developed. This transformation features the use of a tetrasubstituted dienophile in the cycloaddition step.

Cell biology meets biophysics to unveil the different mechanisms of penetratin internalization in cells.

Although cell-penetrating peptides are widely used as molecular devices to cross membranes and transport molecules or nanoparticles inside cells, the underlying internalization mechanism for such behavior is still studied and discussed. One of the reasons for such a debate is the wide panel of chemically different cell-penetrating peptides or cargo that is used. Indeed the intrinsic physico-chemical properties of CPP and conjugates strongly affect the cell membrane recognition and therefore the internalization pathways.

MALDI-TOF mass spectrometry: a powerful tool to study the internalization of cell-penetrating peptides.

This review summarizes the contribution of MALDI-TOF mass spectrometry in the study of cell-penetrating peptide (CPP) internalization in eukaryote cells. This technique was used to measure the efficiency of cell-penetrating peptide cellular uptake and cargo delivery and to analyze carrier and cargo intracellular degradation. The impact of thiol-containing membrane proteins on the internalization of CPP-cargo disulfide conjugates was also evaluated by combining MALDI-TOF MS with simple thiol-specific reactions.

Toward fully synthetic homogeneous beta-human follicle-stimulating hormone (beta-hFSH) with a biantennary N-linked dodecasaccharide. synthesis of beta-hFSH with chitobiose units at the natural linkage sites.

A highly convergent synthesis of the sialic acid-rich biantennary N-linked glycan found in human glycoprotein hormones and its use in the synthesis of a fragment derived from the beta-domain of human Follicle-Stimulating Hormone (hFSH) are described. The synthesis highlights the use of the Sinay radical glycosidation protocol for the simultaneous installation of both biantennary side-chains of the dodecasaccharide as well as the use of glycal chemistry to construct the tetrasaccharide core in an efficient manner. The synthetic glycan was used to prepare the glycosylated 20-27aa domain of the beta-subunit of hFSH under a Lansbury aspartylation protocol.

Modifications in the chemical structure of Trojan carriers: impact on cargo delivery.

Carriers with linear or dendrimeric structures displaying different functional groups were synthesized and their delivery properties were studied.

[Tracking Trojan peptides in cells].

Trojan peptides or cell-penetrating peptides (CPP) are natural or designed peptides identified as cellular membrane-crossing molecules, in particular through their potency to vehiculate various kinds of compounds to the cytoplasm and nucleus of living cells. The indirect methods used so far to detect these peptides in cells led to controversial hypotheses on the mechanism of their cell entry. Therefore, we have developed a MALDI-TOF mass spectrometry-based quantification method to track these peptides inside cells.

Tracking a new cell-penetrating (W/R) nonapeptide, through an enzyme-stable mass spectrometry reporter tag.

We have designed a mass stable reporter (msr) tag with m/z over 500, trifluoroacetyl(alpha,alpha-diethyl)Gly-Lys(Nepsilonbiotin)-(D)Lys-Cys, for the quantification of the uptake and study of the degradation processes of cell-penetrating peptides (CPP), by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This tag was found stable in cell lysis conditions. Using a quantitative MALDI-TOF mass spectrometry analysis based method, an accurate tracking of a new CPP and of its degradation products could be done.

Quantification of the efficiency of cargo delivery by peptidic and pseudo-peptidic Trojan carriers using MALDI-TOF mass spectrometry.

We have measured the efficiencies of two novel pseudo-peptidic carriers and various cell-penetrating peptides (Penetratin, (Arg)9 and the third helix of the homeodomain of Knotted-1) to deliver the same cargo inside cells. The cargo that was studied corresponds to the pseudo-substrate of protein kinase C. Cargo delivery was quantified using a recent method based on isotope labeling and MALDI-TOF MS.