Neutralising properties of peptides derived from CXCR4 extracellular loops towards CXCL12 binding and HIV-1 infection.

The chemokine receptor CXCR4 interacts with a single endogenous chemokine, CXCL12, and regulates a wide variety of physiological and pathological processes including inflammation and metastasis development. CXCR4 also binds the HIV-1 envelope glycoprotein, gp120, resulting in viral entry into host cells. Therefore, CXCR4 and its ligands represent valuable drug targets. In this study, we investigated the inhibitory properties of synthetic peptides derived from CXCR4 extracellular loops (ECL1-X4, ECL2-X4 and ECL3-X4) towards HIV-1 infection and CXCL12-mediated receptor activation. Among these peptides, ECL1-X4 displayed anti-HIV-1 activity against X4, R5/X4 and R5 viruses (IC50=24 to 76µM) in cell viability assay without impairing physiological CXCR4-CXCL12 signalling. In contrast, ECL2-X4 only inhibited X4 and R5/X4 strains, interfering with HIV-entry into cells. At the same time, ECL2-X4 strongly and specifically interacted with CXCL12, blocking its binding to CXCR4 and its second receptor, CXCR7 (IC50=20 and 100µM). Further analysis using mutated and truncated peptides showed that ECL2 of CXCR4 forms multiple contacts with the gp120 protein and the N-terminus of CXCL12. Chemokine neutralisation was mainly driven by four aspartates and the C-terminal residues of ECL2-X4. These results demonstrate that ECL2 represents an important structural determinant in CXCR4 activation. We identified the putative site for the binding of CXCL12 N-terminus and provided new structural elements to explain the recognition of gp120 and dimeric CXCR4 ligands.

Development of PLGA-mannosamine nanoparticles as oral protein carriers.

Here we report the development of polymeric nanoparticles, made of poly(lactide-co-glycolide) (PLGA) chemically modified with mannosamine (MN), intended to specifically interact with the intestinal mucosa and facilitate the intestinal transport of proteins. PLGA-MN nanoparticles displayed nanometric size and a negative zeta potential, which was lower than that of the PLGA nanoparticles. This correlate well with the preferential location of the MN group on the nanoparticles surface obtained by X-ray photoelectron spectroscope (XPS). The presence of MN groups in the polymer chain led to a different surface morphology noted by SEM, an increase of the encapsulation of model proteins, and to help stabilizing the nanoparticles in simulated intestinal fluids. Furthermore, the MN modification significantly enhanced the nanoparticle's interaction with the epithelial cells in human intestinal follicle-associated epithelium cell culture model. Overall, the MN modification significantly modifies the properties of PLGA nanoparticles making them more suitable as nanocarriers for oral protein delivery.

Acylated and unacylated ghrelin binding to membranes and to ghrelin receptor: towards a better understanding of the underlying mechanisms.

The O-octanoylation of human ghrelin is a natural post-translational modification that enhances its binding to model membranes and could potentially play a central role in ghrelin biological activities. Here, we aimed to clarify the mechanisms that drive ghrelin to the membrane and hence to its receptor that mediates most of its endocrinological effects. As the acylation enhances ghrelin lipophilicity and that ghrelin contains many basic residues, we examined the electrostatic attraction and/or hydrophobic interactions with membranes. Using various liposomes and buffer conditions in binding, zeta potential and isothermal titration calorimetry studies, we found that whereas acylated and unacylated ghrelin were both electrostatically attracted towards the membrane, only acylated ghrelin penetrated into the headgroup and the lipid backbone regions of negatively charged membranes. The O-acylation induced a 120-fold increase in ghrelin local concentration in the membrane. However, acylated ghrelin did not deeply penetrate the membrane nor did it perturb its organisation. Conformational studies by circular dichroism and attenuated total reflection Fourier transformed infrared as well as in silico modelling revealed that both forms of ghrelin mainly adopted the same structure in aqueous, micellar and bilayer environments even though acylated ghrelin structure is slightly more α-helical in a lipid bilayer environment. Altogether our results suggest that membrane acts as a "catalyst" in acylated ghrelin binding to the ghrelin receptor and hence could explain why acylated and unacylated ghrelin are both full agonists of this receptor but in the nanomolar and micromolar range, respectively.

CD32+CD4+ memory T cells are enriched for total HIV-1 DNA in tissues from humanized mice.

CD32 has raised conflicting results as a putative marker of the HIV-1 reservoir. We measured CD32 expression in tissues from viremic and virally suppressed humanized mice treated relatively early or late after HIV-1 infection with combined antiretroviral therapy. CD32 was expressed in a small fraction of the memory CD4+ T-cell subsets from different tissues in viremic and aviremic mice, regardless of treatment initiation time. CD32+ memory CD4+ T cells were enriched in cell-associated (CA) HIV-1 DNA but not in CA HIV-1 RNA as compared to the CD32-CD4+ fraction. Using multidimensional reduction analysis, several memory CD4+CD32+ T-cell clusters were identified expressing HLA-DR, TIGIT, or PD-1. Importantly, although tissue-resident CD32+CD4+ memory cells were enriched with translation-competent reservoirs, most of it was detected in memory CD32-CD4+ T cells. Our findings support that CD32 labels highly activated/exhausted memory CD4+ T-cell subsets that contain only a small proportion of the translation-competent reservoir.

Development of Mimokines, chemokine N terminus-based CXCR4 inhibitors optimized by phage display and rational design.

The chemokine receptor CXCR4 (C-X-C chemokine receptor type 4 also known as fusin or CD184 (cluster of differentiation 184)) is implicated in various biological and pathological processes of the hematopoietic and immune systems. CXCR4 is also one of the major coreceptors for HIV-1 entry into target cells and is overexpressed in many cancers, supporting cell survival, proliferation, and migration. CXCR4 is thus an extremely relevant drug target. Among the different strategies to block CXCR4, chemokine-derived peptide inhibitors hold great therapeutic potential. In this study, we used the N-terminus of vCCL2/vMIPII, a viral CXCR4 antagonist chemokine, as a scaffold motif to engineer and select CXCR4 peptide inhibitors, called Mimokines, which imitate the chemokine-binding mode but display an enhanced receptor affinity, antiviral properties, and receptor selectivity. We first engineered a Mimokine phage displayed library based on the first 21 residues of vCCL2, in which cysteine 11 and 12 were fully randomized and screened it against purified CXCR4 stabilized in liposomes. We identified Mimokines displaying up to 4-fold higher affinity for CXCR4 when compared to the reference peptide and fully protected MT-4 cells against HIV-1 infection. These selected Mimokines were then subjected to dimerization, D-amino acid, and aza-ß3-amino acid substitution to further enhance their potency and selectivity. Optimized Mimokines exhibited up to 120-fold enhanced CXCR4 binding (range of 20 nM) and more than 200-fold improved antiviral properties (≤ 1 µM) compared to the parental Mimokines. Interestingly, these optimized Mimokines also showed up to 25-fold weaker affinity for ACKR3/CXCR7 and may therefore serve as lead compounds for further development of more selective CXCR4 peptide inhibitors and probes.

An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells.

An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. The inverted model appears more physiologic, functional and reproducible than the normally oriented model.

Human CD56dimCD16dim Cells As an Individualized Natural Killer Cell Subset.

Human natural killer (NK) cells can be subdivided in several subpopulations on the basis of the relative expression of the adhesion molecule CD56 and the activating receptor CD16. Whereas blood CD56brightCD16dim/- NK cells are classically viewed as immature precursors and cytokine producers, the larger CD56dimCD16bright subset is considered as the most cytotoxic one. In peripheral blood of healthy donors, we noticed the existence of a population of CD56dimCD16dim NK cells that was frequently higher in number than the CD56bright subsets and even expanded in occasional control donors but also in transporter associated with antigen processing-deficient patients, two familial hemophagocytic lymphohistiocytosis type II patients, and several common variable immunodeficiency patients. This population was detected but globally reduced in a longitudinal cohort of 18 HIV-1-infected individuals. Phenotypically, the new subset contained a high percentage of relatively immature cells, as reflected by a significantly stronger representation of NKG2A+ and CD57- cells compared to their CD56dimCD16bright counterparts. The phenotype of the CD56dimCD16dim population was differentially affected by HIV-1 infection as compared to the other NK cell subsets and only partly restored to normal by antiretroviral therapy. From the functional point of view, sorted CD56dimCD16dim cells degranulated more than CD56dimCD16bright cells but less than CD56dimCD16- NK cells. The population was also identified in various organs of immunodeficient mice with a human immune system ("humanized" mice) reconstituted from human cord blood stem cells. In conclusion, the CD56dimCD16dim NK cell subpopulation displays distinct phenotypic and functional features. It remains to be clarified if these cells are the immediate precursors of the CD56dimCD16bright subset or placed somewhere else in the NK cell differentiation and maturation pathway.

Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach.

Peptides and proteins remain poorly bioavailable upon oral administration. One of the most promising strategies to improve their oral delivery relies on their association with colloidal carriers, e.g. polymeric nanoparticles, stable in gastrointestinal tract, protective for encapsulated substances and able to modulate physicochemical characteristics, drug release and biological behavior. The mechanisms of transport of these nanoparticles across intestinal mucosa are reviewed. In particular, the influence of size and surface properties on their non-specific uptake or their targeted uptake by enterocytes and/or M cells is discussed. Enhancement of their uptake by appropriate cells, i.e. M cells by (i) modeling surface properties to optimize access to and transport by M cells (ii) identifying surface markers specific to human M cell allowing targeting to M cells and nanoparticles transcytosis is illustrated. Encouraging results upon in vivo testing are reported but low bioavailability and lack of control on absorbed dose slow down products development. Vaccines are certainly the most promising applications for orally delivered nanoparticles.

Dysfunctions in the migratory phenotype and properties of circulating immature transitional B cells during HIV-1 infection.

OBJECTIVE: The frequency of immature transitional B cells is increased in blood of HIV-1-infected individuals. We investigated whether HIV-1 infection affects expression and function of chemokine receptors important for egress of immature transitional B cells from bone marrow and migration to lymphoid organs. DESIGN: This is a cross-sectional study analysing the migratory phenotype and function of immature transitional B cells in HIV-1-infected individuals, in relation to antiretroviral treatment and age. METHODS: Frequency of blood immature transitional B cells and their phenotypic characteristics, including chemokine receptors and a maturation marker, were determined by immunostainings. Migratory capacities were studied in a migration assay. RESULTS: The increased frequency of immature transitional B cells in untreated HIV-1 infection was normalized in patients receiving antiretroviral treatment; in our cohorts, age did not have an impact on the frequency of circulating immature transitional B cells. Immature transitional B cells from nontreated patients expressed low levels of CD21 molecule. We found an elevated frequency of CXCR3 and CXCR4 expressing immature transitional B cells in treated and nontreated patients. CXCR4 receptor was unresponsive to CXCL12 ligand in in-vitro migration and internalization assays. In addition, CXCR5 expression was downregulated on immature transitional B cells from infected patients, and these cells migrated poorly in response to CXCR5 ligand. CONCLUSION: Circulating immature transitional B cells from HIV-1-infected patients are not fully mature, probably due to premature egress from bone marrow; these cells showed a phenotype which could impair entry into secondary lymphoid organs. Changes in migratory capacity of immature transitional B cells may affect B-cell maturation during HIV-1 infection.

Increased extrafollicular expression of the B-cell stimulatory molecule CD70 in HIV-1-infected individuals.

OBJECTIVE: CD70 molecules expressed by activated T cells provide potent B cell stimulatory signals. We hypothesized that an altered CD70 expression might contribute to B cell abnormalities during HIV-1 infection. DESIGN: CD70 expression and the functional and migratory properties of the CD4CD70 T lymphocytes were analyzed in HIV-1-infected patients and in humanized mice. Correlations were tested between CD70 expression and features of B-cell activation, apoptosis sensitivity and functional exhaustion. METHODS: CD4CD70 T cells were analyzed in cohorts of CD4 T-cell lymphopenic, viremic or nonlymphopenic, nonviremic HIV-1-infected patients and in noninfected individuals. CD70 upregulation was also followed in HIV-1-infected humanized mice. CD38, CD95, LAIR1 and PD-1 expressions were monitored on B-cell subpopulations, Ki67 was assessed to estimate B-cell proliferation and antibody levels were measured in plasma. RESULTS: Blood CD4CD70 T-cell frequencies increased in response to CD4 T-cell depletion or high viremia levels as a possible consequence of increased activation and proliferation in this subset. CD4CD70 T cells produced T-helper 1-type cytokines and expressed chemokine receptors mobilizing toward sites of inflammation but not to lymphoid follicles. High CD70 expression was observed in HIV-1-infected humanized mice at extrafollicular sites (peritoneum, bone-marrow). CD4CD70 T-cell frequencies correlated with the expression of the activation marker CD38 and the death receptor CD95 on various memory B-cell subsets, with B-cell proliferation and with plasma IgG levels. CONCLUSIONS: CD4CD70 T cells may contribute to B cell hyperactivation and accelerated memory B-cell turnover during HIV-1 infection.

Function, diversity and therapeutic potential of the N-terminal domain of human chemokine receptors.

Chemokines and their receptors play fundamental roles in many physiological and pathological processes such as leukocyte trafficking, inflammation, cancer and HIV-1 infection. Chemokine-receptor interactions are particularly intricate and therefore require precise orchestration. The flexible N-terminal domain of human chemokine receptors has regularly been demonstrated to hold a crucial role in the initial recognition and selective binding of the receptor ligands. The length and the amino acid sequences of the N-termini vary considerably among different receptors but they all show a high content of negatively charged residues and are subject to post-translational modifications such as O-sulfation and N- or O-glycosylation. In addition, a conserved cysteine that is most likely engaged in a receptor-stabilizing disulfide bond delimits two functionally distinct parts in the N-terminus, characterized by specific molecular signatures. Structural analyses have shown that the N-terminus of chemokine receptors recognizes a groove on the chemokine surface and that this interaction is stabilized by high-affinity binding to a conserved sulfotyrosine-binding pocket. Altogether, these data provide new insights on the chemokine-receptor molecular interplay and identify the receptor N-terminus-binding site as a new target for the development of therapeutic molecules. This review presents and discusses the diversity and function of human chemokine receptor N-terminal domains and provides a comprehensive annotated inventory of their sequences, laying special emphasis on the presence of post-translational modifications and functional features. Finally, it identifies new molecular signatures and proposes a computational model for the positioning and the conformation of the CXCR4 N-terminus grafted on the first chemokine receptor X-ray structure.

Engineering and screening the N-terminus of chemokines for drug discovery.

Chemokines are small chemoattractive proteins involved in many important physiological and pathological processes such as leukocyte mobilisation, inflammation, cancer and HIV-1 infection. The N-terminus of chemokines was shown to be crucial for interaction and activation with their cognate receptors. Therefore, multiple strategies including elongation, truncation, mutagenesis or chemical modifications of chemokine N-terminus were developed to identify analogues with modified selectivity displaying antagonist or enhanced agonist activities. Library approaches allowed fast screening of a large number of such chemokine variants and led to the identification of promising therapeutic candidates. Additional studies were able to reduce the chemokine to the size of a peptide while retaining its receptor affinity and selectivity. In analogy to full length chemokines, peptides derived from the chemokine N-terminal sequence were improved by mutagenesis, elongation and truncation approaches to develop potential therapeutic molecules used in various clinical trials. Altogether these studies demonstrated the pharmacophore potential of the chemokine N-terminus and its vast modulation properties to develop analogues with great therapeutic value for a large set of pathologies.

Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination.

The presence of RGD on nanoparticles allows the targeting of beta1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of PCL-PEG and incorporated in PLGA-based nanoparticles. RGD and RGDp significantly increased the transport of nanoparticles across an in vitro model of human M cells as compared to enterocytes. RGD, LDVp, LDVd and mannose enhanced nanoparticle uptake by macrophages in vitro. The intraduodenal immunization with RGDp-, LDVd- or mannose-labeled nanoparticles elicited a higher production of IgG antibodies than the intramuscular injection of free ovalbumin or intraduodenal administration of either non-targeted or RGD-nanoparticles. Targeted formulations were also able to induce a cellular immune response. In conclusion, the in vitro transport of nanoparticles, uptake by macrophages and the immune response were positively influenced by the presence of ligands at the surface of nanoparticles. These targeted-nanoparticles could thus represent a promising delivery system for oral immunization.

Helodermin-loaded nanoparticles: characterization and transport across an in vitro model of the follicle-associated epithelium.

M cells represent a potential portal for oral delivery of peptides and proteins due to their high endocytosis abilities. An in vitro model of human FAE (co-cultures) was used to evaluate the influence of M cells on the transport of free and encapsulated helodermin--a model peptide--across the intestinal epithelium. M cells enhanced transport of intact helodermin (18-fold, Papp=3 x 10(-6) cm s(-1)). As pegylation increased nanoparticle transport by M cells, helodermin was encapsulated in 200 nm nanoparticles containing PEG-b-PLA:PLGA 1:1. Stability of the selected formulation was demonstrated in simulated gastric and intestinal fluids. M cells increased the transport of helodermin encapsulated in these nanoparticles by a factor of 415, as compared to Caco-2 cells. Transport of free and encapsulated helodermin occurred most probably by endocytosis. In conclusion, M cells improved helodermin transport across the intestinal epithelium, confirming their high potential for oral delivery of peptides.

PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination.

To improve the efficiency of orally delivered vaccines, PEGylated PLGA-based nanoparticles displaying RGD molecules at their surface were designed to target human M cells. RGD grafting was performed by an original method called "photografting" which covalently linked RGD peptides mainly on the PEG moiety of the PCL-PEG, included in the formulation. First, three non-targeted formulations with size and zeta potential adapted to M cell uptake and stable in gastro-intestinal fluids, were developed. Their transport by an in vitro model of the human Follicle associated epithelium (co-cultures) was largely increased as compared to mono-cultures (Caco-2 cells). RGD-labelling of nanoparticles significantly increased their transport by co-cultures, due to interactions between the RGD ligand and the beta(1) intregrins detected at the apical surface of co-cultures. In vivo studies demonstrated that RGD-labelled nanoparticles particularly concentrated in M cells. Finally, ovalbumin-loaded nanoparticles were orally administrated to mice and induced an IgG response, attesting antigen ability to elicit an immune response after oral delivery.