Estelle Loing

Systemic immune responses induced by mucosal administration of lipopeptides without adjuvant

We recently reported that parenteral injection of malaria palmitoyl‐tailed peptides without adjuvant efficiently induces B, Th and CTL responses. We now show that intranasal (IN) or sub‐lingual (SL) delivery of such lipopeptides induces strong systemic immune responses, as demonstrated by specific Th cell responses from the spleen as well as inguinal lymph nodes, and by the production ofhigh levels of serum antibodies. Overall, both types of responses were significantly higher than in parallel experiments in which the same lipopeptides were delivered by the subcutaneous (s.c.) route. Moreover, the mucosal route resulted in the preferential induction of IFN‐γ producing T cells and of IgG2a antibody production, as compared to the dominant IL‐4 and IgG1 responses obtained by the s.c. route, thus bringing a distinct advantage in the field of many infectious diseases and allergy. Possibly related to this Th1 response, we found that dendritic cells, the principal immune‐competent cells to encounter antigens within mucosal membranes, take up lipopeptide antigens more efficiently than macrophages. Mucosal immunization by lipidated peptides appears therefore as a novel, noninvasive vaccine approach that does not require the use of extraneous adjuvant and which, besides cost‐effectiveness, has attractive practical and immunological features.

Endocytosis of an HIV-derived lipopeptide into human dendritic cells followed by class I-restricted CD8+ T lymphocyte activation

CD8+ T lymphocytes, which are major immune effectors, require primary stimulation by dendritic cells (DC) presenting MHC class I molecule‐bound epitopes. Sensitization to exogenous protein epitopes that are not synthesized in DC, such as cross‐priming, is obtained through pathways leading to their association with MHC class I. To follow class I‐restricted pathways in human DC, we have tracked a lipopeptide derived from the conserved HLA‐A*0201‐restricted HIV‐1 reverse transcriptase 476‐484 epitope, by N‐terminal addition of an Nϵ‐palmytoyl‐lysine. Indeed, lipopeptides elicit cytotoxic responses from CD8+ T lymphocytes, whereas peptides without a lipid moiety do not. The lipopeptide and its parent peptide were labeled unequivocally by rhodamine to study their entry into immature monocyte‐derived human DC by confocal microscopy. The lipid moiety induced endocytosis of the lipopeptide, assessed by rapid entry into vesicles, colocalization with Dextran‐FITC and dependence on energy. Internalization occurred even when actin filaments were depolymerized by Cytochalasin B. This internalization induced functional stimulation of specific CD8+ T lymphocytes in IFN‐γ ELISPOT assays. The peptide alone was not visualized inside the DC and was presented through direct surface association to HLA‐A*0201. Therefore, lipopeptides are a unique opportunity to define precisely the pathways that lead exogenous proteins to associate with MHC class I molecules in DC. The results will also be useful to design lipopeptide vaccines.

Extension of HLA-A*0201-Restricted Minimal Epitope by Nε-Palmitoyl-lysine Increases the Life Span of Functional Presentation to Cytotoxic T Cells

The delineation of the minimal requirements for efficient delivery of functional cytotoxic epitopes into APC could be a step toward the definition of “minimal length” lipopeptides for the modulation of CTL activity. Several analogues of the HLA-A*0201-restricted HIV-1 polymerase (pol476–484) minimal cytotoxic epitope were obtained by modifying P0, P1, or P10 positions by a single Nε-palmitoyl-lysine residue. The use of fluorescent derivatives confirmed the cell-permeating activities and suggested that a P0- and a P1-modified lipopeptide possessing ionizable extremities fulfills the structural requirements for MHC loading. The expressions of HLA-peptide complexes at the surface of TAP-deficient cells incubated with the parent epitope or lipopeptide derivatives were compared, in terms of intensity and stability. Both lipopeptides induced a considerably prolonged expression of conformationally correct complexes, which were dependent on the integrity of the exocytosis pathway, suggesting a dynamic mechanism of formation or reloading of the complexes from an intracellular pool. The agonistic activities of the different HLA-peptide complexes were evaluated using two independent T cell lines from HIV-infected donors. We report that a lipodecapeptide obtained by N-terminal addition of a Nε-palmitoyl-lysine to the pol476–484 epitope was able to increase the life span of functional presentation to cytotoxic T cells specific for the parent peptide.

Two Human Immunodeficiency Virus Vaccinal Lipopeptides Follow Different Cross-Presentation Pathways in Human Dendritic Cells

ABSTRACT An efficient vaccine against human immunodeficiency virus (HIV) must induce good cellular immune responses. To do this, it must be processed and presented by dendritic cells, which are required for primary T-lymphocyte stimulation. We have previously shown that a model lipopeptide containing a short epitopic peptide from HIV-1 was endocytosed and presented in association with major histocompatibility complex class I molecules by human dendritic cells to specific CD8+ T lymphocytes, but the cross-presentation pathway needed to be precisely determined. We have studied a longer lipopeptide (Pol461-484) and another lipopeptide (Nef66-97) currently being used in vaccine trials. Like the shorter lipopeptide, the rhodamine-labeled Pol461-484 lipopeptide was internalized by endocytosis, as assessed by confocal microscopy. The lipopeptides were processed by dendritic cells and presented to CD8+ T cells specific for the HLA-A*0201-restricted Pol476-484 and the HLA-A*0301-restricted Nef73-82 epitope, respectively. Presentation of both lipopeptides was inhibited by brefeldin A. Presentation of the Pol lipopeptide was inhibited by epoxomycin, a proteasome-specific inhibitor, but not by monensin. This shows that it gained access to the cytosol to be digested by the proteasome. In contrast, presentation of the Nef lipopeptide was not inhibited by epoxomycin but was inhibited by monensin, a classical inhibitor of acid-dependent endosomal enzyme activity, indicating an endocytic processing pathway yielding to major histocompatibility complex class I-restricted presentation. Therefore, the two lipopeptides followed different cross-presentation pathways, both resulting in efficient presentation to CD8+ T lymphocytes.

Lipopeptide presentation pathway in dendritic cells

Lipopeptides are currently being evaluated as candidate vaccines in human volunteers. They elicit cytotoxic responses from CD8(+) T lymphocytes, whereas peptides without a lipidic moiety usually do not. The exact processing and presentation pathways leading to association with MHC class I molecules has not yet been defined. This is of particular interest in dendritic cells, which are required for primary T cell stimulation. We have tracked lipopeptides derived from an HLA-A2.1-restricted HIV-1 Reverse Transcriptase epitope, by N-terminal addition of an N-epsilon-palmitoyl-lysine. Entry of the lipopeptides into human monocyte-derived dendritic cells (MDC) was mediated by endocytosis, as assessed by colocalization using analogs labelled with rhodamine, and by confocal microscopy. This internalization in DC induced functional stimulation of CD8(+) T lymphocytes specific for the epitopes, quantified by Interferon-gamma ELISPOT assays. The peptide alone was not visualized inside the DC and was only presented through direct surface association to HLA-A*0201. Therefore, lipopeptides provide a model system to define precisely the cross-presentation pathways that lead exogenous proteins to associate with class I MHC molecules within dendritic cells. Using this approach, cross-presentation pathways can be better defined and vaccine lipopeptides can be further optimized for MHC class I association in human dendritic cells.

Novel Hyperbranched Glycomimetics Recognized by the Human Mannose Receptor: Quinic or Shikimic Acid Derivatives as Mannose Bioisosteres

The mannose receptor mediates the internalization of a wide range of molecules or microorganisms in a pattern recognition manner. Therefore, it represents an attractive entry for specific drug, gene, or antigen delivery to macrophages and dendritic cells. In an attempt to design novel effective synthetic mannose receptor ligands, quinic and shikimic acid were selected as putative mannose mimics on the basis of X‐ray crystallographic data from the related rat mannose‐binding lectin. As the mannose receptor preferentially binds to molecules displaying several sugar residues, fluorescein‐labeled cluster quinic and shikimic acid derivatives with valencies of two to eight were synthesized. Their mannose receptor mediated uptake was assayed on monocyte‐derived human dendritic cells by cytofluorimetric analysis. Mannose‐receptor specificity was further assessed by competitive inhibition assays with mannan, by confocal microscopy analysis, and by expression of the mannose receptor in transfected Cos‐1 cells. Constructs derived from both quinic and shikimic acid were efficiently recognized by the mannose receptor with an optimum affinity for the molecules with a valency of four. As a result, commercially available quinic and shikimic acids appear as stable mannose bioisosteres, which should prove valuable tools for specific cell delivery.

Synthesis of hydrazinopeptides using solid phase N-amination. Application to chemical ligation

Abstract A hydrazinopeptide was synthesized using the solid phase N-electrophilic amination with N-Boc-3-(4-cyanophenyl)oxaziridine and we describe its reactivity towards cyclohexanecarboxaldehyde.

Direct evidence of cytoplasmic delivery of PKC-α, -ϵ and -ζ pseudosubstrate lipopeptides: study of their implication in the induction of apoptosis

Protein kinases C (PKC) are serine/threonine kinase enzymes involved in the mechanism of cell survival. Their pseudosubstrate sequences are autoinhibitory domains, which maintain the enzyme in an inactive state in the absence of allosteric activators, thus representing an attractive tool for the modulation of different PKC isoforms. Here, we report the use of palmitoylated modified PKC‐α, ‐ϵ, and ‐ζ pseudosubstrate peptides, and determine their intracellular distribution together with their respective PKC isoenzymes. Finally, we propose that the differential distribution of the peptides is correlated with a selective induction of apoptosis and therefore argues for different involvement of PKC isoforms in the anti‐apoptotic program.

Grafting of synthetic mannose receptor-ligands onto onion vectors for human dendritic cells targetingElectronic supplementary information (ESI) available: full experimental details. See http://www.rsc.org/suppdata/cc/b2/b206980f/

A practical preparation of onion vesicles targeted to dendritic cells involves the grafting of mannose-mimetic clusters, bearing a hydrazino group, onto the surface of onion vesicles containing an aldehyde functionalized lipid.

A novel dominant-negative mutant form of Epstein–Barr virus latent membrane protein-1 (LMP1) selectively and differentially impairs LMP1 and TNF signaling pathways

The latent membrane protein-1 (LMP1) is an integral membrane molecule expressed by Epstein–Barr virus (EBV) during viral latency and displays properties of a constitutively activated member of the TNF receptor family. LMP1 is required for B-cell or monocyte immortalization induced by EBV and is sufficient to transform rodent fibroblasts. Transforming potential of LMP1 is mediated by its cytoplasmic C-terminal domain, which activates various cellular signaling pathways including NFκB and JNK. In this report, we constructed mutants of LMP1 with preserved membrane spanning domain but mutated in the C-terminal domain and a second truncated C-terminal LMP1 fused to the enhanced green fluorescent protein. This latter mutant, termed LMP1-CT, impairs signaling by ectopic LMP1 as well as endogenous EBV-expressed wild-type (wt) LMP1. In contrast to dominant-negative mutants of LMP1 with preserved membrane spanning domains, LMP1-CT was unable to bind wt LMP1 to form an inactive complex. Its dominant-negative effects were due to binding and sequestration of LMP1 adapters TRAF2 and TRADD as assessed by coimmunoprecipitation experiments and confocal analysis. The effect was selective since LMP1-CT did not inhibit IL-1β-induced signaling, whereas it impaired TNF-triggered NFκB and JNK signals without affecting TNF-induced apoptosis. In addition and in contrast to LMP1 constructs with membrane localization, LMP-CT did not display cytostatic properties in noninfected cells. Importantly, LMP1-CT inhibited survival induced by LMP1 in an EBV-transformed T-cell line expressing the type II viral latency commonly found in the majority of EBV-associated human tumors. These data demonstrate that LMP1-CT is a new tool to explore the differences between LMP1 and TNF signaling and may facilitate the design of molecules with potential therapeutic roles.