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H Gras-Masse

Publications and source records attributed to H Gras-Masse.

At least 19 recordsLinked to original sources
PubMed2001

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.

AIDS Vaccines↗
PubMed2001

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.

Animals↗
PubMed2001

Lipopeptides induce cell-mediated anti-HIV immune responses in seronegative volunteers.

OBJECTIVE: Test the efficacy of a mixture of six NEF (N1, N2, N3), GAG (G1, G2) and ENV (E) lipopeptides in the induction of B- and T-cell anti-HIV responses. DESIGN: A randomized phase I open-label dose-finding trial. Twenty-eight healthy seronegative volunteers received the lipopeptides, with or without the adjuvant QS21. METHODS: Anti-HIV-peptide antibodies were detected by enzyme-linked immunosorbent assay and Western blotting. Induction of cellulary responses was assessed by proliferative test and (51)Cr-release assay. RESULTS: Local and systemic adverse reactions were always mild or moderate. After three injections an antibody response was detected in 25 out of 28 volunteers (89%). T cells from 19 (79%) of the 24 volunteers proliferated in response to at least one peptide. The majority of the volunteers had induced a multispecific proliferative response; that is, cells from volunteers proliferated to two (five of 19), three (five of 19), four (three of 19) or five peptides (one of 19). Cytotoxic responses by anti-HIV CD8+ lymphocytes could be tested in 24 volunteers, 13 (54%) of whom had clear and reproducible responses, with strong activity in the remaining 12 (> 20% of specific lysis), and polyepitopic responses were detected in at least seven of the 13 responders. Cytotoxic responses were found against the whole NEF protein (clade B LAI) in three of four tested volunteers and cross-reactions with the proteins of clade B (MN) and clade A (Bangui) HIV-1 strains, and also HIV-2 ROD, were detected in one of two tested volunteers. CONCLUSIONS: Lipopeptides are promising immunogens for an AIDS vaccine.

AIDS Vaccines↗
PubMed2001

Tartric acid-based linker for the solid-phase synthesis of C-terminal peptide alpha-oxo aldehydes.

A novel linker, based on the anchoring of (+)-dimethyl 2,3-O-isopropylidene-D-tartrate to PEGA or PEG-PS solid supports, was developed for the solid-phase synthesis of C-terminal peptide alpha-oxo aldehydes. Peptide elongation was performed using the 9-fluorenylmethoxycarbonyl/t-Bu chemistry. The peptide and the 1,2-diol were deprotected on the solid phase. Then, a periodic oxidation of the fully deprotected peptidyl-resin led to the simultaneous cleavage of the product from the solid support and to the generation of the alpha-oxo aldehyde moiety. The methodology allowed the distance between the alpha-oxo aldehyde and the peptide to be easily modulated. The C-terminal peptide alpha-oxo aldehydes synthesized in this study were found to be useful partners in hydrazone, thiazolidine, and oxime chemical ligations.

Aldehydes↗
PubMed2001

Synthesis by chemoselective ligation and biological evaluation of novel cell-permeable PKC-zeta pseudosubstrate lipopeptides.

The ability of lipopeptides to passively cross the cell membrane opens new opportunities for the intracellular delivery of bioactive peptides. However, the production of large series of cell-permeable lipopeptides is not trivial due to their generally low solubility. We have evaluated the possibility of associating the fatty acid to the functional cargo using generally applicable ligation chemistries. To this end, we have designed an amphiphilic shuttle in which arginine residues served to solubilize the lipid part in aqueous media, during both the assembly of the lipopeptide and the cellular assays. Our model peptide, the pseudosubstrate sequence of protein kinase C-zeta (PKC-zeta), was associated to the pentapeptide Gly-Arg-Gly-Arg-Lys(Pam)-NH2 through thiazolidine, thioether, disulfide, or hydrazone linkages. The cytoplasm import of the resulting constructs was monitored through the quantification of the apoptosis specifically induced by PKC-zeta inhibition. Our observations suggested the interest of this noninvasive cellular import method to modulate the activity of an intracytoplasmic pharmacological target and showed the influence of a non-amide link created between the functional peptide and the lipidic vector: optimal results, in terms of both specific activity and low basal cytotoxicity, were obtained with the thiazolidine ligation product.

Apoptosis↗
PubMed2001

Chemoselective acylation of fully deprotected hydrazino acetyl peptides. Application to the synthesis of lipopetides.

Fully deprotected N-terminal alpha-hydrazino acetyl peptides were synthesized and chemoselectively acylated on the hydrazine moiety with various fatty acid succinimidyl esters or N-(cholesterylcarbonyloxy) succinimide to give lipopeptides of high purity. The buffer and pH were adjusted in order to minimize the oxidation of the hydrazine moiety and to achieve the best conversion and selectivity. The acylation was performed in a citrate-phosphate buffer/2-methylpropan-2-ol mixture of pH 5.1. The pKa of the alpha-hydrazino acetyl group on our model peptide was found to be 6.45, i.e., about 2 units lower than the pKa of a glycyl residue. The reaction was subsequently applied to the synthesis of a 38AA peptide derivatized by a palmitoyl group.

Acylation↗
PubMed2001

Synthesis of clustered glycoside-antigen conjugates by two one-pot, orthogonal, chemoselective ligation reactions: scope and limitations.

Major histocompatibility class II antigens have been bound to clustered glycosides for selective targeting of the dendritic cell mannose receptor. Di-, tetra-, and octavalent glycoside-antigen conjugates have been obtained after two, orthogonal, hydrazone/thioether ligations, performed by using thio derivatives of D-mannose, D-galactose, or D(-)-quinic acid, glyoxylyl (or hydrazino)-N-chloroacetylated lysinyl trees, and N-terminal hydrazino (or glyoxylyl) peptide antigens. Successful one-pot condensations have been developed to account for the nature of the antigens and the valency of the trees.

Amino Acid Sequence↗
PubMed2001

Single-chain lipopeptide vaccines for the induction of virus-specific cytotoxic T cell responses in randomly selected populations.

Effective vaccine development is now taking advantage of the rapidly accumulating information concerning the molecular basis of a protective immune response. Analysts and medicinal chemists have joined forces with immunologists and taken up the clear challenge of identifying immunologically active structural elements and synthesizing them in pure, reproducible forms. Current literature reveals the growing interest for extremely reductionist approaches aiming at producing totally synthetic vaccines that would be fully defined at the molecular level and particularly safe. The sequential information contained in these formulations tends to be minimized to those epitopes which elicit neutralizing antibodies, or cell-mediated responses. In the following review, we describe some of our results in developing fully synthetic, clinically acceptable lipopeptide vaccines for inducing cytotoxic T lymphocytes (CTL) responses in randomly selected populations.

Antigens, Viral↗
PubMed2000

Temporal loss of Nef-epitope CTL recognition following macaque lipopeptide immunization and SIV challenge.

To address the subtle interactions between antiviral cytotoxic T-cell (CTL) immune responses and the evolution of viral quasispecies variants in vivo, we performed a longitudinal study in a simian immunodeficiency virus (SIV)-infected rhesus macaque that had a long experimental SIV infection before developing simian AIDS. Before being infected with SIV, this animal was immunized with a mixture of seven lipopeptides derived from SIV Nef and Gag proteins and showed a bispecific antiviral CTL response directed toward Nef 169-178 and 211-225 peptides. After SIV infection, CTL activity against the Nef 169-178 epitope was no longer detectable, as assessed from peripheral blood mononuclear cells stimulated by autologous SIV. CTL activity against the 211-225 epitope was lost after 3 months, and an additional CTL response to the amino acids 112-119 Nef epitope emerged. Analysis of the Nef proviral sequence revealed the presence of immune escape variants first in the 211-225 epitope and much later in the 112-119 epitope. In contrast, epitope 169-178 showed only two mutations among all viral sequencing performed. We conclude that in this macaque, bispecific CTL exerted a strong selective pressure and escape virus mutants finally emerged. We identified CTL recognizing a conserved Nef epitope 112-119 (SYKLAIDM), essential for viral replication, which could be associated with a prolonged AIDS-free period. These results stress the importance of the induction of broader multispecific CTLs directed against highly conserved and functional T-cell epitopes by vaccination, with the aim of keeping HIV infection in check.

Amino Acid Sequence↗
PubMed2000

Structural diversity of human class II histocompatibility molecules induced by peptide ligands.

SDS-PAGE analyses of stable HLA-DR1 complexes indicate that the binding of T cell epitopes can lead to multiple conformational variants. Whereas short T epitopes (<14-mer) induce complexes with apparent MW ranging from 47 to 57 kDa, longer peptides form generally high mobility complexes (44-45 kDa). The generation of HLA-DR1 conformational variants appears dependent on core peptide residues fitting inside the groove but can additionally be attributed to the presence of N- and C-terminal flanking residues (PFRs) acting as a complementary mechanism. These PFRs can jointly affect major histocompatibility complex class II conformation and stability, supporting the existence of alternative contacts at a distance from the classical binding site.

Amino Acid Motifs↗
PubMed2000

High immunogenicity in chimpanzees of peptides and lipopeptides derived from four new Plasmodium falciparum pre-erythrocytic molecules.

We have investigated the immunogenicity in chimpanzees of twelve synthetic peptides derived from four new Plasmodium falciparum molecules expressed at pre-erythrocytic stages of the human malaria parasite. These parasite molecules were initially selected through their ability to be recognized by stage restricted human antibodies. Twelve 20- to 41-mer peptides representing potential human B- or T-cell epitopes were selected from these proteins, and synthesized. Six of these were modified by a C-terminal lipidic chain in order to re-inforce their immunogenicity. Strong B- and T-helper cell responses were induced in chimpanzees by lipopeptides injected without adjuvant and by peptides in Montanide. All twelve peptides induced CD4(+) T-cell proliferative responses, as well as the secretion of IFN-gamma (some of them at very high levels) and eleven peptides induced antibody responses. The immune responses elicited in this way were reactive with native parasite proteins, as shown by recall studies with sporozoite stage proteins, and proved to be long-lasting (up to 10 months after immunization). Our results support the strategy employed to select these four new malarial antigens and the corresponding peptides, and suggest that the immunizing formulations are both efficient and clinically acceptable.

Amino Acid Sequence↗
PubMed2000

Extension of HLA-A*0201-restricted minimal epitope by N epsilon-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 N epsilon-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 N epsilon-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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Functionalization of peptides and proteins by aldehyde or keto groups.

The functionalization of peptides and proteins by aldehyde or keto groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes and the advent of protein engineering. The first part of this review focuses upon the tremendous efforts devoted to the solid-phase synthesis of peptide aldehydes as protease inhibitors. The second part describes the utility of the aldehyde or keto functionalities for the site-specific modification of peptides or proteins.

Aldehydes↗
PubMed2000

The deprotection of Lys(Mtt) revisited.

The selective deprotection of Lys(Mtt)-containing peptidyl resins was successfully monitored by RP-HPLC using very short linear gradients. RP-HPLC analyses of the acidic filtrates also revealed the partial cleavage of the Trt groups and of the peptide-resin bond. The absorbance of the Mtt carbocation at 470 nm is only twice that of the Trt cation. Thus, the UV monitoring at 470 nm seems to be inappropriate, especially at the end of the deprotection, when the Mtt and the Trt levels are comparable.

Amino Acid Sequence↗
PubMed2000

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 Nepsilon-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-gamma 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.

Antigen Presentation↗
PubMed2000

Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3.

In humans, sterile immunity against malaria can be consistently induced through exposure to the bites of thousands of irradiated infected mosquitoes. The same level of protection has yet to be achieved using subunit vaccines. Recent studies have indicated an essential function for intrahepatic parasites, the stage after the mosquito bite, and thus for antigens expressed during this stage. We report here the identification of liver-stage antigen 3, which is expressed both in the mosquito and liver-stage parasites. This Plasmodium falciparum 200-kilodalton protein is highly conserved, and showed promising antigenic and immunogenic properties. In chimpanzees (Pan troglodytes), the primates most closely related to humans and that share a similar susceptibility to P. falciparum liver-stage infection, immunization with LSA-3 induced protection against successive heterologous challenges with large numbers of P. falciparum sporozoites.

Animals↗
PubMed2000

Multiepitopic B- and T-cell responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine.

We have attempted to develop an anti-human immunodeficiency virus (HIV) lipopeptide vaccine with several HIV-specific long peptides modified by C-terminal addition of a single palmitoyl chain. A mixture of six lipopeptides derived from regulatory or structural HIV-1 proteins (Nef, Gag, and Env) was prepared. A phase I study was conducted to evaluate immunogenicity and tolerance in lipopeptide vaccination of HIV-1-seronegative volunteers given three injections of either 100, 250, or 500 microg of each lipopeptide, with or without immunoadjuvant (QS21). This report analyzes in detail B- and T-cell responses induced by vaccination. The lipopeptide vaccine elicited strong and multiepitopic B- and T-cell responses. Vaccinated subjects produced specific immunoglobulin G antibodies that recognized the Nef and Gag proteins. After the third injection, helper CD4(+)-T-cell responses as well as specific cytotoxic CD8(+) T cells were also obtained. These CD8(+) T cells were able to recognize naturally processed viral proteins. Finally, specific gamma interferon-secreting CD8(+) T cells were also detected ex vivo.

AIDS Vaccines↗