SIV escape mutants in rhesus macaques vaccinated with NEF-derived lipopeptides and challenged with pathogenic SIVmac251.

BACKGROUND: Emergence of viral variants that escape CTL control is a major hurdle in HIV vaccination unless such variants affect gene regions that are essential for virus replication. Vaccine-induced multispecific CTL could also be able to control viral variants replication. To explore these possibilities, we extensively characterized CTL responses following vaccination with an epitope-based lipopeptide vaccine and challenge with pathogenic SIVmac251. The viral sequences corresponding to the epitopes present in the vaccine as well as the viral loads were then determined in every macaque following SIV inoculation. RESULTS: In most cases, the emergence of several viral variants or mutants within vaccine CTL epitopes after SIV challenge resulted in increased viral loads except for a single macaque, which showed a single escape viral variant within its 6 vaccine-induced CTL epitopes. CONCLUSION: These findings provide a better understanding of the evolution of CD8+ epitope variations after vaccination-induced CTL expansion and might provide new insight for the development of an effective HIV vaccine.

Evaluation of SIV-lipopeptide immunizations administered by the intradermal route in their ability to induce antigen specific T-cell responses in rhesus macaques.

Numerous clinical and experimental observations have shown that cellular immunity, in particular CD8+ T-lymphocytes, plays an important role in the control of HIV infection. We have focused on a lipopeptide vaccination strategy that has been shown to induce polyepitopic T-cell responses in both animals and humans, in order to deliver simian immunodeficiency virus (SIV) antigens to rhesus macaques. Given the relevance of antigen administration route in the development of an effective cellular immune response, this study was designed to assess SIV lipopeptide immunizations administered either by the intradermal (ID) or the intramuscular (IM) routes in their ability to elicit GAG and NEF multispecific T-lymphocytes in the rhesus macaque. Antigen specific T-cell responses were observed between 7 and 11 weeks following vaccination in both groups. Macaques immunized by the IM route yielded antigen-specific IFN-gamma secreting lymphocytes in response to no more than two pools of peptides derived from SIV-NEF. In contrast, among the four ID-immunized macaques, two presented multi-specific T-cell responses to as many as four pools of SIV-NEF and/or GAG peptides. Responses persisted 16 weeks following the vaccination protocol in one of the ID-vaccinated macaques. The induction of such responses is of great clinical relevance in the development of an effective HIV vaccine. Given the crucial role of CD8+ T-lymphocytes in HIV/SIV containment, vaccination through the intradermal route should merit high consideration in the development of an AIDS vaccine.

Structure-activity relationships of imidazole-derived 2-[N-carbamoylmethyl-alkylamino]acetic acids, dual binders of human insulin-degrading enzyme.

Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as amyloid-â and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the understanding of its role in the physiopathology of Alzheimer's disease, amyloid-â clearance, and its validation as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we describe the structure-activity study, that required the synthesis of 48 analogues. We found that while the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was successfully optimized to an amide or a 1,2,4-oxadiazole. Along with improving their activity, compounds were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured in vivo. As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical probes for the exploration of IDE's role.

Human leukocyte antigen class II control of the immune response to p126-derived amino terminal peptide from Plasmodium falciparum.

We investigated the relationships between class II human leukocyte antigens (HLA) and the antibody response to Plasmodium falciparum p126 protein and to its amino-terminal portion (Nt47) in 2 malaria-endemic villages in Brazil, Colina and Ribeirinha. All people from the endemic areas had anti-p126 antibodies, and the frequencies of anti-Nt47 antibodies were similar in both communities (66% for Colina and 75% for Ribeirinha). Typing of HLA showed that Colina and Ribeirinha groups had no significant differences in HLA antigen frequencies. However, in both groups, significant associations between positive response to anti-Nt47 and presence of HLA-DR4, as well as between absence of response and presence of HLA-DR15, were observed. The predominance of positive responses to Nt47 among HLA-DR4 people was independent of the presence of any particular allele. There was no evidence for association between HLA-DQB1 alleles and antibody response to Nt47. Thus, naturally exposed people with different HLA class II antigens seem to respond differently to Nt47, indicating that the choice of relevant peptide sequences may have important consequences for subunit vaccine development.

Grafting of synthetic mannose receptor-ligands onto onion vectors for human dendritic cells targeting.

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.

Inhibition of aggrecanases as a therapeutic strategy in osteoarthritis.

Over the last decade, there has been a large effort to target aggrecanases, which are responsible for the degradation of the aggrecan in the extracellular matrix of joints, in order to hopefully lead to new treatments for osteoarthritis. Only a few inhibitors have been effective in explants or rodent models and thus only a few have reached the clinic, none of which have proven to be effective. In this article, a survey of chemical series is described, covering historical and recent inhibitors and highlighting how some of their problems were resolved, with a critical overview of the challenges encountered. A large effort should be undertaken in designing smaller compounds with higher residence times, defining new interaction sites on the aggrecanases and exploiting target flexibility.

Parallel synthesis of a lipopeptide library by hydrazone-based chemical ligation.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogeneous linear tridecapeptide with potential application for the modulation of skin tanning. To evaluate the interest of introducing a lipid moiety onto this peptide, we developed an efficient chemoselective parallel method to prepare a large series of analogues of alpha-melanocortin with high purity, varying the nature or the relative position of the lipid moiety. Two sets of building blocks containing lipidic alpha-oxo-aldehydes or alpha-hydrazinoacetyl peptides were combined to obtain a 102-membered library of amphiphilic alpha-MSH analogues. This library was pharmacologically tested at 1 x 10(-7) M for the ability to induce AMPc production in M4Be melanoma cell line after stimulation of the human melanocortin MC1 receptor. Among theses lipopeptides, 84 compounds exhibited an AMPc induction higher than Melitane, a patented alpha-MSH agonist. These results provide strong evidence of the interest of introduction of a lipid tail for the pharmacomodulation of bioactive peptides.

Anchorage of synthetic peptides onto liposomes via hydrazone and alpha-oxo hydrazone bonds. preliminary functional investigations.

Synthetic peptidoliposomes have been designed and prepared according to a chemoselective ligation. Two aldehyde-functionalized lipidic anchors were synthesized and incorporated into the lipidic bilayers of unilamellar liposomes during their preparation. Complementary hydrazino acetyl peptides were synthesized on the solid phase using N,N',N'-tri(tert-butyloxycarbonyl)-hydrazino acetic acid and further coupled to the aldehyde groups displayed at the surface of the vesicles. Coupling yields were measured by amino acid hydrolysis following total acid hydrolysis. The ligation methodology proved superior to the simple insertion of lipopeptides, which was performed for comparison in terms of yields, implementation, and reproducibility. To check whether the grafted-peptides were accessible and functional, cytoplasmic sequences of LAMP protein (lysosomal associated membrane protein), which is involved in intracellular membrane trafficking, have been selected. Using this model, we demonstrated in vitro the specific interaction of the synthetic LAMP-peptidoliposomes with the cytoplasmic adaptor protein AP-3, a result that contributes to the understanding of protein sorting in cells. Thus, these results clearly indicate the usefulness of such peptidoliposomes, easily prepared by hydrazone chemoselective ligation, as a tool for biological investigation.

Effect of glycoamphiphiles on the solubilization and dendritic cell uptake of a lipopeptide: a preliminary study.

The selective delivery of antigens to professional antigen-presenting cells represents a promising approach to improve vaccine efficacy. Addition of a glycoamphiphile to a lipopeptide, whose interest for vaccination is now well-established, greatly favors its solubilization in aqueous solutions through the formation of mixed vesicles. Flow cytometry experiments indicate that this formulation does not diminish the uptake of the lipopeptide by the dendritic cells (DCs). These preliminary results suggest a possible straightforward, noncovalent targeting of cocktail-lipopeptide vaccines to the DCs via carbohydrate receptor-mediated endocytosis.

Lipid vector for the delivery of peptides towards intracellular pharmacological targets.

The ability of single-chain lipopeptides to gain access to cellular compartments other than those related to degradation/recycling was first deduced from their capacity to deliver peptide antigens into MHC-class I loading mechanisms. The ability of lipopeptides to escape complete endosome degradation was further illustrated by the selective inhibition of different protein kinase C isoenzymes and, more recently, the presentation of agonistic activity towards the interferon gamma receptor. Taken together, several independent results indicate that modification of a peptide by a single lipid chain confers upon it intracellular trafficking properties that can be used to deliver functional cargo peptides into living cells; the endoplasmic reticulum, cytosolic protease activity, sites of kinase activity, or even the signalling pathway associated with cytokine stimulation, all appear accessible to peptide modified by a single lipidic moiety. In this context, the interferon gamma receptor can be considered as a very discriminative pharmacological model, useful for the comparative evaluation of the cellular delivery of lipopeptides, as it allows the unambiguous tracking of their intact delivery into a wide range of cellular compartments. This model is now being used to probe the influence of the nature of the lipid moiety on the trafficking properties of lipopeptides.

Lipopeptide epitopes extended by an Nepsilon-palmitoyl-lysine moiety increase uptake and maturation of dendritic cells through a Toll-like receptor-2 pathway and trigger a Th1-dependent protective immunity.

Lipopeptides, a form of peptide immunogens, are currently under intense investigation as human vaccines for many infectious pathogens and cancers. However, the cellular and molecular mechanisms of lipopeptide immunogenicity are only partially understood. We have investigated the influence of the lipid content on the immunogenicity of lipopeptides using the herpes simplex virus type 1 (HSV-1) gD(1-23) peptide as a model antigen. Totally synthetic lipopeptides were constructed by covalent attachment to the peptide backbone of either Nepsilon-palmitoyl-lysine (palmitoyl-lipidated peptide, palmitoyl-LP) or cholesterol-lysine (cholesterol-lipidated peptide, cholesterol-LP). Immunization of mice with the palmitoyl-LP, but not with its cholesterol-LP analog, induced a strong T cell-dependent protective immunity against lethal HSV-1 infection. Analysis of cytokine profiles and IgG2a/IgG1 ratios revealed that a dominant Th1-type immune response was stimulated by the palmitoyl-LP, as opposed to a Th2 response generated by its cholesterol-LP analog. The palmitoyl-LP was efficiently taken up in vitro by immature dendritic cells (DC) in a time- and dose-dependent manner, and induced phenotypic maturation and production of pro-inflammatory cytokines by DC. Finally, DC stimulated with the palmitoyl-LP induced antigen-specific T cell responses through the Toll-like receptor-2 pathway. These findings have important implications for the development of effective lipopeptide immunization strategies against infectious pathogens.

Simultaneous lipidation of a characterized peptide mixture by chemoselective ligation.

The modification of a peptide antigen by a fatty acid such as palmitic acid is now recognized as a mean to induce cellular responses. Mixtures of lipopeptides, obtained by combining individually synthesized compounds, were shown to be promising synthetic vaccine candidates. Usually, in lipopeptide synthesis, the fatty acyl moiety is introduced on the crude peptide chain using solid-phase methods. The separation of the target compound from impurities by RP-HPLC is often complicated by the amphiphilic properties of lipopeptides and results in low overall yields. To overcome the difficulties associated with lipopeptide synthesis and mixture preparation, we have developed a method where the fatty acyl moiety is site-specifically and collectively introduced in solution onto a mixture of individually prepurified peptides. The lipidation is based on the quasistoichiometric and high-yielding ligation of a glyoxylyl lipid with hydrazinoacetyl peptides. The hydrazone constructs were prepared in a salt-free medium and could be isolated by direct lyophilization of the reaction mixture. This process is compatible with cysteinyl peptides, and no aggregation nor degradation could be observed.

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-gamma 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.

Synthesis and mannose receptor-mediated uptake of clustered glycomimetics by human dendritic cells: effect of charge.

Effect of charge and shape of multivalent lysine-based cluster glycomimetics on their mannose receptor-mediated uptake by human dendritic cells has been evaluated: The capture is strongly affected by the shape of the ligands. The effect of charge is less pronounced although positive charges on the ligands seem to favor non-specific endocytosis capture.

Peptide arrays for highly sensitive and specific antibody-binding fluorescence assays.

We report a novel generation of peptide arrays fabricated by site-specific ligation of glyoxylyl peptides onto glass slides covered by a semicarbazide sol-gel layer. These arrays allowed the highly sensitive and specific detection of antibodies in very small blood samples from infected individuals using three model peptidic epitopes (HCV Core and NS4, EBV Capsid) in an immunofluorescence assay. Comparison with standard enzyme-linked immunosorbent assays (ELISAs) demonstrated a large gain in sensitivity and specificity. These unique properties, combined with the possibility to immobilize glycoproteins such as antibodies, offer the possibility to perform sandwich immunofluorescent assays in a highly parallel format.

Combined thioether/hydrazone chemoselective ligation reactions for the synthesis of glycocluster-antigen peptide conjugates.

Hydrazone/thioether ligation reactions show promise for the synthesis of clustered glycosides-antigen conjugates. Due to its propensity to aggregate, tetanus toxoid-derived epitopic peptide TT(830-846) was elected to further evaluate this three-component ligation process. This difficult sequence was supplemented by a hydrazine or a glyoxylyl group either at its C- or N-terminus. The peptide-hydrazines or peptide-aldehydes thus obtained were coupled with glyoxylyl- (or hydrazino-) N-chloroacetylated-L-lysinyl trees and 2-thioethyl-alpha-D-mannopyranoside. As anticipated the ligations were controlled by the nature of the peptide and proved difficult for the C-terminal aldehyde derivative. However, when the process was performed in absence of buffer and using mannitol as a dispersing agent, all combinations finally led to the expected glycoconjugates in 40-60% purified yields.

Solid-phase enolate chemistry investigated using HR-MAS NMR spectroscopy.

Supported P4-t-Bu enolate chemistry of phenylacetyloxymethyl polystyrene (PS) resin was investigated using high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Direct analysis of the crude reaction suspensions through the use of a diffusion filter (DF) allowed a rapid selection of the optimal experimental conditions, but also the characterization of the enolate on the solid phase. Comparison with solution experiments and literature data allowed us to address partially the structure of the enolate. HR-MAS NMR spectra of the enolate revealed also a tight interaction of P4-t-Bu base with the polymer matrix.

Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity.

The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4(+) T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD(49-82), gD(146-179), gD(228-257), and gD(332-358)), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4(+) Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2(d) mice with the four newly identified CD4(+) Th1 peptide epitopes but not with four CD4(+) Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.

Alpha-oxo semicarbazone peptide or oligodeoxynucleotide microarrays.

We describe in this paper the preparation and characterization of semicarbazide glass slides and their use for the fabrication of microarrays using site-specific alpha-oxo semicarbazone ligation. The functional density and homogeneity of the semicarbazide glass slides were optimized by analyzing the reactivity of the layer toward a synthetic glyoxylyl fluorescent probe. Oligonucleotide microarrays were prepared by site-specific immobilization of glyoxylyl oligodeoxynucleotides. The slides were directly used in the hybridization assays using fluorescence detection and displayed a significant gain in sensibility as compared to the aldehyde glass slide/amino oligodeoxynucleotide chemistry. Semicarbazide slides were also used for the immobilization of a biotinylated peptide alpha-oxo aldehyde. The peptide microarrays allowed model interaction studies with streptavidin or an anti-biotin antibody.

Peptide-protein microarrays for the simultaneous detection of pathogen infections.

We describe novel peptide-protein microarrays, which were fabricated using semicarbazide glass slides that permitted the immobilization of glyoxylyl peptides by site-specific ligation and the immobilization of proteins by physisorption. The arrays permitted the simultaneous serodetection of antibodies directed against hepatitis C virus (HCV core p21 15-45 peptide, NS4 1925-1947 peptide, core, NS3, NS4, and mixture of core, NS3, NS4, and NS5 antigens), hepatitis B virus (HBc, HBe, and HBs), human immunodeficiency virus (Gp41 and Gp120 for HIV-I and Gp36 for HIV-II), Epstein-Barr virus (VCAp18 153-176 peptide), and syphilis (rTpN47 and rTpN17) antigens using an immunofluorescence assay. Peptide-protein microarrays displayed high signal-to-noise ratios, sensitivities, and specificities for the detection of antibodies as revealed by the analysis of a collection of human sera referenced against these five pathogens.