A novel synthetic lipopeptide is allergy-protective by the induction of LPS-tolerance.

BACKGROUND: Exposure to the environment of traditional farms can protect children from some allergic disease. Due to this exposure, TLR2 expression in these children is increased. TLR2 ligands derived from gram-positive bacteria are found in the dust of these farms. OBJECTIVES: We proved whether a synthetic lipopeptide binding to the TLR1/2 heterodimer is able to protect from allergic disease in two different murine models of allergy. We also investigated the immunological mechanisms underlying the protective properties of the lipopeptide. METHODS: We synthesized a lipopeptide derived from a germination lipoprotein of Bacillus cereus (LPGerD). We evaluated the immunomodulatory activity of LPGerD in a murine model of systemic sensitization (OVA/Alum) and in a model in which mice were sensitized with OVA pulsed bone-marrow-derived dendritic cells (BMDCs) via the airways. Furthermore, the induction of LPS tolerance was studied. RESULTS: Treatment of mice with LPGerD in a mouse model of asthma led to protection against sensitization and airway inflammation. Similarly, bone-marrow-derived dendritic cells (BMDCs) pre-treated with LPGerD were not able to prime mice for allergic immune response. We observed that pre-treatment with LPGerD led to the induction of a LPS-tolerant state in BMDCs. These cells secreted markedly lower amounts of pro-inflammatory cytokines upon LPS stimulation. Furthermore, we observed an up-regulation of IRAK-M mRNA in BMDCs pre-treated with LPGerD. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that induction of a LPS-tolerant state in antigen-presenting cells (APCs) may contribute to the protective effect of a farming environment. TLR2 agonists similar to those appearing in cowshed dust extracts, such as our synthetic LPGerD, lead to the ignorance of the LPS stimulus, which is important for the activation of APCs to mount a Th2 immune response. This substance might be a promising candidate for allergy-preventive treatments as LPGerD had only low pro-inflammatory characteristics.

Synthesis and cellular uptake of a MR contrast agent coupled to an antisense peptide nucleic acid--cell- penetrating peptide conjugate.

In order to image mRNA transcription by in vivo magnetic resonance imaging (MRI), two intracellular MR contrast agents were developed, which are composed of a Gd-DOTA complex, a peptide nucleic acid (PNA) sequence and a cell-penetrating peptide. One was designed to bind to mRNA of dsRed (red fluorescent protein originating from Discosoma coral) by its PNA sequence, whereas the second one contains a nonsense sequence with no natural counterpart. The conjugates were synthesized using a continuous solid-phase synthesis scheme and characterized by ESI-MS. Fluorescence studies showed that both contrast agents could enter efficiently into 3T3 cells in a concentration-dependent manner from 0.5 to 9.0 microM. The contrast agent was located predominantly in vesicles around the nucleus, whereas no uptake into the nucleus was observed. The results of in vitro MR studies showed a statistically significant increase of the intracellular relaxation rate R (1,cell) at a labeling concentration of only 0.5 microM, thus contrast enhancement was detectable too. These results suggest that the synthesized contrast agents could label cells for optical as well as MR imaging and in future might be useful to prove specific accumulation in cells containing target mRNA.

Immunostimulants and Toll-like receptor ligands obtained by screening combinatorial lipopeptide collections.

Synthetic lipopeptides carrying the head group of bacterial lipoproteins are specific ligands of Toll-like receptors (TLR). The three fatty acids containing lipopeptides with the tripalmitoyl-S-glyceryl-cysteinyl N-terminus (Pam(3)Cys) are agonists of TLR2. The structurally related lipopeptides with a head group lacking the fatty acyl residue at the amino-terminus (Pam(2)Cys) stimulate TLR2 and 6. To investigate the influence of the peptide chain of lipohexapeptides with a free N-terminus with regard to their ability to enhance B-cell proliferation, a randomized S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-pentapeptide amide collection Pam(2)CysXXXXX and 5 x 19 subcollections (Pam(2)CysOXXXX, Pam(2)CysXOXXX, Pam(2)CysXXOXX, Pam(2)CysXXXOX, Pam(2)CysXXXXO, O: all protein amino acids except Cys) were prepared by parallel solid-phase synthesis. The collection represents synthetic lipopeptide analogues of the numerous bacterial lipoproteins and of mycoplasma lipoprotein. Each of the 95 subcollections is characterized by one defined and four degenerated amino acid positions thus comprising 19(4) individual lipopeptides with free N-terminal amino groups. High-performance liquid chromatography electrospray mass spectrometry (HPLC-ESI-MS) was applied for the analytical characterization of the lipohexapeptide amide subcollections and for the individual lipohexapeptide amides. The subcollections were tested for polyclonal activation of murine spleen cells, deconvolution led to highly active single S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-pentapeptide amides.

Lipopeptide adjuvants: monitoring and comparison of P3CSK4- and LPS-induced gene transcription.

Bacteria-derived synthetic lipoproteins constitute potent macrophage activators in vivo and are effective stimuli, enhancing the immune response especially with respect to low or non-immunogenic compounds. N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4), exhibiting one of the most effective lipopeptide derivatives, represents a highly efficient immunoadjuvant in parenteral, oral, nasal and genetic immunization either in combination with or after covalent linkage to antigen. In order to further elucidate its molecular mode of action with respect to the transcriptional level, we focused our investigations on the P3CSK4-induced modulation of gene transcription. We could show that P3CSK4 activates/represses an array of at least 140 genes partly involved in signal transduction and regulation of the immune response. P3CSK4 activates the expression of tumor suppressor protein p53 (p53), c-rel, inhibitor of nuclear factor kappa B (NFkappaB) alpha (IkappaB alpha), type 2 (inducible) nitric oxide (NO) synthase (iNOS), CD40-LR, intercellular adhesion molecule-1 (ICAM-1) and interleukin 1/6/15 (IL-1/6/15). We detected no activation of heat shock protein (HSP) 27, 60, 84 and 86, osmotic stress protein 94 (Osp 94), IL-12, extracellular signal-regulated protein kinase 1 (ERK1), p38 mitogen activated protein (MAP)-kinase (p38), c-Jun NH2-terminal kinase (JNK), signal transducer and activator of transcription 1 (STAT1), CD14 and caspase genes. Furthermore, we monitored inhibition of STAT6, Janus kinase 3 (Jak3) and cyclin D1/D3 gene transcription after stimulating bone marrow-derived macrophages (BMDM) with lipopeptide. In addition, we monitored significant differences after lipopeptide and lipopolysaccharide (LPS) stimulation of bone marrow-derived murine macrophages. Our findings are of importance for further optimizing both conventional and genetic immunization, and for the development of novel synthetic vaccines.

AMP deaminase in rat brain: localization in neurons and ependymal cells.

The purine nucleotide cycle enzyme AMP deaminase (AMPD) catalyzes the irreversible hydrolytic deamination of AMP. The physiological function of the purine nucleotide cycle in the brain is unknown. In situ hybridization and immunocytochemical studies were performed to identify the regional and cellular expression of AMPD in rat brain with the goal of elucidating the neural function of the purine nucleotide cycle. AMPD messenger RNA was detected in ventricular ependymal cells and cells of the choroid plexus and in neurons of distinct brain areas. Although only low antibody titers were obtained by immunization with the purified sheep brain AMPD, immunization of mice with synthetic lipopeptide vaccines containing oligopeptides derived from a known partial complementary DNA sequence of the enzyme yielded an antiserum suitable for immunocytochemistry. Immunostaining of cells in culture showed that neurons but not astroglial cells express appreciable amounts of the enzyme. Results of immunocytochemical staining performed on rat brain slices were in accord with the localization of AMPD messenger RNA, thus confirming the expression of AMPD in neurons of the brain stem, hippocampus, cerebellar nuclei and mesencephalic nuclei, as well as in ventricular ependymal cells and their cilia.

Optical biosensors. Monitoring studies of glycopeptide antibiotic fermentation using white light interference.

This paper describes the design, characterization, and use of an optical biosensor suited for the process control of biotechnological processes. The detector principle is based on reflectometric interference spectroscopy (RIfS). RIfS enables a label-free, product-specific monitoring, with a future outline for on-line process control. The potential of the RIfS biosensor is exemplified by the qualitative and quantitative monitoring of the microbial production of vancomycin-type glycopeptide antibiotics.

Peptide vaccines and peptide libraries.

Synthetic immunogens, containing built-in adjuvanticity, B cell, T helper cell and CTL epitopes or mimotopes, are ideal and invaluable tools to study the immune response with respect to antigen processing and presentation. This serves as a basis for the development of complete and minimal vaccines which do not need large carrier proteins, further adjuvants, liposome formulations or other delivery systems. Combinatorial peptide libraries, either completely random or characterized by one or several defined positions, are useful tools for the identification of the critical features of B cell epitopes and of MHC class I and class II binding natural and synthetic epitopes. The complete activity pattern of an O/Xn library with hundreds of peptide collections, each made up from billions of different peptides, represents the ranking of amino acid residues mediating contact to the target proteins of the immune system. Combinatorial libraries support the design of peptides applicable in vaccination against infectious agents as well as therapeutic tumour vaccines. Using the principle of lipopeptide vaccines, strong humoral and cellular immune responses could be elicited. The lipopeptide vaccines are heat-stable, non-toxic, fully biodegradable and can be prepared on the basis of minimized epitopes by modern methods of multiple peptide synthesis. The lipopeptides activate the antigen-presenting macrophages and B cells and have been recently shown to stimulate innate immunity by specific interaction with receptors of the Toll family.

From combinatorial libraries to MHC ligand motifs, T-cell superagonists and antagonists.

Complete experimental data sets of HLA-ligand motifs and T-cell recognition patterns can be derived from combinatorial peptide libraries. These data provide the exact molecular basis for a fast development of synthetic vaccines, T-cell superagonists and non-peptide antagonists. Patient-specific peptides, peptidomimetics and vaccines of highest reactivity can be derived directly from the data sets via our prediction programme EPIPREDICT. The resulting lead structures may be developed into valuable diagnostics and therapeutic tools for the treatment of viral infections, autoimmune diseases and tumors. As one example, antibody and T cell recognition in the intestinal auto-immune disease, coeliac disease was investigated in more detail concerning the deamidation of gamma-gliadin peptides by tissue transglutaminase 9tTG) leading to autoreactive peptides specific for HLA-DQA1*0501, DQB1*0201.

Adjuvant effects of various lipopeptides and interferon-gamma on the humoral immune response of chickens.

The adjuvant effects of various lipopeptides and recombinant chicken interferon gamma (IFN-gamma) on the humoral immune response of laying hens was investigated in four immunization studies. We used the lipopeptide Pam3Cys-Ser-(Lys)4 (PCSL), the conjugate P-Th1 consisting of the lipopeptide P3CS and the T-helper epitope Th1 (FISEAIIHVLHSRHPG), and the conjugate P-Th2 of the lipopeptide P3CSS and the T-helper epitope Th2, which corresponds to the peptide EWEFVNTPPLV, as adjuvants. Human serum albumin (HSA), recombinant bovine somatotropin (RBST), and human immunoglobulin G (IgG) served as antigens in the different experiments. All tested adjuvants enhanced the humoral immune response with various intensities. Chickens showed high antibody titers after the immunization with HSA even without adjuvant, but the adjuvant effects of PCSL and the combination of PCSL and recombinant chicken interferon-gamma (IFN-gamma) were much more pronounced using the antigens RBST and IgG. Especially after the third immunization, higher titers of antibodies were induced by the coadministration of P-Th1 and, to a greater extent, by the combination of PCSL and P-Th1 compared with the use of PCSL. Also, chickens that had received PCSL and P-Th2 showed the highest immune response, even after the second booster. The average concentrations of chicken immunoglobulin Y were significantly higher in 5-mo-old chickens (9.4 mg/mL serum and 10.1 mg/mL egg yolk) compared with 9-mo-old chickens (5.9 mg/mL serum and 5.1 mg/mL egg yolk). The specific serum antibody response was higher in the older chickens than in the younger chickens. Because chicken antibodies are likely to be used increasingly for diagnostic and therapy in the future, lipopeptides and recombinant chicken IFN-gamma may find many applications as adjuvants, thus contributing to the welfare of experimental animals.

An automated prediction of MHC class I-binding peptides based on positional scanning with peptide libraries.

Specificities of three mouse major histocompatibility complex (MHC) class I molecules, Kb, Db, and Ld, were analyzed by positional scanning using combinatorial peptide libraries. The result of the analysis was used to create a scoring program to predict MHC-binding peptides in proteins. The capacity of the scoring was then challenged with a number of peptides by comparing the prediction with the experimental binding. The score and the experimental binding exhibited a linear correlation but with substantial deviations of data points. Statistically, for approximately 80% of randomly chosen peptides, MHC-binding capacity could be predicted within one log concentration of peptides for a half-maximal binding. Known cytotoxic T-lymphocyte epitope peptides could be predicted, with a few exceptions. In addition, frequent findings of MHC-binding peptides with incomplete or no anchor amino acid(s) suggested a substantial bias introduced by natural antigen processing in peptide selection by MHC class I molecules.

Highly potent inhibitors of human cathepsin L identified by screening combinatorial pentapeptide amide collections.

By screening a combinatorial pentapeptide amide collection in an inhibition assay, we systematically evaluated the potential of 19 proteinogenic amino acids and seven nonproteinogenic amino acids to serve as building blocks for inhibitors of human cathepsin L. Particularly efficient were aromatic, bulky, hydrophobic amino-acid residues, especially leucine, and positively charged residues, especially arginine. Building blocks for potential inhibitory peptides were combined by random selection from their activity pattern. This random approach for the design of inhibitors was introduced to compensate for the inaccuracy induced by shifted docking of combinatorial compound collections at the active center of cathepsin L. Thereby, we obtained structurally defined pentapeptide amides which inhibited human cathepsin L at nanomolar concentrations. Among the most potent novel inhibitors, one peptide, RKLLW-NH2, shares the amphiphilic character of the nonamer fragment VMNGLQNRK of the autoinhibitory, substrate-like, but reverse-binding prosegment of human cathepsin L which blocks the active center of the enzyme. Obviously, RKLLW-NH2 carries the functions that are important for enzyme-peptide interaction in a condensed form. This hypothesis was confirmed by structure-activity studies using truncated and modified pentapeptides.

Expression of measles virus antigens in Streptococcus gordonii.

The measles virus proteins haemagglutinin (HA) and fusion protein (F), which together mediate attachment and penetration of the virus in the host cell and can elicit production of neutralising antibodies in the course of natural infection were expressed in the vaccine vector Streptococcus gordonii, a Gram-positive bacterium normally present in the human oral cavity. HA and F were expressed as fusion proteins attached to the bacterial surface, and were both found to be immunogenic when the recombinant S. gordonii were inoculated subcutaneously in mice.

A T-cell epitope determined with random peptide libraries and combinatorial peptide chemistry stimulates T cells specific for cutaneous T-cell lymphoma.

BACKGROUND: Mycosis fungoides is the most frequent T-cell lymphoma of the skin. Despite numerous attempts, no tumour antigens have yet been identified. Only in one case has an idiotype-derived peptide been found to trigger CTL of the respective patient. The identification of natural antigens requires the cultivation of large amounts of tumour cells in vitro, which has been possible in two exceptional cases. The identification of synthetic epitopes for tumour-specific CTL with random peptide libraries can overcome this limitation and is a powerful tool for application in the development of immune therapies for a wide range of patients. MATERIALS AND METHODS: The critical amino acids for the construction of epitopes for the CTCL-specific CTL clone My-La CTL were determined with synthetic peptide libraries in positional scanning OX8 format in a standard 61chromium release assay. Sixteen different peptides could be synthesized from the combinatoric of these amino acids with the canonical anchor amino acids for MHC binding. These peptides were tested for their capacity to stimulate My-La CTL and PBMC of an HLA-matched CTCL patient. RESULTS: A synthetic epitope could be identified for My-La CTL, which was recognized in a HLA-restricted manner. The response towards this epitope was comparable to the response towards their natural target My-La. Using these synthetic epitopes, T cells of a HLA-matched patient could be induced in vitro and led to the establishment of different cell lines and clones. Some of these lines recognized the peptides as well as the allogenic but HLA-matched tumour cell line My-La, indicating that they are specific for a naturally expressed tumour antigen. CONCLUSIONS: The identification of synthetic epitopes for tumour-specific CTL clones can be used for the development of vaccines for immune therapies of cancer; such peptides can be applied inter-individually. Synthetic epitopes must not correspond to the natural ones, but they can be even more potent as stimulation of specific T cells and can be fine-tuned to increase the success of the therapy.

The porcine humoral immune response against pseudorabies virus specifically targets attachment sites on glycoprotein gC.

High titers of virus-neutralizing antibodies directed against glycoprotein gC of Pseudorabies virus (PRV) (Suid herpesvirus 1) are generally observed in the serum of immunized pigs. A known function of the glycoprotein gC is to mediate attachment of PRV to target cells through distinct viral heparin-binding domains (HBDs). Therefore, it was suggested that the virus-neutralizing activity of anti-PRV sera is directed against HBDs on gC. To address this issue, sera with high virus-neutralizing activity against gC were used to characterize the anti-gC response. Epitope mapping demonstrated that amino acids of HBDs are part of an antigenic antibody binding domain which is located in the N-terminal part of gC. Binding of antibodies to this antigenic domain of gC was further shown to interfere with the viral attachment. Therefore, these results show that the viral HBDs are accessible targets for the humoral anti-PRV response even after tolerance induction against self-proteins, which utilize similar HBDs to promote host protein-protein interactions. The findings indicate that the host's immune system can specifically block the attachment function of PRV gC. Since HBDs promote the attachment of a number of herpesviruses, the design of future antiherpesvirus vaccines should aim to induce a humoral immune response that prevents HBD-mediated viral attachment.

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.

Immunostimulation by bacterial components: I. Activation Of macrophages and enhancement of genetic immunization by the lipopeptide P3CSK4.

Synthetic lipopeptides derived from the N-terminus of bacterial lipoprotein constitute potent macrophage activators and polyclonal B-lymphocyte stimulators. They are also efficient immunoadjuvants in parenteral, oral and nasal immunization either in combination with or after covalent linkage to an antigen. Here we show how alterations in the molecular structure influence their biological properties indicating P3CSK4 as one of the most active members of a lipopentapeptide fatty acid library. This compound resulted in a most pronounced macrophage stimulation as indicated by NO release, activation of NFkappaB translocation, and enhancement of tyrosine protein phosphorylation. Furthermore, P3CSK4 activates/represses an array of at least 140 genes partly involved in signal transduction and regulation of the immune response. Finally we have evidence that P3CSK4 constitutes an effective adjuvant for DNA immunizations, especially increasing weak humoral immune responses. Our findings are of importance for further optimizing both conventional and genetic immunization, and for the development of novel synthetic vaccines.

Analysis of peptide length preference of the rat MHC class Ia molecule RT1-A(u), by a modified random peptide library approach.

Using random peptide libraries we have previously shown that both mouse and rat class I molecules can exhibit different peptide length preferences. Such studies required expression of the particular class I molecules in RMA-S, a cell line deficient in the transporter associated with antigen presentation (TAP). For another rat class I molecule called RT1-A(u), however, we found that expression in RMA-S was poor and could not be increased sufficiently by incubation at 26 degrees C. To circumvent this problem we performed our studies on C58, a rat cell line that expresses RT1-A(u) naturally in the presence of a functional TAP transporter. Using C58 cells, cell-surface-expressed class I molecules were 'stripped' of peptides and beta(2)-microglobulin by washing the cells with an acidic citrate buffer (pH 3.3). Peptide stabilization assays, assessed by FACS analysis, were then performed using either specific peptides or synthetic random peptide libraries of different lengths (7-15 amino acids), supplemented with recombinant rat beta(2)-microglobulin. As a positive control an RT1-A(u)-specific nonamer peptide was designed using the previously determined peptide binding motif and this was found to bind to RT1-A(u) at nanomolar concentrations. Both length preference and importance of free N- and C-termini were tested using free base, formylated and acetylated peptide libraries. Results showed that RT1-A(u) was not able to accommodate N- or C-terminally blocked peptides but displayed a preference for peptides of 9-12 amino acids, similar to the preference observed for the RT1-A1(c) allotype, the other rat TAP-B-associated molecule tested thus far. These results suggest that length preference remains a consideration to explain the allelic class I-TAP associations of the RT1-A region.

Quantitative analysis of peptide-MHC class II interaction.

The tremendous progress in the field of basic immunology and immunochemistry made in the last decade has significantly advanced our understanding of antigen processing and presentation by MHC class I and II proteins. In this review different techniques to study peptide interaction with MHC class II molecules are summarized and their impact on the elucidation of quantitative parameters, like affinities or kinetic data, is discussed. A recently introduced method based on synthetic combinatorial peptide libraries allows to quantify the binding contribution of each amino acid residue in a class II ligand and is presented in more detail. As this knowledge is fundamental for current investigations in modern medicine, e.g. for novel immune system based therapy concepts, further aspects like the design of new high affinity MHC class II ligands and the prediction of peptide antigens are discussed.

New ligands for HLA DRB1*0301 by random selection of favourable amino acids ranked by competition studies with undecapeptide amide sublibraries.

An efficient screening procedure for the identification of high affinity HLA class II ligands and their binding pattern has been established to characterize peptide specificities for the HLA allele DRB1*0301. The method is based on the screening of 209 synthetic undecapeptide amide sublibraries O/X10-NH2 representing collections of 19(10) individual peptides in a competition ELISA using HLA DRB1*0301 protein and the biotinylated natural ligand ApoB 2877-2894. Screening results represent the effect on competition induced by an individual amino acid residue in its sequence position of undecapeptide amides. Amino acids clustered as active in their position were randomly selected for the same position of a restricted set of 96 individual undecapeptide amides. This novel approach for the design of ligands was introduced to compensate for the inaccuracy induced by the translational invariance of amino acids in peptide libraries characterized by one defined amino acid. Translational invariance is facilitated by shifted docking of O/X10-NH2 libraries in the binding cleft and protrusion from the ends of the cleft. A second more directed deduced set of 24 peptides was obtained by combination of the most favourable residues in each position. All individual peptides were investigated in the competition assay. The most active HLA DRB1*0301 ligands were obtained by random selection of favourable amino acids and six of them showed improved affinity in comparison to the model ligand alpha AChR 310-325.