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Evidence for specific and direct bacterial product recognition through toll-like receptors (TLRs) has been emphasized recently. We analyzed lipopeptide analogues and enterobacterial lipopolysaccharide (eLPS) for their potential to activate cells through TLR2 and TLR4. Whereas bacterial protein palmitoylated at its N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr(3)CSK(4)) activated cells not only through TLR2 but also through TLR4. Conversely, highly purified eLPS triggered cell activation through overexpressed TLR2 in the absence of TLR4 expression if CD14 was coexpressed. Accordingly, TLR2(-/-) macrophages prepared upon gene targeting responded to Myr(3)CSK(4) challenge, whereas TLR2(-/-)/TLR4(d/d) cells were unresponsive. Through interferon-gamma (IFNgamma) priming, macrophages lacking expression of functional TLR4 and/or MD-2 acquired sensitivity to eLPS, whereas TLR2/TLR4 double deficient cells did not. Not only TLR2(-/-) mice but also TLR4(-/-) mice were resistant to Myr(3)CSK(4) challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven toxemia upon IFNgamma priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr(3)CSK(4) as adjuvant was ineffective in TLR2(-/-)/TLR4(-/-) mice yet effective in wild-type, TLR2(-/-), or TLR4(-/-) mice as shown by analysis of ovalbumin-specific serum Ig concentration. A compound such as Myr(3)CSK(4) whose stimulatory activity is mediated by both TLR2 and TLR4 might constitute a preferable adjuvant. On the other hand, simultaneous blockage of both of the two TLRs might effectively inhibit infection-induced pathology.

Toll-like receptors (TLRs) mediate activation of the immune system upon challenge with microbial agonists, components of disintegrating cells of the body, or metabolic intermediates of lipidic nature. Comparison of murine (m) and human (h) TLR2 primary sequences revealed 65% of identical residues within the extracellular domains in contrast to 84% in the intracellular domains. Comparative analysis of TLR2-driven cell activation by various TLR2 agonists showed that the tri-lauroylated lipopeptide analog (Lau(3)CSK(4)) is recognized efficiently through mTLR2 but not hTLR2. Genetically complemented human embryonic kidney 293 cells and murine TLR2(-/-) embryonic fibroblasts, as well as human and murine macrophage cells, were used for this analysis. In contrast to cellular activation, which depended on blockable access of the TLR2-ligand to TLR2, cellular uptake of Lau(3)CSK(4) and tri-palmitoylated peptide (P(3)CSK(4)) was independent of TLR2. A low-conserved region spanning from leucine-rich repeat (LRR) motif 7 to 10 was found to control TLR2 species-specific cell activation. Exchange of mLRR8 for hLRR8 in mTLR2 abrogated mTLR2-typical cell activation upon cellular challenge with Lau(3)CSK(4) but not P(3)CSK(4), implicating mLRR8 as a central element of Lau(3)CSK(4) recognition. The point mutation L112P within LRR3 abrogated hTLR2-dependent recognition of lipopeptides but merely attenuated mTLR2 function, whereas deletion of the N-terminal third of each LRR-rich domain (LRRs 1 to 7) had the opposite effect on P(3)CSK(4) recognition. Despite similar domain structure of both TLR2 molecules, species-specific properties thus exist. Our results imply distinct susceptibilities of humans and mice to challenge with specific TLR2 ligands.

Toll-like receptors (TLRs) mediate microbial pattern recognition in vertebrates. A broad variety of agonists has been attributed to TLR2 and three TLRs, TLR4, TLR2, and TLR5, have been demonstrated to bind microbial products. Distinct agonists might interact with different subdomains of the TLR2 extracellular domain. The TLR2 extracellular domain sequence includes 10 canonical leucine-rich repeat (LRR) motifs and 8-10 additional and potentially functionally relevant LRR-like motifs. Thus, the transfection of TLR2 LRR/LRR-like motif deletion constructs in human embryonic kidney 293 cells and primary TLR2-deficient mouse fibroblasts was performed for analysis of the role of the regarding domains in specific pattern recognition. Preparations applied as agonists were highly purified soluble peptidoglycan, lipoteichoic acid, outer surface protein A from Borrelia burgdorferi, synthetic mycoplasmal macrophage-activating lipoprotein-2, tripalmitoyl-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4), dipalmitoyl-CSK4 (P2-CSK4), and monopalmitoyl-CSK4 (PCSK4) as well as lipopolysaccharide and inactivated bacteria. We found that a block of the N-terminal seven LRR/LRR-like motifs was not involved in TLR2-mediated cell activation by P3CSK4 and P2CSK4 ligands mimicking triacylated and diacylated bacterial polypeptides, respectively. In contrast, the integrity of the TLR2 holoprotein was compulsory for effective cellular recognition of other TLR2 agonists applied, including PCSK4. The formation of a functionally relevant subdomain by a region including the N-terminal seven LRR/LRR-like motifs rather than by single LRRs is suggested by our results. They further imply that TLR2 contains multiple binding domains for ligands that may contribute to the characterization of its promiscuous molecular pattern recognition.

In previous studies we have shown that lipopeptides constitute potent immunoadjuvants in mice, rabbits and other species: in parenteral immunization, lipopeptide adjuvants were comparable, or in some cases superior to Freund's adjuvant, and were devoid of the side effects of this additive. Here we demonstrate that lipopeptides also constitute adjuvants for mucosal immunizations. The serum antibody responses against the wheat storage protein gliadin, the bee venom constituent melittin, or the hen egg protein ovalbumin could in most cases be enhanced more than 100-fold by the lipopeptide P3CSK4, applied via the nasal route. An enhanced specific antibody level could also be detected in supernatants of cell cultures prepared from spleens, Peyer's patches, lungs and mesenteric lymph nodes of immunized mice. Moreover, the lipopeptide P3CSK4 enhanced chemiluminescence in mouse spleen cells and peritoneal macrophages in vitro, indicating a macrophage-activating effect. Finally, nasal application of lipopeptide increased protection against a lethal infection of influenza. Our findings are of importance for the improvement of immunizations and might lead to more effective vaccines.

To generate conventional or monoclonal antibodies for the serological detection of drugs, antibiotics, toxins and other low molecular mass substances, a suitable and effective adjuvant is needed. Lipopeptides derived from a major component of the bacterial cell wall constitute potent nontoxic and nonpyrogenic immunoadjuvants when mixed with conventional antigens. Here we demonstrate that the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl- serine (P3CS) coupled to a Th-cell epitope (P3CS-Th) can efficiently enhance the specific immune response against low molecular weight compounds in different species. In the presence of the synthetic lipopeptide P3CS-Th, the peptides which are per se non-immunogenic stimulated a specific humoral immune response in mice after intraperitoneal application. Mixtures containing adjuvants without the Th sequence showed no significant antibody induction. A marked enhancement of the humoral immune response was obtained with the low molecular mass antigens Iturin AL, Herbicolin A and Microcystin (MLR) coupled to poly-l-lysin (MLR-PLL), in rabbits and in chickens. Lipopeptide-Th cell epitope conjugates also constituted adjuvants for the in vitro immunization of either human mononuclear cells or mouse B-cells with MLR-PLL; after fusion of the immunized cultures with the heteromyeloma cell lines CB-F7 or the mouse myeloma cell line SP 2/0, respectively, we observed a significantly increased yield of antibody secreting hybridomas.

To evaluate the ability of the lipotripeptide P3CSS to increase peptide-specific immune responses in vivo, we immunized mice from different inbred strains (BALB/c, C3H/HeJ, C57BL/6) with the 22-mer lipopeptide conjugates P3CSS-[RT-(522-543)] and P3CSS-[RT-(528-549)] of HIV-1 reverse transcriptase (RT) which included an immunodominant Th epitope [i.e. RT-(528-543)] characterized previously. Analysis of T and B cell responses to these lipopeptide conjugates indicated that specific Th responses could be readily induced in vivo. The peptide segments could also efficiently prime mice for secondary recognition of native RT. The use of shorter peptides permitted a delineation of the minimal T cell recognition site of this RT C-terminal region [i.e. RT-(528-540)]. Close to this T cell epitope we identified a B cell determinant containing the motif EQVD [RT-(546-549)] which was recognized in three different strains of mice (H-2b, H-2d and H-2k). A comparison with X-ray analysis of the C-terminal region of HIV-1 reverse transcriptase indicated exposed positions of these Th and B cell epitopes. Both the presence of T and B cell sites and its limited polymorphism make the region RT-(528-549) a promising candidate for vaccine design. The use of the P3CSS adjuvant/carrier principle as a nontoxic adjuvant may be of major importance in the development of vaccines applicable to humans.

Various lipopeptides representing the N-terminal part of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas virdis were prepared by solid-phase peptide synthesis. These lipopeptides consisted of a S-[2,3-dihydroxypropyl]-cysteinyl (Dhc) residue N-terminally coupled to the nonapeptide FEPPPATTT. Different numbers of palmitoyl (Pam) chains were attached to Dhc via ester and/or amide bonds. The lipopeptide Dhc(Pam)2-FEPPPATTT containing two ester-bonded palmitoyl residues and a free N-terminus was a potent polyclonal activator of murine (BALB/c) spleen cells at subnanomolar concentrations. The lipopeptide Pam-Dhc(Pam)2-FEPPPATTT containing three palmitoyl residues, the two-chain lipopeptide Pam-Dhc(Pam)-FEPPPATTT containing one amide- and one ester-bonded palmitoyl residue, and the N-terminally elongated lipopeptide SLVAG-Dhc(Pam)2-FEPPPATTT were less active. The nonapeptide FEPPPATTT and the decapeptide Dhc-FEPPPATTT were only marginal splenocyte activators, even at concentrations as high as 1 microM. Thus, lipopeptide Dhc(Pam)2-FEPPPATTT constitutes the first potent splenocyte stimulation Dhc-lipopeptide described so far that contains only two fatty acid residues.

The lipoprotein (LP) from Escherichia coli and other Enterobacteriaceae as well as lipoprotein derived lipopeptides constitute potent B lymphocyte mitogens. Several synthetic peptide segments of the lipoprotein were tested for mitogenic activity towards splenocytes of BALB/c mice. Mitogenic effects were observed for the lipoprotein fragments LP-(2-15), LP-(2-20), LP-(2-25), and LP-(33-47), and a less pronounced effect was determined for LP-(2-10). The mitogenicity of the peptides could be further enhanced by their attachment to the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)- cysteine (Pam3Cys). The additional insertion of 6-aminohexanoic acid (Ahx) between the peptide segments and Pam3Cys further increased mitogenicity. Thus, in addition to the known mitogenic N-terminal lipopeptide of lipoprotein, additional peptide segments of the molecule were shown to exhibit a biological activity.

Synthetic tripalmitoyl-S-glycerylcysteinyl (Pam3Cys) peptides are derived from the N-terminal part of bacterial lipoprotein and constitute polyclonal B-lymphocyte and macrophage activators. In order to elucidate the primary events of leukocyte activation, we investigated the biophysical interaction of lipopeptides containing spin labels or fluorescent markers with phosphatidylcholine vesicles or immune cells. Utilizing fluorescence microscopy and FACS analysis we found, that the surface of cells, after incubation with a fluorescein-labelled lipopeptide, was highly fluorescent. In addition, capping and patching was observed. Furthermore, fluorescence quenching experiments and electron paramagnetic resonance studies using vesicles incubated with lipopeptides suggested, that the peptide moiety and other more polar molecules linked to the lipo-amino acid are exposed to the hydrophilic compartment. These results show that in lipopeptide conjugates the Pam3Cys moiety acts as an efficient membrane anchor for molecules covalently coupled to it. The sequestering of the fatty-acid chains of the lipopeptide within the membrane is an early step of interaction, which might induce the uptake of the lipopeptide into the cell and the stimulation of immunocompetent cells.

Lymphocyte subpopulations involved in the self and nonself recognition processes are antibody producing cells, T-helper cells and T-killer cells. By using lipopeptide adjuvants and lipopeptide-antigen conjugates each of the major pathways of immune response can be specifically addressed on the molecular level of minimized synthetic lipopeptide vaccines. The immunologically active principle of the lipopeptide constructs is the synthetic N-terminus of bacterial lipoprotein, tri-palmitoyl-S-glycerylcysteine, which can be covalently linked to B-, T-helper and CTL epitopes. Methods of multiple peptide synthesis based on Merrifield's solid-phase synthesis allow the economic production of the high numbers of overlapping lipopeptides required for the complete immunological screening of viral proteins.

Lipopeptides, carrying the N-terminal lipoamino acid 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid (Pam3Adh-OH, 1), were obtained by solid-phase synthesis and by synthesis in solution. 2-Amino-6,7-dihydroxyheptanoic acid (Adh) can be regarded as a methylene analogue of S-glycerylcysteine, the N-terminal amino acid of lipoprotein from the outer cell membrane of Escherichia coli (a methylene group substitutes for the sulfur atom). The lipopeptides Pam3Adh-Ser-Ser-Asn-Ala 2a-d, in which the four possible stereoisomers of Pam3Adh-OH (2S,6S)-1 (1a), (2S,6R)-1 (1b), (2R,6S)-1 (1c), and (2R,6R)-1 (1d) are linked to the naturally occurring sequence Ser-Ser-Asn-Ala of the N-terminus of lipoprotein, and also Pam3Adh-Ser-(Lys)4 [2S,6S)-3), with a peptide part rendering the molecule water soluble, were capable of stimulating murine splenocytes polyclonally in vitro, as determined in a proliferation assay and in a hemolytic plaque assay against trinitrophenylated sheep erythrocytes. The diastereomers (2S,6S)-2 and (2R,6S)-2 with S-configurated C-6 were more active than the diastereomers (2S,6R)-2 and (2R,6R)-2 with R-configurated C-6; a change of the configuration at C-2 had less effect on the stimulatory activity. (2S,6S)-2 and (2S,6S)-3 are potent immunoadjuvants. A significantly enhanced primary immune response against trinitrophenylated sheep erythrocytes was obtained in vitro at lipopeptide concentrations of about 5 micrograms/mL and an immunization dose of 10(7) sheep erythrocytes/mL. Balb/c mice, which were immunized with a mixture of ovalbumin and (2S,6S)-2 or (2S,6S)-3, respectively, had a substantially higher antiovalbumin titer 28 days after immunization than mice which had received ovalbumin, (2S,6S)-2 or (2S,6S)-3 alone. Finally, the novel lipopeptides constitute potent macrophage activators: (2S,6S)-3 was able to induce tumor cytotoxicity against the tumor cell line L929 in bone marrow derived macrophages.

Lipopeptide analogues of bacterial lipoprotein activate macrophages and B lymphocytes. The products formed by coupling these lipopeptides to low molecular mass antigens can be used to induce antigen-specific antibodies in mice. In the present work, it is shown that HIV-1 gp160-derived synthetic oligopeptides coupled to the synthetic lipodipeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-s eryl- serine (P3CSS) induce peptide-specific antibodies in mice without adding further adjuvants. Depending on the peptides applied, the conjugates exhibited different lymphocyte stimulatory activity, immunoglobulin isotype patterns, and boost reactions; lipopeptide conjugates inducing a pronounced secondary immune response are considered to possess both B- and T-cell epitopes. Antibodies induced by the lipopeptide-HIV-1-peptide conjugates were also reactive against the recombinant gp160 of HIV-1.

Lipopeptide analogues of the N-terminus of bacterial lipoprotein consisting of N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys) attached to one to five further amino acids [Pam3Cys-Ser-Ser-Asn-Ala, Pam3Cys-Ser-(Lys)4, Pam3Cys-Ala-Gly, and Pam3Cys-Ser] were investigated for biological activity. In vitro, the compounds proved to be potent activators for Balb/c splenocytes as determined by proliferation assays. When given in vivo in combination with SRBC, Pam3Cys-Ser and Pam3Cys-Ala-Gly acted as immunoadjuvants enhancing the antigen specific IgM response after 7, and the IgG response after 14 days. In combination with dinitrophenylated bovine serum albumin (BSA(Dnp)), especially the amphiphilic and water-soluble lipohexapeptide Pam3Cys-Ser-(Lys)4 constituted a potent immune adjuvant. The lipopeptide was able to fully replace Freund's complete adjuvant (FCS) enhancing both anti-Dnp IgM and IgG in Balb/c mice. The hapten Dnp was also coupled directly--or via the spacer molecule 1,6-diaminohexane (HMD)--to the synthetic lipopeptides. The chemically defined low-molecular-mass conjugates obtained were capable of inducing anti-hapten-specific IgM and IgG without further adjuvants or carriers. The anti-hapten responses induced by these chemically uniform lipopeptide-hapten conjugates were, however, less pronounced than the response to the conventional heterogeneous hapten-protein conjugate BSA(Dnp), and only a weak boost effect was observed. Our results show that defined lipopeptides are novel immunoadjuvants either combined with or covalently linked to antigens or haptens.

Synthetic lipopeptides are potent B-lymphocyte and macrophage activators. The role of phosphatidylinositol metabolism and proteinkinase C in lipopeptide induced leukocyte activation were investigated. In murine B-lymphocytes and in bone marrow derived macrophages, lipopeptide failed to induce phosphatidylinositol breakdown, whereas in the macrophage cell line P388D1 formation of inositolphosphates was increased. Translocation of proteinkinase C from a cytosolic to a membrane compartment was only observed in the cell line P388D1 indicating that in the other cells tested lipopeptide acts via different signal transduction pathways.

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 (Pam3Cys) are agonists of TLR2. The structurally related lipopeptides with a head group lacking the fatty acyl residue at the amino-terminus (Pam2Cys) 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 randomizedS-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-pentapeptide amide collection Pam2CysXXXXX and 5 × 19 subcollections (Pam2CysOXXXX, Pam2CysXOXXX, Pam2CysXXOXX, Pam2CysXXXOX, Pam2CysXXXXO, 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 194 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 singleS-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-pentapeptide amides. [ABSTRACT FROM AUTHOR]

We recently showed that synthetic lipopeptides, analogues of the N-terminal region of bacterial lipoprotein, induce DNA synthesis in B lymphocytes in the absence of enhanced phosphatidylinositol 4,5-bisphosphate hydrolysis and protein kinase C translocation. Here we demonstrate that lipopeptides are capable of inducing enhanced expression of MHC class II molecules and early increases in the intracellular free calcium concentration ([Ca2+]1) in B cells. However, they do not effect Tcells. The increase in [Ca2+ seen in B cells is due primarily to Ca2+ release from intracellular stores. Since lipopeptides differ in their capability to induce early increases in [Ca22+]1 and since the calcium response does not correlate with the ability of lipopeptides to induce proliferation and expression of MHC class II molecules, we suggest that this biochemical event may not be essential for lipopeptide-mediated B-cell activation. [ABSTRACT FROM AUTHOR]