Elsevier

Vaccine

Volume 22, Issue 19, 23 June 2004, Pages 2494-2499
Vaccine

Synthetic lipopeptide adjuvants and Toll-like receptor 2—structure–activity relationships

Dedicated to Prof. Dr. Volkmar Braun on the occasion of his 65th birthday
https://doi.org/10.1016/j.vaccine.2003.11.074Get rights and content

Abstract

Bacterial lipoproteins and their synthetic analogues (sLP) are strong immune modulators of the early host responses after infection. Synthetic lipopeptides are strong adjuvants for the adaptive immune system. Lipoproteins and lipopeptides induce signalling in immune cells through Toll-like receptor-TLR2/TLR1 heterodimers. By screening a combinatorial lipohexapeptide amide collection in an in vitro IL-8 induction assay, we systematically evaluated the potential of 19 proteinogenic amino acids in the peptide moiety of Pam3Cys-lipopeptides to interact with TLR2. New Pam3Cys-lipopeptides with high activity were obtained. Different fatty acids were introduced to investigate the influence of the acyl moiety. Lipopeptides with modifications in the core structure of the unusual amino acid S-glycerylcysteine were synthesized and tested for IL-8 induction via TLR2.

Introduction

Lipoproteins are part of the outer membrane of Gram negative bacteria, Gram positive bacteria, Rhodopseudomonas viridis and mycoplasma [1], [2], [3], [4]. Bacterial lipoproteins have no shared sequence homology but are characterized by the N-terminal unusual amino acid S-(2,3-dihydroxypropyl)-l-cysteine acylated by three fatty acids. The prototype of bacterial lipoproteins, Braun’s lipoprotein, was isolated and characterized from Escherichia coli over 30 years ago [5], [6], [7] and in 1975, it was found that the lipoprotein stimulates murine B-cell growth [8]. Lipoproteins from different bacteria activate nuclear factor κB (NF-κB) [9] and cytokine production [10].

Synthetic analogues (sLP) of the N-terminal lipopentapeptide of the lipoprotein of E. coli were synthesized and proved to be as active as the native lipoprotein [11]. They activate B-cells [11], [12], monocytes [13], neutrophils [14] and platelets [15] and act as potent immunoadjuvants in vivo and in vitro [16].

Synthetic lipopeptides with the RR stereoisomer N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R)-propyl]-[R]-cysteine, which reflects the stereochemistry of the N-terminal lipoamino acid of the bacterial lipoprotein, showed higher B-cell mitogenic and protective activity when introduced into vaccines than the mixture of stereoisomers N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteine [16], [17].

Immunisation of guinea pigs and cattle with a totally synthetic vaccine consisting of the built-in lipotripeptide adjuvant Pam3Cys-Ser-Ser and of a B- and T-cell epitope of the foot-and-mouth disease virus lead to a long-lasting protection against virus challenge [17], [18].

The conjugation of MHC class I restricted peptides with Pam3Cys-Ser-Ser resulted for the first time in efficient priming of virus-specific cytotoxic T-cells [19].

A potent hapten-specific immune response was obtained by immunization with a conjugate of lipopeptide, hapten and haplotype-specific T helper-cell epitope [20], [21].

Lipopeptide vaccinations have been carried out in all relevant animal models. So far, no toxic side effects have been observed [22]. The safety, reproducible production and ease of storage and handling of lipopeptide vaccines suggest that they have significant potential for the development of vaccines for humans and domestic animals. Lipopeptides passed phase I vaccination studies without side effects and improve the efficiency of vaccines [23].

The outer membrane lipoprotein (OspA) from Borrelia burgdorferi was used in human vaccine trials [24].

The efficacy of eight adjuvant formulations to prime cytotoxic T lymphocytes in mice was compared and the water soluble lipohexapeptide analogue of bacterial lipoproteins Pam3Cys-SK4, proved to be the most effective additive for eliciting a cellular immune response in mice [25].

Lipopeptide vaccines and the lipopeptide adjuvant Pam3Cys-SKKKK are chemically stable and can be produced in large quantities in reproducible high quality under GMP conditions.

Although bacterial lipoproteins and synthetic analogues were investigated very intensively over decades, it was unclear how the bacterial lipoproteins and their synthetic lipopeptide analogues interact with the target cells. Recently, it was shown, that the signal of lipopeptides and lipoproteins is transduced into a intracellular message via Toll-like receptor 2 [26], [27].

Toll-like receptors (TLR) are prominent pattern recognition receptors (PRR) of the innate immune system recognizing various invading microorganisms through conserved motifs termed “pathogen-associated molecular patterns” (PAMPs) [28], which are essential for and unique in microorganisms. In the 1980s, the first Toll receptor was found in studies of embryonic development in Drosophila melanogaster. To date, 10 mammalian Toll-like receptors (TLR1-TLR10) have been identified [29], and only for TLR10, no ligand has been described. Interaction of PAMPs with TLR induces a variety of host defense responses including the production of pro-inflammatory cytokines and the activation of the adaptive immunity [30], [31], [32].

Each TLR mediates the response to specific PAMPs shared by a set of molecular structures. For example, TLR4 responds to lipopolysaccharides of various Gram negative species in addition to compounds like taxol, HSP60, bacterial glycolipids [33].

Among the TLRs, TLR2 mediates the response to the most diverse set of molecular structures, including peptidoglycan, lipoteichoic acid, lipoarabinomanan, bacterial lipopeptides/proteins, as well as some LPS variants from Gram-negative bacteria, yeast, spirochetes and funghi [33]. Unlike TLR4, which is functionally active as a homodimer [34], TLR2 forms heterodimers with either TLR1 or TLR6 to attain specificity for a given stimulus [35], [36], [37], [38]. Diacyl lipopeptides like macrophage activating lipopeptide from Mycoplasma fermentans (MALP-2, Pam2Cys-GNNDESNISFKEK) contain the diacylated lipoamino acid S-[2,3-bis(palmitoyloxy)-(2R)-propyl]-[R]-cysteine [4]. They are described to require TLR2 and TLR6 for signalling, whereas the triacylated synthetic compound like Pam3Cys-SK4 are able to activate immunocompetent cells independent of TLR6 and mainly through TLR2/TLR1 heterodimers [29], [35], [36], [37], [38]. Also, for MALP-2, it has been shown that the RR stereoisomer is more active than the RS stereoisomer in stimulating the release of cytokines, chemokines and NO [39].

Among the TLR2-dependent PAMPs lipopeptides are primary candidates for analysing the structure–activity relationship of immune modulators. The chemical synthesis of lipopeptides provides diverse collections of defined molecules with variations in the peptide moiety, the number and character of the fatty acids, the stereochemistry of the unusual amino acid S-(2,3-dihydroxy-2(R)-propyl)-R-cysteine and its structural modifications.

In this study, a systematic approach was performed to investigate the influence of the different structural elements of lipopeptides with respect to their ability to induce IL-8 release via activation of TLR2. For this purpose, synthetic combinatorial N-palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl]-cysteine-peptide collections and lipopeptide analogues differing in number, length and structure of amide and ester bound fatty acid residues attached to dihydroxypropylcysteine were synthesized and investigated for biological activity. The dependence of TLR mediated cellular response on the chirality of carbon atom C-2 of the dihydroxypropyl moiety was investigated as well as influences of modifications of the dihydroxypropylcysteine core structure.

The lipohexapeptide amide collection is composed of lipopeptide amide mixtures, which have defined positions O and mixture positions X. The O positions represent 19 individually defined amino acids (all natural l-amino acids excluding cysteine) and the X positions are composed of mixtures containing 19 of all 20 amino acids (excluding cysteine). The collection contains 5 groups of subcollections Pam3Cys-OXXXX-NH2, Pam3Cys-XOXXX-NH2, Pam3Cys-XXOXX-NH2, Pam3Cys-XXXOX-NH2 and Pam3Cys-XXXXO-NH2. Each group is composed of 19 mixtures differing in the defined amino acid O. In total, 95 mixtures (5×19) were screened, each containing 130,321 lipopeptide amides, in total 12,380,495 lipopeptide amides. The screening of this lipopeptide amide collection provides information about the importance of the peptide moiety and the more or less effective residues at each position in respect to the induction of IL-8 release compared to the well described standard Pam3Cys-SKKKK.

Section snippets

Reagents

1-Hydroxybenzotriazole (HOBt), diisopropylethylamine (DIPEA), N,N′-diisopropylcarbodiimide (DIC), piperidine, 4-dimethylaminopyridine (DMAP), tetradecanoic acid, palmitic acid, arachidic acid, benzoic acid, phenylacetic acid, dichloromethane and dimethylformamide (DMF) were from Fluka. Hexanoic acid, octanoic acid, nonanoic acid, decanoic acid, oleic acid, stearic acid, linoleic acid were from Aldrich and trans-2-decenoic acid, dodecanoic acid were provided from Acros.

Protected l-amino acids

Results and discussion

Triacylated lipopeptides like Pam3Cys-SKKKK are described as strong immune modulators that activate the innate immune response via TLR2/1 heterodimers [26], [27], [29], [35], [36], [38]. To study the influence of the different structural elements of lipopeptides with respect to their ability to induce IL-8 release via activation of TLR2 and to identify new potent activators based on the parent lipoprotein structure, a systematic approach was performed. A fully synthetic combinatorial N

Acknowledgments

This study was supported by a grant from the BMBF, BioChance FKZ 0312662.

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