Klaus Mittenbühler

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.

Immunostimulation by the synthetic lipopeptide P3CSK4: TLR4-independent activation of the ERK1/2 signal transduction pathway in macrophages

Synthetic lipopeptides based on bacterial lipoprotein are efficient activators for monocytes/macrophages inducing the release of interleukin (IL)‐1, IL‐6, tumour necrosis factor‐α (TNF‐α), reactive oxygen/nitrogen intermediates, and the translocation of nuclear factor κB (NFκB). In this report we investigate the signal transduction pathways involved in leucocyte activation by the synthetic lipopeptide N‐palmitoyl‐S‐[2,3‐bis(palmitoyloxy)‐(2R,S)‐propyl]‐(R)‐cysteinyl‐seryl‐(lysyl)3‐lysine (P3CSK4). We show that P3CSK4 activates mitogen‐activated protein (MAP)‐kinases ERK1/2 and MAP kinase (MAPK)‐kinases MEK1/2 in bone‐marrow‐derived macrophages (BMDM) and in the macrophage cell line RAW 264·7. Additionally, we could detect differences between the P3CSK4 and lipopolysaccharide (LPS)‐induced phosphorylation of MAP kinases: Different levels in phosphorylation were found both in kinetics and dose–response using RAW 264·7 cells or BMDM from BALB/c and LPS responder mice (C57BL/10ScSn) or LPS non‐responder mice (C57BL/10ScCr). The lipopeptide activated the MAPK‐signalling cascade in both LPS responder and non‐responder macrophages, whereas LPS induced the MAPK signalling pathway only in macrophages derived from LPS responder mice. An approximately 70% decrease of lipopeptide induced NFκB translocation and an about 50% reduction of nitric oxide (NO) release was observed in the presence of anti‐CD14. These data correspond to the reduction of phosphorylation of ERK1/2 after stimulation with P3CSK4 in the presence of anti‐CD14 antibodies. Inhibition of MEK1/2 by PD98059 completely reduced the lipopeptide‐induced phosphorylation of ERK1/2 indicating that MEK1/2 are solely responsible for the phosphorylation of the downstream‐located MAP kinases ERK1/2.

Bacterial cell wall components as immunomodulators—I. lipopeptides as adjuvants for parenteral and oral immunization

We investigated the immunostimulatory properties of synthetically prepared lipopeptides derived from the cell wall of Gram negative bacteria. These compounds constitute potent macrophage activators and polyclonal B-lymphocyte stimulators. They are also immunoadjuvants in parenteral or oral immunization. By coupling the lipopeptides to haptens or low molecular weight antigens which are not immunogenic per se, highly immunogenic conjugates can be prepared. Lipopeptide antigen conjugates as synthetic vaccines give protection by enhancing the antibody-mediated immune response, and they stimulate the cellular immune response in vivo by priming of cytotoxic T-cells.

Generation of antibodies directed against the low-immunogenic peptide-toxins microcystin-LR/RR and nodularin.

The preparation of antibodies against the liver toxin microcystin, as described here, is of major importance for its detection and purification in food and water, and for a therapeutic approach to neutralize the toxin by passive immunization. Microcystin-LR (MLR) and microcystin-RR (MRR) were purified from cyanobacterial cell materials by extraction, Sephadex LH-20-, ODS silica gel-, ionic exchange and RP-HPLC-chromatography. In order to reduce the toxicity for parenteral administration, microcystins were coupled by the carbodiimide method to poly-L-lysine (PLL(50.000)). Mice and rabbits were immunized with the conjugates in the presence of two lipopeptide immunoadjuvants (P(3)CSK(4) and P(3)CS-T(h)). High MLR-specific antibody levels were observed after parenteral coadministration of antigen and lipopeptides, whereas no anti-MLR antibodies were obtained with free microcystin or the microcystin-PLL(50.000)-conjugate in the absence of lipopeptide. In oral immunization, coadministration of antigen and adjuvants resulted in an accelerated development of anti MLR-specific antibodies and high antibody levels. Using the antisera, we could detect different microcystins and nodularin down to a concentration range of 10-50 ng/ml by a competitive inhibition ELISA; detection of microcystins in crude cell preparations was also possible. Furthermore, microcystins from different sources could be detected and discriminated from cyclic cyanopeptolines.

Drug specific antibodies: T-cell epitope-lipopeptide conjugates are potent adjuvants for small antigens in vivo and in vitro

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.

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.

Lipopeptide adjuvants in combination treatment

Synthetic lipopeptides derived from the N-terminus of bacterial lipoprotein constitute potent immunoadjuvants for parenteral and mucosal immunization. When combined with tetanus toxoid (TT) or gliadin as antigens, the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (P(3)CSK(4)) markedly enhanced the specific antibody levels. Lipopeptides also act as macrophage/monocyte activators: P(3)CSK(4) induced nitric oxide release from bone marrow-derived macrophages (BMDM) of LPS responder and nonresponder mice. The antitumoral effect of the lipopeptide was demonstrated by a strong cytostatic activity of the lipopeptide-treated macrophages against the murine B-cell lymphoma cell line Abelson 8-1. The chemically well-defined lipopeptides can be synthesized with high purity and reproducibility and constitute ideal agents to be combined with antigens/vaccines or antitumor treatment.

Modulation of the Th1/Th2 bias by an immunoglobulin histamine complex in the ovalbumin allergy mouse model

Vaccination with the antiallergic drug Histaglobin is used to treat a broad range of human allergic diseases including bronchial asthma, allergic rhinitis, and atopic dermatitis. In order to further elucidate its functional activity, Histaglobin was investigated in an in vivo mouse allergy model. Mice were sensitized with ovalbumin either prior to or after Histaglobin treatment, and its antiallergic potential was evaluated. Ovalbumin-sensitized mice exhibited increased serum levels of IL-4, tumor necrosis factor alpha (TNF-alpha), and an increase of total and ovalbumin-specific IgE; total and ovalbumin-specific IgG levels were also elevated. Subsequent administration (therapeutic treatment) of Histaglobin resulted in a decrease of total and specific serum IgE levels; total and specific IgG1 serum levels were reduced by more than 50% and 45%, respectively; the mice displayed a down-regulation of IL-4 and TNF-alpha serum levels and showed increased levels of IFN-gamma and IgG2a. Mice pretreated with Histaglobin, prior to ovalbumin sensitization (prophylactic treatment), were found to be widely unresponsive to ovalbumin. They exhibited higher serum levels of IFN-gamma and IgG2a (total and specific) compared to saline-treated control mice. The inhibitory effects were still observed 1 month post-immunization. Our data, indicating a Histaglobin-induced modulation of the Th1/Th2 balance in favour of Th1, correspond with the well-known antiallergic activity of Histaglobin observed in patients.

Characterization of different forms of yeast acid trehalase in the secretory pathway

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec 18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.

Lipopeptides: adjuvanticity in conventional and genetic immunization

Synthetic lipopeptides derived from the bacterial cell wall component lipoprotein activate B-lymphocytes and macrophages/monocytes in vitro. In vivo they constitute potent immunoadjuvants for a broad range of different antigens and species comparable or superior to Freunds adjuvant. Here, we demonstrate that P(3)CSK(4), representing a highly active lipopentapeptide derivative in vitro, significantly enhances and accelerates the humoral immune response to tetanus toxoid. P(3)CSK(4) could substitute for up to 90% of the antigen without any decrease in the specific IgG level, and the presence of the lipopeptide resulted in a prolonged production of specific IgG in time. Investigations using P(3)CSK(4) as an adjuvant in genetic immunization confirmed earlier data demonstrating that lipopeptides constitute adjuvants for low-immunogenic DNA constructs and/or for application routes resulting in weak immune responses. We monitored a lipopeptide-dependent shift from a Th1-type to Th2-type response, when DNA immunization was followed by i.p. administration of the lipopeptide adjuvant.