Maria Huber

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.

Lipopeptides as lmmunoadiuvants and lmmunostimulants in Mucosal Immunization

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 Freunds 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, Peyers 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.

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.

Modulation of the Th1/Th2 Bias by Lipopeptide and Saponin Adjuvants in Orally Immunized Mice

We compared the adjuvanticity of the synthetic lipopeptide P3CSK4 of bacterial origin and the plant-derived adjuvant saponin using the wheat storage protein gliadin as antigen. Gluten sensitive BALB/c mice were orally immunized with gliadin in a mixture with either lipopeptide or saponin. The gliadin-specific serum IgG response was markedly enhanced by the saponin adjuvant. The lipopeptide adjuvant enhanced the IgG2a response, but reduced IgG1 production. In contrast, the saponin adjuvant enhanced both IgG2a and IgG1, and the sera showed elevated specific IgE concentrations. Enhanced specific IgA levels were detected in sera and in faeces especially after immunizations with gliadin in combination with P3CSK4 Enhanced specific IgG and IgA levels could also be detected in supernatants of cell cultures prepared from mesenteric lymph nodes and Peyers patches of immunized mice. Our data suggest that both adjuvants enhance the mucosal as well as the systemic immune response; P3CSK4 predominantly elicits the activation of the Th1 subset, whereas saponin activates both the Th1 and Th2 subser. Our findings are of importance for the improvement of mucosal immunizations, and might be a tool for the immunotheraphy of food allergies.

Bacterial cell wall components as immunomodulators—II. the bacterial cell wall extract OM-85 BV as unspecific activator, immunogen and adjuvant in mice

The bacterial extract Broncho-Vaxom used for the prevention and treatment of recurrent respiratory tract infections is an immunomodulator in vitro and in vivo, as determined in a murine model. The extract acts, on the one hand, as macrophage activator and polyclonal B-lymphocyte stimulant. On the other hand, after repeated intraperitoneal or oral immunizations, the extract is immunogenic, inducing serum IgG binding to the bacterial strains used for the preparation of the extract. On bacteria, the sera recognize the cell wall components porin, lipoprotein/lipopeptide and murein. The bacterial extract also exhibits adjuvant properties when applied in mixture with antigens, such as TNP-BSA or an influenza vaccine preparation. The unspecific and the immunospecific stimulatory effect of the extract as well as its adjuvant properties could be of importance for understanding its therapeutic effect.

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.

Passive vaccination with a human monoclonal antibody: Generation of antibodies and studies for efficacy in Bacillus anthracis infections

A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination.

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.

Immunogenicity of an E. coli extract after oral or intraperitoneal administration: induction of antibodies against pathogenic bacterial strains

For the treatment of recurrent infections of the urinary tract, a bacterial extract (OM-89) consisting of immunostimulating components derived from 18 Escherichia coli strains is orally applied to patients. We investigated in a mouse model the immunogenicity of the bacterial extract after intraperitoneal or oral administration. After repeated administration of the extract, serum IgG and IgA responses against the E. coli strains used for the preparation of OM-89 were obtained. This antisera also recognized a number of bacterial strains isolated from patients with urinary tract and enterohemorrhagic E. coli infections, and bound to a variety of other pathogenic strains. Moreover, the supernatants of cell cultures prepared from the urogenital tract of mice immunized with OM-89 contained increased levels of strain specific and of total IgG and IgA. Our findings may contribute to explain the therapeutic effect of OM-89 demonstrated in clinical studies.

Immunomodulating effects of OM-89, a bacterial extract from Escherichia coli, in murine and human leukocytes.

OM-89 (Uro-Vaxom) is a bacterial extract prepared from 18 uropathogenic Escherichia coli strains used for the prevention and treatment of recurrent infections of the urinary tract. The immunomodulating effects of the bacterial extract were investigated in a mouse model. After a single oral administration of OM-89, leukocyte activation was demonstrated ex vivo in blood and liver cells using a chemiluminescence assay. An increase of the production of tumor necrosis factor-alpha (TNF-alpha) in supernatants of peritoneal cells was also observed. After repeated oral administration of OM-89, increased serum immunoglobulin G responses against several E. coli strains were found. Also, adjuvant properties of the extract using ovalbumin as an antigen could be demonstrated. In line with these findings in the mouse system, preliminary in vitro data obtained in the human system showed an increase in TNF-alpha and interleukin-6 production after stimulation of monocyte derived dendritic cells with OM-89. The activation of immune cells is likely to be mediated via Toll like receptors (TLRs); thus, the binding of components of the extract to TLR-4 and marginally to TLR-2 could be shown.