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.

Immunostimulatory properties of the bacterial extract OM-89 in vitro and in vivo.

The therapeutic agent OM-89 (Uro-Vaxom) contains lyophilized immunostimulating fractions from 18 Escherichia coil strains. It has been shown to provide protection against recurrent urinary tract infections in humans and against bacterial infections in mice. Here the immunostimulatory properties of OM-89 were investigated by in vitro and in vivo assays. In vitro the activation of murine spleen cells by the AlamarBlue assay was determined. OM-89 was effective in stimulating the metabolism of spleen cells within a concentration range of 0.625-2.5 mg/ml. The activation of murine bone marrow-derived macrophages by OM-89 was shown by the induction of NO production; OM-89 was a most effective stimulant at concentrations around 6 mg/ml. In the human system, the effect of OM-89 was tested in vitro:metabolic activity of peripheral blood lymphocytes (PBL) was stimulated starting at concentrations of approx. 250 microg/ml, and the spontaneous apoptosis of polymorphonuclear neutrophils (PMN) was reduced starting at OM-89 concentrations of approx. 100 microg/ml. Finally, in a mouse model, the in vivo protection of mice against infection with Salmonella typhimurium after the oral administration of OM-89 was tested (100 mg in a volume of 0.5 ml once a day for 10 consecutive days). The extract proved to be effective: 90% of the OM-89-treated animals survived compared to 58% of the untreated control group.

Human septin-septin interaction: CDCrel-1 partners with KIAA0202.

Septins are evolutionary conserved cytoskeletal GTPases forming heteropolymer complexes involved in cytokinesis and other cellular processes. CDCrel-1 (cell division cycle related-1) is a recently cloned and characterized human septin which is highly expressed in non-dividing cells, such as neurons. Using a yeast two-hybrid system we demonstrate that CDCrel-1 partners with another uncharacterized human septin, KIAA0202. The interaction of CDCrel-1 and KIAA0202 was confirmed in the human leukemia cell line K-562 using pull-down assays with a KIAA0202-glutathione S-transferase fusion protein and by immunoprecipitation of the CDCrel-1-KIAA0202 complex with an anti-KIAA0202 antibody. Expression studies of the two human septins revealed a concomitant expression of both proteins in certain cells.