Study of ribosomal vaccine against experimental Staphylococcal skin infection in rabbit

Il s’agit d'un vaccin constitué d’une partie d'ARN ribosomal de Sta phylococcus (souche humaine de St. aureus, St. epidermidis) et d’une partie et demie environ de protéoglycanes membranal res de Klebsiella {Kl. pneumoniae, biotype a, non pathogène). La vaccination par voie sous-cutanée, deux fois à 15 jours d'intervalle, à la dose de 12,5 mcg (ARNr + protéoglycanes) protège le lapin contre une infection expérimentale, pratiquée 3 semaines après la dernière vaccina tion, par voie intradermique avec une souche pathogène de St. aureus isolée chez le lapin. La protection est appréciée par la réduction (76 %) et par la diminution de la nécrose de la surface réagissante cutanée en comparaison des témoins. La nature de la protection est ensuite discutée ainsi que le mécanisme général d’action des vaccins ribosomaux.The vaccine is composed of one part of Staphylococcus ribosomal RNA (human strains of St. aureus, St. epidermidis) and approximately of one part and half of Klebsiella glycoproteins membranes (Kl. pneumoniae, biotype a, apathogen). 12,5 meg of the vaccine (rRNA + glycoproteins membranes) are injected subcutaneously to each rabbit at 15 days-intervals. The challenge is performed three weeks later, after the second vaccination, by the intra- dermal injection of live pathogen Staphylococcus aureus strain isolated from rabbit. The protection is appreciated by the reduction (76 %) of the surface area of the lesions and by the diminution of the necrosis in comparison with the controls. The nature of the protection is discussed and the general mechanism activity of the ribosomal vaccines

Immunomodulation by microbial ribosomes

Over the past twenty years, many authors have reported evidence of the immunoprotective capacity of ribosomes isolated from bacteria, fungi and parasites. Since 1971 we have explored the protective capacity of ribosomes isolated from a large variety of microorganisms responsible for human and animal diseases. More recently, using monoclonal antibodies raised against ribosomes and then selected for their ability to confer passive immunity to mice, we have studied the mechanism of the protection induced by ribosomes. These studies, in parallel with the development of a technology for the large scale production of ribosomes, have allowed us to achieve a new regard for ribosomal vaccines for use in human. The general concept of ribosomal vaccines in presented and examples of two such vaccines are described with data on the specific protection that they induce in mice against experimental infections with Klebsiella peneumoniae, Streptococcus pneumoniae, S. pyogenes and Haemophilus influenzae for the first one, and against Candida albicans type A and type B for the second one. Because of their high immunogenicity and their innocuity these vaccines represent a decisive improvement over classical microbial vaccines

Role of acyl residues in polyclonal murine B cell activation by acylpoly(1,3)galactosides from Klebsiella pneumoniae.

Abstract Several components of Klebsiella pneumoniae including a membrane proteoglycan (Kp-MPG) were reported to activate macrophages and to induce T-independent polyclonal activation of mouse B cells. Chemically defined derivatives of Kp-MPG were prepared and characterized, enabling us to approach the molecular substructures involved in the binding to lymphocytes and the activation of B cells. Five derivatives were characterized: (i) an acylpoly(1,3)galactoside containing ester-linked fatty acids (EFA-APG) which was obtained by mild alkaline hydrolysis, (ii) a polymer of EFA-APG (APG pol1), (iii) a preparation obtained by drastic alkaline hydrolysis and delipidation which removed the esterified fatty acids (APG), (iv) a polymer of the latter compound (APG pol2), and (v) an APG preparation submitted to mild acid hydrolysis which removed all fatty acids but left the galactose chain of APG (GC-APG) intact. The derivatives were studied for their capacity to bind to and to activate mouse splenocytes. Binding was investigated on BALB/c and C3H/HeJ splenocytes by indirect immunofluorescence using biotinylated F(ab')2 of anti-Kp-MPG antibodies and the streptavidin-phycoerythrin amplification system in flow cytometry and by competition of unlabeled APG with biotinylated APG. Activation was studied by measuring (i) [3H]thymidine incorporation into spleen cells from BALB/c, C3H/HeJ, nude (nu+/nu+) mouse strains, and purified B cells of BALB/c; (ii) immunoglobulin secretion in culture supernatants; and (iii) blastogenesis. The results demonstrate a specific uptake of EFA-APG and APG by T cells as well as by B cells and exclude a contribution of the polygalactose part of the APG molecule (GC-APG) to the binding to spleen lymphocytes. Unlike LPS from the same strain of K. pneumoniae, APG pol1 stimulated B cell activation in the LPS-resistant C3H/HeJ strain as well as in BALB/c mice. The compounds did not activate T cells and were T-independent B cell activators, stimulating nu+/nu+ spleen cells and inducing primarily IgM and IgG3 synthesis. Polymers were more potent activators than monomers and removal of ester-linked fatty acids completely abrogated B cell-activating properties. The monomer APG antagonized B cell activation by Kp-MPG, LPS from K. pneumoniae, and APG pol1. The data indicate that within the EFA-APG molecule, distinct substructures are required for binding and for triggering B cell response.</jats:p

The animal lectin galectin-3 interacts with bacterial lipopolysaccharides via two independent sites.

Abstract Galectin-3 is a beta-galactoside binding protein expressed by activated macrophages, epithelial cells, and certain other cell types. Galectin-3 has a C-terminal carbohydrate binding domain, an N-terminal part consisting of a proline- and glycine-rich repetitive domain, and a small N-terminal domain. Two independent LPS binding sites on galectin-3 were demonstrated by binding of biotinylated LPS to immobilized recombinant galectin-3. One appears to be the carbohydrate binding site in the C-terminal domain that confers binding of LPS from Klebsiella pneumoniae that has a beta-galactoside-containing polysaccharide chain. This binding is best demonstrated using galectin-3 immunocaptured by a mAb to the N-terminal part (M3/38) and is inhibited by lactose. In contrast, Salmonella minnesota R7 LPS (Rd mutant), which is devoid of beta-galactosides, appears to bind to a site within the N-terminal part of galectin-3. This interaction is best demonstrated using galectin-3 directly immobilized in wells, and it is inhibited by the Ab M3/38, but not by lactose. Binding inhibition by polymyxin B and the profile of inhibition by a panel of LPSs with different amounts of the inner and outer cores present indicate that this second binding site recognizes the lipid A/inner core region of LPSs.</jats:p

The animal lectin galectin-3 interacts with bacterial lipopolysaccharides via two independent sites.

International audienceGalectin-3 is a beta-galactoside binding protein expressed by activated macrophages, epithelial cells, and certain other cell types. Galectin-3 has a C-terminal carbohydrate binding domain, an N-terminal part consisting of a proline- and glycine-rich repetitive domain, and a small N-terminal domain. Two independent LPS binding sites on galectin-3 were demonstrated by binding of biotinylated LPS to immobilized recombinant galectin-3. One appears to be the carbohydrate binding site in the C-terminal domain that confers binding of LPS from Klebsiella pneumoniae that has a beta-galactoside-containing polysaccharide chain. This binding is best demonstrated using galectin-3 immunocaptured by a mAb to the N-terminal part (M3/38) and is inhibited by lactose. In contrast, Salmonella minnesota R7 LPS (Rd mutant), which is devoid of beta-galactosides, appears to bind to a site within the N-terminal part of galectin-3. This interaction is best demonstrated using galectin-3 directly immobilized in wells, and it is inhibited by the Ab M3/38, but not by lactose. Binding inhibition by polymyxin B and the profile of inhibition by a panel of LPSs with different amounts of the inner and outer cores present indicate that this second binding site recognizes the lipid A/inner core region of LPSs

Le ciblage des macrophages : un nouveau concept pour l'imagerie des ganglions lymphatiques pathologiques

National audienc

CD14 and CD11b mediate serum-independent binding to human monocytes of an acylpolygalactoside isolated from Klebsiella pneumoniae

A water-soluble acylpolygalactosyl (APG) of 34 kDa was obtained from the Klebsiella pneumoniae membrane by alkaline hydrolysis and delipidation. APG comprises a poly(1,3)galactose chain, a core, and a lipid moiety made of a glucosamine disaccharide with two N-linked beta OH-myristates. The monocyte binding sites for APG were investigated by flow cytometry. Biotin-labelled APG (Biot-APG) bound to monocytes at 4 degrees C in the absence of serum, calcium, and magnesium. The binding was dose dependent, saturable, and displaced by unlabelled APG. Neither the polysaccharide chain present in APG-related molecules nor the PPi group or additional ester-linked myristates and palmitates were required for APG binding. The role of CD11b and CD14 was demonstrated by competitive inhibition with monoclonal antibodies and by the uptake of APG by these solubilized proteins. APG was rapidly internalized into monocytes at 37 degrees C while CD14 and CD11b/CD18 molecules were partially down-modulated. Lipopolysaccharides (LPS) from the same K. pneumoniae strain and from Escherichia coli and Salmonella minnesota partially competed for Biot-APG binding in the absence but not in the presence of serum. When altered by alkaline hydrolysis, those LPS became strong competitors for APG binding. It was concluded that alkaline hydrolysis of the K. pneumoniae membrane yielded molecules structurally related to LPS which bind to LPS membrane receptors in the absence of serum.</jats:p

Study of ribosomal vaccine against experimental Staphylococcal skin infection in rabbit

Il s’agit d'un vaccin constitué d’une partie d'ARN ribosomal de Sta phylococcus (souche humaine de St. aureus, St. epidermidis) et d’une partie et demie environ de protéoglycanes membranal res de Klebsiella {Kl. pneumoniae, biotype a, non pathogène). La vaccination par voie sous-cutanée, deux fois à 15 jours d'intervalle, à la dose de 12,5 mcg (ARNr + protéoglycanes) protège le lapin contre une infection expérimentale, pratiquée 3 semaines après la dernière vaccina tion, par voie intradermique avec une souche pathogène de St. aureus isolée chez le lapin. La protection est appréciée par la réduction (76 %) et par la diminution de la nécrose de la surface réagissante cutanée en comparaison des témoins. La nature de la protection est ensuite discutée ainsi que le mécanisme général d’action des vaccins ribosomaux.The vaccine is composed of one part of Staphylococcus ribosomal RNA (human strains of St. aureus, St. epidermidis) and approximately of one part and half of Klebsiella glycoproteins membranes (Kl. pneumoniae, biotype a, apathogen). 12,5 meg of the vaccine (rRNA + glycoproteins membranes) are injected subcutaneously to each rabbit at 15 days-intervals. The challenge is performed three weeks later, after the second vaccination, by the intra- dermal injection of live pathogen Staphylococcus aureus strain isolated from rabbit. The protection is appreciated by the reduction (76 %) of the surface area of the lesions and by the diminution of the necrosis in comparison with the controls. The nature of the protection is discussed and the general mechanism activity of the ribosomal vaccines