Ribosomes as carriers for antigenic determinants of the surface of micro-organisms.

Over the past twenty-five 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 micro-organisms responsible for human and animal diseases. Accurate biochemical characterization of ribosomes always reveals trace amounts of non-ribosomal components such as short polysaccharides strongly linked to ribosomal RNA after phenol extraction even under denaturing conditions. rRNA-antigen complexes have been purified from Klebsiella pneumoniae ribosomes inducing high level of protection against homologous experimental infection in mice. Monoclonal antibodies raised against ribosomes and then selected for their ability to confer passive immunity to mice have been used to study the mechanism of the protection induced by ribosomes and to characterize their "immunogenic principle". These investigations have clearly shown the presence on ribosomes of epitopes corresponding to antigens normally exposed on the membrane of the bacteria. In the original concept of "ribosomal immunotherapy" that we have developed, ribosomes can be considered as natural carriers for cell surface epitopes, presenting them to the immune system in a highly immunogenic configuration.

Bacterial ribosomal immunostimulants prime alveolar macrophages in vivo to produce interleukin 1 in vitro.

Alveolar macrophages (AMs) may play a key role in human respiratory immune defenses, partially by synthesizing and releasing interleukin 1 (IL = 1). D53 (Ribomunyl), a composite bacterial ribosomal immunostimulant, has been recognized as an efficient prevention of respiratory tract infections. In vitro, D53 enhances the IL-1 production by mouse spleen adherent cells. A thymocyte proliferative response assay was used to evaluate the in vitro IL-1 production by AMs in healthy subjects who received D53 immunostimulant. Twelve nonsmoking healthy subjects took part in a prospective double-blind placebo control study. On day 1, a first bronchoalveolar lavage (BAL) was performed to assess IL-1 production by unstimulated and lipopolysaccharide (LPS) stimulated AM. Then, subjects were randomized to receive D53 (n = 6) or its placebo (n = 6) by both oral and subcutaneous injection routes from day 1 to day 15. On day 15, a second BAL was done and AM IL-1 production was again tested. IL-1 production on day 15 did not significantly differ from day 1 in both D53-treated and placebo groups either when AMs were unstimulated or were stimulated with concentrations of LPS resulting in maximal IL-1 production. However, in the D53-treated group, but not in the placebo group, IL-1 production induced by low LPS concentration (5 mg/L) was significantly higher (mean +/- SEM: 1,238 +/- 287 U/10(6) AM) on day 15 in comparison with day 1 (577 +/- 113 U/10(6) AM; p less than 0.05, Wilcoxon W test) and in comparison with the control group (day 15 IL-1 production induced by 5 mg/L LPS, 758 +/- 175 U/10(6) AM; p less than 0.05, Mann-Whitney U test). Moreover, in the D53-treated group, the optimal LPS concentration (ie, LPS concentration that induced maximal IL-1 production) was significantly lower on day 15 (mean +/- SD: 11 +/- 7 mg/L) than on day 1 (16 +/- 7 mg/L; p less than 0.05 Wilcoxon W test). We conclude that D53 immunostimulant in vivo primes AM to produce IL-1 following low LPS concentration stimulation. This may partially explain the protective effect of D53 immunostimulant against respiratory tract infection.

Phase II study of D.651, an oral vaccine designed to prevent recurrences of vulvovaginal candidiasis.

A vaccine has been prepared with ribosomes of Candida albicans serotypes a and b plus, as adjuvant, membrane proteoglycan from a nonencapsulated Klebsiella pneumoniae. A preliminary phase II trial without placebo control was conducted in 22 women with a history of frequent recurrences of vulvovaginal candidiasis (VVC). Initially, all the patients were treated locally with an antimycotic cream. Beginning at the same time, vaccine was taken orally in capsules containing 0.55 microgram active components. It was administered intermittently over six months, groups of women taking doses of two, three, six or nine capsules. Tolerance was excellent except for mild nausea, probably due to the excipients, in two patients taking nine capsules. Twenty patients completed the study. Only seven of them had a documented recurrence of VVC during the 6 months on vaccine. No recurrence occurred in the eight women taking six or nine capsules per day. Before the study, these 20 patients had had an average of 3.59 attacks of VVC per 6 months. On vaccine, the average rate of recurrence was only 0.55 attacks per 6 months. A multicentre placebo-controlled study of the efficacy of this vaccine is in progress to validate these encouraging preliminary results.

Proliferative response of human T lymphocytes to a vaccinal preparation of ribosomes from Streptococcus pyogenes.

The in vitro lymphocyte-activating properties of a ribosomal preparation of Streptococcus pyogenes were investigated. The preparation was mitogenic for human lymphocytes with a peak of 3H-thymidine incorporation occurring after 3-5 days of culture. The response was abolished by removal of CD3-positive cells and by alteration of accessory cells by exposure to L-leucine methyl ester. Most of the cells synthesizing DNA at the end of the culture expressed CD4 or CD8 but not CD20 antigens. No immunoglobulin synthesis was demonstrable. Although the same preparation was shown to be a T-independent polyclonal B-cell activator of murine cells, it preferentially triggers T cells in humans.

Polyclonal activation of murine B cells by a membrane proteoglycan of Klebsiella pneumoniae.

The lymphocyte activating properties of a membrane proteoglycan (MPG) extracted from a mutant non-encapsulated strain of Klebsiella pneumoniae (Kp) (biotype a I-145) were investigated. Kp MPG induced a strong proliferative response of BALB/c spleen cells and Peyer's patches cells. Thymidine incorporation was dose-related (from 1 to 100 micrograms Kp MPG/ml) and reached a maximum at day 3. It was not reduced by removal of most adherent cells, nor by depletion of Thy1-2 positive cells, but it was abrogated by removal of surface immunoglobulin bearing cells. Spleen cells from nude mice and those from C3H/Hej mice were strongly stimulated by Kp MPG. Conversely Kp MPG did not induce interleukin 2 production and did not trigger the proliferation of thymocytes but stimulated interleukin 1 production by adherent spleen cells. Finally, unfractionated or B-enriched spleen cells cultured with Kp MPG synthesized IgM and, to a lesser extent, IgG and IgA. It is concluded that Kp MPG is a T-independent polyclonal B cell activator and an inducer of interleukin 1 production.

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.

In vitro cell activating properties of the composite ribosomal vaccine D53

D53 (RibomuntyR) is a composite vaccine made of immunogenic ribosomes from 4 bacterial species (Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes and Streptococcus pneumoniae) associated with a membrane proteoglycan from a non encapsulated strain of Klebsiella pneumoniae. D53 is a potent inducer of interleukin-1 production by mouse BALB/c spleen cells as shown by the C3H/HeJ thymocyte co-stimulation assay. Furthermore D53 triggers DNA synthesis by mouse spleen cells and induces the maturation of B lymphocytes into immunoglobulin secreting cells. Polyclonal B cell activation by D53 was readily achieved in the C3H/HeJ strain which is deficient in its response to E. coli lipopolysaccharide. The proliferative response to D53 was abrogated by removal of B cells from the spleen cell suspension, but it was not altered after depletion of T cells or adherent cells. D53 induced polyclonal B cell activation of spleen cells from athymic nude mice and from CBA/N mice. Each component of D53 induced polyclona B cell activation except ribosomes from Streptococcus pneumoniae. Each triggered Interleukin-1 synthesis except ribosomes from Klebsiella penumoniae. These in vitro properties may account for some of the in vivo immunostimulating properties of this composite vaccine.

NK-cell stimulating properties of a membrane proteoglycane from non-capsulated Klebsiella pneumoniae biotype a.

Upon testing the individual fractions of a composite bacterial vaccine for biological activities, a potent immuno-stimulatory capacity could be demonstrated within a crude membrane proteoglycan preparation from Klebsiella pneumoniae. One of its characteristic features was the capacity to induce alpha-type interferon and increased NK activity in vivo in mice, following intraperitoneal or oral administration. A highly purified fraction from the crude preparation was obtained using alkaline hydrolysis, delipidation and size fractionation. This fraction was shown to be a very potent inducer of NK cells in vivo or in vitro, where in the latter systems concentrations as low as 0.1 microgramme per ml were highly efficient.

Role of the polysaccharide and of the lipopolysaccharide in the immunoprotective capacity of Klebsiella pneumoniae ribosomes.

Ribosomes, ribosomal RNA (r-RNA), capsular polysaccharide (PS-K) and lipopolysaccharide (LPS) were isolated and purified from Klebsiella pneumoniae, type I. The protective capacity of these different fractions was investigated in function of their analytical composition. The results show that ribosomes, and in particular, ribosomal RNA have the greatest protective activity at the lowest concentrations. The role of PS-K in the specific immune response to ribosomes was also investigated. Even at very high levels PS-K failed to afford any significant protection. LPS gave no protection. A stable r-RNA/PS-K complex was isolated by means of affinity chromatography. This complex was uncontaminated by LPS and its existence indicated strong bonding between surface polysaccharide antigens and r-RNA. The r-RNA/PS-K complex was not antigenic in itself but conferred good protection when combined with an adjuvant.

Study of the mode of action of ribosomal vaccines from Klebsiella and Streptococcus pneumoniae and their ribonucleic and protein fractions using passive immunization.

The vaccinating potency of ribosomal fractions of Klebsiella pneumoniae and Streptococcus pneumoniae as well as their ribosomal RNA and protein fractions has been studied with respect to their ability to induce cellular or humoral immunity. Experiments with transfer of serum or spleen cells from vaccinated animals have shown that anti-Klebsiella immunity is essentially cellular, while streptococcal immunity is exclusively humoral. Results have been discussed as a function of differential results for the various fractions under study.

An attempt to localize the vaccinating power of Klebsiella pneumoniae ribosomal preparations using saccharose-gradient ultracentrifugation.

The study of the vaccinating power of ribosomal vaccines against Klebsiella pneumoniae led us to define the chemical nature which supports this protective activity. We tried to separate this support and the ribosomes by sucrose gradient ultracentrifugation. We isolated high protective membrane vesicles by this technique applied to salt-washed ribosomal preparations. When the ribosomal preparations were exposed to SDS, the protective activity was conserved all along the gradient, with no correlation with the ribosome concentration. The addition of bovine serum albumin to the ribosomal preparation focused the protective activity on the ribosomal peak. No correlation was observed between the response to capsular polysaccharide and the vaccinating power of the fractions.

[Antigenic similarity between polysaccharide and ribosomal vaccine extracts from Klebsiella pneumonia]. / Similitude antigénique entre des extraits vaccinants polysaccharidiques et ribosomaux de Klebsiella pneumoniae.

We describe in this report the vaccinating power of a polysaccharidic preparation from Klebsiella pneumoniae type I. The essential part played by this extract in the vaccinating capacities of the ribosomal preparations from the same bacterium is demonstrated.

[Experimental infection by "Salmonella typhi-murium": protective role of homologous ribosomal extracts in calves (author's transl)]. / Etude chez le veau, de l'infection expérimentale par Salmonella typhi-murium: rôle protecteur d'extraits ribosomaux homologues.

Eleven calves, 6 months old, vaccinated or not, have been infected experimentally with 10(7) Salmonella typhi-murium, administered by oral route. The control calves had a serious illness, characterized by a severe diarrhoea, hepatic and renal symptoms and a heavy infestation of the main organs. The other five calves, which were orally and subcutaneously vaccinated with ribosomal extracts of S. typhi-murium and S. dublin showed only a moderate alteration of their health while biochemical disorders at the level of liver and kidneys disappeared. However, salmonella were found in mesenteric lymph nodes, but in much lower amounts than in controls.

Ribosomal vaccines: preparation of subcellular fractions.

Methods for isolation and analysis of subcellular bacterial fractions intended to be used in the preparation of ribosomal vaccines are described. Cultivation conditions in fermentors for obtaining maximal growth curve slopes are studied; cultivation is stopped before the end of the exponential growth phase in order to collect biomasses having a high viability rate, which is essential for the quality of the subcellular fractions isolated thereafter. Extraction methods for ribosomes and ribosomal RNA, as well as two proceedings for preparing adjuvant proteoglycans from Klebsiella pneumoniae membranes, are described. The analytical methods used for the control of these preparations and the results obtained for the various fractions are given.

Prevention of bacterial respiratory infection by an association of bacterial ribosomes and membranous proteoglycans. 2. Objective assessment of the response and of the immunological stimulation.

Using a vaccine preparation administered by aerosol for respiratory anti-infectious purposes and corresponding to the original formula of ribosomes and membrane fractions of microbial germs, the authors investigated during a period of nine months whether an objective, transient or lasting stimulation of the total and specific immunoglobulin (Ig) appeared. They provide a statistical analysis based on several serum Ig measurements and demonstrate the stimulant and lasting effect of the vaccine in the production of specific Ig for a sample of patients compared with controls. In those treated they observed apparently disordered variations of the serum levels of the total Ig, which in fact correspond to the initiation of a dynamic equilibrium in relation to the immunogenicity of the vaccine, the initial level of the total Ig and the production of the specific Ig. Finally, after a booster sequence carried out five month after "primary vaccination", they established the restarting of production of specific Ig accompanied this time by a different dynamic response of the total Ig.

Ribosomal vaccines: immunological study.

Ribosomal vaccines prepared from purified bacterial ribosomes induce the production of specific antibodies in female OF1 mice when administered to the animals both with incomplete Freund's adjuvant or purified Klebsiella pneumoniae cell wall proteoglycans. The study of these fractions concerned purified ribosomes extracted from the following bacteria: Klebsiella pneumoniae, Haemophilius influenzae, Steptococcus pneumoniae, Streptococcus pyogenes A12. Specific antibodies determination by immunoelectro diffusion (IED) and passive hemagglutination (PHA) were used to study the immune response.

Immuno-stimulation by a ribosomal vaccine associated with a bacterial cell wall adjuvant in humans.

We have studied a new vaccine of ribosomal nature associated with glycoprotein cell walls from Klebsiella pneumoniae which served as an immunoadjuvant. Thus vaccine was administered by the aerosol route to working men free of any important disease, especially of respiratory disease. A total of 104 men working for the Commissariat à l'Energie Atomique, all volunteers, were randomly placed into two groups. During the first period, 51 patients (group I) were vaccinated three times a week during 5 weeks, and the second group was used as control. During the second period, which started on day 225, the control group received the vaccine, and the first group was revaccinated. Results of this experience show a significant difference in the immunity of the two groups. The specific antibodies increased with vaccination as illustrated by chi-square test (Yates correction), which corresponds to an independent probability equal to 0 (P = 0.5 X 10-4).