The present invention relates to an Immunogenic Complex comprising Ribosomal Complex and Adhesion of a Microbe or Ribosomal Complex and a viral antigen. The Ribosomal Complex is composed of the subunits of ribosomes (50 S and 30 S subunits in bacteria and 60 S and 40 S subunits of eucaryotes), the ribosomal subunits generally retaining sufficient integrity to preserve substantially the double-stranded nature of the large r-RNA's (16 S and 23 S in bacteria; 18 S and 28 S in eukaryotic cytosol) contained in the ribosomal subunits.

Description of the Invention

SUMMARY OF THE INVENTION

The invention provides an immunogenic complex containing at least ribosomal complex and proteineous adhesins of microbes. The unique and deliberate combination of both elements of the immunogenic complex is surprisingly superior in immune induction and immune protection against the target microbial pathogen than either component alone and constitutes active ingredients of superior prophylactic or therapeutic vaccines.

The Immunogenic Complex can be used as a prophylactic vaccine to prevent establishment of diseases but can also be used as a therapeutic agent thanks to its immune-stimulatory effect and effective antigen presenting capability and consequently complements the natural defense against disease. Methods of use, manufacture and administration of pharmaceutical compositions containing above mentioned immunogenic complex are also described.

The present invention thus provides an Immunogenic Complex comprising at least one Ribosomal Complex and (a) at least one Adhesin of a Microbe; or (b) at least one antigen of a virus. Preferably, the Ribosomal Complex is composed of the large and small subunits of ribosomes which are particulate in nature, and/or carries minor fractions of the microbial cellular membrane or cell wall--components. Preferably, the Ribosomal Complex retains sufficient integrity to substantially preserve the double-stranded nature of the large r-RNA's contained in the subunits of ribosomes.

Preferably, an Adhesin is any protein embedded in or on the surface of any Microbe, which protein is involved in the attachment of the Microbe to the host cell surface, optionally any protein structurally similar to, or any polypeptide derived from or corresponding to part of any protein embedded in or on the surface of a Microbe which can still induce an antibody response to said protein. The host cells are typically eukaryotic cells belonging to vertebrate animal groups aves, Pisces and mammalia, including humans, most preferably absorptive enterocytes, M-cells, dendritic cells, macrophages, erythrocytes, fibroblasts and / or epithelial cells.

Preferably, an Immunogenic Complex of the invention comprises Adhesin that binds to components of the extra-cellular matrix that embed host cells. Preferred extracellular matrix components are selected from fibronectin, laminin, collagen, fibrogen, vitronectin or heparin sulfate or analogues, homologues or derivatives of these. In preferred examples, the Adhesin is a protein included in colonization factor antigens present in bacterial fimbriae, or a protein included in colonization factor antigens present in fungal hyphae. Also encompassed is an Immunogenic Complex as descroned comprising Ribosomal Complex and Adhesin which originate from multiple Microbe species, wherein Microbe species is any species belonging to bacteria and/or fungi and/or protozoae. Provided also is an Immunogenic Complex of the invention, where the Ribosomal Complex and Adhesin which originate from multiple Microbe species, whereby from one or more species of Microbes, only Ribosomal Complex but not Adhesin or only Adhesin and not Ribosomal Complex, is included (herein called Heterologous Immunogenic Complex). According to the embodiments of the invention, Ribosomal Complex and/or Adhesin preferably originate from a microbe selected from the group consisting of: Actinobacillus actinomycetemcomitans; Bacille Calmette-Guerin; Blastomyces dermatitidis; Bordetella pertussis; Campylobacter consisus; Campylobacter recta; Candida albicans; Capnocytophaga sp.; Chlamydia trachomatis; Eikenella corrodens; Entamoeba histolitica; Enterococcus sp.; Escherichia coli; Eubacterium sp.; Haemophilus influenzae; Lactobacillus acidophilus; Leishmania sp.; Listeria monocytogenes; Mycobacterium vaccae; Neisseria gonorrhoeae; Neisseria meningitidis; Nocardia sp.; Pasteurella multocida; Plasmodium falciparum; Porphyromonas gingivalis; Prevotella intermedia; Pseudomonas aeruginosa; Rothia dentocarius; Salmonella typhi; Salmonella typhimurium; Serratia marcescens; Shigella dysenteriae; Streptococcus mutants; Streptococcus pneumoniae; Streptococcus pyogenes; Treponema denticola; Trypanosoma cruzi; Vibrio cholera; and Yersinia enterocolitica.

In further aspects, the invention comprises a Bacterio-viral Immunogenic Complex according to the invention, characterized in that viral antigen originates from a virus selected from the group consisting of: Influenza virus; parainfluenza virus; rhinovirus; hepatitis A virus; hepatitis B virus; hepatitis C virus; apthovirus; coxsackievirus; Rubella virus; rotavirus; Denque virus; yellow fever virus; Japanese encephalitis virus; infectious bronchitis virus; Porcine transmissible gastroenteric virus; respiratory syncytial virus; Human immunodeficiency virus; papillomavirus; Herpes simplex virus; varicellovirus; Cytomegalovirus; variolavirus; Vacciniavirus; and suipoxvirus. In a preferred Immunogenic Complex according to the invention, Ribosomal Complex and Adhesin or viral antigen are present in weight ratios respectively from 1 to 20 and 20 to 1.

Preferably, the Ribosomal complex and Adhesin or viral antigen are incorporated in a polymeric matrix, for example a matrix comprising or consisting of chitosan-EDTA Bowman-Birk Inhibitor conjugate. In other embodiments, the Ribosomal complex and Adhesin or viral antigen are incorporated in microparticles. In other embodiments of the Immunogenic Complex according to the invention, Ribosomal Complex and Adhesin or viral antigen which are non-covalently bound to each other by ionic interactions, or are covalently coupled. Coupling can be chemically achieved using N-hydroxysuccinimidyl esters, bis-imido esters or preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) or glutaraldehyde.

Also encompassed by the present invention are Pharmaceutical Composition for prevention and treatment of infectious disease caused by Microbe or virus, comprising Immunogenic Complex according to the invention, wherein the Immunogenic Complex is formulated as a pharmaceutically acceptable delivery form for administration to animals and/or humans. Pharmaceutical composition according to the invention can be used as prophylactic vaccine against a Microbe or a virus, in particular a Microbe as described herein. Preferably an immune response is activated against antigens, including Adhesins, expressed by the Microbes during their established pathogenic phase.

Embodiments according to the invention include Pharmaceutical composition to prevent or control disease caused by toxigenic E. coli, wherein Ribosomal Complex is derived from E. coli and includes as Adhesin, Fae G fimbrial protein, or proteins structurally similar to, or any polypeptide derived from or corresponding to part of Fae G protein, which can still induce an antibody response to Fae G.

Also embodied are Pharmaceutical composition to prevent or control Candida infection, wherein Ribosomal Complex is derived from Candida albicans and includes as Adhesin, ALA1 of C. albicans or a protein structurally similar to, or any polypeptide derived from ALA1, or corresponding to part of the Ala1 gene product, which can still induce an antibody response to ALA1.

Included also in the invention are Pharmaceutical composition to prevent or control Candida infection, wherein Ribosomal Complex is derived from Candida albicans and includes as Adhesin, the HWP1 protein of C. albicans or a protein structurally similar to, or any polypeptide derived from HWP1 or corresponding to part of the hwp1 gene product, which can still induce an antibody response to HWP1.

Included also is a Pharmaceutical composition to prevent or control periodontal disease associated with colonization or infection by Porphyromonas gingivalis, wherein the Ribosomal Complex is derived from P. gingivalis; or a Pharmaceutical composition to prevent or control periodontal disease associated with colonization or infection by Porphyromonas gingivalis and/or Treponema denticola, wherein Ribosomal Complex is derived from P. gingivalis and T. denticola and includes as Adhesin, the Msp protein of T. denticola, or a protein structurally similar to, or any polypeptide derived from Msp or corresponding to part of msp gene product, which can still induce an antibody response to Msp. Embodied also is a Pharmaceutical composition to prevent or control periodontal disease associated with colonization or infection by Porphyromonas gingivalis and/or Campylobacter rectus and/or Treponema denticola, wherein Ribosomal Complex is derived from P. gingivalis, C. rectus and T. denticola and includes as Adhesin, the Msp protein of T. denticola, or a protein structurally similar to Msp, or any polypeptide derived from or corresponding to part of msp gene product, which can still induce an antibody response to Msp.

The Immunogenic Complex or the pharmaceutical composition can be used in the preparation of a medicament for prophylaxis or treatment of infectious diseases in humans or in animals, or for prophylaxis or treatment of toxigenic E. coli infection, for prophylaxis or treatment of Candida infection, for prophylaxis or treatment of periodontal disease, for prophylaxis or treatment of respiratory diseases in humans or in animals, for prophylaxis or treatment of diseases caused by respiratory syncytial virus in humans or in animals, or for prophylaxis or treatment of periodontal disease caused or aggravated by any one of following bacteria: Porphyromonas gingivalis, Campylobacter rectus, Treponema denticola.

Also encompassed is therefore a method of treating infectious diseases in humans or animals, or of providing prophylaxis in respect to said diseases, comprising administrating to said humans or animals an effective amount of the Immunogenic Complex or of the pharmaceutical composition of the invention. Preferably the method is for the treatment or prophylaxis of diseases caused by toxigenic E. coli infection, caused by Candida infection, caused or aggravated by periodontal bacteria, or caused or aggravated by any one of following bacteria: Porphyromonas gingivalis, Campylobacter rectus, Treponema denticola; or caused or aggravated by respiratory syncytial virus.

Also included in this invention are methods for the manufacture of the Immunogenic Complex of the invention comprising admixing Ribosomal Complex and Adhesin of one or multiple Microbes. Preferably the Ribosomal Complex and Adhesin are incorporated in a polymeric matrix containing chitosan-EDTA Bowman-Birk Inhibitor conjugate, or are incorporated in microparticles containing carboxymethylethylcellulose-coated poly[dl-lactide-co-glycolide]. In other methods, the Ribosomal Complex and Adhesin are non-covalently bound to each other by ionic interaction, or are covalently coupled to each other. Coupling can be achieved preferably using N-hydroxysuccinimidyl esters like disuccinimidyl suberate or N-succinimidyl-(3-[2-pyridyl]-dithio)propionate (SPDP), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), bis-imido esters such as dimethyladipimidate or glutaraldehyde. The pharmaceutical composition may also involve admixing a pharmaceutically acceptable carrier, diluent or other excipient.

Also envisioned are methods of administration of the Immunogenic Complex or the pharmaceutical composition according to the invention to humans and/or animals. Examples include oral administration of the Immunogenic Complex or the pharmaceutical composition upon suspension in a drinkable liquid, Topical administration of the Immunogenic Complex or the pharmaceutical composition contained in a liquid, a gel or cream and applied to epithelial cell surfaces, in particular to surfaces of infected or infection-prone areas, nasal administration of the Immunogenic Complex or the pharmaceutical composition contained in a liquid aerosol or droplet dispenser, by inhalation upon containment in a peroral liquid or dry powder aerosol, and Rectal or vaginal or uteral application of the Immunogenic Complex or the pharmaceutical composition contained in a suppository or as a gel or cream.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to immunogenic complexes, the production and formulation thereof, methods of application and the use of immunogenic complexes as either prophylactic vaccines or therapeutic agents in Pharmaceutical compositions. It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

As used herein, the term "Microbes" refers to bacteria, protozoa and fungi.

As used herein, the term "Ribosomal Complex" refers to a complex which is essentially composed of the subunits of ribosomes (50 S and 30 S subunits in bacteria and 60 S and 40 S subunits in eucaryotes) which carry on their surface minor fractions of the microbial cellular membrane or cell wall components.

An important feature of the invention is that the ribosomal subunits in the Ribosomal Complex retain sufficient integrity to preserve substantially the double-stranded nature of the large r-RNA's (16 S and 23 S in bacteria; 18S and 28S in eukaryotic cytosol) contained in the ribosomal subunits.

The Ribosomal Complex is largely particulate in nature. This granular (versus soluble) structure is another important feature of Ribosomal Complex, which plays a role in efficient uptake of the Ribosomal Complex by lymphoid cells such as M cells and Dendritic cells. The effect of Ribosomal Complex is to function as a carrier for additional antigens, to ensure effective endocytosis of the Immunogenic Complex by host lymphoid cells such as M-cells or Dendritic cells at inductive sites of the immune system and to produce an adjuvant effect which boosts the immune response.

As used herein, the term "Adhesin" refers to any protein embedded in or on the surface of Microbes that is involved in the attachment to host cells such as absorptive enterocytes, M-cells, dendritic cells, macrophages, erythrocytes, fibroblasts and epithelial cells or in binding to components of the extra-cellular matrix that embeds host cells such as fibronectin, laminin, collagen, fibrogen, vitronectin, heparin sulfate. "Adhesin" also includes any polypeptide derived from or corresponding to part of such protein that can still induce an immune response against said Adhesin. "Adhesin" also includes the protein complexes of colonization factor antigens such as those present in bacterial fimbriae and fungal hyphae.

The effect of including Adhesin in the immunogenic complex is to enhance the immune response specifically against said adhesins and further contribute to effective immune exclusion of the target micro-organisms antibody-mediated or cellular immune responses. Microbes may express multiple Adhesins on their surface and recognize a variety of host extra-cellular matrix proteins or host cell types.

As used herein, the term "Immunogenic Complex" refers to a complex containing at least following elements: Ribosomal Complex and Adhesin of a Microbe.

An Immunogenic Complex can contain Ribosomal Complex and Adhesins of several species of Microbes. This is particularly advantageous for disease prevention and/or treatment of diseases, which may be caused or aggravated by multiple pathogens (e.g. periodontal disease), or by sequential pathogens (e.g. common cold, angina, bronchitis).

As used herein, the term "Heterologous Immunogenic Complex" refers to an Immunogenic Complex comprising Ribosomal Complex and Adhesin which originate from different, or multiple Microbes, whereby from one or more species of Microbes, only Ribosomal Complex but not Adhesin or only Adhesin and not Ribosomal Complex, is included.

This is particularly advantageous in cases where an immune response is desirable against a given pathogen in a complex of Microbes against which one wishes to use the Heterologous Immunogenic Complex but for which pathogen the use of both Ribosomal Complex and Adhesin is not desirable, and it is preferable to include only Ribosomal Complex or only Adhesin of this Microbe. Concerning Adhesins, this could be the case when, for example, the Adhesin is not or poorly characterized (e.g. Campylobacter rectus) or are difficult or expensive to isolate without loss of their immunogenicity, or induce an immune response which cross-reacts with host tissue. Concerning Ribosomal Complex, examples where it may be desirable to leave this component out for a certain Microbe against which one wishes to produce an immune response, are in cases where the Microbe is difficult or expensive to produce in large quantities (e.g. many oral treponemes associated with periodontal problems); another obvious reason is where an immune response is induced which cross-reacts with host tissue (observed with Streptococcus pyogenes A).

As used herein, the term "Bacterio-viral Immunogenic Complex" refers to an Immunogenic Complex, containing at least following elements: Ribosomal Complex of bacteria and an Antigen of virus.

Similarly to Immunogenic Complex, the Bacterio-viral Immunogenic Complex is advantageous for disease prevention and/or treatment of diseases resulting from infection by several pathogens. In particular are concerned diseases which may initiate as result of viral infection which facilitate colonisation of bacterial pathogens, that super-infect and aggravate and prolong the disease (e.g. Common Cold, Broncheolitis, diarrhoea's, Meningitis caused by Neisseria meningitis following infection by respiratory syncytial virus, etc.).

As used herein, the term "antigen" refers to any macro-molecule including protein, glyco-protein, polypeptide, polysaccharide, lipo-polysaccharide, that is able to interact specifically with an Antigen recognition molecule of the immune system, such as an antibody (immunoglobulin) or T cell-antigen receptor. An antigenic portion of a molecule can be the portion that is immuno-dominant for antibody or T cell receptor recognition, or it can be a portion of such protein, which when fused to a carrier molecule for immunization, is capable of inducing specific Antigen recognition molecules that will bind to it. A molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier molecule.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar undesirable reaction, such as gastric upset, dizziness, fever and the like, when administrated to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means fulfilling the guidelines and approval criteria of a European Community country's Drug Registration Agency concerning products to be used as a drug, or means that the pharmaceutically acceptable compound, composition, method or use, is listed in the European Community country's Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

The term "pharmaceutical carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers include but are not limited to sterile liquids, such as water and oils, including those of petroleum, oil of animal-, vegetable-, or synthetic origin, such as whale oil, sesame oil, soybean oil, mineral oil and the like. Water or, aqueous solutions, saline solutions, and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions, droplet-dispensed solutions and aerosols.

The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response Preferably, the adjuvant is pharmaceutically acceptable.

Protocols describing the preparation of Ribosomal Complex (RC) from Microbes are available in the literature and can be adapted where needed by those skilled in the art. For example, the preparation of RC from bacteria can be done essentially as described by Youmans and Youmans, 1965 and adapted as described by Gregory et al., 1983. Briefly, in the case of RC preparation from bacteria, the bacterial culture is grown in regular broth at a temperature and atmospheric conditions optimal for the species. Subsequently the cells, whilst still in log phase growth, are rapidly cooled to 10.degree. C., harvested by low-speed centrifugation (10.000.times.g for 10 min.), washed three times in a phosphate buffer (0.01M, pH 7.0) containing 0.01 M MgCl.sub.2 (PMB) and frozen at -80.degree. C. In general, but particularly when using virulent Microbes (pathogenic), is recommended to kill the cells prior to further use, for example by treatment with formalin as described by Michalek and McGhee, 1977, and adjust concentrations to 10.sup.8 bacterial or fungal cells/ml or 10.sup.7 protozoa/ml. The preparation can be established to be sterile when no multiplication occurs upon inoculation on Sheep blood and Mitis Salivarius agars (DIFCO) or other adapted rich culture medium. Aliquots are stored at -80.degree. C. Subsequently they are thawed rapidly at 37.degree. C., and 1 g of whole cells is re-suspended with 1 g of micro-glass beads (0.17-0.18 mm) in 1 ml of PMB to which 3 .mu.g/ml Dnase (SIGMA ) is added. The cells are disrupted by shaking for three 2-minute cycles in a Braun homogenizer. Intact cells and debris are removed by two centrifugations (27.000.times.g followed by 47.000.times.g; 10 minutes each).

Preparation of ribosomes from fungi and protozoa follow essentially the same procedure but require adaptation of culture conditions and lysis methods. Given that culture conditions of cultivatable pathogenic Microbes are widely available in published literature, preparation of ribosomes from such Microbes is well within the possibilities of a person skilled in the art.

Integrity of the ribosomal subunits is important. In particular the stabilization of enclosed large ribosomal RNA's by divalent cations such as provided by MgCl.sub.2, concentration which may need adaptation depending on the Microbe and extraction protocol, method which the man skilled in the art shall know to adapt. The ribosomes in the supernatant can be harvested by centrifugation at 180.000 to 250.000.times.g for 2 to 3 hr and then subjected to 5 successive washes in PMB at 180.000 to 250.000.times.g for 2 to 3 hr each. The ribosomal preparation is then clarified twice by two 20-min. centrifugations at 47.000.times.g and the supernatant is filtered through a sterile 0.45 .mu.m Millipore filter (Millipore Filter Corp.). Non-dissociated (=intact) ribosomes can be prepared from gram-negative, Rnase-minus mutant bacteria such as Escherichia coli MRE600 following the method of Staehilin et. al., 1969, with modifications as described by M. M. Yusupov and A. S. Spirin. 1988. The preparations can then adjusted to, for example, 20 mg/ml on the basis of protein content by standard protein quantification methods, using, for example, bovine serum albumin as a standard, and maintained at -80.degree. C. until used. Characterization of the ribosomal fraction and purity can be determined by spectral analysis at 235, 280 and 260 nm in order to determine the contamination of ribosomal RNA by DNA. polyacrylamide gel electrophoresis permits to evaluate the presence of ribosomal proteins and potential contaminating proteins. The degree of intactness can be evaluated by loading a sample of the original homogenate onto a 10% to 40% sucrose gradient, containing an appropriate concentration of Mg Cl.sub.2 and centrifugation. The elusion profile of the sucrose gradient will show the different fractions: 100S=dimers of 70S ribosomes, 70S=intact ribosomes, 60S=interacting 50S and 30S ribosomal subunits, 50S=large ribosomal subunit, 30S=small ribosomal subunit, material less than 30S=degradation products and contaminants. In good preparations that target non-dissociated ribosomes, the 70S peak contains over 80% of all material. Optionally, the 70S peak containing the target non-dissociated ribosomes may constitute at least 50%, 60%, 70% or 90% of all material.

The term "isolated" requires that the material be removed from its original environment (e. g., the natural environment if it is naturally occurring). For example, a naturally-occurring polypeptide, antigen or Ribosomal Complex or ribosomal subunit present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such an antigen, polypeptide, Ribosomal Complex or ribosomal subunit could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.

The term "purified" does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material (e.g. antigen, polypeptides, ribosomal subunits or Ribosomal Complex) to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. As an example, purification from 0.1% concentration to 10% concentration is two orders of magnitude. The term "purified" is further used herein to describe an antigen, polypeptide, ribosomal subunits or Ribosomal Complex which has been separated from other compounds including, but not limited to, polypeptides, antigens, or polynucleotides, carbohydrates, lipids, etc. The term "purified" may be used to specify the separation of monomeric polypeptides of the invention from oligomeric forms such as homo- or hetero-dimers, trimers, etc. A polynucleotide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polynucleotide sequence and conformation. A substantially pure polypeptide, antigen, ribosomal subunit or Ribosomal Complex typically comprises about 50%, preferably 60 to 90% weight/weight of a polypeptide, antigen, ribosomal subunit or ribosomal RNA sample, respectively, more usually about 95%, and preferably is over about 99% pure. Polypeptide, antigen, ribosomal subunit or Ribosomal Complex purity, or homogeneity, is indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.

Adhesins have been widely studied for many Microbes and protocols describing the preparation of Adhesins either directly from Microbes or after cloning of the Adhesin-coding genes in expression vectors, transformation of resulting expression cassettes into appropriate organisms or eukaryotic cells and heterologous production of said Adhesins, are available in the literature and can be adapted where needed by those skilled in the art. A few examples of microbial Adhesins useful in the preparation of Immunogenic Complex are: the products of Staphylococcus aureus genes fnbA and fnbB, encoding 110 and 98 kDa proteins respectively; the porin OmpC protein of Salmonella typhimurium; polypeptide segments PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, corresponding respectively to amino acid sequences of the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin protein are examples of polypeptide Adhesins; Colonization Factor Antigen CFA/II of entero-toxigenic Escherichia coli; full length Streptococcus mutans non-fimbrial cell surface antigen SA I/II; the polypeptide derived from SAI/Il, that spans the residues 1025-1044 in the C-terminal domain, is another example of a polypeptide Adhesin; WI-1 adhesin of Blastomyces dermatitidis yeast; surface Adhesin Fab1 of Streptococcus parasanguis; Porphyromonas gingivalis fimbrial proteins, fibrillous M-protein of Group A Streptococci; filamentous hemagglutinin of Bordetella pertussis.

It will be appreciated that viral antigens to be used in accordance with the present invention can be isolated and purified according to means known in the art. Examples of viral antigens that can be used in the Bacterio-viral Immunogenic Complex include the fusion (F) protein of respiratory syncytial virus (RSV); the attachment G glycoprotein of RSV; the antigen portion comprising the junction of the glycoprotein G with the fusion protein F of RSV; the central conserved domain of the G glycoprotein of (RSV) which spans the amino acids (N-terminal to C-terminal direction) 124 to 230; the hemagglutinin (HA) of influenza virus; the neuraminidase (NA) protein of influenza virus; the nucleoprotein (NP) of influenza virus; the AgD, SgD or CgD antigens of Bovine herpesvirus-1; glycoprotein B (gB) or glycoprotein D (gD); the VP4 antigen of Group A rotavirus; HA or nucleoprotein (NP) of measles virus; the S protein of Hepatitis B virus (HBV); the core protein, (HbcAg), or HbeAg or HbsAg of HBV; HCV proteins such as core, S, E1 and E2; glycoprotein gp 160 or envelope protein or the gag/pol, rev, tat or nef proteins of human immunodeficiency virus (HIV); the nucleoprotein of the lymphocytic choriomeningitis virus; the major capsid protein L1 of papillomavirus; glycoprotein of rabies virus; envelope protein or Vp4 or Vp6 or Vp7 of rotavirus; viral envelope (E) protein, the precursor for membrane (prM) protein and the non-structural protein NS1 of Murray Valley encephalitis virus (MVEV); the prM or E proteins of Japanese encephalitis virus.

To prepare Adhesin or viral antigen in large quantities and pure form, one can purify the Adhesin or viral antigen from the fermented Microbe or virus which naturally expresses said Adhesin or viral antigen. In cases where this is costly, inefficient or is undesirable for public health safety or other reasons, a preferred embodiment of the present invention is to clone and express the gene encoding the Adhesin or viral antigen in a heterologous organism such as E. coli (gram-negative bacterium), Bacillus subtilis (gram-positive bacterium) or Saccharomyces cerevisiae (baker's yeast). Classical recombinant DNA techniques for cloning, expressing a gene in adapted expression vectors and purifying the resulting protein are described in many laboratory manuals available to the person skilled in the art. Alternatively, and particularly when a polypeptide epitope of an adhesin protein or viral antigen is used, well-established peptide synthesis methods can be used to create ultra-pure polypeptides for use in the Immunogenic Complex. In examples below which focus on some other Adhesins, different methods to prepare Adhesins for use in Immunogenic Complex are described.

The optimal ratio of Adhesin or viral antigen/Ribosomal Complex in the Immunogenic Complex or Bacterio-viral Immunogenic Complex depends on several factors including the immunogenicity of the selected adhesin or antigen. Consequently, the optimal ratio of Adhesin or viral antigen to Ribosomal Complex in a vaccine is best determined empirically using appropriate animal models (e.g. mice, rats, rabbits, pigs, monkeys) by comparing immunogenicity and protection against target pathogen of Immunogenic Complex or Bacterio-viral Immunogenic Complex with different Adhesin-to-Ribosomal Complex ratios or viral antigen-to-Ribosomal Complex ratios. For example, the selected ratio's given in the examples which follow and other experiences allows to indicate that the ideal weight ratio of Ribosomal Complex to Adhesin for different Microbes, coupling reactions, delivery systems, pharmaceutical compositions and posologies, lies in the range of 0.05 to 20.

In cases where the Immunogenic Complex or Bacterio-viral Immunogenic Complex is not or is unlikely to be destabilized by solvents nor is to be exposed to excessive degradation such as in the Gastro-intestinal tract (GIT), then the ionic interactions between Ribosomal Complex and Adhesin or viral antigen fractions are generally sufficient for joint delivery to the MALT.

In some cases, in particular if no protective carrier or matrix is used and ionic interaction of RC and Adhesin or viral antigen are considered insufficient for joint delivery to the mucosal immune system, a preferred embodiment of the present invention is to covalently couple the Adhesins or viral antigen to the Ribosomal Complex. This can be achieved by various methods known to the experienced person in the art, such as by use of N-hydroxysuccinimidyl esters like disuccinimidyl suberate or N-Succinimidyl-(3-[2-pyridyl]-dithio)propionate (SPDP) or for example the use of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) or bis imido esters such as dimethyladipimidate. The final choice may also be affected by whether or not the chosen linker is antigenic by itself: following linkers don't behave as hapten by themselves -- see Original Patent.

In a preferred embodiment of present invention for use as oral vaccine, the Immunogenic Complex or Bacterio-viral Immunogenic Complex is protected from the low pH environment and excessive degradation in the gastrointestinal tract (GIT) by enzymes such as pepsins, trypsin, chymotrypsin, elastase and carboxypeptidase, by means of incorporation in a carrier consisting of polymeric matrices such as for example chitosan and collagen or encapsulated in micro-particles such as for example liposomes and poly(lactic-co-glycolic acid) particles or in hydrogels such as for example poly(alginic acid), dextran cellulose, gelatin or albumin. The use of such protective carriers is well documented. The amount of carrier versus Immunogenic Complex or Bacterio-viral Immunogenic Complex employed in such vaccines will vary depending upon the exact carrier used. Adjustment and manipulation of established dosage ranges used with traditional carrier molecules for adaptation to the present invention is well within the ability of those skilled in the art, however it is preferred to keep the size of the micro-particles between 0.1 and 10 .mu.m average diameter.

In one embodiment of the present invention, the Immunogenic Complex, Bacterio-viral Immunogenic Complex or Heterologous Immunogenic Complex are active principles in prophylactic vaccines against microbial species.

In another embodiment of the present invention, the Immunogenic Complexes, Heterologous Immunogenic Complex or Bacterio-viral Immunogenic Complex are derived from multiple Microbes or viruses have therapeutic use against microbial or viral infection, in disease management and in other cases where stimulation of the immune system is desirable. For example, the immuno-stimulatory effect of the Immunogenic Complex on Helper T-cell activation generally enhances the immune reaction of the host against the established pathogen. This may be of paramount importance for recovery of immuno-depressed subjects. In cases where the Micobes' pathogenesis strategy involves impairment of the host's immune defense by, for example, anti-phagocytosis activity or induced macrophage apoptosis or survival in the endocytic vacuole post macropinocytosis followed by cell-to-cell movement in an actin-dependent process, then the use of Adhesins expressed by such Microbes during their virulent phase, in particular those which use M-cells as entry port into host tissues, can be used in Immunogenic Complex of prophylactic as well as of therapeutic vaccines. Examples of Adhesins useful in treatment of such disease are: the Lpf gene product in specialized fimbriae of Salmonella typhimurium (these fimbriae are involved in specific adherence to M cells of murine Follicle Associated Epithelia); the ipa genes products of Shigella flexneri, the major secreted effectors of entry into epithelial cells and protein YadA of enteropathogenic Yersinia. Established gingivitis caused by penetration and colonization of multi-layered gingival pocket epithelial cells by Porphyromonas gingivalis, is an example where Immunogenic Complex, comprising P. gingivalis Ribosomal Complex and/or Adhesin, can be used in a therapeutic vaccine.

Microbes, from which Ribosomal Complexes and/or Adhesins can be prepared, include, but are not limited to, the following: Actinobacillus--Campilobacter consius actinomycetemcomitans--Capnocytophaga sp. Bacille Calmette-Guerin--Chlamydia trachomatis Bordetella pertussis--Eikenella corrodens Enterococcus sp.--Neisseria gonorrhoea Escherichia coli--Neisseria meningitidis Eubacteruim sp.--Nocardia sp. Haemophilus influenzae--Pasteurella multocida Lactobacillus acidophilus--Porphyromonas gingivalis Listeria monocytogenes--Prevotella intermedia Mycobacterium vaccae--Pseudomonas aeruginosa Rothia dentocarius--Streptococcus pyogenes Salmonella typhi--Treponema denticola Salmonella typhimurium--Vibrio cholera Serratia marcescens--Wolinella recta Shigella dysenteriae--Yersinia enterocolitica Streptococcus mutans--Candida albicans Streptococcus pneumoniae--Blastomyces dermatitidis Plasmodium falciparum--Leishmania sp. Trypanosoma cruzi--Entamoeba histolitica

Pharmaceutical compositions can be prepared for prevention and treatment of infectious disease caused by Microbes or viruses. Such pharmaceutical compositions comprise Immunogenic Complex or Bacterio-viral Immunogenic Complex wherein the Immunogenic Complex or Bacterio-viral Immunogenic Complex is formulated with pharmaceutical carriers in pharmaceutically acceptably delivery forms such as liquids, aerosols, lyophilized powders, pills, creams and suppositories; some of which may contain compounds such as erythrosine, titanium dioxide, Fe.sub.2O.sub.2, D-mannitol, magnesium stearate, gelatin, oils, waxes, antibiotics or antiseptics for administration to animals and/or humans.

The dosage and route of administration depends to a large extend on the condition and weight of the subject being treated, as well as on the frequency of treatment. Regiments for boost immunizations, including dose may be influenced by the response of the initial prime inoculation and clinical judgement of the effect. While the above described Immunogenic Complex or Bacterio-viral Immunogenic Complex may be produced and formulated for injection (parenteral or intramuscular), it is particularly suited for delivery to buccal epithelia in a gel, to mucosal tissues of nose, mouth, eye and throat by spray of a liquid suspension, delivery to upper respiratory tract by dry or liquefied aerosol spray, delivery to the gastro-intestinal tract in protective matrix or microparticle, formulated in a pill, and delivery to rectal, vaginal and uteral mucosa incorporated in a gelatinous capsule or suppository.

While the invention has been described and illustrated herein by references to the specific embodiments, various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material combinations of material, and procedures selected for that purpose. Indeed, various modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.
 

Claim 1 of 42 Claims

1. An immunogenic complex comprising: (a) at least one ribosomal complex; and (b) at least one adhesin of a microbe; or (c) at least one viral antigen, wherein the ribosomal complex comprises the large and small ribosomal subunits, corresponding to 50S and 30S subunits in bacteria and 60S and 40S subunits in eucaryotes which subunits are particulate in nature, carry on their surface minor fractions of the microbial cellular membrane or cell wall components and contain large double stranded rRNA's corresponding to 16S and 23S in bacteria, and 18S and 28S in eukaryotic cytosol; wherein the ribosomal complex retain integrity to preserve the double-stranded nature of the large r-RNA's contained in said ribosomal subunits; and wherein the immunogenic complex leads to the production of high levels of IgG antibodies.

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