Link:  Pharm/Biotech Resources

Title:  Vaccines and pharmaceutical compositions using membrane vesicles of microorganisms, and methods for preparing same

United States Patent:  6,916,478

Issued:  July 12, 2005

Inventors:  Kadurugamuwa; Jagath L. (Verona, NJ); Beveridge; Terry J. (Elora, CA)

Assignee:  University of Guelph (Ontario, CA)

Appl. No.:  236557

Filed:  September 6, 2002

The invention relates to novel vaccines and pharmaceutical compositions using membrane vesicles of microorganisms, methods for preparing same, and their use in the prevention and treatment of infectious diseases.

SUMMARY OF THE INVENTION

The present inventors have demonstrated that surface antigens such as lipopolysaccharide can be transferred from a bacteria using membrane vesicles. In particular, the present inventors introduced highly specific antigenic factors from pathogenic bacteria into the surface of avirulent or attenuated strains using membrane vesicles. Membrane vesicles from Shigella flexneri and Pseudomonas aeruginosa were isolated and fused with whole cells of E. coli or S. typhi. The integration of antigens from Shigella flexneri and Pseudomonas aeruginosa into the surface of the avirulent strains was confirmed using electron microscopy of double immunogold-labelled cells, and Western immunoblots. The avirulent strains with integrated surface antigens from the pathogenic bacteria induce immune responses against the antigens. The antigens are stable and continuously expressed on the surface of a carrier strain, and once in the host tissue the carrier strain stops growing (but remains viable) and outer membrane turnover is arrested. The outer membrane antigens will not be released nor replaced once the carrier strain invades the mucosal surface.

The use of membrane vesicles to produce a vaccine in accordance with the present invention has distinct advantages over other methods for generating vaccines. MVs are simply prepared and they readily fuse to carrier strains without complicated mixing formulations. The fusion is thermodynamically stable as it represents a response of two bilayered lipid-protein membranes interacting with one another.

The use of membrane vesicles also permits the simultaneous expression of multiple protective antigens (e.g. LPS and OMPs) from a number of pathogens in a single carrier strain, and this multivalent carrier strain then delivers the heterologous antigens to the immune system. The present invention provides an economical method for inducing protective immunity against a range of serotypes or antigenic variants by fusion of MVs from such pathogens. This eliminates the potential disadvantage of selecting antigenic variants that would become resistant to the antibodies. The present invention also permits the tailoring of vaccines to match differences in serotype distribution of disease in endemic areas.

Broadly stated, the present invention relates to a vaccine against an infectious disease caused by an infectious agent comprising a carrier strain having a membrane vesicle of a microorganism integrated into the cell surface of the carrier strain, wherein the membrane vesicle has an amount of an antigen associated with its cell surface which is effective to provide protection against the infectious agent. The infectious agent may be a microorganism which produces membrane vesicles, or a microorganism which does not produce membrane vesicles.

In accordance with one embodiment of the invention a vaccine against infectious diseases caused by a microorganism which produces membrane vesicles is provided which comprises a carrier strain having a membrane vesicle of the microorganism integrated into the cell surface of the carrier strain. The membrane vesicle may be a natural membrane vesicle of the microorganism, or it may be produced by treating the microorganism with a surface-active agent.

Multivalent vaccines against infectious diseases caused by different infectious agents are also contemplated comprising a carrier strain having membrane vesicles integrated into the cell surface of the carrier strain, wherein the membrane vesicles have amounts of antigens associated with their surfaces which are effective to provide protection against the infectious agents. In an embodiment of the invention, a multivalent vaccine is provided which comprises a carrier strain having at least two membrane vesicles from at least two different microorganisms integrated into the cell surface of the carrier strain, or comprising at least two carrier strains each containing a membrane vesicle from different microorganisms integrated into the cell surface of each of the carrier strains.

The invention also relates to a method of preparing a vaccine against an infectious disease caused by an infectious agent comprising integrating a membrane vesicle of a microorganism into the cell surface of a carrier strain wherein the membrane vesicle has an amount of an antigen associated with its surface which is effective to provide protection against the infectious agent. In an embodiment, the invention relates to a method of preparing a vaccine against infectious diseases caused by a microorganism which produces membrane vesicles which comprises integrating a membrane vesicle produced by the microorganism into the cell surface of a carrier strain.

The invention further relates to a method of preparing a multivalent vaccine against infectious diseases caused by different infectious agents comprising integrating membrane vesicles into the cell surface of a carrier strain, wherein the membrane vesicles have amounts of antigens associated with their surfaces which are effective to provide protection against the infectious agents. The membrane vesicles may be produced by the infectious agents or the membrane vesicles may be obtained from microorganisms which produce membrane vesicles and which are engineered to produce one or more of the antigens so that they are associated with the surface of the membrane vesicles. In an embodiment, the invention relates to a method of preparing a multivalent vaccine against infectious diseases caused by different microorganisms which produce membrane vesicles which comprises integrating membrane vesicles produced by the microorganisms into the cell surface of one or more carrier strains.

The invention still further relates to antibodies against a membrane vesicle of a microorganism for use as a means of passive immunization.

The invention also provides a method for screening for an immunogenic antigen of a pathogen comprising (a) providing a membrane vesicle having a test antigen associated with its surface; (b) vaccinating an animal with the membrane vesicle; and (c) challenging the animal with the pathogen to determine if the test antigen provides protection against the pathogen.

The present inventors have also found that a majority of bacteriolytic enzymes with peptidoglycan hydrolase, lipase, or proteolytic activity are not soluble, and they are concentrated and entrapped within the membrane vesicles of microorganisms. Significantly, the present inventors have shown that these membrane vesicles containing bacteriolytic enzymes are able to lyse gram-negative and gram-positive bacteria. In addition, gentamicin-induced membrane vesicles were found to be more lytic than natural membrane vesicles indicating a synergistic effect of the bacteriolytic enzymes cell-wall degrading activity and the antimicrobial agent's activity.

Therefore, the present invention also contemplates a pharmaceutical composition comprising a membrane vesicle of a microorganism containing one or more enzymes with peptidoglycan hydrolase, lipase, or proteolytic activity, and optionally a therapeutic agent, in an amount effective to have a bactericidal effect on gram-negative and/or gram-positive bacterial pathogens and a pharmaceutically acceptable vehicle or diluent. The membrane vesicle may be a natural membrane vesicle of a microorganism, or it may be produced by treating a microorganism with a surface-active agent. The invention further contemplates a method of treating an infectious disease caused by a gram-negative and/or gram-positive bacterial pathogen comprising administering membrane vesicles of one or more microorganisms containing one or more enzymes with peptidoglycan hydrolase, lipase, or proteolytic activity, and optionally a therapeutic agent, in an amount effective to have a bactericidal effect on the gram-negative and/or gram-positive bacterial pathogens.

The present inventors have also found that impermeable antimicrobial agents such as gentamicin can be introduced into epithelial cells using gentamicin-induced membrane vesicles from Shigella flexneri. Thus, the membrane vesicles may be used for the delivery of antimicrobial agents into a host.

Accordingly, the invention also relates to a composition comprising membrane vesicles of a microorganism containing a therapeutic agent in an amount which is effective to introduce the therapeutic agent into a host. The invention also relates to a method for administering a therapeutic agent to a host comprising administering to the host the therapeutic agent encapsulated in a membrane vesicle of a microorganism.

In an embodiment of the invention, a composition is provided comprising membrane vesicles of a microorganism containing an antimicrobial agent, in an amount which is effective to introduce the antimicrobial agent into a host. The invention also relates to a method for administering an antimicrobial agent into a host comprising administering to the host a membrane vesicle of a microorganism containing the antimicrobial agent.

The invention also relates to a method of inserting nucleic acid molecules into a target cell which comprises encapsulating the nucleic acid in a membrane vesicle of a microorganism, and bringing the membrane vesicle in contact with the target cell whereby the nucleic acid molecule is inserted into the cell.

DETAILED DESCRIPTION OF THE INVENTION

I. Membrane Vesicles

The vaccines, methods and compositions of the invention employ membrane vesicles of microorganisms. Membrane vesicles also known as blebs, are little bud-like protrusions formed in the cell wall, outer membrane, cytoplasmic, and/or plasma membrane of a microorganism. When cultured under selected conditions the membrane vesicles break away from the whole cell into the medium. The membrane vesicles are generally spherical, possess a bilayer, and have a diameter of about 10 to 200 nm, preferably 50-150 nm, most preferably 80 to 100 nm.

The membrane vesicles may be natural membrane vesicles of a microorganism which produces membrane vesicles. Natural membrane vesicles contain outer membrane and periplasm components. Natural membrane vesicles are produced without exposing the microorganism to a surface-active agent. Treatment with a surface active agent produces membrane vesicles which are larger than the natural vesicles. These large membrane vesicles typically contain outer membrane, cytoplasmic membrane or plasma membrane components, and cytoplasm. Membrane vesicles produced by treatment with surface-active agents also include natural membrane vesicles. The membrane vesicles used in the vaccine, methods, and compositions of the invention include both natural membrane vesicles and the larger membrane vesicles.

By way of example, natural membrane vesicles of Pseudomonas aeruginosa contain mainly B-band LPS, mature periplasmic enzymes and secretory enzymes which are in transit. Secretory enzymes may be mature enzymes or proenzymes; the latter being activated once they are liberated from the cell surface. The antimicrobial agent gentamicin increases the incidence of membrane vesicles and frequently results in membrane vesicles which contain outer membrane, cytoplasmic membrane and/or plasma membrane components. Both types of membrane vesicles are enriched with peptidoglycan-hydrolysing enzymes (i.e., autolysins).

While we do not wish to be bound by any particular models, a proposed model for the formation of membrane vesicles in P. aeruginosa is set out in schematic form in FIG. 10 (see original patent). FIG. 10(A) shows the envelope before membrane blebbing is initiated. FIG. 10(B) shows the simplest type of membrane vesicle and is the most frequent natural membrane vesicle. This membrane vesicle is comparatively small, involves only the exfoliation of the outer membrane, and entraps only periplasm. FIG. 10(C) is an extrapolation of FIG. 10(B) in that it includes the entrapment of DNA that has migrated from the cytoplasm to the periplasm and is another possibility for natural membrane vesicles. Although the DNA resembles linear strands, it is possible that both circular or linear complexes could be compartmentalized. FIG. 10(D) shows the production of a more complex membrane vesicle containing both inner and outer membranes as well as some cytoplasmic constituents. Autolysins have been found in both types of membrane vesicles. Surface-active agents such as gentamicin encourage the formation of the membrane vesicles seen in FIG. 10(D).

The membrane vesicles are typically obtained from gram-negative bacteria. Suitable microorganisms for producing the membrane vesicles include Pseudomonas aeruginosa, Escherichia coli, Salmonella gastroenteritis (typhimirium), S. typhi, S. enteriditis, Shigella flexneri, S. sonnie, S dysenteriae, Neisseria gonorrhoeae, N. meningitides, Haemophilus influenzae H. pleuropneumoniae, Pasteurella haemolytica, P. multilocida, Legionella pneumophila, Treponema pallidum, T. denticola, T. orales, Borrelia burgdorferi, Borrelia spp. Leptospira interrogans, Klebsiella pneumoniae, Proteus vulgaris, P. morganii, P. mirabilis, Rickettsia prowazeki, R. typhi, R. richettsii, Porphyromonas (Bacteriodes) gingivalis, Chlamydia psittaci, C. pneumoniae, C. trachomatis, Campylobacter jejuni, C. intermedis, C. fetus, Helicobacter pylori, Francisella tularenisis, Vibrio cholerae, Vibrio parahaemolyticus, Bordetella pertussis, Burkholderie pseudomallei, Brucella abortus, B. susi, B. melitensis, B. canis, Spirillum minus, Pseudomonas mallei, Aeromonas hydrophila, A salmonicida, and Yersinia pestis.

In accordance with preferred embodiments of the invention, the microorganism is selected from the bacterial strains Pseudomonas aeruginosa H103, PAO1, and ATCC 19660, Shigella flexneri, S. dysenteriae, Escherichia coli K12, K30, DH5α, Salmonella typhi, and Neisseria gonorrhoeae CH811, CS19a.

The present inventors are the first to report the release of membrane vesicles from Shigella flexneri. Accordingly, in accordance with one embodiment of the invention, an isolated and purified membrane vesicle of Shigella flexneri is provided.

The membrane vesicles are characterized by having specific antigens associated with their surfaces, and containing specific enzymes, which are native to the microorganism from which the membrane vesicles are derived. Table 1 is a list of microbial pathogens and the antigens and enzymes of the pathogens which can be incorporated into membrane vesicles. For example, membrane vesicles which have endotoxin, outer membrane proteins, pilin, and flagellin associated with the membrane vesicle surface, and which contain protease, phospholipase C, proelastase, and autolysins can be obtained from Pseudomonas aeruginosa, which is a pathogen associated with corneal infections, nosocomial infections etc.

The antimicrobial membrane vesicles described herein may also contain one or more surface active agents which are used to induce formation of the vesicles. Preferably, the membrane vesicles contain a surface-active anti-microbial agent such as polymyxin, or other surface-active agents such as EDTA. Preferably, the membrane vesicles contain aminoglycosides, preferably gentamicin, hygromycin, tobramycin, amakacin, kanamycin, neomycin, paromomycin, and/or streptomycin.

The microorganisms which produce membrane vesicles described herein may also be transfected with one or more nucleotide sequences encoding exogenous proteins in order to provide membrane vesicles have exogenous proteins incorporated into the membrane vesicles or associated with their surface. For example, the exogenous proteins include antigens which are associated with infectious diseases caused by infectious agents which do not produce membrane vesicles including viruses such as human immunodeficiency virus (HIV), influenza (nuriminidase/haemagglutinin), adenovirus, Herpes simplex, measles, simian immunodeficiency virus; fungi such as Histoplasma capsulatum, Cryptococcus neoformans, Blastomyces dermatidis, Candida albicans; protozoa such as Leishmania mexicana, Plasmodium falciparum and Taxoplasma gondii; and, gram-positive bacteria such as Streptococcus mutans, and S. pneumoniae (cell wall antigens). Microorganisms transfected with such antigens may be used to produce membrane vesicles which may be used as vaccines against the infectious agent. The microorganism may also be transfected with a nucleotide sequence encoding an exogenous protein having a known therapeutic or regulatory activity such as hormones preferably insulin, blood clotting factor VIII, growth hormones, hirudin, cytokines such as gamma interferon, tumor necrosis factor, IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, GM-CSF, CSF-1, and G-CSF. Membrane vesicles containing therapeutic or regulatory proteins may be used to deliver the proteins to a host. The microorganisms may also be transfected with proteins which facilitate targeting of a membrane vesicle having the proteins associated with their surfaces to specific target tissues or cells. For example, tumor-associated antigens, CD₄ proteins on T-helper cells, and gp120 in HIV.

II. Preparation of Membrane Vesicles

Suitable microorganisms which may be used to prepare membrane vesicles are described above. The strains of the microorganism used to prepare the membrane vesicles may be reference strains which may be obtained from Research Institutes working in the field, or from public depositories such as the American Type Culture Collection, Bethesda, Md. The microorganism strains may also be obtained from animals, preferably humans suffering from naturally occurring infections.

Nucleotide sequences encoding exogenous proteins may be introduced into microorganisms which produce membrane vesicles using methods well known to those skilled in the art. The necessary elements for the transcription and translation of the inserted nucleotide sequences may be selected depending on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. A reporter gene which facilitates the selection of host cells transformed or transfected with a nucleotide acid sequence may also be incorporated in the microorganism. (See, e.g., Sambrook et al. Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, for transfection/transformation methods and selection of transcription and translation elements, and reporter genes). Sequences which encode exogenous proteins may generally be obtained from a variety of sources, including for example, depositories which contain plasmids encoding sequences including the American Type Culture Collection (ATCC, Rockville Md.), and the British Biotechnology Limited (Cowley, Oxford England).

The microorganisms are grown under suitable conditions that permit natural membrane vesicles to be formed. Suitable growth conditions will be selected having regard to the type of microorganism, and the desired characteristics of the membrane vesicles. Generally, growth mediums suitable for culturing the microorganisms so that they produce membrane vesicles contain a nitrogen source and a carbon source.

Suitable nitrogen sources are nitrogen salts. The initial concentration of the nitrogen source is related to the temperature of the fermentation during the growth phase. There should be enough nitrogen source present to provide a final cell mass of a least about 0.5-1.0 g/l. A useful range of initial nitrogen concentration is selected so that less than 0.1 g/l remains at the conclusion of the growth phase.

As carbon source, sugars such as glucose (or crude glucose such as dextrose), sucrose, fructose, erythrose, mannose, xylose, and ribose, or mixtures of these sugars may be used. Commercial sources of these sugars can conveniently be used. Such sources include liquid sucrose, high fructose corn syrup and dextrose corn syrup. Other carbon sources can be used in combination with these sugars such as mannitol and other sugar derivatives.

The medium preferably includes other components useful in fermentation processes. For example, the medium may include a source of magnesium such as magnesium sulfate, a source of phosphate such as K₂HPO₄, a source of iron such as iron sulfate, and a source of zinc such as zinc sulfate. Useful concentration ranges of magnesium, phosphate, iron and zinc are 2-5 mM, 0.5-5.0 mM, 2-5 mM, 1-5 mM, 0.5 mM and 0.5-5.0 mM, respectively.

The medium may also contain components which support the production of specific enzymes. For example, choline (2-hydroxy methyl-trimethyl ammonium chloride salt) may be added to the medium to support the production of phospholipase C, or chelating compounds such as transferrin to support siderophore production.

By way of example, Pseudomonas aeruginosa may be cultured in a medium containing the following components: 10 mM glucose (or other carbon source); 1.2 mM K₂HPO₄, 3.2 mM MgSO₄.7H₂O, 12 mM (NH₄)₂SO₄, 3 mM NaCl, 3 mM KCl, 3.2 mM FeSO₄.7H₂O, and 50 mM of a suitable buffer (e.g. MOPS).

Commercially available media may be used which favour the production of membrane vesicles. For example, Mueller-Hinton broth, or Trypticase soy broth, may be used for culturing Pseudomonas species; Brain-Heart Infusion may be used for culturing E. coli, Pasteurella, and Neisseria species; and, blood agar may be used for culturing Haemophilus species.

The microorganisms are cultured in two stages. The first stage is carried out at a temperature sufficient to promote the growth phase of the microorganism. After rollover into the stationary phase, the temperature of the growth medium may be reduced to a temperature which promotes production of membrane vesicles. For example, the temperature may be reduced to 20 to 25° C., preferably room temperature.

The final medium is subjected to a variety of steps to recover the desired membrane vesicles. For example, the membrane vesicles may be isolated by precipitation, filtration, and/or differential centrifugation.

Formation of membrane vesicles may be induced using surface-active agents. The release of membrane vesicles typically increases several fold after the microorganism is exposed to an agent. Suitable surface agents include surface-active antimicrobial agents such as polymyxin, atypical metal ions, and EDTA. Preferably, the surface-active agent is an antimicrobial surface-active agent, most preferably an aminoglycoside. Examples of suitable aminoglycosides include gentamicin, hygromycin, tobramycin, amakacin, kanamycin, neomycin, paromomycin, and streptomycin. The method for inducing formation of the membrane vesicles is generally as described above. The microorganism is cultured using the above described conditions, and the surface-active agent is added after the first stage, i.e., after early stationary growth phase. The concentration of antimicrobial agent that is added is about four times the minimal inhibitory concentration (MIC).

By way of example, Pseudomonas aeruginosa can be induced to release membrane vesicles into the medium on exposure of the organism to gentamicin. In particular, Pseudomonas strains are grown in Mueller-Hinton broth to the early stationary phase (10⁶ CFU/ml) at 37° C. Gentamicin at a final concentration of four times the MIC is then added to the bacterial culture in early stationary phase and the culture is incubated at room temperature for about 30 minutes. The cells are removed from the suspension by centrifugation at 4000 to 8000×g for 0.5 to 1 hour, and the supernatant is filtered, preferably through cellulose acetate filters, to remove residual cells. Membrane vesicles are recovered from the filtrates by centrifugation at 100,000 to 170,000×g for 1 to 3 hours. The membrane vesicles are suspended in a suitable buffer, for example HEPES buffer, preferably at a pH of between about 6.8 and 7.4.

The antigens associated with the surface of membrane vesicles may be identified using conventional methods. For example, Western immunoblots of solubilized components of the membrane vesicles can be prepared and specific antigens can be identified using antibodies specific for the antigen (e.g., antibodies specific for LPS, pilin, flagellin etc.). LPS can also be identified using immunogold electron microscopic detection.

Enzymes contained in the membrane vesicles may be identified using conventional enzyme assays. For example, phospholipase C activity may be determined using the synthetic substrate p-nitrophenyl phosphorylcholine (Sigma) as described by Berka et al. (Infect. Immun. 34:1071-1074, 1981); protease may be determined by the assay described by Howe and Iglewski (Infect. Immun. 43:1058-1063, 1984) using Hide powder azure (Sigma); alkaline phosphatase may be assayed using p-nitrophenyl phosphate (pNPP) (Sigma) as described in Tan, A. S. P. and E. A. Worobec (FEMS Microbial. Letts. 106:281-286, 1993); elastase may be determined using elastin Congo red (Sigma) as a substrate in an assay based on the method of Kessler and Safrin (Kessler, E., and M. Safrin, J. Bacteriol. 170:5241-5247, 1988); and hemolysin activity may be measured as described in Bergmann et al. (Infect. Immun. 57:2187-2195, 1989). Peptidoglycan hydrolases may be determined using SDS-PAGE zymogram systems as outlined in Bernadsky, G., et al. (J. Bacteriol. 176:5225-5232, 1994). Immunogold electron microscopic detection may also be used to identify enzymes contained in a membrane vesicle.

III. Vaccines

As hereinbefore mentioned, the present invention relates to a vaccine against an infectious disease caused by an infectious agent comprising a carrier strain having a membrane vesicle of a microorganism integrated into the cell surface of the carrier strain, wherein the membrane vesicle has an amount of an antigen associated with its surface which is effective to provide protection against the infectious agent. The term "integrating" or "integrated" used herein refers to the fusion of the cell membrane of the membrane vesicle with the cell surface of the carrier strain, or the adherence of the membrane vesicle to the cell surface of the carrier strain.

"Infectious disease" refers to any disease or condition due to the action of an infectious agent. The infectious agent may be a microorganism which produces membrane vesicles, or a microorganism which does not produce membrane vesicle. In the former embodiment, the membrane vesicle used in the vaccine is obtained from a microorganism which produces membrane vesicles with one or more antigens associated with the surface of the vesicle.

Therefore, in an embodiment of the invention, a vaccine against infectious diseases caused by a microorganism which produces membrane vesicles is provided which comprises a carrier strain having a membrane vesicle of the microorganism integrated into the cell surface of the carrier strain. The vaccines may be used for the prophylaxis or active immunization and treatment of infectious diseases caused by microorganisms which produce natural membrane vesicles and/or which can be induced to produce membrane vesicles for example using surface-active agents. Examples of pathogenic microorganisms which produce membrane vesicles are listed in Table 1.

In accordance with another embodiment of the invention, a vaccine against infectious diseases caused by an infectious agent which does not produce membrane vesicles is provided which comprises a carrier strain having a membrane vesicle from a microorganism integrated into the cell surface of the carrier strain, wherein the membrane vesicle has an amount of an antigen associated with its surface which is effective to provide protection against the infectious agent. The vaccines may be used for the prophylaxis or active immunization and treatment of infectious diseases caused by microorganisms including viruses such as human immunodeficiency virus (HIV), influenza (nuriminidase/haemagglutinin), adenovirus, Herpes simplex, measles, simian immunodeficiency virus; fungi such as Histoplasma capsulatum, Cryptococcus neoformans, Blastomyces dermatidis, Candida albicans; protozoa such as Leishmania mexicana, Plasmodium falciparum and Taxoplasma gondii; and, gram-positive bacteria such as Streptococcus mutans, and S. pneumoniae. Therefore, the vaccines of the present invention may incorporate membrane vesicles with immunogenic antigens of these microorganisms.

The membrane vesicles employed in the vaccines of the present invention may be natural membrane vesicles of the microorganism or they may be membrane vesicles produced by treating the microorganism with a surface-active agent as described hereinbefore. The membrane vesicles are selected so that they have an amount of an antigen (i.e. immunogen) associated with their surfaces which is effective to provide protection against the pathogenic infectious agent/microorganism. For example, for the pathogens listed in Table 1, membrane vesicles may be selected which contain the specific antigens identified in Table 1. In particular, membrane vesicles may be selected for Pseudomonas aeruginosa which have endotoxin (A- and B-band lipopolysaccharide), outer membrane proteins, pilin, and/or flagellin associated with their surfaces. These membrane vesicles may be fused with a carrier strain to provide a vaccine which is useful for protecting against infections caused by Pseudomonas aeruginosa.

The carrier strain is selected so that it is incapable of multiplying in vivo. Carrier strains are obtained through selection of variants which occur naturally, or using conventional means known to those skilled in the art. Examples of suitable carrier strains are Shigella species, Salmonella species, preferably S. typhi Ty21a, S. typhimurium, Vibrio species, and Escherichia species.

The invention also relates to a method of preparing a vaccine against an infectious disease caused by an infectious agent comprising integrating a membrane vesicle of a microorganism into the cell surface of a carrier strain wherein the membrane vesicle has an amount of an antigen associated with its surface which is effective to provide protection against the infectious agent. In an embodiment, the invention provides a method of preparing a vaccine against infectious diseases caused by a microorganism which produces membrane vesicles which comprises integrating a membrane vesicle produced by the microorganism into the cell surface of a carrier strain.

A membrane vesicle may be integrated into the cell surface of a carrier strain by contacting the membrane vesicle with the carrier strain. By way of example, exponential growth phase cultures of the carrier strain (e.g., S. typhimurium aro A, and S. typhiTy21a) in a suspension of 10⁴ to 10⁹ CFU/ml, preferably 10⁶ CFU/ml, are incubated with membrane vesicles (100 μg/ml of protein) from, for example P. aeruginosa or Shigella flexneri.

The vaccine may be a multivalent vaccine and additionally contain immunogens related to other infectious diseases in a prophylactically or therapeutically effective manner. Multivalent vaccines against infectious diseases caused by different infectious agents may contain a carrier strain having membrane vesicles integrated into the cell surface of the carrier strain, wherein the membrane vesicles have amounts of antigens associated with their surfaces which are effective to provide protection against the infectious agents.

A multivalent vaccine may comprise at least two carrier strains each having membrane vesicles with different immunogens associated with different infectious agents. In an embodiment of the invention a multivalent vaccine is provided comprising at least two carrier strains each having membrane vesicles of different pathogenic microorganisms integrated into the cell surface of the carrier strain For example, a multivalent vaccine may contain a carrier strain having a selected membrane vesicle of P. aeruginosa integrated into its cell surface, and a carrier strain having a selected membrane vesicle of S. flexneri integrated into its cell surface.

A multivalent vaccine may contain a carrier strain having at least two membrane vesicles having different immunogens associated with different infectious agents. In an embodiment of the invention, a multivalent vaccine is provided comprising a carrier strain and membrane vesicles from at least two different microorganisms integrated into the cell surface of the carrier strain. Thus, a carrier strain may contain immunogens relating to more than one pathogenic microorganism. For example, a carrier strain may be contacted with a selected membrane vesicle obtained from P. aeruginosa, and a membrane vesicle obtained from S. flexneri using the methods described herein, to produce a carrier strain having immunogens from both bacteria associated with the cell surface.

Multivalent vaccines are prepared by integrating membrane vesicles into the cell surface of one or more carrier strains as described herein.

The vaccine of the invention contains an immunologically effective amount of the carrier strain(s) with the integrated membrane vesicle(s), for example between 1×10⁹ to 5×10¹⁰ cells per dosage unit, preferably 5×10⁹ to 2×10¹⁰ cells per dosage unit. The optimum amounts of cells depends on the nature of the infection against which protection is required, the characteristics of the animals to be protected, and other factors known to persons skilled in the art.

In addition to the carrier strain(s) with the integrated membrane vesicle(s), the vaccine may comprise an immunologically acceptable carrier such as aqueous diluents, suspending aids, buffers, excipients, and one or more adjuvants known in the art. Suitable adjuvants include aluminum hydroxide, Freund's adjuvant (complete or incomplete), bacteria such as Bordetella pertussis or E coli or bacterium derived matter, immune stimulating complex (iscom), oil, sapronin, oligopeptide, emulsified paraffin-Emulsigen™ (MVP Labs, Ralston, Nebr.), L80 adjuvant containing AL(OH)₃ (Reheis), Quil A (Superphos), or other adjuvants known to the skilled artisan. The vaccine may also contain preservatives such as sodium azide, thimersol, beta propiolactone, and binary ethyleneimine.

The vaccines of the invention can be intended for administration to animals, including mammals, avian species, and fish; preferably humans and various other mammals, including bovines, equines, and swine.

The vaccines of the invention may be administered in a convenient manner, such as intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally or orally. Preferably the vaccine is administered orally, intramuscularly or subcutaneously. The dosage will depend on the nature of the infection, on the desired effect and on the chosen route of administration, and other factors known to persons skilled in the art.

A vaccine prepared using the methods described herein may be tested in in vivo animal systems to confirm their efficacy in the prophylaxis or active immunization and treatment of infectious diseases and to determine appropriate dosages and routes of administration.

The membrane vesicles of the invention are also useful for preparing antibodies which may be used as a means of passive immunization. Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab′)₂ and recombinantly produced binding partners. Polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Monoclonal antibodies may also be readily generated using conventional techniques (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which are incorporated herein by reference; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, William D. Huse et al., "Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281, December 1989; see also L. Sastry et al., "Cloning of the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library," Proc Natl. Acad. Sci USA 86:5728-5732, August 1989; see also Michelle Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas," Strategies in Molecular Biology 3:1-9, January 1990; all of which are incorporated herein by reference). Similarly, binding partners may also be constructed utilizing recombinant DNA techniques.

The membrane vesicles of the present invention additionally are useful for screening for immunogenic antigens of a pathogen which may be used in conventional vaccines or incorporated in a membrane vesicle vaccine as described herein. For example a putative immunogenic antigen of a pathogen may be associated with the surface of the membrane vesicle using the methods described herein. The immunogenicity of the antigen may be determined by vaccinating an animal with the membrane vesicle with the associated antigen, and later challenging the animal with the pathogen to determine the protective effect of the antigen. An antigen showing a protective effect in such a system can be used in conventional vaccines (e.g. by itself or expressed on a carrier strain), or the membrane vesicle with the associated antigen can be used as a vaccine.

III. Use of the Vesicles as Bacteriolytic Agents

As hereinbefore mentioned the present invention also contemplates a pharmaceutical composition comprising a membrane vesicle of a microorganism containing one or more enzymes with peptidoglycan hydrolase, lipase, or proteolytic activity in an amount effective to have a bactericidal effect on gram-negative and/or gram-positive bacterial pathogens, and a pharmaceutically acceptable vehicle or diluent. The membrane vesicle may be a natural membrane vesicle of a microorganism, or it may be produced by treating the microorganism with a surface-active agent as described herein (i.e. large membrane vesicle). Compositions containing the large membrane vesicles therefore may also contain a surface active agent such as an antibiotic.

The invention also contemplates a method of treating an infectious disease caused by a gram-negative and/or gram-positive bacterial pathogen comprising administering an amount of a membrane vesicle containing one or more enzymes with peptidoglycan hydrolase, lipase, or proteolytic activity, effective to have a bactericidal effect on the gram-negative and/or gram-positive bacterial pathogen.

Membrane vesicles for use in these pharmaceutical compositions and methods, may be prepared using the methods described herein. In particular, membrane vesicles containing enzymes with peptidoglycan hydrolase, lipase, and proteolytic activity may be selected using conventional enzyme assays.

Membrane vesicles containing bacteriolytic enzymes and therapeutic agents such as antibiotics i.e. larger membrane vesicles produced after treatment with a surface-active agent as described herein, are particularly useful in the pharmaceutical compositions and methods of the present invention. The therapeutic agent and hydrolytic enzymes in the membrane vesicle act synergistically to provide an enhanced bactericidal effect.

The compositions of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the composition to be administered in which any toxic effects are outweighed by the therapeutic effects of the membrane vesicles.

The composition may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration inhalation, transdermal application, or rectal administration. The pharmaceutical compositions are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets. For parenteral and intracerebral uses, those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral injection can be used, and can therefore be prepared as solutions of the active membrane vesicles or as powders of the vesicles to be mixed with one or more pharmaceutically acceptable excipients or diluents, suitable for the aforesaid uses and with an osmolarity which is compatible with the physiological fluids. For local use, those preparations in the form of creams or ointments for topical use, or in the form of sprays should be considered; for inhalant uses, preparations in the form of sprays, for example nose sprays, should be considered.

The preparations of the invention can be intended for administration to animals, preferably humans and other warm blooded animals.

Administration of an amount effective to have a bactericidal effect is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, an amount effective to have a bactericidal effect may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. Amounts of membrane vesicles effective to have a bactericidal effect on a selected gram-negative and/or gram-positive bacterial pathogen may be determined using conventional in vivo and in vitro tests (see zymogram systems as outlined in Bernadsky, G. et al. supra).

The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active vesicles are combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the pharmaceutical compositions include, albeit not exclusively, solutions of the membrane vesicles in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

The pharmaceutical compositions containing membrane vesicles of a microorganism containing one or more enzymes with peptidoglycan hydrolase, lipase, or proteolytic activity, and optionally a therapeutic agent, and methods of treatment using these compositions, may be used for the prophylaxis and treatment of conditions associated with various gram-negative and gram-positive bacterial pathogens. For example, the compositions and methods are useful in the treatment of conditions associated with the following pathogens:

A. Gram-positive Pathogens