WO2000067784A1 - Bacteriophage isolated from bacterial genomes and extrachromosomal elements and methods of use thereof - Google Patents

Abstract

The present invention relates to compositions, methods, processes, etc., relating to bacteriophage which are encoded by chromosome, plasmids, or an extrachromosomal element of bacteria. The bacteriophage of the present invention are preferably encoded by pathogenicity islands in chromosomes or plasmids of pathogenic bacteria. The bacteriophage can be utilized as a pharmaceutical composition, e.g., to elicit an immune response, e.g., for the purpose of producing antibodies, as vaccines and vaccine vectors to regulate the immune system, e.g., for the prevention and treatment of allergy, disease, and other pathological conditions. The invention finds additional utility in systems and methods for the detection of pathogens comprising bacteriophage and a system and method for the environmental eradication of pathogenic microorganisms.

Description

BACTERIOPHAGE ISOLATED FROM BACTERIAL GENOMES AND EXTRACHROMOSOMAL ELEMENTS AND METHODS OF USE THEREOF

This application claims the benefit of U.S. Provisional Application 60/133,373, filed May 10, 1999, which is hereby incorporated by reference in its entirety. The research described herein was supported by the University of Maryland,

Baltimore, the National Institutes of Health (Grant No. NIH AI-07524-01), the Department of Veterans Affairs, and the Burroughs Wellcome Fund (Grant No. BWF#1669). The government may have certain rights.

BACKGROUND OF THE INVENTION Pathogenic microorganisms are responsible for a wide range of infectious diseases and pathologic infections in humans, animals and plants. Scientists have been studying the science of infection for many years and numerous vaccine strategies for prevention of infection are described in the literature. In order to be successful, the vaccine must balance immunogenicity with safety. Specifically, a vaccine must sufficiently mimic the pathogenic form of the organism so as to stimulate an immune response against it while being sufficiently innocuous so as to prevent the infection itself from presenting in the vaccinee.

Many modern vaccines use recombinatorial genetics techniques to control the expression of the antigen of interest. Certain vaccines use vectors, such as plasmids, to deliver the immunogenic vaccine antigen to the particular immune system site. The plasmid, encoding the antigen of interest, can either be directly injected into the vaccinee or can be carried within another microorganism, such as a non-pathogenic bacterium. Both methods have distinct technical drawbacks associated with them such as plasmid incompatibility, plasmidless takeover of the strain, and limitations on plasmid copy number. Other vaccines involve the administration of a live attenuated form of the pathogenic bacterium. To design such an attenuated mutant, the vaccine developer must have an in-depth understanding of the pathogenic mechanisms of virulence, colonization, and infection so as to allow the mutant to mirror the immunogenic aspects of the live bacterium without mirroring the infectious aspects. The present invention involves a bacteriophage vaccine, an entirely novel and significant improvement over current whole- cell vaccines.

Virulence genes of pathogenic bacteria are those which code for virulence factors such as toxins, adhesins, and invasins. Genes encoding toxins have been found located on transmissible genetic elements such as transposons, plasmids or bacteriophages. Likewise, virulence genes and virulence gene clusters known as "pathogenicity islands" (PAIs) can be part of particular regions on the bacterial chromosome or on plasmids in the cell. PAIs are found in both gram- positive and gram-negative bacteria and are present in the genome of pathogenic strains of a given species but absent or only rarely present in those of nonpathogenic variants of the same or related species. They comprise large DNA regions (up to 200 kb of DNA) and often carry more than one virulence gene and encode proteins which are known to be immunogenic. The unique nucleotide makeup of PAIs often include a %G+C considerably different to the host chromosome (+/- 10%), the presence of specific DNA flanking sequences and cryptic genes with homology to phage integrase genes, plasmid origins of replication, transposases and insertion sequences (IS). PAIs are also associated with tRNA genes, tRNA-like genes and bacteriophage attachment sites in the bacterial chromosome. Taken together, these characteristics suggest PAIs were previously able to spread among bacterial populations by the mechanism of horizontal gene transfer, a process known to contribute to microbial evolution, adaptation, the emergence of pathogens and virulence.

The literature suggested the idea that many pathogenicity islands in bacterial pathogens may be of phage origin. See, e.g., Hacker et al., Molecular Microbiology, 23(6): 1089-1097, 1996. The present invention involves the discovery that certain pathogenicity islands are not merely of 'phage origin' but are phage and contain the complete genome of a bacterial virus (phage or bacteriophage) which has integrated into the chromosome of the host bacteria and which provides the bacterial strain with pathogenic potential.

The first stage of an infection is colonization, the establishment of the pathogen at an appropriate site or surface. Pathogens usually exploit the host sites of environmental contact as points of entry. Sites of entry in human hosts include the urogenital tract, the digestive tract, the respiratory tract and the conjunctiva. Organisms that infect these regions have usually developed tissue adherence mechanisms and some ability to overcome or withstand the constant pressure of the host defenses.

In its simplest form, bacterial adherence or attachment to a eukaryotic cell surface requires the participation of two factors: a receptor and a ligand. Bacterial ligands are called adhesins. The receptors so far defined are usually specific carbohydrate or peptide residues on the eukaryotic cell surface. The bacterial adhesin is typically a cell surface component (capsule, cell wall, fimbriae, pilus, etc.), but can be a carbohydrate or peptide residue on the bacterial cell surface which interacts with the host cell receptor. Adhesins and receptors usually interact in a complementary and specific fashion, in the same way that an enzyme reacts with a specific substrate or antigen reacts with antibody.

Adherence factors involved in colonization by bacterial pathogens include both host factors and bacterial factors. Factors associated with the prokaryotic bacteria include: ligands (surface molecules that exhibits specific binding to a receptor molecule on host surface); adhesins (surface structures or macromolecules that binds the bacterium to a specific surface); fimbriae (filamentous proteins on the surface of bacterial cells that may behave as adhesins for specific adherence); glycocalyx (a layer of exopolysaccharide fibers on the surface of bacterial cells which involved in adherence to a surface); capsules (detectable layer of polysaccharide or polypeptide on the surface of a bacterial cell which mediates specific or nonspecific attachment); and lipopolysaccharide or LPS (distinct cell wall component of the outer membrane of Gram-negative bacteria having structural diversity to mediate specific adherence, functioning as an adhesin); and teichoic acids and lipoteichoic acids or LTA (cell wall components of Gram-positive bacteria involved in nonspecific or specific adherence). Factors associated with the eukaryotic host include: receptors (complementary macromolecular binding sites on host surface that binds specific adhesins or ligands); mucus (the mucopoly saccharide layer of glucosammoglycans covering animal cell mucosal surfaces, often containing neuraminic acid which provides a receptor for many types of bacterial ligands); and fibronectin (protein that coats many epithelial mucosal surfaces and provides specific peptide receptors for adherence of bacterial pathogens) [Todar, Kenneth, "Mechanisms of Bacterial Pathogenicity," University of Wisconsin Department of Bacteriology, http://www.bact.wisc.edu/Bact303/Bact303pathogenesis (1998)] . Pathogenicity islands (PAIs) are virulence gene clusters. The first step of virulence often involves adherent colonization factors. Some colonization factors, particularly fimbriae and pilus subunits, are known to be highly immunogenic and antibodies against these structures have been shown to be protective. Colonization factors have been investigated by various research groups as potential vaccine candidates. For example, intramuscular injection of purified colonization factor antigen I (CFA/I) from E. coli has been demonstrated to induce antigen recognizing antibodies in rhesus monkeys [Cassells et al., Infect Immun 60(6): 2174-81 (6/1992)]. Purified Moraxella bovis pilus (a type IV pilus) vaccines showed protective efficacy against challenge with M. bovis, the bacterial pathogen associated with infectious bovine keratoconjunctivitis [Pugh et al. , Am J. Vet Res, 46(4): 811-815 (4/1985)]. Pilin-based anti Pseudomonas vaccines using the DSL domain of the type IV pilus of Pseudomonas aeruginosa (Pa) as antigen have been shown to provide effective protection against initial colonization and infection with Pa [Hahn et al., Behring Inst Mitt, (98): 315-25 (2/1997)]. Finally, vaccines using purified type I pili from E. coli induced significant protection against colibacillosis in newborn pigs following challenge with E. coli expressing type I pili, demonstrating that type I pili are a virulence factor, as well as an effective vaccine antigen [Jayappa et al. , Infect Immun 48(2): 350-4 (5/1985)].

The pilus-based vaccines are described in the patent literature as well. Lindberg et al. disclose the use of a specific pilus adhesin and adhesia polypeptides as vaccines against the pathogenic pilus-forming bacteria [U.S. Patent No. 5,804,198]. Normark et al. describe specific adhesia polypeptides and antibodies useful in the diagnosis and treatment of pathogenic Neisseria and other type IV pilus presenting microorganisms [U.S. Patent 5,834,591].

Bacterial colonization factors such as those classified in the "type IV pilus" family are found in a wide variety of human_^ animal, and plant pathogens. These "pili" structures are widely regarded as a major virulence factor. PAIs and type IV pili are important to the pathogenic microorganisms in that the presence of these elements are essential for pathogenesis and provide the organism with its ability to cause disease. The proteins encoded by genes on PAIs and proteins of type IV pili are often very immunogenic and antibodies directed against these proteins are often protective. PAIs and type IV pili are found in a wide variety of many bacterial pathogens species which are associated with humans, animal and plants. Examples of these pathogens (and their associated infectious disease or primary symptom) include: Vibrio cholerae (cholera); E. coli (diarrhea), Neisseria gonorrhoea (gonorrhea), Neisseria meningitidis (bacterial meningitis), Pseudomonas aeruginosa (lung infections/cystic fibrosis), Moraxella bovis (bovine keratoconjunctivitis), Legionella pneumophila (pneumonia), Dichelobacter nodosus (cattle foot rot), Eikenella corrodens (periodontal disease and soft tissue infections), and certain species of Bacteroides and Salmonella (diarrhea). The TCP (toxin coregulated pilus) of V. cholerae is highly homologous to the type IV pili of other genuses.

Type IV pilus gene homo logs have been described in the literature. Specifically, a region of the Incll conjugative plasmid R64 contains genes that encode a pilus (Kim et.al. , J. Bacteriology, 179:3594-3603, 1997). These R64 pilus genes have homology to type IV pilin genes, especially those of V. cholerae and E. coli. This suggests that they have evolved from a common ancestral system. Likewise, Eikenella corrodens, a gram negative human pathogen associated with periodontal disease and soft tissue infections, produces a type IV pilus (Hood et al. , Infection and Immunity, 63:3693-6, 1995). Legionella pneumophila which causes pneumonia expresses a type IV pilus (Liles et al. , Infection and Immunity, 66: 1776-82, 1998).

It was recently shown that type IV pilus dependent adhesion is also highly involved in plant- bacteria and fungus-bacteria interactions. The nitrogen fixing endophytic bacteria, Azoarcus spp., can infect roots of rice plants and spread systematically into the shoot without causing symptoms of plant disease. The proteins encoded by the pilus genes are involved in bacterial adhesion to the mycelium of an ascomycete which was isolated from the same rhizosphere as the bacteria. In co culture with the fungus, Azoarcus spp. forms a complex intracytoplasmic membranes, diazosomes, which are required for efficient nitrogen fixation. Adhesion to the mycelium appears to be crucial for this process (Dorr et al., Molecular Microbiology, 30:7-10, 1998). Additionally, the plant growth stimulating bacteria, Pseudomonas putida colonizes plant roots by means of a type IV pilus (de Groot et al., J. Bacteriology, 176:642-50, 1994). Finally, Myxococcus xanthus initiates a multicellular developmental program that culminates in cells aggregating and forming a fruiting body which is similar to the biofilm formed by Pseudomonas aeruginosa. Within this structure, vegetative cells differentiate into spores. This process depends on gliding motility. A component of this motility is due to a type IV pilus (Wall et al, J. Bacteriology, 181:24-33, 1999).

The Vibrio cholerae toxin-co-regulated pilus (TCP) is discussed in the literature as a highly immunogenic virulence factor. It is cited as a critical and essential component for attenuated live bacteria-based vaccines [Voss et al., Microb Pathog. 20(3): 141-53 (3/1996);

Taylor et al, Vaccine, 6(2): 151-4 (4/1998)]. TCP is further described as "highly homologous" to the type IV pili of other genuses in which the type IV pilus protein elicits an immune response.

Several bacterial genuses, such as Pseudomonas, Vibrio, E. coli, Salmonella, Legionella, Dichelobacter and Neisseria, possess structurally and functionally similar colonization factors, known as "type IV pili. " In several of these species, these "pili" contain a fimbrial protein subunit which contains methylated phenylalanine at its amino terminus. These "N-methylphenylalanine pili" have been established as important virulence determinants in the pathogenesis of Pseudomonas aeruginosa lung infection in cystic fibrosis patients. As stated above, these types of fimbriae occur in Neisseria gonorrhoea and their eukaryotic receptor is thought to be an oligosaccharide.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic diagram of the VPI. Arrows above genes show PCR primers used and are identified by the number of the KAR primer series, indicates regions targeted for PCR, indicate common chromosomal flanking DNA, at each end of VPI denotes αtt sites, within the left end of the VPI indicates the defective transposase (xtn). Insertion of the aphA-3 gene encoding Km resistance into a BamHI site is shown between tn and aldA. This Km gene marker was used to demonstrate transfer of the VPI between strains.

Figure 2 shows the nucleotide sequence of AldA (SEQ ID NO: 22). Figure 3 shows the protein sequence of AldA (SEQ ID NO: 23).

Figure 4 shows the nucleotide sequnce of VPI orf2. (SEQ ID NO: 24).

Figure 5 shows the VPI orf2 protein (SEQ ID NO:25). DESCRIPTTON OF THE INVENTION

The present invention relates to compositions, methods, processes, etc., relating to bacteriophage which are encoded by chromosome, plasmids, or other genomic elements of bacteria. The bacteriophage of the present invention are preferably encoded by pathogenicity islands in chromosomes or plasmids of pathogenic bacteria. **The bacteriophage can be utilized as a pharmaceutical composition, e.g., to elicit an immune response, e.g., for the purpose of producing antibodies, as vaccines and vaccine vectors to regulate the immune system, for the prevention and treatment of allergy, disease, and other pathological conditions. The invention finds additional utility in systems and methods for the detection of pathogens comprising bacteriophage and a system and method for the environmental eradication of pathogenic microorganisms.

The present invention relates to an isolated bacteriophage, preferably encoded by a pathogenicity island of a pathogenic bacteria. By the term "isolated," it is meant that it is essentially-free of cells, e.g., separated from a cell culture by centrifugation. An isolated bacteriophage can be further concentrated and/or purified, according to any suitable method, e.g., precipitation, chromatography, etc. By the term "encoded," it is meant that the information necessary to make the phage protein and nucleic acid are present in a pathogenicity island, or other region which is part of a bacterial genome or bacterial extrachromosomal element. The present invention relates to methods of using the bacteriophage to elicit an immune response. By the term "immune response," it is meant any humoral or cell- mediated response of the immune system provoked by an antigen present in the bacteriophage. Such a response includes, antibody production, e.g. , IgG, IgM, etc. , T-cell proliferation, B-cell proliferation, tolerance, anergy, apoptosis, T-cell deletion, production of antibody secreting cells, activation of B-cells and T-cells, etc. See, e.g., Cellular and

Molecular Immunology, Abbas et al. , W.B. Saunders Company, 1997. Such an immune response can be useful for preparing antibodies, e.g., from mice, goats, etc., for allergy and pathogenicity testing, for vaccines, etc. An immune response does not have to be fully protective in order for it be useful. For instance, a less than protective response can be utilized to stimulate antibody production to make polyclonal or monoclonal antibodies, or as a challenge in conventional allergy testing. In addition, a bacteriophage can be administered to treat an infection which is already in progress, e.g., by stimulating the immune system.

An object of the invention is also to provide a bacteriophage-based vaccine against pathogenic bacteria which express virulence factors, such as phage-encoded pathogenicity islands, phage encoded colonization factors (e.g., type IV pili), and/or phage-encoded virulence factors. The phage encoding virulence functions are referred to as "pathophage. " In a preferred embodiment, the pathogenicity island encodes bacterial colonization factors. In a more preferred embodiment, the host colonization factor is a pilus, e.g. , a type I-IV. (Hobbs and Mattick, 1993, Molecular Microbiology 10:233-243; Forest and Trainer, 1997, Gene 192: 165-169). In a preferred embodiment, the PAI encodes for a type IV pilus, a group of proteins described in the art as key colonization factors. In the most preferred embodiment, the pathogenicity island encodes the type IV pilus of Vibrio cholerae. The bacteriophage based vaccines of the invention can contain a mixture of one or more phage and be directed against one or more pathogenic bacteria. Bacteriophage vaccines as described herein possess a number of advantages. For example, bacteriophage are nonliving while outside their bacterial host. Thus, a bacteriophage vaccines is inert, nontoxic, and completely safe, being unable to infect humans, animals or plants, result in disease or revert to virulence (as can be the case when using live attenuated vaccines). Likewise, a bacteriophage can be lyophilized for long term storage and require no cold-chain. In addition, a bacteriophage vaccine can be orally administered. Furthermore, a bacteriophage vaccine can be essentially free of bacteria, or other cells, and thus avoids the toxic effects which are often associated with using live- attenuated bacterial vaccine strains and whose source within the bacteria are unknown. In other words, as the bacteriophage represents a single component (often containing the virulence component(s)) of the whole pathogenic bacteria, the bacteriophage is absolutely incapable of transmitting and producing the infectious disease by itself. Also, most virulence proteins of the bacterial pathogen are encoded by genes on PAIs and are essential to the disease process, are highly immunogenic, and antibodies against these proteins are known to provide protective immunity against subsequent bacterial infection. Finally, the bacteriophage preparation is much less expensive to produce as it relies on an efficient and natural biological system already occurring in nature (i.e, the making of phage by the respective bacteria).

The present invention also relates to the use of the bacteriophage vaccine as a vector, e.g., to transfer DNA sequences from one organism to another, to express additional 'foreign' immunogenic antigens (those not usually associated with the host bacterium) on the surface of the phage as part of its coat proteins, and other purposes for which a vector can be used. A bacteriophage vaccine of the present invention can be used to express foreign antigens from non-bacterial viruses (e.g., such as those viruses that result in human, animal, and plant diseases), antigens from bacterial pathogens and/or their associated phages, antigens from parasites, and any other antigenic moiety, such as moieties that elicit immune responses, allergies, and autoimmune disease. A bacteriophage vaccine of the invention can include and express on its surface several immunogenic and protective antigens, thereby providing protection to a wide range of antigens. Another aspect of the invention involves the mutation or modification of phage. In one embodiment, a phage of the present invention, normally secreted by the bacteria, is genetically engineered to be defective and remain on the surface of the bacteria. They then may function in a manner analogous to 'pili' . The surface expressed phage will intensify the antigenic nature of the bacteria, enhancing its utility as a live attenuated vaccine. In another embodiment, phage that are already defective, being retained on the surface of the bacteria in the wild type, can be reverted to functional phage, thereby allowing the phage to be easily isolated and purified from the bacterial environment and used as phage vaccine.

The present invention also relates to a live bacterial vaccine, e.g., by incorporating a bacteriophage into live bacteria. The bacteria can be naturally non-virulent or non toxigenic or can be a virulent strain attenuated for vaccine purposes. The live bacterial vaccine can produce functional, secretable phage or defective phage retained on the bacterial cell surface or can produce a mixture thereof. The live bacterial vaccine can contain multiple phage, multiple foreign antigens, or a mixture thereof. As mentioned above regarding the bacteriophage based vaccine alone, the live bacterial vaccine can be directed against multiple pathogenic bacteria, viruses or parasites. Another aspect of the invention involves the use of the bacteriophage in rapid detection systems. Such a detection system can involve the use of antibodies, such as those against type IV pilus antigens or other phage encoded proteins. Such a detection system can be used to detect whole cells, free phage, and phage encoded proteins produced by the cells, thereby giving a greater possibility of detecting the presence of pathogen. A bacteriophage-based detection system of the invention can involve a simple, rapid and easily visible agglutination reaction (representing an antigen: antibody complex) if the sample is positive for the bacteriophage component. Such a system could be easily modified to further include a chromogenic marker tagged to the antibody to allow for a color reaction if the sample is positive.

Another embodiment of such a detection system can involved the use of a reporter strain which acts as a recipient for the bacteriophage. The reporter strain can contain a gene which becomes activated by a gene activator encoded on the phage genome. This gene in the reporter strain can be fused to a reporter protein such as green fluorescent protein (GFP). If bacteria making the phage or free phage are present in the sample, the reporter strain becomes infected, and subsequently the reporter gene becomes activated. A simple, rapid, and easily visible color reaction (such as fluorescence) would signify a positive sample.

The present invention also relates to the use of gene sequences contained within the pathogenicity islands and unique to pathogenic strains as genetic markers to identify potentially epidemic strains of bacteria. Another aspect of this invention relates to an eradication strategy wherein the pathophage is used as a means to introduce a suicide message in virulent strains of the bacteria.

It is a further object of this invention to use said phage-encoded pathogenicity islands or "pathophage" as a suicide vector for the elimination of pathogenic and epidemic strains of bacteria. Such an elimination system would involve the introduction of a "kill" gene, such as colicin, into the bacteriophage. The suicide-phage would be introduced into the environment which hosts the pathogen and selectively would infect only those specific bacterial strains with pathogenic and epidemic potential (i.e., those strains having phage- encoded pathogenicity islands and virulence determinants). -lilt is a further object of this invention to use highly conserved genes within the phage encoded pathogenicity island as a genetic marker for identifying potentially epidemic and pandemic strains of bacteria. Such conserved gene sequences located on pathogenicity islands can be used as probes or as targets to detect the presence of pathogenic strains of the bacterial species. Examples of conserved genes in the V. cholerae VPI phage include aldA, tcpR, tcpD, tcpE, acfC, tagE, acfA, and int.

Herein, the term "epitope" refers to an antigenic determinant of a polypeptide in a unique spatial configuration, generally consisting of no less than 3, preferably at least 5, more preferably at least 8 to 10 amino acids, e.g., 8-50 amino acids, 10-35 amino acids, etc.

Herein, the term "immunoreactive" refers to a polypeptide that reacts with an antibody, i.e., binding to an antibody due to epitope recognition. An immunoreactive polypeptide may also be "immunogenic, " referencing the elicitation of a cellular or humoral immune response, in the presence or absence of an adjuvant, whether alone or linked to a carrier.

Herein, the term "antibody" refers to a polypeptide or group of polypeptides which are comprised of at least one antibody combining site or binding domain, said binding domain or combining site formed from the folding of variable domains of an antibody molecule to form three dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigen epitope. The term encompasses immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that contain an antibody combining site or paratope. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those known in the art as Fab, FabB, F(abB).sub.2 and F(v), chimeric antibodies, humanized antibodies, single-chain antibodies, antibody fusions, etc.

As used herein, the term "vaccine antigen" refers to an agent capable of stimulating the immune system of a living organism, inducing the production of an increased level of antibodies, the production of a cellular immune response, or the activation other immune responsive cells involved in the immune response pathway against said antigen. As used herein, the term "vaccine" refers to an agent used to stimulate the immune system of a living organism against a particular infectious agent or organism, capable of inducing range of immune responses, from protection against future infection to the reduction of the severity, mortality or morbidity of subsequent infection. Herein, the native phage or recombinant protein may be given with a carrier as a vaccine. Any carrier may be used which does not itself induce the production of antibodies harmful to the host. Suitable carriers are described in Normark et al., U.S. Patent No. 5,834,591, hereby incorporated by reference in its entirety.

Herein, the term "recombinant polynucleotide" refers to a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin which is distinct in form, linkage or association from the form, linkage, or association in which the polynucleotide exists in nature. The term "recombinant DNA" refers to a DNA molecule produced by operatively linking two DNA segments. Thus, a recombinant DNA molecule is a hybrid molecule comprising at least two nucleotide sequences not normally found together in nature. Recombinant DNA molecules not having a common biological origin (i.e. , evolutionarily different) are said to be "heterologous. "

Herein, the term "fusion polypeptide" refers to a polypeptide comprised of at least two polypeptides and a linking sequence to operatively link the two polypeptides into one continuous polypeptide. The two polypeptides linked in a fusion polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature.

Herein, the term "purified polynucleotide" refers to a polynucleotide which is essentially free, i.e., containing less than about 50%, preferably less than about 70% , even more preferably less than about 90% of polypeptides with which the polynucleotide is naturally associated.

As used herein, the term "vector" refers to a genetic element capable of replication under its own control having 'foreign' polynucleotide segments attached thereto so as to bring about the replication and/or expression of the attached segments. Examples include plasmid, cosmid, chromosome, bacteriophage, virus, bacteria, and other microorganisms. As used herein the term "vaccine vector" refers to a vector encoding for a vaccine antigen segment or capable of functioning as a vaccine. The phage itself may serve as a vector. Heterologous DNA may be inserted into the phage genome by homologous recombination, or by insertion into a restriction site contained in or engineered in the vector. The inserted sequences may be those which encode all or varying segments of the polyprotein, or other open reading frames (ORF) which encode bacterial, viral, phage, or parasitic polypeptides.

As used herein, the abbreviation "PAI" refers to a pathogenicity island, a region of the chromosome or plasmid of a pathogenic bacteria carrying one or more virulence genes, said region being unique to the pathogenic strain of the species as compared to a nonpathogenic strain. As used herein, the term "type IV pilus" or "type IV pilin" refers to a bacteriophage or bacterial filamentous surface appendage formed by pilin subunits involved in pathogen colonization and adhesion and bacterial aggregation, crucial in virulence, and which can serve as a receptor for another bacterial bacteriophage or "pathophage" encoding bacterial virulence factors. Type IV pilins are specifically often defined as pilin which contain a conserved amino acid terminal hydrophobic domain beginning with an amino-terminal phenylalanine that is methylated upon processing and secretion of the pilin. Another characteristic feature of type IV pilins is that in the propilin form they contain similar six- or seven- amino acid long leader peptides, which are much shorter than typical signal sequences. The type IV pilin toxin coregulated pilus (TCP) of V. cholerae is highly homologous to the type IV pilins of other genuses. TCP contains the characteristic amino- terminal hydrophobic domain as well as having a modified N-terminal amino acid that in this case is a modified methionine at the position where some others encode phenylalanine. Precursor TcpA (the major pilin subunit) contains a much longer leader sequence than typical type IV propilins but still retains homology in the region surrounding the processing site.

Herein, the terms "bacteriophage" and "phage" are used interchangeably to mean, e.g. , a bacteria virus which comprises a protein coat surrounding an RNA or DNA core of genetic material but no semipermeable membrane. A bacteriophage is capable of growth and multiplication only in the living cells (bacteria) it infects. Therefore, a bacteriophage is generally regarded as acellular or cell-free and non-living or inert. "Pathophage" refers to bacteriophage which encode virulence or pathogenic potential to a bacterial species. A bacteriophage vaccine in accordance with the present invention comprises a protein coat and RNA or DNA core.

Herein, the abbreviation "VPI" refers to Vibrio cholerae pathogenicity island.

Herein, the abbreviation "VPIΦ" refers to Vibrio cholerae pathogenicity island phage.

Herein, the abbreviation "CTXΦ" refers to cholera toxin phage.

Herein, the abbreviation "CT" refers to cholera toxin.

Herein, the abbreviation "TCP" refers to toxin co-regulated pilus.

The present invention relates to bacteriophage which are encoded by a bacterial chromosome, plasmid, or other extrachromosomal elements, and which are functional phage. By the term "functional phage," it is meant a phage which comprises a protein coat and nucleic acid core. Such a phage can be infectious, i.e. , capable of infecting other bacteria, or it can be infection-incompetent, i.e., not capable of infecting other bacteria, e.g., if it is missing a cork protein which is involved in phage-bacterium adsorption. It can be present in any form, e.g., secreted by the bacteria into the medium, intracellular, or attached to the bacteria surface, e.g., as a pilus, such as a type IV pilus. In preferred embodiments, the bacteriophage is encoded by a pathogenicity island (PAI), hereinafter a "Paiphage."

A PAI can be characterized by one of more of the following criteria: (1) the carriage of often many virulence genes; (2) the presence in pathogenic strains, and absence or sporadic distribution in less-pathogenic strains of one species or related species; (3) a distinct percentage G+C content in comparison to DNA of host bacteria; (4) occupation of large chromosomal or plasmid regions (can be greater than 7 kb up to 200 kb, often about 30kb); (4) a compact, distinct genetic unit, often flanked by direct repeats; (5) an association with tRNA and tRNA-like genes and/or insertion sequences (IS) elements at their boundaries; (6) the presence of (often cryptic) "mobility" genes (IS elements, integrases, transposases, origins of plasmid replication); and (7) instability. See, J. Hacker et al., "Pathogenicity Islands of Virulent Bacteria: Structure, Function and Impact on Microbial Evolution," Molecular Microbiology, 23(6): 1089-1097 (1997). In a preferred embodiment, the PAI encodes for a type IV pilus, a group of proteins described in the art as key colonization factors. These colonization factors are immunogenic and important protective antigens, making them successful vaccine candidates. As the pilus component of the bacteria is known to target the specific site of infection associated with that bacteria (such as the M cells of the intestine in the case of an enteric pathogen), a bacteriophage vaccine representing type IV pili (or encoding other colonization factors) would also target the desired region in order to create an appropriate immune response at that site. Antibodies at that site would be protective against subsequent exposure to the pathogen.

A PAI can be identified routinely, e.g. , using PCR, nucleic acid probing, and other suitable techniques. For instance, a PAI can be identified by sequencing the entire chromosome or plasmid and scanning for the virulence gene cluster characterized by the distinct G/C ratio compared to the rest of the chromosome or plasmid. Alternatively, if the full sequence is not readily available, one can compare the total DNA of pathogenic to nonpathogenic strains of a particular species (by subtractive hybridization techniques) and probing methods and identify those sections that are unique to the pathogen's genome and that are distinct to the pathogenic strain. Those regions unique to the pathogen and those having the distinct G/C ratio are likely PAIs. Likewise, one can use known virulence factors and extend the sequence out looking for additional unique and adjacent genes. In accordance with the present invention, a paiphage, or other chromosomal or extrachromosomal phage, can be identified by any effective method, including, e.g., detecting functional paiphage, or parts thereof (such as nucleic acid or polypeptides) in media in which a bacteria has been cultured. Detection can be accomplished by any suitable method, e.g. , nucleic acid technology to identify phage nucleic acid in the media, such as hybridization, PCR, dot blots, etc., polypeptide detection technology, such as ELISA, immunoprecipitation, etc., functional assays, such as the ability of the media supernatant to cause infection in susceptible bacterial cells, etc. Paiphage can also be identified by a combination of techniques, e.g., scanning a bacterial genome for PAI in a specific bacterial strain and then testing media in which such a strain has been cultured for paiphage, or parts thereof. In addition to PAI, bacteriophage in accordance with the present invention can be isolated from other "islands" or gene clusters in bacterial chromosomes or extrachromosomal elements which heretofore were not recognized to encode functional phage. For instance, bacteria also possess gene clusters which confer non- virulence properties, such as properties which enhance bacterial survival, e.g., genes which encode drug and chemical resistance. Such bacteria can be treated with methods in accordance with the present invention to isolate bacteriophage from it.

In a preferred method of the present invention, the presence of a phage is detected in bacterial supernatant. A preferred method involves centrifuging a bacterial culture to pellet out the cells (whole bacteria). [See detailed section in Methods for Purification steps- -isolation of phage.] Details of the preferred purification method are provided in the example section below. The cell-free supernatant can be removed and tested for the presence of phage. The detection of phage can be performed using PCR or recombinant techniques standard in the art. The bacteriophage contained in the supernatant should be collected and concentrated to ensure that the bacteriophage is in an isolated and purified condition.

A bacteriophage of the present invention can be obtained from any bacteria, including pathogenic bacteria, such as, but not limited to, e.g., gram-negative bacteria, gram-positive, ricketsiae, chlamydiae, mycoplasma, streptococci, diphtheriae, bordetella, neisseria, haemophilus, mycobacterium, klebsiella, legionella, clostridium, staphylococcus, salmonella, shigella, vibrio, escherichia, campy lobacter, brucella, bacillus, yersinia, pleisomonas, aeromonas, leptospira, listeria, pseudomonas, borrelia, rochalimaea, francisella, ehrlichia, treponema, gonococcus, chlamydia, ureaplasma, calymmatobacterium, gardnerella, actinomyces, nocardia, pasteurella, spirillum, bacteriodes, streptobacillus, enterobacter, proteus, amphitrichous, bartonella, corynebacterium, listeria. EPEC, ETEC, rhizobium, helicobacter, myxococcus, azoarcus, acinetobacter, kingella, etc. See, also Fundamentals of Microbiology , Alcamo, I.E. , 1994. In particular, a bacteriophage of the present invention can be obtained from any bacteria comprising a PAI, a pili, or other virulent factor, etc., e.g. , Vibrio cholerae, E. coli, Neisseria gonorrhoea, Neisseria meningitidis , Pseudomonas aeruginosa, Moraxella bovis, Legionella pneumophila, Dichelobacter nodosus , Eikenella corrodens, Bacteroides and Salmonella, Helicobacter spp. , Yersinia spp. , Clostridium spp. , Staphylococcus spp. , Streptococcus spp. , etc. Examples of suitable Escherichia strains which can be employed in the present invention include enterotoxigenic E. coli (ETEC), enteropathogenic E. coli

(EPEC), and E. coli strains O157:H7; 4608-58; 1184-68; 53638-C-17; 13-80; and 6-81 [Sansonetti et al, Ann. Microbiol. (Inst. Pasteur), 132A:351-355 (1982)]. Examples of suitable Neisseria strains which can be employed in the present invention include N. meningitidis (ATCC No. 13077) and N. gonorrhoeae (ATCC No. 19424). Examples of suitable Legionella -strains which can be employed in the present invention include L. pneumophila (ATCC No. 33156). Examples of suitable Pseudomonas strains which can be employed in the present invention include P. aeruginosa (ATCC No. 23267). Examples of suitable Salmonella strains which can be employed in the present invention include Salmonella typhi (ATCC No. 7251) and S. typhimurium (ATCC No. 13311). Examples of suitable Vibrio strains which can be employed in the present invention include V. cholerae N16961; V. cholerae (ATCC No. 14035) and V. cincinnatiensis (ATCC No. 35912).

As described above, it is an object of the invention to use a bacteriophage of the present invention to express one or more "foreign genes. " As used herein, "foreign gene" means a gene encoding a protein or fragment thereof or anti-sense RNA or catalytic RNA, which is foreign to the particular pathogenic bacterial species. The gene may encode for a vaccine antigen, an immunoregulatory agent, or a therapeutic agent. The bacteriophage may also be used as a vaccine vector, an expression cassette encoding foreign genes. The particular foreign gene can be inserted into the PAI while it is integrated in the bacterial chromosome or bacterial plasmid or while in the plasmid replicative form (RF) of the phage using known recombinant techniques (e.g., cloning, allelic exchange, etc.). Alternatively, the foreign antigen or epitope may be incorporated into the major coat protein. In the case of VPI, the major coat protein is TcpA. Other fusion peptides have been expressed on the surface of a filamentous phage particle by fusion to a major coat protein including polypeptides in cpVIII, the major coat protein of the Ff phage (Bichev et al. , Molekulvarnava Biologiva, 24: 530-535 (1990) and Markland et al., Gene, 109: 13-19 (1991)).

The plasmid RF of the phage is preferred in some instances because it can accommodate large pieces of DNA, can be a very useful general cloning vector. The preferred insertion site of the foreign gene is readily determinable by routine experimentation and is not critical to the invention. In a preferred embodiment, the foreign gene (or genes) is introduced into the phage genome so that it is actively transcribed in the host strain; thus, the protein is expressed from a suitable promoter. A possible insertion site in the VPΦ phage is the orβ sequence. Insertion may disrupt release of the phage but not the formation of the structure on the surface of the cell, thereby maintaining the function and operability of the mutant bacteria as a live attenuated vaccine or vaccine vector. Typically, a foreign gene is not inserted such that it disrupts the function of an open reading frame that is essential for subsequent phage expression of the desired immunogenic proteins; rather, it is inserted into intergenic regions or preceding an adjacent nonfunctional gene that may be remnant on the phage genome. In a preferred embodiment, the foreign antigen is itself phage-encoded, thereby allowing it to be successfully expressed on the surface of the phage as one or part of its coat protein and thus be more immunogenic. If the particular foreign gene is not phage encoded, one may have to fuse it with a phage coat protein which is ultimately located on the phage surface to ensure its contact with immune system.

One may further use the bacteriophage to express foreign antigens in a live attenuated bacteria strain as vaccines. A phage that encodes a PAI, type TV pilus, or toxin can multiply infect a particular bacterium. The vaccine antigen expressing phage, either naturally or through bioengineering, can infect a non-toxigenic or non-pathogenic strain of bacteria, thereby creating a live vaccine strain that is naturally attenuated. Alternatively, the phage can infect a virulent strain of bacteria, said strain then attenuated for vaccine purposes. In addition, the genome of the infected_bacteria may undergo a transformation wherein the genome of the phage is integrated with the genome of the host bacteria, resulting in a newly created strain having certain desirable properties. This represents a novel method for modifying the specific properties of a bacterium, useful in introducing mutated genes and creating mutant strains.

In the case of V. cholerae, the CTXΦ which encodes CT is known to naturally multiply infect the same bacterial cell. The bacteria can subsequently be made attenuated so that it is non pathogenic. The proposed system can result in the production, expression or secretion of several independent phage, each with different antigens expressed thereon or of a single phage that expresses the various different antigens on its surface. Alternatively, genetic manipulation of a specific phage encoded gene (e.g. , orβ encoded on the VPI) will render the phage secretion system defective, resulting in the phage remaining on the bacterial cell surface (resembling pili) and thus allows the simultaneous expression of single or multiple foreign antigens on the surface of a single bacterium. A bacteria containing the defective phage, retained as a pili on the cell surface, finds utility as a live vaccine as well as a heat killed vaccine. The latter are generally safer and can be administered with an adjuvant, such as cholera toxin B subunit, to increase immunogenicity. Modifications at orβ may involve the fusion of a foreign gene (antigen) or foreign epitope within the orβ gene so that the foreign antigen is expressed as part of the phage coat protein. It is known in the literature that insertion of epitopes into Orf2-like proteins (called cpIII or Gene III in the case of some filamentous phages) result in expression of foreign antigens on the phage surface. These studies were aimed at making cloning vectors and for making phage libraries, not for vaccines or in any way related to virulence and pathogenesis (McCafferty et al., Science 348:552 554 (1990); and Patent 387874, 1998 Light, II; J.P. and Lerner; R.A.) and McCafferty et al., Protein Eng., 4:955-961 (1991). The expression of a heterodimeric recombinant gene product such as an antibody molecule on the surface of a filamentous phage containing recombinant genes has been described (Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366 (1991) and Barbas et al., Proc.

Natl. Acad. Sci., USA88:7978-7982 (1991)). Human antibodies have been expressed that immunoreact with hepatitis B virus surface antigen. Zebedee et al. , Proc. Natl. Acad. Sci., USA, 89:3175-3179 (1992). i.e. , The heterologous polypeptide(s) could be expressed on the phage surface via one or more of the coat proteins of the phage, i.e., either at the tip of the phage (in the case of Orf2 fusions) or along the entire phage (in the case of the major coat protein). Such would result a single phage expressing multiple antigens on its surface. In addition, a phage having a mutation in orβ mutant (or equivalent thereof) may be capable of being secreted but subsequently unable to infect another bacterial strain. The method of mutating orβ (and hence, other similar proteins) involves well known recombinant techniques described in the literature.

The invention herein contemplates pathogenic bacteria having PAI phage that are functional and secreted (e.g., the 7th pandemic El Tor biotype strains of V. cholerae) as well as those that are defective and retained on the bacterial cell surface (e.g., 6th pandemic classical biotype strains of V. cholerae). The defective strains are likely defective in the 'cork,' 'tip' and/or 'absorption' protein - those proteins similar in function to the Gene III of the non-pathogenic phage M13, for example. These proteins are needed at the final stages for phage assembly and release. An aspect to the invention involves the restoring of function, secretion and release properties to defective phage, those defective phage that are retained on the bacterial cell surface and appear as 'pili. ' Specifically, these defective pathophage may be turned into functional pathophage. This step may involve the introduction of a suitable functional gene, such as genes homologous to Gene III of M13 phage into the bacterium. Alternatively, the restoration may be achieved by minor manipulation of the defective gene(s) involved in the host strain to restore function to the genes involved. These restored pathophage can then be easily isolated and purified and used as bacteriophage-based vaccines. While the findings described in detail herein are directed to V. cholerae, the methods, processes, compositions, etc., can be applied analogously to bacterial strains which comprise paiphage, such as those mentioned above and below, and other known strains, including those deposited with a culture collection, such as ATCC (www.atcc.org), CIP, DSM, FERM, NRRL, etc. Details of this embodiment can be extrapolated from the example described below relating to the use of live attenuated V. cholerae that expresses multiple foreign immunogenic antigens after it has been multiply infected with VPI phage, each of which carry a different antigenic protein. V. cholerae has been successfully used as a vaccine vector to express plasmid carried foreign antigens, such as E. coli (ETEC) colonization factor antigens (CFAs), Clostridium difficile antigens, and E.coli O157:H7 (EHEC) intimin.

Many different serogroups or serotypes of a species can possess the same pathogenicity island, pilus-type, or the like. Thus, it is important to note that the bacteriophage vaccine containing the relevant immunogenic proteins (i.e., antigens) can be effective and provide protection against specific bacterial pathogens regardless of serogroup or serotype. Regarding bacteriophage vaccines against V. cholerae in particular, the newly constructed VPI positive, CTX negative, serogroup O10 strain of V. cholerae, DK239, is a preferred strain in that it secretes only VPI phage and no other phage, making isolation of desired phage less complicated. It is also a good strain in which to study the role of specific virulence factors, i.e. VPI phage alone, then VPI together with CTX, then genetic mutants of these phage, etc. Regarding live attenuated strains of V. cholerae as vaccines, preferred embodiments include VPI positive, CTX negative strains including both Ol (Ogawa and Inaba serotypes) and non-Ol serogroups. While the examples describe the system using V. cholerae and VPI, it is clear that other phage of V. cholerae (e.g., CTXF) and even pathogenic strains such as Salmonella, E. coli, and Shigella and their phage may be used. Indeed, all pathogenic strains from which pathophage (or parts thereof) can be isolated as well as antigens expressed on phage made by these strains are contemplated by the invention herein. These strains can be engineered or mutated to express foreign antigens on the phage encoded structures, said phage either being secreted into the medium or made defective so as to be retained on the bacterial cell surface.

However, the fact that V. cholerae is a good colonizer of the intestine and targets this region well and persists long enough for the body to create an immune response makes it a good candidate for a vaccine strain against enteric infections. By the same token, the fact that Neisseria spp. naturally colonizes the respiratory and genital mucosa makes it a good candidate for an vaccine administered or directed to either of these sites. Obviously, the normally virulent strain must be made non-toxigenic (i.e., attenuated) and must have the cholera toxin genes deleted. However, such attenuation mutations and strains are known in the art and are not critical to the broad aspects of the invention.

Currently, scientists depend on plasmids to allow a single bacteria to express numerous foreign antigens. However, plasmids have limitations because there is a limit on the number and type (compatability) of plasmids a single bacterial cell can possess. Also, plasmids may be unstable and lost by the bacterium under certain conditions. Next, there is a size limitation on the size of DNA which can be incorporated and expressed.

On the other hand, the genome of a filamentous bacteriophage, like the VPIF and other analogous phage ("pathophage") which encode PAIs and virulence factors, is able to incorporate large pieces of DNA into its genome and still be functional as a "phage. " Each copy of the phage genome is specifically manipulated to express a different antigenic protein or proteins, thereby allowing the expression of multiple proteins in a single cell. Multiple copies or "cassettes" of the VPIF in the same cell are possible because the VPI genome incorporates into a specific site, the att site, on the bacterial chromosome. Incorporation of the phage genome into this site restores the αtt site and allows for potentially a subsequent incorporation of an adjacent phage genome. This is referred to as "Pathophage Cassette Vaccine Technology (P AC A VAT). " This initial copy of the phage encoded PAI or type IV pilus (or cassette 1) can encode a foreign protein or proteins and, since it is a lysogenic bacteriophage, whose primary function is the non-lytic infection of bacteria, the phage genome and contents can be stably incorporated into the host bacteria. Thus, the V. cholerae bacterial strain now expresses this foreign protein. The same V. cholerae strain (possessing pathophage cassette 1) can then be infected with a second VPI phage which contains a second foreign antigen (and represents cassette 2). Thus, this strain now expresses two foreign antigens simultaneously. The data to date suggest that multiple phage infection of V. cholerae can occur with CTXF. The data herein suggest that the same is possible for VPIF.

Any antigen can be incorporated into a bacteriophage of the present invention, including, e.g., antigens which elicit immune or allergic responses (e.g., animal dander, pollen, dust, etc.), proteins or antigenic fragment thereof from viral pathogens, bacterial pathogens, and parasitic pathogens. Alternatively, the vaccine antigen may be a synthetic gene, constructed using recombinant DNA methods, which encode antigens or parts thereof from any of the mentioned antigens, especially, viral, bacterial, parasitic pathogens. These pathogens can be infectious in humans, domestic animals or wild animal hosts and plant species. The antigen can be any molecule that is expressed by any viral, bacterial, parasitic pathogen prior to or during entry into, colonization of, or replication in their respective host. Antigens such as colonization factors, invasins, adhesins, outer membrane proteins and the like are frequently most efficacious in vaccine strategies. In addition to immunogenic proteins, the bacteriophage based vaccine may contain certain toxin subunits, particularly the binding subunit of the toxin rather than the subunit associated with disease. Such a vaccine can generate both antibacterial and antitoxic immunity. This combination has been shown to result in synergistic protection.

The viral pathogens, from which the viral antigens are derived, include, but are not limited to, Orthomyxoviruses, such as influenza virus; Retroviruses, such as RSV and SIV, Herpes viruses, such as EBV; CMV or herpes simplex virus; Lentiviruses, such as human immunodeficiency virus; Rhabdoviruses, such as rabies; Picornoviruses, such as poliovirus; Poxviruses, such as vaccinia; Rotavirus; and Parvoviruses. Viruses associated with rubella, influenza, mumps, measles, and poliomyelitis are also contemplated.

Examples of protective antigens of viral pathogens include the human immunodeficiency virus antigens Nef, p24, gpl20, gp41, Tat, Rev, and Pol et al, Nature, 313:277-280 (1985)), T cell and B cell epitopes of gpl20 (Palker et al, J. Immunol. ,

142:3612-3619 (1989)), and the envelope protein; the hepatitis B surface antigen (Wu et al, Proc. Natl. Acad. Sci., USA, 86:4726-4730 (1989)); rotavirus antigens, such as VP4 (Mackow et al, Proc. Natl. Acad. Sci., USA, 87:518-522 (1990)) and VP7 (Green et al, J. Virol. , 62: 1819-1823 (1988)), influenza virus antigens such as hemagglutinin or nucleoprotein (Robinson et al. , Supra; Webster et al, Supra) and herpes simplex virus thymidine kinase (Whitley et al, In: New Generation Vaccines, pages 825-854).

The bacterial pathogens, from which the bacterial antigens are derived, include but are not limited to, Mycobacterium spp. , Helicobacter pylori, Salmonella spp. , Shigella spp. , E. coli, Rickettsia spp. , Listeria spp. , Legionella pneumoniae, Pseudomonas spp. , Acinetobacter spp . , Vibrio spp. , Staphylococcus aureus, Streptococcus pyogenes and Borellia burgdorferi.

Examples of protective antigens of bacterial pathogens include the lipopolysaccharide (or products thereof) from numerous bacterial species such as V. cholerae and Shigella spp. ; the Shigella-like toxin and theShigella sonnei form 1 antigen (Formal et al, Infect. Immun. , 34:746-750 (1981)); the O-antigen of V cholerae Inaba strain 569B (Forrest et al, J. Infect. Dis. , 159: 145-146 (1989); protective antigens of enterotoxigenic E. coli, such as the CFA/I fimbrial antigen (Yamamoto et al, Infect. Immun. , 50:925-928 (1985)) and the nontoxic B-subunit of the heat-labile toxin (Clements et al, 46:564-569 (1984)); pertussis toxin and pertactin of Bordetella pertussis (Roberts et al, Vacc , 10:43-48 (1992)), and adenylate cyclase-hemolysin of 5. pertussis (Guiso et al,

Micro. Path. , 11:423-431 (1991)); and Clostridia botulinum toxin and fragment C of tetanus toxin of Clostridium tetani (Fairweather et al, Infect. Immun. , 58: 1323-1326 (1990)). Antigens against Pseudomonas aeruginosa and Acinetobacter infection can be used for the treatment of burn victims to prevent infection after skin graft. The parasitic pathogens, from which the parasitic antigens are derived, include but are not limited to, Plasmodium spp. , Trypanosome spp. , Giardia spp. , Boophilus spp. , Babesia spp. , Entamoeba spp. , Eimeria spp. , Leishmania spp., Schistosome spp. , Brugia spp. , Fascida spp. , Dirofilaria spp. , Wuchereria spp. , Chlamydia spp. and Onchocerca spp.

Examples of protective antigens of parasitic pathogens include the circumsporozoite antigens of Plasmodium spp. (Sadoff et al, Science, 24Ω:336-337 (1988)), such as the circumsporozoite antigen of P. bergerii or the circumsporozoite antigen of P. falciparum; the merozoite surface antigen of Plasmodium spp. (Spetzler et al, Int. J. Pept. Prot. Res. , 42:351-358 (1994)); the galactose specific lectin of Entamoeba histolytica (Mann et al, Proc. Natl. Acad. Sci., USA, £8:3248-3252 (1991)), gp63 of Leishmania spp. (Russell et al, J. Immunol. , 14Ω: 1274-1278 (1988)), paramyosin of Brugia malayi (Li et al, Mol.

Biochem. Parasitol. , 42:315-323 (1991)), the triose-phosphate isomerase of Schistosoma mansoni (Shoemaker et al, Proc. Natl. Acad. Sci., USA, 82: 1842-1846 (1992)); the secreted globin-like protein of Trichostrongylus colubriformis (Frenkel et al, Mol. Biochem. Parasitol. , 5Ω:27-36 (1992)); the glutathione-S-transferase's of Frasciola hepatica (Hillyer et al, Exp. Parasitol. , 25:176-186 (1992)), Schistosoma bovis and S. japonicum (Bashir et al, Prop. Geog. Med. , 46:255-258 (1994)); and KLH oi Schistosoma bovis and S. japonicum (Bashir et al, supra). Antigens against parasite associated conditions such malaria, pneumocystis, pneumonia, and toxoplasma are also contemplated herein.

As mentioned above, the foreign gene may encode a therapeutic agent to animal cells or animal tissue. For example, the foreign gene may encode a tumor-specific, transplant, or autoimmune antigen or part thereof. Alternatively, the foreign gene may encode a synthetic gene which encode tumor-specific, transplant, or autoimmune antigen or part thereof.

Examples of tumor specific antigens include prostate specific antigen (Gattuso et al, Human Pathol. , 26: 123-126 (1995)), TAG-72 and CEA (Guadagni et al, Int. J. Biol.

Markers, 2:53-60 (1994)), MAGE-1 and yrosinase (Coulie et al, J. Immunothera. , 14: 104- 109 (1993)). Recently it has been shown in mice that immunization with nonmalignant cells expressing a tumor antigen provides a vaccine effect, and also helps the animal mount an immune response to clear malignant tumor cells displaying the same antigen (Koeppen et al, Anal. N. Y. Acad. Sci. , 620:244-255 (1993)). Examples of transplant antigens include the CD3 receptor on T cells (Alegre et al, Digest. Dis. Sci. , 40:58-64 (1995)). Treatment with an antibody to CD3 receptor has been shown to rapidly clear circulating T cells and reverse most rejection episodes (Alegre et al, supra). Examples of autoimmune antigens include IAS a chain (Topham et al, Proc. Natl.

Acad. Sci., USA, 21:8005-8009 (1994)). Vaccination of mice with an 18 amino acid peptide from IAS a chain has been demonstrated to provide protection and treatment to mice with experimental autoimmune encephalomyelitis (Topham et al, supra).

Alternatively, in the present invention, the bacteriophage can express immunoregulatory molecules. These immunoregulatory molecules include, but are not limited to, growth factors, such as M-CSF, GM-CSF; and cytokines, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 or IFN-g. Recently, delivery of cytokines expression cassettes to tumor tissue has been shown to stimulate potent systemic immunity and enhanced tumor antigen presentation without producing a systemic cytokine toxicity (Golumbek et al, Cane. Res. , 52:5841-5844 (1993); Golumbek et al, Immun. Res. , 11: 183-192 (1993); Pardoll, Curr. Opin. Oncol. , 4:1124-1129 (1992); and Pardoll, Curr. Opin. Immunol. , 4:619-623 (1992)).

The particular administration route for the bacteriophage vaccine is not critical to the invention. The vaccine can be administered to the animal recipient by intravenous, intramuscular, intradermal, intraperitoneally, peroral, intranasal, intraocular, intrarectal, intravaginal, oral, immersion and intraurethral inoculation routes. The vaccine may be administered as a single dose or multiple dose. The bacteriophage-encoded vaccine containing the immunogenic antigen(s) may be administered in conjunction with other immunoregulatory agents, such as immune globins. The preparation of vaccines which contain an immunogenic peptide(s) as an active ingredient is known to one skilled in the art. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions. The preparation may also be emulsified, or the protein encapsulated into liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients for example are water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting agents or emulsifying agents, pH buffering agents and/or adjuvants which enhance the effectiveness of the vaccine. Examples of such adjuvants which may be effective are known in the art and include but are not limited to those described at column 18 of Normark et al. , U.S. Patent No. 5,834,591 cited above.

The amount of the bacteriophage vaccine of the present invention to be administered will vary depending on the species of the subject, its age, gender, health, as well as the disease or condition that is being treated or being vaccinated against. Generally, the dosage employed will be about 10³ to 10ⁿ viable phage, preferably about 10⁵ to 10° viable phage. The bacteria or bacteriophage of the present invention are generally administered along with a pharmaceutically acceptable carrier or diluent. In addition, the bacteria or bacteriophage of the present invention can be administered in combination with adjuvants capable of enhancing the immune response. Likewise, the bacteria or bacteriophage of the present invention can be administered in combination with an adhesin or adhesive factor capable of enhancing colonization and persistence.

Any suitable pharmaceutically acceptable carrier or diluent can be employed. Examples of diluents include a phosphate buffered saline, buffer for buffering against gastric acid in the stomach, such as citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) alone (Levine et al, J. Clin. Invest. , 22:888-902 (1987); and Black et al J. Infect. Dis. , 155: 1260-1265 (1987)), or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose, and optionally aspartame (Levine et al, Lancet, 11:467-470 (1988)). Examples of carriers include proteins, e.g., as found in skim milk, sugars, e.g. , sucrose, or polyvinylpyrrolidone. Typically these carriers would be used at a concentration of about 0.1-90% (w/v) but preferably at a range of 1-10% (w/v). The mucosal and systemic immune systems are compartmentalized. Thus, antigens delivered to mucosal surfaces elicit mucosal and systemic responses, whereas parenterally delivered antigens elicit mainly systemic responses but only stimulate poor mucosal responses. Moreover, mucosal stimulation at one mucosal site (for example the intestine) can result in development of immunity at other mucosal surfaces (for example genital/urinary tract). This phenomenon is referred to as the common mucosal system and is well documented. Successful administration requires the presentation of the antigen to the appropriate cell of the immune system. Preferred administration routes include those that target sites of the immune system, such as the mucosa or the lymph tissues. Thus, intranasal, intraocular, intrarectal, and intravaginal are preferred administration routes. The invention herein is particularly suited for vaccines, the preferred vaccinee or recipient or host being an animal. The recipient animal cells employed in the present invention are not critical thereto and include cells present in or derived from all organisms within the kingdom animalia, such as those of the families mammalia, pisces, avian, reptilia. Animal cells are defined as nucleated, non-chloroplast containing cells derived from or present in multicellular organisms whose taxonomic position lies within the kingdom animalia. The cells may be present in the intact animal, a primary cell culture, explant culture or a transformed cell line. The particular tissue source of the cells is not critical to the present invention.

Preferred animal cells are mammalian cells, such as humans, bovine, ovine, porcine, feline, buffalo, canine, goat, equine, donkey, deer, and primate cells. The most preferred animal cells are human cells.

The invention herein is further contemplated to encompass antibodies which react immunologically with VPI phage epitopes or foreign epitopes expressed by the bacteriophage encoded type IV pili or PAI. Monoclonal antibodies directed against VPI phage (or PAI-) encoded epitopes can be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known.

Antibodies, both monoclonal and polyclonal, which are directed against epitopes of the vaccine are particularly useful in diagnosis, and those which are neutralizing, may be useful in passive immunotherapy. Methods for introducing antibodies into an individual to accomplish passive immunotherapy are known in the art. In addition, monoclonal antibodies, in particular may be used to raise anti-idiotypic antibodies.

As described above, an embodiment of the invention concerns immunoassays and diagnostic kits. The epitopes encoded by a bacteriophage of the present invention, such as a phage-encoded PAI or type IV pilus which are immunoreactive with antibodies against epitopes in biological samples are useful in immunoassays to detect the presence of anti- epitope antibodies, or the presence of relevant microorganisms or its antigens in biological samples. Design of the immunoassays is subject to a great deal of variation and many formats are known in the art. For example, an enzyme linked immunosorbent assay (ELISA) may be used to detect and measure antigens (either bound to bacteria or to the phage or free in the environment) in a sample using antibodies made against the phage encoded PAI or type IV pili proteins, or to detect and measure antibodies to proteins in a sample using purified proteins that are encoded by the phage encoded PAI or a pilus.

As described above, another embodiment of a detection system contemplated by the invention herein involves the use of highly conserved genes within the phage-encoded pathogenicity island as a genetic marker for identifying potentially pathogenic, epidemic and pandemic strains of bacteria. Such conserved sequences can be used as probes to detect the presence of the pathogenic strain of bacteria. Examples of such genes found on the VPIF region of V. cholerae include aldA, tcpR, tcpD, tcpE, acfC, tagE, acfk, and int. In the course of the study of the genes found within the VPI phage-encoded pathogenicity island, it was found that the aldA gene is universally present in all epidemic V. cholerae strains. The aldA sequence is identical among strains, whereas other genes of the VPI such as tcpA, the gene encoding the major coat protein of VPIF, vary considerably. Polynucleotide probes for detection may comprise oligomers capable of hybridizing to a sequence in the aldA, wherein the oligomer is comprised of an aldA sequence complementary to at least 6 contiguous nucleotides of aldA. The invention herein contemplates a process for detecting the aldA sequence using a polynucleotide selected to target the aldA sequence or region thereof in a sample. Such a detection process would include the following steps: (1) incubating a suspect sample with an oligomer (or targeting sequence) complementary to the aldA sequence (or target sequence) for a time sufficient to allow specific hybrid duplexes to form between the targeting sequence and the target sequence and (2) detecting hybrids formed.

As mentioned above, another aspect of the invention involves the use of the bacteriophage in rapid detection systems. Specifically, unlike prior art systems that can detect only whole-cell pathogens, the bacteriophage-based detection system of the invention involves a simple, rapid and easily visible agglutination reaction (representing an antigen: antibody complex) if the sample is positive for the bacteriophage component. Such as system can be easily modified to further include a chromogenic marker tagged to the antibody to allow for a color reaction if the sample is positive. Examples of chromogenic markers include but are not limited to fluorochromes such as green fluorescent protein and FITC, peroxidases, and alkaline phosphotases. These and other markers can be purchased from companies such as Sigma. In a second embodiment the detection system would involve the use of a reporter strain which acts as a recipient for the bacteriophage. The reporter strain contains a gene which becomes activated by a gene activator encoded on the phage genome. This gene in the reporter strain would be fused to a reporter protein such as green fluorescent protein (GFP). If bacteria making the phage or free phage are present in the sample, the reporter strain would become infected, and subsequently the reporter gene, would then become activated. A simple, rapid, and easily visible color reaction (such as fluorescence) signifies a positive sample. For example, using the elements of V. cholerae, one would coat microtiter plates with a fusion protein comprised of a 'reporter' strain, such as the CTX phage, fused to a 'reporter' protein, such as green fluorescent protein. The VPI phage, a virulence factor present in pathogenic strains of V. cholerae, contains the receptor for the

CTX phage. A sample would then be added to the plate. If either V. cholerae or VPI phage or both are present in the sample, the CTX phage would become activated thereby activating the reporter protein as well. In the case of GFP, the visible color reaction (e.g., fluorescence) would signify a positive sample. Either embodiment of such a detection system would be not only cheap and rapid but also be able to detect both whole bacteria with phage (or pili) still attached to its surface and additionally free phage in the sample. As mentioned previously, the key to the detection aspect of the invention is the fact that the presence of the PAI bacteriophage is directly related to the presence of pathogenic bacteria. Another utility for the phage as vector for the expression of foreign genes is the use of the phage as a suicide factor. The procedural steps involved in the pathophage-induced pathogen suicide (or PIPS) scheme are described herein, using V. cholerae for example purposes only. It is clear that the same techniques could be applied for other pathogens that contain phage encoded PAIs, including but not limited to Salmonella spp. , ETEC, EPEC, EHEC, and Neisseria gonorrheae. First, one would synthesize or isolate an auxotrophic strain using, for example, a his- V. cholerae Ol (and 0139) recipient strain that is subsequently unable to grow without supplying it with histidine. One would then introduce the his+ gene into the VPI of an independent strain. Next, one subjects the VPI::His containing strain to mutagenesis using UV light, for example and the VPI: : His phage is isolated from this strain. The mutated VPI: : His phage preparation is then mixed with the his-V. cholerae strain. The mixture is then plated on minimal media. In this selection process, V. cholerae colonies that grow are only those in which the VPI: : His phage has infected the his- strain. The UV mutagenesis procedure and subsequent mating will select for a VPI phage that is now adapted to infect normal VPI-containing V. cholerae strains which are typically pathogenic and epidemic strains. Finally, one substitutes a kill gene, such as colicin, for the his gene in this adapted phage and then one isolates the newly created VPI: : kill phage. The suicide phage can be applied to the particular environment where it will then specifically only target and infect pathogenic strains of V. cholerae, forcing these strains to self-destruct or suicide.

Such a system allows for the selective targeting of those strains with pathogenic and/or epidemic potential and not the random targeting of any strain. In addition, the phage can be transferred between animals if this is the reservoir, increasing it effectiveness. In order to be successful, the application should be to the environmental reservoir rather than to an individual. For example, the phage preparation may be infused into the common water supply in certain geographic locations (e.g., apply suicide phage, (e.g. , longus)), to prevent ETEC or suicide BFP to prevent EPEC or VPIF for V. cholerae), applied to high risk animals (e.g., apply suicide Salmonella phage to chickens to prevent Salmonella or feed cows with suicide phage active against EHEC O157:H7).

EXAMPLES

The following examples are provided for illustrative purposes only, and are in no way intended to limit the scope of the present invention.

Bacterial surface structures classified in the type IV pilin family have been observed in a wide variety of human and animal bacterial pathogens which colonize mucosal surfaces including V. cholerae, E. coli, N. gonorrhoea, P. aeruginosa, and Dichelobacter nodosus (20-23). These structures are essential in the colonization of mucosal surfaces. In the case of V. cholerae, it has been shown that the "type IN pilus" also functions as the receptor for another phage (CTXF)4. The results described in detail herein demonstrate that the type IV pilus of V. cholerae is actually a filamentous phage which has been acquired by the bacteria and utilized for pathogenic purposes such as colonization and as receptors for other phages. The data herein also shows that the large pathogenicity island (PAI) in epidemic V. cholerae is actually the genome of a bacterial virus (VPIF) which is able to be transferred to other V. cholerae strains. Strains infected by VPIF are then able to secrete the phage, thereby providing potential for the continual emergence of future novel epidemic and pandemic strains.

Cholera is an ancient and life-threatening diarrheal disease that results in significant morbidity and mortality in millions of people worldwide (7,8). In the last decade of the 20th century, cholera has reached a far wider distribution than at any other time in this century. Cholera is acquired by drinking contaminated food or water which contains toxigenic strains of Vibrio cholerae. Virulent and epidemic strains of V. cholerae require at least two elements to cause disease, the cholera toxin phage (CTXF)4 and the V. cholerae pathogenicity island (VPI)5. CTXF encodes cholera toxin (CT) which is responsible for the severe secretory diarrhea which is characteristic of the disease. The CTXF genome can form a replicative plasmid in the bacterial host or can integrate at a specific site in the V. cholerae chromosome (4,9). The VPI (shown in Figure 1) may represent one of the initial genetic factors required for the emergence of epidemic and pandemic V. cholerae since it contains the toxin coregulated pilus (TCP) gene cluster encoding a "type IV pilus" which functions both as an essential adherence factor (6, 10) and as the CTXF receptor(4). VPI has many properties characteristic of bacterial PAIs; it is large (40-kb in size), contains genes encoding virulence and regulatory factors, integrates into a single preferred chromosomal site (att) adjacent to a tRNA-like gene (ssrA), has a different %G+C content compared to the host chromosome, and contains genes associated with DNA mobility (4-6,11-14).

A V. cholerae strain (E9120) that has lost the VPI has previously been identified (5). This strain contains ctx genes suggesting it previously possessed the VPI to allow CTXF infection, and had an altered att site suggesting VPI was excised. The VPI is flanked by genes with homology to a transposase and a phage-like integrase gene and is found only at phage attachment (att) sites (5,14) suggesting that the VPI is the genome a phage. Other genes of the VPI were examined using a BLAST homology search (15) of the Genbank database. Several other VPI genes whose predicted protein products share some homology with phage or viral proteins were found. TagE has 30% identity, 55% similarity over 188 amino acids to Orflό of Staphylococcus aureus bacteriophage phi (Genbank accession AB009866), OrfZ has 26% identify, 41 % similarity over 97 a.a. to an "early protein" of rat CMV (Genbank accession U62396) and OrfV has 32% identity, 47% similarity over 79 a.a. to the "enhancing" protein of Lymantria dispar nucleopolyhedrovirus (Genbank accession AF019970). Interestingly, even TcpA shows some homology (31 % identity, 48% similarity over 82 a.a.) to the product of a 769 amino acid Orf from TT virus (Genbank accession ABO 11490). Taken together, these results suggested that the VPI is the genome of a phage. The following specific methods were employed in the examples described below. Regarding the particular bacterial strains used in the example-; described below, Strain 395 is a representative of the 6th ("classical") pandemic clone of V. cholerae while strain N 16961 is an isolate from the current 7th ("El Tor") cholera pandemic which began in 1961 in Indonesia and has since spread worldwide. CVD110 is a derivative of a 7th pandemic El Tor strain (E7946) which is deleted in its ctxA, zot, ace, and orfU genes (17). The environmental isolates DK236 and DK237 are nontoxigenic VPI-negative serogroup O10 and Ol strains, respectively and are NalR due to selection of a spontaneous resistant mutant. To construct DK238, the xtn-aldA region of the chromosomal N 16961 VPI was amplified by PCR with primers KAR166 (located in orf I) and KAR167 (located in aldA). This 1.8-kb fragment was ligated into pGEM-T, creating pDK40. This plasmid was digested with BamHI into which aphA-3 (encoding Km Neo resistance) was suitably inserted, creating pDK42. This plasmid was digested with Sphl/Sacl and the xtn-aldA::aphA-3 fragment was ligated into the appropriately digested suicide vector pCVD442, creating pDK43. The plasmid pDK43 was used in allelic exchange to introduce αpb l-3-containing fragment into the homologous region of the N 16961 chromosome, creating strain DK238. Strain TCP2 is derived from 395, has amino acid residues 119-154 deleted from its tcpA gene and does not produce the TCP structured. RT4032 is an in-frame tcpA deletion mutant of 395 received from Ron Taylor. This deletion includes the codon encoding amino acids + 1 of mature TcpA through the TAA stop codon and one additional T nucleotide. Plasmid pRT198 is a pBR322-based plasmid with a 2-kb HindUl fragment containing tcpA of 395 cloned into the HindUl site of pBR322.

Regarding PCR and sequencing procedures described below, PCR was performed according to the method of Saiki et al.25 under the following conditions: (1) denature,

96°C, 3 min; (2) annealing, 48°C, 30 s; (3) extension, 72°C, 2 min, for 1 cycle then (4) denature 96°C, 30 s; (5) annealing, 48°C, 30 s; (6) extension, 72°C, 2 min for 30 cycles.

Primers used to determine the presence and sequence of V. cholerae genes are described as follows: (1) To determine to the presence of orfl:

KAR96 [5'-TGCTACTTACCCAATGGCAC-3' (SEQ ID NO. 1) ] and KAR97 [5'- GAGCCAGGCTTATTTGGGCG-3'(SEQ ID NO. 2)].

(2) To determine to the presence of tcpA :

KAR24 [5'-AAAACCGGTCAAGAGGG-3'(SEQ ID NO. 3)] and KAR25 [5'- CAAAAGCTACTGTGAATGG-3'(SEQ ID NO. 4)] for 7th pandemic strains, and KAR82 [5'-CAAATGCAACGCCGAATGG-3'(SEQ ID NO. 5)] for 6th pandemic strains.

(3) To determine to the presence of toxT:

KAR90 [5'-ATAACTTTACGTGGATGGC-3'(SEQ ID NO. 6)] and KAR91 [5'- AAAATCAGTGATACAATCG-3'(SEQ ID NO. 7)]. (4) To determine to the presence of int:

KAR22 [5'-GATAAAGAGATCAAAGCC-3' (SEQ ID NO. 8)] and KAR23 [5'- ATCTGCTTCCATGTGGG-3'(SEQ ID NO. 9)].

(5) To determine to the presence of the fragment outside the left junction:

KAR94 [5'-TATGATACTGAAAACACCTC-3' (SEQ ID NO. 10] ) and KAR95 [5'- GATGCTAACAGCAGAGCATA-3 ' (SEQ ID NO . 11)] ;

(6) To determine to the presence of the fragment outside the right junction: KAR85 [5'-CGCCTGCGAACCGACACGC-3'(SEQ ID NO. 12)], and KAR86 [5'- GCAGCAAGCCTCCACTCCG-3'(SEQ ID NO. 13)];

(7) To determine to the presence of ompT:

K898 [5'-GAATTCTGTCGGGTTGTAATCCTG-3'(SEQ ID NO. 14)] and K643 [5^*- GCCATACTC AGCATATACAC-3 ' (SEQ ID NO . 15)] .

(8) To determine the presence rfaD:

K371 [5'-CGGGATCCGAGCTCATTACCTACACTAGTG-3'(SEQ ID NO. 16)] and K369 [5'-CGGGATCCGACAGGCTATAATGCGTGCAAC-3'(SEQ ID NO. 17)].

(9) To determine the presence of ctxB: KAR161 [5'-AAAATTCCTTGACGAATACC-3'(SEQ ID NO. 18)] and KAR162 [5'- TTGCTTCTCATCATCGAACC-3'(SEQ ID NO. 19)].

(10) To determine the presence of xtn-aldA region:

KAR166 [5'-GACAGGATTACTGAGATATCTG-3'(SEQ ID NO. 20)] and KAR167 [5'- AACCAAGGTGAGGTTTGTACC-3'(SEQ ID NO. 21)]. Primer synthesis using an Applied Biosystems DNA synthesizer and sequencing of

PCR products using the Taq Dye-Terminator sequencing kit (Perkin-Elmer) and an automated 373A DNA sequencer (Applied Biosystems) was performed by the Biopolymer Laboratory at the University of Maryland.

Regarding isolation of phage and replicative form in the examples described below, isolation and purification of phage from V. cholerae was performed basically as described by Maniatis et all 6 but with modifications. Briefly, 1 litre Luria broth cultures were grown overnight at either 30°C (395) or at 37°C (N16961). Cultures were centrifuged twice at 10,000 x g and the supernatant was passed through 0.45 μm low protein binding filter. DNase I and RNase I (Boehringer Mannheim) were added to the filtrate at a final concentration of 1 μg/ml and incubated at room temperature for 3 hours. NaCl and PEG 8000 were added to a final concentrations of 1 M and 10 % w/v, respectively, and the mixture allowed to precipitate overnight at 4°C. The supernatant was centrifuged at 11,000 x g for 20 min and the pellet resuspended in 4 ml of SM buffer. PEG was removed with an equal volume of chloroform. This supernatant was layered onto a CsC12 step gradient consisting of 2 ml each of CsC12 in SM buffer (d = 1.7, 1.5, and 1.45). After centrifugation at 25,000 rpm for 2 hours in an SW41 rotor (Beckman), the lower phage band (density between 1.45 and 1.5) was extracted and dialyzed against 2 changes of TM buffer. The phage were further concentrated by the addition of PEG 8000 (10% w/v) and placed on ice for 2 hours. The phage preparation was centrifuged at 14,000 x g for 20 min and the resulting pellet (containing phage particles) was resuspended in 100 μl of SM buffer. PEG was removed with an equal volume of chloroform. PCR was performed using 5 μl of the phage preparation.

Isolation of the phage replicative form was performed with 1 L Luria broth cultures according to the method of Maniatis et al. (16). Regarding immunoprecipitation procedures described below, DNase- and RNase-treated filtered supernatant were incubated with rabbit anti-peptide TcpA antibody (1: 10,000) at 37°C and vigorous shaking for 1 hour. Following incubation, mouse anti-rabbit IgG (whole molecule) agarose beads (Sigma) was added and the reaction incubated at 4oC overnight with gentle rotation. After several low speed centrifugation and washes in deionized water, the pellet was finally resuspended in deionized water. Regarding electron microscopy procedures described below, phage preparations of

CVD110 and 395 were placed on a carbon-formvar coated 300 mesh copper grid (Electron Sciences) for 2 min then negatively stained with 1.5% phosphotungstic acid (PTA) for 1 min and analyzed under electron microscopy. In addition, equal volumes of 395 phage preparations were incubated with rabbit anti-peptide TcpA antibody (1 : 10,000) at 37°C for 20 min after which this suspension was placed on a grid for 5 min, and 10 μl of 10 nm colloidal gold-conjugated goat anti-rabbit IgG (ICN Biomedicals, OH) were added prior to being negatively stained.

As mentioned above, the invention described herein began with investigation into the hypothesis that the VPI was a phage. Phage preparations of V. cholerae strains N 16961 and 395 were prepared according to standard methods (16). These cell-free phage preparations were examined for the presence of genes located at the extreme left, center, and extreme right ends of the VPI. Using PCR and primers specific for VPI genes, orf I, tcpA, toxT, and int genes in both 395 and N 16961 preparations were detected and confirmed by sequencing (see Figure 1 and Table 1). In contrast, no PCR products were amplified using primers (KAR94/KAR95 and KAR85/KAR86) specific for flanking chromosomal regions immediately outside the VPI. In addition, no products were obtained for ompT (encoding an outer membrane protein) and rfaD (involved in LPS synthesis) genes which are also located outside the VPI. A phage preparation derived from a VPI-negative strain prepared under identical conditions was also negative for the chromosomal ompT and rfaD genes as well as orf I, tcpA, toxT, and int genes. To show that this technique was able to detect other phage- encoded genes, PCR analysis of the phage preparations detected the ctxB and zot genes which are contained on the CTXF. The finding of VPI genes in DNAase- and RNase-treated phage preparations indicated that the VPI DNA was protected by a protein coat and that this element was most probably a bacteriophage (designated VPIF).

DNA obtained from the phage preparations following standard phenol/chloroform extraction was sensitive to digestion with ssDNA-specific SI nuclease and failed to produce PCR products using VPI-specific primers. The same DNA preparations were resistant to digestion with dsDNA-specific type II restriction endonucleases and yielded VPI PCR products after digestion. In addition, Southern hybridizations on phage DNA preparations from DK238 and CVD110 using forward (KAR24) and reverse (KAR25) primers for tcpA clearly showed hybridization of the phage DNA samples with primer KAR25 only which would hybridize to the positive (+) strand. The positive control, N 16961 chromosome hybridized with both primers. Thus, VPIF, like CTXF, contains positive (+) ssDNA as its genome.

To determine if the VPIF had a plasmid replicative form (RD in the cell, plasmid DNA from N 16961 and 395 was extracted using CsC12 density gradient centrifugation and then analyzed for VPI genes. RF preparations were sensitive to digestion with the double strand-specific restriction enzyme Pv«II (which has a site in the center of tcpA) since PvwII- digested RF preparations failed to generate PCR fragments using tcpA primers. The same RF preparation was resistant to digestion with ssDNA-specific SI nuclease and generated VPI PCR products after SI nuclease-digestion. As a further control to show no chromosomal contamination, PCR on these preparations failed to amplify DNA which is external to the VPIF such as ompT and rfaD (Table 1). In addition, we performed Southern hybridizations on RF preparations to confirm the presence of a VPIF RF. Southern hybridizations on Hαelll-digested RF preparations from DK238 and CVD110 and N16961 chromosome showed hybridization with all lanes with a tcpA PCR product probe but only to N 16961 chromosome when ompT PCR product was used as probe. These results indicated that like other filamentous phages, the VPIF also forms a ds plasmid replicative form in the cell.

Although filamentous phage do not result in lysis of the cell, nor have a "burst size," we attempted to calculate the approximate number of VPIF and CTXF released per cell. Since the amount of DNA (in Fg) can be calculated from the absorbance of DNA in a phage preparation, the number of phage released was calculated by determining the amount of phage DNA present in a 1 liter overnight culture of DK238 and CVDllO containing 1x1012 cells. Strain DK238 is positive for VPIF and CTXF while CVDl lO is positive only for VPIF. Thus, the difference in amount (in Fg) between DK238 and CVDllO should reflect the number of CTXF genomes (7-kb) made per cell and the valve for DK238 should approximate the number of VPIF (40-kb) made per cell. We estimated 280 Fg/L and 200 Fg/L of ssDNA is present in a phage preparation from 1 liter cultures of DK238 and CVDllO, respectively. The following describes the rationale for the calculations. If 1 Fg of l-k6 ssDNA contains 1.8x1012 molecules, then 1 Fg of 7-k6 ssDNA contains 2.6x1011 CTXF molecules, i.e., 1.8x1012/7) and 80 Fg contains 2x1013 molecules. Thus, 2x1013 molecules/ 1x1012 cells suggests that an average of 20 CTXF are produced per cell during an overnight culture. For the estimation of the number of VPIF made per cell, if 1 Fg of 40-kb ssDNA contains 4.5x1010 molecules, then 200 Fg contains 9x1012 molecules. Thus, 9x1012 molecules/ 1x1010 cells suggests that an average of 9 VPIF are produced per cell during an overnight culture.

In addition, we calculated the copy number of RF per cell. Since the amount of DNA (in Fg) can be calculated from the absorbance of plasmid RFDNA, the copy number of RF per cell was calculated by measuring the amount of dsDNA present in a 1 liter overnight culture of DK238 and CVDllO containing 1x1012 cells. Strain DK238 is positive for VPIF and CTXF while CVD110 is positive only for VPIF. Thus, the difference in amount (in Fg) between DK238 and CVDllO should reflect the copy number of CTXF RF (7-kb) made per cell and the value for DK238 should approximate the number of VPIF RF (40-kb) made per cell. We estimated 57 Fg/L and 39 Fg/L of dsDNA is present in a RF preparation from 1 liter cultures of DK238 and CVDllO, respectively. The following described the rationale for the calculations. If 1 Fg of 1-kb dsDNA contains 9.1xlOⁿ molecules, then 1 Fg of 7-kb dsDNA contains 1.3x10" CTXF RF molecules and 18 Fg contains 2.3xl0¹² molecules. Thus, 2.3xl0¹² molecules/ lxlO¹² cells suggests a CTXF RF copy number of 2 per cell during an overnight culture. For the estimation of the copy number of VPIF RF per cell, if 1 Fg of 40-kb dsDNA contains 2.2xl0¹⁰ molecules, then 39 Fg contains 8.6xlO^u molecules. Thus, 8.6xlO^π molecules/ lxlO¹² cells suggests a VPIF RF copy number of 1 per cell from an overnight culture.

Transfer experiments were conducted to determine if the VPIF could be transferred from a VPI-positive donor to a VPI-negative recipient. The VPIF genome of a spontaneously streptomycin (Str) resistant strain (N 16961) was marked with the aphA-3 gene encoding kanamycin (Km) and neomycin (Neo) resistance creating strain DK238. The aphA- 3 gene was inserted into the inter genie region between aldA and xtn which we previously hypothesized was a nonfunctional transposase (see Figure 1) (5). We then performed transfer experiments using whole bacterial cell cultures and phage (cell-free) preparations from donor DK238 to determine whether the VPIF could be transferred into the nontoxigenic VPI-negative strain DK236 (serogroup O10, nalidixic acid (Nal) resistant). Following 1-hour incubation at 37°C (with gentle shaking) of 3xl0⁷ DK236 recipient cells with either 3xl0⁷ whole DK238 donor cells or celLfree phage preparations from 2xl0⁷ donor DK238 cells, cultures were subjected to vigorous pipetting to disrupt bacterial aggregates and then plated onto agar containing Nal/Neo. The use of a NalS donor and NalR recipient allowed us to identify NalR transductants which had acquired the aphA-3 gene via the VPIF. We obtained 1.3xl0⁴ transductants (colonies on Nal/Neo plates) suggesting that on average, about 0.04% of recipient cells were transduced under these conditions. In 1 hour reactions containing a cell-free phage preparation from 2x107 donor cells we similarly obtained > 3x108 transductants per 3x107 recipients, suggesting that the phage made by 1 donor cell, on average, give rise to at least 15 transductants. The successful transfer of the NeoR marker with cell-free DNAased-treated phage preparations, indicated that transduction, rather than conjugation, was the mechanism of DNA transfer. One transductant was selected and designated as DK239. Transfer of VPIF from donor to recipient was confirmed by tests conducted on DK239 including O antigen agglutinations, antibiotic susceptibility testing, ribotyping, and colony hybridizations with radiolabelled aphA-3 and tcpA gene probes. In addition, PCR analysis using primers for the aphA-3, tcpA, toxT, and int genes confirmed transfer (Table 1). PCR analysis on DK239 using primers KAR23/KAR86 for the right VPI junction generated a fragment identical in size to the N 16961 positive control. This showed that the VPIF genome had integrated into the identical att site as other VPI-positive strains. PCR analysis of phage preparations of DK239 suggest that this newly created strain produces and secretes VPIF into the medium. Although DK239 had acquired VPIF, the CTXF was not apparently acquired as shown by PCR (Table 1).

Not all V. cholerae strains tested were capable of serving as donors or recipients of VPIF. Interestingly, although VPIF particles were detected in phage preparations of 395, no transfer of VPIF was observed when a StrR strain of 395 (identically marked by aphA-3 in the VPI) was used as a donor. This result suggests that this 6th pandemic classical strain (and perhaps all classical strains) lacks the appropriate mechanism for efficient transfer or that it transfers the VPIF under different conditions. With El Tor strain N 16961 as donor, transfer into a O10 strain DK236 was observed as noted above but not into the nontoxigenic VPI-negative strain DK237 (serogroup Ol). Since both DK236 and DK237 have a vacant αtt site5, there may be important differences between these strains other than att sites which limit and influence their ability to acquire VPIF. These differences may explain why only a limited number of different toxigenic serogroups are found— epidemic and pandemic cholera has traditionally and consistently been associated with serogroup Ol strains (and recently with 0139 strains). Even though we show that VPI+ non-Ol /non-0139 strains can be created in vitro (and may have the potential to become toxigenic epidemic and pandemic strains), we cannot rule out the possibility that VPI+ CT+ Ol strains possess some advantage in vivo or in the environment over VPI + CT + non-01 strains which explains the predominance of V. cholerae Ol strains in epidemic and pandemic disease. These results do, however, highlight the potential for different serogroups of V. cholerae (other than Ol and 0139) to acquire the VPI and become pathogenic and possibly epidemic and pandemic strains.

Concentrated phage preparations of several VPI-positive V. cholerae strains were obtained and viewed under electron microscopy. Phage preparations from strain 395 bound rabbit anti-peptide TcpA antibodies with colloidal gold-conjugated goat anti-rabbit IgG and revealed numerous gold particles bound to parallel bundles of VPIF particles. Strain

CVDllO is an El Tor strain deleted for ctxA, zot, ace, and orfU genesl7. This strain is unable to produce CTXF. The CVDllO phage preparation was shown to contain VPIF genes but not CTXF- encoded genes (Table 1). Interestingly, the concentrated CVDllO phage preparation demonstrated numerous phage particles some of which formed a "braided" network of filaments and presumably represent VPIF. Immunoelectron microscopy using rabbit anti-peptide TcpA antibodies and colloidal gold-conjugated goat anti-rabbit IgG of phage preparations from the El Tor strain N 16961 showed numerous gold particles bound to filamentous phage.

The data described herein suggesting that the VPI is a bacteriophage, together with previous suggestions that the structure of type IV pili resembles filamentous bacteriophage (18), supports the theory that the TCP and its major pilin subunit TcpA might be in fact the VPIF coat protein. Strain TCP2 is a derivative of the classical strain 395 which has a large internal deletion in the tcpA gene that renders it unable to produce TCP19. PCR analysis did not detect any VPIF genes in phage preparations of TCP2 suggesting that TcpA is required for VPIF production; in contrast, genes of CTXF were readily identified in TCP2 supernatants (see Table 1). Immunoprecipitation experiments were then conducted on these phage preparations. Addition of rabbit anti-peptide TcpA antibodies and mouse anti-rabbit IgG agarose beads bound VPIF thereby allowing selective removal of the complex from the supernatant. PCR analysis on N16961 and 395 strain immunoprecipitates demonstrated the presence of VPIF-encoded genes. The specificity of the antibody for the TcpA subunit and VPIF was demonstrated by the lack of PCR products in these preparations using rfaD and ompT primers. As a further control, no PCR products were detected in similar experiments in the absence of anti-TcpA antibodies presumably because there was no recognition of TCP protein subunit (antigen) on the VPIF surface (Table 2). PCR analysis of phage preparations from an independent tcpA mutant, RT4032, which contains an in-frame (non-polar) deletion in tcpA also failed to generate VPI genes; however, the tcpA mutants TCP2 and RT4032 could be complemented by supplying tcpA on a plasmid (pRT 198) since PCR detected VPI genes in phage preparations of both transformants. The results of PCR, complementation studies, and immunoprecipitation experiments showing DNA associated with the TcpA protein complex are consistent with the hypothesis that TcpA is a major structural protein of VPIF. These findings have important implications for understanding how non-pathogenic bacteria become pathogens. Many bacterial pathogens contain clusters of genes which encode virulence factors responsible for inducing disease. The data described herein demonstrating that the large cluster of virulence genes essential for the epidemic ability of V. cholerae actually represents viral (bacteriophage) DNA that has become incorporated into the bacterial chromosome after viral infection of the bacterial cell. We have demonstrated that this unusually large filamentous bacteriophage, designated VPIF, can be transferred to a VPI-negative V. cholerae strain, thereby conferring new virulence properties on the recipient.

REFERENCES

1. Lee, CA. Infectious Agents and Disease 5, 1-7 (1996).

2. Hacker, J., Blum-Oehler, G., Muhldorfer, I. & Tschape, H. Molecular Microbiology 23, 1089-1097 (1997). 3. Huang, H.C., Lin, R.H., Chang, C.J., Collmer, A. & Deng, W.L. Molecular

Plant Microbe Interactions 8, 733-746 (1995).

4. Waldor, M.K. & Mekalanos, J.J. Science 272, 1910-1914 (1996).

5. Karaolis, D.K.R. et al. Proceedings of the National Academy of Sciences USA 95, 3134-3139 (1998). 6. Taylor, R.K., Miller, V.L. , Furlong, D.B. & Mekalanos, J.J. Proceedings of the

National Academy of Sciences USA 84, 2833-2837 (1987).

7. Pollitzer, R. Monograph Series 43. Geneva: World Health Organization 147-158, (1959).

8. Kaper, J.B. , Morris Jr., J.G. & Levine, M.M. Clinical Microbiology Reviews 8, 48-86 (1995).

9. Pearson, G.D.N., Woods, A., Chiang, S.L. & Mekalanos, J.J. Proceedings of the National Academy of Sciences USA 90, 3750-3754 (1993).

10. Herrington, D.A. et al. Journal of Experimental Medicine 168, 1487-1492 (1988). 11. DiRita, V. , Parsot, C, Jander, G. & Mekalanos, J.J. Proceedings of the

National Academy of Sciences USA 88, 5403-5407 (1991).

12. Carroll, P.A., Tashima, K.T. , Rogers, M.B., DiRita, V.J. & Calderwood, S.B. Molecular Microbiology 25, 1099-1111 (1997).

13. Hase, C.C. & Mekalanos, J.J. Proceedings of the National Academy of Sciences USA 95, 730-734 (1998).

14. Kovach, M.E., Shaffer, M.D. & Peterson, K.M. Microbiology 142, 2165-2174 (1996).

15. Altschul, S.F. et al Nucleic Acids Research 25, 3389-3402 (1997).

16. Maniatis, T., Fritsch, E.F. & Sambrook, J. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1982). 17. Michalski, J., Galen, J.E., Fasano, A. & Kaper, J.B. Infection and Immunity 61, 4462-4468 (1993).

18. Hobbs, M. & Mattick, J.S. Molecular Microbiology 10, 233-243 (1993).

19. Taylor, R., Shaw, C, Peterson, K., Spears, P. & Mekalanos, J. Vaccine 6, 151- 154 (1988).

20. Dalrymple, B. & Mattick, J.S. Journal of Molecular Evolution 25, 261-269 (1987).

21. Shaw, C.E. & Taylor, R.K. Infection and Immunity 58, 3042-3049 (1990).

22. Patel, P. , et al. Infection and Immunity 59, 4674-4676 (1991). 23. Girόn, J.A. , Ho, A.SN. & Schoolnick, G.K. Science 254, 710-713 (1991).

24. Hicks, S. , Frankel, G. , Kaper, J.G., Dougan, G. & Phillips, A.D. Infection and Immunity 66, 1570-1578 (1998).

25. Saiki, R.K. , et al. Science 239, 487-491 (1988).

While the invention has been described in detail, and with reference to specific embodiments thereof, it will be apparent to one with ordinary skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. All references, including publications, patent applications, and patents, are hereby incorporated by reference in their entirety.

Table 1. PCR detection of VPIΦ, CTXΦ, and chromosomal genes from whole cell cultures, cell-free phage preparations and plasmid replicative form preparations.

Strain Sample Detection of Genes

VPIΦ CTXΦ Chromosomal flank _τ r _r, orfl tcpA toxt int ctxB zot Yp_j ompT rfaD

395

Whole Cell + + + + + + + + +

Phage prep + + + + + + - - -

N16961

Whole Cell + + + + + + + + +

Phage prep + + + + + + - - -

CVDl lO

Whole Cell + + + + + + + +

Phage prep + + + + - - - - -

TCP2

Whole Cell + + + + + + + + +

Phage prep - - - - + + - - -

395-RF plasmid + + + + + + -

N 16961-RF plasmid + + + + + + _

DK239

Whole cell + + + + - + + +

Phage prep + + + + - -

Table 2. PCR of phage preparations from 395 (wildtype) and TCP2 (tcpA mutant) immunoprecipitated in the presence or absence of anti-TCP antibody

Claims

WHAT IS CLAIMED:

1. An isolated bacteriophage encoded by a pathogenicity island of a pathogenic bacteria.

2. A bacteriophage of claim 1, wherein said bacteria is a type IV pilus presenting bacteria.

3. A bacteriophage of claim 2, wherein said type IV pilus presenting bacteria is Vibrio cholerae, E. coli, Neisseria gonorrhoea, Neisseria meningitidis, Pseudomonas aeruginosa, Moraxella bovis, Legionella pneumophila, Dichelobacter nodosus, Bacteroides, Salmonella, Eikenella corrodens, or Pseudomonas putida.

4. A bacteriophage of claim 2, wherein said bacteria is Vibrio cholerae and said type

IV pilus is TCP.

5. A method of eliciting an immune response in a subject, comprising: administering an effective amount of an isolated bacteriophage encoded by a pathogenicity island of a pathogenic bacteria.

6. A method of claim 5, wherein said bacteria is a type IV pilus presenting bacteria.

7. A method of claim 6, wherein said type IV pilus presenting bacteria is Vibrio cholerae, E. coli, Neisseria gonorrhoea, Neisseria meningitidis, Pseudomonas aeruginosa, Moraxella bovis, Legionella pneumophila, Dichelobacter nodosus, Bacteroides, Salmonella, Eikenella corrodens, or Pseudomonas putida.

8. A method of claim 6, wherein said bacteria is Vibrio cholerae and said type IV pilus is TCP.

9. A vaccine against an infectious disease caused by a pathogenic bacteria, said vaccine comprising an isolated bacteriophage encoded by a pathogenicity island of said pathogenic bacteria and a pharmaceutically-acceptable carrier.

10. A vaccine of claim 9, wherein said bacteria is a type IV pilus presenting bacteria.

11. A vaccine of claim 10, wherein said type IV pilus presenting bacteria is Vibrio cholerae, E. coli, Neisseria gonorrhoea, Neisseria meningitidis, Pseudomonas aeruginosa, Moraxella bovis, Legionella pneumophila, Dichelobacter nodosus, Bacteroides, Salmonella, Eikenella corrodens, or Pseudomonas putida.

12. A vaccine of claim 11, wherein said bacteria is Vibrio cholerae and said type IV pilus is TCP.

13. A vaccine against more than one infectious disease caused by a pathogenic bacteria or virus, comprising: an isolated bacteriophage encoded by a pathogenicity island of a first pathogenic bacteria, said phage further comprising a foreign nucleotide sequence encoding a vaccine antigen against a second pathogenic bacteria or virus, and a pharmaceutically-acceptable carrier.

14. A method for identifying a phage-encoded pathogenicity islands of a pathogenic bacterial strain comprising:

(a) identifying a virulence gene cluster in the genome of a pathogenic bacteria, and

(b) detecting a bacteriophage encoded by said gene cluster, or a nucleic acid or polypeptide thereof, in the supernatant of a culture media in which said bacteria was cultured.

15. The method of claim 14, wherein identifying a virulence gene cluster comprises scanning genomic DNA sequences for regions having a G+C content which is characteristic of pathogenicity islands.

16. The method of claim 15, wherein said G+C content is at least 10% different from a chromosome of the host bacterium.

17. The method of claim 14, wherein identifying a virulence gene cluster comprises: comparing said pathogenic bacterial genome with the genome of an associated non pathogenic strain to identify those sections of the pathogen genome that are distinct to the pathogenic strain.

18. The method of claim 14, wherein said bacteria is a type IV pilus presenting bacteria.

19. The method of claim 18, wherein said type IV pilus presenting bacteria is Vibrio cholerae, E. coli, Neisseria gonorrhoea, Neisseria meningitidis, Pseudomonas aeruginosa,

Moraxella bovis, Legionella pneumophila, Dichelobacter nodosus , Bacteroide, Salmonella, Eikenella corrodens, or Pseudomonas putida.

20. A method for identifying a bacteriophage encoded by a pathogenicity island of a pathogenic bacteria, comprising: culturing a bacterial strain in a media, said strain comprising a pathogenicity island, and detecting a bacteriophage in said media, wherein said media has been separated from said bacterial strain, and said bacteriophage is encoded by said pathogenicity island.

21. A method of claim 20, wherein said media is centrifuged subsequent to said culturing to remove

22. A method of claim 20, wherein said detecting is performing a PCR reaction on said media separated from said bacterial strain.

23. A method for rapidly detecting the presence of a bacteriophage-encoded, type IV pilus presenting bacterial pathogen, or a bacteriophage nucleic acid or polypeptide thereof, in a sample comprising:

(a) obtaining a reporter strain which acts as a recipient for the type IV pilus bacteriophage, said reporter strain containing a gene activated by a gene activator on the bacteriophage;

(b) fusing said activated gene in reporter strain to a reporter protein; (c) mounting said reporter straining containing fusion reporter protein to a surface;

(d) contacting said surface with said sample, wherein the presence of said pathogen or said pathogen component activates the reporter strain and subsequently the reporter protein, the activation thereof resulting in a rapid and easily visible response; and

(e) determining the presence or absence of said response, the presence thereof indicating the presence of said pathogen or said pathogen component in said sample.

24. The method of claim 23, wherein said reporter protein is green fluorescent protein. If bacteria or phage are present in the sample, the reporter strain, and subsequently reporter protein, would become activated.

25. A kit for rapidly detecting the presence of a bacteriophage-encoded, type IV pilus presenting bacterial pathogen, or a bacteriophage nucleic acid or polypeptide thereof, in a sample comprising:

(a) a fusion protein comprising a reporter strain which acts as a recipient for the type IV pilus bacteriophage, said reporter strain containing a gene activated by a gene activator on the bacteriophage, fused to a reporter protein; and (b) immunoassay components.