Patent Application
20060140975
The invention generally relates to regulated host-vector systems for delivery of genetic vaccine vectors or desired gene products to a eukaryotic host, preparations of bacterial genetic vaccines, genetically engineered microorganisms that are useful for delivery of genetic vaccine vectors or desired gene products, and methods of use for the host-vector system.
Vaccination is a means for preparing the immune system to reduce disease symptoms, prevent horizontal transmission of infectious agents and reduce disease mortality. The immune system of a recipient host will generate an immune response to foreign antigens.
Standard vaccines include the administration of carbohydrates, peptides, polypeptides, proteins, glycosylated polypeptides, lipids, glycolipids and lipoproteins against which an immune response in a host is desired. An alternative to such standard immunization is the passive administration of antibodies to the host.
Passive immunization procedures for humans have limitations. These limitations include the cost of antibody production and the requirement for continuous administration of these antibodies. In addition, although polyclonal or monoclonal antibodies can be readily produced by routine techniques, production and purification of antibody compositions that are sufficiently safe for in vivo delivery is relatively expensive and time consuming.
When soluble proteins or polypeptides (e.g., proteins presented directly into the bloodstream), serve as antigens, the proteins or polypeptides induce a humoral immune response in the form of circulating antibodies. In contrast, cellular immunity is generally elicited by intracellular antigens, such as antigens produced by parasites or viruses, and can also be used by the body to eliminate solid tumors. Cellular immunity involves presentation of the target antigen on the surface of antigen presenting cells by complexing with MHC class I proteins. If an antigen is synthesized in a cell and presented by both Class I and Class II molecules, both antibody production and cell-mediated immunity may result.
Genes from bacteria, viruses, fungi, and parasites have been cloned into a variety of bacteria for the purpose of directing the bacteria to express the foreign antigen or impart on the bacteria certain desired properties for use as a live vaccine. Examples include cloning the genes of Shigella into E. coli rendering the E. coli invasive and therefore more suitable for use as a vaccine strain, or cloning of P. falciparum malaria genes into Salmonella typhimurium which subsequently express these malaria proteins (Sadoff et al., Science 240:336-338(1988); Aggrawal et al., J. Exp. Med. 172:1083-1090 (1990)). All of these bacterial delivery systems require the bacteria itself to produce the antigen or the functional molecule.
Salmonella vector vaccines have been extensively studied. Salmonella vectors are capable of eliciting humoral and cellular immunity against bacterial, viral, fungal, and parasitic antigens (See e.g. Powell et al., U.S. Pat. No. 5,877,159 citing Formal et al., Infect. Immun., 34:746-751 (1981); Aggarwal et al., J. Exp. Med 172:1083-1090 (1990); Cardenas and Clements, Clin. Microbiol. Rev. 5:328-342 (1992); Curtiss et al., Dev. Biol. Stand., 82:23-33 (1994); Chatfield et al., Bio/Technology, 10:888-892 (1992); and Curtiss et al., J. Clin. Invest. 110:1061-1066 (2002)). These humoral responses occur in the mucosal (Cardenas et al., supra), and systemic compartments (Gonzalez et al., J. Infect. Dis., 169:927-931 (1994); Cardenas et al. supra; Curtiss et al., supra). Live oral Salmonella vector vaccines also elicit T cell responses against foreign antigens (Wick et al., Infect. Immun., 62:4542-4548 (1994)). These include antigen-specific cytotoxic CD8+ T cell responses (Gonzalez et al., supra; Aggarwal et al., supra; Flynn et al., Mol. Microbiol., 4:2111-2118 (1990); Turner et al., Infect. Immun., 61:5374-5380 (1993); and Gao et al., Infect. Immun., 60:3780-3789 (1992)).
DNA vaccines have been proposed as a means to induce in vivo cellular immunity by taking advantage of the recipient host's transcription and/or translation machinery. For example, a eukaryotic host is immunized with a eukaryotic expression plasmid encoding the antigen. The eukaryotic host expresses the antigen on the plasmid. DNA vaccination allows for the possibility of co-expressing immunomodulatory molecules like cytokines, co-stimulatory molecules, or antisense RNA to steer the immune response to the preferred direction. It also allows flexibility in manipulation of the DNA encoding the antigen. For additional general discussion of DNA vaccines, see for example, Pardoll and Beckerleg, Immunity, 3:165-169 (1995); Lietner et al., Vaccine 18:765-777 (1999); and Lewis and Babiuk, Adv. Virus Res. 54:129-188 (1999).
Recently, naked DNA vaccines carrying eukaryotic expression cassettes have been used to immunize against influenza both in chickens (Robinson et al., Vacc., 11:957-960 (1993)) and ferrets (Webster et al., Vaccine 12:1495-1498 (1994)); against Plasmodium yoelii in mice (Hoffman et al., Vacc., 12:1529-1533; (1994)); against rabies in mice (Xiang et al., Virol., 199:132-140 (1994)); against human carcinoembryonic antigen in mice (Conry et al., Canc. Res., 54:1164-1168 (1994)) and against hepatitis B in mice (Davis et al., Vacc., 12:1503-1509 (1994); and Davis et al., Hum. Molec. Gen., 2:1847-1851 (1993)).
DNA vaccines may be delivered directly to the host, such as by injection into the muscles of the host. Alternatively, the DNA vaccines may be delivered to the host cells through bacterial carriers, such as Shigella, Listeria, or Salmonella.
Processing of exogenous antigens involves endocytosis, partial degradation within the endocytic vacuole, and binding to class II MHC molecules. Processing of class I-restricted antigens also appears to involve proteolysis and recognition of antigen-derived peptides bound to MHC class I molecules. Antigens synthesized within host cells, or antigens derived from intracellular bacteria that have the ability to exit the endocytic vacuole (e.g., Listeria monocytogenes and Shigella spp.), are processed and then preferentially associate with MHC class I molecules (See e.g. Galan et al., U.S. Pat. No. U.S. Pat. No. 6,306,387 B1).
In the studies by Sizemore et al., Science 270:299-302 (1995) the Shigella flexneri strain delivering the DNA vaccine vector possessed a Deltaasd mutation that imposes a requirement for diaminopimelic acid (DAP), an essential constituent of the rigid layer of the bacterial cell wall. Since DAP is only synthesized by bacteria and is unavailable in animal tissues, these bacteria commence to lyse as a consequence of DAP-less death immediately upon inoculation into an immunized animal host. Thus, many of the bacteria lyse before they enter cells of the immunized animal host. These bacteria, such as Shigella, most likely enter only epithelial cells lining mucosal tissues. See e.g Branstrom et al. (U.S. Pat. No. 5,824,538). DAP-requiring strains of Escherichia coli also lyse too prematurely in the immunized animal host and thus, are unable to systemically colonize the lymphoid tissues. Listeria monocytogenes have been genetically modified to express phage lysis genes, but the Listeria also lyse before the bacteria can gain access to internal lymphoid tissues. Furthermore, L. monocytogenes is not particularly invasive when delivered orally requiring over 109 CFU to cause systemic infection in mice as compared to S. typhimurium cells, which require only 105 CFU.
In previous studies of attenuated Salmonella as a carrier of DNA vaccine vectors, the Salmonella were attenuated by DeltaaroA mutations that enable the bacteria to invade internal lymphoid tissues. However, three or four immunizations were needed to induce a desired level of immunity. Repeated immunizations were required perhaps because a release of the DNA vaccine vector may depend on the host lysing and destroying the attenuated Salmonella, an action that may lead to the degradation of the DNA vaccine vector by endogenous Salmonella nucleases, such as endonuclease I, and/or by host enzymes that are involved in active bacterial degradation.
Once internalized into the host cells, Salmonella are translocated through the epithelial cells to the lamina propria where they are later taken up by macrophages. About 95% of Salmonella bacteria initially gain entry into the internal lymphoid tissues by attachment to and invasion of the M cells overlying the gut associated lymphoid tissue (GALT), nasal associated lymphoid tissue (NALT) and bronchial associated lymphoid tissues (BALT) after which they can gain access to local lymphoid nodes such as the mesenteric lymph nodes and subsequently to the liver and spleen. About 5% of the Salmonella bacteria invade through the epithelial cells to the lamina propria. During the translocation process, Salmonella transit inside endocytic vesicles within infected epithelial cells where they undergo limited replication similar to replication within endocytic vesicles in macrophages. Within infected animal hosts, about half of the bacteria reside outside of the cells in fluids of the interstitial space, in lymph and in blood.
Unlike other facultative intracellular pathogens such as Listeria or Shigella spp., which gain access to the cytosol shortly after entry, Salmonella spp. remain inside the endocytic vesicle in macrophages and epithelial cells but escape late in the cell infection when the cell succumbs either by Salmonella-induced necrosis or apoptosis, and the Salmonella are released to infect other cells. Efficient stimulation of the class-I-restricted immune response, which is known to be important for protection against viruses and a variety of intracellular pathogens (Gao et al., Infect. Immun., 60:3780-3789 (1992); Yang et al., J. Immunol., 145:2281-2285 (1990)), has not been very successful. For example, mice vaccinated with avirulent strains of Salmonella expressing the influenza virus NP failed to mount a significant class I-restricted T cell response against the NP, although they successfully induced class II-restricted responses. (Brett et al., Immunol., 80:306-312 (1993).
Galan et al. used avirulent Salmonella to deliver a peptide epitope fused to a Salmonella effector protein encoded by a nucleic acid molecule such that the chimeric protein containing the antigen was secreted by the Salmonella and translocated into cells within the immunized animal host dependent on a Type III secretion system (TTSS).
The location of antigens in a recombinant attenuated Salmonella vaccine significantly influences the level of induced immune response, especially antibody production, upon immunization of an animal. Thus, if the antigen is retained in the cytoplasm and must be released by the actions of the immunized host, immune responses are poor (Kang and Curtiss, FEMS Immunol. Med. Microbiol. Lett. 37:99-104 (2003)). On the other hand, immune responses, especially antibody responses, are much higher when the antigen is secreted into the periplasm and more so if released into the supernatant fluid (Kang et al., Infect. Immun. 70:1739-1749 (2002)). Thus, the release of an expressed antigen by lysis of the recombinant attenuated antigen delivery strain in lymphoid tissues within the immunized animal would further enhance the immune response to the expressed antigen. This can be readily achieved by using an antigen delivery strain that exhibits a regulated delayed lysis phenotype.
Based on the discussion above, there is a need for a regulated bacterial lysis for delivery of genetic vaccine vectors and for antigen release. The regulated bacterial lysis may be used to produce a live vaccine that can effectively colonize the lymphoid tissues of the eukaryotic host without causing disease and can result in large amounts of antigen, synthesized either by the prokaryote or the eukaryotic host, to induce an effective immune response.
In general, described herein is a host-vector system exhibiting regulated bacterial lysis to enable delivery of genetic vaccine vectors or desired gene products to a eukaryotic host. Also described herein are microorganisms comprising the host-vector system and uses of the host-vector system, such as for immunization of a eukaryotic host.
The host-vector system possesses novel and surprising features for attenuation, biological containment and immunogenicity.
Briefly, therefore, the host-vector system comprises a modified host chromosome and at least one modified vector. The host chromosome comprises an activatable control sequence, which can be activated by an inducer, a sequence that encodes a repressor, and at least one essential gene operably linked to a regulatable promoter sequence. In one embodiment, the essential gene is operably linked to a prokaryotic activator-promoter sequence. The essential gene encodes a polypeptide that is necessary for synthesis of a rigid layer of a cell wall of a prokaryote. In one embodiment, the essential gene is inactivated. In another embodiment, the essential gene is operably linked to an activatible sequence, which may be the same or different from the activatible sequence that is operably linked to the repressor. In one embodiment, where two essential genes Thus, one or more activatible sequences may be included, and one or more inducers may be used. The inducers may be the same or different.
Further, where the eukaryotic host expresses the polynucleotide encoding the desired gene product, the vector of the host-vector system comprises a eukaryotic expression cassette that comprises a eukaryotic promoter sequence, a site for insertion of a gene encoding a desired gene product, and a polyadenylation sequence.
In another embodiment, a microorganism expresses the polynucleotide encoding the desired gene product or a fusion gene product. The polynucleotide is operably linked to a regulatable promoter sequence.
The vector also comprises a prokaryotic activator-promoter sequence, at least one origin of replication (or), a regulatable promoter, at least one essential gene, at least one transcription terminator sequence, and at least one CpG sequence motif that enhances immunogenicity. The regulatable prokaryotic promoter sequence may be regulated by a variety of molecules, including, but not limited to, a repressor, activator, inducer, or enhancers. One or more regulatable prokaryotic promoter sequences is included. The regulatable prokaryotic promoter sequences may be the same or different and can be regulated by the same or different molecules. The essential gene is a gene necessary for synthesis of a rigid layer of a cell wall of a prokaryote. One or more essential genes is included. The essential genes are operably linked to at least one regulatable promoter sequence, which may be the same or different. One or more transcription terminator sequences is included.
In one embodiment, the host-vector system comprises a modified host chromosome and two modified vectors. The first vector comprises i) a eukaryotic expression cassette comprising 1) a eukaryotic promoter sequence; 2) a site for insertion of a gene encoding a desired gene product; and 3) a polyadenylation sequence; ii) a prokaryotic activator-promoter sequence; iii) at least one origin of replication (ori); iv) a regulatable prokaryotic promoter, which is repressible by the repressor; v) an essential gene, wherein the essential gene is necessary for synthesis of a rigid layer of a cell wall of a prokaryote; vi) at least one transcription terminator sequence; and vii) at least one CpG sequence motif, wherein the CpG sequence motif enhances immunogenicity. The second vector comprises i) a prokaryotic activator-promoter sequence; ii) at least one origin of replication (ori); iii) a first regulatable prokaryotic promotor sequence, wherein the first regulatable prokaryotic promotor sequence is repressible by a first repressor; iv) a second regulatable prokaryotic promotor sequence, wherein the second regulatable prokaryotic promotor sequence is repressible by a second repressor; v) at least one essential gene, wherein the essential gene is necessary for synthesis of a rigid layer of a cell wall of a prokaryote; vi) at least one transcription terminator sequence; and vii) a site for insertion of a gene encoding a desired gene product.
The host-vector system may further comprise a polynucleotide encoding a desired gene product. The desired gene product may be an antigen. The antigen may derive from a variety of pathogens, including, but not limited to, a bacterium, a virus, a fungus, or a parasite. Eimeria, HBV and Streptococcus are non-limiting examples. The antigen may be a protein, polypeptide, or epitope encoded by a polynucleotide derived from the pathogen.
Further, a microorganism, such as a bacterium, preferably, Salmonella, may comprise the host-vector system to deliver the genetic vaccine vector or desired gene product to a vertebrate, such as a mouse, rat, bird, or human. The microorganism may deliver the genetic vaccine vector to a eukaryotic host, who then expresses the polynucleotide encoding the antigen. Alternatively, the microorganism expresses the polynucleotide encoding the desired gene product and upon lysis, delivers the desired gene product to the eukaryotic host. In an embodiment, where at least two vectors is used in the host-vector system, both the eukaryotic host expresses the desired gene product from the first vector and a bacterial host express the desired gene product from the second vector. The gene product may be an antigen.
In either instance, the microorganism, possessing the host-vector system, may be used in a prophylactic or a therapeutic composition, such as a vaccine. The vaccine may be administered to a eukaryotic host, such as a vertebrate. The vertebrate may be a mouse, rat, bird, or human. Methods of delivery of a polynucleotide encoding a desired gene product and methods of delivery of a desired gene product are also provided herein. For example, the host-vector system may be used to vaccinate poultry against coccidiosis or humans against pneumonia.
In addition to the attenuation and containment features described throughout, the host-vector system comprises additional advantageous features that enhance its effectiveness and efficiency in delivery of genetic vaccine vectors or desired gene products. For example, a modification or multiple modifications (no particular order is required) to the host-vector system may be made to delay lysis of the microorganism, to preserve the vector after lysis of the microorganism, to deprive the microorganism of other unintended means of survival, to allow protein synthesis to either continue or be inhibited while the microorganism undergoes lysis, to reduce undesirable effects associated with vaccination, to eliminate or minimize toxicity to the eukaryotic host, to enhance means for the microorganism to escape the endosomes, and to enhance desired means of antigen processing and presentation. It is intended that the host-vector system and uses thereof may contain any number of these and other features.
In addition to using the host-vector system to confer protective immunity to bacterial, viral, fungal and parasite pathogens, it can be appreciated that the host-vector system may be used for other applications, including, but not limited to, development of anti-cancer vaccines, anti-fertility treatment, and growth enhancing effects on agriculturally important animals, such as, but not limited to, poultry, swine, sheep, and cattle.
In the description that follows, a number of terms used in recombinant nucleic acids research and in the fields of bacteriology, virology, microbiology, immunology, genetics and animal science are utilized. In order to provide a clear and consistent understanding of the disclosure herein, including the scope to be given such terms, the following definitions are provided.
Definitions.
An “activator” refers to a DNA-binding molecule that regulates one or more genes by increasing the rate of transcription.
“Attenuated” refers to a pathogen having mutations that reduce the ability of the pathogen to elicit disease symptoms and disease in an animal, but which do not eliminate the potential of the attenuated bacterium to attach to, invade and persist in appropriate lymphoid tissues within the animal. Attenuated microbes are useful, for example, to expose an organism to a particular genetic vaccine vector or gene product, such as an antigen or a therapeutic protein, over an extended time period. “Attenuated” does not mean that a microbe of that genus or species cannot ever function as a pathogen, but that the particular microbe being used is attenuated with respect to the particular animal being tested. Attenuated strains are incapable of inducing a full suite of symptoms of the disease that is normally associated with its pathogenic counterpart. Sometimes “avirulent” is used as a substitute term for attenuated.
A “Bacterial Artificial Chromosome” (BAC) vector is a vector derived from a low copy number plasmid (one to two copies per chromosome DNA equivalent), such as mini-F, that can be stably replicated and maintained in a bacterial host. A BAC vector can be used for insertion of up to about 600 kb of DNA using gene cloning methods. This inserted DNA may come from a single source or multiple sources.
“Biological containment”, as used herein, refers to a restriction on the survival of a microorganism to a targeted environment, such as within a eukaryotic host, due to modifications to the microorganism provided by the host-vector system.
A polynucleotide sequence is “codon optimized” when the codons for amino acids in the original polynucleotide that the host-vector strain rarely used for expression of highly expressed genes are replaced by codons that the host-vector strain preferentially used for expression of highly expressed genes. Usually, the codons for the amino acids arginine, leucine, isoleucine, glycine and proline are changed.
In general, “control sequences” are DNA sequences that are necessary to effect the expression of coding sequences to which they are operably linked. As such, control sequences provide sites for the action of repressors, activators, enhancers, RNA polymerase, and other transcription factors. Nonlimiting examples of such control sequences are promoters and ribosome binding sites.
Control sequences permitting expression of gene products in bacteria are distinctly different from control sequences necessary for gene expression in eukaryotic organisms such that prokaryotic control sequences generally do not function in eukaryotic cells and vice versa. The term “control sequence” can encompass those sequences from prokaryotes or eukaryotes.
An “activatible control sequence” refers to a DNA sequence that can be activated by an inducer.
Cytotoxic T lymphocytes (CTL), also known as killer T lymphocytes, are able to kill target cells that are infected with intracellular pathogens that can be bacteria, viruses, fungi, parasites or tumor cells. Macrophages, dendritic cells, and similar cells that present T-cell epitopes in association with MHC class I antigens stimulate production of CTLs.
A “derivative” is a molecule, plasmid, vector or strain, including a host-vector strain, that is derived from a parental molecule, plasmid, vector or strain, including a host-vector strain. A derivative may have less or more genetic information than the parental element from which it is derived. In some instances, a derivative might be a hybrid derived from two or more parental elements.
An “essential gene”, as used herein, refers to a gene that is necessary for the prokaryote to synthesize the rigid layer of its cell wall. Examples of essential genes include, but are not limited to, murA, murB, murC, murD, murE, murF, murG, murH and, murI (which are necessary for synthesis of the murein rigid cell wall layer), dapA, dapB, dapC, dapD, dapE, dapF and asd (which are necessary for synthesis of diaminopimelic acid (DAP)), air and dadB (which are necessary for synthesis of D-alanine), ddlA, and ddlB (which are necessary for synthesis of D-alanyl-D-alanine). Muramic acid, DAP and D-alanine are unique constituents of the rigid layer of the cell wall and are not incorporated into any other bacterial structure or component. Even though gram-positive bacteria, other than Mycobacterium species and several other acid-fast gram-positive bacterial genera, lack DAP in the rigid layer of their cell wall, there are numerous other genes for synthesis of muramic acid, D-alanine or D-alanyl-D-alanine to select for regulation using a regulatory sequence such as the araC PBAD regulatory system to achieve bacterial cell tysis in vivo.
The term “genetic vaccine vector” may be a DNA or RNA vaccine vector. The DNA or RNA encodes at least one antigen. The antigen may derive from various sources, including, but not limited to bacteria, viruses, fungi, and parasites and from animals, including humans. “DNA vaccine vector” refers to a plasmid DNA molecule propagated in a bacterial cell that has a gene sequence encoding a desired gene product operably linked to a eukaryotic control sequence, so that the desired gene product is expressed only after introduction of the DNA vaccine vector internally into eukaryotic cells by vaccination (immunization). The DNA vaccine vector can be administered to individuals to be immunized by injection, air gun, or preferably, by use of attenuated bacteria that liberate the DNA vaccine vector on entrance into host cells of the immunized individual.
A “gene” is a biological unit of heredity. Generally, a gene is a polynucleotide sequence that encodes an RNA molecule or a polypeptide, or a mutation of said polynucleotide sequence. The gene may be a naturally occurring sequence that is capable of being expressed into an active or inactive polypeptide. The gene may also comprise a mutation, for example a point mutation, insertion, or deletion, such that it is not capable of being expressed, or such that it expresses an altered or truncated polypeptide or RNA molecule. A gene may be created by recombinant DNA methodologies. Alternatively, the gene may be synthesized by well-known synthetic methods.
A gene may be “inactivated” by any method that eliminates or substantially impairs the expression of its gene product. For example, inactivation can be achieved by mutations.
As used herein, “immune system” refers to anatomical features and mechanisms by which a multicellular animal reacts to an antigen. Such a system has both humoral and cellular components. The humoral component produces antibodies that specifically bind to the antigen. There are five types of antibodies or immunoglobulins (Ig), i.e., IgA, IgD, IgE, IgG or IgM. The cellular component or response to antigens has been well studied and widely reported. T cells can either directly kill pathogens or secrete cellular hormones, cytokines, to direct other cells to kill pathogens. A more complete description of both the humoral and cellular components is given in Roitt, Brostoff and Male, Immunology: Fourth Edition, C.V. Mosby International Ltd., London(1998).
By using vaccines, physicians and veterinarians have successfully induced humoral and cellular immunity to human pathogens, e.g. smallpox, rubella, etc., and to animal pathogens such Marek's disease virus, Newcastle disease virus, etc. Some vaccines may include live bacteria that express antigens or carry gene material encoding antigens to which immune responses are desired.
To initiate an immune response to pathogens or vaccines, the human body often requires dendritic cells (DC). Related to macrophages, these cells express large numbers of MHC class I/II-peptide complexes which prime helper and killer-T lymphocytes in vivo to the offending pathogens. DC's become even more effective in the presence of inflammatory cytokines, including interferon-gamma. Such cytokines cause DCs to upregulate adhesion and costimulatory molecules and, thus, to become more potent, terminally differentiated stimulators of T-cell immunity. DC's can acquire antigens along mucosal surfaces, travel to T cell-rich lymphoid tissue (such as GALT, MALT, spleen, etc.(Chen et. al., Mol. Microbiol. 21:1101-1115 (1996)), and stimulate T cell activation, proliferation, and differentiation. Because of unique surface markers and monoclonal antibodies against them, activities of DC's can be discerned in populations of other cells and can also be purified for use in various studies.
“Immunization” is a process of inducing in an animal an immune response to an antigen by administration of a vaccine. The term “vaccination” is often used interchangeably with the term immunization.
“Incompatibility” is an attribute of plasmids that governs their ability or inability to be stably maintained within the same bacterial host cell. This property can be governed by one or more genes and/or non-coding DNA sequences on the plasmid. The abbreviation “Inc”, with a capital letter modifier, is used to designate the “incompatibility group” of the particular plasmid.
An “individual” treated with a vaccine of the invention is defined herein as including all vertebrates, for example, mammals, including domestic animals and humans, various species of birds, including domestic birds, particularly those of agricultural importance. In addition, mollusks and certain other invertebrates have a primitive immune system, and are included as an “individual”.
As used herein, an “inducer” is an extracellular stimulus that causes an activatible control sequence to become active. The inducer provides the signal that allows the DNA sequence to be transcribed into mRNA. Non-limiting examples of inducers are small molecules (e.g. arabinose and lactose), inorganic molecules (e.g. Fe, Mg, or P), temperature alteration, osmotic stress, and starvation.
As used herein, “microbe” or “microorganism” includes bacteria, viruses, protozoa, and fungi.
A “mutation” is an alteration of a polynucleotide sequence, characterized either by an alteration in one or more nucleotide bases, or by an insertion of one or more nucleotides into the sequence, or by a deletion of one or more nucleotides from the sequence, or a combination of these.
The term “operably linked”, as used herein when describing the relationship between two nucleic acid or polypeptide regions simply means that they are functionally related to each other. For example, a pre-sequence is operably linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
“Pathogen-associated molecular patterns” (PAMPs) are compounds like lipopolysaccharide (LPS), teichoib acid, lipoteichoic acid, peptidoglycan, flagella, bacterial CpG containing oligonucleotides, etc. that are recognized by “Toll-like receptors” (TLRs) and serve to stimulate the innate immune system, which is necessary to induce acquired immunity in an immunized individual.
A “polynucleotide” refers to a covalently-linked sequence of nucleotides in which the 3′ and 5′ ends on each nucleotide are joined by phosphodiester bonds. The polynucleotide may encode a gene or fragments of a gene. For example, a polynucleotide may encode the HBV pre SI and S2 epitopes or the M. tuberculosis ESAT-6 T-cell epitopes.
The term “promoter sequence” as used herein refers to the minimal sequence sufficient to direct transcription. Also included in the invention is an enhancer sequence which may or may not be contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5′ or 3′ regions of the native gene. Optionally, expression is cell-type specific, tissue-specific, or species specific.
A “prokaryotic activator-promoter” is a promoter that is derived from a prokaryote and/or can be utilized by a prokaryote to transcribe the DNA sequence into RNA. Activation can be achieved, for example, by an inducer.
A “regulatable promoter” is any promoter whose activity is affected by a cis or trans acting factor (e.g., an inducible promoter, such as an external signal or agent). The regulatable promoter is considered a “repressible promoter” if it can be repressed by a repressor. Non-limiting examples of repressible promoters are lambda PL, lambda PR, P22 PL, P22 PR, Plac, Ptac and Ptrc.
A “replicon” is a unit of replication that possesses an origin of replication termed ori and one or more sequences that encode proteins and/or RNA molecules that regulate initiation of replication.
As used herein, a “repressor” is a protein that is synthesized by a regulator gene and binds to an operator locus, blocking transcription of that operon.
A “transcription terminator” is an element or sequence at the 3′ end of a gene that causes transcription with synthesis of RNA to cease and often is accomplished by the RNA taking on a stem-loop structure followed by a short sequence of U's.
“Transfection” is a process for the delivery of nucleic acids, usually from viruses, into cells to result in infection of those cells and the production of viruses. If the virus nucleic acid has mutations or other modifications, the transfection may not produce viruses.
“Ubiqutination” is a multi-step process whereby proteins or peptides are covalently attached to “ubiqutin” and, thus, marked for degradation in the proteosome in eukaryotic cells. This process, if rapid, can enhance presentation of T-cell epitopes to facilitate induction of a strong CTL response. If slow or absent, the process can result in higher antibody titers to a delivered or synthesized antigen.
By “vaccine” is meant an agent designed to stimulate the immune system of a living organism so that protection against future harm is provided. A particular vaccine may or may not be effective in any particular animal. Immunization refers to the process of rendering an organism immune to a disease.
“Vector” as used herein denotes a genetically engineered nucleic acid construct capable of being modified by gene recombinant techniques to incorporate any desired foreign nucleic acid sequence, which may be used as a means to introduce said sequence in a host cell, replicate it, clone it, and/or express said nucleic acid sequence, wherein said vector comprises all the necessary sequence information to enable the vector to be replicated in host cells, and/or to enable the nucleic acid sequence to be expressed, and/or to enable recombination to take place, and/or to enable the vector to be packaged in viral particles. This recitation of the properties of a vector is not meant to be exhaustive. Those skilled in the art will understand that the use of the term “vector”, and its plural “vectors”, can be used interchangeably, and where appropriate refer to one or more vectors as described herein.
Vectors, optionally containing a foreign nucleic acid, may be “introduced” into a host cell, tissue or organism in accordance with known techniques such as transformation, transfection using calcium-phosophate precipitated DNA, electroporation, particle bombardment, transfection with a recombinant virus or phagemid, infection with an infective viral particle, injection into tissues or microinjection of the DNA into cells or the like. Both prokaryotic and eukaryotic hosts may be employed, which may include bacteria, yeast, plants and animals, including human cells.
The term “expression vector”, as used herein refers to a nucleic acid construct containing a nucleic acid sequence, which is operably linked to a suitable control sequence capable of effecting the expression of said nucleic acid in a suitable subject. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control termination of translation and transcription. The vector may be a plasmid, a phage, or a combination of sequences from both plasmids and phages, such as cosmids and phasmids. Once transformed into a suitable subject, the vector may replicate and function independently of the subject's genome, or may, in some instances, integrate into the genome itself. In the present specification, “plasmid” and “vector” are sometimes used interchangeably as a DNA plasmid vector is the most commonly used form of vector at present. However, the invention includes such other forms of expression vectors, which serve equivalent functions and which are, or become, known in the art.
“Virus-like particles” (VLPs) are particles that assemble, usually in bacteria, due to expression of one or more viral genes whose proteins self-assemble into multi-protein structures that resemble virus particles. These VPLs are non-infectious because they lack the viral genome and one or more gene products that would be necessary for infection of eukaryotic cells or animals.
The term “pathogen” as used herein refers to viruses, bacteria, fungi, protozoa, parasites, or other microbes, organisms and agents that infect cell(s) and tissues thereby causing disease or other adverse symptoms. As particularly used herein, the term “pathogen” preferably refers to agents that have the capability to infect, or avoid destruction by, macrophages. Examples of such agents include, but are not limited to, E. acervulina, E. tenella, E. maxima, P. acnes, L. monocytogenes, S. typhimurium, N. gonorrhoea, M. avium, M. tuberculosis, M. leprae, B. abortus, C. albicans, L. major. Plasmodium falciparum, Marek's disease virus, influenza virus and HBV.
Description
We commenced to develop a DNA vaccine delivery system using Salmonella typhimurium UK-1 that would colonize internal lymphoid tissues before commencing to lyse to liberate a DNA vaccine vector, without degradation, into the cytoplasm of host cells capable of active protein synthesis. We genetically modified genes encoding enzymes needed for synthesis of essential constituents of the rigid layer of the bacterial cell wall so that their expression would be turned off in vivo with dilution of enzyme concentration at each cell division to result in lysis after 5 to 10 cell division cycles. First, we determined whether we could use the araC PBAD activator-promoter system on a multicopy plasmid vector to regulate synthesis of the asd gene product. We, thus, constructed the plasmid vector pYA3450 (
When pYA3450 was introduced into Chi8276 (Table 1), a S. typhimurium UK-1 strain with a DeltaasdA16 mutation (see
Because translation efficiency is known to be influenced by the start codon specifying the N-terminal methionine, we used site-directed mutagenesis to change the start codon for the asd gene from ATG to GTG in pYA3450. GTG is used to initiate translation by some 8% of the genes in E. coli and Salmonella. This generated plasmid pYA3530 (
We next determined whether use of the araC PBAD activator-promoter to regulate expression of a chromosomal gene encoding an enzyme for synthesis of an essential constituent of the rigid layer of the bacterial cell wall might not result in a strain that would exhibit arabinose-dependent growth with lysis occurring in media devoid of arabinose. We chose a construction in which the promoter for the murA gene was deleted and replaced by the araC PBAD activator-promoter. The murA gene encodes the enzyme UDP-N-acetylglucosamine enolpyruvyltransferase (EC 2.5.1.7), an enzyme critical for the biosynthesis of muramic acid that serves as one of two repeating sugars of the rigid layer of the bacterial cell wall. The resulting strain Chi8645 (Table 1) with the DeltaPmurA7::araC PBAD murA construction (see
Chi8645 was quite attenuated since 3 of 4 female BALB/c mice survived a dose of 1×109 CFU with the one mouse dying on day 14, which is relatively late compared to deaths caused by wild-type S. typhimurium UK-1 Chi3761. In challenge studies of mice immunized with Chi8645, 8 of 13 mice survived challenge with 1×109 CFU of Chi3761. Thus Chi8645 exhibited some ability to induce protective immunity to challenge with virulent S. typhimurium. We next examined the ability of Chi8645 to colonize lymphoid tissues following oral inoculation of female BALB/c mice. In an experiment in which the inoculating dose was 7.9×108 CFU, the total mean titers of Chi8645 recovered from Peyer's patches (8 to 11/mouse) on days 1, 2 and 3 following oral inoculation were 1.1×102, 63, and 3.3 CFU, respectively. Viable Chi8645 cells were never recovered from the spleen or any other internal lymphoid organs. Elimination of the ability to metabolize arabinose and alteration of its uptake and retention can prolong the time when arabinose is maintained in the cytoplasm of the cell to activate the AraC gene product to cause transcription of genes under the control of the PBAD promoter. We introduced the DeltaaraE25 mutation that eliminates a low-affinity arabinose uptake protein and the DeltaaraBAD1923 mutation preventing utilization and breakdown of arabinose into Chi8645. Both the DeltaaraE25 and DeltaaraBAD1923 mutations (see
The results with strains possessing an arabinose-dependent regulatable chromosomal essential gene for cell wall biosynthesis indicated the need for a higher number of copies of the essential gene so that cells would have a sufficient enzyme content to permit 5 to 10 generations of growth prior to commencing cell wall-less death leading to lysis. In other words, the bacterial strain had to be capable of a sufficient number of generations of growth in the absence of arabinose to enable colonization of internal lymphoid organs such as the liver and spleen before lysis and liberation of the DNA vaccine vector into the cytoplasm of cells within the immunized host. We considered two approaches to enable creating a DNA vaccine delivery strain with these desired attributes. In one approach, the araC PBAD activator-promoter could be modified to achieve less leakiness and a decreased occurrence of transcription under control of the PBAD promoter in the absence of arabinose. Alternatively, we could screen other bacterial strains taken from the various species within the Enterobacteriaceae capable of arabinose utilization and therefore possessing an arabinose operon including the araC PBAD activator-promoter to identify one with a greater dependence on arabinose for inducing transcription. Although we discovered a number of potential candidates, we used PCR cloning to recover the araC PBAD activator-promoter from the selected E. coli K-12 strain Chi289. As a second means to preclude low-level expression of plasmid genes encoding essential cell wall enzymes, we decided to use a regulatable promoter to synthesize antisense mRNA to interfere with transcription of and/or block translation of mRNA transcribed from the plasmid genes encoding essential cell wall enzymes. As an additional means to ensure lysis after 5 to 10 generations of growth in the absence of arabinose, we included two essential genes necessary for synthesis of the rigid layer of the bacterial cell wall, murA and asd. As an added safety feature, we retained in the bacterial host strain for the DNA vaccine plasmid vector the chromosomal DeltaPmurA::araC PBAD murA deletion-insertion mutation (
To deliver a DNA vaccine vector, we engineered the bacterial delivery strain to inhibit or preclude degradative damage to the circular DNA vaccine vector plasmid. This was accomplished by the introduction of a defined deletion mutation Deltaend42311 (see
We have determined that for optimal lysis of the bacterial DNA vaccine vector delivery strain it is important that the concentrations of both enzymes encoded by the murA and asd genes be depleted in the same or nearly same generation of bacterial growth. Since in two-gene operons there can be differences in the translation efficiency of the mRNA and it is also possible that transcription of a two-gene operon ceases before the second gene sequence is fully transcribed, we considered that having the asd gene with an ATG start would enhance the likelihood that the amounts of the asd and murA encoded enzymes would be nearly the same. The synthesis of anti-sense mRNA also might be more effective in preventing expression of the proximal asd gene that the distal murA gene. Currently, optimal results are achieved with the GTG codon for the murA gene and either the GTG or ATG start codon for the asd gene. Both plasmids have the P22 PR that is repressed by the C2 repressor whose synthesis is regulated by the araC PBAD c2 construction inserted within the inactivated chromosomal asdA19 mutation in Chi8854. This results in synthesis of anti-sense mRNA for the asd and murA genes when arabinose is absent. Both plasmids possess three transcription terminator sequences to preclude interference in expression of gene information in one domain by expression in another domain. The pUC ori domain for replication, the prokaryotic regulatory domain with the araC, murA and asd genes, and the eukaryotic expression domain are all separated by transcription terminators. The two DNA vaccine plasmid vectors pYA3650 and pYA3651 (
Described herein is a regulated bacterial lysis for delivery of genetic vaccine vectors and desired gene products to a eukaryotic host. It is discovered that a host-vector system can be used to efficiently and effectively deliver gene vectors and desired gene products to a eukaryotic host.
As described herein, a host-vector system provides a means for regulated lysis of bacteria, which involves a novel and unobvious means for delivery of genetic vaccine vectors and desired gene products to eukaryotic hosts. The host-vector system involves both attenuation and biological containment of the bacteria. The attenuated bacteria allows for preparation of vaccines that are avirulent in eukaryotic hosts, including mice, chickens, and presumably humans. Some researchers attenuate bacteria by introducing mutations that permit environmental regulation of surface molecule synthesis such as lipopolysaccharides in gram-negative microorganisms as affected by a galE mutation. Bacteria can also be attenuated by introduction of mutations that impose specific nutritional requirements, such as for constituents of nucleic acids such as purines, constituents of the cell wall such as diaminopimelic acid (DAP) or that impose requirements for aromatic amino acids and vitamins derived therefrom, such as caused by aro mutations. Still other means of attenuation are achieved by mutating genes affecting global regulation of other genes. Through biological containment, the vaccine shed, for example, iP feces is unable to survive in nature to cause immunization of other unintended individuals.
In an embodiment, the host-vector system has a number of desirable features. First, the host-vector system comprises a host chromosome that has been modified to comprise an activatible control sequence, such as araC PBAD, that controls the synthesis of a regulatable gene. The araC PBAD activator-promoter drives the arabinose-dependent expression of regulatory genes at this chromosome location. The regulatable gene encodes, for example, a repressor that represses a regulatable control sequence on a vector. In the presence of an inducer, such as arabinose (which can be supplemented in the culture media), the activatible control sequence is activated to transcribe a gene encoding the repressor, such as C2 or Lacd. In one aspect, the sequence that encodes the repressor is operably-linked to the activatible control sequence. The repressor represses a regulatable promoter sequence, such as P22 PR or Ptrc, thereby preventing synthesis of the genes under the control of the regulatable promoter sequence. In the absence of the inducer, the activatible control sequence is not activated, and the repressor is not produced. Without the repressor, the regulatable promoter sequence is not repressed. Thus, synthesis of mRNA for genes (or its anti-sense strand) under the control of the regulatable promoter can occur.
In the host-vector system, the host chromosome has at least one essential gene that has been modified. In one embodiment, two essential genes are modified. In one embodiment, the essential gene is inactivated. In another embodiment, the essential gene is operably linked to an activatible control sequence, which may be the same as or different from the activatible control sequence that controls expression of the repressor. Thus, there may be multiple activatible control sequences. These activatible control sequences may be activated by the same or different inducers. For example, a first activatible control sequence is operably linked to the sequence that encodes the repressor. The inducer may be arabinose or lactose. A second activatible control sequence may be operably linked to the essential gene.
The essential gene encodes a polypeptide that is necessary for synthesis of a rigid layer of a cell wall of a prokaryote. Any number of essential genes may be modified, such as by inactivation or regulation under the control of an activatible control sequence. For example, murA, murB, murC, murD, murE, murF, murG, murH, and mur are necessary for synthesis of the murein rigid cell wall layer. dapA, dapB, dapC, dapD, dapE, dapF and asd are necessary for synthesis of diaminopimelic acid (DAP)), alr and dadB (which are necessary for synthesis of D-alanine), ddlA, and ddlB (which are necessary for synthesis of D-alanyl-D-alanine). Muramic acid, DAP and D-alanine are unique to bacteria and are not either synthesized by or available in animals, including humans.
In one embodiment, the essential gene is asd. The asd gene encodes aspartic semialdehyde dehydrogenase, an enzyme necessary for the synthesis of diaminopimelic acid (DAP), which is an essential constituent of the rigid layer of the bacterial cell wall (Nakayama et al., 1988, Bio/Tech. 6:693-697). Inactivation of asd prevents the bacteria from synthesizing the rigid layer of its cell wall, which leads to lysis of the bacteria, unless either DAP is supplied in the growth medium or a wild-type asd+ gene is present on a plasmid within the mutant bacteria
In another embodiment, the essential genes are alr and dadB. The alr gene encodes alanine racemase whereas the dadB gene encodes a catabolic enzyme enabling bacteria to grow on D-alanine but which in the reverse direction can cause synthesis of D-alanine from L-alanine. D-alanine is a unique constituent of the rigid layer of the bacterial cell wall. Inactivation of both the alr and dadB genes prevents the bacteria from synthesizing the rigid layer of the cell wall, which leads to lysis of the bacteria, unless either D-alanine is supplied in the growth medium or a wild-type alr+ or a wild-type dadB+ gene is present on a plasmid within the mutant bacteria.
The vector of the host-vector system comprises several advantageous features for delivery of a genetic vaccine vector. For example, the vector comprises a eukaryotic expression cassette, which comprises a eukaryotic promoter sequence, a site for insertion of a polynucleotide encoding a desired gene product, and a polyadenylation sequence. The eukaryotic promoter sequence, for example, may be PCMV, which is derived from the human cytomegalovirus. A polynucleotide encoding a desired gene product may be inserted into a site for insertion of a polynucleotide encoding a desired gene product. Insertion of a polynucleotide at this site places the polynucleotide under the control of the eukaryotic promoter. If a sequence to be expressed lacks a Kozak sequence to facilitate mRNA binding to ribosomes, one is provided immediately upstream of the start codon. Through the eukaryotic promoter, the eukaryotic host expresses the desired gene product. The polyadenylation sequence may derive from a variety of sources, such as from the gene for bovine growth hormone. The polyadenylation sequence enhances the stability of mRNA and prevents its breakdown by ribonuclease.
In one embodiment, the prokaryote expresses the polypeptide encoding the desired gene product using a vector with a prokaryotic expression cassette. In this instance, the eukaryotic expression cassette is not used. Thus, the eukaryotic expression cassette need not be in the vector.
In another embodiment, the vector of the host-vector system has a prokaryotic activator-promoter sequence, at least one origin of replication (ori), a regulatable promoter, which is repressible by a repressor, at least one essential gene, at least one transcription terminator sequence, and at least one CpG sequence motif.
When the prokaryotic activator-promoter is activated by, for example, an inducer, such as arabinose or lactose, the prokaryote expresses a gene under the control of the prokaryotic activator-promoter. The prokaryotic activator-promoter may be araC PBAD. In one embodiment, the prokaryotic activator-promoter is operably linked to at least one essential gene.
Further, replication of the vector can commence at the origin of replication. The or may be pUC, pBR, p15A, pSC101, or pBAC. One can also include an or from plasmids, such as BAC vectors, with only one or two plasmid copies per chromosomal DNA equivalent. In one embodiment, the orn is a pUC ori.
Still further, the regulatable promoter of the vector may be regulated by a repressor, such as C2 or LacI. In one embodiment, a regulatable promoter sequence may be repressed by a repressor. In another embodiment, multiple regulatable promoter sequences may be used. The regulatable promoter sequences may be repressed by the same or different repressors. In still another embodiment, a first regulatable promoter sequence is operably linked to a site for insertion of a polynucleotide encoding a desired gene product. In this way, expression of the polynucleotide inserted at this site would be under the control of the regulatable promoter sequence. The regulatable promoter may a phage P22 PR or Ptrc.
In another embodiment, the regulatable promoter sequence allows synthesis of anti-sense mRNA for the essential genes, such as asd and murA genes, in the vector, when the phage P22 repressor protein C2 is absent. This repressor protein is synthesized under the control of the araC PBAD activator-promoter as described above. To increase repression of the regulatable promoter, multiple copies of the gene encoding the repressors may be present. For example, the amount of C2 repressor specified by a single chromosomal gene might be insufficient to repress completely a plasmid localized PR such that low-level transcription leading to anti-sense mRNA for the asd and murA genes might occur. Thus, multiple copies of regulatable c2 and/or lacI genes may be inserted into the chromosome.
The essential gene on the vector may complement the essential gene that has been modified on the chromosome. For example, the asd gene may be inactivated in the bacterial chromosome. The bacteria can no longer make DAP, which is necessary for the bacteria to synthesize a rigid layer of its cell wall. Thus, when the asd gene is inactivated, the bacteria undergo DAP-less death through lysis. Bacterial lysis releases genetic vaccine vectors or desired gene products to the eukaryotic host. As explained further below, this lysis may be delayed to allow the bacteria to first effectively colonize the lymphoid tissues, where macrophages and dendritic cells are abundant, and allow the bacteria to undergo 5 to 10 generations of replication, which produces a large number of genetic vaccine vectors or amounts of desired gene products. With a large number of genetic vaccine vectors, the eukaryotic host can express a large amount of antigen, which enhances the induction of a desired immune response. In another embodiment, the prokaryote expresses the gene encoding the desired gene products. Delayed lysis would also allow the bacteria time to colonize the lymphoid tissue, replicate the vector, and express the desired gene product, such as an antigen, in large amount before lysis. Upon lysis, a large amount of the desired gene product is presented to the cells of the eukaryotic host.
The essential gene on the vector allows the bacteria to survive in culture. The essential gene may be operably linked to a prokaryotic activator-promoter sequence on the vector.
In vaccination, whether the eukaryotic host or the prokaryote expresses the antigen on the vector of the host-vector system, delayed lysis allows production of a large amount of antigen for presentation to the immune system of the eukaryotic host to elicit an effective immune response. After expression of the antigen, the cells (e.g. dendritic cells) of the eukaryotic host processes (e.g. by complexing the antigen to MHC I and MHC II proteins) and presents the antigen to the immune system of the eukaryotic hosts, thereby eliciting an efficient and effective immune response. In most cases, Class I molecules present foreign proteins synthesized in a cell. For presentation by Class II, the foreign protein either can be synthesized in the cell or taken up by the cell from the outside (i.e., presented in the form of a free protein or peptide). If an antigen is synthesized in a cell and presented by both Class I and Class II molecules, both antibody producing B cells and cytotoxic T cells are produced. However, if an antigen originated outside of a cell and is presented only by Class II, the specific immune response is largely limited to T helper cells and antibody production (Robinson, et al., Eds., THE SCIENTIFIC FUTURE OF DNA FOR IMMUNIZATION, American Academy of Microbiology, pp. 1-29, (1997)). For example, Salmonella can enter the host cell and lyse within the endosome or in the cytoplasm. The antigens released in the endosome are processed and presented with MHC class II molecules to recruit Th2 cells to enhance induction of antibodies by B cells whereas antigens released into the cytoplasm are processed and presented with MHC I proteins to Th1 cells to enhance induction of cellular immunity and CTLs. Alternatively, Salmonella can enter the host cell, lyse, and release gene vectors. Using the released gene vectors, the host cell expresses the antigens, which are processed and presented with MHC I proteins to Th1 cells. Still further, Salmonella can exist outside of the host cells, such as in body fluids. Through regulated lysis, Salmonella can release the antigens outside of the host cells. The antigens are then taken up by endosomes, which can lead to a Class II presentation for recruitment to Th2 cells that enhances antibody production.
Still further, the host-vector system comprises transcription terminator sequences. As transcription of the several genes on the vector may interfere with the effectiveness of the host-vector system, transcription terminator sequences are used to keep separate the transcription of the genes under the different promoters. For example, the transcription terminator sequence avoids araC PBAD driven transcription beyond the inserted gene, such as the c2 and/or lacI gene, that might attenuate or alter the physiology of the bacteria. The transcription terminator sequence may be rrfG.
CpG sequence motifs may be used to enhance immunogenicity of the host-vector system. (Krieg, Annu. Rev. Immunol. 20:709-760 (2002)). The motifs are prevalent in bacteria, but are absent in vertebrates. The motifs are unmethylated in bacteria, which makes them appear foreign to vertebrates, which lack such sequences. These CpG sequence motifs enhance inununogenicity by acting like adjuvants and stimulate the human immune system. In humans, preferably GTCGTT is used to activate the human immune cells. The GTCGTT motif is also optimal for stimulation of lymphocyte proliferation in several species including cattle, sheep, goats, horses, pigs, dogs, cats, and chickens, while the GACGTT motif is preferred for rabbits and mice (Rankin et al., Antisense Nucl.Acid Drug Devel. 11:333-340 (2001)). CpG DNA sequences within bacterial DNA possess adjuvant activity that enhance induction of immunity by DNA vaccines.
Any microorganism may be used to carry the host-vector system for delivery of the genetic vaccine vector or desired gene product. Preferably, the microorganism has an ability to invade and colonize the lymphoid tissue. Salmonella is an example of such a microorganism. Shigella, enteroinvasive E. coli, Yersinia and Listeria are other examples of bacteria that could be modified to exhibit regulated delayed lysis for the delivery of antigens or genetic vaccines. The lymphoid tissue may be in a liver, spleen, or a regional lymph node.
A number of advantageous modifications may be made to the host-vector system to further enhance its effectiveness and efficiency. The host-vector system may have one modification or multiple modifications, which can occur in any order.
For example, after growth in an arabinose-enriched environment and then transferred to an environment without arabinose, the bacterium still retains some arabinose. The limited amount of arabinose allows the bacterium to survive for a short period of time. This delay allows the bacterium time to replicate and colonize the lymphoid tissue. In one embodiment, to further delay lysis of the bacterium, the araBAD gene is mutated. The enzymes encoded by the araBAD genes degrades arabinose. For example, inclusion of the DeltaaraBAD1923 mutation as diagrammed in
In another embodiment, the araE gene is mutated. The araE gene encodes a transport protein. For example, the mutation of araE further delays onset of lysis because the arabinose taken up into cells during permissive growth does not leak out of cells via the AraE transport protein.
In still another embodiment, the genes encoding endonuclease I enzyme may be mutated. Endonuclease I enzyme degrades nucleic acids, especially circular plasmid DNA molecules that can be digested by endonucleases but not by exonucleases. When the host strain possesses a mutation that inactivates the periplasmic endonuclease I enzyme, recovery of circular plasmid DNA is improved and the efficiency of plasmid transfer to a host strain by either transformation or electroporation is enhanced. It would also not be subject to attack and breakdown when released by a bacterium undergoing regulated lysis in an immunized animal or individual.
In yet another embodiment, the gind or gmd-fcl gene may be mutated. Bacteria experiencing perturbations in the synthesis of their cell walls, due to either inhibition of cell wall synthesis by antibiotics or by metabolic limitations as a result of mutational alterations, frequently respond to this type of stress by synthesizing the extracellular polysaccharide colanic acid. Deletion mutation of the gmd gene precludes colanic acid synthesis that could enable bacterial cell to survive, while undergoing death by lysis due to the presence of mutations in its essential genes. The mutation would block the conversion of GDP-mannose to GDP-fucose. GDP-fucose is necessary for the synthesis of colanic acid since fucose represents one-third of the sugar mass of colanic acid.
In a further embodiment, the relA gene is mutated. Bacterial strains that undergo lysis as a result of their inability to synthesize the rigid layer of the bacterial cell wall may cease to lyse if protein synthesis is arrested. To uncouple this interdependence for continued protein synthesis for lysis to occur, the relA gene was inactivated by mutation. Although protein synthesis within bacteria in vivo is not likely to impair genetic vaccine vector delivery by lysis, the bacterial strain might encounter some environments, following excretion in feces, for example, in which protein synthesis might be inhibited. In this instance, bacterial cells for genetic vaccine or antigen delivery might survive. For example,
In another embodiment, the msbB gene is mutated. The lysis of gram-negative bacteria in vivo leads to the release of lipopolysaccharide (LPS) that contains endotoxin due to the presence of lipid A. Although lipid A serves as an adjuvant to stimulate the innate immune system to produce interferon-gamma, which enhances the induction of a desired cellular immune response, this inflammatory response can also induce fever and have an adverse consequence on an immunized animal or human host. There are numerous mutations that can alter the myristillation of lipid A with alterations of attached fatty acids to alter the degree of toxicity (Raetz and Whitfield, Annu. Rev. Biochem. 71:635-700 (2002)). Mutation of the msbB gene decreases LPS toxicity without altering the virulence of the bacterial strain possessing this mutation.
In another embodiment, it is contemplated that the host-vector system may be modified to encode a SipB protein. SipB has homology to the Shigella flexneri IpaB protein (Hume et al., Mol. Microbiol. 49:425439 (2003)) that is responsible for enabling Shigella to rapidly exit the endosome and replicate freely in the cytoplasm. Therefore, since SipB can partially substitute for IpaB in Shigella, an increased amount of SipB production by Salmonella may permit endosome escape to enhance induction of a CD8-dependent CTL response. It is further contemplated, that the sifA gene may be inactivated to facilitate Salmonella's exit from the endosome. The sifA gene product is required to maintain Salmonella cells within the endosome, and inactivation of this gene enables Salmonella cells to escape the endosome and multiply in the cytoplasm (Brummel et al., Infect. Immun. 70:3264-3270 (2002); Beuzon et al., Microbiology 148:2705-2715 (2002)). Because the bacteria entering the cytoplasm are often killed, preferably, the promoter for the sifA gene is deleted and replaced with the araC PBAD activator-promoter so that non-expression of the sifA gene is delayed until after the DNA vaccine delivery host-vector has undergone some 5 to 10 generations of growth and adequately colonized lymphoid tissues. The DeltaPsif196::TT araC PBAD sifA deletion-insertion construction diagrammed in
The host-vector system has multiple advantageous applications. In one embodiment, the host-vector system further comprises a polynucleotide encoding a desired gene product. The polynucleotide may encode an auto-antigen, such as a sperm-specific or egg-specific antigen to induce an immunity that may confer immunocontraception or infertility. The antigens may represent protective antigens encoded by genes from a pathogen that render the pathogen virulent. Other antigens may be tumor antigens that can be used to develop a vaccine against cancer. Depending on the type of immunity desired, any person of ordinary skill in the art would know the appropriate antigen to select to develop a vaccine or vaccination regimen that would stimulate that type of immunity. Antigens can be specified by polynucleotides cloned from pathogens or as cDNA clones from animals, including humans. The polynucleotides can be synthesized with codons to optimize expression either in bacteria or in vertebrate animals, including humans. In developing a vaccine against HBV, for example, the protective pre S1 and pre S2 epitopes can be expressed as fusions to the HBV core, which self-assembles into a particle in bacteria (Schodel et al., Infect. Immun. 62:1669-1676 (1994)). These particles are highly immunogenic and can be delivered to the immunized individual by regulated delayed lysis of the bacterial host-vector system. Because the expression of the core genes for the woodchuck and duck hepatitis viruses (that are closely related to HBV) occurs in bacteria and the synthesized core proteins self-assemble into core particles, it would be feasible to use core particles from these viruses for presentation of B-cell and T-cell epitopes representing protective antigens from various bacterial, viral, fungal, and parasite pathogens.
The host-vector system displaying regulated delayed lysis can be used to deliver to an immunized individual a bolus of antigen synthesized in the host-vector strain prior to its lysis in vivo. Such antigen synthesis can be controlled using a regulatable promoter such as Ptrc. Ptrc is regulated by the production of a LacI repressor which is encoded by an araC PBAD lacI that exists either in the chromosome or on a low copy number plasmid in the host-vector strain. Growth of such a strain in the presence of arabinose causes synthesis of the LacI repressor to repress antigen synthesis controlled by Ptrc. However, LacI synthesis ceases following immunization since arabinose is not present. The amount of LacI is diluted out with each cell division of the host-vector strain in the immunized animal or individual. This leads to a gradual de-repression of Ptrc-controlled antigen synthesis. Because the plasmid copy number encoding essential genes for cell wall synthesis outnumbers the copy number encoding the araC PBAD lacI, synthesis of antigen encoded by the gene regulated by Ptrc commences several generations (i. e., cell divisions) prior to the onset of cell lysis. Enhancement of the immunogenicity of the antigen synthesized by the host-vector system can be achieved by partial exportation of the antigen out of the host-vector cells, either as soluble antigen or contained in membrane vesicles. Export of antigens outside of host-vector cells can be accomplished by fusing the sequence encoding the antigen to a sequence encoding the beta-lactamase signal sequence and additional amino acids to facilitate secretion (Kang et. al, Infect. Immun. 70:1739-1749 (2002)). This type of construction has been accomplished for the alpha-helical portion of the S. pneumoniae PspA antigen encoded by the original sequence from S. pneumoniae strain RX1 and a codon sequence with codons optimized for high-level expression in Salmonella, Shigella or E. coli.
In an embodiment, the host-vector strain delivers the antigen(s) by utilizing the Type m secretion system, which allows delivery of T-cell epitopes into the cytoplasm of cells within the immunized animal or individual. This type of antigen delivery results in MHC class I presentation and induction of a Th1 -dependent CTL immune response. To achieve this result, an additional plasmid vector system is used. Salmonella strains with the air and dadB mutations require D-alanine for growth. D-alanine is an essential constituent of the rigid layer of the bacterial cell wall and is not present in animals, including humans. A wild-type alr+ gene in a plasmid vector enables selective maintenance of the plasmid in a bacterial strain in an immunized animal that has mutations in the air and dadB genes. Use of such a plasmid vector encoding a fusion between a Type III effector protein, e.g. SptP, SopE or SopE2, and an antigen sequence containing T-cell epitopes, e.g. the ESAT-6 antigen from M. tuberculosis or the LLO antigen from L. monocytogenes, enables induction of CTL responses to protect against infection with M. tuberculosis and L. monocytogenes, respectively.
Other combination vaccines are also contemplated. A fusion between codon-optimized sequences encoding the alpha-helical domains of PspA antigens from S. pneumoniae strains (e.g. RX1 and EF5668) ensure induction of immunity to the majority of S. pneumoniae strains of diverse serotypes (Hollingshead et. al., Infect. Immun. 68:5889-5900 (2000)). This fusion can be fused to beta-lactamase secretion sequence and placed under the Ptrc control in the regulated delayed lysis vectors, such as pYA3681 or pYA3682. PspA antigens is an adhesin necessary for S. pneumoniae colonization. Psa A is specified as a fusion to the beta-lactamase signal sequence with the sequence inserted in an Alr+ vector. The strain also possesses mutations in the air and dadB genes to impose a requirement for D-alanine. The psaA sequence could be PCR amplified from S. pneumoniae TIGR-4. As a result, a vaccine displaying regulated delayed cell lysis can be engineered to synthesize two protective pneumococcal antigens to stimulate immunity that could both block colonization and systemic infection by diverse S. pneumoniae serotypes. The antigens would initially be secreted or released in membrane vesicles during cell divisions in vivo after immunization and then released as a bolus of antigens when the cells commence to lyse.
Other potential protective antigens are from viruses, such as HIV, influenza, and papilloma. HIV antigenic proteins that could be expressed in a DNA vaccine vector to be delivered to an immunized host by the host-vector system with regulatable lysis in vivo as described comprises gp120, gp140, gp160 (Env), Nef, Pol, Tat, Rev and Gag/Pol. These antigens or peptide epitopes with B-cell and/or T-cell stimulatory properties derived therefrom could also be presented by HBV core particles to be released from a host-vector system having the regulatable lysis in vivo phenotype as described to stimulate an immune response in a vaccinated individual to these HIV epitopes. Thus, it is contemplated that the papillomavirus gene for the L1 core protein, which assemble into virus-like particles (VLPs) in the cytoplasm of Salmonella vaccine strains, can be expressed. (Benyacoub et al., Infect. Immun. 67:3674-3679 (1999)). These VLPs could be released by a host-vector system with regulated lysis occurring in vivo. The delivery system may be further modified to deliver a protective T-cell epitope encoded by the papillomavirus E7 gene (Nonn et al., J. Cancer Res. Clin. Oncol. 2:381-389 (2003). This can be accomplished with an Alr+ plasmid vector designed to deliver the N-terminal portion of the SptP, SopE or SopE2 proteins, which can be delivered by the TTSS, a protein encoded by genes in Salmonella Pathogenicity Island 1 (SPI-1). Such a vaccine should be useful to prevent papilloma-induced cervical cancer.
The host-vector system with regulated delayed lysis can have additional uses in addition to those described. Vaccines against many viral diseases of animals, including humans, make use of live attenuated viruses. These viruses must each be propagated in specific cell lines or animal hosts, including embryonated eggs. A producer of such vaccines must maintain all these diverse cell lines and propagation systems. This can result in considerable expenses because cell line storage and cell culture media are expensive. The complexities and costs increase further when one also considers that many of these live viral vaccines use attenuated virus derivatives that protect against a particular strain of a given virus that are most prevalent in a portion of the world. This is particularly true in the poultry industry. Thus, the propagation and delivery of attenuated viral genomes in a bacterial host-vector system of the present invention would be at a considerable savings in reduced vaccine production and delivery costs. Such bacterial strains can be stored in −70° C. freezers, grown in inexpensive media and preserved by lyophilization.
Artificial bacterial chromosomes (BACs) generated from mini-F plasmids have been very useful for cloning (and sequencing) large segments of DNA (up to 600 kb) from various sources into strains of E. coli (Shizuya et al., Proc. Natl. Acad. Sci. USA 89:8794-8797 (1992)). BACs have been used to clone the entire genome of a herpesvirus and transfected into eukaryotic cells to cause a productive infection with release of infectious virus particles (Messerle et al., Proc. Natl. Acad. Sci. USA 94:14759-14763 (1997)). More recently, Tischer et al. (J. Gen. Virol. 83:2367-2376 (2002)) have generated a DNA vaccine based on the cloning of the entire genome of an attenuated Marek's disease virus vaccine into a BACs vector and have been able to deliver this recombinant viral DNA vaccine to chickens. Some chickens (depending on the delivery means, dose and timing of immunization) developed immunity and did not develop tumors after challenge with a virulent Marek's disease virus strain. A much more cost-effective and efficacious means to deliver such a recombinant BAC to chickens is to introduce the recombinant BAC into a S. typhimurium host-vector system with regulated delayed lysis, and Chi8888 is further modified to display a regulated delayed means to escape the endosome to release the recombinant BACs into the cytoplasm of cells within the immunized chickens. The Delta PsifA196::TTaraCPBADsifA mutation in Chi8925 is introduced into Chi8888 or its derivative using the suicide vector pYA3719 (
Currently available BAC vectors are derived from the mini-F plasmid, which possesses IncF genes that are incompatible for maintenance of this plasmid in a strain that possesses another plasmid with the same or closely similar incompatibility genes. The S. typhimurium virulence plasmid is a stable low-copy number plasmid that is also IncF (Tinge and Curtiss, J. Bacteriol. 72:5266-5277 (1990)) and is, therefore, incompatible with the currently available IncF BAC vectors. To circumvent this problem, the genes for the IncF in the BAC vector can be replaced with IncI-alpha (Nozue et al., Plasmid 19:46-56 (1988)). This generates an IncI-alpha BAC vector with a low copy number replicon that will be compatible with the S. typhimurium virulence plasmid. This IncI-alpha BAC vector with the attenuated Marek's virus genome could then be efficiently and cost effectively delivered to day-of-hatch chicks using the bacterial host-vector system of the present invention. The vaccine may be administered by course spray. (Sandra Kelly, WO 00/04920 or PCT/US99/15843) DNA copies of RNA virus genomes can be used in the present invention.
Another use of a modified IncI-alpha BAC would be to specify synthesis of a large number of protective antigens from one or more pathogens. One could employ such a system as a means to screen for genes encoding protective antigens from uncharacterized pathogens.
The antigens may also derive from Plasmodium species that cause malaria, Eimeria species that cause coccidiosis, Shistosoma species that cause shistosomiasis, and Trypanosome species that cause a variety of diseases. These antigens can be encoded by a DNA vaccine vector to be expressed by the immunized host to induce systemic and cellular immunities or epitopes derived from these antigens can be delivered either as B-cell epitopes by fusion to HBV cores to stimulate antibody production or as T-cell epitopes by fusion to effector proteins such as SptP, SopE or SopE2 to be delivered by Type III secretion to stimulate cellular immunity.
In an embodiment, the host-vector system is used in a vaccine. The vaccine may be administered to a eukaryotic host, such as a vertebrate. The vaccine may be administered via oral, rectal, intravaginal and intrapenile routes of administration. Still further, the vaccine can be administered by intranasal inoculation or by injection intradermally, intramuscular, subcutaneously, intraveinously and intraperitonealy. The vaccine is generally suspended in a suitable excipient and is administered at appropriate doses. Optimal doses are determined by clinical trials. The vertebrate may be, for example, a mouse, rat, bird, or human.
The host-vector system can be applied to develop vaccines to protect against a diversity of pathogens, which may be bacterial, viral, fungal, or parasitic. In an embodiment, the host-vector system is applied to immunize a eukaryotic host against coccidiosis. Here, the eukaryotic host is typically a poultry species, such as a chicken. Eimeria species cause coccidiosis in poultry, a bloody diarrhea with severe negative economic consequences for the poultry industry. The industry spends some $500,000,000 annually for anti-coccidial drugs. No effective safe vaccine presently exists. Since coccidiosis is a parasitic disease with a one-week life cycle with oocysts that germinate into sporozoites that differentiate into merozoites, it is important to induce cellular immunity to both the sporozoite and merozoite stages. Since Eimeria is an intracellular pathogen, induction of a CD8+ dependant response generating CTLs would be most protective. This can be achieved by DNA vaccines since protein antigens are synthesized in the cytoplasm of host cells, which leads to MHC class I presentation that results in inducing a CTL response. The delivery of a DNA vaccine would stimulate other branches of the immune system to result in production of mucosal and systemic antibody production and also stimulate CD4+ T cells. The nucleotide and amino acid sequences of many Eimeria antigens are known and can be inserted in the host-vector system for delivery of the DNA sequences encoding these antigens to the poultry to elicit an immune response for protection against coccidiosis.
All references cited herein are hereby incorporated by reference in their entirety.
The following Materials and Methods were used, where appropriate, throughout the examples described below.
a. Bacterial strains media and bacterial growth. Vaccine strains for testing in mice and chickens were derived from S. typhimurium UTK-1, which has the highest virulence for chickens and mice of any S. typhimurium strains we have so far tested (Curtiss, New Generation Vaccines, p. 715-740 (1990)). Table 1 lists the bacterial strains along with strains of S. paratyphi A and S. typhi to develop DNA vaccine vector and/or antigen delivery vaccines for humans. Table 2 lists the plasmids.
*araCPBAD fragment from χ289
b. Molecular and gene procedures. Unless indicated otherwise, standard methods for DNA isolation, restriction enzyme digestion, DNA cloning and use of PCR for construction and verification of vectors were used. (See e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second ed (1989)). DNA sequence analysis was performed in the Department of Biology DNA Sequence Laboratory at Washington University. Synthesis of oligonucleotide and/or gene segment were performed by commercial entities. Phage P22HTint (Schmieger, Molec. Gen. Genet. 119:75-88 (1972)) was used to transduce mutations of a selectable phenotype from one S. typhimurium strain into other strains. Conjugational transfer of suicide vectors was performed by standard methods (Miller and Mekalanos, J. Bacteriol. 170:2575-2583 (1988)) using the suicide vector donor strain MGN-617 (also referred to as Chi7213) (Table 1). Many of the plasmids constructed possess the wild-type asd gene, and these recombinant constructs were introduced into Chi6212 (Table 1) by selecting for DAP-independent isolates. These constructs were sequenced and evaluated for their ability to complement various S. typhimurium mutant strains (Table 1) and for their ability to specify synthesis of Asd and MurA proteins using gel electrophoresis and western blot analysis. Chi6212, containing pYA3542 (Table 2), produced the His-tagged Asd, which was used to obtain anti-Asd rabbit antisera for western blot analyses. Table 3 lists the oligonucleotide primers used for constructions of the plasmid, for PCR reactions to confirm presence of mutations/insertions, and for DNA sequence analysis.
c. Strain characterization. Experimental vaccine strains were fully characterized prior to use for immunization studies. Those strains were compared with vector control strains for stability of plasmid maintenance when strains are grown in the presence of DAP over a 50 generation period. Molecular gene attributes were confirmed by use of PCR and/or Southern blot analyses with appropriate probes. Measurement of lipopolysaccharide or its absence was performed after electrophoresis using silver stained gels (Hitchcock and Brown, J. Bacteriol. 154:269-277 (1983)). Motility tests and use of specific antisera for given flagellar antigens were used to reveal presence or absence of flagella. Presence of fimbrial adhesins was assayed using agglutination of yeast and red blood cells in the presence and absence of mannose as a function of growth conditions, Congo red binding assays and by transmission electron microscopy (TEM) using negative staining with phosphotungstic acid.
d. Cell biology. The ability of various constructed Salmonella strains to attach to, invade into and survive in various cells in culture are quantified using well established methods (Chen et al., Mol. Micriobiol. 21:1101-1115 (1996)). Murine and human macrophage cell lines and murine bone marrow derived and human peripheral blood monocyte-derived macrophages were used. To evaluate whether engineered Salmonella strains are capable of escaping the endosome, we fixed infected cells, created thin sections, and examined the sections by transmission electron microscopy. These methods were previously described by Miller et al. (Alpuche-Arunda, et al., Proc. Nat. Acad. Sci. USA. 85:7972-7976 (1992)) and Finlay et al. (Mol. Microbiol. 20:151-164 (1996)).
e. Animal experimentation. BALB/c and C57BL/6 female mice, six to eight weeks of age, were used for most experiments. Inbred mice with other MHC haplotypes and Swiss Webster outbred mice were also used in some studies. Mice were held in quarantine one-week before use in experiments. They were deprived of food and water six hours before per oral immunization. No bicarbonate was administered. Food and water were returned 30 min after immunization. In initial experiments comparing multiple strains, only four to five mice were included per group. A second boosting immunization is given four weeks after the first dose, but after a retro-orbital bleed to collect serum and collection of feces and/or vaginal secretions for quantitation of SIGA. In some studies, to maximize induction of antibodies, an immunization regimen of multiple (low or increasing) doses was used during an initial seven to ten day period. In other experiments, candidate vaccine strains were quantitatively enumerated in various tissues as a function of time after inoculation. Generally, three mice were used per time point. The inoculation procedures were the same as in the immunization studies. All animals were housed in BL2 containment with filter bonnet covered cages. If high inununogenicity was observed in initial tests after primary immunization, subsequent studies were done to determine the lowest level of vaccine inocula to induce a significant immune response. Important experiments have been repeated with larger groups of mice for a given dose.
One-day-old white leghorn chicks (hatched from fertile eggs from Spafas or Sunrise Farms in our animal facility) were used for experiments in St. Louis. For infection of chicks, bacterial strains were grown as standing overnights and then diluted into appropriate prewarmed media and grown with mild aeration on a rotary shaker, generally to an OD600 of about 0.8. Cells were sedimented by centrifugation and suspended in buffered saline with gelatin (BSG) before administration to chicks orally, intranasally or by course spray. Course spray is a method used in the hatchery to immunize day-of-hatch chicks (Kelly WO 00/04920 or PCT/US99/15843). Chicks in a box are sprayed by a regulated spray nozzle suspended above the box. Birds were euthanized as a function of time after infection and the titers of bacteria recovered from various tissues, organs and organ contents quantitated by plating on suitable media. In the case of too few to count, detection was by inoculation of 100 microl tissue samples into selenite broth for enrichment, after which cultures were streaked on MacConkey agar for identification as Salmonella. These methods have been previously described (Curtiss and Kelly, Infect. Immun. 55:3035-3043 (1987)). Strains recovered from birds were tested to verify the maintenance of relevant attributes, including presence of plasmids. Birds were housed in either small isolators or Horsefall isolators under BSL2 containment in our animal facility and cared for by approved procedures.
Clinical trials with human volunteers are conducted as previously described (Tacket et al., Infect. Immun. 60:536-541 (1992); Nardelli-Haefliger et al., Infect. Immun. 64:5219-5224 (1996);Tacket et al., Infect. Immun. 65:3381-3385 (1997); Frey et al., Clin. Immunol. 101:32-37 (2001)).
f. Monitoring Immune Responses.
i. Antigen Preparation: We have purified antigens as His-tagged proteins from recombinant E. coli. Salmonella LPS O-antigen was obtained commercially. We have prepared a S. typhimurium outer membrane protein (SOMP) fraction and a heat killed extract from a galE mutant derived from the wild-type S. typhimurium UK-1 strain Chi3761. This strain grown in the absence of galactose in the medium yields SOMPs uncontaminated with LPS O-antigens. These antigens were used as controls in western blots as well as for immunoassays as described below. We isolated HBV core particles with and without inserted epitopes such as the pre S1, S2 epitopes and also the HBV S antigen to monitor immune responses to these antigens. Synthetic peptides for various T-cell epitopes to use in T-cell proliferation assays were prepared commercially. In some cases, Dr. Mark Jenkins provided the cell free extracts of Eimeria acervulina sporozoite antigens.
ii. ELISA: Serum antibodies are measured in blood collected by retro-orbital bleeding in mice and by veinipuncture in chickens and mucosal antibodies as extracted copro antibodies from feces (chickens) or in vaginal secretions (mice). Sera and secretions were initially pooled from all mice in a group. In later studies with successful constructs, antibody titers were monitored in individual mice. We employ a doubling dilution method with the end point titer being the dilution giving an OD410 three times that for the reagent or unimmunized animal control. SIgA titers against the various antigens were monitored by ELISA (Hassan and Curtiss, Infect. Immun. 62:2027-2036 (1994); Hassan and Curtiss, Infect. Immun. 64:938-944 (1996)) in the same way. Since we are often interested in distinguishing between a Th1 and Th2 response, the titers of IgG1 versus Ig2A are determined. These methods have been described in previous manuscripts (Dogett, et al., Infect. Immun. 61:1859-1866 (1993); Nayak et al., Infect. Immun. 66:3744-3751 (1998); Hassan, et al., Avian Dis. 37:19-26 (1993)).
iii. ELISPOT Analysis: We routinely use ELISPOT analysis (Czerkinsky, et al., J. Immunol. Methods. 65:109-121 (1983)) to determine the titers of circulating cells secreting antibodies that are antigen-specific in human trials and occasionally, in experiments with mice and chickens.
iv. T-cell Proliferation Assays: These are performed on spleenocyte preparations obtained from groups of mice or chickens at one, two and four weeks post-vaccination. Proliferative responses to the nickel column-purified His-tagged antigens are measured by a non-radioactive calorimetric assay (Promega) with which we have had much success. Stimulation with specific peptides containing T-cell epitopes are also employed. Stimulation with S. typhimurium outer membrane proteins and concanavalin A serve as controls.
v. CTL Assays: CTL responses to T-cell epitopes are quantitated using the appropriate murine cell lines (P815 or EL-4) transfected with plasmids (Wu and Kipps, J. Immunol. 159:6037-6043 (1997); Denis et al., Infect. Immun. 66:1527-1533 (1998)) encoding the epitopes to serve as targets for assays of BALB/c or C57BL/6 immunized mice. Effector cells are obtained from spleens of non-immunized and immunized mice. Part of the vector cell population was used immediately in a Cytotox 96 non-radioactive CTL assay (Promega). The remaining cells are re-stimulated with the appropriate antigen for five to seven days prior to use in the CTL assay. The CTL assay measures lactate dehydrogenase released due to lysis of target cells. The values obtained are used to calculate the percentage of target cells that lyse relative to the quantity of effector cells added. A 51Cr release assay can also be used (see below).
CTL responses to Eimeria sporozoite and merozoite antigens are quantitated using syngeneic and allogeneic primary chicken kidney target cells are infected with a Simbis virus vector expressing the relevant sporozoite or merozoite antigen. The target cells are labeled with 200-300 microCi of Na2 51CrO4 for 90 min, washed three times and suspended in assay medium at a final concentration of 2×105 cells/ml (99, 100). Effector cells are obtained from spleens of Eimeria-infected unimmunized chickens as well as from chickens immunized with attenuated Salmonella delivering a DNA vaccine. The CTL assay measures 51Cr released due to lysis of target cells and values obtained were used to calculate the percentage of target cells lysed relative to quantity of effector cells added. Mark Jenkins provided Eimeria oocysts for infections.
g. Statistical analysis. The results were analyzed using the appropriate statistical test from the SAS program to evaluate their relative significance.
Example 1 demonstrates a method to construct bacterial strains with markerless deletion mutations. In vaccine strains for animals and humans, gene information specifying resistance to antibiotics is not preferred. Accordingly, we devised strategies for making recombinant vaccines that use a balanced-lethal host-vector system that did not rely on use of antibiotic resistance determinants (Nakayama et al., Bio/Technol. 6:155-160 (1988)). Further, we devised strategies using suicide vectors to introduce markerless deletion mutations into the chromosome of bacterial vaccine strains. This has been accomplished by first generating a deletion mutation for a specific gene or genes using PCR methodology. A fragment of DNA possessing the defined deletion is then cloned into a suicide vector. These suicide vectors generally possess genes for antibiotic resistance, such as for tetracycline, chloramphenicol and/or ampicillin, a gene that confers sensitivity to the sugar sucrose to cells that possess the suicide vector, a sequence enabling mobilization of plasmid transfer to a recipient cell due to the presence of a chromosomally integrated IncP plasmid in the donor bacterium, and the R6K ori that enables replication only in bacterial host cells possessing the pir gene inserted into the chromosome (Miller and Mekalanos, J. Bacteriol 70:2575-2583 (1988)). The suicide vector was generally maintained in the suicide vector donor strain MGN-617 that possesses the lambdapir and IncP conjugative plasmid in the chromosome and a Deltaasd mutation that enables selection against it on agar medium lacking DAP. Mating of MGN-617, which possesses the suicide vector, with a suitable recipient was conducted on L agar containing DAP and an antibiotic to which the suicide vector confers resistance. Suicide vectors that integrated into the chromosome by a homologous reciprocal crossover event between sequences flanking the deleted gene on the suicide vector and homologous sequences on the bacterial chromosome survived. The mating mixture was then transferred to L agar containing the same antibiotic but lacking DAP which lead to the death of the donor cells. Antibiotic-resistant isolates were selected and grown as small cultures that were appropriately diluted and then plated on Luria agar with and without 5% sucrose. Sucrose-resistant isolates were screened for a phenotype associated with the presence of the defined markerless deletion mutation, such as for a requirement for DAP. In cases in which a discernible phenotype is unavailable, DNA from sucrose-resistant isolates was evaluated by PCR to identify isolates that possess the deletion of the gene in question. A wild-type strain served as a control. Table 1 lists many bacterial strains with markerless defined deletion mutations. Table 2 lists the suicide vectors for their introduction into these strains or into strains not yet possessing the defined deletion mutation.
Example 2 shows the generation of suicide vectors for introduction of defined deletion and deletion-insertion mutations to contribute desirable features to bacterial strains to display regulated lysis for delivery of DNA vaccine vectors or attenuated viral vaccines or for release of antigens.
In order to develop a host-vector system with regulated delayed lysis to use in the immunization of humans, in those cases in which the DNA sequence for S. typhi and/or S. paratyphi A gene in question and its flanking sequences are different than in S. typhimurium, we construct suicide vectors to introduce defined deletion and deletion-insertion mutations into the chromosomes of S. typhi and S. paratyphi A This is the case for the asd gene and its flanking sequences. We designed suicide vectors for introducing DeltaasdA33 and DeltaasdA183::TT araC PBAD c2 mutations into the chromosomes of S. typhi and S. paratyphi A (see
The DeltaaraBAD1923 mutation (
As depicted in
When the host strain possesses a mutation that inactivates the periplasmic endonuclease I enzyme, recovery of circular plasmid DNA is improved and the efficiency of plasmid transfer to a host strain by either transformation or electroporation is enhanced. It therefore follows that the integrity of DNA vaccine vectors released from bacteria undergoing regulated lysis in vivo would be superior from strains lacking endonuclease I. Thus, we designed and constructed the suicide vector pYA3652 for the introduction of the Deltaend42311 mutation (
Bacteria experiencing perturbations in the synthesis of their cell walls, due to either inhibition of cell wall synthesis by antibiotics or by metabolic limitations as a result of mutational alterations, frequently respond to this type of stress by synthesizing the extracellular polysaccharide colanic acid. This behavior was observed in the design of Escherichia coil K-12 bacterial strains to exhibit biological containment for safe conduct of recombinant DNA research. Accordingly, we introduced mutations, present in Chi1776, to preclude colanic acid synthesis that could enable bacterial cell to survive, while undergoing death by lysis due to the presence of asd4 and dapD mutations, which block DAP biosynthesis that result in DAP-less death by cell lysis. We, therefore, have designed and generated deletion mutations that would block the conversion of GDP-mannose to GDP-fucose. GDP-fucose is necessary for the synthesis of colanic acid since fucose represents one-third of the sugar mass of colanic acid.
Bacterial strains that undergo lysis as a result of their inability to synthesize the rigid layer of the bacterial cell wall would cease to lyse if protein synthesis is arrested for any reason. To uncouple this interdependence for continued protein synthesis for lysis to occur, the relA gene was inactivated by mutation. Although protein synthesis within bacteria in vivo is not likely to impair DNA vaccine vector delivery by lysis, the bacterial strain might encounter some environments, following excretion in feces, for example, in which protein synthesis might be inhibited. In this case, a DNA vaccine or antigen delivery host-vector system might survive. Thus, inclusion of a DeltarelA1123 mutation would uncouple the dependence of lysis on protein synthesis and ensure the ultimate lysis and death of the DNA vaccine vector delivery host strain.
The lysis of Gram-negative bacteria in vivo leads to the release of lipopolysaccharide (LPS) that contains endotoxin due to the presence of lipid A. Although lipid A serves as an adjuvant to stimulate the innate immune system to produce interferon gamma, which enhances the induction of a desired cellular immune response, this inflammatory response can also induce fever and have an adverse consequence on an immunized animal or human host. There are numerous mutations that can alter the myristillation of lipid A with alterations of attached fatty acids to alter the degree of toxicity (Raetz and Whiffeld, Annu. Rev. Biochem. 71:635-700 (2002)). We chose to delete the msbB gene to decrease LPS toxicity without altering the virulence of the Salmonella strain possessing this mutation.
A goal in the delivery of a DNA vaccine vector by a bacterial strain exhibiting regulated lysis would be to use a bacterial strain that would efficiently deliver the DNA vaccine vector to stimulate a strong desired immune response to the antigen specified within the eukaryotic expression cassette in the DNA vaccine vector and to diminish the immune responses induced to the bacterial carrier strain. Since the flagellar antigens of Salmonella induce a dominant T cell immune response (Cookson and Bevan, J. Immunol. 158:4310-4319 (1997)) and their absence has no influence on Salmonella invasiveness to lymphoid tissues or virulence (Lockman and Curtiss, Infect. Immun. 60:491-496 (1992)), we generated the DeltafliC825 (
One means to enable Salmonella to escape from the endosome is to delete the sifA gene (Stein et al., Mol. Microbiol. 20:151-164 (1996); Brumell et al, Traffic 2002:3:407-415 (2002)).
D-alanine is an essential constituent of the rigid layer of the cell wall in all bacteria and is incorporated into the peptidoglycan as D-alanyl-D-alanine. After covalent linkage to the L-alanine-D-glutamate-DAP tripeptide that is attached to the muramic acid-N-acetyl-glucosamine carbohydrate backbone of the murein layer, the terminal D-alanine is cleaved off to create the crosslink for the next muramic acid-N-acetyl-glucosamine chain. The synthesis of D-alanine in gram-negative bacteria is carried out by two genes, air encoding alanine racemase, which interconverts D-alanine and L-alanine, and dadB which encodes a catabolic enzyme that enables metabolism of D-alanine as a carbon source but which can synthesize D-alanine when acting in the reverse reaction. We constructed the suicide vector pYA 3667 (
Alternatively, the Deltaalr-3 and DeltadadB4 mutations can be introduced into the chromosome of a host-vector strain to enable use of an Alr+ or DadB+ plasmid to specify synthesis of an antigen that can be delivered to an immunized host. Various uses of this system are described in subsequent Examples to facilitate immunization of individuals with multiple antigens to induce immunity to a pathogen, to accomplish induction of infertility, or to develop a therapeutic vaccine against a particular cancer. The delivery of the different antigens by such a combination host-vector strain can be designed to stimulate specific types of mucosal, systemic and/or cellular immunities.
Example 3 demonstrates the virulence and invasive properties of S. typhimurium strains with single mutations that are or can be included in strains exhibiting regulated lysis for DNA vaccine delivery and/or antigen release.
Table 5 provides data from a number of determinations of the relative virulence of S. typhimurium strains. Strains are grown in LB broth to an OD600 of ˜0.8, concentrated in BSG with 20 microl samples administered orally to mice that had been fasted for food and water for about 4 h. Food and water were returned 30 min after infection. The LD50 for the wild-type S. typhimurium UK-1 strain Chi3761 is about 5×104 based on multiple experiments. Strains such as Chi8276 and Chi8290 that possess Deltaasd mutations are total avirulent independent of dose, a result supported in the literature. The DeltaPmurA7::araC PBAD murA mutant strain Chi8645 caused some lethal infections in one experiment but no deaths in another (Table 5). Chi8897 with the DeltadadB4 mutation and Chi8898 with the Deltaalr-3 mutation have the same virulence and LD50 as the wild-type parent Chi3761. Chi8899, on the other hand, is totally avirulent when used to orally inoculate mice with or without supplementing D-alanine or D-alanyl-D-alanine in the drinking water (Table 5). S. typhimurium strains with the DeltaaraBAD1923, DeltaaraE25, DeltaendA2311, Deltagmd-11, Delta(gmd-fcl)-26, DeltarelA4, DeltarelA1123, DeltamsbB48 and DeltafliC825 DeltafljB217 mutations all display virulence that is essentially the same, considering statistical noise, as that exhibited by the wild-type parents of these strains, Chi3761 and Chi3339 (Table 1). In regard to mutations that delete genes for arabinose utilization, numerous strains of Salmonella including S. typhi are unable to ferment or utilize arabinose with no impact on virulence.
Example 4 shows construction of the DNA vaccine vector delivery strain Chi8854.The S. typhimurium UK-1 strain Chi8645 with the DeltaPmurA7::araC PBAD murA deletion-insertion mutation, which was derived from the wild-type strain Chi3761, was the starting strain. This strain is arabinose-dependent, so we added 0.1% arabinose to LB and L broth or agar media because they both contain yeast extract which contains some arabinose. The suicide vector pYA3485 (
We next introduced the DeltaaraBAD1923 mutation by mating Chi8653 with MGN-617 (pYA3484). The suicide vector pYA3484 (
We next introduced the DeltaasdA19::TT araC PBAD c2 deletion-insertion mutation into Chi8655. DAP was present during all steps. To do this, the pMEG-611 suicide vector (
We next introduced the DeltaendA2311 mutation. The suicide vector pYA3652 (
Example 5 shows modifications of Chi8854 to provide features to further ensure complete lysis of the host-vector system. Either the Deltagmd-11 or Delta(gmd-fcl)-26 mutation was introduced using Chi8829 and pYA3628 (
We next introduced the DeltarelA1123 mutation present in Chi8882 into Chi8885 using the suicide vector pYA3679 (
Example 6 shows further modifications of strain Chi8854 or strain Chi8888 to enhance efficacy and safety as a DNA vaccine vector delivery host.
A diversity of genotypic alterations of Chi8854 and/or Chi8888 is possible using available strains with defined deletion mutations as listed in Table 1 and suicide vectors as listed in Table 2. These constructions make use of the transductional method of strain construction with markerless deletion mutations as described in Examples 1 and 2. The DeltamsbB48 mutation changes the myristillation of fatty acids on lipid A of LPS and decreases the toxicity of the LPS endotoxin. This may be important if the release of endotoxin due to cell lysis in vivo causes adverse symptoms. The presence of the DeltamsbB48 mutation is without effect on the virulence of S. typhimurium (see Table 5). Using the suicide vector pYA3529 (
If additional sustained growth in vivo prior to onset of lysis is beneficial, the DeltaaraBAD]923 mutation in Chi8888 can be replaced with the DeltaaraBAD23 c2::rrfG TT mutation using the suicide vector diagrammed in
The DeltafliC825 mutation in Chi8600 can be introduced into Chi8888 using the suicide vector pYA3547 (
The use of additional plasmid vectors to specify synthesis or delivery of additional antigens that encode the genetic information for the regulated delayed lysis phenotype can be developed using the alr+ or dadB+ wild-type genes in a bacterial strain that have both the Deltaalr-3 and DeltadadB4 mutations. Such a host-vector combination ensures the stable maintenance of the plasmid vector in the host-vector strain in vivo. The P22-mediated transduction system described in Example 1 is used to transfer the Deltaalr-3 mutation from Chi8898 (Table 1) through the suicide vector pYA3667 (
Example 7 shows construction of strains derived from S. typhi and S. paratyphi A to display regulated lysis for the delivery of either DNA vaccine vectors or protective antigens to humans.
The construction of host-vector systems for vaccination of humans would preferably use host-adapted invasive strains of Salmonella such as either S. paratyphi A or S. typhi. We would use the S. paratyphi A strain Chi8387, which is derived from ATCC 9281 by curing of a low molecular weight Col-like plasmid that interferes with replication of cloning vectors with pBR ori or pUC ori. The S. typhi strains would either be Chi8438, an RpoS+ derivative of S. typhi Ty2, or the S. typhi ISP1820 strain Chi3744 which also has the RpoS+ phenotype. We have previously shown that the RpoS+ phenotype is important for initial colonization of the GALT and is therefore necessary for high immunogenicity. These wild-type human-virulent Salmonella strains can be genetically modified in the same manner using the methods described in Example 1 and used for the construction of Chi8858 (Example 5) and further gene modifications as described in Example 6. One exception is the absence of deleting genes for arabinose breakdown in S. typhi since it lacks these genes but it may still be necessary to delete the araE gene.
As already described in Examples 2 and 4, we designed Deltaasd and Deltaasd::TT araC PBAD c2 deletion and deletion-insertion mutations (
The deletion and deletion-insertion mutants (e.g. Chi8888 or its derivatives as identified and described in Examples 2-7) and the plasmid vectors described in the following Examples together can impart, a regulated delayed lysis phenotype which can be used to provide attenuation and biological containment properties to constructed derivatives of numerous bacterial pathogens, such as strains of Yersinia, (e.g. Y. pestis), strains of Shigella, (e.g. S. flexneri), strains of E. coli, strains of Francisella, (e.g F. tularensis), strains of Listeria, (e.g. L. monocytogenes), and other bacterial strains that are naturally invasive or genetically modified to be invasive in an immunzed individual. Table 1 lists strains of bacteria that can be used for these constructions. Occasionally, the constructions need to be modified to accommodate differences in nucleotide sequences, which a person of ordinary skill in the art can readily perform. These same bacterial strains can be designed to serve as hosts for the delivery of antigens synthesized by the host-vector strain, for delivery of a genetic vaccine to specify synthesis of antigens by the immunized individual, or for delivery of attenuated viral genomes as an anti-virus vaccine.
Example 9 shows the steps toward eventual construction of plasmids to enable regulated lysis of bacterial strains for DNA vaccine delivery and/or protective antigen release. As stated in the introduction, the plasmid vectors pYA3450 (
To construct pYA3607, isolated plasmid pMEG-104 (
Example 10 shows construction of pYA3646, which allows the S. typhimurium cells to exhibit arabinose-dependent growth and to lyse after several cell divisions in the absence of arabinose.
To eliminate the P22 c2 gene fragment in pYA3608 (
Example 11 shows construction of a system with regulation of two genes encoding enzymes for essential constituents of the rigid layer of the bacterial cell wall. We had already determined that the DeltaPmurA7::araC PBAD murA mutation (
The araC PBAD activator-promoter on the plasmids permits a low basal level of transcription in the absence of arabinose. (Guzman et al., J. Bacteriol. 177:4121-4130 (1995)). This sequence is derived from E. coli B/r. We used computer search analyses to examine sequences of many araC PBAD activator-promoters in various gram-negative bacteria and used PCR to amplify the sequence selected from E. coli K-12 strain Chi289 (Table 1). This is diagrammed in
We further synthesized and evaluated various combinations of asd and murA genes with ATG, GTG and TTG start codons. Little or no difference was observed with either TTG or GTG start codons. Both enabled us to construct plasmids that conferred arabinose-dependant growth and strains that could undergo a suitable number of cell divisions in media without arabinose.
Example 12 shows construction of pYA3647. To enhance regulated lysis, we used a regulated araC PBAD activator-promoter, the synthesis of anti-sense mRNA and alteration in the start codons for the murA and asd genes from ATG to GTG. Since the asd gene is distal from the PBAD promoter (the murA gene is proximal), it is likely transcribed less well than the murA gene. The asd gene is also proximal to the P22 PR. Vectors with an SD-ATG asd provides similar results in regulated lysis experiments as in animals.
Example 13 shows construction of pYA3650. The DNA vaccine vector pYA3650 (
To establish a balanced-lethal host-vector system, the drug-resistance marker present in pVAX1 was replaced with a regulatable araC PBAD asd cassette (see
In the absence of the inducer arabinose, AraC is capable of activating PBAD transcription to about 1% of the induced level when using the E. coli B/r araC PBAD sequences. The transcriptional level of asd under the regulation of the B/r PBAD in pYA3611 without inducer resulted in sufficient Asd to permit construction of balanced-lethal systems with strains such as Chi8289 (Table 1) which could grow in the absence of either arabinose or DAP. A new araC PBAD fragment from E. coli K-12 strain Chi289 that is present in pYA3624 (
We introduced the murA sequence derived from E. coli K-12 strain Chi289 into pYA3614 to enhance the arabinose-dependent growth attributes of strains with the plasmid vector. As diagrammed in
Example 14 shows construction of pYA3651. To generate a DNA vaccine vector with the SD-ATG asd sequence, pYA3614 (
CpG sequences (Krieg, Annu. Rev. Immunol. 20:709-760 (2002)) can enhance the immunogenicity of DNA that are present in bacterial but not in mammalian DNA. These immune enhancing unmethylated CpG sequences act like adjuvants. The DNA vaccine vectors pYA3650 and pYA3651 have a higher number (15) of such sequences to stimulate immune responses in both mice and humans than commercially-available vectors such as pVAX1. Table 6 lists the 15 immune -enhancing CpG sequences in pYA3650 and pYA3651.
*GTCGTT motif is optimal for stimulation of lymphocyte proliferation in several species, including cattle, sheep, goats, horses, pigs, dogs, cats and chickens.
Example 15 shows the influence of ATG, GTG, and TTG start codons in the asd gene on synthesis of Asd protein in S. typhimurium and E. coli K-12.
Example 16 shows phenotypic properties of recombinant host-vector strains displaying arabinose-dependant growth and regulated cell lysis in the absence of arabinose. As a routine procedure, bacterial strains with or without plasmids were grown overnight at 37° C. on a roller drum in LB broth supplemented with 0.1% arabinose and with 50 microg DAP/mi, if necessary. These cultures were diluted 1:1000 in BSG and plated on Minimal agar supplemented with lysine, threonine and methionine (to satisfy the nutritional requirements, other than for DAP, due to the asd mutation) and 0.4% glycerol which were supplemented with (a) 50 microg DAP/ml plus 0.1% arabinose, (b) 0.1% arabinose with no DAP, (c) 50 microg DAP/ml with no arabinose, and (d) without DAP and arabinose but with 0.1% glucose. Plates were incubated at 37° C. for up to four days, with growth or no growth usually recorded after 48 h of incubation. Results of these tests are presented in Table 7.
*++: growth +: growth slowly +/−: very poor growth −: no growth
Chi8854 containing any of the plasmids with the B/r araC PBAD sequence (prior to introducing the E. coli K-12 araC PBAD sequence present in pYA3624) demonstrated arabinose-independent growth and grew on medium (c). In general, lysis plasmids with the lower copy number p15A ori demonstrated arabinose-dependant growth except for those plasmids such as pYA3613 and pYA3643 that possessed SD-ATG murA genes (Table 7). Chi8854 with either pYA3646 or pYA3647 was unable to grow on medium without arabinose independent of the presence of DAP. For the higher copy number DNA vaccine vectors, the most stringent arabinose-dependent growth was observed with pYA3650 in Chi8854. This plasmid has both SD-GTG murA and SD-GTG asd genes. When the plasmid, such as pYA3615 or pYA3649, has the SD-ATG murA sequence poor growth is observed in the absence of arabinose. Collectively, these results lead one to conclude that the chromosomal DeltaPmurA7::araC PBAD murA mutation-insertion is somewhat leaky and permits very low-level transcription in the absence of arabinose. To remedy this minor problem, we can readily substitute the non (less) leaky E. coli K-12 Chi289 araC PBAD sequence in place of the E. coli B/r sequence in the DeltaPmurA7::TT araC PBAD murA construction using pYA3624 (
Example 17 shows the virulence and colonizing ability of recombinant strains with DNA vaccine vectors in mice.
Bacteria were grown in Luria broth supplemented with 0.2% arabinose to OD600 of about 0.8. Bacterial cells were collected by centrifugation and suspended in buffered saline with gelatin (BSG) for oral inoculation of mice. Morbidity and mortality were observed for 30 days. Surviving mice were challenged 30 days after the initial inoculation with virulent wild-type UK-1 Chi3761 grown in Luria broth. Morbidity and mortality observations were recorded daily for an additional 30 days post challenge. Both inoculating and challenge doses were measured in CFU.
Genotype of Strains:
Chi8804 (DeltaaraE25, DeltaasdA19::TTaraC PBAD c2 5ST1T2,DeltaPmurA7::araC PBAD murA).
The results in Table 8 reveal that the recombinant strains are totally attenuated and cause no deaths when the inoculating doses were 10,000 times what would be a lethal dose for the wild-type Chi3761 strain. Partial protective immunity against subsequent challenge is apparent with some strains but not with the arabinose-utilizing strain Chi8806, at least at the high challenge dose used. This lower immunogenicity can be due to rapid breakdown of arabinose to cause cell wall-less death to commence more rapidly to induce less of an immune response to Salmonella antigens. These results are very desirable, however, since the objective is to induce maximal immune responses to antigens specified by the DNA vaccine vector or released by lysing Salmonella cells. Some immunity to Salmonella is a plus but is not the objective and a very strong induction of immunity to Salmonella antigens could compete in the induction of desired immune responses to specified or delivered protective antigens.
Table 9 presents data demonstrating that Chi8888, Chi8888(with pYA3650) and Chi8888(with pYA3651) were avirulent when orally administered at high doses to female BALB/c mice. Chi8888 with either pYA3650 or pYA3651 conferred a modest level of immunity to challenge with the wild-type virulent S. typhimurium UK-1 strain Chi3761, at a dose 10,000 times its mean lethal dose (Table 5). In contrast, Chi8888 without a vector did not induce a detectable level of immunity to challenge.
MDD*: Mean number of days to death
Colonization of 8-week old female BALB/c mice orally inoculated with Chi8854 (pYA3650) was investigated. The results presented in Table 10 indicate that the constructed DNA vaccine delivery host-vector systems are able to transiently colonize lymphoid tissues but at very modest numbers. These results probably represent a significant underestimation of the bacterial titers achieved in various tissues. Since these bacterial strains are being stressed by being caused to lyse in vivo, it is likely that cells are probably killed in the course of tissue maceration, vortex mixing, dilution and plating by spreading on plates. We have observed this problem before, as have others. We therefore decided to evaluate a clinical parameter indicative of systemic infection. We thus chose to measure temperatures of mice rectally (Stiles et al., Infect. Immun. 67:1521-1525 (1999); Li et al., Infect. Immun. 70:2519-2525 (2002)) under the expectation that infection and eventual lysis would cause a significant increase in temperature but that little or no temperature increase would be observed if in vivo proliferation and colonization of lymphoid tissues was minimal. We used the commercially available rectal temperature probe YSI 402. As revealed in
Example 18 shows construction of DNA vaccine vectors specifying the Eimeria acervulina sporozoite antigen EASZ240 and the merozoite antigen EAMZ250 with FLAG epitope fusions.
Chi8888 containing pYA3674 that was derived from pYA3650 with the GTG start codons for the asd and murA genes and encodes the Eimeria acervulina EASZ240 sporozoite antigen (
Example 19 shows construction of strains of bacteria with in vivo regulated lysis with gene attributes that enhance exit from the endosome and entry into the cytoplasm of host cells for release and function of genetic vaccines to induce CTL immune responses. We have devised two means to enhance the ability of Salmonella to exit the endosome.
In one example, the Type III effector protein SipB is over-expressed on a plasmid such as that diagrammed in
In another example, a means to cause Salmonella to escape from the endosome is to delete the sifA gene (Stein et al., Mol. Microbiol. 20:151-164 (1996); Brumell et al., Traffic 2002:3:407-415 (2002)).
Example 20 shows construction of vectors to enable regulated lytic release of protective antigens specified by cloned genes and synthesized by the bacterial delivery host. The plasmids pYA3646 (
To evaluate the efficacy of a host-vector system for release of a protective antigen synthesized within the bacterial host prior to lysis in vivo after colonization of lymphoid tissues, we constructed recombinant plasmids to specify synthesis of the S. pneumoniae protective PspA antigen (Kang et al. (2002)). Specifically, we specified the synthesis of the alpha-helical domain of PspA that contained the protective B-cell epitopes fused to the signal sequence for beta-lactamase which caused export of some PspA antigen to the periplasmic space and resulted in enhanced immunogenicity (Kang et al. (2002)). We further used gene splicing techniques to insert a PspA-encoding sequence that had been codon optimized. That is, we selected codons that are used in highly expressed genes in E. coli and Salmonella to result in pYA3712 (
Because expression of protective antigens during growth of a vaccine strain and during the initial phases of infection after orally administration of the vaccine to a host may potentially interfere with successful colonization of the internal lymphoid tissues, we subjected the antigen expression to regulation. Since the Pft promoter, which is used to control expression of the beta-lactamase-PspA fusions in pYA3712 and pYA3713 (
Example 22 shows construction of a recombinant vaccine strain with regulated expression of cloned genes for the hepatitis B virus core with inserted pre S 1, S2 epitopes and regulated in vivo lysis to release the synthesized HBV core pre S1, S2 antigen. We have extensively used the hepatitis B virus core as a particulate antigen produced by attenuated Salmonella typhimurium for mice (Schodel et al., Infect. Immun. 62:1669-1676 (1994)) and in S. typhi for humans (Nardelli-Haefliger et al., Infect. Immun. 64:5219-5224 (1996)). The HBV core gene product self assembles when synthesized in bacteria and the core gene can be engineered to express protective B-cell and T-cell epitopes from various pathogens including HBV preS1, S2 and Plasmodium falciparum circumsporozoite antigens. They can also be used to express protective antigens of Eimeria species.
The HBV surface (S) antigen is a protective antigen present in current commercially-available HBV vaccines. The S antigen is glycosylated and must be synthesized in eukaryotic cells to elicit protective immunity, and a DNA vaccine specifying the HBV S antigen can stimulate immune responses. (Oka et al., Immunology 103:90-97 (2001)). It follows that a DNA sequence encoding the HBV S antigen could be inserted into the DNA vaccine vector pYA3650 (
As described in Example 6, we are able to introduce both the Deltaalr-3 and DeltadadB4 mutations into Chi8888 or a derivative to impose a requirement for D-alanine. An Alr+ or DadB+ plasmid is introduced into the Chi8888 derivative. The Alr+ or DadB+ plasmid contains a replicon that is compatible with the pBR ori and pUC ori plasmids, such as pYA3681 and pYA3682 for synthesis of antigens within the bacterial host or pYA3650 and pYA3651 for synthesis of antigens in the immunized host. This is accomplished by using a mini-F ori, a mini-I-alpha ori, the pSC101 ori or the p15A ori as the replicon for the Alr+ or DadB+ plasmid. The design of Alr+ plasmids with the pSC101 ori and p15A ori is diagrammed in
The inventors contemplate that an improved vaccine against HBV can be made using the HBV core and preS1 and preS2 fusions as the antigen. The fusion is placed in an Alr+ plasmid with the p15A ori. The S antigen derives from pYA3650 (pUC ori). Both plasmids are introduced into a Chi8888 derivative having at a minimum both the Deltaalr-3 and DeltadadB4 mutations. Other desired mutations to provide gene regulation functions and the like as described in the preceding Examples can also be included. This strain would display regulated delayed lysis in vivo after colonization of lymphoid tissues to release a bolus of recombinant HBV core particles, which would be synthesized and assembled in the bacterial host prior to lysis. This would stimulate protective immune responses to the preS1 and preS2 antigens, and the release of the DNA vaccine vector that would direct the synthesis of S antigen within cells in the immunized host to also induce a protective immune response to the S antigen.
The inventors contemplate that the host-vector system of the present invention can also be used to develop a vaccine against M. tuberculosis. The Mtb 39A antigen is a 39 kDa protein belonging to the PPE family of conserved proteins of M. tuberculosis. Human T-cell epitopes have been identified in this protein, and the gene encoding this antigen has been inserted in a DNA vaccine vector, delivered by injection, to elicit protective immune responses in mice (Dillon et al.,Infect. Immun. 67:2941-2950 (1999)) and monkeys. Through PCR methods, we insert the DNA sequence encoding the Mtb 39A antigen with a Kozak sequence into the DNA vaccine vector pYA3650 (
The ESAT-6 antigen and its co-expressed Cfp-10 antigen (and probably their homologs) contain human T-cell epitopes. We used the SPI-1 encoded TTSS of Salmonella to deliver these antigens to the cytoplasm of cells within an immunized individual. This means of delivery leads to MHC class I presentation to enhance induction of a CD8+-dependent CTL response. Sequences encoding ESAT-6 or Cfp-10 are introduced into an Alr+ vector derived from the construct shown in
The host-vector strains with the regulated delayed lysis in vivo phenotype can also be used to deliver attenuated viral vaccines. This can be accomplished by having the entire genome of the attenuated virus cloned into a BAC vector such that the viral genome sequence excises from the BAC vector in the cytoplasm of cells to replicate and express viral antigens to induce protective immunity. This type of construction has been successfully developed for the delivery of a BAC containing an attenuated Marek's disease virus delivered as a DNA vaccine to chickens (Tischer et al., J. Gen. Virol. 83:2367-2376 (2002)). Marek's disease virus vaccination is universally used in the poultry industry. Because currently available BAC vectors are derived from the mini-F plasmid and these plasmids are incompatible with the virulence plasmids of Salmonella, it is necessary to engineer the BAC vector to replace the IncF encoding genes with the IncI encoding genes for IncI-alpha, IncII and IncI-beta from the mini-Rtsl plasmid. In addition, to avoid use of antibiotic resistance genes in live bacterial vaccines, we replace the drug resistance genes prevalent in BAC vectors with the wild-type alr+ gene. The construction of this IncI Alr+ BAC plasmid is diagrammed in
Table 11 shows the bacterial strains and host-vector systems that have been deposited with ATCC.
The examples provided above are for illustrative purposes only, and not to limit the scope of the present invention. In light of the present disclosure, numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art.
This application claims priority to provisional application No. 60/407,522, entitled “Regulated Bacterial Lysis for Genetic Vector Delivery and Antigen Release”, filed on Sep. 1, 2002.
This invention was made with government support under Grant Numbers NIH DE06669 and USDA 099-35204-8572. The government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US03/26883 | 8/29/2003 | WO | 11/15/2005 |
| Number | Date | Country | |
|---|---|---|---|
| 60407522 | Sep 2002 | US |
