Free-living microbes are provided in which the nucleic acid has been modified so that the microbe is attenuated for proliferation and/or which comprise genetic mutations that attenuate the ability of the microbe to repair its nucleic acid. Methods of using the modified microbes for the loading, activation, and/or maturation of antigen-presenting cells are also provided. Vaccine compositions comprising the modified microbes and/or the antigen-presenting cells and methods of using the vaccines are also provided. The microbes may be further modified to include heterologous antigens, such as tumor antigens or infectious disease antigens, for use as a vaccine against cancer or infectious diseases.

Description of the Invention

The present invention involves modified free-living microbes and the use of modified free-living microbes in vaccine compositions, wherein the nucleic acid of the microbe is modified so that proliferation of the microbe is attenuated. In some embodiments, the microbial gene expression is substantially unaffected by the modification. The present invention also involves the use of the modified microbes for antigen loading and induction of the activation/maturation of antigen presenting cells (APCs), in vitro or ex vivo. The antigen may be either an antigen produced naturally by the modified microbe, or may be a heterologous antigen expressed by a recombinant microbe. The resulting antigen presenting cells are suitable for use in vaccine compositions and for immunotherapy. The immune response stimulated by administration of the resulting vaccine compositions may be a CD4.sup.+ or a CD8.sup.+ immune response.

One such modified microbe is Listeria monocytognes. The inventors have engineered Listeria to be particularly sensitive to inactivation by psoralens, a group of compounds that form irreversible cross-links in the genomes of bacteria after illumination with ultraviolet A (UVA) light, so that they are non-viable. (See Example 3, below.) The attenuation of proliferation of wild-type and modified Listeria while maintaining expression of model antigens has now been shown (see Example 1-2 and 11, below). The modified Listeria is also shown to provide an anti-tumor response (Examples 4 and 14-16, below) and induce antigen-specific T-cell responses (Example 5) and in vivo cytotoxic responses (Example 20). Listeria is rapidly phagocytosed by DC and transported into the phagolysosomal compartment. This encounter results in the phenotypic maturation of the DC and subsequent secretion of a broad profile of immunostimulatory cytokines, including IFN-.gamma., IL-12, and TNF-.alpha.. The inventors have now demonstrated that infection of immature DC with recombinant Listeria results in rapid DC activation/maturation, together with MHC class I-restricted presentation of an encoded heterologous antigen. Additionally, degradation of Listeria vaccines within the phagolysosome results in presentation of encoded antigen via the MHC class II pathway. (See Examples, below)

Another such modified microbe is Bacillus anthracis. The inventors have also engineered attenuated strain of Bacillus anthracis which are particularly sensitive to inactivation by psoralens (see Example 21, below).

Accordingly, the invention provides a vaccine comprising a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation. In some embodiments, the attenuation of the proliferation of the microbe is controllable in a dose-dependent manner. In some embodiments, microbial gene expression in the microbe is substantially unaffected by attenuation of the proliferation of the microbe. In some embodiments, the microbe in the vaccine expresses an antigen at a sufficient level to induce an immune response to the antigen in an individual upon administration of the vaccine to the individual. In some embodiments, the nucleic acid has been modified by reaction with a nucleic acid targeted compound which reacts directly with the nucleic acid. In one embodiment, the nucleic acid target compound is an alkylator such as .beta.-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester. In other embodiments, the nucleic acid targeted compound is a psoralen compound (e.g., 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen, also referred to herein as "S-59") activated by UVA irradiation. In some embodiments, the microbe in the vaccine comprises a genetic mutation that attenuates the ability of the microbe to repair its nucleic acid that has been modified. In some embodiments, the microbe is a bacterium, such as Bacillus anthracis or Listeria monocytogenes. In some embodiments, the microbe comprises a heterologous nucleic acid sequence encoding an antigen. In some embodiments, the vaccine further comprises a pharmaceutically acceptable carrier and/or an adjuvant. The invention further provides a method of preventing or treating a disease in a host, comprising administering to the host an effective amount of the vaccine. The invention also provides a method of inducing an immune response in a host to an antigen comprising administering to the host an effective amount of the vaccine, wherein the microbe expresses the antigen.

The invention also provides an isolated mutant Listeria strain, such as a mutant Listeria monoxytogenes strain, comprising a genetic mutation that attenuates its ability to repair its nucleic acid. In some embodiments, the mutant Listeria strain is defective with respect to at least one DNA repair enzyme (such as UvrA and/or UvrB). In some embodiments, the mutant Listeria strain comprises a genetic mutation in the uvrA gene and/or the uvrB gene. In some embodiments, the mutant strain is the Listeria monocytogenes .DELTA.actA/.DELTA.uvrAB strain deposited with the American Type Culture Collection (ATCC) and identified by accession number PTA-5563. In other embodiments, the strain is a mutant of the Listeria monoxytogenes .DELTA.actA/.DELTA.uvrAB strain deposited with the American Type Culture Collection (ATCC) and identified by accession number PTA-5563, wherein the mutant of the deposited strain is defective with respect to UvrA, UvrB, and ActA. The invention further provides vaccines and professional antigen-presenting cells comprising the mutant Listeria strain. Methods of using the modified Listeria strain to induce immune responses and to prevent or treat disease are also provided.

The invention provides an isolated mutant Bacillus anthracis strain, comprising a genetic mutation that attenuates its ability to repair its nucleic acid. In some embodiments, the mutant strain is defective with respect to at least one DNA repair enzyme (such as UvrA and/or UvrB). In some embodiments, the mutant strain comprises a genetic mutation in the uvrA gene and/or the uvrB gene. In some embodiments, the mutant strain is attenuated with respect to RecA. In some embodiments, the mutant strain comprises a genetic mutation in the recA gene. In some embodiments, the mutant strain comprises one or more mutations in the lef gene, cya gene, or both genes, that decreases the toxicity of the strain. The invention further provides vaccines and professional antigen-presenting cells comprising the mutant strain. Methods of using the modified Bacillus anthracis strain to induce immune responses and to prevent or treat disease are also provided.

In addition, the invention provides a professional antigen-presenting cell (e.g., a dendritic cell) comprising a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation. In some embodiments, the attenuation of the proliferation of the microbe is controllable in a dose-dependent manner. In some embodiments, microbial gene expression in the microbe is substantially unaffected by attenuation of the proliferation of the microbe. In some embodiments, the microbe in the vaccine expresses an antigen at a sufficient level to induce an immune response to the antigen in an individual upon administration of the vaccine to the individual. In some embodiments, the nucleic acid has been modified by reaction with a nucleic acid targeted compound which reacts directly with the nucleic acid. In one embodiment, the nucleic acid target compound is an alkylator such as .beta.-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester. In other embodiments, the nucleic acid targeted compound is a psoralen compound activated by UVA irradiation. In some embodiments, the microbe in the vaccine comprises a genetic mutation that attenuates the ability of the microbe to repair its nucleic acid that has been modified. In some embodiments, the microbe is a bacterium. In some embodiments, the microbe comprises a heterologous nucleic acid sequence encoding an antigen. The invention also provides a vaccine comprising the antigen-presenting cell. The invention further provides a method of preventing or treating a disease in a host, comprising administering to the host an effective amount of the antigen-presenting cell. The invention also provides a method of inducing an immune response in a host to an antigen comprising administering to the host an effective amount of the antigen-presenting cell, wherein the microbe expresses the antigen. The invention further provides a method of activating naive T cells ex vivo or in vitro, comprising contacting the naive T cells with the professional antigen-presenting cell under suitable conditions and for a sufficient time to activate the naive T-cells.

The invention provides a method of loading professional antigen-presenting cells with an antigen comprising contacting the professional antigen-presenting cells with a free-living microbe that comprises a nucleic acid sequence encoding the antigen, under suitable conditions and for a sufficient time to load the professional antigen-presenting cells, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation.

The invention also provides a method of activating and/or maturing professional antigen-presenting cells comprising contacting the professional antigen-presenting cells with a free-living microbe that comprises a nucleic acid sequence encoding an antigen, under suitable conditions and for a sufficient time to load the professional antigen-presenting cells, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation.

The invention further provides a method of preventing or treating a disease in a host, comprising the following steps. (a) loading professional antigen-presenting cells with an antigen by contacting the cells with a free-living microbe that comprises a nucleic acid sequence encoding an antigen, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation; and (b) administering an effective amount of a composition comprising the loaded professional antigen-presenting cells to the host.

The invention also provides a method of loading antigen-presenting cells, such as dendritic cells, with an antigen, comprising contacting the cells in vitro or ex vivo with a modified microbe expressing the antigen, under suitable conditions and for a time sufficient to load the antigen-presenting cells.

The invention provides a method of activating and/or maturing antigen-presenting cells comprising contacting the antigen-presenting cells in vitro or ex vivo with a modified microbe under suitable conditions and for a time sufficient to effect activation and/or maturation of the dendritic cells and/or to allow the antigen-presenting cells to mature.

The invention provides a method of inducing an immune response to an antigen, comprising administering to the host an effective amount of an immunogenic composition comprising an antigen presenting cell presenting the antigen, wherein the antigen-presenting cell comprises a modified microbe.

In addition, the invention provides a method of inducing an immune response to an antigen, comprising the following steps: (a) contacting antigen-presenting cells in vitro or ex vivo with Listeria expressing the antigen under suitable conditions and for a time sufficient to load the antigen-presenting cells with the antigen and to effect activation and/or maturation of the antigen-presenting cells; and (b) administering an effective amount of the antigen-presenting cells to the host. In one embodiment, proliferation of the microbe is attenuated.

The invention also provides an ex vivo or in vitro professional antigen-presenting cell comprising a modified microbe, wherein proliferation of the microbe is attenuated.

Additionally, the invention provides a vaccine comprising an antigen-presenting cell, wherein the antigen-presenting cell comprises a modified microbe and a pharmaceutical composition comprising a antigen-presenting cell and a pharmaceutically acceptable carrier, wherein the antigen-presenting cell comprises Listeria.

Microbe-Based Vaccines

The present invention involves modified free-living microbes and the use of modified free-living microbes in a vaccine composition, wherein the nucleic acid of the microbe is modified so that proliferation of the microbe is attenuated. In some embodiments, the microbial gene expression is substantially unaffected by the modification.

It has been observed that killed microbial vaccines are often inferior to live attenuated microbial vaccines [Lauvau et al., Science 294:1735-1739 (2001)]. In completely killed microbes, the de novo microbial gene expression is essentially eliminated. Therefore, the modification of the microbial nucleic acid to an appropriate level such that proliferation is attenuated while maintaining a sufficient level of microbial gene expression may be more effective than a killed microbial vaccine and provides an approach to vaccine preparation that can be applied to any microbial vector, whether the vaccine targets the prevention of infectious disease caused by the microbial vector, or the vector is used to deliver a heterologous antigen. It is to be understood that the use of the term microbes as it relates to all embodiments of the present invention is intended to mean free-living microbes and is not intended to include viruses. Such a microbe-based vaccine may be used to deliver a specific antigen to an individual. In one embodiment, the vaccine delivers more than one antigen. Such vaccines are designed to stimulate an immune response to one or more antigens, resulting in an individual who is immunized against the antigen or antigens. The immune response that is generated can be either an antibody mediated response, a cell mediated response, or both. The term vaccine is intended to encompass a preventative vaccine, i.e. one that is given to stimulate an immune response so that if the individual subsequently is exposed to the antigen in nature, the pre-formed immune response will increase the individual's ability to fight off the agent or cells carrying the antigen. The term vaccine is also intended to encompass a therapeutic vaccine, i.e. one that is given to an individual who already has a disease associated with the vaccine antigen, wherein the vaccine can elicit an immune response or boost the individual's existing immune response to the antigen to provide an increased ability to fight the agent or cells carrying the antigen. This includes an immune response to a diseased cell, such as a cancer cell, as well as an immune response to a disease associated protein such as a prion. In one embodiment, the free-living microbe is selected from the group consisting of bacteria, protozoa, and fungi. In one embodiment, the free-living microbe is a bacteria selected from the group consisting of Gram positive bacteria, Gram negative bacteria, intracellular bacteria and mycobacteria. The present invention includes various levels of modification of the nucleic acid of microbes. It is understood that the metabolism of the microbial nucleic acid occurs in several ways. Replication of the microbe involves the copying of the DNA of the entire microbial genome in order to replicate the microbe and the subsequent partitioning of the DNA molecules into separate cells, i.e. the cell divides with the resulting cells both having a complete copy of the DNA of the microbial genome. Microbial nucleic acid metabolism also involves the combination of transcription of DNA into RNA and translation of RNA to produce proteins. The transcription of the microbial genome involves the copying of portions of the DNA of the microbial genome into RNA, either messenger or transfer RNA. The translation of the messenger RNA involves the reading of this RNA in order to produce a specific protein or portion of a protein. In the present invention the nucleic acid of a population of microbes is modified to a desired extent based upon the nature of the microbe and its intended use. In some embodiments, the desired extent of modification is such that replication of the microbe's genome is significantly attenuated while the production of proteins remains sufficiently active (i.e. the microbe is metabolically active). It is to be understood that whatever the nature of the modification, the level of modification can be represented in terms of the number of modifications on average per base pair of the microbial genome. For example, if the modification is due to covalent binding of a compound to the nucleic acid (adducts), the modification can be represented in terms of the average number of base pairs between adducts. The microbes of the invention can be modified to levels of about 1 modification per 10.sup.4-10.sup.8 base pairs, also about 1 modification per 10.sup.4-10.sup.7, also about 1 modification per 10.sup.5-10.sup.7, or about 1 modification per 10.sup.5-10.sup.6 base pairs. In one embodiment, the level of modification is adjusted to the minimum amount required to block DNA replication in the microbial population, such that the population shows no observable proliferation, while maintaining sufficient activity of transcription and translation of individual genes (i.e. maintains some metabolic activity) to achieve a safe and effective vaccine.

In one aspect, the invention provides a vaccine comprising a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation. In some embodiments, the attenuation of the proliferation of the microbe is controllable in a dose-dependent manner. In some embodiments, microbial gene expression in the microbe is substantially unaffected by attenuation of the proliferation of the microbe. In some embodiments, the microbe in the vaccine expresses an antigen at a sufficient level to induce an immune response to the antigen in an individual upon administration of the vaccine to the individual. In some embodiments, the nucleic acid has been modified by reaction with a nucleic acid targeted compound which reacts directly with the nucleic acid. In one embodiment, the nucleic acid target compound is an alkylator such as .beta.-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester. In other embodiments, the nucleic acid targeted compound is a psoralen compound (e.g., 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen, also referred to herein as "S-59") activated by UVA irradiation. In some embodiments, the microbe in the vaccine comprises a genetic mutation that attenuates the ability of the microbe to repair its nucleic acid that has been modified. In some embodiments, the microbe is a bacterium, such as Bacillus anthracis or Listeria monocytogenes. In some embodiments, the microbe comprises a heterologous nucleic acid sequence encoding an antigen. In some embodiments, the vaccine further comprises a pharmaceutically acceptable carrier and/or an adjuvant. The invention further provides a method of preventing or treating a disease in a host, comprising administering to the host an effective amount of the vaccine. The invention also provides a method of inducing an immune response in a host to an antigen comprising administering to the host an effective amount of the vaccine, wherein the microbe expresses the antigen.

The invention further provides vaccines comprising a mutant Listeria monocytogenes strain or a mutant Bacillus anthracis strain, wherein the mutant Listeria monocytogenes strain or Bacillus anthracis strain comprises a genetic mutation that attenuates its ability to repair its nucleic acid.

Antigen-presenting Cell Vaccines.

The present invention involves modified free-living microbes and the use of modified free-living microbes in the preparation of vaccine compositions based on antigen-presenting cells, wherein the nucleic acid of the microbe is modified so that proliferation of the microbe is attenuated. In some embodiments, the microbial gene expression of the modified microbe is substantially unaffected.

In one embodiment of the invention, the antigen-presenting cells used in the vaccines are professional antigen presenting cells. Professional antigen-presenting cells include macrophages, dendritic cells and B cells. Other professional antigen-presenting cells include monocytes, marginal zone Kupffer cells, microglia, Langerhans' cells, interdigitating dendritic cells, follicular dendritic cells, and T cells. In one embodiment, the professional antigen-presenting cells are dendritic cells. In another embodiment, the professional antigen-presenting cells are macrophages or dendritic cells (DCs). In one embodiment the antigen-presenting cells are human cells.

In one embodiment, immature antigen-presenting cells, such as DCs, are isolated from a patient and infected with a modified microbe expressing an antigen. The resulting, loaded, antigen-presenting cells are then transferred back into the patient as an autologous APC vaccine, thereby inducing either a CD4+ or a CD8+ immune response.

Accordingly, one example of a method of preparing and using an antigen-presenting cell vaccine of the invention is as follows: Immature DCs are isolated from colon cancer patients and infected with S-59/UVA-inactivated, non-viable, metabolically active recombinant Listeria-CEA vaccines. DC Infection with Listeria results in efficient loading of CEA tumor antigen into the MHC class I and II pathways. Listeria infection stimulates DC to undergo rapid activation and maturation, critical for DC to become potent APCs capable of inducing primary T cell responses in vivo. Mature DC upregulate the expression of CD83, co-stimulatory molecules such as CD80, CD86, as well as MHC molecules. Listeria vaccine-loaded DCs are washed and infused back into the patient as an autologous DC vaccine to stimulate a CEA-specific T cell response.

Particular embodiments are exemplified in the specific Examples listed below. It is understood, however, that the general methods and techniques described herein may be more broadly applied to a wide variety of modified microbes, antigens, and diseases. One of ordinary skill in the art will be able to readily adapt the teachings described herein.

In an alternative embodiment, immature antigen-presenting cells, such as DCs, are infected in vitro with a modified microbe expressing an antigen. The resulting, loaded, antigen-presenting cells are then used to prime a T-cell population which is then transferred into the patient, thereby inducing either a CD4+ or a CD8+ immune response to the antigen.

In another aspect, the invention provides a professional antigen-presenting cell (e.g., a dendritic cell) comprising a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation. In some embodiments, the attenuation of the proliferation of the microbe is controllable in a dose-dependent manner. In some embodiments, microbial gene expression in the microbe is substantially unaffected by attenuation of the proliferation of the microbe. In some embodiments, the microbe in the vaccine expresses an antigen at a sufficient level to induce an immune response to the antigen in an individual upon administration of the vaccine to the individual. In some embodiments, the nucleic acid has been modified by reaction with a nucleic acid targeted compound which reacts directly with the nucleic acid. In one embodiment, the nucleic acid target compound is an alkylator such as .beta.-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester. In other embodiments, the nucleic acid targeted compound is a psoralen compound activated by UVA irradiation. In some embodiments, the microbe in the vaccine comprises a genetic mutation that attenuates the ability of the microbe to repair its nucleic acid that has been modified. In some embodiments, the microbe is a bacterium. In some embodiments, the microbe comprises a heterologous nucleic acid sequence encoding an antigen. The invention also provides a vaccine comprising the antigen-presenting cell. The invention further provides a method of preventing or treating a disease in a host, comprising administering to the host an effective amount of the antigen-presenting cell. The invention also provides a method of inducing an immune response in a host to an antigen comprising administering to the host an effective amount of the antigen-presenting cell, wherein the microbe expresses the antigen. The invention further provides a method of activating naive T cells ex vivo or in vitro, comprising contacting the naive T cells with the professional antigen-presenting cell under suitable conditions and for a sufficient time to activate the naive T-cells.

Attenuation of Microbial Replication.

The present invention involves the modification of microbial nucleic acid in order to attenuate replication of the microbe. This attenuation in replication can be used to increase the level of safety upon administration of the microbes to individuals. The ability of a microbe to proliferate can be measured by culturing a population of microbes under conditions that provide normal growth. The normal growth of a population of microbes is considered to be the growth of microbes having no modifications to the nucleic acid of the microbe. The modification of the microbial genome will result in some attenuation so that the microbe will not undergo normal growth. Some microbes will form colonies that can be counted on solidified growth medium. Attenuation of the replication of the microbe can thus be measured as a reduction in the number of colony forming units (CFU). A stock solution of the microbe colony will be serially diluted until the number of colony forming units can be easily measured (e.g. 50-500 CFU). Typically, dilutions are 10-fold and the number of colonies counted for one or more of the diluted samples is used to estimate the log titer of the sample. For example, an aliquot of diluted microbe stock is plated on growth media and the resulting colonies are counted. The colony forming units per mL (CFU/mL) of the dilution is calculated, and the colony forming units per mL of the original stock (known as the titer) is calculated from the dilution. The log number is known as the log titer. As an example, 24 colony forming units on plating a 0.2 mL aliquot of a 1.times.10.sup.5 dilution gives a 1.2.times.10.sup.7 titer, or 7.08 log titer stock. The attenuation can be measured as the comparison of microbial titer prior to modification of the microbial nucleic acid to that after modification of the microbial nucleic acid. The log of the ratio of the titer of unmodified microbe to the titer of microbe after modification represents the log attenuation (or simply the difference in log titer of the two). For example, if an unmodified microbe titer measures 1.2.times.10.sup.7 and a modified microbe titer measures 4.3.times.10.sup.2, the resulting level of attenuation is 4.45 log. This method can be used to assess the attenuation of any microbe, whether pathogenic or non-pathogenic. For some microbes, rather than measuring the growth of the microbe directly, a plaque assay that measures the microbe by its ability to kill infected cells can be used. For example, certain intracellular bacteria can be grown on a lawn of mammalian cells that it can infect. After appropriate incubation conditions, the lawn can be observed for plaques (clear areas in the cell layer that represent killed cells). The above calculations are similar, where the number of plaque forming units is substituted for colony forming units to assess attenuation of the number of plaque forming units by modification of the nucleic acid of the microbe. For embodiments of the invention, the desired amount of attenuation can range from a two-fold reduction to much greater levels of attenuation, including a level where essentially no proliferation is observed, depending on the desired level of safety and the intended application of the microbe. A two-fold attenuation in replication would be observed if for a given dilution, there are half as many colonies (or plaques) in the population of a microbe where the nucleic acid is modified as there are in an unmodified population of the microbe (about 0.3 log attenuation). In some embodiments, the attenuation is at least about 0.3 log, about 1 log, about 2 log, about 3 log, about 4 log about 5 log, about 6 log, or at least about 8 log. In some embodiments, the attenuation is in the range of about 0.3 to >10 log, about 2 to >10 log, about 4 to >10 log, about 6 to >10 log, about 0.3-8 log, also about 0.3-7 log, also about 0.3-6 log, also about 0.3-5 log. In also about 0.3-4 log, also about 0.3-3 log, also about 0.3-2 log, also about 0.3-1 log. In some embodiments, the attenuation is in the range of about 1 to >10 log, 1-8 log, 1-6 log, also about 2-6 log, also about 2-5 log, also about 3-5 log. In one embodiment of the invention, the attenuation results in essentially complete inactivation (e.g. where no colonies or plaques are observed to the limit of detection), wherein the microbial gene expression is sufficiently active. Such a population of microbes can be achieved by titrating the concentration of the agent used to modify the microbial nucleic acid to find the lowest concentration at which no colonies or plaques are observed at the limit of detection.

In the case of pathogenic microbes, it is also possible to assess the attenuation in terms of biological effects of the microbe. For example, the pathogenicity of a microbe can be assessed by measurement of the median lethality (LD.sub.50) in mice or other vertebrates. The LD.sub.50 is the amount (e.g. CFU) of microbe injected into the vertebrate that would result in the death of half of the population of the vertebrate. The LD.sub.50 values can be compared for modified and unmodified microbes as a measure of the amount of attenuation. For example, if an unmodified population of microbes has an LD.sub.50 of 10.sup.3 microbes and the population of microbes in which the nucleic acid has been modified has an LD.sub.50 of 10.sup.5 microbes, the microbe has been attenuated so that its LD.sub.50 is increased 100-fold, or by 2 log. In some embodiments, the LD.sub.50 is 2-fold to 1000-fold higher. In some embodiments, an attenuated strain is used that already has a relatively high LD.sub.50. In such cases, the modified microbes increase in LD.sub.50 will be limited by how much material can be infused without causing harm. For example, the LD.sub.50 of a heat killed organism would not be much higher than about 1-5.times.10.sup.9 simply because of the loading of biological material into the mice and/or the inflammatory reaction to the bacterial wall components. The degree of attenuation may also be measured qualitatively by other biological effects, such as the extent of tissue pathology or serum liver enzyme levels. Typically, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, and billirubin levels in the serum are determined at a clinical laboratory for mice injected with microbes of the present invention. Comparisons of these effects in mice or other vertebrates would be made for unmodified and modified microbe as a way to assess the attenuation of the microbe. In addition to measuring the effects of the microbes on the tissues, the amount of viable microbe that can be recovered from infected tissues such as liver or spleen as a function of time could also be used as a measure of attenuation by comparing these values in mice injected with unmodified vs. modified microbes.

Expression of Proteins by Microbes of the Invention.

The modification of the nucleic acid of the microbe, in addition to attenuating proliferation of the microbe, is controlled so that microbial gene expression is substantially unaffected. To be substantially unaffected, the microbial gene expression need not be completely active upon modification of the nucleic acid. It is only necessary that in a population of a microbe in which the nucleic acid is modified to attenuate replication, microbial gene expression is sufficiently active to provide an adequate level of expression of the desired protein by the microbe. An adequate level of expression depends to some extent on the intended use of the microbe. For example, if the microbe contains a particular antigen that is to be used as a vaccine, adequate expression would be determined as the minimum level of expression that provides an effective protective or therapeutic immune response to the vaccine. The microbial gene expression can also be assessed by both in vitro and in vivo methods in order to assess whether such a vaccine might provide an effective immune response. In general, a population of a microbe in which the nucleic acid has been modified can be compared to an unmodified population of the microbe with respect to a particular antigen.

One possibility is to measure the presentation of the antigen of interest by an antigen presenting cell that has been mixed with a population of the microbe. The microbes may be mixed with a suitable antigen presenting cell or cell line, for example a dendritic cell, and the antigen presentation by the dendritic cell to a T cell that recognizes the antigen can be measured. If the microbes are expressing the antigen at a sufficient level, it will be processed into peptide fragments by the dendritic cells and presented in the context of MHC class I or class II to CD8+ or CD4+ T cells, respectively. For the purpose of detecting the presented antigen, a T cell clone or T cell line responsive to the particular antigen may be used. The T cell may also be a T cell hybridoma, where the T cell is immortalized by fusion with a cancer cell line. Such T cell hybridomas, T cell clones, or T cell lines can comprise either CD8+ or CD4+ T cells. The antigen presenting cell can present to either CD8+ or CD4+ T cells, depending on the pathway by which the antigens are processed. CD8+ T cells recognize antigens in the context of MHC class I while CD4+ T cells recognize antigens in the context of MHC class II. The T cell will be stimulated by the presented antigen through specific recognition by its T cell receptor, resulting in the production of certain proteins, such as IL-2 or interferon-.gamma. (IFN-.gamma.), that can be quantitatively measured (for example using an ELISA assay). Alternatively, a hybridoma can be designed to include a reporter gene, such as .beta.-galactosidase, that is activated upon stimulation of the T cell hybridoma by the presented antigens. The increase in the production of .beta.-galactosidase can be readily measured by its activity on a substrate, such as chlorophenolred-.beta.-D-galactopyranoside, which results in a color change. The color change can be directly measured as an indicator of specific antigen presentation (Examples 1, 2 and 11). Additional in vitro and in vivo methods for assessing the antigen expression of microbial vaccines of the present invention can be found in Example 5. It is also possible to directly measure the expression of a particular protein by microbes of the present invention. For example, a radioactively labeled amino acid can be added to a cell population and the amount of radioactivity incorporated into a particular protein can be determined. The proteins synthesized by the cell population can be isolated, for example by gel electrophoresis or capillary electrophoresis, identified as the protein of interest, e.g. by binding with an antibody-specific for the protein, and the amount of radioactivity can be quantitatively measured to assess the expression level of the particular protein. Alternatively, the proteins can be expressed without radioactivity and detected by various methods, such as an ELISA assay or by gel electrophoresis and Western blot with detection using an enzyme linked antibody or fluorescently labeled antibody.

While it is possible that the modification of the microbial nucleic acid reduces the level of protein expression as compared to an unmodified microbe, it is to be understood that this may still provide an effective vaccine. It is the combination of attenuation of proliferation with adequate protein expression that is important in some embodiments of the invention. The efficacy of a vaccine is generally related to the dose of antigen that can be delivered by the microbe, and in some instances, some level of active gene expression by the microbe is necessary. The attenuation of replication of the microbe may be several log while the microbial gene expression is still sufficiently maintained. If the same dose of an attenuated microbe is compared to that of an unmodified microbe, the resulting antigen expression (as assessed by the methods discussed above) in the attenuated microbe population is at least about 1%, about 5%, about 10%, about 25%, about 50%, about 75% or at least about 90% of the antigen expression in the unmodified microbe population. Since there may be several log attenuation in replication, the dose of the modified microbe may be safely increased by up to several log, resulting in an equivalent or greater amount of the antigen presented by the attenuated microbes relative to unmodified microbes upon vaccination.

In some embodiments, a heterologous nucleic acid sequence encoding a protein may be codon-optimized to match the codon preference of the bacterial host expressing the protein. In addition, the sequence encoding a signal peptide fused to the expressed protein may also be codon-optimized to match the codon preference of the bacterial host. In preferred embodiments, the bacterial host is Listeria and either or both of the heterologous protein encoding sequence and the sequence encoding a signal peptide may be codon-optimized. For further information on codon optimization of antigens and signal sequences in Listeria, see U.S. application Ser. No. 60/532,598, incorporated by reference herein.

Microbial Nucleic Acid Modification.

The nucleic acid of a population of a microbe can be modified by a variety of methods. The nucleic acid of the microbe can be modified by physical means, e.g. irradiation with ultraviolet light or ionizing radiation. Ionizing radiation, such as x-rays or .gamma.-rays, may be used to cause single-strand or double-strand breaks in the nucleic acid. Ultraviolet radiation may be used to cause pyrimidine dimers in the nucleic acid. The appropriate dose of radiation is determined by assessing the effects of the radiation on replication and protein expression as detailed above.

The nucleic acid of the microbe can also be modified by chemical means, e.g. by reaction with a nucleic acid targeted compound. In one embodiment, the microbe is treated with a nucleic acid targeted compound that can modify the nucleic acid such that the proliferation of the microbe is attenuated, wherein the microbial population is still able to express a desired protein antigen to a degree sufficient to elicit an immune response. The nucleic acid targeted compound is not limited to a particular mechanism of modifying the nucleic acid. Such compounds modify the nucleic acid either by reacting directly with the nucleic acid (i.e. all or some portion of the compound covalently binds to the nucleic acid), or by indirectly causing the modification of the nucleic acid (e.g. by causing oxygen damage via generation of singlet oxygen or oxygen radicals, by generating radicals of the compound that cause damage, or by other mechanisms of reduction or oxidation of the nucleic acid). Enediynes are an example of a class of compounds that form radical species that result in the cleavage of DNA double strands [Nicolaou et al., Proc. Natl. Acad. Sci. USA, 90:5881-5888 (1993)]. Compounds that react directly with the nucleic acid may react upon activation of the compound, for example upon radiation of the compound. Compounds that react indirectly to cause modification of the nucleic acid may require similar activation to generate either an activated species of the compound or to generate some other active species. While not being limited to the means for activation of nucleic acid targeted compounds, one embodiment of the invention includes the use of photoactivated compounds that either react directly with the nucleic acid or that generate a reactive species such as a reactive oxygen species (e.g. singlet oxygen) which then reacts with the nucleic acid.

The nucleic acid targeted compounds preferentially modify nucleic acids without significantly modifying other components of a biological sample. Such compounds provide adequate modification of the nucleic acid without significantly altering or damaging cell membranes, proteins, and lipids. Such compounds may modify these other cell components to some degree that is not significant. These cell components such as cell membranes, proteins and lipids are not significantly altered if their biological function is sufficiently maintained. In the case of treating a microbe with a nucleic acid targeted compound, the nucleic acid modification is such that the replication of the microbe is attenuated while the cell membranes, proteins and lipids of the microbe are essentially unaffected such that microbial gene expression is active (e.g. the enzymes required for this are not significantly affected), and the surface of the microbe maintains essentially the same antigenicity as a microbe that has not been treated with the compound. As a result, such compounds are useful in preparing an inactivated microbe for use as a vaccine since the proliferation of the microbe is sufficiently attenuated while maintaining sufficient antigenicity or immunogenicity to be useful as a vaccine. Because the compounds specifically modify nucleic acids, the modification can be controlled to a desired level so that replication is attenuated while maintaining a sufficient level of protein expression. The modification can be controlled by varying the parameters of the reaction, such as compound concentration, reaction media, controlling compound activation factors such as light dose or pH, or controlling compounds that cause oxygen damage by controlling the oxygen concentration (either physically, e.g. by degassing, or chemically, by use of oxygen scavengers). A nucleic acid targeted compound is any compound that has a tendency to preferentially bind nucleic acid, i.e. has a measurable affinity for nucleic acid. Such compounds have a stronger affinity for nucleic acids than for most other components of a biological sample, especially components such as proteins, enzymes, lipids and membranes. The nucleic acid targeting provides specificity for the modification of nucleic acids without significantly affecting other components of the biological sample, such as the machinery for gene transcription and protein translation.

Compounds can be targeted to nucleic acids in a number of modes. Compounds which bind by any of the following modes or combinations of them are considered nucleic acid targeted compounds. Intercalation, minor groove binding, major groove binding, electrostatic binding (e.g. phosphate backbone binding), and sequence-specific binding (via sequence recognition in the major or minor groove) are all non-covalent modes of binding to nucleic acids. Compounds that include one or more of these modes of binding will have a high affinity for nucleic acids. While the invention is not limited to the following compounds, some examples of compounds having these modes of binding to nucleic acid are as follows: intercalators are exemplified by acridines, acridones, proflavin, acriflavine, actinomycins, anthracyclinones, beta-rhodomycin A, daunamycin, thiaxanthenones, miracil D, anthramycin, mitomycin, echinomycin, quinomycin, triostin, diacridines, ellipticene (including dimers, trimers and analogs), norphilin A, fluorenes and flourenones, fluorenodiamines, quinacrine, benzacridines, phenazines, phenanthradines, phenothiazines, chlorpromazine, phenoxazines, benzothiazoles, xanthenes and thio-xanthenes, anthraquinones, anthrapyrazoles, benzothiopyranoindoles, 3,4-benzpyrene, benzopyrene diol epoxidie, 1-pyrenyloxirane, benzanthracene-5,6-oxide, benzodipyrones, benzothiazoles, quinolones, chloroquine, quinine, phenylquinoline carboxamides, furocoumarins (e.g. psoralens, isopsoralens, and sulfur analogs thereof), ethidium salts, propidium, coralyne, ellipticine catinn and derivatives, polycyclic hydrocarbons and their oxirane derivatives, and echinimycin; minor groove binders are exemplified by distamycin, mitomycin, netropsin, other lexitropsins, Hoechst 33258 and other Hoechst dyes, DAPI (4',6'-diamidine-2-phenylindole), berenil, and triarylmethane dyes; major groove binders are exemplified by aflatoxins; electrostatic binders are exemplified by spermine, spermidine, and other polyamines; and sequence-specific binders are exemplified by nucleic acids or analogues which bind by such sequence-specific interactions as triple helix formation, D-loop formation, and direct base pairing to single stranded targets. Other sequence-specific binding compounds include poly pyrrole compounds, poly pyrrrole imidazole compounds, cyclopropylpyrroloindole compounds and related minor groove binding compounds [Wemmer, Nature Structural Biology, 5(3):169-171 (1998), Wurtz et al., Chemistry & Biology 7(3):153-161 (2000), Anthoney et al., Am. J. Pharmacogenomics 1(1):67-81 (2001)].

In addition to targeting nucleic acids, the compounds are also able to react with the nucleic acid, resulting in covalent binding to the nucleic acid. Nucleic acid alkylators are a class of compounds that can react covalently with nucleic acid and include, but are not limited to, mustards (e.g. mono or bis haloethylamine groups, and mono haloethylsulfide groups), mustard equivalents (e.g. epoxides, alpha-halo ketones) and mustard intermediates (e.g. aziridines, aziridiniums and their sulfur analogs), methanesulphonate esters, and nitroso ureas. The nucleic acid alkylators typically react with a nucleophilic group on the nucleic acid. It is the combination of the nucleic acid alkylating activity and the nucleic acid targeting ability of these compounds that gives them the ability to covalently react specifically with nucleic acids, providing the desired modification of the nucleic acid of microbes for use in the present invention. The specificity of these compounds may be further enhanced by the use of a quencher that will not enter the microbe. Such a quencher will quench reactions with the surface of the microbe while still allowing the nucleic acid targeted compounds to react with the microbial nucleic acid. A discussion of such quenching can be found in U.S. Pat. No. 6,270,952, the disclosure of which is hereby incorporated by reference. The modification of the microbial nucleic acid can be controlled by adjusting the compound concentration and reaction conditions. The appropriate concentration and reaction conditions are determined by assessing their effects on replication and protein expression as detailed above. The compounds used in the present invention are effective at concentrations of about 10 pM to 10 mM, also about 100 pM to 1 mM, also about 1 nM to 10 .mu.M, also about 1-500 nM, also about 1-200 nM or about 1-100 nM. A discussion of nucleic acid targeted, nucleic acid reactive compounds for specific reaction with nucleic acids, in particular microbial nucleic acids, can be found in U.S. Pat. Nos. 6,143,490 and 6,093,725, the disclosures of which are hereby incorporated by reference.

The nucleic acid can be modified by using a nucleic acid targeted compound that requires activation with radiation in order to cause the nucleic acid modification. Such compounds are targeted to nucleic acids as discussed above. These compounds include, but are not limited to, acridines, acridones, anthyrl derivatives, alloxazines (e.g. riboflavin), benzotriazole derivatives, planar aromatic diazo derivatives, planar aromatic cyano derivatives, toluidines, flavines, phenothiazines (e.g. methylene blue), furocoumarins, angelicins, psoralens, sulfur analogs of psoralens, quinolones, quinolines, quinoxalines, napthyridines, fluoroquinolones, anthraquinones, and anthracenes. Many of these compounds are used as DNA photocleavage agents [Da Ros et al., Current Pharmaceutical Design 7:1781 (2001)]. While the invention is not limited to the method of activation of the nucleic acid targeted compounds, typically, the compounds can be activated with light of particular wavelengths. The effective wavelength of light depends on the nature of the compound and can range anywhere from approximately 200 to 1200 nm. For some of these compounds, activation causes modification of the nucleic acid without direct binding of the compound to the nucleic acid, for example by generating reactive oxygen species in the vicinity of the nucleic acid. For some of these compounds, activation results in binding of the compound directly to the nucleic acid (i.e. the compound binds covalently). Some of these compounds can react with the nucleic acid to form an interstrand crosslink. Psoralens are an example of a class of compounds that crosslink nucleic acids. These compounds are typically activated with UVA light (320-400 nm). Psoralen compounds for use in the present invention are exemplified in U.S. Pat. Nos. 6,133,460 and 5,593,823, the disclosures of which are hereby incorporated by reference. Again, it is the combination of nucleic acid targeting and the ability to modify the nucleic acid upon activation that provide specific reactivity with nucleic acids. The modification of the microbial nucleic acid can be controlled by adjusting the compound concentration, reaction conditions and light dose. The appropriate concentration and light dose are determined by assessing their effects on replication and protein expression as detailed above. In addition to compound concentration and level of light exposure, the reaction is affected by the conditions under which the sample is dosed with UVA light. For example, the required overall concentration for irradiating a population of microbes in a buffered media is going to vary from a population that is cultured in a growth media (e.g. BHI, Triptase Soy Broth). The photoreaction may be affected by the contents of the growth media, which may interact with the psoralen, thereby requiring a higher overall concentration of the psoralen. In addition, the effective dosing of the microbes may depend on the growth phase of the organism and the presence or absence of compound during the growth phase. In one embodiment, the population of microbes comprises growth media during the psoralen UVA treatment. In one embodiment, the psoralen is added to the population of microbes, the population is cultured to grow the microbes in the presence of psoralen and growth media, and the UVA treatment is performed at some point in the growth phase of the microbes. In one embodiment, the population is grown to an OD of 0.5-1 (1.times.10.sup.7 to 1.times.10.sup.9 CFU/mL) in the presence of the psoralen prior to irradiation with an appropriate dose of UVA light. Psoralen compounds are effective at concentrations of about 10 pM to 10 mM, also about 100 pM to 1 mM, also about 1 nM to 10 .mu.M, also about 1-500 nM, also about 1-200 nM or about 1-100 nM, with the UVA light dose ranging from about 0.1-100 J/cm.sup.2, also about 0.1-20 J/cm.sup.2, or about 0.5-10 J/cm.sup.2, 0.5-6 J/cm.sup.2 or about 2-6 J/cm.sup.2. In one embodiment, the microbe is treated in the presence of growth media at psoralen concentrations of about 10 pM to 10 mM, also about 1-5000 nM, also about 1-500 nM, also about 5-500 nM, or about 10-400 nM. In one embodiment, the microbe treated in the presence of growth media is grown to an OD of 0.5-1 in the presence of psoralen at concentrations of about 10 pM to 10 mM, also about 1-5000 nM, also about 1-500 nM, also about 5-500 nM, or about 10-400 nM. Following the growth to an OD of 0.5-1, the microbe population is irradiated with UVA light at a dose ranging from about 0.1-100 J/cm.sup.2, also about 0.1-20 J/cm.sup.2, or about 0.5-10 J/cm.sup.2, 0.5-6 J/cm.sup.2 or about 2-6 J/cm.sup.2.

Microbes Containing Heterologous Nucleic Acid Sequences

Microbes can be altered to include a heterologous nucleic acid sequence that can be expressed by the microbe. The heterologous sequence can encode at least one specific protein antigen. The microbes may be altered by methods known to one skilled in the art [Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, (2000)]. The microbes can be altered to contain one or more sequences that encode one or more antigens. The heterologous nucleic acid sequence encoding a specific antigen is not limited to an exact nucleic acid sequence but is of a sequence that is sufficient to provide the expression of an antigen that will elicit the desired immune response when administered to an individual. The heterologous sequence can be expressed as an antigen related to a particular disease. The microbe expressing such antigens can be used as a vaccine, wherein the vaccine may be used as a preventative treatment or a therapeutic treatment. Diseases that can be treated by such vaccines include infectious diseases, autoimmune diseases, allergies, cancers and other hyperproliferative diseases.

The microbes of the invention may be altered to contain a heterologous nucleic acid sequence encoding a specific tumor antigen. A large number of tumor specific antigens that are recognized by T cells have been identified [Renkvist et al., Cancer Immunol Innumother 50:3-15 (2001)]. These tumor antigens may be differentiation antigens (e.g., PSMA, Tyrosinase, gp100), tissue-specific antigens (e.g. PAP, PSA), developmental antigens, tumor-associated viral antigens (e.g. HPV 16 E7), cancer-testis antigens (e.g. MAGE, BAGE, NY-ESO-1), embryonic antigens (e.g. CEA, alpha-fetoprotein), oncoprotein antigens (e.g. Ras, p53), over-expressed protein antigens (e.g. ErbB2 (Her2/Neu), MUC1), or mutated protein antigens. The tumor antigens that may be encoded by the heterologous nucleic acid sequence include, but are not limited to, 707-AP, Annexin II, AFP, ART-4, BAGE, .beta.-catenin/m, BCL-2, bcr-abl, bcr-abl p190, bcr-abl p210, BRCA-1, BRCA-2, CAMEL, CAP-1, CASP-8, CDC27/m, CDK-4/m, CEA, CT9, CT10, Cyp-B, Dek-cain, DAM-6 (MAGE-B2), DAM-10 (MAGE-B1), ELF2M, EphA2, ETV6-AML1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GnT-V, gp100, HAGE, HER2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT, hTRT, iCE, inhibitors of apoptosis (e.g. survivin), KIAA0205, LAGE, LAGE-1, LDLR/FUT, MAGE-1, MAGE-2, MAGE-3, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-B5, MAGE-B6, MAGE-C2, MAGE-C3, MAGE-D, MART-1, MART-1/Melan-A, MC1R, MDM-2, mesothelin, Myosin/m, MUC1, MUC2, MUM-1, MUM-2, MUM-3, neo-polyA polymerase, NA88-A, NY-ESO-1, NY-ESO-1a (CAG-3), PAGE-4, PAP, Proteinase 3 (PR3), P15, p190, Pm1/RAR.alpha., PRAME, PSA, PSM, PSMA, RAGE, RAS, RCAS1, RU1, RU2, SAGE, SART-1, SART-2, SART-3, SP17, SPAS-1, TEL/AML1, TPI/m, Tyrosinase, TARP, TRP-1 (gp75), TRP-2, TRP-2/INT2, WT-1, and alternatively translated NY-ESO-ORF2 and CAMEL proteins, derived from the NY-ESO-1 and LAGE-1 genes. The microbes of the present invention encompass any tumor antigen that can elicit a tumor-specific immune response, including antigens yet to be identified. The microbes may be altered to contain more than one heterologous sequence encoding more than one tumor antigen. Preferred antigens include mesothelin [Argani et al., Clin Cancer Res. 7(12):3862-8 (2001)], Sp17 [Lim et al., Blood. 97(5):1508-10 (2001)], gp100 [Kawakami et al., Proc. Natl. Acad. Sci. USA 91:6458 (1994)], PAGE-4 [Brinkmann et al., Cancer Res. 59(7):1445-8 (1999)], TARP [Wolfgang et al., Proc. Natl. Acad. Sci. USA 97(17):9437-42 (2000)], EphA2 [Tatsumi et al., Cancer Res. 63(15):4481-9 (2003)], PR3 [Muller-Berat et al., Clin. Immunol. Immunopath. 70(1):51-9 (1994)] and SPAS-1 [U.S. patent application Publication No. 20020150588].

In one embodiment of the invention, the heterologous antigen expressed by the modified microbe is CEA. CEA is a 180-kDA membrane intercellular adhesion glycoprotein that is over-expressed in a significant proportion of human tumors, including 90% of colorectal, gastric, and pancreatic, 70% of non-small cell lung caner, and 50% of breast cancer (Hammarstrom, Semin. Cancer Biol., 9:67-81). A variety of immunotherapeutics such as anti-idiotype monoclonal antibody mimicking CEA (Foon et al., Clin. Cancer Res., 87:982-90 (1995), or vaccination using a recombinant vaccinia virus expressing CEA (Tsang et al., J. Natl. Cancer Inst., 87:982-90 (1995)) have been investigated, unfortunately, however, with limited success. Nonetheless, investigators have identified a HLA*0201-restricted epitope, CAP-1(CEA605-613), that is recognized by human T cell lines that were generated from vaccinated patients. Vaccination of patients with DC pulsed with this epitope failed to induce clinical responses (Morse et al., Clin. Cancer Res., 5:1331-8 (1999)). Recently, a CEA605-613 peptide agonist was identified with a heteroclitic aspartate to asparagine substitution at position 610 (CAP1-6D). Although this amino acid substitution did not alter MHC binding affinity of this peptide, the use of the altered peptide ligand (APL) resulted in improved generation of CEA-specific cytotoxic T lymphocytes (CTL) in vitro. CAP1-6D-specific CTL maintained their ability to recognize and lyse tumor cells expressing native CEA (Zaremba et al., Cancer Res., 57: 4570-7 (1997); Salazar et al., Int. J. Cancer, 85:829-38 (2000)). Fong et al. demonstrated induction of CEA-specific immunity in patients with colon cancer vaccinated with Flt3-ligand expanded DC incubated with this APL. Encouragingly, 2 of 12 patients after vaccination experienced dramatic tumor regressions that correlated with the induction of peptide-MHC tetramer.sup.+ T cells (Fong et al., Proc. Natl. Acad. Sci. U.S.A., 98:8809-14 (2001)). Taken together, this work provides significant validation for CEA-targeted immunotherapy for colorectal cancer.

In another embodiment, the heterologous antigen expressed by the modified microbe is proteinase-3 or is derived from proteinase-3. For instance, in one embodiment, the antigen comprises the HLA-A2.1-restricted peptide PR1 (aa 169-177; VLQELNVTV (SEQ ID NO:50)). Information on proteinase-3 and/or the PR1 epitope is publicly available in the following references: U.S. Pat. No. 5,180,819, Molldrem, et al., Blood, 90:2529-2534 (1997); Molldrem et al., Cancer Research, 59:2675-2681 (1999); Molldrem, et al., Nature Medicine, 6:1018-1023 (2000); and Molldrem et al., Oncogene, 21: 8668-8673. (2002).

Accordingly, in some embodiments, the modified microbe comprises a nucleic acid molecule encoding an antigen such as mesothelin, SPAS-1, proteinase-3, EphA2, SP-17, gp100, PAGE-4, TARP, Her-2/neu, WT-1, NY-ESO-1, PSMA, K-ras, or CEA, or an antigen derived from one of those proteins. In some embodiments, the modified microbe comprises a nucleic acid molecule encoding an antigen such as mesothelin, SPAS-1, proteinase-3, SP-17, gp100, PAGE-4, TARP, WT-1, NY-ESO-1 or CEA, or an antigen derived from one of those proteins. In some embodiments, the modified microbe comprises a nucleic acid molecule encoding human mesothelin, or an antigen derived from human mesothelin. In other embodiments, the modified microbe comprises a nucleic acid molecule encoding human EphA2, or derived from human EphA2.

The microbes of the invention may be altered to contain a heterologous nucleic acid sequence encoding a specific infectious disease antigen. In one embodiment, the antigen is derived from a human or animal pathogen. The pathogen is optionally a virus, bacterium, fungus, or a protozoan. For instance, the antigen may be a viral or fungal or bacterial antigen.

For instance, the antigen may be derived from Human Immunodeficiency virus (such as gp 120, gp 160, gp41, gag antigens such as p24gag and p55gag, as well as proteins derived from the pol, env, tat, vif, rev, nef, vpr, vpu and LTR regions of HIV), Feline Immunodeficiency virus, or human or animal herpes viruses. In one embodiment, the antigen is derived from herpes simplex virus (HSV) types 1 and 2 (such as gD, gB, gH, Immediate Early protein such as ICP27), from cytomegalovirus (such as gB and gH), from Human Metapneumovirus, from Epstein-Barr virus or from Varicella Zoster Virus (such as gpI, II or III). (See, e. g. Chee et al. (1990) Cytomegaloviruses (J. K. McDougall, ed., Springer Verlag, pp. 125-169; McGeoch et al. (1988) J. Gen. Virol. 69: 1531-1574; U.S. Pat. No. 5,171,568; Baer et al. (1984) Nature 310: 207-211; and Davison et al. (1986) J. Gen. Virol. 67: 1759-1816.)

In another embodiment, the antigen is derived from a hepatitis virus such as hepatitis B virus (for example, Hepatitis B Surface antigen), hepatitis A virus, hepatitis C virus, delta hepatitis virus, hepatitis E virus, or hepatitis G virus. See, e. g., WO 89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodes several viral proteins, including E1 and E2. See, e. g., Houghton et al., Hepatology 14: 381-388(1991).

An antigen that is a viral antigen is optionally derived from a virus from any one of the families Picornaviridae (e. g., polioviruses, rhinoviruses, etc.); Caliciviridae; Togaviridae (e. g., rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae (e. g., rotavirus, etc.); Birnaviridae; Rhabodoviridae (e. g., rabies virus, etc.); Orthomyxoviridae (e. g., influenza virus types A, B and C, etc.); Filoviridae; Paramyxoviridae (e. g., mumps virus, measles virus, respiratory syncytial virus, parainfluenza virus, etc.); Bunyaviridae; Arenaviridae; Retroviradae (e. g., HTLV-I; HTLV-11; HIV-1 (also known as HTLV-111, LAV, ARV, hTLR, etc.)), including but not limited to antigens from the isolates HIVI11b, HIVSF2, HTVLAV, HIVLAI, HIVMN); HIV-1 CM235, HIV-1; HIV-2, among others; simian immunodeficiency virus (SIV); Papillomavirus, the tick-borne encephalitis viruses; and the like. See, e. g. Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2.sup.nd Edition (B. N. Fields and D. M. Knipe, eds. 1991), for a description of these and other viruses.

In some alternative embodiments, the antigen is derived from bacterial pathogens such as Mycobacterium, Bacillus, Yersinia, Salmonella, Neisseria, Borrelia (for example, OspA or OspB or derivatives thereof), Chlamydia, or Bordetella (for example, P.69, PT and FHA), or derived from parasites such as plasmodium or Toxoplasma. In one embodiment, the antigen is derived from Mycobacterium tuberculosis (e.g. ESAT-6, 85A, 85B, 72F), Bacillus anthracis (e.g. PA), or Yersinia pestis (e.g. F1, V). In addition, antigens suitable for use in the present invention can be obtained or derived from known causative agents responsible for diseases including, but not limited to, Diptheria, Pertussis, Tetanus, Tuberculosis, Bacterial or Fungal Pneumonia, Otitis Media, Gonorrhea, Cholera, Typhoid, Meningitis, Mononucleosis, Plague, Shigellosis or Salmonellosis, Legionaire's Disease, Lyme Disease, Leprosy, Malaria, Hookworm, Onchocerciasis, Schistosomiasis, Trypanosomiasis, Leishmaniasis, Giardia, Amoebiasis, Filariasis, Borelia, and Trichinosis.

The microbes of the invention may be altered to contain a heterologous nucleic acid sequence encoding an autoimmune disease-specific antigen. In a T cell mediated autoimmune disease, a T cell response to self antigens results in the autoimmune disease. The type of antigen for use in treating an autoimmune disease with the vaccines of the present invention might target the specific T cells responsible for the autoimmune response. For example, the antigen may be part of a T cell receptor, the idiotype, specific to those T cells causing an autoimmune response, wherein the antigen incorporated into a vaccine of the invention would elicit an immune response specific to those T cells causing the autoimmune response. Eliminating those T cells would be the therapeutic mechanism to alleviating the autoimmune disease. Another possibility would be to incorporate an antigen that will result in an immune response targeting the antibodies that are generated to self antigens in an autoimmune disease or targeting the specific B cell clones that secrete the antibodies. For example, an idiotype antigen may be incorporated into the microbe that will result in an anti-idiotype immune response to such B cells and/or the antibodies reacting with self antigens in an autoimmune disease. Autoimmune diseases that may be treatable with vaccine microbes of the present invention include, but are not limited to, rheumatoid arthritis, multiple sclerosis, Crohn's disease, lupus, myasthenia gravis, vitiligo, scleroderma, psoriasis, pemphigus vulgaris, fibromyalgia, colitis and diabetes. A similar approach may be taken for treating allergic responses, where the antigens incorporated into the vaccine microbe target either T cells, B cells or antibodies that are effective in modulating the allergic reaction. In some autoimmune diseases, such as psoriasis, the disease results in hyperproliferative cell growth with expression of antigens that may be targeted as well. Such an antigen that will result in an immune response to the hyperproliferative cells is considered.

Rather than targeting the malfunctioning cells of a disease, the microbes of the present invention comprise antigens that target unique disease associated protein structures. One example of this is the targeting of antibodies, B cells or T cells using idiotype antigens as discussed above. Another possibility is to target unique protein structures resulting from a particular disease. An example of this would be to incorporate an antigen that will generate an immune response to proteins that cause the amyloid plaques observed in diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease (CJD) and Bovine Spongiform Encephalopathy (BSE). While this approach may only provide for a reduction in plaque formation, it may be possible to provide a curative vaccine in the case of diseases like CJD. This disease is caused by an infectious form of a prion protein. The vaccine incorporates an antigen to the infectious form of the prion protein such that the immune response generated by the vaccine may eliminate, reduce, or control the infectious proteins that cause CJD.

Microbes Containing Mutations

In one embodiment, the invention includes a vaccine comprising a microbe wherein the nucleic acid of the microbe is modified so that the proliferation of the microbe is attenuated, wherein the microbial population is still able to express a desired antigen to an extent that is sufficient to elicit an immune response, and wherein the microbe is further attenuated by at least one genetic mutation. The mutation in the microbe may affect a variety of features of the microbe. In some cases, the mutation affects the ability of the microbe to invade certain cells. For example, certain intracellular bacteria can invade a variety of cell types depending on receptors present on the bacteria. The mutation may alter the expression of certain receptors so that the bacteria is taken up by some cell types but not others. As an example of this, Listeria is typically taken up by phagocytic cells and also actively invades non-phagocytic cells (e.g. hepatic cells). A mutation of Listeria may be used in which the invasion of non-phagocytic cells is significantly reduced or eliminated while the uptake by phagocytic cells is sufficiently active. Such a mutation may provide for a better immune response as the vaccine would be preferentially taken up by phagocytic cells, which are important in presenting the bacterial antigens to the immune system. It is understood that the mutation can be to any gene that results in an attenuation of the ability of the microbe to invade certain cell types, and that this is exemplified by mutations to intemalin genes in Listeria (e.g. inlA, inlB). Similar genes may exist (e.g. invasin genes in Salmonella, Bacillus anthracis, and Yersinia) in other bacteria, and mutations in these genes are encompassed by the present invention. The mutation might impact other features of the microbe, such as a virulence factor or a gene that allows for growth and spreading, thereby reducing the virulence of the microbe. For example, a mutation in the actA gene of Listeria causes a deficiency in the polymerization of host cell actin, which inhibits the ability of the Listeria to spread to other cells. A mutation in the hly gene of Listeria (listeriolysin (LLO) protein) impacts the ability of the Listeria to escape the phagolysosome of an infected cell. A mutation in either the plcA or plcB genes of Listeria impacts the ability of the Listeria to spread from cell to cell. A mutation in the yop gene of Yersinia affects the ability of the Yersinia to prevent phagocytosis by macrophages. In another embodiment the genetic mutation attenuates the expression of certain antigens, for example, antigens that would normally result in an immune response to the microbe itself. Such a mutation may be useful if the microbe is used as a vaccine comprising a heterologous antigen in order to stimulate a strong immune response to the heterologous antigen but with a reduced immune response to the delivery microbe compared to the non-mutated microbe. In one embodiment, the microbe is attenuated by a mutation in more than one gene. In one embodiment, one of the mutations is in an intemalin gene of Listeria or a similar gene in other bacteria. In one embodiment, the mutation is in one or more of an internalin gene of Listeria or similar gene in other bacteria. In one embodiment, one of the mutations is in the actA gene. In one embodiment, the microbe comprises Listeria monoxytogenes with mutations in the actA gene and one or more intemalin genes. In a preferred embodiment, the Listeria monoxytogenes comprises a mutation in the actA gene and the inlB gene, preferably the Listeria monoxytogenes comprises an actA/inlB deletion mutant (which is alternatively referred to herein as either .DELTA.actA.DELTA.inlB or actA.sup.-inlB.sup.-). The sequences of a variety of Listeria genes including those described herein are found in Genbank accession no. NC.sub.--003210.

The microbe might contain a mutation that significantly reduces the ability of the microbe to repair modifications to their nucleic acid. Such a mutation could be in any of a variety of genes that are involved in the DNA repair mechanisms of microbes [Aravind et al., Nucleic Acids Research 27(5):1223-1242 (1999)]. Microbes that are deficient in their ability to repair damage to their nucleic acid provide an added level of safety and efficacy to the use of the microbes of the present invention. Using the appropriate repair deficient mutants, the microbes are exquisitely sensitive to nucleic acid modification. The nucleic acid of the microbes may be modified to a lesser degree yet still ensure the desired amount of attenuation of proliferation. This provides a larger window of efficacy in which to operate so that the expression of the microbial nucleic acid is sufficient to generate the desired proteins. In the case where de novo antigen expression is required, this provides a vaccine that will elicit an effective immune response. It also provides an added level of safety as the level of attenuation of proliferation achieved can not be compromised by repair of the modified nucleic acid. In another embodiment, the genetic mutation alters the susceptibility of the microbe to treatment with a nucleic acid targeted compound, for example by altering the permeability of the microbe to the compound or by altering the ability of the compound to access and bind the microbial nucleic acid. Such mutations may also impact the efficacy of the process of attenuating proliferation while leaving microbial gene expression substantially unaffected.

To illustrate the advantages of using a repair deficient mutant, one can consider the mechanism of the attenuation of microbial proliferation. The microbial nucleic acid is modified either by strand breakage or pyrimidine dimers, or by chemical modifications such as monoadducts or crosslinks. If the mechanisms for repair of these modifications are intact, a certain number of modifications will be required in order to achieve sufficient attenuation of proliferation. The greater the modification of nucleic acid, the greater the reduction in protein expression. Even though the levels of modification required to attenuate proliferation are much lower than the levels required to stop protein expression, protein expression will still be reduced to some extent, possibly to an unacceptable level. The use of repair deficient mutants significantly reduces the levels needed to attenuate proliferation such that a lower modification level will result in adequate attenuation of proliferation. Since the nucleic acid modification is much lower, the expression of proteins will be less affected, providing for a higher level of expression of the protein of interest. Such repair deficient mutants may be particularly useful in the preparation of vaccines, such as vaccines to the microbe itself, where the safety of the vaccine can be increased by a slight modification of the nucleic acid, leaving a sufficiently high level of protein expression, in particular the antigen to which the immune response is targeted. In one embodiment the repair deficient mutant lacks the ability to make PhrB (a photolyase), which repairs pyrimidine dimers. For example, the mutation may be in the phrB gene, or a functionally equivalent gene, depending on the genus and species of the microbe. Such a mutant could be used in conduction with ultraviolet irradiation (e.g. UVB, UVC) of the microbe to produce pyrimidine dimers in the microbial nucleic acid. In one embodiment the repair deficient mutant is unable to repair interstand crosslinks. Such mutants include, but are not limited to, mutations in uvr genes, i.e. uvrA, uvrB, uvrC, and uvrD genes as well as recA genes, or functionally equivalent genes, depending on the genus and species of the microbe. The mutations may be in one or more of these genes. These mutations result in attenuation in the activity of the corresponding enzymes UvrA (an ATPase), UvrB (a helicase), UvrC (a nuclease), UvrD (a helicase II) and RecA (a recombinase). These mutants would be used in conjunction with a crosslinking compound, such as a psoralen. Since the microbial nucleic acid is crosslinked in some locations, and these crosslinks can not be repaired, the microbe is unable to replicate as the original strands of nucleic acid can not be separated. Since they can not be repaired, very few crosslinks are needed, the microbial nucleic acid is for the most part accessible for transcription, and protein expression is not altered significantly. In a preferred embodiment, a population of repair deficient microbial mutants that are unable to repair interstrand crosslinks are suitably crosslinked such that essentially every microbe in the population contains at least one crosslink, such that attenuation of replication is essentially complete, wherein the microbial gene expression of the population is sufficiently active. In one embodiment, a mutation in the recA gene is a conditional mutation. In such a mutation, the mutation in the recA gene results in the attenuation in the activity of recA only under certain conditions (i.e. non-permissive conditions), such as a suitable pH or temperature of the microbial population. A microbe comprising a conditional recA mutation can be cultured under permissive conditions in order to grow sufficient levels of the microbe and then placed under non-permissive conditions for treatment to modify the nucleic acid, then stored under non-permissive conditions such that the nucleic acid damage is not adequately repaired. As an example of this, a recA temperature sensitive mutant is grown at 30.degree. C., where it grows well, and is treated to modify the nucleic acid at 42.degree. C., which is non-permissive for recA such that it is very sensitive to treatment, such as psoralen crosslinking. While the treated microbe may be stored under non-permissive conditions, it is possible that upon vaccination, the conditions may permit expression of recA, resulting in some repair and presenting a safety issue. It is possible to construct the microbe such that the recA is under the control of the lac repressor, such that growth of the strain can be induced by isopropyl-.beta.-D-thiogalactopyranoside (IPTG) when growth is desirable, prior to the inactivation and/or immunization steps. The possibility of recA expression can then be eliminated for the inactivation and/or immunization steps by withholding further IPTG from the strain and/or eliminating IPTG from the strain's environment.

In one embodiment, the microbe comprises at least one mutation that significantly reduces the ability of the microbe to repair modifications to their nucleic acid in combination with at least one mutation not related to repair mechanisms. The mutation that is not related to repair mechanisms may affect a variety of features of the microbe, such as the ability of the microbe to invade certain cells, a mutation in a virulence factor or a gene that allows for growth and spreading, or a mutation that attenuates the expression of certain antigens. Such mutations are discussed above and include, but are not limited to, mutations in intemalin genes (e.g. inlB), actA gene, hly gene, plcA gene, or plcB gene of Listeria, invasion genes (e.g. Salmonella, Bacillus anthracis, and Yersinia) or the yop gene of Yersinia. In one embodiment, the microbe comprises Listeria monoxytogenes having a mutation in the actA gene. In one embodiment, the Listeria monoxytogenes comprises a mutation in the actA gene and in an internalin gene. In one embodiment, the Listeria monoxytogenes comprises an actA mutation and a uvrAB mutation, preferably actA/uvrAB deletion mutations (which may be referred to as either .DELTA.actA.DELTA.uvrAB or actA.sup.-uvrAB.sup.-). In one embodiment, the Listeria monocytogenes comprises an actA mutation, an inlB mutation, and a uvrAB mutation, preferably actA/inlB/uvrAB deletion mutations. In some other embodiments, the microbe comprises Bacillus anthracis having a uvrAB mutation, such as a deletion.

In another embodiment, the invention provides an isolated mutant Listeria strain, such as a mutant Listeria monoxytogenes strain, comprising a genetic mutation that attenuates its ability to repair its nucleic acid. In some embodiments, the mutant Listeria strain is defective with respect to at least one DNA repair enzyme (such as UvrA and/or UvrB). In some embodiments, the mutant Listeria strain comprises a genetic mutation in the uvrA gene and/or the uvrB gene. In some embodiments, the mutant strain is the Listeria monoxytogenes .DELTA.actA.DELTA.uvrAB strain deposited with the American Type Culture Collection (ATCC) and identified by accession number PTA-5563. In other embodiments, strain is a mutant of the Listeria monoxytogenes .DELTA.actA.DELTA.uvrAB strain deposited with the American Type Culture Collection (ATCC) and identified by accession number PTA-5563, wherein the mutant of the deposited strain is defective with respect to UvrA, UvrB, and ActA.

In some embodiments, the invention provides a free-living microbe which is defective with respect to at least one DNA repair enzyme (relative to wild type). In some embodiments, the microbe that is defective with respect to at least one DNA repair enzyme is attenuated for DNA repair relative to wild type. In some embodiments, the capacity of the microbe for DNA repair is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 90% relative to wild type. Methods for assessing the ability of a microbe to effect DNA repair are well known to those of ordinary skill in the art. In some embodiments, the microbe is defective with respect to one or more of the following enzymes: PhrB, UvrA, UvrB, UvrC, UvrD, and RecA. In some embodiments, the microbe is defective with respect to UvrA, UvrB, or both enzymes. In some embodiments, the microbe is defective with respect to RecA, or a functional equivalent of Rec A. In some embodiments, the microbe comprise a genetic mutation in one or more gene selected from the group consisting of phrB, uvrA, uvrB, uvrC, uvrD and recA, or in a functional equivalent of one or more gene selected from the group consisting of phrB, uvrA, uvrB, uvrC, uvrD and recA. In some embodiments, the microbe comprises genetic mutations in both uvrA and uvrB, or in functional equivalents of both uvrA and uvrB. In some embodiments, the microbe comprises a genetic mutation in recA. In some embodiments, the microbe is a bacterium. For instance, in some embodiments, the microbe is Mycobacterium tuberculosis, Listeria monocytognes, or Bacillus anthracis.

The invention also provides an isolated mutant Listeria monocytogenes strain, comprising a genetic mutation that attenuates its ability to repair its nucleic acid. In some embodiments, the mutant strain is defective with respect to at least one DNA repair enzyme (such as UvrA and/or UvrB). In some embodiments, the mutant strain comprises a genetic mutation in the uvrA gene and/or the uvrB gene. In some embodiments, the uvrA gene, the uvrB gene, or both genes are deleted. In some embodiments, the mutant strain is attenuated with respect to RecA. In some embodiments, the mutant strain comprises a genetic mutation in the recA gene. In some embodiments, the mutant microbe is the Listeria monoxytogenes actA.sup.-/uvrAB.sup.- strain deposited with the American Type Culture Collection (ATCC) and identified by accession number PTA-5563, or a mutant of the deposited strain which is defective with respect to UvrA, UvrB, and ActA.

The invention also provides an isolated mutant Bacillus anthracis strain, comprising a genetic mutation that attenuates its ability to repair its nucleic acid. In some embodiments, the mutant strain is defective with respect to at least one DNA repair enzyme (such as UvrA and/or UvrB). In some embodiments, the mutant strain comprises a genetic mutation in the uvrA gene and/or the uvrB gene. In some embodiments, the uvrA gene (SEQ ID NO:18), the uvrB gene (SEQ ID NO:19), or both genes are deleted. In some embodiments, the mutant strain is attenuated with respect to RecA. In some embodiments, the mutant strain comprises a genetic mutation in the recA gene. In some embodiments, the mutant strain comprises a mutation in the recA gene that makes expression of the recA protein temperature sensitive. In some alternative embodiments, a mutant strain of B. anthracis is constructed which is under control of the lac repressor (inducible by IPTG), permitting expression of recA during growth, but not during inactivation (such as with S-59/UVA) and/or post-immunization. In some embodiments, the mutant strain comprises one or more mutations in the lef gene, cya gene, or both genes, that decreases the toxicity of the strain.

As with any microbe of the invention, the modification of the DNA of the repair deficient (e.g. uvr deficient) bacteria with psoralen can be controlled by adjusting the compound concentration, reaction conditions and light dose. The appropriate concentration, reaction conditions and light dose are determined by assessing their effects on replication and protein expression as detailed above. The use of repair deficient mutants provides an additional level of control of proliferation while maintaining adequate protein expression such that the parameters of concentration, reaction conditions and light dose can be adjusted over a wider range of conditions to provide a suitable population of microbes. For example, there will be a broader range of nucleic acid modification density over which proliferation can be completely inhibited without significantly affecting protein expression. The minimum level of modification required to completely inhibit repair deficient strains is much less than for non-repair deficient strains (see Examples 3, 7, 11, and 21). As a result, the modification level can be higher than the minimum level required to stop proliferation (ensuring complete inactivation) yet still be below a level that is detrimental to protein expression. Thus, while the invention is effective for non-repair deficient strains, uvr deficient strains provide greater flexibility in preparing a desirable population of microbes that would be effective as a vaccine. Psoralen compounds are effective at concentrations of about 10 pM to 10 mM, also about 100 pM to 1 mM, also about 1 nM to 10 .mu.M, also about 1-500 nM, also about 1-200 nM or about 1-100 nM, with the UVA light dose ranging from about 0.1-100 J/cm.sup.2, also about 0.1-20 J/cm.sup.2, also about 0.5-10 J/cm.sup.2, or about 0.5-6 J/cm.sup.2 or about 2-6 J/cm.sup.2. In one embodiment, the microbe is treated in the presence of growth media at psoralen concentrations of about 10 pM to 10 mM, also about 1-5000 nM, also about 1-500 nM, also about 5-500 nM, or about 10-400 nM. In one embodiment, the microbe treated in the presence of growth media is grown to an OD of 0.5-1 in the presence of psoralen at concentrations of about 10 pM to 10 mM, also about 1-5000 nM, also about 1-500 nM, also about 5-500 nM, or about 10-400 nM. Following the growth to an OD of 0.5-1, the microbe population is irradiated with UVA light at a dose ranging from about 0.1-100 J/cm.sup.2, also about 0.1-20 J/cm.sup.2, or about 0.5-10 J/cm.sup.2, 0.5-6 J/cm.sup.2 or about 2-6 J/cm.sup.2.

In order to generate primarily psoralen crosslinks in any microbe, particularly uvr deficient mutant bacteria, it is possible to dose the psoralen and UVA light initially to form adducts and follow this with a second dose of UVA light alone to convert some or most of the monoadducts to crosslinks. The psoralen photochemistry is such that absorption of a photon of appropriate energy will first form a monoadduct. Absorption of an additional photon will convert this monoadduct to a crosslink when a furan side monoadduct is appropriately situated in the DNA double helix [Tessman et al., Biochemistry 24:1669-1676 (1985)]. The sample can be dosed with a lower UVA dose at a desired concentration of psoralen and the unreacted psoralen can be removed, e.g. by washing, dialysis or ultrafiltration of the bacteria. The bacteria containing psoralen adducts (monoadducts and crosslinks) can be further dosed with UVA light to convert some or most of the monoadducts to crosslinks without resulting in significant additional adducts to the bacteria. This allows for the controlled addition of a low number of psoralen adducts with the initial light dose, then converting a substantial number of any monoadducts to crosslink with the second dose. This provides for modification of the microbial genome at sufficiently low levels wherein a majority of the adducts formed will be crosslinks. This is particularly effective for blocking replication with uvr deficient mutants. In such embodiments, psoralen compounds are effective at concentrations of about 10 pM to 10 mM, also about 100 pM to 1 mM, also about 1-500 nM, also about 1-200 nM or about 1-100 nM, with the UVA light dose ranging from about 0.1-10 J/cm.sup.2, also about 0.1-2 J/cm.sup.2, or about 0.5-2 J/cm.sup.2. Following removal of most of the unreacted psoralen by washing, dialysis or ultrafiltration of the bacteria, the bacteria may be dosed with UVA light ranging from 0.1-100 J/cm.sup.2, also about 0.1-20 J/cm.sup.2, or about 0.5-10 J/cm.sup.2 or about 2-6 J/cm.sup.2.

Vaccine Compositions and in vivo Efficacy

Vaccine compositions of the invention comprise a microbe in which the microbial nucleic acid is modified and/or comprise an antigen-presenting cell which has been antigen-loaded and/or activated/matured by infection with a microbe in which the microbial nucleic acid is modified so that the proliferation of the microbe is attenuated, wherein the microbial gene expression is substantially unaffected, as discussed above. The vaccine compositions of the present invention can be used to stimulate an immune response in an individual. The formulations can be administered to an individual by a variety of administration routes. Methods of administration of such a vaccine composition are known in the art, and include oral, nasal, intraveneous, intradermal, intraperitoneal, intramuscular, intralymphatic and subcutaneous routes of administration. The vaccine compositions may further comprise additional components known in the art to improve the immune response to a vaccine, such as adjuvants or T cell co-stimulatory molecules. The invention also includes medicaments comprising the pharmaceutical compositions of the invention. An individual to be treated with such vaccines, is any vertebrate, preferably a mammal, including domestic animals, sport animals, and primates, including humans. The vaccine may be administered as a prophylactic, where the individual is vaccinated in order to immunize the individual against a particular disease. While the vaccine can be given to any individual, in some instances, such as with cancer vaccines, the individual treated might be limited to those individuals at higher risk of developing a cancer. The vaccine may also be administered as a therapeutic, where the individual having a particular disease is vaccinated in order to improve the immune response to the disease or a disease related protein. In this embodiment, the vaccine may result in a lessening of the physical symptoms associated with the disease. For example, with cancer vaccines, the vaccination may result in stopping the growth of a tumor, preferably a lessening of the mean tumor volume, more preferably elimination of any tumors. In one embodiment, the mean tumor volume decreases by at least about 5%, also about 10%, also about 25%, also about 50%, also about 75%, also about 90% or about 100%. Similarly, the vaccination may result in stopping the metastases of a tumor, preferably resulting in a reduction in the number of tumor metastases. An additional effect of a cancer vaccine would be an extension of the median survival of the individual. In humans, the median survival may be extended by at least about 3 months, also at least about 6 months, or at least about 12 months.

Vaccine formulations are known in the art and include numerous additives, such as preservatives, stabilizers, adjuvants, antibiotics, and other substances. Preservatives, such as thimerosal or 2-phenoxy ethanol, are added to slow or stop the growth of bacteria or fungi resulting from inadvertent contamination, especially as might occur with vaccine vials intended for multiple uses or doses. Stabilizers, such as lactose or monosodium glutamate (MSG), are added to stabilize the vaccine formulation against a variety of conditions, such as temperature variations or a freeze-drying process. Adjuvants, such as aluminum hydroxide or aluminum phosphate, are added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response. Additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences, like CpG, are also potential vaccine adjuvants. Antibiotics, such as neomycin and streptomycin, are added to prevent the potentially harmful growth of germs. Vaccines may also include a suspending fluid such as sterile water or saline. Vaccines may also contain small amounts of residual materials from the manufacturing process, such as cell or bacterial proteins, egg proteins (from vaccines that are produced in eggs), DNA or RNA, or formaldehyde from a toxoiding process.

The efficacy of the vaccines can be evaluated in an individual, for example in mice. A mouse model is recognized as a model for efficacy in humans and is useful in assessing and defining the vaccines of the present invention. The mouse model is used to demonstrate the potential for the effectiveness of the vaccines in any individual. Vaccines can be evaluated for their ability to provide either a prophylactic or therapeutic effect against a particular disease. For example, in the case of infectious diseases, a population of mice can be vaccinated with a desired amount of the appropriate vaccine of the invention, where the microbe expresses an infectious disease associated antigen. This antigen can be from the delivery microbe itself or can be a heterologous antigen. The mice can be subsequently infected with the infectious agent related to the vaccine antigen and assessed for protection against infection. The progression of the infectious disease can be observed relative to a control population (either non vaccinated or vaccinated with vehicle only or a microbe that does not contain the appropriate antigen).

In the case of cancer vaccines, tumor cell models are available, where a tumor cell line expressing a desired tumor antigen can be injected into a population of mice either before (therapeutic model) or after (prophylactic model) vaccination with a microbe of the invention containing the desired tumor antigen. Vaccination with a microbe containing the tumor antigen can be compared to control populations that are either not vaccinated, vaccinated with vehicle, or with a microbe that expresses an irrelevant antigen. In addition, the relative efficacy of the vaccines of the invention can be compared to a population of microbe in which the microbial nucleic acid has not been modified. The effectiveness of the vaccine in such models can be evaluated in terms of tumor volume as a function of time after tumor injection or in terms of survival populations as a function of time after tumor injection (e.g. Example 4). In one embodiment, the tumor volume in mice vaccinated with nucleic acid modified microbe is about 5%, about 10%, about 25%, about 50%, about 75%, about 90% or about 100% less than the tumor volume in mice that are either not vaccinated or are vaccinated with vehicle or a microbe that expresses an irrevelant antigen. In another embodiment, this differential in tumor volume is observed at least about 10, about 17, or about 24 days following the implant of the tumors into the mice. In one embodiment, the median survival time in the mice vaccinated with nucleic acid modified microbe is at least about 2, about 5, about 7 or at least about 10 days longer than in mice that are either not vaccinated or are vaccinated with vehicle or a microbe that expresses an irrelevant antigen. In addition to an effective immune response to the vaccines of the present invention, the modified microbes provide an added level of safety such that a higher dose of the microbe may be administered relative to the corresponding unmodified microbe. In one embodiment of the invention, the vaccination with the nucleic acid modified microbe is done at a dose of microbes that is the same as the dose of the corresponding unmodified microbe. In another embodiment, the vaccination of nucleic acid modified microbe is safely dosed at a level that is at least about 2, about 5, about 10, about 10.sup.2, about 10.sup.3, or at least about 10.sup.4 fold higher than the vaccination dose of the corresponding unmodified microbe, wherein the resulting tumor volume and median survival times discussed above are observed for the nucleic acid modified microbe.

Methods of Use

A variety of methods of using the modified microbes, antigen-presenting cells, vaccines, and pharmaceutical compositions described herein are provided by the present invention. For instance, methods of using the modified microbes, antigen-presenting cells, vaccines, and pharmaceutical compositions described herein to induce immune responses and/or to treat or prevent disease are provided. Method of using the modified microbes and/or mutant strain to prepare vaccines and other compositions are also provided.

For instance, in one aspect, the invention provides a method of inducing an immune response in a host to an antigen, comprising administering to the host an effective amount of a composition comprising a free-living microbe that expresses the antigen, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation. In some embodiments, the composition comprising the microbe is a vaccine. In some embodiments, the composition comprising the microbe is a professional antigen-presenting cell. The antigen may be heterologous or autologous to the microbe as described above. In some embodiments, the nucleic acid of the microbe has been modified by reaction with a nucleic acid targeted compound that reacts directly with the nucleic acid.

The invention also provides a method of inducing an immune response in a host to an antigen, comprising administering to the host an effective amount of a composition comprising a mutant strain of Listeria monoxytogenes that expresses the antigen, wherein the mutant strain comprises a genetic mutation that attenuates its ability to repair its nucleic acid. The antigen may be a Listerial or non-Listerial antigen. In some embodiments, the nucleic acid of the Listeria has been modified so that the microbe is attenuated for proliferation (e.g., by S-59/UVA treatment).

The invention also provides a method of inducing an immune response in a host to an antigen, comprising administering to the host an effective amount of a composition comprising a mutant strain of Bacillus anthracis that expresses the antigen, wherein the mutant strain comprises a genetic mutation that attenuates its ability to repair its nucleic acid. In some embodiments, the nucleic acid of the Bacillus has been modified so that the microbe is attenuated for proliferation (e.g., by S-59/UVA treatment).

The invention also provides a method of preventing or treating a disease in a host, comprising administering to the host an effective amount of a composition comprising a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation. In some embodiments, the composition comprising the microbe is a vaccine. In some embodiments, the composition comprising the microbe is a professional antigen-presenting cell.

The invention also provides a method of preventing or treating a disease in a host, comprising administering to the host an effective amount of a composition comprising a mutant strain of Listeria monocytognes, wherein the mutant strain comprises a genetic mutation that attenuates its ability to repair its nucleic acid. In some embodiments, the nucleic acid of the Listeria has been modified so that the microbe is attenuated for proliferation (e.g., by S-59/UVA treatment). In some embodiments, the disease is an infectious disease. In other embodiments, the disease is cancer.

The invention also provides a method of preventing or treating disease in a host, comprising administering to the host an effective amount of a composition comprising a mutant strain of Bacillus anthracis, wherein the mutant strain comprises a genetic mutation that attenuates its ability to repair its nucleic acid. In some embodiments, the nucleic acid of the Bacillus has been modified so that the microbe is attenuated for proliferation (e.g., by S-59/UVA treatment).

The invention also provides a free-living microbe for medical use, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation and/or the microbe is defective with respect to a DNA repair enzyme. It is understood that medical use encompasses both therapeutic and preventative medical applications (e.g., for use as a vaccine). In some embodiments, the microbe has been modified by reaction with a nucleic acid targeted compound that reacts directly with the nucleic acid so that the microbe is attenuated for proliferation. In some embodiments, the microbe is Listeria monoxytogenes or Bacillus anthracis.

In other aspects, the invention provides a professional antigen-presenting cell for medical use, wherein the antigen-presenting cell comprises a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation and/or the microbe is defective with respect to a DNA repair enzyme. In some embodiments, the microbe has been modified by reaction with a nucleic acid targeted compound that reacts directly with the nucleic acid so that the microbe is attenuated for proliferation. In some embodiments, the microbe is Listeria monocytogenes or Bacillus anthracis.

The invention also provides a mutant Listeria monoxytogenes strain for medical use, wherein the mutant Listeria monoxytogenes strain comprises a genetic mutation that attenuates its ability to repair its nucleic acid.

In addition, the invention provides a mutant Bacillus anthracis strain for medical use, wherein the mutant Bacillus anthracis strain comprises a genetic mutation that attenuates its ability to repair its nucleic acid.

The invention further provides the use of a free-living microbe, wherein the nucleic acid has been modified so that the microbe is attenuated for proliferation, for the manufacture of a medicament for a disease unrelated and/or not caused by the free-living microbe. In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease unrelated to the free-living microbe.

The invention further provides the use of a free-living microbe for the manufacture of a medicament for a disease unrelated and/or not caused the microbe, wherein the microbe is defective with respect to at least one DNA repair enzyme. In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease unrelated to the microbe.

Additionally, the invention provides the use of a professional antigen-presenting cell, wherein the antigen-presenting cell comprises a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation and/or wherein the microbe is defective with respect to at least one DNA repair enzyme, for the manufacture of a medicament for a disease unrelated and/or not caused by the free-living microbe. In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease unrelated to the free-living microbe.

The invention further provides the use of a mutant strain of Listeria monocytogenes, wherein the mutant Listeria monoxytogenes strain comprises a genetic mutation that attenuates its ability to repair its nucleic acid, for the manufacture of a medicament for a disease unrelated and/or not caused by Listeria monocytognes. In some embodiments, the disease is cancer. In some embodiments, the disease is an infectious disease unrelated to the Listeria monocytognes.

In another aspect, the invention provides a method of activating naive T cells, comprising contacting the naive T cells with a professional antigen-presenting cell under suitable conditions and for a sufficient time to activate the naive T cells, wherein the antigen-presenting cell comprises a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation. The contacting step of this method may be performed either in vitro or in vivo. Suitable conditions and a sufficient time for activating the naive T-cells would be known to one of ordinary skill of the art. In addition, examples of such conditions are provided in the specific Examples, below.

A method of loading professional antigen-presenting cells with an antigen is also provided. The method comprises contacting the professional antigen-presenting cells with a free-living microbe that comprises a nucleic acid sequence encoding the antigen, under suitable conditions and for a sufficient time to load the professional antigen-presenting cells (e.g., dendritic cells), wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation and/or the microbe is defective with respect to at least one DNA repair enzyme. The contacting step of the method may be performed in vitro, ex vivo, or in vivo. The antigen may be heterologous or autologous to the microbe. Suitable conditions and a sufficient time for loading antigen-presenting cells would generally be known to one of ordinary skill of the art. In addition, examples of such conditions are provided in the specific Examples, below.

In another aspect, the invention provides a method of activating and/or maturing professional antigen-presenting cells comprising contacting the professional antigen-presenting cells (in vitro, ex vivo, and/or in vivo) with a free-living microbe that comprises a nucleic acid sequence encoding an antigen, under suitable conditions and for a sufficient time to activate and/or bring to maturation the professional antigen-presenting cells, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation. The contacting step of the method may be performed either in vitro or in vivo. The antigen may be heterologous or autologous to the microbe. Suitable conditions and a sufficient time for activating antigen-presenting cells and/or bringing antigen-presenting cells to maturation would generally be known to one of ordinary skill of the art. In addition, examples of such conditions are provided in the specific Examples, below.

In another aspect, the invention provides a method of preventing or treating a disease in a host, comprising the following steps. (a) loading professional antigen-presenting cells with an antigen by contacting the cells with a free-living microbe that comprises a nucleic acid sequence encoding an antigen, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation; and (b) administering an effective amount of a composition comprising the loaded professional antigen-presenting cells to the host.

In still another aspect, the invention provides a method of inducing an immune response to an antigen in a host, comprising the following steps. (a) loading professional antigen-presenting cells with the antigen by contacting the cells with a free-living microbe that comprises a nucleic acid sequence encoding the antigen, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation; and (b) administering an effective amount of a composition comprising the loaded professional antigen-presenting cells to the host.

Kits

The invention further provides kits (or articles of manufacture) comprising the modified microbes and mutant strains of the present invention.

In one aspect, the invention provides a kit comprising both (a) a composition comprising a mutant Listeria monoxytogenes strain comprising a genetic mutation that attenuates its ability to repair its nucleic acid, a mutant Bacillus anthracis strain comprising a genetic mutation that attenuates its ability to repair its nucleic acid, or a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation; and (b) instructions for the use of the composition in the prevention or treatment of a disease in a host. In some embodiments, the instructions are on a label. In other embodiments, the instructions are on an insert contained within the kit.

In another aspect, the invention provides a kit comprising both (a) a composition comprising a mutant Listeria monoxytogenes strain comprising a genetic mutation that attenuates its ability to repair its nucleic acid, a mutant Bacillus anthracis strain comprising a genetic mutation that attenuates its ability to repair its nucleic acid, or a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation; and (b) instructions for the administration of the composition to a host. In some embodiments, the instructions are on a label. In other embodiments, the instructions are on an insert contained within the kit.

In another aspect, the invention provides a kit comprising both (a) a composition comprising a mutant Listeria monoxytogenes strain comprising a genetic mutation that attenuates its ability to repair its nucleic acid, a mutant Bacillus anthracis strain comprising a genetic mutation that attenuates its ability to repair its nucleic acid, or a free-living microbe, wherein the nucleic acid of the microbe is modified so that the microbe is attenuated for proliferation; and (b) instructions for selecting a host to which the composition is to be administered. In some embodiments, the instructions are on a label. In other embodiments, the instructions are on an insert contained within the kit.

In some embodiments of each of the aforementioned aspects, the composition is a vaccine. In some embodiments of each of the aforementioned aspects, the composition is a professional-antigen-presenting cell. In some embodiments of each of the aforementioned aspects, the nucleic acid of the free-living microbe has been modified by reaction with a nucleic acid targeted compound that reacts directly with the nucleic acid. In some embodiments, the microbe has been s-59/UVA treated. In some embodiments, the microbe is defective with respect to a DNA repair enzyme.
 

Claim 1 of 17 Claims

1. A method of preventing or treating a disease in a host, comprising administering to the host an effective amount of a vaccine comprising a modified Listeria monocytogenes bacterium, wherein the modified bacterium comprises (i) psoralen-induced interstrand crosslinks introduced between the strands of genomic DNA double helix, said interstrand crosslinks inhibiting replication of said modified bacterium (ii) one or more genetic mutations in uvrA and uvrB genes inhibiting excision repair of said interstrand crosslinks, and (iii) a nucleic acid sequence encoding a polypeptide heterologous to said Listeria monocytogenes bacterium operably linked to a promoter sequence directing expression of the heterologous polypeptide by the modified bacterium.
 

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