Recombinant nucleic acid molecules, expression cassettes, and bacteria, and methods of use thereof

#The field of this invention relates generally to novel polynucleotides and expression cassettes useful for expression of polypeptides, including heterologous polypeptides, in recombinant bacteria. In particular, this invention relates to recombinant bacteria comprising the novel expression cassettes and/or nucleic acid molecules which are useful in vaccine compositions.

#Microbes have begun to be developed for use as vaccines that deliver heterologous antigens. Heterologous antigen delivery is provided by microbes that have been modified to contain nucleic acid sequences encoding a protein or antigen originating from a different species. Heterologous antigen delivery is especially advantageous for treating or preventing diseases or conditions that result from especially virulent or lethal sources, such as cancer and pathogenic agents (for example, HIV or Hepatitis B), wherein injection of a native infectious agent or cancer cell is potentially deleterious to the recipient organism, and administration of attenuated or killed agent or cell has proven unsuccessful in eliciting an effective immune response, or where sufficient attenuation of the infectious agent or cancer cell cannot be assured with acceptable certainty. Recently, certain bacterial strains have been developed as recombinant vaccines. For instance, an oral vaccine of attenuated Salmonella modified to express Plasmodium berghei circumsporozite antigen has been shown to protect mice against malaria (Aggarwal et al. 1990. J. Exp. Med. 172:1083).

#Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular bacterium that is being developed for use in antigen-specific vaccines due to its ability to prime a potent CD4+/CD8+ T-cell mediated response via both MHC class I and class II antigen presentation pathways. See, for instance, U.S. Pat. Nos. 6,051,237, 6,565,852, and 5,830,702.

#FIG. 1 shows the hly promoter alignment for the Listeria monocytogenes DP-L4056 (SEQ ID NO:1) (bottom sequence) and EGD strains (SEQ ID NO:2) (top sequence).

#FIG. 2 shows the sequence (SEQ ID NO:3) of a polynucleotide encoding a fusion protein comprising the LLO signal peptide, LLO PEST sequence, and the full-length human EphA2 antigen.

#FIG. 3 shows the sequence (SEQ ID NO:4) of the fusion protein encoded by the polynucleotide shown in FIG. 2.

#The present invention provides a variety of polynucleotides including recombinant nucleic acid molecules, expression cassettes, and expression vectors useful for expression and/or secretion of polypeptides, including heterologous polypeptides (e.g. antigens and/or mammalian proteins), in bacteria, such as Listeria. In some embodiments, these polynucleotides can be used for enhanced expression and/or secretion of polypeptides in bacteria. Some of the expression cassettes comprise codon-optimized coding sequences for the polypeptide and/or for the signal peptide. In addition, some of the expression cassettes for use in bacteria contain signal peptide sequences derived from other bacterial sources and/or from a variety of different secretory pathways. Bacteria comprising the expression cassettes are also provided, as are compositions, such as vaccines, containing the bacteria. Methods of using the polynucleotides, bacteria, and compositions to induce an immune response and/or to prevent or treat a condition, such as a disease (e.g. cancer), in a host are also provided.

#The invention is based, in part, on the discovery that codon-optimization of the signal peptide sequence in an expression cassette enhances the expression and/or secretion of a heterologous polypeptide (such as an antigen) from recombinant bacteria (particularly in combination with codon-optimization of the heterologous polypeptide), even when the signal peptide sequence is native to the bacteria (see, e.g., Examples 19 and 27, below). Additionally, it has been discovered that signal peptide sequences from non-secA1 secretory pathways and/or signal peptide sequences from non-Listerial bacterial sources can also be used to effect efficient expression and/or secretion of heterologous polypeptides from Listeria (see, e.g., Examples 19, 27, and 30 below). The invention is also based, in part, on the additional discovery that codon-optimization of the coding sequences of heterologous polypeptides enhances expression and/or secretion of the heterologous polypeptides in Listeria (see e.g., Example 19, below). Enhanced expression and/or secretion of the heterologous protein obtained through optimization of the expression cassette has also been shown to lead to enhanced immunogenicity of the bacteria comprising the optimized expression cassettes (see, e.g., Example 20, below). In addition, expression cassettes encoding protein chimeras comprising a heterologous antigen embedded within an autolysin have been shown to useful in effecting efficient expression and secretion of a heterologous antigen in Listeria (see, e.g., Example 29, below). The autolysin protein chimeras have also been shown to be immunogenic (see, e.g., Example 31A, below). In addition, Listeria comprising codon-optimized expression cassettes and/or expression cassettes comprising non-Listerial signal peptides have also been shown to be immunogenic, reduce tumor volume, and increase survival in a mouse model (see, e.g., Examples 31B-E, below).

#Accordingly, in one aspect, the invention provides a recombinant nucleic acid molecule, comprising a first polynucleotide encoding a signal peptide, wherein the first polynucleotide is codon-optimized for expression in a bacterium, and a second polynucleotide encoding a polypeptide (e.g., an antigen), wherein the second polynucleotide is in the same translational reading frame as the first polynucleotide, and wherein the recombinant nucleic acid molecule encodes a fusion protein comprising the signal peptide and the polypeptide. In some embodiments, the second polynucleotide is codon-optimized as well (typically for expression in the same type of bacteria as the first polynucleotide). In some embodiments, the first polynucleotide or the first and second polynucleotides are codon-optimized for expression in Listeria, Bacillus, Yersinia pestis, Salmonella, Shigella, Brucella, mycobacteria or E. coli. In some embodiments, the polynucleotide(s) is codon-optimized for expression in Listeria, such as Listeria monocytogenes. In some embodiments, the polypeptide encoded by the second polynucleotide is (or comprises) an antigen, which, in some instances, may be a non-bacterial antigen. For instance, the antigen is, in some embodiments a tumor-associated antigen or is derived from such a tumor-associated antigen. For instance, in some embodiments, the antigen is K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA, or is derived from K-Ras, H-Ras, N-Ras, 12-K-Ras, mesothelin, PSCA, NY-ESO-1, WT-1, survivin, gp100, PAP, proteinase 3, SPAS-1, SP-17, PAGE-4, TARP, or CEA. For instance, in some embodiments, the antigen is mesothelin, or an antigenic fragment or antigenic variant of mesothelin. In some other embodiments, the antigen is NY-ESO-1, or an antigenic fragment or antigenic variant of NY-ESO-1. In some embodiments, the antigen is an infectious disease antigen or is derived from an infectious disease antigen. In some embodiments, the signal peptide is bacterial (Listerial or non-Listerial). In some embodiments, the signal peptide encoded by the codon-optimized first polynucleotide is native to the bacterium. In other embodiments, the signal peptide encoded by the codon-optimized first polynucleotide is foreign to the bacterium. In some embodiments, the signal peptide is a secA1 signal peptide, such as an LLO signal peptide from Listeria monocytogenes, a Usp45 signal peptide from Lactococcus lactis, or a Protective Antigen signal peptide from Bacillus anthracis. In some embodiments, the signal peptide is a secA2 signal peptide. For instance, the signal peptide may be the p60 signal peptide from Listeria monocytogenes. In addition, the recombinant nucleic acid molecule optionally comprises a third polynucleotide sequence encoding p60, or a fragment thereof, in the same translational reading frame as the first and second polynucleotides, wherein the second polynucleotide is positioned within the third polynucleotide or between the first and third polynucleotides. In still further embodiments, the signal peptide is a Tat signal peptide, such as a B. subtilis Tat signal peptide (e.g., PhoD). The invention also provides expression cassettes comprising the recombinant nucleic acid molecule and further comprising a promoter operably linked to the recombinant nucleic acid molecule (e.g., to the first and second polynucleotides (and third polynucleotide, if present)). Expression vectors and recombinant bacteria (e.g. Listeria) comprising the expression cassette are also provided, as are pharmaceutical compositions, immunogenic compositions, and vaccines, comprising the bacteria. Methods of using the bacteria or compositions comprising the bacteria to induce an immune response and/or prevent or treat a condition, such as a disease, are also provided. The use of the bacterium in the manufacture of a medicament for inducing an immune response in a host to an antigen, wherein the polypeptide encoded by the second polynucleotide comprises the antigen is also provided.