HK1215260B - Signal peptide fusion partners facilitating listerial expression of antigenic sequences and methods of preparation and use thereof - Google Patents

Description

Signal peptide fusion partners that promote Listeria expression of antigenic sequences and methods for their preparation and use

This application claims priority to U.S. Provisional Patent Application No. 61/746,237, filed December 27, 2012, and U.S. Provisional Patent Application No. 61/780,744, filed March 13, 2013, each of which is hereby incorporated by reference in its entirety, including all tables, figures, and claims.

Background of the Invention

The following discussion of the background technology of the present invention is provided only to help readers understand the present invention, and is not admitted to describe or constitute prior art of the present invention.

Listeria monocytogenes (Lm) is a facultative intracellular bacterium characterized by its ability to induce a profound innate immune response, resulting in robust and highly functional CD4 and CD8 T cell immunity specific for the vaccine-encoded antigen. Lm is a foodborne bacterium with increased pathogenicity in immunocompromised individuals, including patients with cancer or other virally induced immunodeficiencies, pregnant women, the elderly, and infants.

Recombinantly modified Lm vaccine platforms engineered to encode designated antigens associated with selected target pathogens or malignancies have formed the basis of several human clinical trials. Since Listeria can be a pathogenic organism and is particularly so in immunocompromised settings, it is preferred that the administration step include administering an attenuated Listeria that encodes an immunologically active portion of the target antigen. "Attenuation" refers to the process of modifying bacteria to reduce or eliminate their pathogenicity but retain their ability to act as a preventive or therapeutic agent for the target disease. For example, a genetically defined attenuated live LmΔactAΔinlB that is missing two virulence genes and attenuated by >3 log in a mouse model of listeriosis maintains its immunological efficacy and has been shown to induce robust CD4 and CD8 T cell immunity in mouse models of human disease as well as in humans, and has been shown to be safe and well tolerated in clinical settings in patients with different solid tumor malignancies.

Listeria strains are most commonly engineered to secrete tumor antigens as fusions with all or part of a secreted Listeria protein, such as listeriolysin O (LLO) or ActA. It has been suggested that a possible reason for the efficacy of the vaccine constructs may be the presence of an amino acid sequence known as the "PEST" motif within LLO and ActA. The PEST region (P, proline; E, glutamic acid; S, serine; T, threonine) is a hydrophilic amino acid sequence present near the NH2 or COOH terminus of certain proteins. It is believed that it targets proteins that are rapidly degraded by the cellular proteasome. In order to be recognized by T lymphocytes, protein antigens must be converted into short peptides that bind to MHC molecules displayed on the surface of antigen-presenting cells. And indeed, the PEST region of LLO has been shown to be crucial to the success of Listeria vaccines because it can increase the supply of peptides available for presentation by MHC class I molecules by shortening the cellular half-life of the protein.

Summary of the Invention

The present invention provides nucleic acids, expression systems and vaccine strains that provide efficient expression and secretion of target antigens into the cytosol of host cells and that elicit effective CD4 and CD8 T cell responses by functionally linking a signal peptide/secretion chaperone of Listeria or other bacteria as an N-terminal fusion partner in a translation reading frame with a selected recombinantly encoded protein antigen. These bacterial N-terminal signal peptide/secretion chaperone fusion partners direct the secretion of fusion proteins synthesized from recombinant bacteria in infected host mammalian cells. As described below, these N-terminal fusion partners are deleted (by actual deletion, by mutation or by a combination of these approaches) of any PEST sequences originally present in the sequence.

Bacterial N-terminal signal peptide/secretion chaperone fusion partners are modified relative to the native polypeptide sequence in terms of modification of the PEST sequence and optionally in terms of length and/or the presence of a hydrophobic motif outside the signal sequence. For example, ActA can be truncated to delete the C-terminal membrane binding domain, and in certain embodiments, the number of non-antigenic residues in the fusion protein is even further reduced. In addition, one or more hydrophobic residues that are not part of the signal sequence and that form a hydrophobic motif in the polypeptide sequence are deleted from these N-terminal fusion partners (again by actual deletion, by mutation, or by a combination of these approaches). The resulting fusion protein is expressed at high levels and generates a robust immune response to the target antigen contained in the fusion protein.

In a first aspect, the present invention relates to a polynucleotide comprising:

(a) a promoter; and

(b) a nucleic acid operably linked to the promoter, wherein the nucleic acid encodes a fusion protein comprising:

a polypeptide derived by recombinant modification of a secreted Listeria protein sequence comprising, in its unmodified form, a signal sequence and one or more PEST motifs, said modification comprising removal of each of said PEST motifs by or substitution of one or more residues such that the polypeptide lacks any PEST motifs; and

Non-Listeria antigens.

In certain embodiments, the N-terminal signal peptide/secretion chaperone fusion partner can be derived from ActA or LLO. One or more P, E, S, and T residues, and preferably each P, E, S, and T residue, in the PEST motif of the ActA or LLO polypeptide sequence are replaced with residues other than P, E, S, and T. As described below, even the removal of a single residue can make this motif less "PEST-like." Alternatively, one or more P, E, S, and T residues, and preferably each P, E, S, and T residue, in the PEST motif of the ActA or LLO polypeptide sequence can be simply deleted. For example, each P, E, S, and T residue in the PEST motif can be replaced with K or R. The derived polypeptide most preferably retains the signal sequence of the secreted Listeria protein sequence (e.g., ActA or LLO) in unmodified form.

In cases where the secreted Listeria protein sequence is an ActA sequence, it is preferred to delete at least 75% of the PEST motif KTEEQPSEVNTGP. In certain preferred embodiments, the sequence KTEEQPSEVNTGP or KTEEQPSEVNTGPR is deleted. In cases where the secreted Listeria protein sequence is an LLO sequence, it is preferred to delete at least 75% of the PEST motif KENSISSMAPPASPPASPK. In certain preferred embodiments, the sequence KENSISSMAPPASPPASPK or NSISSMAPPASPPASPKTPIEKKHAD is deleted.

Optionally, the sequence forming the hydrophobic motif can be replaced with one or more non-hydrophobic amino acids. Thus, modification of the N-terminal signal peptide/secretion chaperone fusion partner can further include removing one or more hydrophobic domains that are not part of the signal sequence in the secreted Listeria protein sequence; and/or replacing one or more residues within one or more hydrophobic domains that are not part of the signal sequence in the secreted Listeria protein sequence with non-hydrophobic amino acids. For the examples described below, the sequence LIAML in ActA can be replaced with the sequence QDNKR.

As described herein, the N-terminal signal peptide/secretion chaperone fusion partner is optionally truncated relative to the native length of the parent protein (e.g., ActA or LLO). For example, ActA can be truncated to delete the C-terminal membrane binding domain, and in certain embodiments, even further reduce the number of non-antigenic residues in the fusion protein. Similarly, LLO can be truncated before approximately residue 484 to eliminate cholesterol binding, and in certain embodiments, even further reduce the number of non-antigenic residues in the fusion protein.

In preferred embodiments, the secreted Listeria protein sequence is derived from an ActA sequence and the polypeptide comprises at least the first 95 residues of one of the sequences designated dlPEST and dlPESTqdnkr in Figure 2 .

In preferred embodiments, the secreted Listeria protein sequence is derived from an LLO sequence and the polypeptide comprises at least the first 95 residues of one of the sequences designated LLO dlPEST and LLO dl26 in Figure 2 .

In certain embodiments, the promoter provides regulatory sequences that induce expression of the fusion protein in the host cell after the bacterium is introduced into the host organism. By way of example only, the promoter is a PrfA-dependent Listeria monocytogenes promoter. The PrfA-dependent promoter can be selected from the group consisting of inlA promoter, inlB promoter, inlC promoter, hpt promoter, hly promoter, plcA promoter, mpl promoter, and actA promoter.

The non-Listerial antigen portion of the fusion protein of the present invention comprises one or more sequences selected to induce a desired immune response specific for the encoded heterologous antigen (i.e., to reduce, prevent, or ameliorate the symptoms of the condition being treated). In certain embodiments, the non-Listerial antigen comprises one or more sequences encoding a cancer cell, tumor, or infectious agent antigen.

In a related aspect, the polynucleotides of the invention are provided as a component of a plasmid, vector, or the like.

In another related aspect, the present invention provides recombinant Listeria bacteria that have been modified to comprise a polynucleotide of the present invention. In various embodiments, the polynucleotide can be provided in an additional manner or can be integrated into the bacterial genome. The recombinant Listeria bacteria can be further modified to attenuate, for example, by functional deletion of the genomic actA and/or inlB genes of the bacteria. In certain embodiments, the polynucleotide of the present invention is inserted into the genomic actA or inlB gene of the bacteria. The bacteria of the present invention can be used as an expression platform for expressing one or more heterologous genes of the bacteria, for example, for use in generating an immune response to heterologous proteins expressed from those genes. Thus, this aspect can provide a vaccine comprising a recombinant Listeria bacterium and a pharmacologically acceptable excipient.

In yet another related aspect, the invention provides a method for stimulating an immune response to a non-Listerial antigen in a mammal, comprising administering to the mammal an effective amount of a Listeria bacterium described herein, wherein the non-Listerial antigen is expressed in one or more cells of the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 schematically illustrates some of the functional properties of ActA.

FIG2 depicts various modifications of the sequences of ActA and LLO.

FIG3 depicts the locations of PEST motifs in LLO sequences scored using the epestfind algorithm.

FIG4 depicts four PEST motifs in the ActA sequence scored using the epestfind algorithm.

Figure 5 depicts the results of a B3Z T-cell activation assay following immunization with Listeria monocytogenes expressing fusion constructs with various modified ActA and LLO fusion partners.

Figure 6 depicts responses from certain LLO441 (A) and ActAN100 (B) vaccine strains.

Figure 7 depicts several substitutions and deletions for deleting the PEST motif using ActA as a model system.

FIG8 shows the results of modifying the hydrophobic motif LIAML on the hydrophilicity map of ActAN100.

Figure 9 depicts the percentage survival of animals immunized with Listeria monocytogenes expressing a fusion construct having a modified ActAN100 sequence fused to human mesothelin residues 35-621 following challenge with CT-26 tumor cells.

FIG10 depicts a schematic representation of the EGFRvIII _20-40 /NY-ESO-1 _1-165 fusion construct of the present invention and expression of the fusion construct as determined by Western blot.

Figure 11 depicts EGFR- _specific T cell responses determined by intracellular cytokine staining as (A) the percentage of IFN-γ-positive EGFRvIII-specific CD8+ T cells and (B) the absolute number of IFN-γ-positive EGFRvIII-specific _CD8 + T cells per spleen after immunization with Listeria monocytogenes expressing a fusion construct with a modified ActAN100 sequence fused to EGFRvIII 20-40/NY-ESO-1 1-165.

FIG. 12 depicts NY-ESO-1-specific CD8+ T cell responses following immunization with Listeria monocytogenes expressing a fusion construct with a modified ActAN100 sequence fused to EGFRvIII _20-40 /NY-ESO-1 _1-165 .

Detailed Description of the Invention

The present invention relates to compositions and methods for preparing antigenic fusion proteins for expression in Listeria bacteria. The present invention can provide attenuated bacterial vaccine strains with favorable safety profiles for treating or preventing diseases with risk-benefit profiles unsuitable for attenuated live vaccines. While Listeria monocytogenes is described in detail below, those skilled in the art will appreciate that the methods and compositions described herein are generally applicable to Listeria species.

Listeria monocytogenes (Lm) is a facultative intracellular bacterium characterized by its ability to induce a profound innate immune response, resulting in robust and highly functional CD4 and CD8 T cell immunity specific for the vaccine-encoded antigen. Lm is a foodborne bacterium with increased pathogenicity in immunocompromised individuals, including patients with cancer or other virally induced immunodeficiencies, pregnant women, the elderly, and infants. To elicit the desired CD8 T cell response, Lm-based vaccines must retain the ability to escape from the vacuole of infected dendritic cells (DCs) in a process mediated by the expression of a pore-forming cytolysin called listeriolysin O (LLO), and the desired antigens must be engineered to be expressed and secreted from the bacteria into the cytoplasm, where they are subsequently processed and presented on MHC class I molecules.

In the development of Lm vaccine strains, a discrepancy clearly exists between the level of antigen expression and the requirement for antigen processing. While the immunological efficacy of Lm-based vaccines is directly related to the level of antigen expression and secretion in host cells, it has been shown that effective MHC class I and class II priming and induction of antigen-specific immune responses depends on rapid turnover of the antigen by the cell's proteolytic machinery.

Provided herein are antigen expression cassettes that cause efficient expression and secretion of the encoded antigen into the cytosol of the host cell, and by functionally linking the signal peptide/secretion chaperone of Listeria or other bacteria as an N-terminal fusion partner with the secreted recombinant encoded protein antigen in the translation reading frame to induce effective CD4 and CD8 T cell responses. These bacterial N-terminal signal peptide/secretion chaperone molecule fusion partners guide the secretion of the fusion protein synthesized from the recombinant bacteria in infected host mammalian cells. As described below, any PEST sequence originally present in these N-terminal fusion partners is deleted (by actual deletion, by mutation, or by a combination of these approaches). Optionally, hydrophobic residues that are not part of the signal sequence in these N-terminal fusion partners are deleted (again by actual deletion, by mutation, or by a combination of these approaches). The resulting fusion protein is expressed at a high level and produces a robust immune response to the target antigen contained in the fusion protein.

In a preferred embodiment, the fusion protein is functionally linked to the Lm PrfA inducible promoter. Preferred non-limiting examples are the hly promoter, which drives expression of the listeriolysin O (LLO) protein, and the actA promoter, which drives expression of the ActA protein, in wild-type Listeria monocytogenes. The PrfA-dependent promoter is induced in infected mammalian host cells and the functionally linked protein is synthesized at high levels. The temporally regulated high-level expression of the encoded fusion protein comprising the selected antigen functionally linked to the PrfA-dependent promoter in the host cells promotes antigen processing and presentation, thereby eliciting an optimal Lm vaccine-induced immune response.

As described below, preferred non-limiting examples of N-terminal signal peptide/secretion chaperone fusion partners are modified LLO or ActA proteins derived from Listeria monocytogenes. LLO and ActA N-terminal signal peptide/secretion chaperone fusion partners can be functionally linked to a Listeria PrfA-dependent promoter (e.g., hly promoter or actA promoter). In a preferred embodiment, ActA and LLO N-terminal signal peptide/secretion chaperone fusion partners lacking any PEST-like sequence motifs are provided for in-frame fusion with any selected antigen sequence. The PEST ^- N-terminal fusion partners are referred to herein as PEST ^- ActA and PEST ^- LLO.

Figure 1 schematically illustrates certain functional properties of ActA. The underlined region illustrates the location of the PEST sequence within the native ActA sequence. In certain embodiments, the N-terminal signal peptide/secretion chaperone is derived from ActA in that it comprises the signal sequence of ActA and is truncated at approximately amino acid residue 389 of ActA to delete the C-terminal domain containing the transmembrane region. As used herein, the term "approximately" in this context refers to +/- 25 amino acid residues.

As used herein, the term "derived" refers to the removal of the native PEST sequence of the Listeria protein and, optionally, truncated relative to the native length and/or modified to one or more hydrophobic motifs outside the signal sequence. For example, ActA can be truncated to delete the C-terminal membrane binding domain, and in certain embodiments, to even further reduce the number of non-antigenic residues in the fusion protein. In addition, one or more hydrophobic residues that are not part of the signal sequence and that form a hydrophobic motif in the polypeptide sequence are deleted from these N-terminal fusion partners (again, by actual deletion, by mutation, or by a combination of these approaches). As described below, the resulting fusion proteins are expressed at high levels and generate a robust immune response to the target antigen contained in the fusion protein.

Similarly, native LLO contains 529 residues and includes a 25-residue signal sequence followed by four domains. Domain 4 is approximately residues 415-529 and contains the cholesterol binding region. Domain 1 contains a single PEST sequence. In certain embodiments, the N-terminal signal peptide/secretion chaperone is derived from LLO in that it contains the signal sequence of LLO and is truncated approximately before residue 484 to eliminate cholesterol binding. As used herein, the term "derived" in this context refers to being modified relative to the native LLO sequence in terms of length, the presence of the PEST sequence, and the presence of a hydrophobic motif outside the signal sequence. Preferably, the modified ActA is truncated approximately at residue 441.

As demonstrated below, the PEST sequence and hydrophobic domain can be functionally deleted by removing them or by non-conservative substitution of residues or by a combination of these approaches. By way of example only, the following examples demonstrate the substitution of the LIAML hydrophobic motif in ActA by the sequence QDNKR; and the actual deletion of all or part of the ActA PEST sequence.

It should be understood that the present invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the accompanying drawings. The present invention is capable of embodiments other than those described and can be practiced and implemented in various ways. It should also be understood that the phraseology and terminology used herein and in the abstract are for illustrative purposes only and should not be construed as limiting.

Therefore, those skilled in the art will appreciate that the concept on which this disclosure is based can be readily utilized as a basis for designing other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions as long as they do not depart from the spirit and scope of the present invention.

1. Definition

Abbreviations used to indicate mutations in genes or in bacteria containing such genes are as follows. For example, the abbreviation "Listeria monocytogenes ΔactA" means a deletion of part or all of the actA gene. The delta symbol (Δ) means a deletion. Abbreviations including a superscript minus sign (Listeria ^ActA- ) mean that the actA gene has been mutated, for example, by deletion, point mutation, or frameshift mutation, but not limited to these types of mutations.

As used herein, "administering" refers to, but is not limited to, contacting an exogenous ligand, reagent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition with a subject, cell, tissue, organ, or biological fluid, etc. "Administering" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, placebo methods, and experimental methods. Treatment of cells encompasses contacting an agent with a cell as well as contacting an agent with a fluid, wherein the fluid is in contact with the cell. "Administering" also encompasses in vitro and ex vivo treatment of a cell, for example, by a reagent, diagnostic agent, binding composition, or by another cell.

When referring to a ligand and a receptor, an "agonist" encompasses a molecule, combination of molecules, complex, or combination of agents that stimulates a receptor. For example, a granulocyte-macrophage colony-stimulating factor (GM-CSF) agonist can encompass GM-CSF, a mutein or derivative of GM-CSF, a peptide mimetic of GM-CSF, a small molecule that mimics the biological function of GM-CSF, or an antibody that stimulates the GM-CSF receptor.

When referring to ligands and receptors, "antagonists" encompass molecules, combinations of molecules, or complexes that inhibit, counteract, downregulate, and/or desensitize a receptor. An "antagonist" encompasses any agent that inhibits the constitutive activity of a receptor. Constitutive activity is activity that occurs in the absence of a ligand/receptor interaction. An "antagonist" also encompasses any agent that inhibits or prevents the stimulated (or regulated) activity of a receptor. For example, GM-CSF receptor antagonists include (without intending any limitation) antibodies that bind to the ligand (GM-CSF) and prevent it from binding to the receptor, or antibodies that bind to the receptor and prevent the ligand from binding to the receptor, or wherein the antibody locks the receptor into an inactive conformation.

As used herein, an "analog" or "derivative" with respect to a peptide, polypeptide, or protein refers to another peptide, polypeptide, or protein that has similar or identical functions as the original peptide, polypeptide, or protein, but does not necessarily contain a similar or identical amino acid sequence or structure of the original peptide, polypeptide, or protein. Analogs preferably meet at least one of the following requirements: (a) a proteinaceous agent having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the original amino acid sequence; (b) a proteinaceous agent encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding the original amino acid sequence; and (c) a proteinaceous agent encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to a nucleotide sequence encoding the original amino acid sequence.

"Antigen presenting cells" (APCs) are cells of the immune system that present antigens to T cells. APCs include dendritic cells, monocytes, macrophages, marginal zone Kupffer cells, microglia, Langerhans cells, T cells, and B cells. Dendritic cells occur in at least two lineages. The first lineage encompasses pre-DC1, bone marrow DC1, and mature DC1. The second lineage encompasses CD34 ⁺ CD45RA ^- early multipotent progenitor cells, CD34 ⁺ CD45RA ⁺ cells, CD34 ⁺ CD45RA ⁺ CD4 ⁺ IL-3Rα ⁺ pre-DC2 cells, CD4 ⁺ CD11c ^- plasmacytoid pre-DC2 cells, human lymphoid DC2-like plasmacytoid DC2, and mature DC2.

"Attenuation" and "attenuated" encompass genes in bacteria, viruses, parasites, infectious organisms, prions, tumor cells, infectious organisms, etc. that are modified to reduce the toxicity to the host. The host can be a human or animal host, or an organ, tissue, or cell. As a non-limiting example, bacteria can be attenuated to reduce binding to a host cell, reduce propagation from one host cell to another host cell, reduce extracellular growth, or reduce intracellular growth in a host cell. Attenuation can be assessed by measuring, for example, one or more signs of toxicity, LD ₅₀ , clearance from an organ, or a competitive index (see, for example, Auerbuch et al. (2001) Infect. Immunity 69:5953-5957). Typically, attenuation results in an increase in _LD50 and/or an increase in clearance rate of at least 25%; more typically, an increase of at least 50%; most typically, an increase of at least 100% (1-fold); normally, an increase of at least 5-fold; more typically, an increase of at least 10-fold; most typically, an increase of at least 50-fold; frequently, an increase of at least 100-fold; more typically, an increase of at least 500-fold; and most typically, an increase of at least 1000-fold; typically, an increase of at least 5000-fold; more typically, an increase of at least 10,000-fold; and most typically, an increase of at least 50,000-fold; and most typically, an increase of at least 100,000-fold.

"Attenuated gene" encompasses genes that mediate toxicity, pathology or virulence to the host, grow in the host or survive in the host, wherein the gene mutates in a manner to alleviate, reduce or eliminate toxicity, pathology or virulence. The reduction or elimination can be assessed by comparing the toxicity or toxicity mediated by the mutant gene with the toxicity or toxicity mediated by the non-mutant (or parent) gene. "Mutant gene" encompasses deletions, point mutations and frameshift mutations in gene regulatory regions, gene coding regions, gene non-coding regions or any combination thereof.

"Conservatively modified variants" apply to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids encoding the same amino acid sequence or an amino acid sequence having one or more conservative substitutions. An example of a conservative substitution is exchanging an amino acid from one of the following groups for another amino acid from the same group (U.S. Patent No. 5,767,063 issued to Lee et al.; Kyte and Doolittle (1982) J. Mol. Biol. 157: 105-132). In contrast, a non-conservative substitution is exchanging an amino acid from one of the following groups for another amino acid from a different group.

(1) Hydrophobicity: norleucine, Ile, Val, Leu, Phe, Cys, Met;

(2) Neutral hydrophilicity: Cys, Ser, Thr;

(3) Acidic: Asp, Glu;

(4) Basic: Asn, Gln, His, Lys, Arg;

(5) Residues that affect chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe; and

(7) Small amino acids: Gly, Ala, Ser.

An "effective amount" encompasses, but is not limited to, an amount that can improve, reverse, alleviate, prevent, or diagnose the symptoms or signs of a medical condition or disorder. Unless expressly or otherwise required by context, an "effective amount" is not limited to the minimum amount sufficient to improve the condition.

"Extracellular fluid" encompasses, for example, serum, plasma, blood, interstitial fluid, cerebrospinal fluid, secretions, lymph, bile, sweat, fecal matter, and urine."Extracellular fluid" may include colloids or suspensions, such as whole blood or coagulated blood.

In the context of polypeptides, the term "fragment" includes peptides or polypeptides that comprise an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of a larger polypeptide.

"Gene" refers to a nucleic acid sequence encoding an oligopeptide or polypeptide. An oligopeptide or polypeptide may be biologically active, antigenically active, inactive, or antigenically inactive, etc. The term gene encompasses, for example, the sum of an open reading frame (ORF) encoding a specific oligopeptide or polypeptide; the sum of an ORF plus nucleic acids encoding introns; the sum of an ORF and an operably linked promoter; the sum of an ORF and an operably linked promoter and any introns; the sum of an ORF and an operably linked promoter, introns, and promoter, as well as other regulatory elements (e.g., enhancers). In certain embodiments, "gene" encompasses any cis sequence required for regulating gene expression. The term gene may also refer to a nucleic acid encoding an antigenic peptide or an antigenically active fragment of a peptide, oligopeptide, polypeptide, or protein. The term gene does not necessarily mean that the encoded peptide or protein has any biological activity, or even that the peptide or protein has antigenic activity. Nucleic acid sequences encoding non-expressible sequences are generally considered pseudogenes. The term gene also encompasses nucleic acid sequences encoding ribonucleic acids such as rRNA, tRNA, or ribozymes.

"Growth" of bacteria such as Listeria encompasses, without limitation, bacterial physiological functions and gene functions related to colonization, replication, increased protein content, and/or increased lipid content. Unless explicitly or otherwise indicated by the context, the growth of Listeria encompasses the growth of bacteria outside the host cell as well as the growth within the host cell. Growth-related genes include (without implication of any limitation) genes that mediate energy production (e.g., glycolysis, Krebs cycle, cytochrome), assimilation and/or dissimilation of amino acids, carbohydrates, lipids, minerals, purines, and pyrimidines, nutrient transport, transcription, translation, and/or replication. In some embodiments, the "growth" of Listeria refers to the intracellular growth of Listeria, i.e., growth within a host cell such as a mammalian cell. Although the intracellular growth of Listeria can be measured by optical microscopy or colony forming unit (CFU) assay, growth is not limited by any measurement technique. Biochemical parameters such as the amount of Listeria antigens, Listeria nucleic acid sequences, or lipids specific to Listeria can be used to assess growth. In some embodiments, the gene that mediates growth is a gene that specifically mediates intracellular growth. In some embodiments, genes that specifically mediate intracellular growth include but are not limited to genes whose inactivation reduces the intracellular growth rate, but does not significantly, substantially or significantly reduce the rate of extracellular growth (e.g., growth in broth), or genes whose inactivation reduces the intracellular growth rate to a higher degree than that of reducing the extracellular growth rate. To provide non-limiting examples, in some embodiments, genes whose inactivation reduces the intracellular growth rate to a higher degree than that of extracellular growth include cases where inactivation reduces intracellular growth to less than 50% of a normal value or maximum value, while reducing extracellular growth to only 1-5%, 5-10% or 10-15% of the maximum value. In certain aspects, the present invention encompasses Listeria that is attenuated in intracellular growth but not attenuated in extracellular growth, Listeria that is not attenuated in intracellular growth and not attenuated in extracellular growth, and Listeria that is not attenuated in intracellular growth but attenuated in extracellular growth.

A "labeled" composition can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical, isotopic, or chemical methods. For example, useful labels include ³² P, ³³ P, ³⁵ S, ¹⁴ C, ³ H, ¹²⁵ I, stable isotopes, epitope tags, fluorescent dyes, electron-dense reagents, substrates or enzymes (e.g., for enzyme-linked immunosorbent assays), or fluorettes (see, e.g., Rozinov and Nolan (1998) Chem. Biol. 5:713-728).

As used herein, "hydrophobic motif" refers to a group of continuous amino acid residues that exhibit hydrophobic characteristics by hydrophilicity analysis in the context of being a part of the entire protein. "Hydrophilicity analysis" refers to the analysis of polypeptide sequences by the method of Kyte and Doolittle ("A Simple Method for Displaying the Hydropathic Character of a Protein". J. Mol. Biol. 157 (1982) 105-132). In this method, a hydrophobicity score of 4.6 to -4.6 is given to each amino acid. A score of 4.6 is the most hydrophobic, and a score of -4.6 is the most hydrophilic. The window size is then set. The window size is the number of amino acids that will be averaged and assigned to the first amino acid in the window for the hydrophobicity score. The calculation begins at the first amino acid window and calculates the average value of all hydrophobicity scores in the window. The window then moves down one amino acid and calculates the average value of all hydrophobicity scores in the second window. This pattern continues to the end of the protein, calculating the average score of each window and assigning it to the first amino acid in the window. The average value is then plotted on the graph. The y-axis represents the hydrophobicity score and the x-axis represents the window number. The following hydrophobicity scores were used for the 20 common amino acids.

"Ligand" refers to a small molecule, peptide, polypeptide, or membrane-associated or membrane-bound molecule that is an agonist or antagonist of a receptor. "Ligand" also encompasses binding agents that are not agonists or antagonists and that do not have agonist or antagonist properties. Conventionally, when a ligand is bound to a membrane on a first cell, a receptor typically appears on a second cell. The second cell may have the same identity (homonym) as the first cell, or it may have a different identity (synonym). The ligand or receptor may be completely intracellular, i.e., it may remain in the cytosol, the nucleus, or in some other intracellular compartment. The ligand or receptor may change its position, for example, from an intracellular compartment to the outside of the plasma membrane. The complex of a ligand and a receptor is called a "ligand receptor complex." When a ligand and a receptor participate in a signal transduction pathway, the ligand appears at an upstream position in the signal transduction pathway, while the receptor appears at a downstream position in the signal transduction pathway.

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded, double-stranded, or multi-stranded form. Non-limiting examples of nucleic acids are, for example, cDNA, mRNA, oligonucleotides, and polynucleotides. A particular nucleic acid sequence may also implicitly encompass "allelic variants" and "splice variants."

"Operably linked," in the context of a promoter and a nucleic acid encoding mRNA, means that the promoter is available to initiate transcription of the nucleic acid.

The terms "percent sequence identity" and "% sequence identity" refer to the percentage of sequence similarity found by comparing or aligning two or more amino acid or nucleic acid sequences. Percent identity can be determined by directly comparing the sequence information between the two molecules by aligning the sequences, counting the number of exact matches between the two aligned sequences, dividing by the length of the shorter sequence and multiplying the result by 100. The algorithm used to calculate sequence identity is the Smith-Waterman homology search algorithm (see, e.g., Kann and Goldstein (2002) Proteins 48:367-376; Arslan et al. (2001) Bioinformatics 17:327-337).

When referring to a polypeptide, "purified" and "isolated" mean that the polypeptide is present in the substantial absence of other biomacromolecules with which it is associated in nature. As used herein, the term "purified" means that the identified polypeptide often comprises at least 50% by weight of the polypeptide present, more often at least 60% by weight, usually at least 70% by weight, more usually at least 75% by weight, most usually at least 80% by weight, typically at least 85% by weight, more usually at least 90% by weight, most usually at least 95% by weight, and routinely at least 98% by weight or greater. Water, buffers, salts, detergents, reducing agents, protease inhibitors, stabilizers (including added proteins, such as albumin), and excipients, as well as the weight of molecules with a molecular weight of less than 1000, are not generally used in determining the purity of a polypeptide. See, for example, U.S. Patent No. 6,090,611 issued to Covacci et al. for a discussion of purity.

"Peptide" refers to a short amino acid sequence in which amino acids are linked to each other by peptide bonds. The peptide can be free or in combination with another part (e.g., a macromolecule, a lipid, an oligosaccharide or polysaccharide, and/or a polypeptide). When the peptide is incorporated into a polypeptide chain, the term "peptide" can still be used to specifically refer to the short amino acid sequence. A "peptide" can be linked to another part by a peptide bond or some other type of bonding. The length of a peptide is at least two amino acids and is typically less than about 25 amino acids in length, with the maximum length depending on convention or context. The terms "peptide" and "oligopeptide" are used interchangeably.

" PEST motif " is defined herein as a hydrophilic segment of at least 12 amino acid lengths, which has high local concentrations of P, E, S and T amino acids, and is scored as effective PEST motifs according to the epestfind algorithm. Negatively charged amino acids are clustered within these motifs, while positively charged amino acids arginine (R), histidine (H) and lysine (K) are typically prohibited. The epestfind algorithm more strictly defines the final criterion because the PEST motif side joints are positively charged amino acids. All amino acids between the positively charged side portions are counted and only those containing a large number of amino acid motifs equal to or higher than the window size parameter are further considered. In addition, all ' effective ' PEST districts are required to contain at least one proline (P), an aspartic acid (D) or glutamic acid (E) and at least one serine (S) or threonine (T). Sequences that do not meet the above criteria are classified as ' invalid ' PEST motifs.

" Effective " PEST motifs are refined by means of a scoring parameter based on the local enrichment of key amino acids and the hydrophobicity of the motif. The enrichment of D, E, P, S and T is expressed as mass percent (w/w) and corrected for 1 equivalent of D or E, 1 equivalent of P and 1 equivalent of S or T. The calculation of hydrophobicity follows the method of J.Kyte and R.F.Doolittle in principle. In order to simplify the calculation, the Kyte-Doolittle hydrophilicity index initially in the range of -4.5 (for arginine) to +4.5 (for isoleucine) is converted to a positive integer. This is achieved by the following linear transformation, thereby obtaining a value of 0 (for arginine) to 90 (for isoleucine).

Hydrophilicity index = 10 * Kyte-Doolittle hydrophilicity index + 45

The hydrophobicity of the motif is calculated as the sum of the product of the molar percentage of each amino acid species and the hydrophobicity index. The desired PEST score is obtained as a combination of the local enrichment term and the hydrophobicity term, as expressed by the following formula:

PEST score = 0.55*DEPST-0.5*Hydrophobicity Index.

In addition, the epestfind algorithm includes a correction for the tyrosine hydrophilicity index proposed by Robert H. Stellwagen of the University of Southern California. However, PEST scores can range from -45 (for polyisoleucine) to approximately +50 (for polyaspartic acid plus one proline and one serine). 'Valid' PEST motifs are those with a score above a threshold of 5.0 and are considered to have real biological value.

"Protein" generally refers to the amino acid sequence that makes up a polypeptide chain. Protein can also refer to the three-dimensional structure of a polypeptide. "Denatured protein" refers to a partially denatured polypeptide with some residual three-dimensional structure, or alternatively, refers to a substantially random three-dimensional structure, i.e., completely denatured. The present invention encompasses reagents for and methods of using polypeptide variants, including, for example, glycosylation, phosphorylation, sulfation, disulfide bond formation, deamidation, isomerization, cleavage points in signal or leader sequence processing, covalently and non-covalently bound cofactors, oxidation variants, and the like. The formation of disulfide-linked proteins has been described (see, e.g., Woycechowsky and Raines (2000) Curr. Opin. Chem. Biol. 4: 533-539; Creighton et al. (1995) Trends Biotechnol. 13: 18-23).

"Recombinant" as used in reference to, for example, nucleic acids, cells, animals, viruses, plasmids, vectors, etc., indicates modification by introduction of exogenous non-natural nucleic acids, alteration of natural nucleic acids, or by derivatization in whole or in part from recombinant nucleic acids, cells, viruses, plasmids, or vectors. Recombinant proteins refer to proteins derived from recombinant nucleic acids, viruses, plasmids, vectors, etc. "Recombinant bacteria" encompass bacteria in which the genome has been engineered by recombinant methods, such as by mutation, deletion, insertion, and/or rearrangement. "Recombinant bacteria" also encompass bacteria that have been modified to include a recombinant extragenomic nucleic acid, such as a plasmid or a second chromosome, or bacteria in which an existing extragenomic nucleic acid has been altered.

"Sample" refers to a sample from a human, animal, placebo, or research sample, such as a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid, or solidified material. A "sample" can be tested in vivo, e.g., without removal from the human or animal body, or can be tested in vitro. For example, a sample can be tested after processing by a histological method. A "sample" also refers to, for example, cells comprising a fluid or tissue sample, or cells separated from a fluid or tissue sample. A "sample" can also refer to cells, tissues, organs, or fluids that have just been collected from a human or animal, or to processed or stored cells, tissues, organs, or fluids.

"Selectable markers" encompass nucleic acids that allow one to select or eliminate cells containing the selectable marker. Examples of selectable markers include, but are not limited to, for example: (1) nucleic acids encoding products that confer resistance to otherwise toxic compounds (e.g., antibiotics) or encoding sensitivity to otherwise harmless compounds (e.g., sucrose); (2) nucleic acids encoding products that are otherwise deficient in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) nucleic acids encoding products that inhibit the activity of a gene product; (4) nucleic acids encoding products that can be readily identified (e.g., phenotypic markers, e.g., β-galactosidase, green fluorescent protein (GFP), cell surface proteins, epitope tags, FLAG tags); (5) nucleic acids encoding products that can be identified by hybridization techniques (e.g., PCR or molecular beacons).

When referring to a ligand/receptor, nucleic acid/complementary nucleic acid, antibody/antigen or other binding pair (e.g., a cytokine and a cytokine receptor), "specific" or "selective" binding indicates a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biological agents. Thus, under specified conditions, a specified ligand binds to a specific receptor without binding to other proteins present in the sample in significant amounts. Specific binding can also mean that a binding compound, nucleic acid ligand, antibody or binding composition, for example, derived from an antigen binding site of an antibody of the intended method, binds to its target with an affinity that is often at least 25% greater, more often at least 50% greater, most often at least 100% greater (1-fold), normally at least 9-fold greater, more normally at least 19-fold greater, and most normally at least 99-fold greater than the affinity for any other binding compound.

In typical embodiments, the affinity of the antibody is greater than about 10 ⁹ liters/mol, as determined, for example, by Scatchard analysis (Munsen et al. (1980) Analyt. Biochem. 107: 220-239). Those skilled in the art recognize that some binding compounds can specifically bind to more than one target, for example, an antibody specifically binds to its antigen, specifically binds to a lectin through the antibody's oligosaccharides, and/or specifically binds to an Fc receptor through the antibody's Fc region.

"Spreading" of bacteria encompasses "cell-to-cell spread" such as, for example, vesicle-mediated, i.e., the transfer of bacteria from a first host cell to a second host cell. Functions involved in spreading include, but are not limited to, for example, the formation of actin tails, the formation of pseudopodia-like extensions, and the formation of double-membrane vacuoles.

As used herein, the term "subject" refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary diseases. In certain embodiments, the subject is a "patient," i.e., a living human receiving medical care for a disease or condition. This includes humans undergoing investigation for signs of pathology in an undefined disease.

A "target site" of a recombinase is a nucleic acid sequence or region that is recognized, bound to, and/or acted upon by the recombinase (see, e.g., U.S. Patent No. 6,379,943 issued to Graham et al.; Smith and Thorpe (2002) Mol. Microbiol. 44:299-307; Groth and Calos (2004) J. Mol. Biol. 335:667-678; Nunes-Duby et al. (1998) Nucleic Acids Res. 26:391-406).

A "therapeutically effective amount" is defined as an amount of an agent or pharmaceutical composition sufficient to induce a desired immune response specific for the encoded heterologous antigen, to demonstrate a patient benefit, i.e., to reduce, inhibit, or ameliorate the symptoms of the condition being treated. When the agent or pharmaceutical composition comprises a diagnostic agent, a "diagnostically effective amount" is defined as an amount sufficient to produce a signal, image, or other diagnostic parameter. The effective amount of a pharmaceutical formulation will vary depending on a variety of factors, such as the sensitivity of the subject, the age, sex, and weight of the subject, and the subject's idiosyncratic responses (see, e.g., U.S. Pat. No. 5,888,530 issued to Netti et al.).

"Treatment" (with respect to a condition or disease) is a method for obtaining a beneficial or expected result, preferably including a clinical result. For the purposes of the present invention, a beneficial or expected result with respect to a disease includes, but is not limited to, one or more of the following: improving the condition associated with the disease, curing the disease, reducing the severity of the disease, slowing the progression of the disease, alleviating one or more symptoms associated with the disease, improving the quality of life of the patient, and/or prolonging survival. Similarly, for the purposes of the present invention, a beneficial or expected result with respect to a condition includes, but is not limited to, one or more of the following: improving the condition, curing the condition, reducing the severity of the condition, slowing the progression of the condition, alleviating one or more symptoms associated with the condition, improving the quality of life of the patient, and/or prolonging survival.

"Vaccine" covers preventive vaccines. Vaccines also cover therapeutic vaccines, such as vaccines administered to mammals containing conditions or diseases associated with the antigens or epitopes provided by the vaccine. Many bacterial species have been developed for use as vaccines and can be used in the present invention, including but not limited to Shigella flexneri, Escherichia coli, Listeria monocytogenes, Yersinia enterocolitica, Salmonella typhimurium, Salmonella typhi or mycobacterium species. This list is not intended to be limiting. See, for example, WO04/006837; WO07/103225; and WO07/117371, each of which is hereby incorporated by reference in its entirety (including all tables, figures and claims). The bacterial vector used in the vaccine composition can be a facultative intracellular bacterial vector. The bacteria can be used to deliver the polypeptides described herein to antigen presenting cells in a host organism.As described herein, Listeria monocytogenes provides a preferred vaccine platform for the expression of the antigens of the present invention.

Antigen construct

Target antigen

A preferred feature of the fusion proteins described herein is that they are capable of eliciting an innate immune response to the antigen and an antigen-specific T cell response when recombinantly expressed in a host via a Listeria monocytogenes vaccine platform. For example, Listeria monocytogenes expressing antigens as described herein can induce type 1 interferon (IFN-α/β) and a series of co-regulated chemokines and cytokine proteins that shape the nature of the vaccine-induced immune response. In response to this immune stimulation, NK cells and antigen-presenting cells (APCs) are recruited to the liver according to an intravenous inoculation route, or alternatively, to the inoculation site according to other inoculation routes (e.g., by muscle, subcutaneous, or intradermal immunization routes). In certain embodiments, 24 hours after the vaccine platform of the present invention is delivered to a subject, the vaccine platform induces an increase in serum concentrations of one or more, preferably all, cytokines and chemokines selected from IL-12p70, IFN-γ, IL-6, TNFα, and MCP-1; and induces CD4+ and/or CD8+ antigen-specific T cell responses to one or more antigens expressed by the vaccine platform. In other embodiments, the vaccine platform of the present invention also induces the maturation of resident immature hepatic NK cells, as demonstrated by upregulation of activation markers such as DX5, CD11b, and CD43 in a mouse model system, or by measuring NK cell-mediated cytolytic activity using ⁵¹ Cr-labeled YAC-1 cells as target cells.

The ability of Listeria monocytogenes to act as a vaccine vector has been reviewed in Wesikirch et al., Immunol. Rev. 158: 159-169 (1997). Many desirable natural biological features of Listeria monocytogenes make it an attractive platform for therapeutic vaccines. The main rationale is that the intracellular life cycle of Listeria monocytogenes enables effective stimulation of CD4+ and CD8+ T cell immunity. A variety of pathogen-associated molecular pattern (PAMP) receptors (including TLRs (TLR2, TLR5, TLR9)), nucleotide-binding oligomerization domains (NODs), and stimulators of interferon genes (STINGs) are triggered in response to post-infection interactions with Listeria monocytogenes macromolecules, leading to full activation of innate immune effectors and release of Th-1 polarized cytokines, thereby profoundly affecting the occurrence of CD4+ and CD8+ T cell responses to expressed antigens.

Recently, Listeria monocytogenes strains have been developed as efficient intracellular delivery vehicles for heterologous proteins, providing delivery of antigens to the immune system to induce immune responses to clinical conditions where injection of pathogenic agents is not permitted (e.g., cancer and HIV). See, for example, U.S. Patent No. 6,051,237; Gunn et al., J. Immunol., 167:6471-6479 (2001); Liau et al., Cancer Research, 62:2287-2293 (2002); U.S. Patent No. 6,099,848; WO99/25376; WO96/14087; and U.S. Patent No. 5,830,702), each of which is hereby incorporated by reference in its entirety (including all tables, figures, and claims). It has also been demonstrated that a recombinant Listeria monocytogenes vaccine expressing lymphocytic choriomeningitis virus (LCMV) antigen was shown to induce protective cell-mediated immunity to the antigen (Shen et al., Proc. Natl. Acad. Sci. USA, 92:3987-3991 (1995).

In certain embodiments, the Listeria monocytogenes used in the vaccine compositions of the present invention comprises attenuating mutations in actA and/or inlB, and preferably a complete or partial deletion of actA and inlB (referred to herein as "LmΔactA/ΔinlB"), and contains recombinant DNA encoding the expression of one or more antigens of interest. The antigens are preferably under the control of bacterial expression sequences and stably integrated into the Listeria monocytogenes genome.

The present invention also encompasses Listeria species attenuated by at least one regulatory factor (e.g., a promoter or transcription factor). The following is about promoters. ActA expression is regulated by two different promoters (Vazwuez-Boland et al. (1992) Infect. Immun. 60:219-230). InlA and InlB expression are co-regulated by five promoters (Lingnau et al. (1995) Infect. Immun. 63:3896-3903). Transcription of many Listeria monocytogenes genes (e.g., hly, plcA, ActA, mpl, prfA, and iap) requires the transcription factor prfA. The regulatory properties of PrfA are mediated by, for example, the PrfA-dependent promoter (PinlC) and the PrfA cassette. In certain embodiments, the present invention provides nucleic acids encoding an inactivated, mutated, or deleted ActA promoter, inlB promoter, PrfA, PinlC, PrfA cassette, and the like (see, e.g., Lalic Mullthaler et al. (2001) Mol. Microbiol. 42: 111-120; Shetron-Rama et al. (2003) Mol. Microbiol. 48: 1537-1551; Luo et al. (2004) Mol. Microbiol. 52: 39-52). PrfA can be made constitutively active by a Gly145Ser mutation, a Gly155Ser mutation, or a Glu77Lys mutation (see, e.g., Mueller and Freitag (2005) Infect. Immun. 73: 1917-1926; Wong and Freitag (2004) J. Bacteriol. 186: 6265-6276; Ripio et al. (1997) J. Bacteriol. 179: 1533-1540).

Examples of target antigens that can be used in the present invention are listed in the table below. The target antigen can also be a fragment or fusion polypeptide comprising an immunologically active portion of the antigen listed in the table. This list is not intended to be limiting.

Table 1. Antigens.

Other organisms known in the art as suitable antigens include, but are not limited to, Chlamydia trachomatis, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrheae, Vibrio cholerae, Salmonella species (including Salmonella typhi and Salmonella typhimurium), Enterica (including Helicobacter pylori, Shigella flexneri and other Group D Shigella species), Burkholderia mallei, mallei), Burkholderia pseudomallei, Klebsiella pneumonia, Clostridium species (including C. difficile), Vibrio parahaemolyticus, and V. vulnificus. This list is not intended to be limiting.

The antigenic sequences described herein are preferably expressed as a single polypeptide fused in frame to the modified amino-terminal portion of the Listeria monocytogenes ActA or LLO protein with an ActA or LLO secretory signal sequence. The ActA signal sequence is MGLNRFMRAMMVVFITANCITINPDIIFA; the LLO signal sequence is MKKIMLVFIT LILVSLPIAQ QTE. Preferably, the native signal sequence used is unmodified in the construct.

In some embodiments, the modified ActA comprises a modified form of about the first 100 amino acids of ActA, referred to herein as ActA-N100. ActA-N100 has the following sequence:

VGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEEKTEE50

QPSEVNTGPR YETAREVSSR DIEELEKSNK VKNTNKADLI AMLKAKAEKG

100

In this sequence, the first residue is depicted as valine; the polypeptide synthesized by Listeria monocytogenes has a methionine at this position. Thus, ActA-N100 may also have the following sequence:

MGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEEKTEE

50

QPSEVNTGPR YETAREVSSR DIEELEKSNK VKNTNKADLI AMLKAKAEKG

100

The constructs of the present invention may also comprise one or more additional non-ActA residues located between the C-terminal residue of the modified ActA and the antigen sequence. In the following sequence, ActA-N100 is extended by two residues added by the introduction of the BamH1 site:

VGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEEKTEE50

QPSEVNTGPR YETAREVSSR DIEELEKSNK VKNTNKADLI AMLKAKAEKG

100

GS

When synthesized with a methionine as the first residue, it has the sequence:

MGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEEKTEE

50

QPSEVNTGPR YETAREVSSR DIEELEKSNK VKNTNKADLI AMLKAKAEKG

100

GS.

These sequences can then serve as a basis for modification by deletion (actual or functional) of the PEST motif and any existing hydrophobic motifs. Thus, a modified ActA of the present invention may comprise or consist of the following sequence (dashed lines indicate deletions and bold text indicates substitutions):

VGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEE----

50

----------YETAREVSSR DIEELEKSNK VKNTNKADQDNKRKAKAEKG 100

In this sequence, the first residue is depicted as valine; the polypeptide is synthesized by Listeria and has a methionine at this position. Therefore, the modified one may also comprise or consist of the following sequence (SEQ ID NO: 4):

MGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEE---- 50

----------YETAREVSSR DIEELEKSNK VKNTNKADQDNKRKAKAEKG 100

In these cases, substitution with QDNKR and deletion of the PEST motif are optionally included, and as described above, these constructs of the invention may also comprise one or more additional non-ActA residues between the C-terminal residue of the modified ActA and the antigen sequence.

Alternatively, the antigenic sequence is preferably expressed as a single polypeptide fused to a modified amino-terminal portion of the Listeria monocytogenes LLO protein, which allows the fusion protein from the bacterium to be expressed and secreted within the inoculated host. In these embodiments, the antigenic construct can be a polynucleotide comprising a promoter operably linked to a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (a) the modified LLO and (b) one or more antigenic epitopes to be expressed as a fusion protein following the modified LLO sequence. The LLO signal sequence is MKKIMLVFIT LILVSLPIAQ QTEAK. In some embodiments, the promoter is the hly promoter.

In some embodiments, the modified LLO comprises a modified form of approximately the first 441 amino acids of LLO, referred to herein as LLO-N441. LLO-N441 has the following sequence:

In this sequence, the PEST motif is represented by KENSISSMA PPASPPASPK. This motif can be replaced by the following sequence (dashed lines indicate deletions and bold text indicates substitutions):

KE----------------- is functionally absent by substitution or by its complete absence. This is meant to be an example only.

Because sequences encoded by one organism may not necessarily be codon optimized for optimal expression in the chosen vaccine platform bacterial strain, the invention also provides nucleic acids that utilize codon alterations optimized for expression by bacteria such as Listeria monocytogenes.

In various embodiments, at least 1% of any non-optimal codons are altered to provide optimal codons, more typically at least 5% are altered, most typically at least 10% are altered, frequently at least 20% are altered, more typically at least 30% are altered, most typically at least 40% are altered, typically at least 50% are altered, more typically at least 60% are altered, most typically at least 70% are altered, preferably at least 80% are altered, more preferably at least 90% are altered, optimally at least 95% are altered, and routinely 100% of any non-optimal codons are codon-optimized for Listeria expression (Table 2).

Table 2. Optimal codons for expression in Listeria.

The present invention provides a large number of Listeria species and strains for preparing or engineering the bacteria of the present invention. The Listeria of the present invention is not limited to the species and strains disclosed in Table 3.

Table 3. Listeria strains suitable for use in the present invention (e.g., as vaccines or as nucleic acid sources).

Therapeutic compositions.

The bacterial compositions described herein can be administered to a host alone or in combination with a pharmaceutically acceptable excipient in an amount sufficient to induce an appropriate immune response. The immune response may include, but is not limited to, a specific immune response, a nonspecific immune response, both specific and nonspecific responses, an innate response, a primary immune response, an adaptive immunity, a secondary immune response, a memory immune response, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression. The vaccines of the present invention can be stored, for example, frozen, lyophilized, as a suspension, as a cell paste, or in combination with a solid matrix or gel matrix.

In certain embodiments, after an effective dose of the first vaccine is administered to a subject to elicit an immune response, a second vaccine is administered. This is known in the art as a "prime-boost" regimen. In such regimens, the compositions and methods of the present invention can be delivered as a "prime," as a "boost," or as both a "prime" and a "boost." Any number of "boost" immunizations can be delivered to maintain the magnitude or effectiveness of the vaccine-induced immune response.

For example, a first vaccine comprising killed but metabolically active Listeria that encodes and expresses an antigenic polypeptide can be delivered as a "prime," and a second vaccine comprising an attenuated (live or killed but metabolically active) Listeria that encodes an antigenic polypeptide can be delivered as a "boost." However, it should be understood that each of the priming and boosting does not necessarily utilize the methods and compositions of the present invention. Rather, the present invention encompasses the use of other vaccine forms and the bacterial vaccine methods and compositions of the present invention. The following are examples of suitable mixed priming-boosting regimens: DNA (e.g., plasmid) vaccine priming/bacterial vaccine boosting; viral vaccine priming/bacterial vaccine boosting; protein vaccine priming/bacterial vaccine boosting; DNA priming/bacterial vaccine boosting plus protein vaccine boosting; bacterial vaccine priming/DNA vaccine boosting; bacterial vaccine priming/viral vaccine boosting; bacterial vaccine priming/protein vaccine boosting; bacterial vaccine priming/bacterial vaccine boosting plus protein vaccine boosting, etc. This list is not intended to be limiting.

The priming vaccine and the boosting vaccine can be administered by the same route or by different routes. The term "different routes" encompasses, but is not limited to, different body parts, such as oral, parenteral, enteral, parenteral, rectal, intrasegmental (lymph node), intravenous, arterial, subcutaneous, intradermal, intramuscular, intratumoral, peritumoral, infusion, mucosal, nasal, in the cerebrospinal space or cerebrospinal fluid, and by different modes, such as oral, intravenous, and intramuscular.

The effective amount of the initial exemption or booster vaccine can be given in a single dose, but is not limited to a single dose. Therefore, administration can be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times or more vaccine administration. If there is more than one or more vaccine administrations in the inventive method, the time interval of administration can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes or more minutes, and the interval can be about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours etc. In the context of hours, the term "about" means any time within plus or minus 30 minutes. The time intervals for administration can also be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinations thereof. The present invention is not limited to dosing intervals that are equally spaced apart in time, but rather encompasses administration at unequal intervals, such as a primary immunization regimen consisting of administrations on 1 day, 4 days, 7 days, and 25 days, just to provide non-limiting examples.

In certain embodiments, administration of a booster vaccination may begin about 5 days after the start of the primary vaccination; about 10 days after the start of the primary vaccination; about 15 days after the start of the primary vaccination; about 20 days; about 25 days; about 30 days; about 35 days; about 40 days; about 45 days; about 50 days; about 55 days; about 60 days; about 65 days; about 70 days; about 75 days; about 80 days; about 6 months and about 1 year after the start of the primary vaccination. Preferably, one or both of the primary and booster vaccinations comprise delivery of a composition of the invention.

"Pharmaceutically acceptable excipients" or "diagnostally acceptable excipients" include, but are not limited to, sterile distilled water, saline, phosphate buffered saline, amino acid-based buffers, or bicarbonate buffered saline. The excipient selected and the amount of excipient used will depend on the mode of administration. Administration can be oral, intravenous, subcutaneous, cutaneous, intradermal, intramuscular, mucosal, parenteral, intraorgan, intralesional, intranasal, inhalation, intraocular, intramuscular, intravascular, intranodal, by scarification, rectal, intraperitoneal, or any one or combination of a variety of well-known routes of administration. Administration can include injection, infusion, or a combination thereof.

Administration of the vaccine of the present invention by non-oral routes can avoid tolerance. Methods for administration in the following ways are known in the art: intravenous, subcutaneous, intradermal, intramuscular, intraperitoneal, oral, mucosal, via the urethra, via the reproductive tract, via the gastrointestinal tract or by inhalation.

The effective amount for a particular patient may vary depending on factors such as the condition being treated, the patient's general health, the route and dosage of administration, and the severity of side effects. Guidance for methods of treatment and diagnosis is available (see, e.g., Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

The vaccines of the present invention can be administered at a dosage or dosage regimen in which each dose comprises at least 100 bacterial cells/kg body weight or more; in certain embodiments 1000 bacterial cells/kg body weight or more; normally at least 10,000 cells/kg body weight; more normally at least 100,000 cells/kg body weight; most normally at least 1 million cells/kg body weight; often at least 10 million cells/kg body weight; more often at least 100 million cells/kg body weight; usually at least 10 billion cells/kg body weight; typically at least 10 billion cells/kg body weight; routinely at least 100 billion cells/kg body weight; and sometimes at least 1 trillion cells/kg body weight. The present invention provides the above dosages in which the bacterial administration unit is colony forming units (CFU) (the equivalent of CFU before psoralen treatment) or in which the unit is the number of bacterial cells.

The vaccine of the present invention can be administered in one or more doses, wherein each dose comprises 10 ⁷ to 10 ⁸ bacteria/70 kg body weight (or/1.7 square meters surface area; or/1.5 kg liver weight); 2×10 ⁷ to 2×10 ⁸ bacteria/70 kg body weight (or/1.7 square meters surface area; or/1.5 kg liver weight); 5×10 ⁷ to 5×10 ⁸ bacteria/70 kg body weight (or/1.7 square meters surface area; or/1.5 kg liver weight); 10 ⁸ to 10 9 bacteria/70 kg body weight (or/1.7 square meters surface area; or/1.5 kg liver weight); 2.0×10 ⁸ to 2.0×10 ⁹ bacteria/70 kg (or/1.7 square meters surface area, or/1.5 kg liver weight); 5.0×10 ⁸ to 5.0×10 ⁸ bacteria/70 kg (or/1.7 square meters surface area, or/1.5 kg liver weight); ⁹ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 10 ⁹ to 10 ¹⁰ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 2×10 ⁹ to 2×10 ¹⁰ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 5×10 ⁹ to 5×10 ¹⁰ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 10 ¹¹ to 10 ¹² bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 2×10 ¹¹ to 2×10 ¹² bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 5×10 ¹¹ to 5×10 ¹² bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 10 ¹² to 10 ¹³ bacteria/70 kg (or/1.7 m2 surface area); 2×10 ¹² to 2×10 ¹³ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 5×10 ¹² to 5×10 ¹³ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 10 ¹³ to 10 ¹⁴ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 2×10 ¹³ to 2×10 ¹⁴ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 5×10 ¹³ to 5×10 ¹⁴ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 10 ¹⁴ to 10 ¹⁵ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight); 2×10 ¹⁴ to 2×10 ¹⁵ bacteria/70 kg (or/1.7 m2 surface area, or/1.5 kg liver weight), etc. (wet weight).

Also provided are one or more of the above dosages, wherein the dosage is administered by injection once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once every 7 days, wherein the injection regimen is maintained for, for example, only 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer. The present invention also includes combinations of the above dosages and regimens, for example, a relatively large initial bacterial dose followed by relatively small subsequent doses, or a relatively small initial dose followed by a large dose.

For example, dosing schedules of once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, etc., can be used with the present invention. The dosing schedule encompasses a total period of administration of, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and 12 months.

The above dosing regimen cycle is provided. The cycle can be repeated about, for example, every 7 days; every 14 days; every 21 days; every 28 days; every 35 days; 42 days; every 49 days; every 56 days; every 63 days; every 70 days, etc. Non-dosing intervals can occur between cycles, where the intervals can be about, for example, 7 days; 14 days; 21 days; 28 days; 35 days; 42 days; 49 days; 56 days; 63 days; 70 days, etc. In this context, the term "about" means plus or minus 1 day, plus or minus 2 days, plus or minus 3 days, plus or minus 4 days, plus or minus 5 days, plus or minus 6 days, or plus or minus 7 days.

The present invention encompasses methods for administering Listeria orally. Methods for administering Listeria intravenously are also provided. In addition, methods for administering Listeria orally, intramuscularly, intravenously, intradermally, and/or subcutaneously are provided. The present invention provides Listeria bacteria, or cultures or suspensions of Listeria bacteria, prepared by growing in a culture medium that is meat-based or contains polypeptides derived from meat or animal products. The present invention also provides Listeria bacteria, or cultures or suspensions of Listeria bacteria, prepared by growing in a culture medium that does not contain meat or animal products, prepared by growing on a culture medium containing plant polypeptides, prepared by growing on a culture medium that is not based on yeast products, or prepared by growing on a culture medium containing yeast polypeptides.

Methods of co-administration with other therapeutic agents are well known in the art (Hardman et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Pactice: A Practical Approach, Lippincott, Williams & Wilkins, Philadelphia, PA; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Philadelphia, PA).

Other reagents that are beneficial to improving cytolytic T cell reactions can also be used. The reagent is referred to as a carrier in this article. These include but are not limited to B7 costimulatory molecules, interleukin-2, interferon-γ, GM-CSF, CTLA-4 antagonists, OX-40/OX-40 ligands, CD40/CD40 ligands, sargramostim, levamisole, vaccinia virus, bacillus Calmette-Guérin (BCG), liposomes, alum, Freund's complete or incomplete adjuvant, detoxified endotoxin, mineral oil, surfactant (such as lecithin (lipolecithin)), pluronic polyol (pluronic polyol), polyanion, peptide and oil or hydrocarbon emulsion. Preferably, the carrier for inducing T cell immune response that preferentially stimulates cytolytic T cell reaction relative to antibody reaction, but those that stimulate two types of reactions can also be used. In the case where the reagent is a polypeptide, the polypeptide itself or the polynucleotide encoding the polypeptide can be applied. The carrier can be a cell, such as an antigen presenting cell (APC) or a dendritic cell. Antigen presenting cells include cell types such as 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. Facultative antigen presenting cells can also be used. Examples of facultative antigen presenting cells include astrocytes, follicular cells, endothelial cells, and fibroblasts. The vector can be a bacterial cell transformed to express a polypeptide or deliver a polynucleotide, which is then expressed in the cells of the inoculated subject. Adjuvants such as aluminum hydroxide or aluminum phosphate can be added to improve the ability of the vaccine to trigger, enhance, or prolong the immune response. Other substances used alone or in combination with the compositions, such as cytokines, chemokines and bacterial nucleic acid sequences (such as CpG), toll-like receptor (TLR) 9 agonists and other agonists for TLR 2, TLR 4, TLR 5, TLR 7, TLR 8, TLR 9, including lipoproteins, LPS, monophosphoryl lipid A, lipoteichoic acid, imiquimod, resiquimod and other similar immunomodulators such as cyclic dinucleotide STING agonists (including c-di-GMP, c-di-AMP, c-di-IMP and c-AMP-GMP) are also potential adjuvants. Other representative examples of adjuvants include the synthetic adjuvant QS-21 containing homogeneous saponins purified from the bark of Quillaja saponaria and Corynebacterium parvum (McCune et al., Cancer, 1979; 43: 1619). It should be understood that the adjuvant is optimized. In other words, one skilled in the art can perform routine experimentation to determine the optimal adjuvant to use.

An effective amount of a therapeutic agent is one that reduces or improves symptoms by at least 10% of normal, more typically at least 20%, most typically at least 30%, usually at least 40%, more typically at least 50%, most typically at least 60%, frequently at least 70%, more typically at least 80%, and most typically at least 90%, routinely at least 95%, more typically at least 99%, and most typically at least 99.9%.

The reagents and methods of the present invention provide vaccines that comprise only one vaccination, or comprise a first vaccination, or comprise at least one booster vaccination, at least two booster vaccinations, or at least three booster vaccinations. Parameter guidance for booster vaccinations is available. See, e.g., Marth (1997) Biologicals 25:199-203; Ramsay et al. (1997) Immunol. Cell Biol. 75:382-388; Gherardi et al. (2001) Histol. Histopathol. 16:655-667; Leroux-Roels et al. (2001) Act A Clin. Belg. 56:209-219; Greiner et al. (2002) Cancer Res. 62:6944-6951; Smith et al. (2003) J. Med. Virol. 70: Suppl 1: S38-S41; Sepulveda-Amor et al. (2002) Vaccine 20:2790-2795).

Therapeutic formulations can be prepared for storage by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, for example, a lyophilized powder, a slurry, an aqueous solution, or a suspension (see, e.g., Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

Example

The following examples are provided to illustrate the present invention. These examples are in no way intended to limit the scope of the present invention.

Example 1.

Figure 2 depicts various modifications of the sequences of ActA and LLO tested in the following examples.

With respect to the modified ActA sequence, the parent ActA sequence was truncated at residue 100 and extended by two residues added by the introduction of the BamH1 site, which are shown in the figure as residues G101 and S102. The modifications of the PEST sequence involved the deletions shown in the figure, and the optional replacement of QDNKR with the hydrophobic motif LIAML is also depicted. With respect to the modified LLO sequence, the parent LLO sequence was truncated at residue 441. The modifications of the PEST sequence involved the deletions shown in the figure. In each case, the depicted signal peptide/secretion chaperone element was functionally linked in frame to the selected antigen sequence.

Example 2.

Figure 3 depicts the location of PEST motifs in LLO sequences scored using the epestfind algorithm. A single motif with a score of +4.72 was modified as described above in Example 1. A hydrophilicity plot is also shown at the top of the figure. Multiple hydrophobic motifs (rising peaks above the sequence diagram) that can be modified as described herein are shown.

Similarly, Figure 4 depicts four PEST motifs in the ActA sequence scored using the epestfind algorithm. The first of these motifs has a score of +10.27 and was modified as described above in Example 1. The remaining PEST motifs were deleted by truncating the ActA sequence at residue 100. A hydrophilicity plot is also shown at the top of the figure. The hydrophobic motif LIAML is clearly visible as the rising peak above the sequence diagram in ActAN100.

Example 3.

Figure 5 shows the results of a B3Z T cell activation assay following immunization with the constructs shown in the figure. In each antigen construct, HIV gag was expressed fused to the SIINFEKL ("SL8") epitope tag and inserted into the genome of the host LmΔactAΔinlB vaccine strain. DC2.4 cells were infected with the selected strains and incubated with the OVA _257-264 -specific T cell hybridoma B3Z. Presentation of the SIINFEKL epitope on H-2K ^b class I molecules was assessed by measuring β-galactosidase expression using a chromogenic substrate. As shown in the figure, the absence of the PEST sequence had a positive or neutral effect on the assay results.

Example 4.

Figure 6 shows responses from LLO441 (A) and ActAN100 vaccine strains. BALB/c mice were vaccinated once intravenously with 5×10 ⁶ colony-forming units (cfu) of the indicated vaccine strains containing either an N-terminal fusion partner containing or lacking the PEST motif to directly compare the immunogenicity of these isogenic strains, which differ only in the composition of the N-terminal LLO or ActA fusion partner. At the peak of the Lm vaccine response 7 days after vaccination, mouse spleens were harvested and HIV-Gag CD8 T cell responses specific for the H2K ^d -restricted HIV Gag _197-205 epitope AMQMLKETI were determined by IFN-γ ELISpot assay using lymphocytes isolated from mouse whole blood using Lympholyte-Mammal (Cedarlane Labs, Burlington, NC) and murine IFN-γ ELISpot (BD Biosciences, San Jose, CA). At the end of the experiment, splenocytes were subjected to ELISpot assays. 2 × ¹⁰ cells/well were incubated with the appropriate peptide overnight at 37°C in anti-murine IFN-γ-coated ELISpot plates (Millipore, Billerica, MA). As a negative control, cells were not incubated with peptide. Murine ELISpots were developed using alkaline phosphatase detection reagent (Invitrogen, Carlsbad, CA) and scanned and quantified using an Immunospot plate reader and software (CTL Ltd, Cleveland, OH).

Mice vaccinated with the Lm vaccine strain containing the PEST ^- LLO ₄₄₁ N-terminal secretion/chaperone element generated HIV Gag-specific CD8 T cell responses that were higher than those generated by mice vaccinated with the isogenic Lm vaccine strain containing the LLO ₄₄₁ N-terminal secretion/chaperone element with the PEST motif. Mice vaccinated with the Lm vaccine strain containing the PEST ^- ActAN100 N-terminal secretion/chaperone element generated HIV Gag-specific CD8 T cell responses that were at least equivalent to those generated by mice vaccinated with the isogenic Lm vaccine strain containing the ActAN100 N-terminal secretion/chaperone element with the PEST motif.

Example 5.

Figure 7 depicts several alternative substitutions and deletions for deleting the PEST motif using ActA as a model system. Substitution of any of the five P, E, S, and T amino acids (E50, P52, S53, E54, T57) in the ActAN100 sequence for a positively charged residue (R, K, or H) is sufficient to eliminate the positive score obtained using the pestfinder algorithm.

Figure 8. More detailed depiction of the results of modifying the hydrophobic motif LIAML on the resulting hydropathy map. Non-conservative substitutions to QDNKR are sufficient to remove the hydrophobic nature of this sequence.

Example 6.

ActA-N100 (MGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEEKTEQPSEVNTGPR YETAREVSSR DIEELEKSNK VKNTNKADLI AMLKAKAEKG gs) and a modified version thereof in which the PEST motif has been deleted and contains a non-conservative QDNKR substitution (MGLNRFMRAM MVVFITANCI TINPDIIFAATDSEDSSLNT DEWEEEYETA REVSSRDIEE LEKSNKVKNT NKADQDNKRK AKAEKgl; referred to herein as ActA-N100*) were used to prepare a fusion construct with human mesothelin residues 35-621 (restriction sites for making in-frame fusions are included between the ActA and mesothelin sequences in lowercase). The construct was integrated into the chromosomal tRNA ^Arg locus of Listeria monocytogenes ΔactAΔinlB. Balb/c mice were challenged with 2×10 ⁵ human mesothelin-expressing CT-26 tumor cells on day 0. Mice were therapeutically vaccinated with a Listeria vaccine strain on days 4 and 17. The results of this experiment are plotted as the percentage survival of the vaccinated animals in Figure 9. As shown, there was no difference in efficacy between the ActA-N100-based vaccine and the ActA-N100*-based vaccine.

Example 7.

Prepare Listeria monocytogenes ΔactAΔinlB similar to Example 6, in which the mesothelin antigen sequence is replaced by 5 copies of the EGFRvIII _20-40 sequence and NY-ESO-1 _1-165 . The DNA and protein sequences used in the antigen construct are as follows (lowercase, not underlined: actA promoter; lowercase, underlined: restriction site; uppercase, bold: ActAN100* sequence; uppercase, underlined: EGFRvIII20-40x5; uppercase, italic: NY-ESO-1 (1-165) (each EGFRvIII _20-40 repeat in the peptide sequence is double underlined, and the leading Val codon is used to encode Met):

Fusion construct is schematically depicted in the left figure of Figure 10. Mouse dendritic cell line DC2.4 was infected with LmΔactA/ΔinlB (Figure 10, right figure, lane 1), BH3763 (EGFRvIII _20-40 /NY-ESO-1 _1-165 ) or BH3816 (clinical strain with EGFRvIII _20-40 /NY-ESO-1 _1-165 wherein the selection marker has been deleted). After 7 hours, the cells were washed, dissolved, run on SDS-PAGE, and transferred to nitrocellulose. Western blot was probed with a rabbit polyclonal antibody produced for the amino terminus of ActA protein and expression levels were normalized to the Listeria P60 protein associated with the bacterial count of infected cells. High levels of fusion construct expression were made by both research strains and clinical strains.

Female B10.Br mice (n=5/group) were intravenously inoculated with different doses of BH3816 (LmΔactAΔinlBEGFRvIII-NY-ESO-1). EGFR-specific T cell responses were determined by intracellular cytokine staining and are shown in Figure 11 as (A) the percentage of IFN-γ-positive EGFRvIII-specific CD8+ T cells; and (B) the absolute number of IFN-γ-positive EGFRvIII-specific CD8+ T cells per spleen. A robust EGFR T cell response was observed. As shown in Figure 12, NY-ESO-1-specific CD8+ T cell responses were also observed, as determined by intracellular cytokine staining 7 days after primary vaccination using the established H-2 ^d restricted epitope ARGPESRLL.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the purposes and advantages mentioned, as well as those inherent therein. The examples provided herein represent preferred embodiments, are illustrative, and are not intended as limitations on the scope of the invention.

It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the spirit and scope of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The inventions described herein, as appropriate and illustratively, may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by either of the other two terms. The terms and expressions employed are used in descriptive and non-limiting terms, and the use of these terms and expressions is not intended to exclude any equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications may be made within the scope of the claimed invention. Therefore, it should be understood that, although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

Claims (13)

1. A polynucleotide comprising: (a) promoter; and (b) A nucleic acid operably linked to the promoter, wherein the nucleic acid encodes a fusion protein, the fusion protein comprising: A polypeptide derived by recombination modification of the Listeria ActA protein sequence, wherein the sequence of the polypeptide is MGLNRFMRAMMVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEEYETA REVSSRDIEE LEKSNKVKNT NKADQDNKRKAKAEKG; and Non-Listeria antigens. 2. The polynucleotide of claim 1, wherein the polypeptide retains the signal sequence of the Listeria ActA protein sequence in its unmodified form. 3. The polynucleotide according to any one of claims 1-2, wherein the promoter is an actA or hly promoter. 4. The polynucleotide according to any one of claims 1-2, wherein the non-Listeria antigen is a cancer cell, tumor, or infectious agent antigen. 5. A plasmid comprising a polynucleotide according to any one of claims 1-4. 6. A Listeria bacterium comprising a polynucleotide according to any one of claims 1-4. 7. The Listeria bacterium according to claim 6, wherein it is Listeria monocytogenes. 8. The Listeria bacteria according to claim 7, wherein the bacteria are attenuated by the loss of function of the actA gene in the bacterial genome. 9. Listeria bacteria according to any one of claims 6-8, wherein the polynucleotide according to any one of claims 1-4 is inserted into the actA or inlB gene of the genome of the bacteria. 10. Use of an effective amount of Listeria bacteria according to any one of claims 6-9 in the preparation of a medicament for stimulating an immune response to a non-Listeria antigen in a mammal, wherein the non-Listeria antigen is expressed in one or more cells of the mammal. 11. A vaccine comprising Listeria bacteria according to any one of claims 6-9 and a pharmacologically acceptable excipient. 12. A method for producing Listeria bacteria for use in a vaccine, comprising: The polynucleotide according to any one of claims 1-4 is integrated into the genome of the Listeria spp. bacteria. 13. The method of claim 12, wherein the polynucleotide of any one of claims 1-4 is inserted into the actA or inlB gene of the bacterial genome.