METHODS AND COMPOSITIONS FOR INCREASING EFFICACY OF

VACCINES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Application No. 62/472,555, filed March 16, 2017 and US Application No. 62/564,117, filed September 27, 2017, each of which is herein incorporated by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

[0002] The Sequence Listing written in file 510749SEQLIST.txt is 90,980 bytes, was created on March 15, 2018, and is hereby incorporated by reference.

BACKGROUND

[0003] Listeria monocytogenes (Lm) is a gram-positive facultative intracellular pathogen that causes listeriosis. In its intercellular lifecycle, Lm enters host cells by phagocytosis or by active invasion of non-phagocytic cells. Following internalization, Lm may mediate its escape from the membrane bound phagosome/vacuole by secretion of several bacterial virulence factors, primarily the pore-forming protein listeriolysin O (LLO), enabling the bacteria to enter the host cell cytoplasm. In the cytoplasm, Lm replicates and spreads to adjacent cells based on the mobility facilitated by the bacterial actin-polymerizing protein (ActA). In the cytoplasm, Lm-secreted proteins are degraded by the proteasome and processed into peptides that associate with MHC class I molecules in the endoplasmic reticulum. This unique characteristic makes it a very attractive cancer vaccine vector in that tumor antigen can be presented with MHC class I molecules to activate tumor-specific cytotoxic T lymphocytes (CTLs). While residing in the cytosol, the bacteria can be recognized by various intercellular receptors, for example by recognition of peptidoglycan by nuclear oligomerization domain-like receptors and Lm DNA by DNA sensor, AIM2, and activate inflammatory and immune-modulatory cascades.

[0004] In addition, once internalized, Lm may then be processed in the phagosomal compartment and peptides presented on MHC Class II for activation of Lm-specific CD4- T cell responses. This combination of inflammatory responses and efficient delivery of antigens to the MHC I and MHC II pathways makes Lm a powerful vaccine vector in treating, protecting against, and inducing an immune response against a tumor.

[0005] Presently, there remains a need to identify patients who will respond best to such vaccines and increasing the therapeutic benefit of such vaccines. The present invention addresses this need by providing methods and compositions for screening patients who are likely to experience a survival benefit of such vaccines, lowering the level of serum expression of SERPINA1 before administering such vaccines to increase the therapeutic benefit of such vaccines, and increase the overall survival rate of a subject having a disease.

SUMMARY

[0006] Methods and compositions are provided for inducing an immune response against a disease in a subject which may include a cancer or tumor. In one aspect, provided herein are immunogenic compositions comprising vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides and an inhibitor of SERPINA1 for treating a disease in a subject. In other aspect, provided herein are recombinant Listeria strains comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to disease-associate antigenic peptide. Also provided are such fusion polypeptides and nucleic acids encoding such fusion polypeptides.

[0007] In another aspect, provided herein are immunogenic compositions,

pharmaceutical compositions, or vaccines comprising a recombinant Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to disease-associate antigenic peptide. Also provided are immunogenic compositions, pharmaceutical compositions, or vaccines comprising the fusion polypeptide or a nucleic acid encoding the fusion polypeptide.

[0008] In another aspect, provided herein are methods of inducing an immune response against a disease in a subject by obtaining a biological sample from the subject, determining that the serum expression of SERPINA1 in the biological sample is below 3.5 mg/ml, and administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease- associated antigenic peptides. In another aspect, provided herein are methods of inducing an immune response against a disease in a subject, by obtaining a biological sample from the subject, determining that the serum expression of SERPINA1 in the biological sample is above 3.5 mg/ml, and administering to the subject a composition comprising an inhibitor of SERPINA1, administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides. In another aspect, provided herein are methods of inducing an immune response against a disease in a subject comprising, administering to a subject an effective amount of a combination comprising, i. a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides, and ii. an effective amount of a composition comprising an inhibitor of SERPINA1. In another aspect, provided herein are methods of increasing the overall survival rate of a subject having a disease by obtaining a biological sample from the subject, determining that the serum expression of SERPINA1 in the biological sample is above 3.5 mg/ml, administering to the subject a composition comprising an inhibitor of SERPINA1, and administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides. In another aspect, provided herein are methods of inducing an immune response against a disease in a subject by obtaining a biological sample from the subject, determining that the serum expression of SERPINA1 in the biological sample is above 3.5 mg/ml, lowering the level of serum expression of SERPINA1 to below 3.5 mg/ml, and administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides.

[0009] In another aspect, provided herein are methods of inducing an immune response against a disease, method of inducing an enhanced immune response against a disease, method of increasing the overall survival rate of a subject having a disease, methods of inducing an immune response against a tumor or cancer in a subject, comprising administering to the subject a recombinant Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to disease-associate antigenic peptide. Also provided are methods of inducing an immune response against a tumor or cancer in a subject, comprising administering to the subject an immunogenic composition, a pharmaceutical composition, or a vaccine comprising such a recombinant Listeria strain. Also provided are methods of inducing an immune response against a tumor or cancer in a subject, comprising administering to the subject the fusion

polypeptide or a nucleic acid encoding the fusion polypeptide, an immunogenic composition comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide, a pharmaceutical composition comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide, or a vaccine comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide.

[0010] In another aspect, provided herein are methods of preventing or treating a tumor or cancer in a subject, comprising administering to the subject a recombinant Listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to disease-associate antigenic peptide. Also provided are methods of preventing or treating a tumor or cancer in a subject, comprising administering to the subject an immunogenic composition, a pharmaceutical composition, or a vaccine comprising such a recombinant Listeria strain. Also provided are methods of preventing or treating a tumor or cancer in a subject, comprising administering to the subject the fusion polypeptide, a nucleic acid encoding the fusion polypeptide, an immunogenic composition comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide, a

pharmaceutical composition comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide, or a vaccine comprising the fusion polypeptide or the nucleic acid encoding the fusion polypeptide.

[0011] In another aspect, provided herein are kits, comprising a first container and a second container, wherein the first container comprises at least one dose of a composition comprising an inhibitor of SERPINA1, the second container comprises at least one dose of a composition comprising vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides.

[0012] In another aspect, provided herein are cell banks comprising one or more recombinant Listeria strains comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST- containing peptide fused to disease-associate antigenic peptide.

DEFINITIONS

[0013] The terms "protein," "polypeptide," and "peptide," used interchangeably herein, refer to polymeric forms of amino acids of any length, including coded and non- coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms include polymers that have been modified, such as polypeptides having modified peptide backbones.

[0014] Proteins are said to have an "N-terminus" and a "C-terminus." The term "N- terminus" relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (- H2). The term "C-terminus" relates to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).

[0015] The term "fusion protein" refers to a protein comprising two or more peptides linked together by peptide bonds or other chemical bonds. The peptides can be linked together directly by a peptide or other chemical bond. For example, a chimeric molecule can be recombinantly expressed as a single-chain fusion protein. Alternatively, the peptides can be linked together by a "linker" such as one or more amino acids or another suitable linker between the two or more peptides.

[0016] The terms "nucleic acid" and "polynucleotide," used interchangeably herein, refer to polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0017] Nucleic acids are said to have "5' ends" and "3' ends" because

mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3 ' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3 ' elements.

[0018] "Codon optimization" refers to a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a polynucleotide encoding a fusion polypeptide can be modified to substitute codons having a higher frequency of usage in a given Listeria cell or any other host cell as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at the "Codon Usage Database." The optimal codons utilized by L.

monocytogenes for each amino acid are shown US 2007/0207170, herein incorporated by reference in its entirety for all purposes. These tables can be adapted in a number of ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available {see, e.g., Gene Forge).

[0019] The term "plasmid" or "vector" includes any known delivery vector including a bacterial delivery vector, a viral vector delivery vector, a peptide immunotherapy delivery vector, a DNA immunotherapy delivery vector, an episomal plasmid, an integrative plasmid, or a phage vector. The term "vector" refers to a construct which is capable of delivering, and, optionally, expressing, one or more fusion polypeptides in a host cell.

[0020] The term "episomal plasmid" or "extrachromosomal plasmid" refers to a nucleic acid vector that is physically separate from chromosomal DNA (i.e., episomal or extrachromosomal and does not integrated into a host cell's genome) and replicates independently of chromosomal DNA. A plasmid may be linear or circular, and it may be single-stranded or double-stranded. Episomal plasmids may optionally persist in multiple copies in a host cell's cytoplasm (e.g., Listeria), resulting in amplification of any genes of interest within the episomal plasmid.

[0021] The term "genomically integrated" refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence integrates into the genome of the cell and is capable of being inherited by progeny thereof. Any protocol may be used for the stable incorporation of a nucleic acid into the genome of a cell.

[0022] The term "stably maintained" refers to maintenance of a nucleic acid molecule or plasmid in the absence of selection (e.g., antibiotic selection) for at least 10 generations without detectable loss. For example, the period can be at least 15 generations, 20 generations, at least 25 generations, at least 30 generations, at least 40 generations, at least 50 generations, at least 60 generations, at least 80 generations, at least 100 generations, at least 150 generations, at least 200 generations, at least 300 generations, or at least 500 generations. Stably maintained can refer to a nucleic acid molecule or plasmid being maintained stably in cells in vitro (e.g., in culture), being maintained stably in vivo, or both. [0023] An "open reading frame" or "ORF" is a portion of a DNA which contains a sequence of bases that could potentially encode a protein. As an example, an ORF can be located between the start-code sequence (initiation codon) and the stop-codon sequence (termination codon) of a gene.

[0024] A "promoter" is a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked polynucleotide. A promoter can be active in one or more of the cell types disclosed herein (e.g., a eukaryotic cell, a non-human mammalian cell, a human cell, a rodent cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013/176772, herein incorporated by reference in its entirety.

[0025] "Operable linkage" or being "operably linked" refers to the juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence).

[0026] "Sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).

[0027] "Percentage of sequence identity" refers to the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the

comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0028] Unless otherwise stated, sequence identity/similarity values refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. "Equivalent program" includes any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0029] The term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non- polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue. Typical amino acid categorizations are summarized below.

Alanine Ala A Nonpolar Neutral 1.8

Arginine Arg R Polar Positive -4.5

Asparagine Asn N Polar Neutral -3.5

Aspartic acid Asp D Polar Negative -3.5

Cysteine Cys C Nonpolar Neutral 2.5

Glutamic acid Glu E Polar Negative -3.5

Glutamine Gin Q Polar Neutral -3.5

Glycine Gly G Nonpolar Neutral -0.4

Histidine His H Polar Positive -3.2

Isoleucine lie I Nonpolar Neutral 4.5

Leucine Leu L Nonpolar Neutral 3.8

Lysine Lys K Polar Positive -3.9

Methionine Met M Nonpolar Neutral 1.9

Phenylalanine Phe F Nonpolar Neutral 2.8

Proline Pro P Nonpolar Neutral -1.6

Serine Ser S Polar Neutral -0.8

Threonine Thr T Polar Neutral -0.7

Tryptophan Trp w Nonpolar Neutral -0.9

Tyrosine Tyr Y Polar Neutral -1.3

Valine Val V Nonpolar Neutral 4.2

[0030] A "homologous" sequence (e.g., nucleic acid sequence) refers to a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, 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%, at least 96%, at least 97%, at least 98%), at least 99%, or 100% identical to the known reference sequence. [0031] The term "wild type" refers to entities having a structure and/or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type gene and polypeptides often exist in multiple different forms (e.g., alleles).

[0032] The term "isolated" with respect to proteins and nucleic acid refers to proteins and nucleic acids that are relatively purified with respect to other bacterial, viral or cellular components that may normally be present in situ, up to and including a substantially pure preparation of the protein and the polynucleotide. The term "isolated" also includes proteins and nucleic acids that have no naturally occurring counterpart, have been chemically synthesized and are thus substantially uncontaminated by other proteins or nucleic acids, or has been separated or purified from most other cellular components with which they are naturally accompanied (e.g., other cellular proteins, polynucleotides, or cellular components).

[0033] "Exogenous" or "heterologous" molecules or sequences are molecules or sequences that are not normally expressed in a cell or are not normally present in a cell in that form. Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. An exogenous or heterologous molecule or sequence, for example, can include a mutated version of a corresponding endogenous sequence within the cell or can include a sequence corresponding to an endogenous sequence within the cell but in a different form (i.e., not within a chromosome). An exogenous or heterologous molecule or sequence in a particular cell can also be a molecule or sequence derived from a different species than a reference species of the cell or from a different organism within the same species. For example, in the case of a Listeria strain expressing a heterologous polypeptide, the heterologous polypeptide could be a polypeptide that is not native or endogenous to the Listeria strain, that is not normally expressed by the Listeria strain, from a source other than the Listeria strain, derived from a different organism within the same species.

[0034] In contrast, "endogenous" molecules or sequences or "native" molecules or sequences are molecules or sequences that are normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0035] The term "variant" refers to an amino acid or nucleic acid sequence (or an organism or tissue) that is different from the majority of the population but is still sufficiently similar to the common mode to be considered to be one of them (e.g., splice variants). [0036] The term "isoform" refers to a version of a molecule (e.g., a protein) with only slight differences compared to another isoform, or version (e.g., of the same protein). For example, protein isoforms may be produced from different but related genes, they may arise from the same gene by alternative splicing, or they may arise from single nucleotide polymorphisms.

[0037] The term "fragment" when referring to a protein means a protein that is shorter or has fewer amino acids than the full length protein. The term "fragment" when referring to a nucleic acid means a nucleic acid that is shorter or has fewer nucleotides than the full length nucleic acid. A fragment can be, for example, an N-terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment. A fragment can also be, for example, a functional fragment or an immunogenic fragment.

[0038] The term "analog" when referring to a protein means a protein that differs from a naturally occurring protein by conservative amino acid differences, by modifications which do not affect amino acid sequence, or by both.

[0039] The term "functional" refers to the innate ability of a protein or nucleic acid (or a fragment, isoform, or variant thereof) to exhibit a biological activity or function. Such biological activities or functions can include, for example, the ability to elicit an immune response when administered to a subject. Such biological activities or functions can also include, for example, binding to an interaction partner. In the case of functional fragments, isoforms, or variants, these biological functions may in fact be changed (e.g., with respect to their specificity or selectivity), but with retention of the basic biological function.

[0040] The terms "immunogenicity" or "immunogenic" refer to the innate ability of a molecule (e.g., a protein, a nucleic acid, an antigen, or an organism) to elicit an immune response in a subject when administered to the subject. Immunogenicity can be measured, for example, by a greater number of antibodies to the molecule, a greater diversity of antibodies to the molecule, a greater number of T-cells specific for the molecule, a greater cytotoxic or helper T-cell response to the molecule, and the like.

[0041] The term "antigen" is used herein to refer to a substance that, when placed in contact with a subject or organism (e.g., when present in or when detected by the subject or organism), results in a detectable immune response from the subject or organism. An antigen may be, for example, a lipid, a protein, a carbohydrate, a nucleic acid, or combinations and variations thereof. For example, an "antigenic peptide" refers to a peptide that leads to the mounting of an immune response in a subject or organism when present in or detected by the subject or organism. For example, such an "antigenic peptide" may encompass proteins that are loaded onto and presented on MHC class I and/or class II molecules on a host cell's surface and can be recognized or detected by an immune cell of the host, thereby leading to the mounting of an immune response against the protein. Such an immune response may also extend to other cells within the host, such as diseased cells (e.g., tumor or cancer cells) that express the same protein.

[0042] The term "epitope" refers to a site on an antigen that is recognized by the immune system (e.g., to which an antibody binds). An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996), herein incorporated by reference in its entirety for all purposes.

[0043] The term "mutation" refers to the any change of the structure of a gene or a protein. For example, a mutation can result from a deletion, an insertion, a substitution, or a rearrangement of chromosome or a protein. An "insertion" changes the number of nucleotides in a gene or the number of amino acids in a protein by adding one or more additional nucleotides or amino acids. A "deletion" changes the number of nucleotides in a gene or the number of amino acids in a protein by reducing one or more additional nucleotides or amino acids.

[0044] A "frameshift" mutation in DNA occurs when the addition or loss of nucleotides changes a gene's reading frame. A reading frame consists of groups of 3 bases that each code for one amino acid. A frameshift mutation shifts the grouping of these bases and changes the code for amino acids. The resulting protein is usually nonfunctional. Insertions and deletions can each be frameshift mutations.

[0045] A "missense" mutation or substitution refers to a change in one amino acid of a protein or a point mutation in a single nucleotide resulting in a change in an encoded amino acid. A point mutation in a single nucleotide that results in a change in one amino acid is a "nonsynonymous" substitution in the DNA sequence. Nonsynonymous substitutions can also result in a "nonsense" mutation in which a codon is changed to a premature stop codon that results in truncation of the resulting protein. In contrast, a "synonymous" mutation in a DNA is one that does not alter the amino acid sequence of a protein (due to codon degeneracy).

[0046] The term "somatic mutation" includes genetic alterations acquired by a cell other than a germ cell (e.g., sperm or egg). Such mutations can be passed on to progeny of the mutated cell in the course of cell division but are not inheritable. In contrast, a germinal mutation occurs in the germ line and can be passed on to the next generation of offspring.

[0047] The term "in vitro" refers to artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube).

[0048] The term "in vivo" refers to natural environments (e.g., a cell or organism or body) and to processes or reactions that occur within a natural environment.

[0049] The terms " AXAL", "axalimogene filolisbac", " ADXS-HPV" used

interchangeably herein, refer to attenuated live Listeria encoding HPV 16 E7 vaccine.

[0050] Compositions or methods "comprising" or "including" one or more recited elements may include other elements not specifically recited. For example, a composition that "comprises" or "includes" a protein may contain the protein alone or in combination with other ingredients.

[0051] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.

[0052] Unless otherwise apparent from the context, the term "about" encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations ± 0.5%, 1%, 5%, or 10% from a specified value.

[0053] The singular forms of the articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an antigen" or "at least one antigen" can include a plurality of antigens, including mixtures thereof.

[0054] Statistically significant means p <0.05.

[0055] The term "reference level" refers to a baseline cutoff wherein values below the cutoff associate significantly with increased OS and/or >12 month survival in AXAL- treated patients and/or wherein values above the cutoff associate significantly with decreased OS and/or decreased >12 month survival in AXAL-treated patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 shows survival between top and bottom quantiles of AAT gene expression in The Cancer Genome Atlas (TCGA) cervical cancer. Serum AAT gene transcription level does not generally correlate with cervical cancer overall survival. TCGA cervical cancer dataset, all stages of disease similarly represented.

[0057] Figure 2 shows AAT levels do not predict overall survival in HPV+ head and neck cancer.

[0058] Figure 3 shows AAT level does not predict response to PD-1 in melanoma.

[0059] Figure 4 shows GOG-265 baseline AAT levels vs. 12 month overall survival. Lower levels of AAT significantly associated with better survival on AXAL treatment in GOG-265.

[0060] Figure 5 shows GOG-265 baseline AAT levels vs. 12 month overall survival. Lower levels of AAT significantly associated with better survival on AXAL treatment in GOG-265.

[0061] Figure 6 shows KM curves for GOG-0265 with baseline serum AAT </> 3.5 mg/ml applied.

[0062] Figure 7 shows KM curve proj ection of GOG-265 +/- AAT </> 3.5 vs. GOG- 127 series trial in recurrent/refractory cervical cancer.

[0063] Figure 8 shows the study design for GOG/NRG-0265.

[0064] Figure 9 shows the median overall survival and a 12-month OS results for the GOG/NRG-0265 trial.

[0065] Figure 10 shows high baseline levels of 4 serum proteins negatively associate with OS in PRmCC patients participating in the GOG/NRG-0265 trial.

[0066] Figure 11 shows patients who survived >12 months have significantly lower baseline levels of the 4 serum proteins than patients who survived <12 months.

[0067] Figure 12 shows two dimensional hierarchical clustering identifies 2 patient clusters, distinguishable by low (cluster 1) or high (cluster 2) baseline levels of the 4 serum proteins

[0068] Figure 13 shows Kaplan-Meier estimates of OS for patient clusters 1 and 2.

[0069] Figures 14A and 14B show (A) the majority of patients who survived >12 months are found in Cluster 1 and (B) Kaplan-Meier estimates of OS for patients in clusters 1 and 2 who survived <12 months. DETAILED DESCRIPTION

I. Overview

[0070] Provided herein are methods and compositions to screen out patients who are not likely to experience a survival benefit on vaccine treatment and would eliminate the risk of exposing patients to vaccine treatment when they are not likely to obtain clinical benefit. The methods and compositions provided herein also provide a way to lowering the level of serum expression of SERPINAl, CRP, TIMP-1, andor VEGF or inhibiting SERPINAl, CRP, TIMP-1, andor VEGF activity to increase the clinical benefit of vaccine treatment and increase the overall survival rate of a subject having a disease.

Additionally, the methods and compositions provided herein enhances the therapeutic potential of AXAL in cervical cancer.

[0071] Cervical cancer is the most common human papillomavirus (HPV)-associated cancer and the fourth most common cancer in women worldwide. Axalimogene filolisbac (AXAL) - a live attenuated Listeria monocytogenes (Lm)-based immunotherapy that expresses and secretes the full length E7 protein of UPV 16 - was developed as a vaccine- based immunotherapy for the treatment of cervical cancer as well as of other UPV- associated cancers. Without being bound to theory, Lm-based immunotherapies act by stimulating innate immunity through multiple mechanisms including the STING pathway, by reducing the frequency and functions of immunosuppressive cells in the tumor microenvironment, and by inducing the generation of tumor antigen-specific T cells that infiltrate and destroy the tumor.

[0072] Also provided herein are vaccines comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides. These vaccines may be DNA-based vaccines, peptide-based vaccines, viral vaccines, dendritic cell vaccines, virus-like particles, or bacterial vaccines. Also provided herein are recombinant fusion polypeptides comprising disease-associated antigenic peptides (e.g., fused to a PEST-containing peptide). Also provided herein are nucleic acids encoding such fusion polypeptides; recombinant bacteria or Listeria strains comprising such fusion polypeptides or such nucleic acids; cell banks comprising such recombinant bacteria or Listeria strains; immunogenic compositions, pharmaceutical compositions, and vaccines comprising such fusion polypeptides, such nucleic acids, or such recombinant bacteria or Listeria strains; and methods of generating such fusion polypeptides, such nucleic acids, and such recombinant bacteria or Listeria strains. Also provided are methods of inducing an immune response against a disease, method of inducing an enhanced immune response against a disease, method of increasing the overall survival rate of a subject having a disease inducing an anti-tumor-associated-antigen immune response in a subject, methods of inducing an anti -tumor or anti-cancer immune response in a subject, methods of treating a tumor or cancer in a subject, methods of preventing a tumor or cancer in a subject, and methods of protecting a subject against a tumor or cancer using such recombinant fusion polypeptides, nucleic acids, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines.

[0073] The Lm technology has a mechanism of action that incorporates potent innate immune stimulation, delivery of a target peptide directly into the cytosol of dendritic cells and antigen presenting cells, generation of a targeted T cell response, and reduced immune suppression by regulatory T cells and myeloid-derived suppressor cells in the tumor microenvironment. Multiple treatments can be given and/or combined without neutralizing antibodies. The Lm technology can use, for example, live, attenuated, bioengineered Lm bacteria to stimulate the immune system to view tumor cells as potentially bacterial-infected cells and target them for elimination. The technology process can start with a live, attenuated strain of Listeria and can add, for example, multiple copies of a plasmid that encodes a fusion protein sequence including a fragment of, for example, the LLO (listen olysin O) molecule joined to the antigen of interest. This fusion protein is secreted by the Listeria inside antigen-presenting cells. This results in a stimulation of both the innate and adaptive arms of the immune system that reduces tumor defense mechanisms and makes it easier for the immune system to attack and destroy the cancer cells. II. Immunogenic Compositions, Pharmaceutical Compositions, and Vaccines

[0074] Provided herein are immunogenic compositions, pharmaceutical compositions, or vaccines comprising a disease-associated antigenic peptide (e.g., a disease-specific antigenic peptide) or a nucleic acid encoding a disease-associated antigenic peptide. For example, provided herein are immunogenic compositions, pharmaceutical compositions, or vaccines comprising a recombinant fusion polypeptide as disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide as disclosed herein, or a recombinant bacteria or Listeria strain as disclosed herein. An immunogenic composition comprising a Listeria strain can be inherently immunogenic by virtue of its comprising a Listeria strain and/or the composition can also further comprise an adjuvant. Other immunogenic compositions can comprise DNA immunotherapy or peptide immunotherapy compositions or other vaccines. These vaccines may be DNA-based vaccines, peptide-based vaccines, viral vaccines, dendritic cell vaccines, virus-like particles, or bacterial vaccines.

[0075] The term "immunogenic composition" refers to any composition containing an antigen that elicits an immune response against the antigen in a subject upon exposure to the composition. The immune response elicited by an immunogenic composition can be to a particular antigen or to a particular epitope on the antigen.

[0076] An immunogenic composition can comprise a single recombinant fusion polypeptide as disclosed herein, nucleic acid encoding a recombinant fusion polypeptide as disclosed herein, or recombinant bacteria or Listeria strain as disclosed herein, or it can comprise multiple different recombinant fusion polypeptides as disclosed herein, nucleic acids encoding recombinant fusion polypeptides as disclosed herein, or recombinant bacteria or Listeria strains as disclosed herein. A first recombinant fusion polypeptide is different from a second recombinant fusion polypeptide, for example, if it includes one antigenic peptide that the second recombinant fusion polypeptide does not. The two recombinant fusion polypeptides can include some of the same antigenic peptides and still be considered different. Such different recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, or recombinant bacteria or Listeria strains can be administered concomitantly to a subject or sequentially to a subject. Sequential administration can be particularly useful when a drug substance comprising a recombinant Listeria strain (or recombinant fusion polypeptide or nucleic acid) disclosed herein is in different dosage forms (e.g., one agent is a tablet or capsule and another agent is a sterile liquid) and/or is administered on different dosing schedules (e.g., one composition from the mixture is administered at least daily and another is administered less frequently, such as once weekly, once every two weeks, or once every three weeks). The multiple recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, or recombinant bacteria or Listeria strains can each comprise a different set of antigenic peptides. Alternatively, two or more of the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, or recombinant bacteria or Listeria strains can comprise the same set of antigenic peptides (e.g., the same set of antigenic peptides in a different order).

[0077] An immunogenic composition can additionally comprise an adjuvant (e.g., two or more adjuvants), a cytokine, a chemokine, or combination thereof. Optionally, an immunogenic composition can additionally comprises antigen presenting cells (APCs), which can be autologous or can be allogeneic to the subject.

[0078] The term adjuvant includes compounds or mixtures that enhance the immune response to an antigen. For example, an adjuvant can be a non-specific stimulator of an immune response or substances that allow generation of a depot in a subject which when combined with an immunogenic composition disclosed herein provides for an even more enhanced and/or prolonged immune response. An adjuvant can favor, for example, a predominantly Thl -mediated immune response, a Thl-type immune response, or a Thl- mediated immune response. Likewise, an adjuvant can favor a cell-mediated immune response over an antibody-mediated response. Alternatively, an adjuvant can favor an antibody-mediated response. Some adjuvants can enhance the immune response by slowly releasing the antigen, while other adjuvants can mediate their effects by any of the following mechanisms: increasing cellular infiltration, inflammation, and trafficking to the injection site, particularly for antigen-presenting cells (APC); promoting the activation state of APCs by upregulating costimulatory signals or major histocompatibility complex (MHC) expression; enhancing antigen presentation; or inducing cytokine release for indirect effect.

[0079] Examples of adjuvants include saponin QS21, CpG oligonucleotides, unmethylated CpG-containing oligonucleotides, MPL, TLR agonists, TLR4 agonists, TLR9 agonists, Resiquimod ^® , imiquimod, cytokines or nucleic acids encoding the same, chemokines or nucleic acids encoding same, IL-12 or a nucleic acid encoding the same, IL-6 or a nucleic acid encoding the same, and lipopolysaccharides. Another example of a suitable adjuvant is Montanide ISA 51. Montanide ISA 51 contains a natural

metabolizable oil and a refined emulsifier. Other examples of a suitable adjuvant include granulocyte/macrophage colony-stimulating factor (GM-CSF) or a nucleic acid encoding the same and keyhole limpet hemocyanin (KLH) proteins or nucleic acids encoding the same. The GM-CSF can be, for example, a human protein grown in a yeast (S. cerevisiae) vector. GM-CSF promotes clonal expansion and differentiation of hematopoietic progenitor cells, antigen presenting cells (APCs), dendritic cells, and T cells. Yet other examples of adjuvants include growth factors or nucleic acids encoding the same, cell populations, Freund's incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG (bacille Calmette-Guerin), alum, interleukins or nucleic acids encoding the same, quill glycosides, monophosphoryl lipid A, liposomes, bacterial mitogens, bacterial toxins, or any other type of known adjuvant (see, e.g., Fundamental Immunology, 5th ed. (August 2003): William E. Paul (Editor); Lippincott Williams & Wilkins Publishers; Chapter 43 : Vaccines, GJV Nossal, which is herein incorporated by reference in its entirety for all purposes).

[0080] An immunogenic composition can further comprise one or more

immunomodulatory molecules. Examples include interferon gamma, a cytokine, a chemokine, and a T bell stimulant.

[0081] An immunogenic composition can be in the form of a vaccine or

pharmaceutical composition. The terms "vaccine" and "pharmaceutical composition" are interchangeable and refer to an immunogenic composition in a pharmaceutically acceptable carrier for in vivo administration to a subject. A vaccine may be, for example, a peptide vaccine (e.g., comprising a recombinant fusion polypeptide as disclosed herein), a DNA vaccine (e.g., comprising a nucleic acid encoding a recombinant fusion polypeptide as disclosed herein), or a vaccine contained within and delivered by a cell (e.g., a recombinant Listeria as disclosed herein). A vaccine may prevent a subject from contracting or developing a disease or condition and/or a vaccine may be therapeutic to a subject having a disease or condition. Methods for preparing peptide vaccines are well known and are described, for example, in EP 1408048, US 2007/0154953, and Ogasawara et al. (1992) Proc. Natl Acad Sci USA 89:8995-8999, each of which is herein incorporated by reference in its entirety for all purposes. Optionally, peptide evolution techniques can be used to create an antigen with higher immunogenicity. Techniques for peptide evolution are well known and are described, for example, in US 6,773,900, herein incorporated by reference in its entirety for all purposes.

[0082] A "pharmaceutically acceptable carrier" refers to a vehicle for containing an immunogenic composition that can be introduced into a subject without significant adverse effects and without having deleterious effects on the immunogenic composition. That is, "pharmaceutically acceptable" refers to any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of at least one immunogenic composition for use in the methods disclosed herein. Pharmaceutically acceptable carriers or vehicles or excipients are well known.

Descriptions of suitable pharmaceutically acceptable carriers, and factors involved in their selection, are found in a variety of readily available sources such as, for example, Remington 's Pharmaceutical Sciences, 18th ed., 1990, herein incorporated by reference in its entirety for all purposes. Such carriers can be suitable for any route of administration (e.g., parenteral, enteral (e.g., oral), or topical application). Such pharmaceutical compositions can be buffered, for example, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the immunogenic compositions and route of administration.

[0083] Suitable pharmaceutically acceptable carriers include, for example, sterile water, salt solutions such as saline, glucose, buffered solutions such as phosphate buffered solutions or bicarbonate buffered solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates (e.g., lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, and the like. Pharmaceutical compositions or vaccines may also include auxiliary agents including, for example, diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffering agents, viscosity enhancing additives, lubricants, salts for influencing osmotic pressure, buffers, vitamins, coloring, flavoring, aromatic substances, and the like which do not deleteriously react with the immunogenic composition.

[0084] For liquid formulations, for example, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils include those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solid carriers/diluents include, for example, a gum, a starch (e.g., corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, or dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0085] Optionally, sustained or directed release pharmaceutical compositions or vaccines can be formulated. This can be accomplished, for example, through use of liposomes or compositions wherein the active compound is protected with differentially degradable coatings (e.g., by microencapsulation, multiple coatings, and so forth). Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the compositions and use the lyophilisates obtained (e.g., for the preparation of products for injection). [0086] An immunogenic composition, pharmaceutical composition, or vaccine disclosed herein may also comprise one or more additional compounds effective in preventing or treating cancer. For example, the additional compound may comprise a compound useful in chemotherapy, such as amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine, gliadelimplants, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomaldoxorubicin, liposomaldaunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel (Taxol), pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. The additional compound can also comprise other biologies, including Herceptin ^® (trastuzumab) against the FIER2 antigen, Avastin ^® (bevacizumab) against VEGF, or antibodies to the EGF receptor, such as Erbitux ^® (cetuximab), and Vectibix ^® (panitumumab). The additional compound can also comprise, and inhibitor of SERPF A1 or another compound that lowers the level of serum expression of SERPINA1 to below 3.5 mg/ml. The additional compound can also comprise, for example, an additional immunotherapy.

[0087] An additional compound can also comprise an immune checkpoint inhibitor antagonist, such as a PD-1 signaling pathway inhibitor, a CD-80/86 and CTLA-4 signaling pathway inhibitor, a T cell membrane protein 3 (TEVI3) signaling pathway inhibitor, an adenosine A2a receptor (A2aR) signaling pathway inhibitor, a lymphocyte activation gene 3 (LAG3) signaling pathway inhibitor, a killer immunoglobulin receptor (KIR) signaling pathway inhibitor, a CD40 signaling pathway inhibitor, or any other antigen-presenting cell/T cell signaling pathway inhibitor. Examples of immune checkpoint inhibitor antagonists include an anti-PD-Ll/PD-L2 antibody or fragment thereof, an anti-PD-1 antibody or fragment thereof, an anti-CTLA-4 antibody or fragment thereof, or an anti-B7- H4 antibody or fragment thereof. An additional compound can also comprise a T cell stimulator, such as an antibody or functional fragment thereof binding to a T-cell receptor co-stimulatory molecule, an antigen presenting cell receptor binding co-stimulatory molecule, or a member of the TNF receptor superfamily. The T-cell receptor co- stimulatory molecule can comprise, for example, CD28 or ICOS. The antigen presenting cell receptor binding co-stimulatory molecule can comprise, for example, a CD80 receptor, a CD86 receptor, or a CD46 receptor. The TNF receptor superfamily member can comprise, for example, glucocorticoid-induced TNF receptor (GITR), OX40 (CD 134 receptor), 4- IBB (CD137 receptor), or TNFR25. See, e.g., WO2016100929,

WO2016011362, and WO2016011357, each of which is incorporated by reference in its entirety for all purposes.

A. Recombinant Fusion Polypeptides

[0088] Disclosed herein are recombinant fusion polypeptides comprising a PEST- containing peptide fused to disease-associated antigenic peptides.

[0089] Also disclosed herein are recombinant fusion polypeptides comprising disease- associated antigenic peptides, and wherein the fusion polypeptide does not comprise a PEST-containing peptide.

[0090] Disclosed herein are recombinant fusion polypeptides comprising a PEST- containing peptide fused to one or more antigenic peptides (i.e., in tandem, such as PEST- peptide l-peptide2), wherein each antigenic peptide comprises a neoepitope present in a cancer sample or tumor sample from a subject (e.g., an altered amino acid sequence encoded by a nonsynonymous mutation in a gene) that is not present in a healthy biological sample (e.g., a healthy biological sample from the subject). PEST-containing peptides suitable for inclusion in the fusion polypeptides are disclosed elsewhere herein. Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own PEST-containing peptide (e.g., PEST 1 -peptide 1; PEST2-peptide2).

[0091] Also disclosed herein are recombinant fusion polypeptides comprising one or more antigenic peptides, wherein each antigenic peptide comprises a neoepitope present in a cancer cell or tumor cell from a subject that is not present in a healthy cell from the subject, and wherein the fusion polypeptide does not comprise a PEST-containing peptide.

[0092] The fusion polypeptides disclosed herein comprise antigenic peptides comprising neoepitopes. These neoepitopes can be patient-specific (i.e., subject-specific) cancer mutations. A process of for creating a personalized immunotherapy may comprise use of extracted nucleic acid a cancer sample from a subject and extracted nucleic acid from a normal or healthy reference sample in order to identify somatic mutations or sequence differences present in the cancer sample as compared with the normal or healthy sample, wherein these sequence having somatic mutations or differences encode an expressed amino acid sequence. A peptide expressing such somatic mutations or sequence differences can be referred to as a "neoepitope." A cancer-specific neoepitope may refer to an epitope that is not present in a reference sample (such as a normal non-cancerous or germline cell or tissue) but is found in a cancer sample. This includes, for example, situations wherein in a normal non-cancerous or germline cell a corresponding epitope is found; however, due to one or more mutations in a cancer cell, the sequence of the epitope is changed so as to result in the neoepitope. A neoepitope can comprise a mutated epitope, and can comprise non-mutated sequence on either or both sides of the mutation.

[0093] Disclosed herein are recombinant fusion polypeptides comprising a PEST- containing peptide fused to two or more antigenic peptides (i.e., in tandem, such as PEST- peptidel-peptide2), wherein each antigenic peptide comprises a single, recurrent cancer mutation (i.e., a single, recurrent change in the amino acid sequence of a protein, or a sequence encoded by a single, different, nonsynonymous, recurrent cancer mutation in a gene), and wherein at least two of the antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein. Alternatively, each of the antigenic peptides comprises a different recurrent cancer mutation from a different cancer-associated protein. Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own PEST- containing peptide (e.g., PEST 1 -peptide 1; PEST2-peptide2). Optionally, some or all of the fragments are non-contiguous fragments of the same cancer-associated protein. Noncontiguous fragments are fragments that do not occur sequentially in a protein sequence (e.g., the first fragment consists of residues 10-30, and the second fragment consists of residues 100-120; or the first fragment consists of residues 10-30, and the second fragment consists of residues 20-40). Optionally, each of the antigenic peptides comprises a different recurrent cancer mutation from a single type of cancer.

[0094] Also disclosed herein are recombinant fusion polypeptides comprising two or more antigenic peptides, wherein each antigenic peptide comprises a single, recurrent cancer mutation (i.e., a single, recurrent change in the amino acid sequence of a protein, or a sequence encoded by a single, different, nonsynonymous, recurrent cancer mutation in a gene), wherein at least two of the antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein, and wherein the fusion polypeptide does not comprise a PEST-containing peptide. Alternatively, each of the antigenic peptides comprises a different recurrent cancer mutation from a different cancer- associated protein. Optionally, some or all of the fragments are non-contiguous fragments of the same cancer-associated protein. Optionally, each of the antigenic peptides comprises a different recurrent cancer mutation from a single type of cancer.

[0095] The fusion polypeptides disclosed herein comprise antigenic peptides comprising recurrent cancer mutations from cancer-associated proteins. The term "cancer- associated protein" includes proteins having mutations that occur in multiple types of cancer, that occur in multiple subjects having a particular type of cancer, or that are correlated with the occurrence or progression of one or more types of cancer. For example, a cancer-associated protein can be an oncogenic protein (i.e., a protein with activity that can contribute to cancer progression, such as proteins that regulate cell growth), or it can be a tumor-suppressor protein (i.e., a protein that typically acts to alleviate the potential for cancer formation, such as through negative regulation of the cell cycle or by promoting apoptosis). Preferably, a cancer-associated protein has a

"mutational hotspot." A mutational hotspot is an amino acid position in a protein-coding gene that is mutated (preferably by somatic substitutions rather than other somatic abnormalities, such as translocations, amplifications, and deletions) more frequently than would be expected in the absence of selection. Such hotspot mutations can occur across multiple types of cancer and/or can be shared among multiple cancer patients. Mutational hotspots indicate selective pressure across a population of tumor samples. Tumor genomes contain recurrent cancer mutations that "drive" tumorigenesis by affecting genes (i.e., tumor driver genes) that confer selective growth advantages to the tumor cells upon alteration. Such tumor driver genes can be identified, for example, by identifying genes that are mutated more frequently than expected from the background mutation rate (i.e., recurrence); by identifying genes that exhibit other signals of positive selection across tumor samples (e.g., a high rate of non-silent mutations compared to silent mutations, or a bias towards the accumulation of functional mutations); by exploiting the tendency to sustain mutations in certain regions of the protein sequence based on the knowledge that whereas inactivating mutations are distributed along the sequence of the protein, gain-of- function mutations tend to occur specifically in particular residues or domains; or by exploiting the overrepresentation of mutations in specific functional residues, such as phosphorylation sites. Many of these mutations frequently occur in the functional regions of biologically active proteins (for example, kinase domains or binding domains) or interrupt active sites (for example, phosphorylation sites) resulting in loss-of-function or gain-of-function mutations, or they can occur in such a way that the three-dimensional structure and/or charge balance of the protein is perturbed sufficiently to interfere with normal function. Genomic analysis of large numbers of tumors reveals that mutations often occur at a limited number of amino acid positions. Therefore, a majority of the common mutations can be represented by a relatively small number of potential tumor- associated antigens or T cell epitopes.

[0096] A "recurrent cancer mutation" is a change in the amino acid sequence of a protein that occurs in multiple types of cancer and/or in multiple subjects having a particular types of cancer. Such mutations associated with a cancer can result in tumor- associated antigens that are not normally present in corresponding healthy tissue.

[0097] Tumor-driver genes and cancer-associated proteins having common mutations that occur across multiple cancers or among multiple cancer patients are known, and sequencing data across multiple tumor samples and multiple tumor types exists. See, e.g., Chang et al. (2016) Nat Biotechnol 34(2)A55-163 Tamborero et al. (2013) Sci Rep 3 :2650, each of which is herein incorporated by reference in its entirety.

[0098] Also provided herein are recombinant fusion polypeptides comprising from N- terminal end to C-terminal end a bacterial secretion sequence, a ubiquitin (Ub) protein, and two or more antigenic peptides (i.e., in tandem, such as Ub-peptidel-peptide2), wherein disease-associated antigenic peptides. Alternatively, a combination of separate fusion polypeptides can be used in which each antigenic peptide is fused to its own secretion sequence and Ub protein (e.g., Ubl-peptidel; Ub2-peptide2).

[0099] Nucleic acids (termed minigene constructs) encoding such recombinant fusion polypeptides are also disclosed. Such minigene nucleic acid constructs can further comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a minigene nucleic acid construct can further comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acid constructs, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.

[0100] The bacterial signal sequence can be a Listerial signal sequence, such as an Hly or an ActA signal sequence, or any other known signal sequence. In other cases, the signal sequence can be an LLO signal sequence. The signal sequence can be bacterial, can be native to a host bacterium (e.g., Listeria monocytogenes, such as a secAl signal peptide), or can be foreign to a host bacterium. Specific examples of signal peptides include an Usp45 signal peptide from Lactococcus lactis, a Protective Antigen signal peptide from Bacillus anthracis, a secA2 signal peptide such the p60 signal peptide from Listeria monocytogenes, and a Tat signal peptide such as a B. subtilis Tat signal peptide (e.g., PhoD). In specific examples, the secretion signal sequence is from a Listeria protein, such as an ActA ₃ oo secretion signal or an ActAioo secretion signal.

[0101] The ubiquitin can be, for example, a full-length protein. The ubiquitin expressed from the nucleic acid construct provided herein can be cleaved at the carboxy terminus from the rest of the recombinant fusion polypeptide expressed from the nucleic acid construct through the action of hydrolases upon entry to the host cell cytosol. This liberates the amino terminus of the fusion polypeptide, producing a peptide in the host cell cytosol.

[0102] Selection of, variations of, and arrangement of antigenic peptides within a fusion polypeptide are discussed in detail elsewhere herein, and Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen_are discussed in more detail elsewhere herein.

[0103] The recombinant fusion polypeptides can comprise one or more tags. For example, the recombinant fusion polypeptides can comprise one or more peptide tags N- terminal and/or C-terminal to the combination of the two or more antigenic peptides. A tag can be fused directly to an antigenic peptide or linked to an antigenic peptide via a linker (examples of which are disclosed elsewhere herein). Examples of tags include the following: FLAG tag, 3xFLAG tag; His tag, 6xHis tag; and SIINFEKL tag. An exemplary SIINFEKL tag is set forth in SEQ ID NO: 16 (encoded by any one of the nucleic acids set forth in SEQ ID NOS: 1-15). An exemplary 3xFLAG tag is set forth in SEQ ID NO: 32 (encoded by any one of the nucleic acids set forth in SEQ ID NOS: 17- 31). Other tags include chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX), and poly(NANP). Particular recombinant fusion polypeptides comprise a C-terminal SIINFEKL tag. Such tags can allow for easy detection of the recombinant fusion protein, confirmation of secretion of the recombinant fusion protein, or for following the immunogenicity of the secreted fusion polypeptide by following immune responses to these "tag" sequence peptides. Such immune response can be monitored using a number of reagents including, for example, monoclonal antibodies and DNA or RNA probes specific for these tags.

[0104] The recombinant fusion polypeptides disclosed herein can be expressed by recombinant Listeria strains or can be expressed and isolated from other vectors and cell systems used for protein expression and isolation. Recombinant Listeria strains comprising expressing such antigenic peptides can be used, for example in immunogenic compositions comprising such recombinant Listeria and in vaccines comprising the recombinant Listeria strain and an adjuvant. Expression of one or more antigenic peptides as a fusion polypeptides with a nonhemolytic truncated form of LLO, ActA, or a PEST- like sequence in host cell systems in Listeria strains and host cell systems other than Listeria can result in enhanced immunogenicity of the antigenic peptides.

[0105] Nucleic acids encoding such recombinant fusion polypeptides are also disclosed. The nucleic acid can be in any form. The nucleic acid can comprise or consist of DNA or RNA, and can be single-stranded or double-stranded. The nucleic acid can be in the form of a plasmid, such as an episomal plasmid, a multicopy episomal plasmid, or an integrative plasmid. Alternatively, the nucleic acid can be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome. Such nucleic acids can have one open reading frame or can have two or more open reading frames (e.g., an open reading frame encoding the recombinant fusion polypeptide and a second open reading frame encoding a metabolic enzyme). In one example, such nucleic acids can comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. For example, a nucleic acid can comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different polypeptide. In some nucleic acids, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.

(1) Antigenic Peptides

[0106] Disease-associated antigenic peptides can include Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen. The Human Papilloma Virus can be HPV 16 or HPV 18. The antigenic peptide can also include HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 antigens operably linked in tandem or HPV16 antigenic peptide operably linked in tandem to an HPV antigenic peptide.

[0107] The fusion polypeptide can include a single antigenic peptide or can includes two or more antigenic peptides. Each antigenic peptide can be of any length sufficient to induce an immune response, and each antigenic peptide can be the same length or the antigenic peptides can have different lengths.

[0108] Each antigenic peptide can also be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria monocytogenes or another bacteria of interest. For example, antigenic peptides can be scored by a Kyte and Doolittle hydropathy index 21 amino acid window, and all scoring above a cutoff (around 1.6) can be excluded as they are unlikely to be secretable by Listeria monocytogenes. Likewise, the combination of antigenic peptides or the fusion polypeptide can be hydrophilic or can score up to or below a certain hydropathy threshold, which can be predictive of secretability in Listeria monocytogenes or another bacteria of interest.

[0109] The antigenic peptides can be linked together in any manner. For example, the antigenic peptides can be fused directly to each other with no intervening sequence.

Alternatively, the antigenic peptides can be linked to each other indirectly via one or more linkers, such as peptide linkers. In some cases, some pairs of adjacent antigenic peptides can be fused directly to each other, and other pairs of antigenic peptides can be linked to each other indirectly via one or more linkers. The same linker can be used between each pair of adjacent antigenic peptides, or any number of different linkers can be used between different pairs of adjacent antigenic peptides. In addition, one linker can be used between a pair of adjacent antigenic peptides, or multiple linkers can be used between a pair of adjacent antigenic peptides.

[0110] Any suitable sequence can be used for a peptide linker. As an example, a linker sequence may be, for example, from 1 to about 50 amino acids in length. Some linkers may be hydrophilic. The linkers can serve varying purposes. For example, the linkers can serve to increase bacterial secretion, to facilitate antigen processing, to increase flexibility of the fusion polypeptide, to increase rigidity of the fusion polypeptide, or any other purpose. In some cases, different amino acid linker sequences are distributed between the antigenic peptides or different nucleic acids encoding the same amino acid linker sequence are distributed between the antigenic peptides (e.g., SEQ ID NOS: 84-94) in order to minimize repeats. This can also serve to reduce secondary structures, thereby allowing efficient transcription, translation, secretion, maintenance, or stabilization of the nucleic acid (e.g., plasmid) encoding the fusion polypeptide within a Lm recombinant vector strain population. Other suitable peptide linker sequences may be chosen, for example, based on one or more of the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the antigenic peptides; and (3) the lack of hydrophobic or charged residues that might react with the functional epitopes. For example, peptide linker sequences may contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al. (1985) Gene 40:39-46; Murphy et al. (1986) Proc Natl Acad Sci USA 83 :8258-8262; US 4,935,233; and US 4,751, 180, each of which is herein incorporated by reference in its entirety for all purposes. Specific examples of linkers include those in the following table (each of which can be used by itself as a linker, in a linker comprising repeats of the sequence, or in a linker further comprising one or more of the other sequences in the table), although others can also be envisioned {see, e.g., Reddy Chichili et al. (2013) Protein Science 22: 153-167, herein incorporated by reference in its entirety for all purposes). Unless specified, "n" represents an undetermined number of repeats in the listed linker.

(2) PEST-Containing Peptides

[0111] The recombinant fusion proteins disclosed herein comprise a PEST-containing peptide. The PEST-containing peptide may at the amino terminal (N-terminal) end of the fusion polypeptide (i.e., N-terminal to the antigenic peptides), may be at the carboxy terminal (C -terminal) end of the fusion polypeptide (i.e., C-terminal to the antigenic peptides), or may be embedded within the antigenic peptides. In some recombinant Listeria strains and methods, a PEST containing peptide is not part of and is separate from the fusion polypeptide. Fusion of an antigenic peptides to a PEST-like sequence, such as an LLO peptide, can enhance the immunogenicity of the antigenic peptides and can increase cell-mediated and antitumor immune responses (i.e., increase cell-mediated and anti-tumor immunity). See, e.g. , Singh et al. (2005) J Immunol 175(6):3663-3673, herein incorporated by reference in its entirety for all purposes.

[0112] A PEST-containing peptide is one that comprises a PEST sequence or a PEST- like sequence. PEST sequences in eukaryotic proteins have long been identified. For example, proteins containing amino acid sequences that are rich in prolines (P), glutamic acids (E), serines (S) and threonines (T) (PEST), generally, but not always, flanked by clusters containing several positively charged amino acids, have rapid intracellular half- lives (Rogers et al. (1986) Science 234:364-369, herein incorporated by reference in its entirety for all purposes). Further, it has been reported that these sequences target the protein to the ubiquitin-proteosome pathway for degradation (Rechsteiner and Rogers (1996) Trends Biochem. Sci. 21 :267-271, herein incorporated by reference in its entirety for all purposes). This pathway is also used by eukaryotic cells to generate immunogenic peptides that bind to MHC class I and it has been hypothesized that PEST sequences are abundant among eukaryotic proteins that give rise to immunogenic peptides (Realini et al. (1994) FEBS Lett. 348: 109-1 13, herein incorporated by reference in its entirety for all purposes). Prokaryotic proteins do not normally contain PEST sequences because they do not have this enzymatic pathway. However, a PEST-like sequence rich in the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T) has been reported at the amino terminus of LLO and has been reported to be essential for J. monocytogenes pathogenicity (Decatur and Portnoy (2000) Science 290:992-995, herein incorporated by reference in its entirety for all purposes). The presence of this PEST-like sequence in LLO targets the protein for destruction by proteolytic machinery of the host cell so that once the LLO has served its function and facilitated the escape of L. monocytogenes from the phagosomal or phagolysosomal vacuole, it is destroyed before it can damage the cells.

[0113] Identification of PEST and PEST-like sequences is well known in the art and is described, for example, in Rogers et al. (1986) Science 234(4774):364-378 and in

Rechsteiner and Rogers (1996) Trends Biochem. Sci. 21 :267-271, each of which is herein incorporated by reference in its entirety for all purposes. A PEST or PEST-like sequence can be identified using the PEST-find program. For example, a PEST-like sequence can be a region rich in proline (P), glutamic acid (E), serine (S), and threonine (T) residues. Optionally, the PEST-like sequence can be flanked by one or more clusters containing several positively charged amino acids. For example, a PEST-like sequence can be defined as a hydrophilic stretch of at least 12 amino acids in length with a high local concentration of proline (P), aspartate (D), glutamate (E), serine (S), and/or threonine (T) residues. In some cases, a PEST-like sequence contains no positively charged amino acids, namely arginine (R), histidine (H), and lysine (K). Some PEST-like sequences can contain one or more internal phosphorylation sites, and phosphorylation at these sites precedes protein degradation.

[0114] In one example, the PEST-like sequence fits an algorithm disclosed in Rogers et al. In another example, the PEST-like sequence fits an algorithm disclosed in

Rechsteiner and Rogers. PEST-like sequences can also be identified by an initial scan for positively charged amino acids R, H, and K within the specified protein sequence. All amino acids between the positively charged flanks are counted, and only those motifs containing a number of amino acids equal to or higher than the window-size parameter are considered further. Optionally, a PEST-like sequence must contain at least one P, at least one D or E, and at least one S or T.

[0115] The quality of a PEST motif can be refined by means of a scoring parameter based on the local enrichment of critical amino acids as well as the motifs hydrophobicity. Enrichment of D, E, P, S, and T is expressed in mass percent (w/w) and corrected for one equivalent of D or E, onel of P, and one of S or T. Calculation of hydrophobicity can also follow in principle the method of Kyte and Doolittle (1982) J. Mol. Biol. 157: 105, herein incorporated by reference in its entirety for all purposes. For simplified calculations, Kyte-Doolittle hydropathy indices, which originally ranged from -4.5 for arginine to +4.5 for isoleucine, are converted to positive integers, using the following linear

transformation, which yielded values from 0 for arginine to 90 for isoleucine: Hydropathy index = 10 * Kyte-Doolittle hydropathy index + 45.

[0116] A potential PEST motif s hydrophobicity can also be calculated as the sum over the products of mole percent and hydrophobicity index for each amino acid species. The desired PEST score is obtained as combination of local enrichment term and hydrophobicity term as expressed by the following equation: PEST score = 0.55 * DEPST - 0.5 * hydrophobicity index.

[0117] Thus, a PEST-containing peptide can refer to a peptide having a score of at least +5 using the above algorithm. Alternatively, it can refer to a peptide having a score of at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 35, at least 38, at least 40, or at least 45. [0118] Any other available methods or algorithms known in the art can also be used to identify PEST-like sequences. See, e.g. , the CaSPredictor (Garay-Malpartida et al. (2005) Bioinformatics 21 Suppl l :i 169-76, herein incorporated by reference in its entirety for all purposes). Another method that can be used is the following: a PEST index is calculated for each stretch of appropriate length (e.g. a 30-35 amino acid stretch) by assigning a value of one to the amino acids Ser, Thr, Pro, Glu, Asp, Asn, or Gin. The coefficient value (CV) for each of the PEST residues is one and the CV for each of the other AA (non-PEST) is zero.

[0119] Examples of PEST-like amino acid sequences are those set forth in SEQ ID NOS: 43-51. One example of a PEST-like sequence is

KENSIS SM APP ASPP ASPKTPIEKKHADEIDK (SEQ ID NO: 43). Another example of a PEST-like sequence is KENSIS SMAPP ASPP ASPK (SEQ ID NO: 44). However, any PEST or PEST-like amino acid sequence can be used. PEST sequence peptides are known and are described, for example, in US 7,635,479; US 7,665,238; and US 2014/0186387, each of which is herein incorporated by reference in its entirety for all purposes.

[0120] The PEST-like sequence can be from a Listeria species, such as from Listeria monocytogenes. For example, the Listeria monocytogenes ActA protein contains at least four such sequences (SEQ ID NOS: 45-48), any of which are suitable for use in the compositions and methods disclosed herein. Other similar PEST-like sequences include SEQ ID NOS: 52-54. Streptolysin O proteins from Streptococcus sp. also contain a PEST sequence. For example, Streptococcus pyogenes streptolysin O comprises the PEST sequence KQNTASTETTTTNEQPK (SEQ ID NO: 49) at amino acids 35-51 and

Streptococcus equisimilis streptolysin O comprises the PEST-like sequence

KQNTANTETTTTNEQPK (SEQ ID NO: 50) at amino acids 38-54. Another example of a PEST-like sequence is from Listeria seeligeri cytolysin, encoded by the Iso gene:

RSEVTISPAETPESPPATP (e.g., SEQ ID NO: 51).

[0121] Alternatively, the PEST-like sequence can be derived from other prokaryotic organisms. Other prokaryotic organisms wherein PEST-like amino acid sequences would be expected include, for example, other Listeria species.

(a) Listeriolysin O (LLO)

[0122] One example of a PEST-containing peptide that can be utilized in the compositions and methods disclosed herein is a listeriolysin O (LLO) peptide. An example of an LLO protein is the protein assigned GenBank Accession No. P13128 (SEQ ID NO: 55; nucleic acid sequence is set forth in GenBank Accession No. XI 5127). SEQ ID NO: 55 is a proprotein including a signal sequence. The first 25 amino acids of the proprotein is the signal sequence and is cleaved from LLO when it is secreted by the bacterium, thereby resulting in the full-length active LLO protein of 504 amino acids without the signal sequence. An LLO peptide disclosed herein can comprise the signal sequence or can comprise a peptide that does not include the signal sequence. Exemplary LLO proteins that can be used comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 55 or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of SEQ ID NO: 55. Any sequence that encodes a fragment of an LLO protein or a homologue, variant, isoform, analog, fragment of a homologue, fragment of a variant, or fragment of an analog of an LLO protein can be used. A homologous LLO protein can have a sequence identity with a reference LLO protein, for example, of greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%.

[0123] Another example of an LLO protein is set forth in SEQ ID NO: 56. LLO proteins that can be used can comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 56 or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of SEQ ID NO: 56.

[0124] Another example of an LLO protein is an LLO protein from the Listeria monocytogenes 10403S strain, as set forth in GenBank Accession No.: ZP 01942330 or EBA21833, or as encoded by the nucleic acid sequence as set forth in GenBank Accession No. : NZ_AARZ01000015 or AARZ01000015.1. Another example of an LLO protein is an LLO protein from the Listeria monocytogenes 4b F2365 strain {see, e.g., GenBank Accession No. : YP_012823), EGD-e strain {see, e.g., GenBank Accession No. :

NP 463733), or any other strain of Listeria monocytogenes. Yet another example of an LLO protein is an LLO protein from Flavobacteriales bacterium HTCC2170 {see, e.g., GenBank Accession No.: ZP 01106747 or EAR01433, or encoded by GenBank

Accession No. : NZ_AAOC01000003). LLO proteins that can be used can comprise, consist essentially of, or consist of any of the above LLO proteins or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of the above LLO proteins. [0125] Proteins that are homologous to LLO, or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms thereof, can also be used. One such example is alveolysin, which can be found, for example, in Paenibacillus alvei (see, e.g., GenBank Accession No. : P23564 or AAA22224, or encoded by GenBank Accession No. : M62709). Other such homologous proteins are known.

[0126] The LLO peptide can be a full-length LLO protein or a truncated LLO protein or LLO fragment. Likewise, the LLO peptide can be one that retains one or more functionalities of a native LLO protein or lacks one or more functionalities of a native LLO protein. For example, the retained LLO functionality can be allowing a bacteria (e.g., Listeria) to escape from a phagosome or phagolysosome, or enhancing the immunogenicity of a peptide to which it is fused. The retained functionality can also be hemolytic function or antigenic function. Alternatively, the LLO peptide can be a nonhemolytic LLO. Other functions of LLO are known, as are methods and assays for evaluating LLO functionality.

[0127] An LLO fragment can be a PEST-like sequence or can comprise a PEST-like sequence. LLO fragments can comprise one or more of an internal deletion, a truncation from the C-terminal end, and a truncation from the N-terminal end. In some cases, an LLO fragment can comprise more than one internal deletion. Other LLO peptides can be full-length LLO proteins with one or more mutations.

[0128] Some LLO proteins or fragments have reduced hemolytic activity relative to wild type LLO or are non-hemolytic fragments. For example, an LLO protein can be rendered non-hemolytic by deletion or mutation of the activation domain at the carboxy terminus, by deletion or mutation of cysteine 484, or by deletion or mutation at another location.

[0129] Other LLO proteins are rendered non-hemolytic by a deletion or mutation of the cholesterol binding domain (CBD) as detailed in US 8,771,702, herein incorporated by reference in its entirety for all purposes. The mutations can comprise, for example, a substitution or a deletion. The entire CBD can be mutated, portions of the CBD can be mutated, or specific residues within the CBD can be mutated. For example, the LLO protein can comprise a mutation of one or more of residues C484, W491, and W492 (e.g., C484, W491, W492, C484 and W491, C484 and W492, W491 and W492, or all three residues) of SEQ ID NO: 55 or corresponding residues when optimally aligned with SEQ ID NO: 55 (e.g., a corresponding cysteine or tryptophan residue). As an example, a mutant LLO protein can be created wherein residues C484, W491, and W492 of LLO are substituted with alanine residues, which will substantially reduce hemolytic activity relative to wild type LLO. The mutant LLO protein with C484A, W491A, and W492A mutations is termed "mutLLO."

[0130] As another example, a mutant LLO protein can be created with an internal deletion comprising the cholesterol-binding domain. The sequence of the cholesterol- binding domain of SEQ ID NO: 55 set forth in SEQ ID NO: 74. For example, the internal deletion can be a 1-11 amino acid deletion, an 11-50 amino acid deletion, or longer.

Likewise, the mutated region can be 1-11 amino acids, 11-50 amino acids, or longer (e.g., 1-50, 1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 1-2, 1-3, 1-4, 1-5, 1-6, 1- 7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-20, 11-25, 11-30, 11-35, 11-40, 11-50, 11-60, 11-70, 11-80, 11-90, 11- 100, 11-150, 15-20, 15-25, 15-30, 15-35, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90, 15- 100, 15-150, 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20- 150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100, or 30-150 amino acids). For example, a mutated region consisting of residues 470-500, 470-510, or 480-500 of SEQ ID NO: 55 will result in a deleted sequence comprising the CBD (residues 483-493 of SEQ ID NO: 55). However, the mutated region can also be a fragment of the CBD or can overlap with a portion of the CBD. For example, the mutated region can consist of residues 470-490, 480-488, 485-490, 486-488, 490-500, or 486-510 of SEQ ID NO: 55. For example, a fragment of the CBD (residues 484-492) can be replaced with a

heterologous sequence, which will substantially reduce hemolytic activity relative to wild type LLO. For example, the CBD (ECTGLAWEWWR; SEQ ID NO: 74) can be replaced with a CTL epitope from the antigen NY-ESO-1 (ESLLMWITQCR; SEQ ID NO: 75), which contains the HLA-A2 restricted epitope 157-165 from NY-ESO-1. The resulting LLO is termed "ctLLO."

[0131] In some mutated LLO proteins, the mutated region can be replaced by a heterologous sequence. For example, the mutated region can be replaced by an equal number of heterologous amino acids, a smaller number of heterologous amino acids, or a larger number of amino acids (e.g., 1-50, 1-11, 2-1 1, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9- 11, 10-11, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-20, 11-25, 11-30, 11-35, 11-40, 11-50, 11-60, 11-70, 11-80, 11-90, 11-100, 11-150, 15-20, 15-25, 15-30, 15-35, 15-40, 15-50, 15- 60, 15-70, 15-80, 15-90, 15-100, 15-150, 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100, or 30- 150 amino acids). Other mutated LLO proteins have one or more point mutations (e.g., a point mutation of 1 residue, 2 residues, 3 residues, or more). The mutated residues can be contiguous or not contiguous.

[0132] In one example embodiment, an LLO peptide may have a deletion in the signal sequence and a mutation or substitution in the CBD.

[0133] Some LLO peptides are N-terminal LLO fragments (i.e., LLO proteins with a C-terminal deletion). Some LLO peptides are at least 494, 489, 492, 493, 500, 505, 510, 515, 520, or 525 amino acids in length or 492-528 amino acids in length. For example, the LLO fragment can consist of about the first 440 or 441 amino acids of an LLO protein (e.g., the first 441 amino acids of SEQ ID NO: 55 or 56, or a corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO: 55 or 56). Other N- terminal LLO fragments can consist of the first 420 amino acids of an LLO protein (e.g., the first 420 amino acids of SEQ ID NO: 55 or 56, or a corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO: 55 or 56). Other N-terminal fragments can consist of about amino acids 20-442 of an LLO protein (e.g., amino acids 20-442 of SEQ ID NO: 55 or 56, or a corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO: 55 or 56). Other N-terminal LLO fragments comprise any ALLO without the activation domain comprising cysteine 484, and in particular without cysteine 484. For example, the N-terminal LLO fragment can correspond to the first 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, or 25 amino acids of an LLO protein (e.g., the first 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, or 25 amino acids of SEQ ID NO: 55 or 56, or a corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO: 55 or 56). Preferably, the fragment comprises one or more PEST-like sequences. LLO fragments and truncated LLO proteins can contain residues of a homologous LLO protein that correspond to any one of the above specific amino acid ranges. The residue numbers need not correspond exactly with the residue numbers enumerated above (e.g., if the homologous LLO protein has an insertion or deletion relative to a specific LLO protein disclosed herein). Examples of N-terminal LLO fragments include SEQ ID NOS: 57, 58, and 59. LLO proteins that can be used comprise, consist essentially of, or consist of the sequence set forth in SEQ ID NO: 57, 58, or 59 or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of SEQ ID NO: 57, 58, or 59. In some compositions and methods, the N-terminal LLO fragment set forth in SEQ ID NO: 59 is used. An example of a nucleic acid encoding the N-terminal LLO fragment set forth in SEQ ID NO: 59 is SEQ ID NO: 60. (b) ActA

[0134] Another example of a PEST-containing peptide that can be utilized in the compositions and methods disclosed herein is an ActA peptide. ActA is a surface- associated protein and acts as a scaffold in infected host cells to facilitate the

polymerization, assembly, and activation of host actin polymers in order to propel a Listeria monocytogenes through the cytoplasm. Shortly after entry into the mammalian cell cytosol, L. monocytogenes induces the polymerization of host actin filaments and uses the force generated by actin polymerization to move, first intracellularly and then from cell to cell. ActA is responsible for mediating actin nucleation and actin-based motility. The ActA protein provides multiple binding sites for host cytoskeletal components, thereby acting as a scaffold to assemble the cellular actin polymerization machinery. The N-terminus of ActA binds to monomeric actin and acts as a constitutively active nucleation promoting factor by stimulating the intrinsic actin nucleation activity. The actA and hly genes are both members of the 10-kb gene cluster regulated by the transcriptional activator PrfA, and actA is upregulated approximately 226-fold in the mammalian cytosol. Any sequence that encodes an ActA protein or a homologue, variant, isoform, analog, fragment of a homologue, fragment of a variant, or fragment of an analog of an ActA protein can be used. A homologous ActA protein can have a sequence identity with a reference ActA protein, for example, of greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%.

[0135] One example of an ActA protein comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 61. Another example of an ActA protein comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 62. The first 29 amino acid of the proprotein corresponding to either of these sequences are the signal sequence and are cleaved from ActA protein when it is secreted by the bacterium. An ActA peptide can comprise the signal sequence (e.g., amino acids 1-29 of SEQ ID NO: 61 or 62), or can comprise a peptide that does not include the signal sequence. Other examples of ActA proteins comprise, consist essentially of, or consist of homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of isoforms, or fragments of analogs of SEQ ID NO: 61 or 62.

[0136] Another example of an ActA protein is an ActA protein from the Listeria monocytogenes 10403S strain (GenBank Accession No. : DQ054585) the NICPBP 54002 strain (GenBank Accession No. : EU394959), the S3 strain (GenBank Accession No. : EU394960), NCTC 5348 strain (GenBank Accession No.: EU394961), NICPBP 54006 strain (GenBank Accession No. : EU394962), M7 strain (GenBank Accession No. :

EU394963), S19 strain (GenBank Accession No.: EU394964), or any other strain of Listeria monocytogenes. LLO proteins that can be used can comprise, consist essentially of, or consist of any of the above LLO proteins or homologues, variants, isoforms, analogs, fragments, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of the above LLO proteins.

[0137] ActA peptides can be full-length ActA proteins or truncated ActA proteins or ActA fragments (e.g., N-terminal ActA fragments in which a C-terminal portion is removed). Preferably, truncated ActA proteins comprise at least one PEST sequence (e.g., more than one PEST sequence). In addition, truncated ActA proteins can optionally comprise an ActA signal peptide. Examples of PEST-like sequences contained in truncated ActA proteins include SEQ ID NOS: 45-48. Some such truncated ActA proteins comprise at least two of the PEST-like sequences set forth in SEQ ID NOS: 45-48 or homologs thereof, at least three of the PEST-like sequences set forth in SEQ ID NOS: 45- 48 or homologs thereof, or all four of the PEST-like sequences set forth in SEQ ID NOS: 45-48 or homologs thereof. Examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of about residues 30-122, about residues 30-229, about residues 30-332, about residues 30-200, or about residues 30-399 of a full length ActA protein sequence (e.g., SEQ ID NO: 62). Other examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of about the first 50, 100, 150, 200, 233, 250, 300, 390, 400, or 418 residues of a full length ActA protein sequence (e.g., SEQ ID NO: 62). Other examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of about residues 200-300 or residues 300-400 of a full length ActA protein sequence (e.g., SEQ ID NO:

62). For example, the truncated ActA consists of the first 390 amino acids of the wild type ActA protein as described in US 7,655,238, herein incorporated by reference in its entirety for all purposes. As another example, the truncated ActA can be an ActA-NlOO or a modified version thereof (referred to as ActA-NlOO*) in which a PEST motif has been deleted and containing the nonconservative QDNKR (SEQ ID NO: 73) substitution as described in US 2014/0186387, herein incorporated by references in its entirety for all purposes. Alternatively, truncated ActA proteins can contain residues of a homologous ActA protein that corresponds to one of the above amino acid ranges or the amino acid ranges of any of the ActA peptides disclosed herein. The residue numbers need not correspond exactly with the residue numbers enumerated herein (e.g., if the homologous ActA protein has an insertion or deletion, relative to an ActA protein utilized herein, then the residue numbers can be adjusted accordingly).

[0138] Examples of truncated ActA proteins include, for example, proteins comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 63, 64, 65, or 66 or homologues, variants, isoforms, analogs, fragments of variants, fragments of isoforms, or fragments of analogs of SEQ ID NO: 63, 64, 65, or 66. SEQ ID NO: 63 referred to as ActA/PESTl and consists of amino acids 30-122 of the full length ActA sequence set forth in SEQ ID NO: 62. SEQ ID NO: 64 is referred to as

ActA/PEST2 or LA229 and consists of amino acids 30-229 of the full length ActA sequence set forth in the full-length ActA sequence set forth in SEQ ID NO: 62. SEQ ID NO: 65 is referred to as ActA/PEST3 and consists of amino acids 30-332 of the full-length ActA sequence set forth in SEQ ID NO: 62. SEQ ID NO: 66 is referred to as

ActA/PEST4 and consists of amino acids 30-399 of the full-length ActA sequence set forth in SEQ ID NO: 62. As a specific example, the truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 64 can be used.

[0139] Examples of truncated ActA proteins include, for example, proteins comprising, consisting essentially of, or consisting of the sequence set forth in SEQ ID NO: 67, 69, 70, or 72 or homologues, variants, isoforms, analogs, fragments of variants, fragments of isoforms, or fragments of analogs of SEQ ID NO: 67, 69, 70, or 72. As a specific example, the truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 67 (encoded by the nucleic acid set forth in SEQ ID NO: 68) can be used. As another specific example, the truncated ActA protein consisting of the sequence set forth in SEQ ID NO: 70 (encoded by the nucleic acid set forth in SEQ ID NO: 71) can be used. SEQ ID NO: 71 is the first 1170 nucleotides encoding ActA in the Listeria monocytogenes 10403S strain. In some cases, the ActA fragment can be fused to a heterologous signal peptide. For example, SEQ ID NO: 72 sets forth an ActA fragment fused to an Hly signal peptide. (3) Generating Immunotherapy Constructs Encoding Recombinant Fusion Polypeptides

[0140] Also provided herein are methods for generating immunotherapy constructs encoding or compositions comprising the recombinant fusion polypeptides disclosed herein. For example, such methods can comprise selecting and designing antigenic peptides to include in the immunotherapy construct (and, for example, testing the hydropathy of the each antigenic peptide, and modifying or deselecting an antigenic peptide if it scores above a selected hydropathy index threshold value), designing one or more fusion polypeptides comprising each of the selected antigenic peptides, and generating a nucleic acid construct encoding the fusion polypeptide.

[0141] The antigenic peptides can be screened for hydrophobicity or hydrophilicity. Antigenic peptides can be selected, for example, if they are hydrophilic or if they score up to or below a certain hydropathy threshold, which can be predictive of secretability in a particular bacteria of interest (e.g., Listeria monocytogenes). For example, antigenic peptides can be scored by Kyte and Doolittle hydropathy index with a 21 amino acid window, all scoring above cutoff (around 1.6) are excluded as they are unlikely to be secretable by Listeria monocytogenes. See, e.g., Kyte-Doolittle (1982) JMol Biol 157(1): 105-132; herein incorporated by reference in its entirety for all purposes.

Alternatively, an antigenic peptide scoring about a selected cutoff can be altered (e.g., changing the length of the antigenic peptide). Other sliding window sizes that can be used include, for example, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or more amino acids. For example, the sliding window size can be 9-11 amino acids, 11-13 amino acids, 13-15 amino acids, 15-17 amino acids, 17-19 amino acids, 19-21 amino acids, 21-23 amino acids, 23-25 amino acids, or 25-27 amino acids. Other cutoffs that can be used include, for example, the following ranges 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2.0-2.2 2.2-2.5, 2.5-

3.0, 3.0-3.5, 3.5-4.0, or 4.0-4.5, or the cutoff can be 1.4, 1.5, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,

2.3, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,

4.4, or 4.5. The cutoff can vary, for example, depending on the genus or species of the bacteria being used to deliver the fusion polypeptide.

[0142] Other suitable hydropathy plots or other appropriate scales include, for example, those reported in Rose et al. (1993) Annu Rev Biomol Struct 22:381-415; Biswas et al. (2003) Journal of Chromatography A 1000:637-655; Eisenberg (1984) Ann Rev Biochem 53 :595-623; Abraham and Leo (1987) Proteins: Structure, Function and

Genetics 2: 130-152; Sweet and Eisenberg (1983) Mol Biol 171 :479-488; Bull and Breese (191 '4) Arch Biochem Biophys 161 :665-670; Guy (1985) Biophys J 47:61-70; Miyazawa et al. (1985) Macromolecules 18:534-552; Roseman (1988) JMolBiol 200:513-522;

Wolfenden et al. (1981) Biochemistry 20:849-855; Wilson (1981) Biochem J 199:31-41; Cowan and Whittaker (1990) Peptide Research 3 :75-80; Aboderin (1971) Int J Biochem 2:537-544; Eisenberg et al. (1984) JMolBiol 179: 125-142; Hopp and Woods (1981) Proc Natl Acad Sci USA 78:3824-3828; Manavalan and Ponnuswamy (1978) Nature 275:673- 674; Black and Mould (1991) Anal Biochem 193 :72-82; Fauchere and Pliska (1983) Eur J Med Chem 18:369-375; Janin (1979) Nature 277:491-492; Rao and Argos (1986) Biochim Biophys Acta 869: 197-214; Tanford (1962) Am Chem Soc 84:4240-4274; Welling et al. (1985) FEBS Lett 188:215-218; Parker et al. (1986) Biochemistry 25:5425-5431; and Cowan and Whittaker (1990) Peptide Research 3 :75-80, each of which is herein incorporated by reference in its entirety for all purposes.

[0143] Optionally, the antigenic peptides can be scored for their ability to bind to the subject human leukocyte antigen (HLA) type (for example by using the Immune Epitope Database (IED) available at www.iedb.org, which includes netMHCpan, ANN,

SMMPMBEC. SMM, CombLib_Sidney2008, PickPocket, and netMHCcons) and ranked by best MHC binding score from each antigenic peptide. Other sources include TEpredict (tepredict.sourceforge.net/help.html) or other available MHC binding measurement scales. Cutoffs may be different for different expression vectors such as Salmonella.

[0144] Optionally, the antigenic peptides can be screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL-10-inducing T helper epitopes, and so forth) to deselect antigenic peptides or to avoid immunosuppressive influences.

[0145] Optionally, a predicative algorithm for immunogenicity of the epitopes can be used to screen the antigenic peptides. However, these algorithms are at best 20% accurate in predicting which peptide will generate a T cell response. Alternatively, no

screening/predictive algorithms are used. Alternatively, the antigenic peptides can be screened for immunogenicity. For example, this can comprise contacting one or more T cells with an antigenic peptide, and analyzing for an immunogenic T cell response, wherein an immunogenic T cell response identifies the peptide as an immunogenic peptide. This can also comprise using an immunogenic assay to measure secretion of at least one of CD25, CD44, or CD69 or to measure secretion of a cytokine selected from the group comprising IFN-γ, TNF-a, IL-1, and IL-2 upon contacting the one or more T cells with the peptide, wherein increased secretion identifies the peptide as comprising one or more T cell epitopes. [0146] The selected antigenic peptides can be arranged into one or more candidate orders for a potential fusion polypeptide. If there are more usable antigenic peptides than can fit into a single plasmid, different antigenic peptides can be assigned priority ranks as needed/desired and/or split up into different fusion polypeptides (e.g., for inclusion in different recombinant Listeria strains). Priority rank can be determined by factors such as relative size, priority of transcription, and/or overall hydrophobicity of the translated polypeptide. The antigenic peptides can be arranged so that they are joined directly together without linkers, or any combination of linkers between any number of pairs of antigenic peptides, as disclosed in more detail elsewhere herein. The number of linear antigenic peptides to be included can be determined based on consideration of the number of constructs needed versus the mutational burden, the efficiency of translation and secretion of multiple epitopes from a single plasmid, and the MOI needed for each bacteria or Lm comprising a plasmid.

[0147] The combination of antigenic peptides or the entire fusion polypeptide (i.e., comprising the antigenic peptides and the PEST-containing peptide and any tags) also be scored for hydrophobicity. For example, the entirety of the fused antigenic peptides or the entire fusion polypeptide can be scored for hydropathy by a Kyte and Doolittle hydropathy index with a sliding 21 amino acid window. If any region scores above a cutoff (e.g., around 1.6), the antigenic peptides can be reordered or shuffled within the fusion polypeptide until an acceptable order of antigenic peptides is found (i.e., one in which no region scores above the cutoff). Alternatively, any problematic antigenic peptides can be removed or redesigned to be of a different size. Alternatively or additionally, one or more linkers between antigenic peptides as disclosed elsewhere herein can be added or modified to change the hydrophobicity. As with hydropathy testing for the individual antigenic peptides, other window sizes can be used, or other cutoffs can be used (e.g., depending on the genus or species of the bacteria being used to deliver the fusion polypeptide). In addition, other suitable hydropathy plots or other appropriate scales could be used.

[0148] Optionally, the combination of antigenic peptides or the entire fusion polypeptide can be further screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL-10-inducing T helper epitopes, and so forth) to deselect antigenic peptides or to avoid immunosuppressive influences.

[0149] A nucleic acid encoding a candidate combination of antigenic peptides or fusion polypeptide can then be designed and optimized. For example, the sequence can be optimized for increased levels of translation, duration of expression, levels of secretion, levels of transcription, and any combination thereof. For example, the increase can be 2- fold to 1000-fold, 2-fold to 500-fold, 2-fold to 100-fold, 2-fold to 50-fold, 2-fold to 20- fold, 2-fold to 10-fold, or 3-fold to 5-fold relative to a control, non-optimized sequence.

[0150] For example, the fusion polypeptide or nucleic acid encoding the fusion polypeptide can be optimized for decreased levels of secondary structures possibly formed in the oligonucleotide sequence, or alternatively optimized to prevent attachment of any enzyme that may modify the sequence. Expression in bacterial cells can be hampered, for example, by transcriptional silencing, low mRNA half-life, secondary structure formation, attachment sites of oligonucleotide binding molecules such as repressors and inhibitors, and availability of rare tRNAs pools. The source of many problems in bacterial expressions is found within the original sequence. The optimization of RNAs may include modification of cis acting elements, adaptation of its GC-content, modifying codon bias with respect to non-limiting tRNAs pools of the bacterial cell, and avoiding internal homologous regions. Thus, optimizing a sequence can entail, for example, adjusting regions of very high (> 80%) or very low (< 30%) GC content. Optimizing a sequence can also entail, for example, avoiding one or more of the following cis-acting sequence motifs: internal TATA-boxes, chi-sites, and ribosomal entry sites; AT -rich or GC-rich sequence stretches; repeat sequences and RNA secondary structures; (cryptic) splice donor and acceptor sites; branch points; or a combination thereof. Optimizing expression can also entail adding sequence elements to flanking regions of a gene and/or elsewhere in the plasmid.

[0151] Optimizing a sequence can also entail, for example, adapting the codon usage to the codon bias of host genes (e.g., Listeria monocytogenes genes). For example, the codons below can be used for Listeria monocytogenes.

into a delivery vehicle such as a bacteria strain or Listeria strain. Other delivery vehicles may be suitable for DNA immunotherapy or peptide immunotherapy, such as a vaccinia virus or virus-like particle. Once a plasmid encoding a fusion polypeptide is generated and introduced into a bacteria strain or Listeria strain, the bacteria or Listeria strain can be cultured and characterized to confirm expression and secretion of the fusion polypeptide comprising the antigenic peptides.

B. Recombinant Bacteria or Listeria Strains

[0153] Also provided herein are recombinant bacterial strains, such as a Listeria strain, comprising a recombinant fusion polypeptide disclosed herein or a nucleic acid encoding the recombinant fusion polypeptide as disclosed elsewhere herein. Preferably, the bacterial strain is a Listeria strain, such as a Listeria monocytogenes (Lm) strain. Lm has a number of inherent advantages as a vaccine vector. The bacterium grows very efficiently in vitro without special requirements, and it lacks LPS, which is a major toxicity factor in gram-negative bacteria, such as Salmonella. Genetically attenuated Lm vectors also offer additional safety as they can be readily eliminated with antibiotics, in case of serious adverse effects, and unlike some viral vectors, no integration of genetic material into the host genome occurs.

[0154] The recombinant Listeria strain can be any Listeria strain. Examples of suitable Listeria strains include Listeria seeligeri, Listeria grayi, Listeria ivanovii, Listeria murrayi, Listeria welshimeri, Listeria monocytogenes (Lm), or any other Listeria species known in the art. Preferably, the recombinant listeria strain is a strain of the species Listeria monocytogenes. Examples of Listeria monocytogenes strains include the following: L. monocytogenes 10403S wild type (see, e.g., Bishop and Hinrichs (1987) J Immunol 139:2005-2009; Lauer et al. (2002) JBact 184:4177-4186); L. monocytogenes DP-L4056, which is phage cured (see, e.g., Lauer et al. (2002) JBact 184:4177-4186); L. monocytogenes DP-L4027, which is phage cured and has an hly gene deletion (see, e.g., Lauer et al. (2002) J act 184:4177- 4186; Jones and Portnoy (1994) Infect Immunity 65:5608-5613); L. monocytogenes DP-L4029, which is phage cured and has an actA gene deletion (see, e.g., Lauer et al. (2002) JBact 184:4177-4186; Skoble et al. (2000) J Cell Biol 150:527- 538); L. monocytogenes DP-L4042 (delta PEST) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci. USA 101 : 13832-13837 and supporting information); L.

monocytogenes DP-L4097 (LLO-S44A) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101 : 13832-13837 and supporting information); L. monocytogenes DP- L4364 (delta IplA; lipoate protein ligase) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101 : 13832-13837 and supporting information); L. monocytogenes DP-L4405 (delta MA) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101 : 13832-13837 and supporting information); L. monocytogenes DP-L4406 (delta inlE) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101 : 13832-13837 and supporting information); L. monocytogenes CS-LOOOl (delta actA; delta inlB) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101 : 13832-13837 and supporting information); L. monocytogenes CS- L0002 (delta actA; delta IplA) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101 : 13832-13837 and supporting information); L. monocytogenes CS-L0003 (LLO L461T; delta IplA) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101 : 13832- 13837 and supporting information); L. monocytogenes DP-L4038 (delta actA; LLO L461T) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101 : 13832-13837 and supporting information); L. monocytogenes DP-L4384 (LLO S44A; LLO L461T) {see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101 : 13832-13837 and supporting information); a J. monocytogenes strain with an IplAl deletion (encoding lipoate protein ligase LplAl) {see, e.g., O'Riordan et al. (2003) Science 302:462-464); L. monocytogenes DP-L4017 (10403S with LLO L461T) {see, e.g., US 7,691,393); L. monocytogenes EGD {see, e.g., GenBank Accession No. AL591824). In another embodiment, the Listeria strain is L. monocytogenes EGD-e (see GenBank Accession No. NC_003210; ATCC Accession No. BAA-679); L. monocytogenes DP-L4029 {actA deletion, optionally in combination with uvrAB deletion (DP-L4029uvrAB) {see, e.g., US 7,691,393); L. monocytogenes actA- linlB - double mutant {see, e.g., ATCC Accession No. PTA-5562); L. monocytogenes IplA mutant or hly mutant {see, e.g., US 2004/0013690); L. monocytogenes dalldat double mutant {see, e.g., US 2005/0048081). Other J. monocytogenes strains includes those that are modified (e.g., by a plasmid and/or by genomic integration) to contain a nucleic acid encoding one of, or any combination of, the following genes: hly (LLO; listeriolysin); iap (p60); inlA; inlB; inlC; dal (alanine racemase); dat (D-amino acid aminotransferase); plcA; plcB; actA; or any nucleic acid that mediates growth, spread, breakdown of a single walled vesicle, breakdown of a double walled vesicle, binding to a host cell, or uptake by a host cell. Each of the above references is herein incorporated by reference in its entirety for all purposes.

[0155] The recombinant bacteria or Listeria can have wild-type virulence, can have attenuated virulence, or can be avirulent. For example, a recombinant Listeria of can be sufficiently virulent to escape the phagosome or phagolysosome and enter the cytosol. Such Listeria strains can also be live-attenuated Listeria strains, which comprise at least one attenuating mutation, deletion, or inactivation as disclosed elsewhere herein.

Preferably, the recombinant Listeria is an attenuated auxotrophic strain. An auxotrophic strain is one that is unable to synthesize a particular organic compound required for its growth. Examples of such strains are described in US 8, 114,414, herein incorporated by reference in its entirety for all purposes.

[0156] Preferably, the recombinant Listeria strain lacks antibiotic resistance genes. For example, such recombinant Listeria strains can comprise a plasmid that does not encode an antibiotic resistance gene. However, some recombinant Listeria strains provided herein comprise a plasmid comprising a nucleic acid encoding an antibiotic resistance gene. Antibiotic resistance genes may be used in the conventional selection and cloning processes commonly employed in molecular biology and vaccine preparation. Exemplary antibiotic resistance genes include gene products that confer resistance to ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, chloramphenicol (CAT), neomycin, hygromycin, and gentamicin.

(1) Bacteria or Listeria Strains Comprising

Recombinant Fusion Polypeptides or Nucleic Acids Encoding

Recombinant Fusion Polypeptides

[0157] The recombinant bacterial strains (e.g., Listeria strains) disclosed herein comprise a recombinant fusion polypeptide disclosed herein or a nucleic acid encoding the recombinant fusion polypeptide as disclosed elsewhere herein.

[0158] In bacteria or Listeria strains comprising a nucleic acid encoding a recombinant fusion protein, the nucleic acid can be codon optimized. Examples of optimal codons utilized by L. monocytogenes for each amino acid are shown US 2007/0207170, herein incorporated by reference in its entirety for all purposes. A nucleic acid is codon- optimized if at least one codon in the nucleic acid is replaced with a codon that is more frequently used by L. monocytogenes for that amino acid than the codon in the original sequence.

[0159] The nucleic acid can be present in an episomal plasmid within the bacteria or Listeria strain and/or the nucleic acid can be genomically integrated in the bacteria or Listeria strain. Some recombinant bacteria or Listeria strains comprise two separate nucleic acids encoding two recombinant fusion polypeptides as disclosed herein: one nucleic acid in an episomal plasmid, and one genomically integrated in the bacteria or Listeria strain.

[0160] The episomal plasmid can be one that is stably maintained in vitro (in cell culture), in vivo (in a host), or both in vitro and in vivo. If in an episomal plasmid, the open reading frame encoding the recombinant fusion polypeptide can be operably linked to a promoter/regulatory sequence in the plasmid. If genomically integrated in the bacteria or Listeria strain, the open reading frame encoding the recombinant fusion polypeptide can be operably linked to an exogenous promoter/regulatory sequence or to an endogenous promoter/regulatory sequence. Examples of promoters/regulatory sequences useful for driving constitutive expression of a gene are well known and include, for example, an hly, hlyA, actA, pr/A, and p60 promoters of Listeria, the Streptococcus bac promoter, the Streptomyces griseus sgiA promoter, and the B. thuringiensis phaZ promoter. In some cases, an inserted gene of interest is not interrupted or subjected to regulatory constraints which often occur from integration into genomic DNA, and in some cases, the presence of the inserted heterologous gene does not lead to rearrangement or interruption of the cell's own important regions.

[0161] Such recombinant bacteria or Listeria strains can be made by transforming a bacteria or Listeria strain or an attenuated bacteria or Listeria strain described elsewhere herein with a plasmid or vector comprising a nucleic acid encoding the recombinant fusion polypeptide. The plasmid can be an episomal plasmid that does not integrate into a host chromosome. Alternatively, the plasmid can be an integrative plasmid that integrates into a chromosome of the bacteria or Listeria strain. The plasmids used herein can also be multicopy plasmids. Methods for transforming bacteria are well known, and include calcium-chloride competent cell-based methods, electroporation methods, bacteriophage- mediated transduction, chemical transformation techniques, and physical transformation techniques. See, e.g., de Boer et al. (1989) Cell 56:641-649; Miller et al. (1995) FASEB J. 9: 190-199; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al. (1997) Current Protocols in Molecular Biology, John Wiley & Sons, New York; Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, D.C.; and Miller, 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., each of which is herein incorporated by reference in its entirety for all purposes.

[0162] Bacteria or Listeria strains with genomically integrated heterologous nucleic acids can be made, for example, by using a site-specific integration vector, whereby the bacteria or Listeria comprising the integrated gene is created using homologous recombination. The integration vector can be any site-specific integration vector that is capable of infecting a bacteria or Listeria strain. Such an integration vector can comprise, for example, a PSA attPP' site, a gene encoding a PSA integrase, a U153 attPP' site, a gene encoding a U153 integrase, an Al 18 attPP' site, a gene encoding an Al 18 integrase, or any other known attPP' site or any other phage integrase.

[0163] Such bacteria or Listeria strains comprising an integrated gene can also be created using any other known method for integrating a heterologous nucleic acid into a bacteria or Listeria chromosome. Techniques for homologous recombination are well known, and are described, for example, in Baloglu et al. (2005) Vet Microbiol 109(1- 2): 11-17); Jiang et al. 2005) Acta Biochim Biophys Sin (Shanghai) 37(1): 19-24), and US 6,855,320, each of which is herein incorporated by reference in its entirety for all purposes.

[00100] Integration into a bacteria or Listerial chromosome can also be achieved using transposon insertion. Techniques for transposon insertion are well known, and are described, for example, for the construction of DP-L967 by Sun et al. (1990) Infection and Immunity 58: 3770-3778, herein incorporated by reference in its entirety for all purposes. Transposon mutagenesis can achieve stable genomic insertion, but the position in the genome where the heterologous nucleic acids has been inserted is unknown.

[0164] Integration into a bacterial or Listerial chromosome can also be achieved using phage integration sites {see, e.g., Lauer et al. (2002) J Bacteriol 184(15):4177-4186, herein incorporated by reference in its entirety for all purposes). For example, an integrase gene and attachment site of a bacteriophage (e.g., U153 or PSA listeriophage) can be used to insert a heterologous gene into the corresponding attachment site, which may be any appropriate site in the genome (e.g. comK or the 3' end of the org tRNA gene). Endogenous prophages can be cured from the utilized attachment site prior to integration of the heterologous nucleic acid. Such methods can result, for example, in single-copy integrants. In order to avoid a "phage curing step," a phage integration system based on PSA phage can be used (see, e.g., Lauer et al. (2002) J Bacteriol 184:4177-4186, herein incorporated by reference in its entirety for all purposes). Maintaining the integrated gene can require, for example, continuous selection by antibiotics.

Alternatively, a phage-based chromosomal integration system can be established that does not require selection with antibiotics. Instead, an auxotrophic host strain can be complemented. For example, a phage-based chromosomal integration system for clinical applications can be used, where a host strain that is auxotrophic for essential enzymes, including, for example, D-alanine racemase is used (e.g., Lm dal(-)dat(-)). [0165] Conjugation can also be used to introduce genetic material and/or plasmids into bacteria. Methods for conjugation are well known, and are described, for example, in Nikodinovic et al. (2006) Plasmid 56(3):223-227 and Auchtung et al. (2005) Proc Natl Acad Sci USA 102(35): 12554-12559, each of which is herein incorporated by reference in its entirety for all purposes.

[0166] In a specific example, a recombinant bacteria or Listeria strain can comprise a nucleic acid encoding a recombinant fusion polypeptide genomically integrated into the bacteria or Listeria genome as an open reading frame with an endogenous actA sequence (encoding an ActA protein) or an endogenous hly sequence (encoding an LLO protein). For example, the expression and secretion of the fusion polypeptide can be under the control of the endogenous actA promoter and ActA signal sequence or can be under the control of the endogenous hly promoter and LLO signal sequence. As another example, the nucleic acid encoding a recombinant fusion polypeptide can replace an actA sequence encoding an ActA protein or an hly sequence encoding an LLO protein. As another example, a recombinant Listeria strain, the strain comrpsing a nucleic acid sequence encoding at least one fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to one or more disease-associated antigenic peptides, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA and inlB, wherein the nucleic acid is genomically integrated, and wherein the PEST-containing peptide is an ActA protein or a fragment thereof.

[0167] Selection of recombinant bacteria or Listeria strains can be achieved by any means. For example, antibiotic selection can be used. Antibiotic resistance genes may be used in the conventional selection and cloning processes commonly employed in molecular biology and vaccine preparation. Exemplary antibiotic resistance genes include gene products that confer resistance to ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, chloramphenicol (CAT), neomycin, hygromycin, and gentamicin. Alternatively, auxotrophic strains can be used, and an exogenous metabolic gene can be used for selection instead of or in addition to an antibiotic resistance gene. As an example, in order to select for auxotrophic bacteria comprising a plasmid encoding a metabolic enzyme or a complementing gene provided herein, transformed auxotrophic bacteria can be grown in a medium that will select for expression of the gene encoding the metabolic enzyme (e.g., amino acid metabolism gene) or the complementing gene. Alternatively, a temperature-sensitive plasmid can be used to select recombinants or any other known means for selecting recombinants.

(2) Attenuation of Bacteria or Listeria Strains

[0168] The recombinant bacteria strains (e.g., recombinant Listeria strains) disclosed herein can be attenuated. The term "attenuation" encompasses a diminution in the ability of the bacterium to cause disease in a host animal. For example, the pathogenic characteristics of an attenuated Listeria strain may be lessened compared with wild-type Listeria, although the attenuated Listeria is capable of growth and maintenance in culture. Using as an example the intravenous inoculation of BALB/c mice with an attenuated

Listeria, the lethal dose at which 50% of inoculated animals survive (LD50) is preferably increased above the LD50 of wild-type Listeria by at least about 10-fold, more preferably by at least about 100-fold, more preferably at least about 1,000 fold, even more preferably at least about 10,000 fold, and most preferably at least about 100,000-fold. An attenuated strain of Listeria is thus one that does not kill an animal to which it is administered, or is one that kills the animal only when the number of bacteria administered is vastly greater than the number of wild-type non-attenuated bacteria which would be required to kill the same animal. An attenuated bacterium should also be construed to mean one which is incapable of replication in the general environment because the nutrient required for its growth is not present therein. Thus, the bacterium is limited to replication in a controlled environment wherein the required nutrient is provided. Attenuated strains are

environmentally safe in that they are incapable of uncontrolled replication

(a) Methods of Attenuating Bacteria and Listeria Strains

[0169] Attenuation can be accomplished by any known means. For example, such attenuated strains can be deficient in one or more endogenous virulence genes or one or more endogenous metabolic genes. Examples of such genes are disclosed herein, and attenuation can be achieved by inactivation of any one of or any combination of the genes disclosed herein. Inactivation can be achieved, for example, through deletion or through mutation (e.g., an inactivating mutation). The term "mutation" includes any type of mutation or modification to the sequence (nucleic acid or amino acid sequence) and may encompass a deletion, a truncation, an insertion, a substitution, a disruption, or a translocation. For example, a mutation can include a frameshift mutation, a mutation which causes premature termination of a protein, or a mutation of regulatory sequences which affect gene expression. Mutagenesis can be accomplished using recombinant DNA techniques or using traditional mutagenesis technology using mutagenic chemicals or radiation and subsequent selection of mutants. Deletion mutants may be preferred because of the accompanying low probability of reversion. The term "metabolic gene" refers to a gene encoding an enzyme involved in or required for synthesis of a nutrient utilized or required by a host bacteria. For example, the enzyme can be involved in or required for the synthesis of a nutrient required for sustained growth of the host bacteria. The term "virulence" gene includes a gene whose presence or activity in an organism's genome that contributes to the pathogenicity of the organism (e.g., enabling the organism to achieve colonization of a niche in the host (including attachment to cells), immunoevasion

(evasion of host's immune response), immunosuppression (inhibition of host's immune response), entry into and exit out of cells, or obtaining nutrition from the host).

[0170] A specific example of such an attenuated strain is Listeria monocytogenes {Lm) dal(-)dat(-) (Lmdd). Another example of such an attenuated strain is Lm dal(-)dat(-)AactA (LmddA). See, e.g., US 2011/0142791, herein incorporated by references in its entirety for all purposes. LmddA is based on a Listeria strain which is attenuated due to the deletion of the endogenous virulence gene actA. Such strains can retain a plasmid for antigen expression in vivo and in vitro by complementation of the dal gene. Alternatively, the LmddA can be a dal/dat/actA Listeria having mutations in the endogenous dal, dat, and actA genes. Such mutations can be, for example, a deletion or other inactivating mutation.

[0171] Another specific example of an attenuated strain is Lm prfA(-) or a strain having a partial deletion or inactivating mutation in the prfA gene. The PrfA protein controls the expression of a regulon comprising essential virulence genes required by Lm to colonize its vertebrate hosts; hence the prfA mutation strongly impairs PrfA ability to activate expression of PrfA-dependent virulence genes.

[0172] Yet another specific example of an attenuated strain is Lm inlB(-)actA(-) in which two genes critical to the bacterium's natural virulence— internalin B and actA— are deleted.

[0173] Other examples of attenuated bacteria or Listeria strains include bacteria or Listeria strains deficient in one or more endogenous virulence genes. Examples of such genes include actA, prfA, plcB, plcA, inlA, inlB, inlC, inlJ, and bsh in Listeria. Attenuated Listeria strains can also be the double mutant or triple mutant of any of the above- mentioned strains. Attenuated Listeria strains can comprise a mutation or deletion of each one of the genes, or comprise a mutation or deletion of, for example, up to ten of any of the genes provided herein (e.g., including the actA, pr/A, and dal/dat genes). For example, an attenuated Listeria strain can comprise a mutation or deletion of an endogenous internalin C (inlC) gene and/or a mutation or deletion of an endogenous actA gene.

Alternatively, an attenuated Listeria strain can comprise a mutation or deletion of an endogenous internalin B (inlB) gene and/or a mutation or deletion of an endogenous actA gene. Alternatively, an attenuated Listeria strain can comprise a mutation or deletion of endogenous inlB, inlC, and actA genes. Translocation of Listeria to adjacent cells is inhibited by the deletion of the endogenous actA gene and/or the endogenous inlC gene or endogenous MB gene, which are involved in the process, thereby resulting in high levels of attenuation with increased immunogenicity and utility as a strain backbone. An attenuated Listeria strain can also be a double mutant comprising mutations or deletions of both plcA and plcB. In some cases, the strain can be constructed from the EGD Listeria backbone.

[0174] A bacteria or Listeria strain can also be an auxotrophic strain having a mutation in a metabolic gene. As one example, the strain can be deficient in one or more endogenous amino acid metabolism genes. For example, the generation of auxotrophic strains of Listeria deficient in D-alanine, for example, may be accomplished in a number of ways that are well known, including deletion mutations, insertion mutations, frameshift mutations, mutations which cause premature termination of a protein, or mutation of regulatory sequences which affect gene expression. Deletion mutants may be preferred because of the accompanying low probability of reversion of the auxotrophic phenotype. As an example, mutants of D-alanine which are generated according to the protocols presented herein may be tested for the ability to grow in the absence of D-alanine in a simple laboratory culture assay. Those mutants which are unable to grow in the absence of this compound can be selected.

[0175] Examples of endogenous amino acid metabolism genes include a vitamin synthesis gene, a gene encoding pantothenic acid synthase, a D-glutamic acid synthase gene, a D-alanine amino transferase (dat) gene, a D-alanine racemase (dal) gene, dga, a gene involved in the synthesis of diaminopimelic acid (DAP), a gene involved in the synthesis of Cysteine synthase A (cysK), a vitamin-B12 independent methionine synthase, trpA, trpB, trpE, asnB, gltD, gltB, leuA, argG, and thrC. The Listeria strain can be deficient in two or more such genes (e.g., dat and dal). D-glutamic acid synthesis is controlled in part by the dal gene, which is involved in the conversion of D-glu + pyr to alpha-ketoglutarate + D-ala, and the reverse reaction.

[0176] As another example, an attenuated Listeria strain can be deficient in an endogenous synthase gene, such as an amino acid synthesis gene. Examples of such genes include folP, a gene encoding a dihydrouridine synthase family protein, ispD, ispF, a gene encoding a phosphoenolpyruvate synthase, hisF, hisH,fliI, a gene encoding a ribosomal large subunit pseudouridine synthase, ispD, a gene encoding a bifunctional GMP synthase/glutamine amidotransferase protein, cobS, cobB, cbiD, a gene encoding a uroporphyrin-III C-methyltransferase/uroporphyrinogen-III synthase, cobQ, uppS, truB, dxs, mvaS, dapA, ispG,folC, a gene encoding a citrate synthase, argJ, a gene encoding a 3-deoxy-7-phosphoheptulonate synthase, a gene encoding an indole-3 -glycerol -phosphate synthase, a gene encoding an anthranilate synthase/glutamine amidotransferase component, menB, a gene encoding a menaquinone-specific isochorismate synthase, a gene encoding a phosphoribosylformylglycinamidine synthase I or II, a gene encoding a phosphoribosylaminoimidazole-succinocarboxamide synthase, carB, car A, thyA, mgsA, aroB, hepB, rluB, ilvB, ilvN, alsS,fabF,fabH, a gene encoding a pseudouridine synthase, pyrG, truA, pabB, and an atp synthase gene (e.g., atpC, atpD-2, aptG, atpA-2, and so forth).

[0177] Attenuated Listeria strains can be deficient in endogenous phoP, aroA, aroC, aroD, or plcB. As yet another example, an attenuated Listeria strain can be deficient in an endogenous peptide transporter. Examples include genes encoding an ABC

transporter/ ATP-binding/permease protein, an oligopeptide ABC transporter/oligopeptide- binding protein, an oligopeptide ABC transporter/permease protein, a zinc ABC transporter/zinc-binding protein, a sugar ABC transporter, a phosphate transporter, a ZIP zinc transporter, a drug resistance transporter of the EmrBIQacA family, a sulfate transporter, a proton-dependent oligopeptide transporter, a magnesium transporter, a formate/nitrite transporter, a spermidine/putrescine ABC transporter, a Na/Pi- cotransporter, a sugar phosphate transporter, a glutamine ABC transporter, a major facilitator family transporter, a glycine betaine/L-proline ABC transporter, a molybdenum ABC transporter, a techoic acid ABC transporter, a cobalt ABC transporter, an ammonium transporter, an amino acid ABC transporter, a cell division ABC transporter, a manganese ABC transporter, an iron compound ABC transporter, a maltose/maltodextrin ABC transporter, a drug resistance transporter of the Bcr/CflA family, and a subunit of one of the above proteins. [0178] Other attenuated bacteria and Listeria strains can be deficient in an endogenous metabolic enzyme that metabolizes an amino acid that is used for a bacterial growth process, a replication process, cell wall synthesis, protein synthesis, metabolism of a fatty acid, or for any other growth or replication process. Likewise, an attenuated strain can be deficient in an endogenous metabolic enzyme that can catalyze the formation of an amino acid used in cell wall synthesis, can catalyze the synthesis of an amino acid used in cell wall synthesis, or can be involved in synthesis of an amino acid used in cell wall synthesis. Alternatively, the amino acid can be used in cell wall biogenesis. Alternatively, the metabolic enzyme is a synthetic enzyme for D-glutamic acid, a cell wall component.

[0179] Other attenuated Listeria strains can be deficient in metabolic enzymes encoded by a D-glutamic acid synthesis gene, dga, an air (alanine racemase) gene, or any other enzymes that are involved in alanine synthesis. Yet other examples of metabolic enzymes for which the Listeria strain can be deficient include enzymes encoded by serC (a phosphoserine aminotransferase), asd (aspartate betasemialdehyde dehydrogenase; involved in synthesis of the cell wall constituent diaminopimelic acid), the gene encoding gsaB- glutamate-l-semialdehyde aminotransferase (catalyzes the formation of 5- aminolevulinate from (S)-4-amino-5-oxopentanoate), hemL (catalyzes the formation of 5- aminolevulinate from (S)-4-amino-5-oxopentanoate), aspB (an aspartate aminotransferase that catalyzes the formation of oxalozcetate and L-glutamate from L-aspartate and 2- oxoglutarate), argF-1 (involved in arginine biosynthesis), aroE (involved in amino acid biosynthesis), aroB (involved in 3-dehydroquinate biosynthesis), aroD (involved in amino acid biosynthesis), aroC (involved in amino acid biosynthesis), hisB (involved in histidine biosynthesis), hisD (involved in histidine biosynthesis), hisG (involved in histidine biosynthesis), metX (involved in methionine biosynthesis), proB (involved in proline biosynthesis), argR (involved in arginine biosynthesis), argj (involved in arginine biosynthesis), thil (involved in thiamine biosynthesis), LMO/2365 1652 (involved in tryptophan biosynthesis), aroA (involved in tryptophan biosynthesis), ilvD (involved in valine and isoleucine biosynthesis), ilvC (involved in valine and isoleucine biosynthesis), leuA (involved in leucine biosynthesis), dapF (involved in lysine biosynthesis), and thrB (involved in threonine biosynthesis) (all GenBank Accession No. NC_002973).

[0180] An attenuated Listeria strain can be generated by mutation of other metabolic enzymes, such as a tRNA synthetase. For example, the metabolic enzyme can be encoded by the trpS gene, encoding tryptophanyltRNA synthetase. For example, the host strain bacteria can be A(trpS aroA), and both markers can be contained in an integration vector. [0181] Other examples of metabolic enzymes that can be mutated to generate an attenuated Listeria strain include an enzyme encoded by murE (involved in synthesis of diaminopimelic acid; GenBank Accession No: NC_003485), LMOf2365_2494 (involved in teichoic acid biosynthesis), WecE (Lipopolysaccharide biosynthesis protein rffA;

GenBank Accession No: AE014075.1), or amiA (an N-acetylmuramoyl-L-alanine amidase). Yet other examples of metabolic enzymes include aspartate aminotransferase, histidinol-phosphate aminotransferase (GenBank Accession No. NP 466347), or the cell wall teichoic acid glycosylation protein GtcA.

[0182] Other examples of metabolic enzymes that can be mutated to generate an attenuated Listeria strain include a synthetic enzyme for a peptidoglycan component or precursor. The component can be, for example, UDP-N-acetylmuramylpentapeptide, UDP-N-acetylglucosamine, MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol, GlcNAc-p-(l,4)-MurNAc-(pentapeptide)-pyrophosphorylundecapre nol, or any other peptidoglycan component or precursor.

[0183] Yet other examples of metabolic enzymes that can be mutated to generate an attenuated Listeria strain include metabolic enzymes encoded by murG, murD, murA-1, or murA-2 (all set forth in GenBank Accession No. NC_002973). Alternatively, the metabolic enzyme can be any other synthetic enzyme for a peptidoglycan component or precursor. The metabolic enzyme can also be a trans-glycosylase, a trans-peptidase, a carboxy-peptidase, any other class of metabolic enzyme, or any other metabolic enzyme. For example, the metabolic enzyme can be any other Listeria metabolic enzyme or any other Listeria monocytogenes metabolic enzyme.

[0184] Other bacterial strains can be attenuated as described above for Listeria by mutating the corresponding orthologous genes in the other bacterial strains.

(b) Methods of Complementing Attenuated Bacteria and Listeria Strains

[0185] The attenuated bacteria or Listeria strains disclosed herein can further comprise a nucleic acid comprising a complementing gene or encoding a metabolic enzyme that complements an attenuating mutation (e.g., complements the auxotrophy of the auxotrophic Listeria strain). For example, a nucleic acid having a first open reading frame encoding a fusion polypeptide as disclosed herein can further comprise a second open reading frame comprising the complementing gene or encoding the complementing metabolic enzyme. Alternatively, a first nucleic acid can encode the fusion polypeptide and a separate second nucleic acid can comprise the complementing gene or encode the complementing metabolic enzyme.

[0186] The complementing gene can be extrachromosomal or can be integrated into the bacteria or Listeria genome. For example, the auxotrophic Listeria strain can comprise an episomal plasmid comprising a nucleic acid encoding a metabolic enzyme. Such plasmids will be contained in the Listeria in an episomal or extrachromosomal fashion. Alternatively, the auxotrophic Listeria strain can comprise an integrative plasmid (i.e., integration vector) comprising a nucleic acid encoding a metabolic enzyme. Such integrative plasmids can be used for integration into a Listeria chromosome. Preferably, the episomal plasmid or the integrative plasmid lacks an antibiotic resistance marker.

[0187] The metabolic gene can be used for selection instead of or in addition to an antibiotic resistance gene. As an example, in order to select for auxotrophic bacteria comprising a plasmid encoding a metabolic enzyme or a complementing gene provided herein, transformed auxotrophic bacteria can be grown in a medium that will select for expression of the gene encoding the metabolic enzyme (e.g., amino acid metabolism gene) or the complementing gene. For example, a bacteria auxotrophic for D-glutamic acid synthesis can be transformed with a plasmid comprising a gene for D-glutamic acid synthesis, and the auxotrophic bacteria will grow in the absence of D-glutamic acid, whereas auxotrophic bacteria that have not been transformed with the plasmid, or are not expressing the plasmid encoding a protein for D-glutamic acid synthesis, will not grow. Similarly, a bacterium auxotrophic for D-alanine synthesis will grow in the absence of D- alanine when transformed and expressing a plasmid comprising a nucleic acid encoding an amino acid metabolism enzyme for D-alanine synthesis. Such methods for making appropriate media comprising or lacking necessary growth factors, supplements, amino acids, vitamins, antibiotics, and the like are well-known and are available commercially.

[0188] Once the auxotrophic bacteria comprising the plasmid encoding a metabolic enzyme or a complementing gene provided herein have been selected in appropriate medium, the bacteria can be propagated in the presence of a selective pressure. Such propagation can comprise growing the bacteria in media without the auxotrophic factor. The presence of the plasmid expressing the metabolic enzyme or the complementing gene in the auxotrophic bacteria ensures that the plasmid will replicate along with the bacteria, thus continually selecting for bacteria harboring the plasmid. Production of the bacteria or Listeria strain can be readily scaled up by adjusting the volume of the medium in which the auxotrophic bacteria comprising the plasmid are growing. [0189] In one specific example, the attenuated strain is a strain having a deletion of or an inactivating mutation in dal and dat (e.g., Listeria monocytogenes {Lm) dal(-)dat(-) (Lmdd) or Lm dal(-)dat(-)AactA (LmddA)), and the complementing gene encodes an alanine racemase enzyme (e.g., encoded by dal gene) or a D-amino acid aminotransferase enzyme (e.g., encoded by dat gene). An exemplary alanine racemase protein can have the sequence set forth in SEQ ID NO: 76 (encoded by SEQ ID NO: 78; GenBank Accession No: AF038438) or can be a homologue, variant, isoform, analog, fragment, fragment of a homologue, fragment of a variant, fragment of an analog, or fragment of an isoform of SEQ ID NO: 76 . The alanine racemase protein can also be any other Listeria alanine racemase protein. Alternatively, the alanine racemase protein can be any other gram- positive alanine racemase protein or any other alanine racemase protein. An exemplary D- amino acid aminotransferase protein can have the sequence set forth in SEQ ID NO: 77 (encoded by SEQ ID NO: 79; GenBank Accession No: AF038439) or can be a

homologue, variant, isoform, analog, fragment, fragment of a homologue, fragment of a variant, fragment of an analog, or fragment of an isoform of SEQ ID NO: 77. The D- amino acid aminotransferase protein can also be any other Listeria D-amino acid aminotransferase protein. Alternatively, the D-amino acid aminotransferase protein can be any other gram-positive D-amino acid aminotransferase protein or any other D-amino acid aminotransferase protein.

[0190] In another specific example, the attenuated strain is a strain having a deletion of or an inactivating mutation in prfA (e.g., Lm pr/A(-)), and the complementing gene encodes a PrfA protein. For example, the complementing gene can encode a mutant PrfA (D133V) protein that restores partial PrfA function. An example of a wild type PrfA protein is set forth in SEQ ID NO: 80 (encoded by nucleic acid set forth in SEQ ID NO: 81), and an example of a D133V mutant PrfA protein is set forth in SEQ ID NO: 82

(encoded by nucleic acid set forth in SEQ ID NO: 83). The complementing PrfA protein can be a homologue, variant, isoform, analog, fragment, fragment of a homologue, fragment of a variant, fragment of an analog, or fragment of an isoform of SEQ ID NO: 80 or 82. The PrfA protein can also be any other Listeria PrfA protein. Alternatively, the PrfA protein can be any other gram-positive PrfA protein or any other PrfA protein.

[0191] In another example, the bacteria strain or Listeria strain can comprise a deletion of or an inactivating mutation in an actA gene, and the complementing gene can comprise an actA gene to complement the mutation and restore function to the Listeria strain. [0192] Other auxotroph strains and complementation systems can also be adopted for the use with the methods and compositions provided herein.

(3) Preparation and Storage of Bacteria or Listeria

Strains

[0193] The recombinant bacteria strain (e.g., Listeria strain) optionally has been passaged through an animal host. Such passaging can maximize efficacy of the Listeria strain as a vaccine vector, can stabilize the immunogenicity of the Listeria strain, can stabilize the virulence of the Listeria strain, can increase the immunogenicity of the Listeria strain, can increase the virulence of the Listeria strain, can remove unstable substrains of the Listeria strain, or can reduce the prevalence of unstable sub-strains of the Listeria strain. Methods for passaging a recombinant Listeria strain through an animal host are well known in the art and are described, for example, in US 2006/0233835, herein incorporated by reference in its entirety for all purposes.

[0194] The recombinant bacteria strain (e.g., Listeria strain) can be stored in a frozen cell bank or stored in a lyophilized cell bank. Such a cell bank can be, for example, a master cell bank, a working cell bank, or a Good Manufacturing Practice (GMP) cell bank. Examples of "Good Manufacturing Practices" include those defined by 21 CFR 210-211 of the United States Code of Federal Regulations. However, "Good Manufacturing Practices" can also be defined by other standards for production of clinical-grade material or for human consumption, such as standards of a country other than the United States. Such cell banks can be intended for production of clinical-grade material or can conform to regulatory practices for human use.

[0195] Recombinant bacteria strains (e.g., Listeria strains) can also be from a batch of vaccine doses, from a frozen stock, or from a lyophilized stock.

[0196] Such cell banks, frozen stocks, or batches of vaccine doses can, for example, exhibit viability upon thawing of greater than 90%. The thawing, for example, can follow storage for cryopreservation or frozen storage for 24 hours. Alternatively, the storage can last, for example, for 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 5 months, 6 months, 9 months, or 1 year.

[0197] The cell bank, frozen stock, or batch of vaccine doses can be cryopreserved, for example, by a method that comprises growing a culture of the bacteria strain (e.g., Listeria strain) in a nutrient media, freezing the culture in a solution comprising glycerol, and storing the Listeria strain at below -20°C. The temperature can be, for example, about - 70°C or between about -70 to about -80°C. Alternatively, the cell bank, frozen stock, or batch of vaccine doses can be cryopreserved by a method that comprises growing a culture of the Listeria strain in a defined medium, freezing the culture in a solution comprising glycerol, and storing the Listeria strain at below -20°C. The temperature can be, for example, about -70°C or between about -70 to about -80°C. Any defined microbiological medium may be used in this method.

[0198] The culture (e.g., the culture of a Listeria vaccine strain that is used to produce a batch of Listeria vaccine doses) can be inoculated, for example, from a cell bank, from a frozen stock, from a starter culture, or from a colony. The culture can be inoculated, for example, at mid-log growth phase, at approximately mid-log growth phase, or at another growth phase.

[0199] The solution used for freezing optionally contain another colligative additive or additive with anti -freeze properties in place of glycerol or in addition to glycerol.

Examples of such additives include, for example, mannitol, DMSO, sucrose, or any other colligative additive or additive with anti-freeze properties.

[0200] The nutrient medium utilized for growing a culture of a bacteria strain (e.g., a Listeria strain) can be any suitable nutrient medium. Examples of suitable media include, for example, LB; TB; a modified, animal-product-free Terrific Broth; or a defined medium.

[0201] The step of growing can be performed by any known means of growing bacteria. For example, the step of growing can be performed with a shake flask (such as a baffled shake flask), a batch fermenter, a stirred tank or flask, an airlift fermenter, a fed batch, a continuous cell reactor, an immobilized cell reactor, or any other means of growing bacteria.

[0202] Optionally, a constant pH is maintained during growth of the culture (e.g. in a batch fermenter). For example, the pH can be maintained at about 6.0, at about 6.5, at about 7.0, at about 7.5, or about 8.0. Likewise, the pH can be, for example, from about 6.5 to about 7.5, from about 6.0 to about 8.0, from about 6.0 to about 7.0, from about 6.0 to about 7.0, or from about 6.5 to about 7.5.

[0203] Optionally, a constant temperature can be maintained during growth of the culture. For example, the temperature can be maintained at about 37°C or at 37°C.

Alternatively, the temperature can be maintained at 25°C, 27°C, 28°C, 30°C, 32°C, 34°C, 35°C, 36°C, 38°C, or 39°C. [0204] Optionally, a constant dissolved oxygen concentration can be maintained during growth of the culture. For example, the dissolved oxygen concentration can be maintained at 20% of saturation, 15% of saturation, 16% of saturation, 18% of saturation, 22%) of saturation, 25% of saturation, 30%> of saturation, 35% of saturation, 40% of saturation, 45% of saturation, 50% of saturation, 55% of saturation, 60% of saturation, 65% of saturation, 70% of saturation, 75% of saturation, 80% of saturation, 85% of saturation, 90% of saturation, 95% of saturation, 100% of saturation, or near 100%) of saturation.

[0205] Methods for lyophilization and cryopreservation of recombinant bacteria strains (e.g., Listeria strains are known. For example, a Listeria culture can be flash- frozen in liquid nitrogen, followed by storage at the final freezing temperature.

Alternatively, the culture can be frozen in a more gradual manner (e.g., by placing in a vial of the culture in the final storage temperature). The culture can also be frozen by any other known method for freezing a bacterial culture.

[0206] The storage temperature of the culture can be, for example, between -20 and - 80°C. For example, the temperature can be significantly below -20°C or not warmer than -70°C. Alternatively, the temperature can be about -70°C, -20°C, -30°C, -40°C, -50°C, - 60°C, -80°C, -30 to -70°C, -40 to -70°C, -50 to -70°C, -60 to -70°C, -30 to -80°C, -40 to - 80°C, -50 to -80°C, -60 to -80°C, or -70 to -80°C. Alternatively, the temperature can be colder than 70°C or colder than -80°C.

C. Inhibitor of SERPINAl, CRP, TIMP-1, and/or VEGF

[0207] The methods and compositions herein can comprise inhibitors of SERPINAl . "SERPINAl", "AAT" or "Alpha- 1 Antitrypsin", used interchangeably herein, is a serine protease inhibitor (serpins) and is frequently elevated in some cancers (i.e., cervical, CRC, lung cancer, thyroid, ovarian, bladder, gastric, breast). Protein sequences for SERPINAl may include Uniprot Reference Number P01009 and a amino acid sequence for Homo sapiens alpha- 1 -antitrypsin precursor may include Genbank Accession No.

NP OOl 121179 (SEQ ID NO. 96). A nucleic acid sequence for Homo sapiens SERPF A1 gene for alpha- 1 -antitrypsin may include Genbank Accession No. AM048838 (SEQ ID NO: 95). AAT has distinct anti-inflammatory and immunosuppressive properties: Inhibits Dendritic cell and macrophage activation, antigen presentation, generation for CD8+ Tcells vs. new targets, tolerogenic: Increases Tregs, IL-lr antagonist, IL-10 secretion, Inhibits Tumor Apoptosis, inhibits release of inflammatory cytokines IL-lb, TNFa, Neutrophil de-granulation, inhibits Chemokines IL-8, MCP1 that drive macrophage migration and myelopoeisis,inhibits Infiltration of activated macrophages, neutrophils, and T cells into tumors, used clinically to rescue graft-versus-host disease (GvHD) patients who resistant to corticosteroids. Serum AAT levels do not generally correlate with overall survival in cervical cancer (Figure 1). Additionally, AAT levels do not predict overall survival in HPV+ head and neck cancer (Figure 2) and do not predict response to nonspecific immunotherapies like anti-PD-1 in Melanoma (Figure 3). Non-specific immunotherapies do not require dendritic cell activation to present new targets.

[0208] An "inhibitor of SERPF Al" refers to a composition that inhibits the immunological activity of SERPFNAl or decreases serum expression of SERPINA1. Compositions that inhibits the immunological activity of SERPFNAl may include blocking antibodies, chemical inhibitors, blocking peptides, and inhibitory peptides.

Compositions that decrease expression of SERPINA1 may include RNAi shRNA, siRNA, dsRNA, and artificial transcription factors (e.g., transcriptional repressors designed to decrease expression of SERPINA1 gene). Additional inhibitors of SERPINA1 may include small molecules and nanoparticles.

[0209] The methods and compositions herein can comprise inhibitors of CRP. An "inhibitor of CRP" refers to a composition that inhibits the immunological activity of CRP or decreases serum expression of CRP. Compositions that inhibits the immunological activity of CRP may include blocking antibodies, chemical inhibitors, blocking peptides, and inhibitory peptides. Compositions that decrease expression of CRP may include RNAi shRNA, siRNA, dsRNA, and artificial transcription factors (e.g., transcriptional repressors designed to decrease expression of CRP gene). Additional inhibitors of CRP may include small molecules and nanoparticles.

[0210] The methods and compositions herein can comprise inhibitors of TF P-l . An "inhibitor of TFMP-l" refers to a composition that inhibits the immunological activity of TFMP-l or decreases serum expression of TFMP-l . Compositions that inhibits the immunological activity of TFMP-l may include blocking antibodies, chemical inhibitors, blocking peptides, and inhibitory peptides. Compositions that decrease expression of TFMP-l may include RNAi shRNA, siRNA, dsRNA, and artificial transcription factors (e.g., transcriptional repressors designed to decrease expression of TFMP-l gene).

Additional inhibitors of TFMP-l may include small molecules and nanoparticles.

[0211] The methods and compositions herein can comprise inhibitors of VEGF. An "inhibitor of VEGF" refers to a composition that inhibits the immunological activity of VEGF or decreases serum expression of VEGF. Compositions that inhibits the

immunological activity of VEGF may include blocking antibodies, chemical inhibitors, blocking peptides, and inhibitory peptides. Compositions that decrease expression of VEGF may include RNAi shRNA, siRNA, dsRNA, and artificial transcription factors (e.g., transcriptional repressors designed to decrease expression of VEGF gene).

Additional inhibitors of VEGF may include small molecules and nanoparticles.

[0212] Expression of SERPF A1, CRP, TEVIP-1, andor VEGF can be determined by measuring, for example, mRNA levels or protein levels in a biological sample from a subject. In embodiments, the methods disclosed herein comprise companion diagnostic steps utilizing any known methods for determining the level of SERPINAl, CRP, TEVIP-1, andor VEGF in combination with therapeutic steps.

[0213] A baseline AAT cut-off at 3.5 mg/ml would screen out patients who are not likely to experience a survival benefit on AXAL. Baseline AAT <3.5 mg/ml can be used as biomarker for clinical benefit in AXAL treatment. Additionally, AAT <3.5 mg/ml as an eligibility criteria would eliminate the risk of exposing patients to AXAL treatment when they are not likely to obtain clinical benefit.

[0214] The cutoff for the serum expression of SERPINAl in a patient may be less than 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2 mg/ml, 2.1 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml, 2.5 mg/ml, 2.6 mg/ml, 2.7 mg/ml, 2.8 mg/ml, 2.9 mg/ml, 3 mg/ml, 3.1 mg/ml, 3.2 mg/ml, 3.3 mg/ml,

3.4 mg/ml, 3.5 mg/ml, 3.6 mg/ml, 3.7 mg/ml, 3.8 mg/ml, 3.9 mg/ml, or 4.0 mg/ml. The serum expression of SERPINAl in a patient may be between about 1 mg/ml and about 4 mg/ml, about 1.5 mg/ml and about 4 mg/ml, about 2 mg/ml and about 4 mg/ml, about 2.5 mg/ml and about 4 mg/ml, about 3 mg/ml and about 4 mg/ml, or about 3.5 mg/ml and about 4 mg/ml. In another embodiment, the cutoff is between about 1 mg/ml and about

3.5 mg/ml, about 1 mg/ml and about 3 mg/ml, about 1 mg/ml and about 2.5 mg/ml, about 1 mg/ml and about 2 mg/ml about 1 mg/ml and about 1.5 mg/ml.

V. Therapeutic Methods

[0215] The recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, and vaccines disclosed herein can be used in various methods. For example, they can be used in methods of inducing an immune response against a disease, in methods of inducing an enhanced immune response against a disease, in method of increasing the overall survival rate of a subject having a disease, in methods of inducing an anti-tumor-associated-antigen immune response in a subject, in methods of inducing an anti-tumor or anti-cancer immune response in a subject, in methods of treating a tumor or cancer in a subject, in methods of preventing a tumor or cancer in a subject, or in methods of protecting a subject against a tumor or cancer. They can also be used in methods of increasing the ratio of T effector cells to regulatory T cells (Tregs) in the spleen and tumor of a subject, wherein the T effector cells are targeted to a tumor- associated antigen. They can also be used in methods for increasing tumor-associated- antigen T cells in a subject, increasing survival time of a subject having a tumor or cancer, delaying the onset of cancer in a subject, or reducing tumor or metastasis size in a subject.

[0216] A method of inducing an immune response against a disease in a subject, the method comprising the steps of: obtaining a biological sample from the subject, determining that the serum expression of SERPINA1 in the biological sample is below 3.5 mg/ml, and administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease- associated antigenic peptides. For example, the composition comprising a recombinant Listeria strain may be administered to the subject about 2-4 hours after the composition comprising an inhibitor of SERPINA1. The serum expression of SERPINA1 may be less than, for example, 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2 mg/ml, 2.1 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml, 2.5 mg/ml, 2.6 mg/ml, 2.7 mg/ml, 2.8 mg/ml, 2.9 mg/ml, 3 mg/ml, 3.1 mg/ml, 3.2 mg/ml, 3.3 mg/ml, 3.4 mg/ml, 3.5 mg/ml, 3.6 mg/ml, 3.7 mg/ml, 3.8 mg/ml, 3.9 mg/ml, or 4.0 mg/ml. The serum expression of SERPINA1 may be for example, between about 1 mg/ml and about 4 mg/ml, about 1.5 mg/ml and about 4 mg/ml, about 2 mg/ml and about 4 mg/ml, about 2.5 mg/ml and about 4 mg/ml, about 3 mg/ml and about 4 mg/ml, or about 3.5 mg/ml and about 4 mg/ml. In another embodiment, the cutoff is between about 1 mg/ml and about 3.5 mg/ml, about 1 mg/ml and about 3 mg/ml, about 1 mg/ml and about 2.5 mg/ml, about 1 mg/ml and about 2 mg/ml about 1 mg/ml and about 1.5 mg/ml.

[0217] A method of inducing an immune response against a disease in a subject or a method of increasing the overall survival rate of a subject having a disease, the method comprising the steps of: obtaining a biological sample from the subject, determining that the serum expression of SERPINA1 in the biological sample is above 3.5 mg/ml, and administering to the subject a composition comprising an inhibitor of SERPINA1, administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides. For example, the composition comprising a recombinant Listeria strain may be administered to the subject about 2-4 hours after the composition comprising an inhibitor of SERPFNAl . The serum expression of SERPINAl may be, for example, above 3.6 mg/ml, 3.7 mg/ml, 3.8 mg/ml, 3.9 mg/ml, or 4.0 mg/ml.

[0218] A method of inducing an immune response against a disease in a subject by obtaining a biological sample from the subject, determining that the serum expression of SERPINAl in the biological sample is above 3.5 mg/ml, lowering the level of serum expression of SERPINAl to below 3.5 mg/ml, and administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides. The serum expression of SERPINAl, for example, may be lowered to less than 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2 mg/ml, 2.1 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml, 2.5 mg/ml, 2.6 mg/ml, 2.7 mg/ml, 2.8 mg/ml, 2.9 mg/ml, 3 mg/ml, 3.1 mg/ml, 3.2 mg/ml, 3.3 mg/ml, 3.4 mg/ml, 3.5 mg/ml, 3.6 mg/ml, 3.7 mg/ml, 3.8 mg/ml, 3.9 mg/ml, or 4.0 mg/ml. The serum expression of SERPINAl, for example, may lowered to between about 1 mg/ml and about 4 mg/ml, about 1.5 mg/ml and about 4 mg/ml, about 2 mg/ml and about 4 mg/ml, about 2.5 mg/ml and about 4 mg/ml, about 3 mg/ml and about 4 mg/ml, or about 3.5 mg/ml and about 4 mg/ml. In another embodiment, the cutoff is between about 1 mg/ml and about 3.5 mg/ml, about 1 mg/ml and about 3 mg/ml, about 1 mg/ml and about 2.5 mg/ml, about 1 mg/ml and about 2 mg/ml about 1 mg/ml and about 1.5 mg/ml.

[0219] A composition comprising, i. a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease- associated antigenic peptides, and ii. an effective amount of a composition comprising an inhibitor of SERPFNAL. For example, the composition comprising a vaccine and the composition comprising an inhibitor of SERPFNAl are administered to the subject simultaneously or alternatively the composition comprising a vaccine and the composition comprising an inhibitor of SERPFNAl are administered to the subject sequentially.

[0220] A method of inducing an immune response against one or more disease- associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides or a method of inducing an anti-Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, recurrent cancer mutation, or cancer-specific neoepitope immune response in a subject can comprise, for example, administering to the subject a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein (e.g., that comprises a recombinant fusion polypeptide comprising the Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, recurrent cancer mutation, or cancer-specific neoepitope or a nucleic acid encoding the recombinant fusion polypeptide). An anti- Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, recurrent cancer mutation, or cancer-specific neoepitope immune response can thereby be induced in the subject. For example, in the case of a recombinant Listeria strain, the Listeria strain can express the fusion

polypeptide, thereby eliciting an immune response in the subject. The immune response can comprise, for example, a T-cell response, such as a CD4+FoxP3- T cell response, a CD8+ T cell response, or a CD4+FoxP3- and CD8+ T cell response. Such methods can also increase the ratio of T effector cells to regulatory T cells (Tregs) in the spleen and tumor microenvironments of the subject, allowing for a more profound anti-tumor response in the subject.

[0221] A method of inducing an immune response against one or more disease- associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides or a method of inducing an anti-tumor or anti-cancer immune response in a subject can comprise, for example, administering to the subject a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein. An anti-tumor or anti-cancer immune response can thereby be induced in the subject. For example, in the case of a recombinant Listeria strain, the Listeria strain can express the fusion polypeptide, thereby eliciting an anti-tumor or anticancer response in the subject.

[0222] A method of inducing an immune response against one or more disease- associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides or a method of treating a tumor or cancer in a subject (e.g., wherein the tumor or cancer expresses Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, recurrent cancer mutation, or cancer-specific neoepitope), can comprise, for example, administering to the subject a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion

polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein. The subject can then mount an immune response against the tumor or cancer expressing the Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, recurrent cancer mutation, or cancer-specific neoepitope, thereby treating the tumor or cancer in the subject.

[0223] A method of inducing an immune response against one or more disease- associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides or a method of preventing a tumor or cancer in a subject or protecting a subject against developing a tumor or cancer (e.g., wherein the tumor or cancer is associated with expression of Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, recurrent cancer mutation, or cancer-specific neoepitope), can comprise, for example, administering to the subject a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion

polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein. The subject can then mount an immune response against the Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, recurrent cancer mutation, or cancer-specific neoepitope, thereby preventing a tumor or cancer or protecting the subject against developing a tumor or cancer.

[0224] In some of the above methods, two or more recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines are administered. The multiple recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can be administered sequentially in any order or combination, or can be administered simultaneously in any combination. As an example, if four different Listeria strains are being administered, they can be administered sequentially, they can be administered simultaneously, or they can be administered in any combination (e.g., administering the first and second strains simultaneously and subsequently administering the third and fourth strains

simultaneously). Optionally, in the case of sequential administration, the compositions can be administered during the same immune response, preferably within 0-10 or 3-7 days of each other. The multiple recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can each comprise a different set of antigenic peptides. Alternatively, two or more can comprise the same set of antigenic peptides (e.g., the same set of antigenic peptides in a different order).

[0225] Cancer is a physiological condition in mammals that is typically characterized by unregulated cell growth and proliferation. Cancers can be hematopoietic malignancies or solid tumors (i.e., masses of cells that result from excessive cell growth or proliferation, including pre-cancerous legions). Metastatic cancer refers to a cancer that has spread from the place where it first started to another place in the body. Tumors formed by metastatic cancer cells are called a metastatic tumor or a metastasis, which is a term also used to refer to the process by which cancer cells spread to other parts of the body. In general, metastatic cancer has the same name and same type of cancer cells as the original, or primary, cancer. Examples of solid tumors include melanoma, carcinoma, blastoma, and sarcoma. Hematologic malignancies include, for example, leukemia or lymphoid malignancies, such as lymphoma. Exemplary categories of cancers include brain, breast, gastrointestinal, genitourinary, gynecologic, head and neck, heme, skin and thoracic. Brain malignancies include, for example, glioblastoma, high-grade pontine glioma, low- grade glioma, medulloblastoma, neuroblastoma, and pilocytic astrocytoma.

Gastrointestinal cancers include, for example, colorectal, gallbladder, hepatocellular, pancreas, P ET, gastric, and esophageal. Genitourinary cancers include, for example, adrenocortical, bladder, kidney chromophobe, renal (clear cell), renal (papillary), rhabdoid cancers, and prostate. Gynecologic cancers include, for example, uterine carcinosarcoma, uterine endometrial, serous ovarian, and cervical. Head and neck cancers include, for example, thyroid, nasopharyngeal, head and neck, and adenoid cystic. Heme cancers include, for example, multiple myeloma, myelodysplasia, mantle-cell lymphoma, acute lymphoblastic leukemia (ALL), non-lymphoma, chronic lymphocytic leukemia (CLL), and acute myeloid leukemia (AML). Skin cancers includes, for example, cutaneous melanoma and squamous cell carcinoma. Thoracic cancers include, for example, squamous lung, small-cell lung, and lung adenocarcinoma.

[0226] More particular examples of such cancers include squamous cell cancer or carcinoma (e.g., oral squamous cell carcinoma), myeloma, oral cancer, juvenile nasopharyngeal angiofibroma, neuroendocrine tumors, lung cancer, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, glial tumors, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, breast cancer, triple-negative breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer or carcinoma, salivary gland carcinoma, kidney or renal cancer (e.g., renal cell carcinoma), prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, fibrosarcoma, gallbladder cancer, osteosarcoma, mesothelioma, as well as head and neck cancer. A cancer can also be a brain cancer or another type of CNS or intracranial tumor. For example, a subject can have an astrocytic tumor (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, pilocytic astrocytoma, subependymal giant cell astrocytoma, pleomorphic

xanthoastrocytoma), oligodendroglial tumor (e.g., oligodendroglioma, anaplastic oligodendroglioma), ependymal cell tumor (e.g., ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma), mixed glioma (e.g., mixed

oligoastrocytoma, anaplastic oligoastrocytoma), neuroepithelial tumor of uncertain origin (e.g., polar spongioblastoma, astroblastoma, gliomatosis cerebri), tumor of the choroid plexus (e.g., choroid plexus papilloma, choroid plexus carcinoma), neuronal or mixed neuronal-glial tumor (e.g., gangliocytoma, dyplastic gangliocytoma of cerebellum, ganglioglioma, anaplastic ganglioglioma, desmoplastic infantile ganglioma, central neurocytoma, dysembryoplastic neuroepthelial tumor, olfactory neuroblastoma), pineal parenchyma tumor (e.g., pineocytoma, pineoblastoma, mixed

pineocytoma/pineoblastoma), or tumor with mixed neuroblastic or glioblastic elements (e.g., medulloepithelioma, medulloblastoma, neuroblastoma, retinoblastoma,

ependymoblastoma).

[0227] The term "treat" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted tumor or cancer. Treating may include one or more of directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, slowing the progression of, stabilizing the progression of, inducing remission of, preventing or delaying the metastasis of, reducing/ameliorating symptoms associated with the tumor or cancer, or a combination thereof. For example, treating may include increasing expected survival time or decreasing tumor or metastasis size. The effect (e.g., suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, slowing the progression of, stabilizing the progression of, inducing remission of, preventing or delaying the metastasis of, reducing/ameliorating symptoms of, and so forth, can be relative to a control subject not receiving a treatment or receiving a placebo treatment. The term "treat" or "treating" can also refer to increasing percent chance of survival or increasing expected time of survival for a subject with the tumor or cancer (e.g., relative to a control subject not receiving a treatment or receiving a placebo treatment). In one example, "treating" refers to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of alternative therapeutics, decreasing resistance to alternative therapeutics, or a combination thereof (e.g., relative to a control subject not receiving a treatment or receiving a placebo treatment). The terms "preventing" or "impeding" can refer, for example to delaying the onset of symptoms, preventing relapse of a tumor or cancer, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, preventing metastasis of a tumor or cancer, or a combination thereof. The terms

"suppressing" or "inhibiting" can refer, for example, to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

[0228] The term "subject" refers to a mammal (e.g., a human) in need of therapy for, or susceptible to developing, a tumor or a cancer. The term subject also refers to a mammal (e.g., a human) that receives either prophylactic or therapeutic treatment. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, non-human mammals, and humans. The term "subject" does not necessarily exclude an individual that is healthy in all respects and does not have or show signs of cancer or a tumor.

[0229] An individual is at increased risk of developing a tumor or a cancer if the subject has at least one known risk-factor (e.g., genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the tumor or cancer than individuals without the risk factor.

[0230] A "symptom" or "sign" refers to objective evidence of a disease as observed by a physician or subjective evidence of a disease, such as altered gait, as perceived by the subject. A symptom or sign may be any manifestation of a disease. Symptoms can be primary or secondary. The term "primary" refers to a symptom that is a direct result of a particular disease or disorder (e.g., a tumor or cancer), while the term "secondary" refers to a symptom that is derived from or consequent to a primary cause. The recombinant fusion polypeptides, nucleic acids encoding the recombinant fusion polypeptides, the immunogenic compositions, the pharmaceutical compositions, and the vaccines disclosed herein can treat primary or secondary symptoms or secondary complications.

[0231] The recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines are administered in an effective regime, meaning a dosage, route of administration, and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of the tumor or cancer. Alternatively, the recombinant fusion

polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines are administered in an effective regime, meaning a dosage, route of

administration, and frequency of administration that induces an immune response to a heterologous antigen in the recombinant fusion polypeptide (or encoded by the nucleic acid), the recombinant bacteria or Listeria strain, the immunogenic composition, the pharmaceutical composition, or the vaccine, or in the case of recombinant bacteria or

Listeria strains, that induces an immune response to the bacteria or Listeria strain itself. If a subject is already suffering from the tumor or cancer, the regime can be referred to as a therapeutically effective regime. If the subject is at elevated risk of developing the tumor or cancer relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients. For example, a regime can be considered therapeutically or prophylactically effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods described herein, or if a more favorable outcome is demonstrated in treated patients versus control patients in a controlled clinical trial (e.g., a phase II, phase II/III or phase III trial) at the p < 0.05 or 0.01 or even 0.001 level.

[0232] Exemplary dosages for a recombinant Listeria strain are, for example, 1 x 10 ⁶ - 1 x 10 ⁷ CFU, 1 x 10 ⁷ - 1 x 10 ⁸ CFU, 1 x 10 ⁸ - 3.31 x 10 ¹⁰ CFU, 1 x 10 ⁹ - 3.31 x 10 ¹⁰ CFU, 5-500 x 10 ⁸ CFU, 7-500 x 10 ⁸ CFU, 10-500 x 10 ⁸ CFU, 20-500 x 10 ⁸ CFU, 30-500 x 10 ⁸ CFU, 50-500 x 10 ⁸ CFU, 70-500 x 10 ⁸ CFU, 100-500 x 10 ⁸ CFU, 150-500 x 10 ⁸ CFU, 5- 300 x 10 ⁸ CFU, 5-200 x 10 ⁸ CFU, 5-15 x 10 ⁸ CFU, 5-100 x 10 ⁸ CFU, 5-70 x 10 ⁸ CFU, 5- 50 x 10 ⁸ CFU, 5-30 x 10 ⁸ CFU, 5-20 x 10 ⁸ CFU, 1-30 x 10 ⁹ CFU, 1-20 x 10 ⁹ CFU, 2-30 x 10 ⁹ CFU, 1-10 x 10 ⁹ CFU, 2-10 x 10 ⁹ CFU, 3-10 x 10 ⁹ CFU, 2-7 x 10 ⁹ CFU, 2-5 x 10 ⁹ CFU, and 3-5 x 10 ⁹ CFU. Other exemplary dosages for a recombinant Listeria strain are, for example, 1 x 10 ⁷ organisms, 1.5 x 10 ⁷ organisms, 2 x 10 ⁸ organisms, 3 x 10 ⁷ organisms, 4 x 10 ⁷ organisms, 5 x 10 ⁷ organisms, 6 x 10 ⁷ organisms, 7 x 10 ⁷ organisms, 8 x 10 ⁷ organisms, 10 x 10 ⁷ organisms, 1.5 x 10 ⁸ organisms, 2 x 10 ⁸ organisms, 2.5 x 10 ⁸ organisms, 3 x 10 ⁸ organisms, 3.3 x 10 ⁸ organisms, 4 x 10 ⁸ organisms, 5 x 10 ⁸ organisms, 1 x 10 ⁹ organisms, 1.5 x 10 ⁹ organisms, 2 x 10 ⁹ organisms, 3 x 10 ⁹ organisms, 4 x 10 ⁹ organisms, 5 x 10 ⁹ organisms, 6 x 10 ⁹ organisms, 7 x 10 ⁹ organisms, 8 x 10 ⁹ organisms, 10 x 10 ⁹ organisms, 1.5 x 10 ¹⁰ organisms, 2 x 10 ¹⁰ organisms, 2.5 x 10 ¹⁰ organisms, 3 x 10 ¹⁰ organisms, 3.3 x 10 ¹⁰ organisms, 4 x 10 ¹⁰ organisms, and 5 x 10 ¹⁰ organisms. The dosage can depend on the condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic, and other factors.

[0233] Administration can be by any suitable means. For example, administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intracerebroventricular, intraperitoneal, topical, intranasal, intramuscular, intra-ocular, intrarectal, conjunctival, transdermal, intradermal, vaginal, rectal, intratumoral, parcanceral, transmucosal, intravascular, intraventricular, inhalation (aerosol), nasal aspiration (spray), sublingual, aerosol, suppository, or a combination thereof. For intranasal administration or application by inhalation, solutions or suspensions of the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines mixed and aerosolized or nebulized in the presence of the appropriate carrier are suitable. Such an aerosol may comprise any recombinant fusion polypeptide, nucleic acids encoding a recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine described herein. Administration may also be in the form of a suppository (e.g., rectal suppository or urethral suppository), in the form of a pellet for subcutaneous implantation (e.g., providing for controlled release over a period of time), or in the form of a capsule. Administration may also be via injection into a tumor site or into a tumor. Regimens of administration can be readily determined based on factors such as exact nature and type of the tumor or cancer being treated, the severity of the tumor or cancer, the age and general physical condition of the subject, body weight of the subject, response of the individual subject, and the like.

[0234] The frequency of administration can depend on the half-life of the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines in the subject, the condition of the subject, and the route of administration, among other factors. The frequency can be, for example, daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the subject's condition or progression of the tumor or cancer being treated. The course of treatment can depend on the condition of the subject and other factors. For example, the course of treatment can be several weeks, several months, or several years (e.g., up to 2 years). For example, repeat administrations (doses) may be undertaken immediately following the first course of treatment or after an interval of days, weeks or months to achieve tumor regression or suppression of tumor growth. Assessment may be determined by any known technique, including diagnostic methods such as imaging techniques, analysis of serum tumor markers, biopsy, or the presence, absence, or amelioration of tumor-associated symptoms. As a specific example, the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can be administered every 3 weeks for up to 2 years. In one example, a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein is administered in increasing doses in order to increase the T-effector cell to regulatory T cell ratio and generate a more potent anti-tumor immune response. Anti-tumor immune responses can be further strengthened by providing the subject with cytokines including, for example, IFN-γ, T F-a, and other cytokines known to enhance cellular immune response. See, e.g., US 6,991,785, herein incorporated by reference in its entirety for all purposes.

[0235] Some methods may further comprise "boosting" the subject with additional recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines or administering the recombinant fusion polypeptides, nucleic acids encoding recombinant fusion polypeptides, recombinant bacteria or Listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines multiple times. "Boosting" refers to administering an additional dose to a subject. For example, in some methods, 2 boosts (or a total of 3 inoculations) are administered, 3 boosts are

administered, 4 boosts are administered, 5 boosts are administered, or 6 or more boosts are administered. The number of dosages administered can depend on, for example, the response of the tumor or cancer to the treatment.

[0236] Optionally, the recombinant fusion polypeptide, nucleic acids encoding a recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine used in the booster inoculation is the same as the recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine used in the initial "priming" inoculation. Alternatively, the booster recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine is different from the priming recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine. Optionally, the same dosages are used in the priming and boosting inoculations. Alternatively, a larger dosage is used in the booster, or a smaller dosage is used in the booster. The period between priming and boosting inoculations can be experimentally determined. For example, the period between priming and boosting inoculations can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6-8 weeks, or 8-10 weeks.

[0237] Heterologous prime boost strategies have been effective for enhancing immune responses and protection against numerous pathogens. See, e.g., Schneider et al. (1999) Immunol. Rev. 170:29-38; Robinson (2002) Nat. Rev. Immunol. 2:239-250; Gonzalo et al. (2002) Vaccine 20: 1226-1231; and Tanghe (2001) Infect. Immun. 69:3041-3047, each of which is herein incorporated by reference in its entirety for all purposes. Providing antigen in different forms in the prime and the boost injections can maximize the immune response to the antigen. DNA vaccine priming followed by boosting with protein in adjuvant or by viral vector delivery of DNA encoding antigen is one effective way of improving antigen-specific antibody and CD4 ⁺ T-cell responses or CD8 ⁺ T-cell responses. See, e.g. , Shiver et al . (2002) Nature 415: 331-335; Gilbert et al . (2002) Vaccine 20: 1039- 1045; Billaut-Mulot et al. (2000) Vaccine 19:95-102; and Sin et al. (1999) DNA Cell Biol. 18:771-779, each of which is herein incorporated by reference in its entirety for all purposes. As one example, adding CRL1005 poloxamer (12 kDa, 5% POE) to DNA encoding an antigen can enhance T-cell responses when subjects are vaccinated with a DNA prime followed by a boost with an adenoviral vector expressing the antigen. See, e.g., Shiver et al. (2002) Nature 415:331-335, herein incorporated by reference in its entirety for all purposes. As another example, a vector construct encoding an

immunogenic portion of an antigen and a protein comprising the immunogenic portion of the antigen can be administered. See, e.g., US 2002/0165172, herein incorporated by reference in its entirety for all purposes. Similarly, an immune response of nucleic acid vaccination can be enhanced by simultaneous administration of (e.g., during the same immune response, preferably within 0-10 or 3-7 days of each other) a polynucleotide and polypeptide of interest. See, e.g., US 6,500,432, herein incorporated by reference in its entirety for all purposes.

[0238] The therapeutic methods disclosed herein can also comprise administering one or more additional compounds effective in preventing or treating cancer. For example, an additional compound may comprise a compound useful in chemotherapy, such as amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine, gliadelimplants, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomaldoxorubicin,

liposomaldaunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel (Taxol), pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or a combination thereof. Alternatively, an additional compound can also comprise other biologies, including Herceptin ^® (trastuzumab) against the HER2 antigen, Avastin ^® (bevacizumab) against VEGF, or antibodies to the EGF receptor, such as Erbitux ^® (cetuximab), and Vectibix ^® (panitumumab). Alternatively, an additional compound can comprise other immunotherapies. Alternatively, the additional compound can be an indoleamine 2,3-dioxygenase (IDO) pathway inhibitor, such as 1- m ethyl tryptophan (1MT), 1 -methyl tryptophan (1MT), Necrostatin-1, Pyridoxal

Isonicotinoyl Hydrazone, Ebselen, 5-Methylindole-3-carboxaldehyde, CAY10581, an anti-IDO antibody, or a small molecule IDO inhibitor. IDO inhibition can enhance the efficacy of chemotherapeutic agents. The therapeutic methods disclosed herein can also be combined with radiation, stem cell treatment, surgery, or any other treatment. [0239] Such additional compounds or treatments can precede the administration of a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion

polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein, follow the administration of a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion

polypeptide, a recombinant bacteria or Listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein, or be simultaneous to the administration of a recombinant fusion polypeptide, a nucleic acid encoding a recombinant fusion polypeptide, a recombinant bacteria or Listeria strain, an immunogenic

composition, a pharmaceutical composition, or a vaccine disclosed herein.

[0240] Targeted immunomodulatory therapy is focused primarily on the activation of costimulatory receptors, for example by using agonist antibodies that target members of the tumor necrosis factor receptor superfamily, including 4-1BB, OX40, and GITR (glucocorticoid-induced TNF receptor-related). The modulation of GITR has

demonstrated potential in both antitumor and vaccine settings. Another target for agonist antibodies are co-stimulatory signal molecules for T cell activation. Targeting

costimulatory signal molecules may lead to enhanced activation of T cells and facilitation of a more potent immune response. Co-stimulation may also help prevent inhibitory influences from checkpoint inhibition and increase antigen-specific T cell proliferation.

[0241] Listeria-based immunotherapy acts by inducing the de novo generation of tumor antigen-specific T cells that infiltrate and destroy the tumor and by reducing the numbers and activities of immunosuppressive regulatory T cells (Tregs) and myeloid- derived suppressor cells (MDSCs) in the tumor microenvironment. Antibodies (or functional fragments thereof) for T cell co-inhibitory or co-stimulatory receptors (e.g., checkpoint inhibitors CTLA-4, PD-1, TEVI-3, LAG3 and co-stimulators CD 137, OX40, GITR, and CD40) can have synergy with Listeria-based immunotherapy.

[0242] Thus, some methods can comprise further administering a composition comprising an immune checkpoint inhibitor antagonist, such as a PD-1 signaling pathway inhibitor, a CD-80/86 and CTLA-4 signaling pathway inhibitor, a T cell membrane protein 3 (TEVI3) signaling pathway inhibitor, an adenosine A2a receptor (A2aR) signaling pathway inhibitor, a lymphocyte activation gene 3 (LAG3) signaling pathway inhibitor, a killer immunoglobulin receptor (KIR) signaling pathway inhibitor, a CD40 signaling pathway inhibitor, or any other antigen-presenting cell/T cell signaling pathway inhibitor. Examples of immune checkpoint inhibitor antagonists include an anti-PD-Ll/PD-L2 antibody or fragment thereof, an anti-PD-1 antibody or fragment thereof, an anti-CTLA-4 antibody or fragment thereof, or an anti-B7-H4 antibody or fragment thereof. For example, an anti PD-1 antibody can be administered to a subject at 5-10 mg/kg every 2 weeks, 5-10 mg/kg every 3 weeks, 1-2 mg/kg every 3 weeks, 1-10 mg/kg every week, 1- 10 mg/kg every 2 weeks, 1-10 mg/kg every 3 weeks, or 1-10 mg/kg every 4 weeks.

[0243] Likewise, some methods can further comprise administering a T cell stimulator, such as an antibody or functional fragment thereof binding to a T-cell receptor co-stimulatory molecule, an antigen presenting cell receptor binding co-stimulatory molecule, or a member of the TNF receptor superfamily. The T-cell receptor co- stimulatory molecule can comprise, for example, CD28 or ICOS. The antigen presenting cell receptor binding co-stimulatory molecule can comprise, for example, a CD80 receptor, a CD86 receptor, or a CD46 receptor. The TNF receptor superfamily member can comprise, for example, glucocorticoid-induced TNF receptor (GITR), OX40 (CD 134 receptor), 4- IBB (CD137 receptor), or TNFR25.

[0244] For example, some methods can further comprise administering an effective amount of a composition comprising an antibody or functional fragment thereof binding to a T-cell receptor co-stimulatory molecule or an antibody or functional fragment thereof binding to an antigen presenting cell receptor binding a co-stimulatory molecule. The antibody can be, for example, an anti-TNF receptor antibody or antigen-binding fragment thereof (e.g., TNF receptor superfamily member glucocorticoid-induced TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor), or TNFR25), an anti-OX40 antibody or antigen-binding fragment thereof, or an anti-GITR antibody or antigen binding fragment thereof. Alternatively, other agonistic molecules can be administered (e.g., GITRL, an active fragment of GITRL, a fusion protein containing GITRL, a fusion protein containing an active fragment of GITRL, an antigen presenting cell (APC)/T cell agonist, CD 134 or a ligand or fragment thereof, CD 137 or a ligand or fragment thereof, or an inducible T cell costimulatory (ICOS) or a ligand or fragment thereof, or an agonistic small molecule).

[0245] In a specific example, some methods can further comprise administering an anti-CTLA-4 antibody or a functional fragment thereof and/or an anti-CD137 antibody or functional fragment thereof. For example, the anti-CTLA-4 antibody or a functional fragment thereof or the anti-CD 137 antibody or functional fragment thereof can be administered about 72 hours after the first dose of recombinant fusion polypeptide, nucleic acids encoding a recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine, or about 48 hours after the first dose of recombinant fusion polypeptide, nucleic acids encoding a recombinant fusion polypeptide, recombinant bacteria or Listeria strain, immunogenic composition, pharmaceutical composition, or vaccine. The anti-CTLA-4 antibody or a functional fragment thereof or anti-CD 137 antibody or functional fragment thereof can be administered at a dose, for example, of about 0.05 mg/kg and about 5 mg/kg. A recombinant Listeria strain or immunogenic composition comprising a recombinant Listeria strain can be administered at a dose, for example, of about 1 x 10 ⁹ CFU. Some such methods can further comprise administering an effective amount of an anti-PD-1 antibody or functional fragment thereof.

[0246] Methods for assessing efficacy of cancer immunotherapies are well known and are described, for example, in Dzojic et al. (2006) Prostate 66(8):831-838; Naruishi et al. (2006) Cancer Gene Ther. 13(7):658-663, Sehgal et al. (2006) Cancer Cell Int. 6:21), and Heinrich et al. (2007) Cancer Immunol Immunother 56(5):725-730, each of which is herein incorporated by reference in its entirety for all purposes. As one example, for prostate cancer, a prostate cancer model can be to test methods and compositions disclosed herein, such as a TRAMP-C2 mouse model, a 178-2 BMA cell model, a PAIII

adenocarcinoma cells model, a PC-3M model, or any other prostate cancer model.

[0247] Alternatively or additionally, the immunotherapy can be tested in human subjects, and efficacy can be monitored using known. Such methods can include, for example, directly measuring CD4+ and CD8+ T cell responses, or measuring disease progression (e.g., by determining the number or size of tumor metastases, or monitoring disease symptoms such as cough, chest pain, weight loss, and so forth). Methods for assessing the efficacy of a cancer immunotherapy in human subjects are well known and are described, for example, in Uenaka et al. (2007) Cancer Immun. 7:9 and Thomas-

Kaskel et al. (2006) Int J Cancer 119(10):2428-2434, each of which is herein incorporated by reference in its entirety for all purposes.

VI. Kits

[0248] Also provided are kits comprising a reagent utilized in performing a method disclosed herein or kits comprising a composition, tool, or instrument disclosed herein.

[0249] For example, such kits can comprise a recombinant fusion polypeptide disclosed herein, a nucleic acid encoding a recombinant fusion polypeptide disclosed herein, a recombinant bacteria or Listeria strain disclosed herein, an immunogenic composition disclosed herein, a pharmaceutical composition disclosed herein, or a vaccine disclosed herein. Such kits can additionally comprise an instructional material which describes use of the recombinant fusion polypeptide, the nucleic acid encoding the recombinant fusion polypeptide, the recombinant Listeria strain, the immunogenic composition, the pharmaceutical composition, or the vaccine to perform the methods disclosed herein. Such kits can optionally further comprise an applicator. In one embodiment, a kit, comprising a first container, a second container, and a package insert, wherein the first container comprises at least one dose of a composition comprising an inhibitor of SERPINAl, the second container comprises at least one dose of a composition comprising a recombinant Listeria strain, the strain comprising a nucleic acid sequence encoding at least one fusion polypeptide, and the package insert comprises instructions for treating a patient for a disease using the composition. Although model kits are described below, the contents of other useful kits will be apparent in light of the present disclosure.

[0250] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

LISTING OF EMBODIMENTS

[0251] The subject matter disclosed herein includes, but is not limited to, the following embodiments: 1. A method of inducing an immune response against a disease in a subject, the method comprising the steps of: a) obtaining a biological sample from the subject, b) determining that the serum expression of SERPINA1 in the biological sample is below 3.5 mg/ml, and c) administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides.

2. A method of inducing an immune response against a disease in a subject, the method comprising the steps of: a) obtaining a biological sample from the subject, b) determining that the serum expression of SERPINA1 in the biological sample is above 3.5 mg/ml, and c) administering to the subject a composition comprising an inhibitor of SERPINA1, d) administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides.

3. The method of any one of embodiments 1 and 2, wherein serum expression of SERPINA1 is determined by measuring mRNA levels in the biological sample.

4. The method of any one of embodiments 1 and 2, wherein serum expression of SERPINA1 is determined by measuring protein levels in the biological sample.

5. The method of embodiment 2, wherein the composition comprising a recombinant Listeria strain is administered to the subject about 2-4 hours after the composition comprising an inhibitor of SERPINA1.

6. A method of inducing an immune response against a disease in a subject comprising, administering to a subject an effective amount of a combination comprising, i. a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease- associated antigenic peptides, and ii. an effective amount of a composition comprising an inhibitor of SERPINAl .

7. The method of embodiment 6, wherein the composition comprising a vaccine and the composition comprising an inhibitor of SERPINAl are

administered to the subject simultaneously.

8. The method of embodiment 6, wherein the composition comprising a vaccine and the composition comprising an inhibitor of SERPINAl are

administered to the subject sequentially.

9. The method of any one of embodiments 1-8, wherein the vaccine comprises a recombinant Listeria strain, the strain comprising a nucleic acid sequence encoding at least one fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to the disease-associated antigenic peptides.

10. An immunogenic composition comprising vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease- associated antigenic peptides and an inhibitor of SERPINAl for treating a disease in a subject.

11. A kit, comprising a first container and a second container, wherein the first container comprises at least one dose of a composition comprising an inhibitor of SERPINAl, the second container comprises at least one dose of a composition comprising vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides.

12. The method of any one of embodiments 1-8, wherein the inhibitor of SERPAINA1 inhibits the immunological activity of SERPINAl .

13. The method of any one of embodiments 1-8, wherein the inhibitor of SERPAINA1 decreases serum expression of SERPINAl .

14. The method of embodiment 9, wherein the PEST-containing peptide is a listeriolysin O (LLO) protein or a fragment thereof or an ActA protein or a fragment thereof.

15. The method of embodiment 14, wherein the PEST-containing peptide is an N-terminal fragment of LLO . 16. The method of any one of embodiments 9 and 14-15, wherein the recombinant Listeria strain is an auxotrophic Listeria strain.

17. The method of any one of embodiments 9 and 14-16, wherein the nucleic acid sequence is operably integrated into the Listeria genome. 18. The method of any one of embodiments 9 and 14-16, wherein the nucleic acid sequence is in an episomal plasmid.

19. The method of any one of embodiments 9 and 14-18, wherein the recombinant Listeria strain is attenuated and comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes. 20. The method of embodiment 19, wherein the one or more endogenous genes comprise prfA.

21. The method of embodiment 20, wherein the nucleic acid comprises a second open reading frame encoding a D133 V PrfA mutant protein.

22. The method of embodiments 19, wherein the one or more endogenous genes comprise actA, dal, and dat.

23. The method of any one of embodiments 9 and 14-22, wherein the nucleic acid comprises a second open reading frame encoding a metabolic enzyme.

24. The method of embodiment 23, wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid aminotransferase enzyme. 25. The method of any one of embodiments 9 and 14-24, wherein the disease- associated antigenic peptides is Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, or a fragment thereof.

26. The method of embodiment 25, wherein the Human Papilloma Virus is HPV 16 or HPV 18.

27. The method of any one of embodiments 9 and 14-26, wherein the disease- associated antigenic peptides comprises HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 antigens operably linked in tandem.

28. The method of any one of embodiments 1-8, wherein the fusion

polypeptide comprises a PEST-containing peptide fused to two or more disease- associated antigenic peptides, wherein each of the disease-associated antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the disease-associated antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein. 29. The method of any one of embodiments 1-8, wherein the fusion

polypeptide comprises a PEST-containing peptide fused to one or more disease- associated antigenic peptides, wherein each of the disease-associated antigenic peptides comprises a cancer-specific neoepitope comprising a cancer-specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject.

30. The method of embodiment 9, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in pr/A, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

31. The method of embodiment 9, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

32. The method of any one of embodiments 1-9 and 14-31, wherein the disease is tumor or cancer.

33. The method of embodiment 32, wherein the tumor or cancer is a cervical tumor or cancer, an anal tumor or cancer, a head and neck tumor or cancer, or an oropharyngeal tumor or cancer.

34. The immunogenic composition of embodiment 10, wherein the vaccine comprises a recombinant Listeria strain, the strain comprising a nucleic acid sequence encoding at least one fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to the disease-associated antigenic peptides. 35. The immunogenic composition of any one of embodiments 10 and 34, wherein the inhibitor of SERPAINAl inhibits the immunological activity of SERPINAl .

36. The immunogenic composition of any one of embodiments 10 and 34-35, wherein the inhibitor of SERPAINAl decreases serum expression of SERPINAl .

37. The immunogenic composition of embodiment 34, wherein the PEST- containing peptide is a listeriolysin O (LLO) protein or a fragment thereof or an ActA protein or a fragment thereof.

38. The immunogenic composition of embodiment 37, wherein the PEST- containing peptide is an N-terminal fragment of LLO.

39. The immunogenic composition of any one of embodiments 34 and 37-38, wherein the recombinant Listeria strain is an auxotrophic Listeria strain.

40. The immunogenic composition of embodiments 34 and 37-39, wherein the nucleic acid sequence is operably integrated into the Listeria genome. 41. The immunogenic composition of embodiments 34 and 37-39, wherein the nucleic acid sequence is in an episomal plasmid.

42. The immunogenic composition of embodiments 34 and 37-41, wherein the recombinant Listeria strain is attenuated and comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes. 43. The immunogenic composition of embodiment 42, wherein the one or more endogenous genes comprise prfA.

44. The immunogenic composition of embodiment 43, wherein the nucleic acid comprises a second open reading frame encoding a D133V PrfA mutant protein.

45. The immunogenic composition of embodiment 42, wherein the one or more endogenous genes comprise actA, dal, and dat.

46. The immunogenic composition of any one of embodiments 34 and 37-45, wherein the nucleic acid comprises a second open reading frame encoding a metabolic enzyme. 47. The immunogenic composition of embodiment 46, wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid aminotransferase enzyme.

48. The immunogenic composition of any one of embodiments 34 and 37-47, wherein the disease-associated antigenic peptides is Human Papilloma Virus

(HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, or a fragment thereof.

49. The immunogenic composition of embodiment 48, wherein the Human Papilloma Virus is HPV 16 or HPV 18. 50. The immunogenic composition of any one of embodiments 34 and 37-49, wherein the disease-associated antigenic peptides comprises HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 antigens operably linked in tandem.

51. The immunogenic composition of embodiment 34, wherein the fusion polypeptide comprises a PEST-containing peptide fused to two or more disease- associated antigenic peptides, wherein each of the disease-associated antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the disease-associated antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein.

52. The immunogenic composition of embodiment 34, wherein the fusion polypeptide comprises a PEST-containing peptide fused to one or more disease- associated antigenic peptides, wherein each of the disease-associated antigenic peptides comprises a cancer-specific neoepitope comprising a cancer-specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject. 53. The immunogenic composition of embodiment 34, wherein the

recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in pr/A, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein, and wherein the PEST-containing peptide is an N- terminal fragment of LLO.

54. The immunogenic composition of embodiment 34, wherein the

recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

55. The immunogenic composition of any one of embodiments 34-54, wherein the disease is tumor or cancer.

56. The immunogenic composition of embodiment 55, wherein the tumor or cancer is a cervical tumor or cancer, an anal tumor or cancer, a head and neck tumor or cancer, or an oropharyngeal tumor or cancer.

57. A method of increasing the overall survival rate of a subject having a disease, the method comprising the steps of: a) obtaining a biological sample from the subject, b) determining that the serum expression of SERPINA1 in the biological sample is above 3.5 mg/ml, c) administering to the subject a composition comprising an inhibitor of SERPINA1, and d) administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides.

58. A method of inducing an immune response against a disease in a subject, the method comprising the steps of: a) obtaining a biological sample from the subject, b) determining that the serum expression of SERPINA1 in the biological sample is above 3.5 mg/ml, c) lowering the level of serum expression of SERPINA1 to below 3.5 mg/ml, and d) administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides. 59. A method of inducing an immune response against a disease in a subject, the method comprising the steps of: a) obtaining a biological sample from the subject, b) determining that the serum expression of SERPINAI, CRP, TIMP-1 and/or VEGF in the biological sample is below a reference level, and c) administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides.

60. A method of inducing an immune response against a disease in a subject, the method comprising the steps of: a) obtaining a biological sample from the subject, b) determining that the serum expression of SERPINAI, CRP, TIMP-1 and/or VEGF in the biological sample is above a reference level, and c) administering to the subject a composition comprising an inhibitor of SERPINAI, CRP, TFMP-1 and/or VEGF, d) administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides.

61. The method of any one of embodiments 59 and 60, wherein serum expression of SERPINAI, CRP, TFMP-1 and/or VEGF is determined by measuring mRNA levels in the biological sample.

62. The method of any one of embodiments 59 and 60, wherein serum expression of SERPINAI, CRP, TFMP-1 and/or VEGF is determined by measuring protein levels in the biological sample.

63. The method of embodiment 60, wherein the composition comprising a recombinant Listeria strain is administered to the subject about 2-4 hours after the composition comprising an inhibitor of SERPINAI, CRP, TFMP-1 and/or VEGF.

64. A method of inducing an immune response against a disease in a subject comprising, administering to a subject an effective amount of a combination comprising, i. a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease- associated antigenic peptides, and ii. an effective amount of a composition comprising an inhibitor of SERPINAl .

65. The method of embodiment 64 wherein the composition comprising a vaccine and the composition comprising an inhibitor of SERPINAl, CRP, TEVIP-1 and/or VEGF are administered to the subject simultaneously.

66. The method of embodiment 64, wherein the composition comprising a vaccine and the composition comprising an inhibitor of SERPINAl, CRP, TEVIP-1 and/or VEGF are administered to the subject sequentially. 67. The method of any one of embodiments 59-67, wherein the vaccine comprises a recombinant Listeria strain, the strain comprising a nucleic acid sequence encoding at least one fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to the disease-associated antigenic peptides. 68. An immunogenic composition comprising vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease- associated antigenic peptides and an inhibitor of SERPINAl, CRP, TEVIP-1 and/or VEGF for treating a disease in a subject.

69. A kit, comprising a first container and a second container, wherein the first container comprises at least one dose of a composition comprising an inhibitor of

SERPINAl, CRP, TEVIP-1 and/or VEGF, the second container comprises at least one dose of a composition comprising vaccine comprising one or more disease- associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides. 70. The method of any one of embodiments 59-67, wherein the inhibitor of

SERPINAl, CRP, TEVIP-1 and/or VEGF inhibits the immunological activity of SERPINAl, CRP, TEVIP-1 and/or VEGF.

71. The method of any one of embodiments 59-67, wherein the inhibitor of SERPINAl, CRP, TEVIP-1 and/or VEGF decreases serum expression of

SERPINAl, CRP, TEMP-1 and/or VEGF. 72. The method of embodiment 67, wherein the PEST-containing peptide is a listeriolysin O (LLO) protein or a fragment thereof or an ActA protein or a fragment thereof.

73. The method of embodiment 72, wherein the PEST-containing peptide is an N-terminal fragment of LLO.

74. The method of any one of embodiments 67 and 72-73, wherein the recombinant Listeria strain is an auxotrophic Listeria strain.

75. The method of any one of embodiments 67 and 72-74, wherein the nucleic acid sequence is operably integrated into the Listeria genome. 76. The method of any one of embodiments 67 and 72-74, wherein the nucleic acid sequence is in an episomal plasmid.

77. The method of any one of embodiments 67 and 72-76, wherein the recombinant Listeria strain is attenuated and comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes. 78. The method of embodiment 77, wherein the one or more endogenous genes comprise prfA.

79. The method of embodiment 78, wherein the nucleic acid comprises a second open reading frame encoding a D133 V PrfA mutant protein.

80. The method of embodiments 79, wherein the one or more endogenous genes comprise actA, dal, and dat.

81. The method of any one of embodiments 67 and 72-80, wherein the nucleic acid comprises a second open reading frame encoding a metabolic enzyme.

82. The method of embodiment 81, wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid aminotransferase enzyme. 83. The method of any one of embodiments 67 and 72-82, wherein the disease- associated antigenic peptides is Human Papilloma Virus (HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, or a fragment thereof.

84. The method of embodiment 83, wherein the Human Papilloma Virus is HPV 16 or HPV 18. 85. The method of any one of embodiments 67 and 72-84, wherein the disease- associated antigenic peptides comprises HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 antigens operably linked in tandem.

86. The method of any one of embodiments 59-66, wherein the fusion polypeptide comprises a PEST-containing peptide fused to two or more disease- associated antigenic peptides, wherein each of the disease-associated antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the disease-associated antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein. 87. The method of any one of embodiments 59-66, wherein the fusion polypeptide comprises a PEST-containing peptide fused to one or more disease- associated antigenic peptides, wherein each of the disease-associated antigenic peptides comprises a cancer-specific neoepitope comprising a cancer-specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject.

88. The method of embodiment 67, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in pr/A, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein, and wherein the PEST-containing peptide is an N-terminal fragment of

LLO.

89. The method of embodiment 67, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

90. The method of any one of embodiments 59-67 and 72-89, wherein the disease is tumor or cancer. 91. The method of embodiment 90, wherein the tumor or cancer is a cervical tumor or cancer, an anal tumor or cancer, a head and neck tumor or cancer, or an oropharyngeal tumor or cancer. 92. The immunogenic composition of embodiment 68, wherein the vaccine comprises a recombinant Listeria strain, the strain comprising a nucleic acid sequence encoding at least one fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to the disease-associated antigenic peptides.

93. The immunogenic composition of any one of embodiments 68 and 92, wherein the inhibitor of SERPINAl, CRP, TEVIP-1 and/or VEGF inhibits the immunological activity of SERPINAl, CRP, TEVIP-1 and/or VEGF.

94. The immunogenic composition of any one of embodiments 68 and 92-93, wherein the inhibitor of SERPINAl , CRP, TEVIP- 1 and/or VEGF decreases serum expression of SERPINAl, CRP, TEVIP-1 and/or VEGF.

95. The immunogenic composition of embodiment 92, wherein the PEST- containing peptide is a listeriolysin O (LLO) protein or a fragment thereof or an ActA protein or a fragment thereof. 96. The immunogenic composition of embodiment 95, wherein the PEST- containing peptide is an N-terminal fragment of LLO.

97. The immunogenic composition of any one of embodiments 92 and 95-96, wherein the recombinant Listeria strain is an auxotrophic Listeria strain.

98. The immunogenic composition of embodiments 92 and 95-97, wherein the nucleic acid sequence is operably integrated into the Listeria genome.

99. The immunogenic composition of embodiments 92 and 95-97, wherein the nucleic acid sequence is in an episomal plasmid.

100. The immunogenic composition of embodiments 92 and 95-99, wherein the recombinant Listeria strain is attenuated and comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes.

101. The immunogenic composition of embodiment 100, wherein the one or more endogenous genes comprise prfA.

102. The immunogenic composition of embodiment 101, wherein the nucleic acid comprises a second open reading frame encoding a D133V PrfA mutant protein. 103. The immunogenic composition of embodiment 100, wherein the one or more endogenous genes comprise actA, dal, and dat.

104. The immunogenic composition of any one of embodiments 92 and 95-103, wherein the nucleic acid comprises a second open reading frame encoding a metabolic enzyme.

105. The immunogenic composition of embodiment 104, wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid aminotransferase enzyme.

106. The immunogenic composition of any one of embodiments 92 and 95-105, wherein the disease-associated antigenic peptides is Human Papilloma Virus

(HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2 antigen, Her2/neu chimeric antigen, or a fragment thereof.

107. The immunogenic composition of embodiment 106, wherein the Human Papilloma Virus is HPV 16 or HPV 18. 108. The immunogenic composition of any one of embodiments 92 and 95-107, wherein the disease-associated antigenic peptides comprises HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 antigens operably linked in tandem.

109. The immunogenic composition of embodiment 92, wherein the fusion polypeptide comprises a PEST-containing peptide fused to two or more disease- associated antigenic peptides, wherein each of the disease-associated antigenic peptides comprises a recurrent cancer mutation, and wherein at least two of the disease-associated antigenic peptides comprise different recurrent cancer mutations and are fragments of the same cancer-associated protein.

110. The immunogenic composition of embodiment 92, wherein the fusion polypeptide comprises a PEST-containing peptide fused to one or more disease- associated antigenic peptides, wherein each of the disease-associated antigenic peptides comprises a cancer-specific neoepitope comprising a cancer-specific mutation found in a cancer sample from the subject but not in a healthy biological sample from the subject. 111. The immunogenic composition of embodiment 92, wherein the

recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in pr/A, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding a D133V PrfA mutant protein, and wherein the PEST-containing peptide is an N- terminal fragment of LLO.

112. The immunogenic composition of embodiment 92, wherein the

recombinant Listeria strain is an attenuated Listeria monocytogenes strain comprising a deletion of or inactivating mutation in actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase enzyme or a D-amino acid aminotransferase enzyme, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.

113. The immunogenic composition of any one of embodiments 92-112, wherein the disease is tumor or cancer.

114. The immunogenic composition of embodiment 113, wherein the tumor or cancer is a cervical tumor or cancer, an anal tumor or cancer, a head and neck tumor or cancer, or an oropharyngeal tumor or cancer.

115. A method of increasing the overall survival rate of a subject having a disease, the method comprising the steps of: a) obtaining a biological sample from the subject, b) determining that the serum expression of SERPINA1, CRP, TIMP-1 and/or VEGF in the biological sample, c) administering to the subject a composition comprising an inhibitor of SERPINA1, CRP, TIMP-1 and/or VEGF, and d) administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides.

116. A method of inducing an immune response against a disease in a subject, the method comprising the steps of: a) obtaining a biological sample from the subject, b) determining that the serum expression of SERPINA1, CRP, TIMP-1 and/or VEGF in the biological sample, c) lowering the level of serum expression of SERPINA1, CRP, TEVIP- 1 and/or VEGF to below a reference level, e.g. 3.5 mg/ml for SERPF A1, and d) administering to the subject a composition comprising a vaccine comprising one or more disease-associated antigenic peptides or nucleic acid encoding the disease-associated antigenic peptides.

BRIEF DESCRIPTION OF THE SEQUENCES

[0252] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5' end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.

SEQ

ID Type Description NO

23 DNA 3xFLAG Tag v7

24 DNA 3xFLAG Tag v8

25 DNA 3xFLAG Tag v9

26 DNA 3xFLAG Tag vlO

27 DNA 3xFLAG Tag vl l

28 DNA 3xFLAG Tag vl2

29 DNA 3xFLAG Tag vl3

30 DNA 3xFLAG Tag vl4

31 DNA 3xFLAG Tag vl5

32 Protein 3xFLAG Tag

33 Protein Peptide Linker vl

34 Protein Peptide Linker v2

35 Protein Peptide Linker v3

36 Protein Peptide Linker v4

37 Protein Peptide Linker v5

38 Protein Peptide Linker v6

39 Protein Peptide Linker v7

40 Protein Peptide Linker v8

41 Protein Peptide Linker v9

42 Protein Peptide Linker v 10

43 Protein PEST-Like Sequence vl

44 Protein PEST-Like Sequence v2

45 Protein PEST-Like Sequence v3

46 Protein PEST-Like Sequence v4

47 Protein PEST-Like Sequence v5

48 Protein PEST-Like Sequence v6

49 Protein PEST-Like Sequence v7

50 Protein PEST-Like Sequence v8

51 Protein PEST-Like Sequence v9

52 Protein PEST-Like Sequence vlO

53 Protein PEST-Like Sequence vl l

54 Protein PEST-Like Sequence vl2

55 Protein LLO Protein vl

56 Protein LLO Protein v2

57 Protein N-Terminal Truncated LLO vl

58 Protein N-Terminal Truncated LLO v2

59 Protein N-Terminal Truncated LLO v3

60 DNA Nucleic Acid Encoding N-Terminal Truncated LLO v3

61 Protein ActA Protein vl

62 Protein ActA Protein v2

63 Protein ActA Fragment vl

64 Protein ActA Fragment v2

65 Protein ActA Fragment v3

66 Protein ActA Fragment v4

67 Protein ActA Fragment v5

68 DNA Nucleic Acid Encoding ActA Fragment v5

69 Protein ActA Fragment v6

70 Protein ActA Fragment v7

71 DNA Nucleic Acid Encoding ActA Fragment v7

72 Protein ActA Fragment Fused to Hly Signal Peptide

73 Protein ActA Substitution

74 Protein Cholesterol-Binding Domain of LLO

75 Protein HLA-A2 restricted Epitope from NY-ESO-1

76 Protein Lm Alanine Racemase

77 Protein Lm D-Amino Acid Aminotransferase

rote n omo sap ens a p a- -ant tryps n precursor en an ccess on o.

SIINFEKL Tag

DNA v.l (SEQ ID NO: 1):

GCACGTAGTATAATCAACTTTGAAAAACTGTAATAA

DNA v.2 (SEQ ID NO: 2):

G C AC GT T C T AT T AT C AAC T T C GAAAAAC T AT AAT AA

DNA v.3 (SEQ ID NO: 3):

G C C C G C AGT AT T AT C AAT T T C GAAAAAT TAT AAT AA DNA v.4 (SEQ ID NO: 4):

GCGCGCTCTATAATTAACTTCGAAAAACTTTAATAA

DNA v.5 (SEQ ID NO: 5):

GCACGCTCCATTATTAACTTTGAAAAACTTTAATAA

DNA v.6 (SEQ ID NO: 6):

GCTCGCTCTATCATCAATTTCGAAAAACTTTAATAA

DNA v.7 (SEQ ID NO: 7):

GCAC GT AGT AT TAT T AACT T C GAAAAGT TAT AAT AA

DNA v.8 (SEQ ID NO: 8):

G C AC GT T C CAT CAT T AAC T T T GAAAAAC TAT AAT AA DNA v.9 (SEQ ID NO: 9):

GCT CGCT CAAT CAT CAACTTT GAAAAGCTATAATAA

DNA v.lO (SEQ ID NO: 10):

GCT CGCT CTAT CAT CAACTT CGAAAAATT GTAATAA DNA v.ll (SEQ ID NO: 11):

GCTCGCTCTATTATCAATTTTGAAAAATTATAATAA

DNA v.l2 (SEQ ID NO: 12):

GCTCGTAGTATTATTAATTTCGAAAAATTATAATAA

DNA v.l3 (SEQ ID NO: 13):

GCTCGTTCGATTATCAACTTCGAAAAACTGTAATAA

DNA v.l4 (SEQ ID NO: 14):

GCAAGAAGCATCATCAACTTCGAAAAACTGTAATAA

DNA v.l5 (SEQ ID NO: 15):

GCGCGTTCTATTATTAATTTTGAAAAATTATAATAA

Protein (SEQ ID NO: 16):

ARSIINFEKL

3xFLAG Tag

DNA v.l (SEQ ID NO: 17):

GATTATAAAGATCATGACGGAGACTATAAAGACCATGACATTGATTACAAAGACGACGAT GACAAA

DNA v.2 (SEQ ID NO: 18):

GACTATAAAGACCACGATGGCGATTATAAAGACCATGATATTGACTACAAAGATGATGAT GATAAG

DNA v.3 (SEQ ID NO: 19):

GATTATAAAGATCATGATGGCGACTATAAAGATCATGATATCGATTACAAAGATGACGAT GACAAA

DNA v.4 (SEQ ID NO: 20):

GACTACAAAGATCACGATGGTGACTACAAAGATCACGACATTGATTATAAAGACGATGAT GACAAA

DNA v.5 (SEQ ID NO: 21):

GATTACAAAGATCACGATGGTGATTATAAGGATCACGATATTGATTACAAAGACGACGAC GATAAA

DNA v.6 (SEQ ID NO: 22):

GATTACAAAGATCACGATGGCGATTACAAAGATCATGACATTGACTACAAAGACGATGAT GATAAA

DNA v.7 (SEQ ID NO: 23):

GATTACAAGGATCATGATGGTGATTACAAAGATCACGATATCGACTACAAAGATGATGAC GATAAA

DNA v.8 (SEQ ID NO: 24):

GACTACAAAGATCATGATGGTGATTACAAAGATCATGACATTGATTATAAAGATGATGAT GACAAA

DNA v.9 (SEQ ID NO: 25):

GATTATAAAGACCATGATGGTGATTATAAGGATCATGATATCGATTATAAGGATGACGAC GATAAA

DNA v.lO (SEQ ID NO: 26):

GATTATAAAGATCACGATGGCGATTATAAAGACCACGATATTGATTATAAAGACGACGAT GACAAA

DNA v.ll (SEQ ID NO: 27):

GACTATAAAGACCACGATGGTGATTATAAAGATCACGACATCGACTACAAAGACGATGAT GATAAA DNA v.l2 (SEQ ID NO: 28):

GACTACAAAGATCACGACGGCGATTATAAAGATCACGATATTGACTATAAAGATGACGAT GATAAA

DNA v.l3 (SEQ ID NO: 29):

GATTATAAAGACCATGATGGAGATTACAAAGATCATGATATTGACTATAAAGACGACGAC GATAAA

DNA v.l4 (SEQ ID NO: 30):

GATTATAAAGATCACGATGGTGACTACAAAGATCACGATATCGATTATAAAGACGATGAC GATAAA

DNA v.l5 (SEQ ID NO: 31):

GACTACAAAGATCACGATGGTGATTATAAAGACCATGATATTGATTACAAAGATGATGAT GACAAA

Protein (SEQ ID NO: 32):

DYKDHDGDYKDHDIDYKDDDDK

Peptide Linkers

Peptide Linker v.l (SEQ ID NO: 33):

( GAS ) _n

Peptide Linker v.2 (SEQ ID NO: 34):

(GSA) n

Peptide Linker v.3 (SEQ ID NO: 35):

(G) _n ; n = 4-8

Peptide Linker v.4 (SEQ ID NO: 36):

( GGGGS ) _n ; n = 1-3

Peptide Linker v.5 (SEQ ID NO: 37):

VGKGGSGG

Peptide Linker v.6 (SEQ ID NO: 38):

( PAPAP ) _n

Peptide Linker v.7 (SEQ ID NO: 39):

( EAAAK) _n ; n=l-3

Peptide Linker v.8 (SEQ ID NO: 40):

(AYL) n

Peptide Linker v.9 (SEQ ID NO: 41):

(LRA) n

Peptide Linker v.lO (SEQ ID NO: 42):

(RLRA) n

PEST-Like Sequences

PEST-Like Sequence v.l (SEQ ID NO: 43):

KENSISSMAPPASPPASPKTPIEKKHADEIDK PEST-Like Sequence v.2 (SEQ ID NO: 44):

KENSI SSMAPPASPPASPK

PEST-Like Sequence v.3 (SEQ ID NO: 45):

KTEEQPSEVNTGPR

PEST-Like Sequence v.4 (SEQ ID NO: 46):

KESWDASESDLDSSMQSADESTPQPLK

PEST-Like Sequence v.5 (SEQ ID NO: 47):

KSEEVNASDFPPPPTDEELR

PEST-Like Sequence v.6 (SEQ ID NO: 48):

RGGRPTSEEFSSLNSGDFTDDENSETTEEEIDR

PEST-Like Sequence v.7 (SEQ ID NO: 49):

KQNTASTETTTTNEQPK

PEST-Like Sequence v.8 (SEQ ID NO: 50):

KQNTANTETTTTNEQPK

PEST-Like Sequence v.9 (SEQ ID NO: 51):

RSEVTISPAETPESPPATP

PEST-Like Sequence v.lO (SEQ ID NO: 52):

KASVTDTSEGDLDSSMQSADESTPQPLK

PEST-Like Sequence v.ll (SEQ ID NO: 53):

KNEEVNASDFPPPPTDEELR

PEST-Like Sequence v.12 (SEQ ID NO: 54):

RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR

LLO Proteins

LLO Protein v.l (SEQ ID NO: 55):

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADE IDKYIQGLDYNK NNVLVYHGDAVTNVPPRKGYKDGNEYIWEKKKKSINQNNADIQV AISSLTYPGALVKANSELVENQPDV LPVKRDSLTLSIDLPGMTNQDNKIWKNATKSN NA TLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQ LIAKFGTAFKA NSLN FGAI SEGKMQEEVISFKQIYYN EPTRPSRFFGKAVTKEQLQALGVNAEN PPAYI SSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAK DEVQIID GNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVI KNNSEYIETTSKAYTDGKINIDHSGGYVAQFN ISWDE YDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDR NLPLV KNRNI SIWGTTLYPKYSNKVDNPIE

LLO Protein v.2 (SEQ ID NO: 56):

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADE IDKYIQGLDYNK NNVLVYHGDAVTNVPPRKGYKDGNEYIWEKKKKSINQNNADIQV AISSLTYPGALVKANSELVENQPDV LPVKRDSLTLSIDLPGMTNQDNKIWKNATKSN NA TLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQ LIAKFGTAFKAWNSLNWFGAI SEGKMQEEVISFKQIYYN EPTRPSRFFGKAVTKEQLQALG AEN PPAYI SSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAK DEVQIID GNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKIN IDHSGGYVAQFN ISWDEWYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWR TVIDDRNLPLV KNRNI SIWGTTLYPKYSNKVDNPIE N-Terminal Truncated LLO Protein v,l (SEQ ID NO: 57):

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADE IDKYIQGLDYNK NNVLVYHGDAVTNVPPRKGYKDGNEYIWEKKKKSINQNNADIQV AISSLTYPGALVKANSELVENQPDV LPVKRDSLTLSIDLPGMTNQDNKIWKNATKSN NA TLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQ LIAKFGTAFKA NSLN FGAISEGKMQEEVISFKQIYYN EPTRPSRFFGKAVTKEQLQALG AEN PPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIY GGSAKDEVQIID GNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKIN IDHSGGYVAQFN ISWDE YD

N-Terminal Truncated LLO Protein v.2 (SEQ ID NO: 58):

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASPKTPIEKKHADE IDKYIQGLDYNK NNVLVYHGDAVTNVPPRKGYKDGNEYIWEKKKKSINQNNADIQV AISSLTYPGALVKANSELVENQPDV LPVKRDSLTLSIDLPGMTNQDNKIWKNATKSN NA TLVERWNEKYAQAYSNVSAKIDYDDEMAYSESQ LIAKFGTAFKA NSLN FGAISEGKMQEEVISFKQIYYN EPTRPSRFFGKAVTKEQLQALG AEN PPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIY GGSAKDEVQIID GNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTD

N-Terminal Truncated LLO Protein v.3 (SEQ ID NO: 59):

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADE IDKYIQGLDYNK NNVLVYHGDAVTNVPPRKGYKDGNEYIWEKKKKSINQNNADIQV AISSLTYPGALVKANSELVENQPDV LPVKRDSLTLSIDLPGMTNQDNKIWKNATKSN NA TLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQ LIAKFGTAFKA NSLN FGAISEGKMQEEVISFKQIYYN EPTRPSRFFGKAVTKEQLQALG AEN PPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIY GGSAKDEVQIID GNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKIN IDHSGGYVAQFN ISWDEWYD

Nucleic Acid Encoding N-Terminal Truncated LLO Protein v.3 (SEQ ID NO: 60):

AT GAAAAAAAT AAT GCTAGTTTT TAT T AC AC T TAT AT TAGTTAGTCTAC C AAT T G C G C AAC AAAC T GAAG C A AAGGATGCATCTGCATTCAATAAAGAAAATTCAATTTCATCCATGGCACCACCAGCATCT CCGCCTGCAAGT C C T AAGAC G C C AAT C GAAAAGAAAC AC G C G GAT GAAAT C GAT AAGT AT AT AC AAG GAT T G GAT T AC AAT AAA AACAATGTATTAGTATACCACGGAGATGCAGTGACAAATGTGCCGCCAAGAAAAGGTTAC AAAGATGGAAAT GAAT AT AT T GT T GT GGAGAAAAAGAAGAAAT C CAT CAAT CAAAAT AAT GCAGACAT T CAAGT T GT GAATGCA ATTTCGAGCCTAACCTATCCAGGTGCTCTCGTAAAAGCGAATTCGGAATTAGTAGAAAAT CAACCAGATGTT C T C C C T GT AAAAC GT GAT T CAT T AAC AC T C AG CAT T GAT T T G C C AG GT AT GAC T AAT C AAGAC AAT AAAAT A GTTGTAAAAAATGCCACTAAATCAAACGTTAACAACGCAGTAAATACATTAGTGGAAAGA TGGAATGAAAAA TAT G C T C AAG C T TAT C C AAAT GT AAGT G C AAAAAT T GAT TAT GAT GAC GAAAT G G C T T AC AGT GAAT C AC AA TTAATTGCGAAATTTGGTACAGCATTTAAAGCTGTAAATAATAGCTTGAATGTAAACTTC GGCGCAATCAGT GAAG G GAAAAT G C AAGAAGAAGT CAT T AGT T T T AAAC AAAT T T AC TAT AAC GT GAAT GT T AAT GAAC C T AC A AGACCTTCCAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTG AATGCAGAAAAT CCTCCTGCATATATCTCAAGTGTGGCGTATGGCCGTCAAGTTTATTTGAAATTATCAACT AATTCCCATAGT ACTAAAGTAAAAGCTGCTTTTGATGCTGCCGTAAGCGGAAAATCTGTCTCAGGTGATGTA GAACTAACAAAT ATCATCAAAAATTCTTCCTTCAAAGCCGTAATTTACGGAGGTTCCGCAAAAGATGAAGTT CAAATCATCGAC GGCAACCTCGGAGACTTACGCGATATTTTGAAAAAAGGCGCTACTTTTAATCGAGAAACA CCAGGAGTTCCC AT T G C T TAT AC AAC AAAC T T C C T AAAAGAC AAT GAAT T AG C T GT TAT T AAAAAC AAC T C AGAAT AT AT T GAA AC AAC T T C AAAAG C T T AT AC AGAT G GAAAAAT T AAC AT C GAT C AC T C T G GAG GAT AC GT T G C T CAAT T C AAC AT T T C T T G G GAT GAAGT AAAT TAT GAT

ActA Proteins

ActA Protein v.l (SEQ ID NO: 61)

MRAMMWFITANCITINPDIIFAATDSEDSSLNTDEWEEEKTEEQPSEWTGPRYETAREVS SRDIEELEKS NKVKNTNKADLIAMLKAKAEKGPNNNNNNGEQTGNVAINEEASGVDRPTLQVERRHPGLS SDSAAEIKKRRK AIASSDSELESLTYPDKPTKANKRKVAKESWDASESDLDSSMQSADESTPQPLKANQKPF FPKVFKKIKDA GKWVRDKIDENPEVKKAIVDKSAGLIDQLLTKKKSEEWASDFPPPPTDEELRLALPETPM LLGFNAPTPSE PSSFEFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTEDELEIMRETAPSL DSSFTSGDLASL RSAINRHSENFSDFPLIPTEEELNGRGGRPTSEEFSSLNSGDFTDDENSETTEEEIDRLA DLRDRGTGKHSR NAGFLPLNPFISSPVPSLTPKVPKISAPALISDITKKAPFKNPSQPLNVFNKKTTTKTVT KKPTPVKTAPKL AELPATKPQETVLRENKTPFIEKQAETNKQSINMPSLPVIQKEATESDKEEMKPQTEEKM VEESESANNANG KNRSAGIEEGKLIAKSAEDEKAKEEPGNHTTLILAMLAIGVFSLGAFIKIIQLRKNN ActA Protein v.2 (SEQ ID NO: 62)

MGLNRFMRAMMWFITANCITINPDIIFAATDSEDSSLNTDEWEEEKTEEQPSE TGPRYETAREVSSRDI EELEKSNKVKNTNKADLIAMLKAKAEKGPNNNNNNGEQTGNVAINEEASGVDRPTLQVER RHPGLSSDSAAE IKKRRKAIASSDSELESLTYPDKPTKANKRKVAKESWDASESDLDSSMQSADESTPQPLK ANQKPFFPKVF KKIKDAGKWVRDKIDENPEVKKAIVDKSAGLIDQLLTKKKSEE ASDFPPPPTDEELRLALPETPMLLGFN APTPSEPSSFEFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTEDELEIMR ETAPSLDSSFTS GDLASLRSAINRHSENFSDFPLIPTEEELNGRGGRPTSEEFSSLNSGDFTDDENSETTEE EIDRLADLRDRG TGKHSRNAGFLPLNPFISSPVPSLTPKVPKISAPALISDITKKAPFKNPSQPLNVFNKKT TTKTVTKKPTPV KTAPKLAELPATKPQETVLRENKTPFIEKQAETNKQSINMPSLPVIQKEATESDKEEMKP QTEEKMVEESES ANNANGKNRSAGIEEGKLIAKSAEDEKAKEEPGNHTTLILAMLAIGVFSLGAFIKIIQLR KNN

ActA Fragment v.l (SEQ ID NO: 63)

ATDSEDSSLNTDEWEEEKTEEQPSE TGPRYETAREVSSRDIEELEKSNKVKNTNKADLIAMLKAKAEKGP NNNNNNGEQTGNVAINEEASG

ActA Fragment v.2 (SEQ ID NO: 64)

ATDSEDSSLNTDEWEEEKTEEQPSE TGPRYETAREVSSRDIEELEKSNKVKNTNKADLIAMLKAKAEKGP NNNNNNGEQTGNVAINEEASGVDRPTLQVERRHPGLSSDSAAEIKKRRKAIASSDSELES LTYPDKPTKANK RKVAKESWDASESDLDSSMQSADESTPQPLKANQKPFFPKVFKKIKDAGKWVRDK

ActA Fragment v.3 (SEQ ID NO: 65)

ATDSEDSSLNTDEWEEEKTEEQPSE TGPRYETAREVSSRDIEELEKSNKVKNTNKADLIAMLKAKAEKGP NNNNNNGEQTGNVAINEEASGVDRPTLQVERRHPGLSSDSAAEIKKRRKAIASSDSELES LTYPDKPTKANK RKVAKESWDASESDLDSSMQSADESTPQPLKANQKPFFPKVFKKIKDAGKWVRDKIDENP EVKKAIVDKSA GLIDQLLTKKKSEE ASDFPPPPTDEELRLALPETPMLLGFNAPTPSEPSSFEFPPPPTDEELRLALPETP MLLGFNAPATSEPSS

ActA Fragment v.4 (SEQ ID NO: 66)

ATDSEDSSLNTDEWEEEKTEEQPSE TGPRYETAREVSSRDIEELEKSNKVKNTNKADLIAMLKAKAEKGP NNNNNNGEQTGNVAINEEASGVDRPTLQVERRHPGLSSDSAAEIKKRRKAIASSDSELES LTYPDKPTKANK RKVAKESWDASESDLDSSMQSADESTPQPLKANQKPFFPKVFKKIKDAGKWVRDKIDENP EVKKAIVDKSA GLIDQLLTKKKSEE ASDFPPPPTDEELRLALPETPMLLGFNAPTPSEPSSFEFPPPPTDEELRLALPETP MLLGFNAPATSEPSSFEFPPPPTEDELEIMRETAPSLDSSFTSGDLASLRSAINRHSENF SDFPLIPTEEEL NGRGGRPTSE

ActA Fragment v.5 (SEQ ID NO: 67)

MRAMMWFITANCITINPDIIFAATDSEDSSLNTDEWEEEKTEEQPSE TGPRYETAREVSSRDIKELEKS NKVRNTNKADLIAMLKEKAEKGPNINNNNSEQTENAAINEEASGADRPAIQVERRHPGLP SDSAAEIKKRRK AIASSDSELESLTYPDKPTK KKKVAKESVADASESDLDSSMQSADESSPQPLKANQQPFFPKVFKKIKDA GKWVRDKIDENPEVKKAIVDKSAGLIDQLLTKKKSEE ASDFPPPPTDEELRLALPETPMLLGFNAPATSE PSSFEFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTEDELEIIRETASSL DSSFTRGDLASL RNAINRHSQNFSDFPPIPTEEELNGRGGRP

Nucleic Acid Encoding ActA Fragment v.5 (SEQ ID NO: 68)

ATGCGTGCGATGATGGTGGTTTTCATTACTGCCAATTGCATTACGATTAACCCCGACATA ATATTTGCAGCG AC AGAT AG C GAAGAT T C T AGT C T AAAC AC AGAT GAAT G G GAAGAAGAAAAAAC AGAAGAG C AAC C AAG C GAG GTAAATACGGGACCAAGATACGAAACTGCACGTGAAGTAAGTTCACGTGATATTAAAGAA CTAGAAAAATCG AAT AAAGT GAGAAAT AC GAAC AAAG C AGAC C T AAT AG C AAT GT T GAAAGAAAAAG C AGAAAAAG GT C C AAAT AT C AAT AAT AAC AAC AGT GAAC AAAC T GAGAAT G C G G C T AT AAAT GAAGAG G C T T C AG GAG C C GAC C GAC C A G C T AT AC AAGT G GAG C GT C GT CAT C C AG GAT T G C CAT C G GAT AG C G C AG C G GAAAT T AAAAAAAGAAG GAAA G C CAT AG CAT CAT C G GAT AGT GAG C T T GAAAG C C T T AC T TAT C C G GAT AAAC C AAC AAAAGT AAAT AAGAAA AAAGTGGCGAAAGAGTCAGTTGCGGATGCTTCTGAAAGTGACTTAGATTCTAGCATGCAG TCAGCAGATGAG T C T T C AC C AC AAC C T T T AAAAG C AAAC C AAC AAC CAT T T T T C C C T AAAGT AT T T AAAAAAAT AAAAGAT G C G GGGAAATGGGTACGTGATAAAATCGACGAAAATCCTGAAGTAAAGAAAGCGATTGTTGAT AAAAGTGCAGGG T T AAT T GAC C AAT TAT T AAC C AAAAAGAAAAGT GAAGAG GT AAAT G C T T C G GAC T T C C C G C C AC C AC C T AC G GATGAAGAGTTAAGACTTGCTTTGCCAGAGACACCAATGCTTCTTGGTTTTAATGCTCCT GCTACATCAGAA CCGAGCTCATTCGAATTTCCACCACCACCTACGGATGAAGAGTTAAGACTTGCTTTGCCA GAGACGCCAATG CTTCTTGGTTTTAATGCTCCTGCTACATCGGAACCGAGCTCGTTCGAATTTCCACCGCCT CCAACAGAAGAT GAAC T AGAAAT CAT C C G G GAAAC AG CAT C C T C G C T AGAT TCTAGTTT T AC AAGAG G G GAT TTAGCTAGTTTG AGAAAT G C TAT T AAT C G C C AT AGT C AAAAT T T C T C T GAT T T C C C AC C AAT C C C AAC AGAAGAAGAGT T GAAC G G GAGAG G C G GT AGAC C A

ActA Fragment v.6 (SEQ ID NO: 69)

MGLNRFMRAMMWFITANCITINPDI I FAATDSEDS SLNTDEWEEEKTEEQPSE TGPRYETAREVS SRDI KELEKSNKVRNTNKADLIAMLKEKAEKG

ActA Fragment v.7 (SEQ ID NO: 70)

MRAMMWFITANCIT INPDI I FAATDSEDS SLNTDEWEEEKTEEQPSE TGPRYETAREVS S RDI EELEKS NKVKNTNKADLIAMLKAKAEKGPNNNNNNGEQTGNVAINEEASGVDRPTLQVERRHPGLS SDSAAEI KKRRK AIAS S DSELESLTYPDKPTKANKRKVAKESWDASES DLDS SMQSADEST PQPLKANQKPFFPKVFKKI KDA GKWVRDKI DENPEVKKAIVDKSAGLI DQLLTKKKSEE ASDFPP PPTDEELRLALPET PMLLGFNAPTPSE PS S FEFPPPPTDEELRLALPETPMLLGFNAPATSEPS S FEFPPPPTEDELEIMRETAPS LDS S FTSGDLASL RSAINRHSENFSDFPLI PTEEELNGRGGRP

Nucleic Acid Encoding ActA Fragment v.7 (SEQ ID NO: 71)

ATGCGTGCGATGATGGTAGTTTTCATTACTGCCAACTGCATTACGATTAACCCCGACATA ATATTTGCAGCG AC AGAT AG C GAAGAT T C C AGT C T AAAC AC AGAT GAAT G G GAAGAAGAAAAAAC AGAAGAG C AG C C AAG C GAG GTAAATACGGGACCAAGATACGAAACTGCACGTGAAGTAAGTTCACGTGATATTGAGGAA CTAGAAAAATCG AAT AAAGT GAAAAAT AC GAAC AAAG C AGAC C T AAT AG C AAT GT T GAAAG C AAAAG C AGAGAAAG GT C C GAAT AAC AAT AAT AAC AAC G GT GAG C AAAC AG GAAAT GT G G C T AT AAAT GAAGAG G C T T C AG GAGT C GAC C GAC C A ACTCTGCAAGTGGAGCGTCGTCATCCAGGTCTGTCATCGGATAGCGCAGCGGAAATTAAA AAAAGAAGAAAA G C CAT AG CGTCGTCG GAT AGT GAG C T T GAAAG C C T T AC T TAT C C AGAT AAAC C AAC AAAAG C AAAT AAGAGA AAAGTGGCGAAAGAGTCAGTTGTGGATGCTTCTGAAAGTGACTTAGATTCTAGCATGCAG TCAGCAGACGAG T C T AC AC C AC AAC C T T T AAAAG C AAAT C AAAAAC CAT T T T T C C C T AAAGT AT T T AAAAAAAT AAAAGAT G C G GGGAAATGGGTACGTGATAAAATCGACGAAAATCCTGAAGTAAAGAAAGCGATTGTTGAT AAAAGTGCAGGG T T AAT T GAC C AAT TAT T AAC C AAAAAGAAAAGT GAAGAG GT AAAT G C T T C G GAC T T C C C G C C AC C AC C T AC G GATGAAGAGTTAAGACTTGCTTTGCCAGAGACACCGATGCTTCTCGGTTTTAATGCTCCT ACTCCATCGGAA CCGAGCTCATTCGAATTTCCGCCGCCACCTACGGATGAAGAGTTAAGACTTGCTTTGCCA GAGACGCCAATG CTTCTTGGTTTTAATGCTCCTGCTACATCGGAACCGAGCTCATTCGAATTTCCACCGCCT CCAACAGAAGAT GAACTAGAAATTATGCGGGAAACAGCACCTTCGCTAGATTCTAGTTTTACAAGCGGGGAT TTAGCTAGTTTG AGAAGT G C TAT T AAT C G C CAT AG C GAAAAT T T C T C T GAT T T C C C AC T AAT C C C AAC AGAAGAAGAGT T GAAC G G GAGAG G C G GT AGAC C A

ActA Fragment Fused to Hly Signal Peptide (SEQ ID NO: 72)

MKKIMLVFITLI LVSLPIAQQTEASRATDSEDS SLNTDEWEEEKTEEQPSE TGPRYETAREVS SRDI EEL EKSNKVKNTNKADLIAMLKAKAEKGPNNNNNNGEQTGNVAINEEASGVDRPTLQVERRHP GLS SDSAAEI KK RRKAIAS SDSELESLTYPDKPTKANKRKVAKESWDASESDLDS SMQSADESTPQPLKANQKPFFPKVFKKI KDAGKWVRDK

ActA Substitution (SEQ ID NO: 73)

QDNKR

LLO Mutations

Cholesterol-Binding Domain of LLO (SEQ ID NO: 74)

ECTGLAWEWWR

HLA-A2 restricted Epitope from NY-ESO-1 (SEQ ID NO: 75)

ESLLMWITQCR

Dal and Dat

Lm Alanine Racemase (SEQ ID NO: 76)

MVTGWHRPTWI EI DRAAI RENI KNEQNKLPESVDLWAWKANAYGHGI I EVARTAKEAGAKGFCVAI LDEAL ALREAGFQDDFI LVLGATRKEDANLAAKNHI SLTVFREDWLENLTLEATLRIHLKVDSGMGRLGI RTTEEAR RIEATSTNDHQLQLEGIYTHFATADQLETSYFEQQLAKFQTILTSLKKRPTYVHTANSAA SLLQPQIGFDAI RFGISMYGLTPSTEIKTSLPFELKPALALYTEMVHVKELAPGDSVSYGATYTATEREWVA TLPIGYADGLIR HYSGFHVLVDGEPAPIIGRVCMDQTIIKLPREFQTGSKVTIIGKDHGNTVTADDAAQYLD TINYEVTCLLNE RIPRKYIH

Lm D-Amino Acid Aminotransferase (SEQ ID NO: 77)

MKVL NHLVEREDATVDIEDRGYQFGDGVYEWRLYNGKFFTYNEHIDRLYASAAKIDLVIPYSKE ELREL LEKLVAENNINTGNVYLQVTRGVQNPRNHVIPDDFPLEGVLTAAAREVPRNERQFVEGGT AITEEDVRWLRC DIKSLNLLGNILAKNKAHQQNALEAILHRGEQVTECSASNVSIIKDGVLWTHAADNLILN GITRQVIIDVAK KNGIPVKEADFTLTDLREADEVFISSTTIEITPITHIDGVQVADGKRGPITAQLHQYFVE EITRACGELEFA K

Nucleic Acid Encoding Lm Alanine Racemase (SEQ ID NO: 78)

ATGGTGACAGGCTGGCATCGTCCAACATGGATTGAAATAGACCGCGCAGCAATTCGCGAA AATATAAAAAAT GAACAAAATAAACTCCCGGAAAGTGTCGACTTATGGGCAGTAGTCAAAGCTAATGCATAT GGTCACGGAATT ATCGAAGTTGCTAGGACGGCGAAAGAAGCTGGAGCAAAAGGTTTCTGCGTAGCCATTTTA GATGAGGCACTG GCTCTTAGAGAAGCTGGATTTCAAGATGACTTTATTCTTGTGCTTGGTGCAACCAGAAAA GAAGATGCTAAT CTGGCAGCCAAAAACCACATTTCACTTACTGTTTTTAGAGAAGATTGGCTAGAGAATCTA ACGCTAGAAGCA ACACTTCGAATTCATTTAAAAGTAGATAGCGGTATGGGGCGTCTCGGTATTCGTACGACT GAAGAAGCACGG CGAATTGAAGCAACCAGTACTAATGATCACCAATTACAACTGGAAGGTATTTACACGCAT TTTGCAACAGCC GACCAGCTAGAAACTAGTTATTTTGAACAACAATTAGCTAAGTTCCAAACGATTTTAACG AGTTTAAAAAAA CGACCAACTTATGTTCATACAGCCAATTCAGCTGCTTCATTGTTACAGCCACAAATCGGG TTTGATGCGATT CGCTTTGGTATTTCGATGTATGGATTAACTCCCTCCACAGAAATCAAAACTAGCTTGCCG TTTGAGCTTAAA CCTGCACTTGCACTCTATACCGAGATGGTTCATGTGAAAGAACTTGCACCAGGCGATAGC GTTAGCTACGGA GCAACTTATACAGCAACAGAGCGAGAATGGGTTGCGACATTACCAATTGGCTATGCGGAT GGATTGATTCGT CATTACAGTGGTTTCCATGTTTTAGTAGACGGTGAACCAGCTCCAATCATTGGTCGAGTT TGTATGGATCAA ACCATCATAAAACTACCACGTGAATTTCAAACTGGTTCAAAAGTAACGATAATTGGCAAA GATCATGGTAAC ACGGTAACAGCAGATGATGCCGCTCAATATTTAGATACAATTAATTATGAGGTAACTTGT TTGTTAAATGAG CGCATACCTAGAAAATACATCCATTAG

Nucleic Acid Encoding L/w D-Amino Acid Aminotransferase (SEQ ID NO: 79)

ATGAAAGTATTAGTAAATAACCATTTAGTTGAAAGAGAAGATGCCACAGTTGACATTGAA GACCGCGGATAT CAGTTTGGTGATGGTGTATATGAAGTAGTTCGTCTATATAATGGAAAATTCTTTACTTAT AATGAACACATT GATCGCTTATATGCTAGTGCAGCAAAAATTGACTTAGTTATTCCTTATTCCAAAGAAGAG CTACGTGAATTA CTTGAAAAATTAGTTGCCGAAAATAATATCAATACAGGGAATGTCTATTTACAAGTGACT CGTGGTGTTCAA AACCCACGTAATCATGTAATCCCTGATGATTTCCCTCTAGAAGGCGTTTTAACAGCAGCA GCTCGTGAAGTA CCTAGAAACGAGCGTCAATTCGTTGAAGGTGGAACGGCGATTACAGAAGAAGATGTGCGC TGGTTACGCTGT GATATTAAGAGCTTAAACCTTTTAGGAAATATTCTAGCAAAAAATAAAGCACATCAACAA AATGCTTTGGAA GCTATTTTACATCGCGGGGAACAAGTAACAGAATGTTCTGCTTCAAACGTTTCTATTATT AAAGATGGTGTA TTATGGACGCATGCGGCAGATAACTTAATCTTAAATGGTATCACTCGTCAAGTTATCATT GATGTTGCGAAA AAGAATGGCATTCCTGTTAAAGAAGCGGATTTCACTTTAACAGACCTTCGTGAAGCGGAT GAAGTGTTCATT TCAAGTACAACTATTGAAATTACACCTATTACGCATATTGACGGAGTTCAAGTAGCTGAC GGAAAACGTGGA CCAATTACAGCGCAACTTCATCAATATTTTGTAGAAGAAATCACTCGTGCATGTGGCGAA TTAGAGTTTGCA AAATAA

PrfA

Wild Type PrfA (SEQ ID NO: 80)

MNAQAEEFKKYLETNGIKPKQFHKKELIFNQWDPQEYCIFLYDGITKLTSISENGTIMNL QYYKGAFVIMSG FIDTETSVGYYNLEVISEQATAYVIKINELKELLSKNLTHFFYVFQTLQKQVSYSLAKFN DFSINGKLGSIC GQLLILTYVYGKETPDGIKITLDNLTMQELGYSSGIAHSSAVSRIISKLKQEKVIVYKNS CFYVQNLDYLKR YAPKLDEWFYLACPATWGKLN

Nucleic Acid Encoding Wild Type PrfA (SEQ ID NO: 81)

ATGAACGCTCAAGCAGAAGAATTCAAAAAATATTTAGAAACTAACGGGATAAAACCAAAA CAATTTCATAAA AAAGAACTTATTTTTAACCAATGGGATCCACAAGAATATTGTATTTTTCTATATGATGGT ATCACAAAGCTC ACGAGTATTAGCGAGAACGGGACCATCATGAATTTACAATACTACAAAGGGGCTTTCGTT ATAATGTCTGGC TTTATTGATACAGAAACATCGGTTGGCTATTATAATTTAGAAGTCATTAGCGAGCAGGCT ACCGCATACGTT ATCAAAATAAACGAACTAAAAGAACTACTGAGCAAAAATCTTACGCACTTTTTCTATGTT TTCCAAACCCTA CAAAAACAAGTTTCATACAGCCTAGCTAAATTTAATGATTTTTCGATTAACGGGAAGCTT GGCTCTATTTGC GGTCAACTTTTAATCCTGACCTATGTGTATGGTAAAGAAACTCCTGATGGCATCAAGATT ACACTGGATAAT TTAACAATGCAGGAGTTAGGATATTCAAGTGGCATCGCACATAGCTCAGCTGTTAGCAGA ATTATTTCCAAA TTAAAGCAAGAGAAAGTTATCGTGTATAAAAATTCATGCTTTTATGTACAAAATCTTGAT TATCTCAAAAGA TATGCCCCTAAATTAGATGAATGGTTTTATTTAGCATGTCCTGCTACTTGGGGAAAATTA AATTAA

D133V PrfA (SEQ ID NO: 82)

MNAQAEEFKKYLETNGIKPKQFHKKELIFNQWDPQEYCIFLYDGITKLTSISENGTIMNL QYYKGAFVIMSG FIDTETSVGYYNLEVISEQATAYVIKINELKELLSKNLTHFFYVFQTLQKQVSYSLAKFN VFSINGKLGSIC GQLLILTYVYGKETPDGIKITLDNLTMQELGYSSGIAHSSAVSRIISKLKQEKVIVYKNS CFYVQNRDYLKR YAPKLDEWFYLACPATWGKLN

Nucleic Acid Encoding D133V PrfA (SEQ ID NO: 83)

ATGAACGCTCAAGCAGAAGAATTCAAAAAATATTTAGAAACTAACGGGATAAAACCAAAA CAATTTCATAAA AAAGAACTTATTTTTAACCAATGGGATCCACAAGAATATTGTATTTTTCTATATGATGGT ATCACAAAGCTC ACGAGTATTAGCGAGAACGGGACCATCATGAATTTACAATACTACAAAGGGGCTTTCGTT ATAATGTCTGGC TTTATTGATACAGAAACATCGGTTGGCTATTATAATTTAGAAGTCATTAGCGAGCAGGCT ACCGCATACGTT ATCAAAATAAACGAACTAAAAGAACTACTGAGCAAAAATCTTACGCACTTTTTCTATGTT TTCCAAACCCTA CAAAAACAAGTTTCATACAGCCTAGCTAAATTTAATGTTTTTTCGATTAACGGGAAGCTT GGCTCTATTTGC GGTCAACTTTTAATCCTGACCTATGTGTATGGTAAAGAAACTCCTGATGGCATCAAGATT ACACTGGATAAT TTAACAATGCAGGAGTTAGGATATTCAAGTGGCATCGCACATAGCTCAGCTGTTAGCAGA ATTATTTCCAAA TTAAAGCAAGAGAAAGTTATCGTGTATAAAAATTCATGCTTTTATGTACAAAATCTGATT ATCTCAAAAGAT ATGCCCCTAAATTAGATGAATGGTTTTATTTAGCATGTCCTGCTACTTGGGGAAAATTAA ATTAA

4X Glycine Linker DNA Sequences

Gl (SEQ ID NO: 84)

GGTGGTGGAGGA

G2 (SEQ ID NO: 85)

GGTGGAGGTGGA

G3 (SEQ ID NO: 86)

GGTGGAGGAGGT

G4 (SEQ ID NO: 87)

GGAGGTGGTGGA

G5 (SEQ ID NO: 88)

GGAGGAGGTGGT

G6 (SEQ ID NO: 89)

GGAGGTGGAGGT

G7 (SEQ ID NO: 90)

GGAGGAGGAGGT

G8 (SEQ ID NO: 91)

GGAGGAGGTGGA G9 (SEQ ID NO: 92)

GGAGGTGGAGGA

G10 (SEQ ID NO: 93)

GGTGGAGGAGGA

Gil (SEQ ID NO: 94)

G GAG GAG GAG GA

SERPINA1

Homo sapiens SERPINA1 gene for alpha-l-antitrypsin (Genbank Acession No. AM048838) (SEQ ID NO: 95)

ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGGCCTGTGCTGCCTGGTCCCT GTCTCCCTGGCT GAG GAT C C C C AG G GAGAT G C T G C C C AGAAGAC AGAT AC AT C C C AC CAT GAT C AG GAT C AC C C AAC C T T C AAC AAGATCACCCCCAACCTGGCTGAGTTCGCCTTCAGCCTATACCGCCAGCTGGCACACCAG TCCAACAGCACC AATATCTTCTTCTCCCCAGTGAGCATCGCTACAGCCTTTGCAATGCTCTCCCTGGGGACC AAGGCTGACACT CACGATGAAATCCTGGAGGGCCTGAATTTCAACCTCACGGAGATTCCGGAGGCTCAGATC CATGAAGGCTTC CAGGAACTCCTCCGTACCCTCAACCAGCCAGACAGCCAGCTCCAGCTGACCACCGGCAAT GGCCTGTTCCTC AGCGAGGGCCTGAAGCTAGTGGATAAATTTTTGGAGGATGTTAAAAAGTTGTACCACTCA GAAGCCTTCACT GT C AAC T T C T G G GAC AC C GAAGAG G C C AAGAAAC AGAT C AAC GAT T AC GT G GAGAAG G GT AC T C AAG G GAAA ATTGTGGATTTGGTCAAGGAGCTTGACAGAGACACAGTTTTTGCTCTGGTGAATTACATC TTCTTTAAAGGC AAAT G G GAGAGAC C C T T T GAAGT C AAG GAC AC C GAG GAAGAG GAC T T C C AC GT G GAC C AG G C GAC C AC C GT G AAGGTGCCTATGATGAAGCGTTTAGGCATGTTTAACATCCAGCACTGTAAGAAGCTGTCC AGCTGGGTGCTG CTGATGAAATACCTGGGCAATGCCACCGCCATCTTCTTCCTGCCTGATGAGGGGAAACTA CAGCACCTGGAA AAT GAAC T C AC C C AC GAT AT CAT C AC C AAGT T C C T G GAAAAT GAAGAC AGAAG GT C T G C C AG C T T AC AT T T A CCCAAACTGTCCATTACTGGAACCTATGATCTGAAGAGCGTCCTGGGTCAACTGGGCATC ACTAAGGTCTTC AGCAATGGGGCTGACCTCTCCGGGGTCACAGAGGAGGCACCCCTGAAGCTCTCCAAGGCC GTGCATAAGGCT GTGCTGACCATCGACAAGAAAGGGACTGAAGCTGCTGGGGCCATGTTTTTAGAGGCCATA CCCATGTCTATC CCCCCCGAGGTCAAGTTCAACAAACCCTTTGTCTTCTTAATGATTGAACAAAATACCAAG TCTCCCCTCTTC AT G G GAAAAGT G GT GAAT C C C AC C C AAAAAT AA

Homo sapiens alpha-l-antitrypsin precursor (Genbank Accession No.

NP_001121179) (SEQ ID NO: 96)

MPS SVSWGI LLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQS NST NI FFS PVS IATAFAMLSLGTKADTHDEI LEGLNFNLTEI PEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFL SEGLKLVDKFLEDVKKLYHSEAFT FGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFAL YI FFKG KWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLS SWVLLMKYLGNATAI FFLPDEGKLQHLE NELTHDI ITKFLENEDRRSASLHLPKLS ITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKA VLTI DEKGTEAAGAMFLEAI PMS I PPEVKFNKPFVFLMI EQNTKS PLFMGKWNPTQK EXAMPLES

Example 1. Serum AAT expression levels in GOG-265 patients with Persistent or Recurrent Cervical Cancer

[0253] Serum AAT expression levels were analyzed for patients of GOG-265

(NCT01266460) Vaccine Therapy in Treating Patients With Persistent or Recurrent Cervical Cancer. GOG 0265 is an open-label, single arm 2-stage study designed to evaluate the safety, tolerability and efficacy of ADXS-HPV in approximately 67 patients. Patients receive one cycle (three doses) of ADXS-HPV at 1 x 109 cfu every 28 days. The primary efficacy endpoint is 12-month survival, with secondary efficacy endpoints of progression-free survival, overall survival and objective tumor response. The primary safety endpoints are the number of patients with dose-limiting toxicities and the frequency and severity of adverse effects. Stage 1 enrolled 29 patients and stage 2 is set to enroll another 38.

[0254] It was shown that the majority of long term survivors with AXAL (>12 Mo.) in GOG-265 (NCT01266460) had lower AAT at baseline (Figure 4 and Figure 5).

[0255] Listeria-based vaccines deliver targets directly into dendritic cells, activating them and stimulating them to present new targets and generate T cell immunity. High serum levels of AAT specifically inhibits dendritic cell activation, antigen presentation, and subsequently prevent the generate of CD8+ T cell responses. AAT also increases the generation of immunosuppressive Tregs, inhibits immune cell infiltration into tumor tissue, and reduces apoptotic death of tumor cells blocking downstream effects on the tumor. High serum AAT functions as a negative regulator (checkpoint) for dendritic cell activation, antigen presentation, and preempts the generation of CD8+ Tcells that are the key mediators of listeria-based immunotherapy (e.g., AXAL) efficacy.

[0256] Therefore, employing a baseline AAT cut-off at 3.5 mg/ml would screen out patients who are not likely to experience a survival benefit on AXAL. Baseline AAT <3.5 mg/ml can be used as biomarker for clinical benefit in AXAL treatment. Additionally, AAT <3.5 mg/ml as an eligibility criteria would eliminate the risk of exposing patients to AXAL treatment when they are not likely to obtain clinical benefit.

[0257] 12 month survival is a good surrogate for overall survival. AXAL

monotherapy compares favorably with 127C series for 12 Mo. and overall survival.

Setting an eligibility criteria for AXAL treatment of AAT <3.5 may increase 12 month survival from 38% to 49% and median survival from 6.2 months to 10.3 months in advanced cervical cancer (Figures 6 and 7). Example 2: Identification of Potential Therapeutic Targets Using Baseline Serum Protein Levels Associated with Survival in Axalimogene Filolisbac (AXAL)-Treated Metastatic Cervical Cancer Patients [0258] Prognostic biomarkers that identify high-risk patients may guide treatment decisions and thus improve clinical outcomes for patients with persistent, recurrent or metastatic cervical cancer (PRmCC).

[0259] To identify such biomarkers, the association between baseline inflammation- related serum protein levels and overall survival (OS) in 45 of the 50 AXAL-treated PRmCC patients who participated in the phase 2 GOG/NRG-0265 trial was evaluated.

The objective of this study was to evaluate the association between baseline inflammation- related serum protein levels and OS in AXAL-treated PRmCC patients in order to identify candidate prognostic biomarkers of clinical outcome.

Materials and Methods

[0260] The GOG/NRG-0265 trial is a phase 2 evaluation of AXAL in the treatment of persistent or recurrent squamous or non-squamous cell carcinoma of the cervix. The study design for GOG/NRG-0265 is summarized in Figure 8. Baseline demographics and prior treatment history of all patients participating in GOG/NRG-0265 and the 45 patients with baseline serum are shown below in Table 1.

Table 1

Prior bevacizumab treatment, n

30 (60) 27 (60)

(%)

aGOG PS, Gynecologic Oncology Group performance status

bFIGO, International Federation of Gynecology and Obstetrics

[0261] Baseline levels of 54 serum analytes were measured using custom multiplex immunoassays (Myriad RBM, Austin, TX). Only those serum proteins whose

measurements were above the level of detection for all patients were analyzed further. Linear regression analysis, with a cut-off P value < 01, was used to assess the association between baseline serum protein levels and OS. Unsupervised hierarchical clustering with complete linkage was used to group or cluster patients based on their baseline patterns of the 4 identified serum proteins that associated with OS. Kaplan-Meier analysis with log- rank test was used to compare the survival curves of the two patient clusters identified by unsupervised hierarchical clustering.

[0262] The GOG/NRG-0265 trial, which evaluated the efficacy of AXAL in the treatment of PRmCC, AXAL demonstrated a median overall survival of 6.2 months and a 12-month OS rate of 38% (19/50) (Figure 9).

Results

[0263] Of the 54 serum proteins tested in 45 of the 50 AXAL-treated PRmCC patients who participated in the GOG/NRG-0265 trial, baseline levels of only 4 serum proteins - alpha- 1 anti -trypsin (AAT), C-reactive protein (CRP), tissue inhibitor of

metalloproteinases 1 (TIMP-1) and vascular endothelial growth factor (VEGF) were found to associate significantly with OS (P< 01) (Table 2). The levels of all 4 serum proteins were negatively associated with OS (Figure 10). In addition, baseline levels of the AAT, CRP, TFMP-1 and VEGF were significantly lower in patients who survived >12 months than in those patients who survived <12 months (Figure 11).

Table 2

Significant positive correlations exist between serum

AAT levels and tumor stage and between serum AAT levels and tumor growth and progression

CRP • Liver C-reactive protein, an acute phase reactant protein; serum

Tumor tissue CRP levels rise in response to elevated levels of IL-6 and other proinflammatory cytokines

• Relationship exists between elevated serum/tumor CRP levels and prognosis in patients with solid tumors

TIMP-1 • Macrophages Tissue inhibitor of metalloproteinases-1, a natural

Variety of inhibitor of matrix metalloproteinases (MMPs) which tissues regulates their ability to degrade extracellular matrix including Inhibits apoptosis and promotes cell growth as well as tumor tissue angiogenesis

• High serum/tumor tissue levels in cancer patients

correlate with poor prognosis

VEGF • Mast cells Vascular endothelial growth factor, promotes

• Macrophages angiogenesis and vasculogenesis

Variety of Overexpression is not only observed in multiple tumor tissues types but is also associated with tumor stage and with a including poor prognosis

tumor tissue

[0264] In addition to evaluating the association of baseline levels of individual serums proteins with OS, we employed unsupervised two-dimensional hierarchical clustering with complete linkage to subset PRmCC patients based on the expression levels of this set of 4 serum proteins. The resulting dendogram identified 2 patient clusters, which are

distinguishable by a low expression pattern (cluster 1) or a high expression pattern (cluster 2) of the baseline levels of the 4 serum proteins (Figure 12).

[0265] Survival analysis for both patient clusters after AXAL treatment revealed that: cluster 1 exhibited a 12-month OS rate of 56%, whereas cluster 2 exhibited a rate of 15% (HR=0.23; 95% CI: 0.10-0.48; P< 001) (Figure 13) and cluster 1 exhibited a median OS of 12.32 months, whereas cluster 2 exhibited a median OS of 4.08 months (P< 0003) (Figure 14).

[0266] Further analysis of the two patient clusters demonstrated that 82% of the patients surviving >12 months and 39% of the patients surviving <12 months were found in patient cluster 1 (Figure 15 A) and 18% of the patients surviving >12 months and 61% of the patients surviving <12 months were found in patient cluster 2 (Figure 15 A).

[00101] After AXAL treatment, patients who survived <12 months and who have a low expression pattern of the 4 serum proteins (ie, cluster 1 criteria) have a significantly higher median OS than patients who survived <12 months and who have a high expression pattern of the 4 serum proteins (ie, cluster 2 criteria) [median OS: 5.36 months versus 3.85 months (P=.02)] (Figure 15B).

[0267] Together, these findings suggest that the baseline levels of these 4 serum proteins (AAT, CRP, TIMP-1 and VEGF) have prognostic value for OS in AXAL-treated PRmCC patients and cluster 1 criteria may also identify PRmCC patients most likely to benefit from AXAL treatment. Furthermore, these 4 serum proteins may be potential therapeutic targets as well.