Listeria variants and methods of use thereof

Abstract

The present disclosure provides variant Listeria bacteria comprising a mutation in a purA gene encoding adenylosuccinate synthetase, wherein the variant Listeria bacteria are adenosine auxotrophs. The present disclosure provides compositions comprising a subject variant Listeria bacterium, including compositions further comprising a multispecific antibody. Also provided are immunogenic compositions comprising a variant Listeria bacterium of the present disclosure and kits comprising a unit dose thereof. The present disclosure additionally provides methods of inducing an immune response in an individual, the methods comprising administering to the individual an effective amount of an immunogenic composition of the present disclosure.

Description

LISTERIA VARIANTS AND METHODS OF USE THEREOFCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 598,660 filed November 14, 2023. which application is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Numbers AI027655 andAI063302 awarded by the National Institutes of Health. Tire government has certain rights in the invention.INCORPORATION-BY- REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML,“BERK-492PRV SEQ LIST” created on October 24, 2023 and having a size of 7,956 bytes. Hie contents of the Sequence Listing XML are incorporated by reference herein in their entirety.INTRODUCTION

[0004] Listeria monocytogenes is a rapidly growing, Gram-positive, facultative intracellular bacterial pathogen of humans and animals. This bacterium is easy to manipulate genetically and the cell biology of its infection has been extensively characterized. L. monocytogenes is able to invade host cells, including professional antigen-presenting cells (APCs), rapidly escape from phagosomes, and grow in the host cytosol where it delivers antigens and metabolites that induce robust host cell-mediated immunity (CMI). In addition, L. monocytogenes does not cause rapid cell death of host APCs, another feature that favors the development of CMI. Due to its ability to induce strong antigen-specific T-cell responses, L. monocytogenes has been used as a therapeutic vaccine in more than 20 cancer clinical trials and administered to more than 1800 patients. However, in contrast to a strong stimulation of antigenspecific CD8+cytotoxic T-cells observed in preclinical mouse studies, L. monocytogenes vaccines have been less immunogenic in clinical trials. In rare cases, live bacteria were found in patients’ blood or on implants, after the administration of live vaccines. Additionally, even attenuated vaccine strains still caused severe adverse events and consequently put clinical trials on hold.SUMMARY

[0005] Due, in part, to the safety and efficacy concerns of using L. monocytogenes as a live vector for cancer immunotherapy, the inventors have identified a need for safer and more potent strainsof L. monocytogenes. Accordingly, the present disclosure provides variant Listeria bacteria comprising a mutation in a purA gene encoding adenylosuccinate synthetase, wherein the variant Listera bacteria are adenosine auxotrophs. Tire present disclosure provides compositions comprising a subject variant Listeria bacterium, including compositions further comprising a multispecific antibody. Also provided are immunogenic compositions comprising a variant Listeria bacterium of the present disclosure and kits comprising a unit dose thereof. The present disclosure additionally provides methods of inducing an immune response in an individual, the methods comprising administering to the individual an effective amount of an immunogenic composition of the present disclosure.

[0006] The present disclosure provides variant Listeria bacteria comprising a mutation in a purA gene encoding adenylosuccinate synthetase, wherein the variant Listeria bacteria are adenosine auxotrophs. In some cases, the mutation is a deletion of all or a portion of the purA gene. In some embodiments, a variant Listeria bacterium does not grow extracellularly in a mammal. In some cases, a variant Listeria bacterium requires from lOOpM to 500pM adenosine supplementation to grow. In certain embodiments, a variant Listeria bacterium further comprises a deletion of all or a portion of an endogenous ribC gene and / or an endogenous ribF gene.

[0007] In certain embodiments, a variant Listeria bacterium is genetically modified to comprise one or more heterologous nucleic acids. In some cases, the one or more heterologous nucleic acids are integrated into the bacterial genome. In some embodiments, the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the nonmevalonate pathway, wherein the variant Listeria bacterium grows aerobically. In some cases, the one or more heterologous nucleic acids comprise nucleotide sequences encoding IspE, GcpE, and IspA polypeptides. In some cases, the one or more heterologous nucleic acids comprise nucleotide sequences encoding Dxs, IspD, IspF, IspE, GcpE, and IspA polypeptides. A subject variant Listeria bacterium, in some embodiments, may further comprise a loss of function mutation in an endogenous LytB gene. In some embodiments, a variant Listeria bacterium further comprises one or more additional mutations that confers enhanced function of the non-mevalonate pathway under aerobic conditions. In some cases, the one or more additional mutations that confers enhanced function of the non-mevalonate pathway under aerobic conditions comprises a mutation in an endogenous gene selected from lmol694,fur,flgE, ribF. uracil-DNA glycosylase, DNA-directed RNA polymerase subunit a, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, and heptaprenyl diphosphate synthase component I. In certain embodiments, at least one of the one or more heterologous nucleic acids comprises a nucleotide sequence encoding an antigen polypeptide. In some cases, the antigen is a cancer-associated antigen.

[0008] In certain embodiments, a variant Listeria bacterium further comprises one or more additional mutations that confers an attenuated phenotype on the bacterium and / or one or more additional mutations that provide a growth advantage. In some cases, the one or more additional mutations thatconfers an attenuated phenotype comprises a mutation in a gene selected from actA and in / B. and wherein tire one or more additional mutations that provide a rowth advantage comprises a mutation in an eetB gene.

[0009] In some embodiments, a variant Listeria bacterium is a variant Listeria monocytogenes bacterium.

[0010] The present disclosure provides compositions comprising a subject variant Listeria bacterium. In certain embodiments, the composition further comprises a multispecific antibody that comprises a first antigen-binding site specific for a cancer-associated antigen and a second antigenbinding site specific for a T cell. In some embodiments, the T cell may be a mucosal-associated invariant T (MAIT) cell, a y / 8 T cell, a CD8+T cell, or a natural killer (NK) cell. Hie present disclosure additionally provides immunogenic compositions comprising a subject variant Listeria bacterium.

[0011] The present disclosure provides a method of inducing an immune response in an individual, the methods comprising administering to the individual an effective amount of an immunogenic composition of the present disclosure. In certain embodiments of the methods, the variant Listeria bacterium is genetically modified to comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway, wherein the variant Listeria bacterium grows aerobically, and wherein said immune response is induced to a phosphoantigen produced by the variant Listeria bacterium. In some cases, the phosphoantigen is (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP). In certain embodiments of the methods, the variant Listeria bacterium is genetically modified to comprise a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous polypeptide, and wherein said immune response is induced to the heterologous polypeptide. In some cases, the heterologous polypeptide is an antigen. In some cases, the antigen is a cancer-associated antigen. In some embodiments, the immune response comprises a gamma-delta T cell response.

[0012] The present disclosure additionally provides kits comprising a unit dose of a subject immunogenic composition. In certain embodiments, the unit dose is an oral dose. In certain embodiments, the unit dose is injectable. In some embodiments, the kit further comprises a recombinant expression vector comprising a nucleotide sequence encoding a heterologous polypeptide. In some cases, the heterologous polypeptide is an antigen. In some cases, the antigen is a cancer-associated antigen.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 depicts growth curves of the purA mutants in chemically defined media supplemented w ith indicated concentrations of adenosine. Overnight bacterial cultures were diluted to OD600 of 0.05 to start the growth curve then incubated at 37°C w ith agitation. Growth was monitored spectrophotometrically.

[0014] FIG. 2A-2B depict growth of the purA mutants in blood and serum. Mid-log bacteria were inoculated into 3 ml culture at the density of 1 x 106CFUs per milliliter. Growth was measured by plating on BHI agar 24 hours after incubation in a 37°C shaker. (FIG. 2A) Growth in defibrinated sheep blood. (FIG. 2B) Growth in sterile human serum buffered at pH 7.0 with 5 mM HEPES. Three independent experiments were combined. Student t-test, ***P < 0.001.

[0015] FIG. 3A-3B show that the purA mutants do not grow in the cytosol of bone marrow- derived macrophages (BMMs). (A) Phagosome escape. BMMs were infected with mid-log phase bacteria at an MOI of 5-10 for 30 minutes in the presence of cytochalasin D as described previously. Relative escape was measured by the percentage of p62+bacteria of the total intracellular bacteria in relative to that of wild type (WT). Data are mean ± SD. Three independent experiments w ere combined with more than 100 bacteria analyzed for each strain per experiment. Student t-test. *P < 0.05. (B) Intracellular growth curve in BMMs. BMMs were infected at an MOI of 0.25 for 30 minutes. Adenosine w as added into the BMM medium at 1-hour post-infection.

[0016] FIG. 4A-4C show7that the purA mutants are severely attenuated in mice. Eight-week- old CD-I mice (Charles River) were infected intravenously with 1 x 1()' CFUs of indicated strains. Bacterial burdens in livers, spleens (A) and gallbladders (B) were measured 48-hour post infection by plating homogenized organs. Each circle represents an individual mouse. Lines present medians. Two biological repeats were combined with a total of 6-10 mice per strain. P values of unpaired student t-test were indicated. L.o.d, limit of detection. (C) The in vivo growth curve in mice. Eight-w eek-old CD-I mice (Charles River) were infected intravenously with 1 x 105colony forming units (CFUs) of indicated strains. Bacterial burdens in livers (left) and spleens (right) were measured 0.5-hour, 5-hour, 10-hour. 24- hour, and 48-hour post-infection. Two biological repeats were combined with a total of 7-9 mice per strain. L.o.d, limit of detection.

[0017] FIG. 5A-5B show that the purA mutants elicit potent cell-mediated immunity. (A) Vaccination with the purA mutants confers long-term protection against WT L. monocytogenes . C57BL / 6J mice were vaccinated intravenously with 103or 105CFUs of L. monocytogenes . Four weeks post-vaccination, mice were challenged intravenously with 5 x 104CFUs of WT L. monocytogenes. Three days post-challenge, WT CFUs were enumerated from the spleens. Data is pooled from three independent experiments for the 103group, and two experiments for the 105group. L.o.d, limit of detection. (B) The fspurA mutants induced a potent cytotoxic CD8+T cell response. C57BL / 6J mice were infected intravenously with 103CFUs of indicated attenuated strains. Single cell suspensions of splenocytes were stimulated w ith OVA-specific epitopes in the presence of the protein transportation inhibitor. Antigen-specific CD8+T cell response was assessed by intracellular cytokine staining on day 7 after a single vaccination. Data is pooled from three independent experiments. One-way ANOVA, multiple comparisons; ns, not significant; *P < 0.05; ***P < 0.001; ****P < 0.0001.DEFINITIONS

[0018] “Heterologous,” as used herein, refers to a nucleic acid or polypeptide that is not found in a naturally-occurring bacterium (e.g.. a Listeria bacterium). For example, a “heterologous” nucleic acid comprising nucleotide sequences encoding polypeptides in a non-mevalonate pathway is a nucleic acid that is not found in a naturally-occurring bacterium (e.g., a Listeria bacterium). “Heterologous,” as used herein, also refers to a nucleic acid or polypeptide that is not found in a native nucleic acid or polypeptide, respectively. For example, a promoter sequence that is heterologous to a non-mevalonate pathway gene is a promoter sequence that is not found associated with the non-mevalonate pathway gene in nature.

[0019] The tenn “naturally-occurring” as used herein as applied to a nucleic acid, a protein, a cell, or an organism, refers to a nucleic acid, cell, protein, or organism that is found in nature.

[0020] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this tenn includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA. DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiment being described, singlestranded (such as sense or antisense) and double-stranded polynucleotides.

[0021] The terms "polypeptide," "peptide," and "protein", are used interchangeably herein, refer to a polymeric fonn of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The tenn includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

[0022] As used herein the tenn “isolated” is meant to describe a polynucleotide, a polypeptide, or a cell (e.g., a Listeria bacterium) that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified cell may be present in a mixed population of genetically modified cells.

[0023] ‘Recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of syntheticoligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the fomi of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5’ or 3’ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “DNA regulatory sequences”, below).

[0024] Tirus, e.g., the tenn “recombinant” polynucleotide or “recombinant” nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding tire same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0025] Similarly, the tenn “recombinant” polypeptide refers to a polypeptide which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention. Thus, e.g., a polypeptide that comprises a heterologous amino acid sequence is recombinant.

[0026] By “construct” or “vector” is meant a recombinant nucleic acid, generally recombinantDNA, which has been generated for the purpose of the expression and / or propagation of a specific nucleotide sequence(s), or is to be used in the construction of other recombinant nucleotide sequences.

[0027] Tire terms “DNA regulatory sequences.” “control elements,” and “regulatory elements,” used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate expression of a coding sequence and / or production of an encoded polypeptide in a host cell.

[0028] The term “transformation” is used interchangeably herein with “genetic modification” and refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (e.g., DNA exogenous to the cell) into the cell. Genetic change (“modification”) can be accomplished either by incorporation of the new nucleic acid into the genome of the host cell, or bytransient or stable maintenance of the new nucleic acid as an episomal element. Where the cell is a prokaryotic cell, permanent changes can be introduced into the chromosome or via extrachromosomal elements such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, and the like. A general discussion of these methods can be found in Ausubel, et aL, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0029] “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. As used herein, the terms “heterologous promoter” and “heterologous control regions” refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature. For example, a “transcriptional control region heterologous to a coding region” is a transcriptional control region that is not nonnally associated with the coding region in nature.

[0030] A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nhn.nih.gov / BLAST. See, e.g., Altschul et al. (1990), Afo / . Biol. 215:403-10. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that pennits gaps in sequence alignments. ee Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0031] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0032] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper andlower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0034] It must be noted that as used herein and in the appended claims, the singular forms '‘a,” ■‘an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a variant Listeria bacterium” includes a plurality of such bacteria and reference to “a mutation in a purA gene” includes reference to one or more mutations in a purA gene and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0035] The use of the terms “a,” “an,” and “the,” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising.” “having.” “including,” and “containing” are to be construed as open-ended terms (z.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10- 15 is disclosed, then 11. 12. 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments of the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.

[0036] As used herein, the term “about” used in connection with an amount indicates that the amount can vary by 10% of the stated amount. For example, “about 100” means an amount of from 90- 110. Where about is used in the context of a range, the “about” used in reference to the lower amount ofthe range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and “about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range. For example, from about 100 to about 1000 means that the range extends from 90 to 1100.

[0037] The term “and / or” as used herein a phrase such as “A and / or B” is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used herein a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0038] It is understood that aspects and embodiments of the present disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0039] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

[0040] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.DETAILED DESCRIPTION

[0041] Hie present disclosure provides variant Listeria bacteria comprising a mutation in a purA gene encoding adenylosuccinate synthetase, wherein the variant Listera bacteria are adenosine auxotrophs. The present disclosure provides compositions comprising a subject variant Listeria bacterium, including compositions further comprising a multispecific antibody. Also provided are immunogenic compositions comprising a variant Listeria bacterium of the present disclosure and kits comprising a unit dose thereof. The present disclosure additionally provides methods of inducing an immune response in an individual, the methods comprising administering to the individual an effective amount of an immunogenic composition of the present disclosure.V RI NT LISTERIA BACTERIA

[0042] The de novo synthesis and / or acquisition of purine and pyrimidine nucleotides is required for various bacterial cellular functions such as energy storage, second messenger signaling, and genome replication and is thus critical for growth. However, the average concentration of purine bases and nucleosides (e.g.. adenosine, guanosine) in extracellular fluids is low (e.g., in the range of 0.4-6pM). Consequently, de novo nucleotide biosynthesis is a critical metabolic function required for bacterial proliferation in extracellular environments (e.g., blood). Facultative intracellular pathogens, such as L. monocytogenes, can survive and proliferate in extracellular environments during systemic infections, limiting their therapeutic potential. Exemplary extracellular growth niches of Listeria bacteria (e.g., L. monocytogenes) include blood and gallbladders. Notably, adenosine is abundant in tumor microenvironments. As such, an engineered adenosine auxotroph of a Listeria bacterium would lose the ability to thrive in extracellular niches low in adenosine and would prove advantageous for use in a therapy (e.g., a cancer immunotherapy). For example, an engineered adenosine auxotroph of a Listeria bacterium may be useful in induction of CMI within a tumor microenvironment where adenosine is abundant but be unable to cause systemic infection due to poor adenosine availability in other extracellular niches.

[0043] Thus, the present disclosure provides a variant Listeria bacterium comprising a mutation in a purA gene encoding adenylosuccinate synthetase, wherein the variant Listera bacteria are adenosine auxotrophs.

[0044] Adenylosuccinate synthetase catalyzes the conversion of inosine monophosphate (IMP) and L-aspartate to adenylosuccinate, coupled to the hydrolysis of GTP. Hie reaction catalyzed by adenylosuccinate synthetase is the first committed step in the de novo synthesis of the purine nucleotide adenosine monophosphate (AMP) and plays a key role in the regulation of purine nucleotide interconversion. Suitable mutations in a pur A gene may include any mutation that disrupts the activity of the adenylosuccinate synthetase encoded by the purA gene, such that the subject the Listeria bacterium is rendered an adenosine auxotroph. Examples of suitable mutations include, but are not limited to, missense mutations, in-frame deletions, in-frame insertions, out-of-frame deletions, out-of-frame insertions, and premature stop codons. In some cases, the subject mutation is an in-frame deletion. In some cases, the subject mutation is a deletion of all or a portion of the purA gene. In some cases, the subject mutation is a deletion of all of the purA gene. In some cases, the subject mutation is a deletion of a portion of the purA gene. In some cases, the subject mutation is an in-frame deletion within the purA gene. In some embodiments, the mutation is not a transposon insertion (i.e., the subject listeria bacterium does not include a transposon insertion that renders it an adenosine auxotroph by disrupting the purA gene).

[0045] By '‘adenosine auxotroph”, it is meant that the subject variant Listeria bacterium requires additional supplementation of adenosine relative to a wi Id-type Listeria bacterium to grow and / or proliferate. Adenosine auxotrophy may be assessed by measuring the growth and / or proliferation rate of a subject Listeria bacterium relative to a wild-type Listeria bacterium in a chemically defined medium with varying levels of supplemented adenosine (e.g., in a chemically defined medium supplemented with OpM adenosine, in a chemically defined medium supplemented with lOOpM adenosine). For example, where a wild-type Listeria may exhibit a given growth rate in a chemically defined medium without adenosine, a Listeria bacterium that is an adenosine auxotroph may fail to grow in a chemically defined medium without adenosine and only exhibit a comparable growth rate in a chemically defined medium supplemented with lOOpM adenosine. Methods of measuring the growth rate of bacterial cultures (e.g., measurement of the optical density of a liquid culture inoculated with said bacteria) are well known in the art.

[0046] In certain embodiments, a variant Listeria of the present disclosure requires from lOOpM to 500pM adenosine supplementation to grow, e.g., in some cases the variant Listeria bacterium requires from lOOpM to 150pM adenosine supplementation, from 150pM to 200pM adenosine supplementation, from 250pM to 300pM adenosine supplementation, from 300pM to 350pM adenosine supplementation, from 350pM to 400pM adenosine supplementation, from 400pM to 450pM adenosine supplementation, and from 450pM to 500pM adenosine supplementation. A wild-type Listeria bacterium, for example, may require no adenosine supplementation to grow (e.g., in some cases, a wild-type Listeria may require no exogenous adenosine to grow). In some embodiments, a subject variant Listeria bacterium can be grown (cultured) in vitro in a culture medium supplemented with adenosine, it does not grow in vivo extracellularly. In some cases, a subject Listeria bacterium does not grow in vivo extracellularly in a mammal. In some embodiments, a variant Listeria bacterium of the present disclosure requires adenosine supplementation to grow, and thus is a conditionally obligate intracellular bacterium when in vivo (e.g., when in a mammal or other animal host). In some embodiments, a subject variant Listeria bacterium of the present disclosure may survive in tumors (e.g., the subject variant Listeria bacterium may survive extracellularly in the tumor microenvironment).

[0047] In some embodiments, a variant Listeria bacterium of tire present disclosure further comprises a mutation in a ribC gene and / or a rib gene. In some cases, the mutation comprises a deletion of all or a portion of the ribC gene and / or the ribF gene. The ribC and ribF genes are required for flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) biosynthesis. RibC is a bifunctional enzyme that catalyzes tire phosphorylation of riboflavin to FMN and the adenylylation of FMN to form FAD. RibF also converts FMN to FAD by adenylylation. In some embodiments, a variant Listeria bacterium of the present disclosure comprising a mutation in a ribC gene and / or a ribF gene can be grown (cultured) in vitro in a culture medium supplemented with FMN and FAD. it cannot grow in vivo(e.g., inside a mammal) extracellularly. In some embodiments, a variant Listeria bacterium of the present disclosure comprising a mutation in a ribC gene and / or a ribF gene requires flavin mononucleotide and flavin adenine dinucleotide supplementation to grow, and thus is a conditionally obligate intracellular bacterium when in vivo (e.g., when in a mammal or other animal host).

[0048] A variant Listeria bacterium of the present disclosure may include, in addition to the ribC / ribF modification described above, a deletion of all or apart of the eetB gene. Deletion of all or a part of the eetB gene can provide a growth advantage. In some cases, such a variant strain exhibits increased growth rate in broth in vitro, compared to the growth rate in broth of a Listeria bacterium comprising the ribC / ribF modification without the deletion of the eetB gene.

[0049] In certain embodiments, a subject variant Listeria bacterium is genetically modified to comprise one or more heterologous nucleic acids. In some embodiments, the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway, wherein the variant Listeria bacterium grows aerobically. The mevalonate pathway for isoprenoid biosynthesis is an essential metabolic pathway for the biosynthesis of isoprenoids in eukaryotes, archaea, and some bacteria. The non-mevalonate pathway represents an alternative metabolic pathway for the synthesis of isoprenoids unique to bacteria and some parasites. Tightly regulated expression of non-mevalonate pathway genes (e.g.. by grouping into an operon under control of a single promoter) is required to avoid toxicity from accumulation of intermediate products. Although most organisms encode either the mevalonate or non-mevalonate pathway of isoprenoid biosynthesis, Listeria represents some of the very few bacteria that encode both pathways. Enzymes of the non-mevalonate pathway include Dxs, IspD, IspF, LytB, IspE, GcpE, and IspA proteins. Dxs catalyzes the conversion of pyruvate and D-glyceraldehyde 3-phosphate to 1-deoxy-D-xylulose 5- phosphate (DOXP). IspD catalyzes the conversion of 2-C-methyl-D-erythritol 4-phosphate (MEP) to 4- diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME). IspF catalyzes the conversion of 4- diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (CDP-MEP) to 2-C-methyl-D-erythritol-2, 4- cyclodiphosphate (MEcPP). LytB (i.e., IspH) catalyzes the conversion of (E)-4-hydroxy-3 -methyl -but-2- enyl pyrophosphate (HMBPP) to dimethylallyl pyrophosphate (DMAPP). IspE catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME) to 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (CDP-MEP). GcpE catalyzes the conversion of 2-C-methyl-D-erythritol-2. 4- cyclodiphosphate (MEcPP) to (E)-4-hydroxy-3 -methyl -but-2-enyl pyrophosphate (HMBPP). IspA catalyzes the conversion of dimethylallyl pyrophosphate (DMAPP) to Famesyl pyrophosphate (FPP). However, the native non-mevalonate pathway can only be used anaerobically in Listeria bacteria and therefore production of isoprenoids through the native non-mevalonate pathway is limited to anaerobic conditions.

[0050] In some embodiments of a subject variant Listeria bacterium, all polypeptides required for isoprenoid synthesis through the non-mevalonate pathway are encoded on a single heterologous nucleic acid and the nucleotide sequences encoding all of the polypeptides are operably linked to a single transcriptional control element (e.g., a single promoter). In some cases, all polypeptides required for isoprenoid synthesis through the non-mevalonate pathway are encoded in separate heterologous nucleic acids. In some embodiments, the one or more heterologous nucleic acids comprise nucleotide sequences coding for IspE, GcpE, and IspA polypeptides. In some embodiments, the one or more heterologous nucleic acids comprise nucleotide sequences encoding Dxs, IspD, IspF, IspE, GcpE, and IspA polypeptides.

[0051] In some embodiments, a heterologous nucleic acid may comprise a nucleotide sequence encoding a Dxs polypeptide with 80% or more (e.g., at least 80%, at least 85%. at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) amino acid sequence identity to the amino acid sequence:

[0052] MDLLSIQDPSFLKNMSIDELEKLSDEIRQFLITSLSASGGHIGPNLGVVELTVALH KEFNSPKDKFLWDVGHQSYVHKLLTGRGKEFATLRQYKGLCGFPKRSESEHDVWETGHSSTSL SGAMGMAAARDIKGTDEYIIPIIGDGALTGGMALEALNHIGDEKKDMIVILNDNEMSIAPNVGAI HSMLGRLRTAGKYQWVKDELEYLFKKIPAVGGKLAATAERVKDSLKYMLVSGMFFEELGFTY LGPVDGHSYHELIENLQYAKKTKGPVLLHVITKKGKGYKPAETDTIGTWHGTGPYKINTGDFVK PKAAAPSWSGLVSGTVQRMAREDGRIVAITPAMPVGSKLEGFAKEFPDRMFDVGIAEQHAATM AAAMAMQGMKPFLAIYSTFLQRAYDQVVHDICRQNANVFIGIDRAGLVGADGETHQGVFDIAF MRHIPNMVLMMPKDENEGQHMVHTALSYDEGPIAMRFPRGNGLGVKMDEQLKTIPIGTWEVL RPGNDAVILTFGTTIEMAIEAAEELQKEGLSVRVVNARFIKPIDEKMMKSILKEGLPILTIEEAVLE GGFGSSILEFAHDQGEYHTPIDRMG1PDRFIEHGSVTALLEEIGLTKQQVANR1RLLMPPKTHKGI GSHFL (SEQ ID NO: 1).

[0053] In some embodiments, a heterologous nucleic acid may comprise a nucleotide sequence encoding an IspD polypeptide with 80% or more (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) amino acid sequence identity to the amino acid sequence:

[0054] MSYDVV1PAAGQGKRMKAGRNKLFIELKGDPV11HTLRVFDSHRQCDKI1LV1NE QEREHFQQLLSDYPFQTSIELVAGGDERQHSVYKGLKAVKQEKIVLVHDGARPFIKHEQIDELIA EAEQTGAAILAVPVKDTIKRVQDLQVSETIERSSLWAVQTPQAFRLSLLMKAHAEAERKGFLGT DDASLVEQMEGGSVRVVEGSYTNIKLTTPDDLTSAEAIMESESGNKHVHLG (SEQ ID NO: 2).

[0055] In some embodiments, a heterologous nucleic acid may comprise a nucleotide sequence encoding an IspF polypeptide with 80% or more (e.g., at least 80%. at least 85%, at least 90%, at least95%, at least 97%, at least 98%, at least 99%, or 100%) amino acid sequence identity to the amino acid sequence:

[0056] MFRIGQGFDVHQLVEGRPLIIGGIEIPYEKGLLGHSDADVLLHTVADACLGAVGE GDIGKHFPDTDPEFKDADSFKLLQHVWGIVKQKGYVLGNIDCTIIAQKPKMLPYIEDMRKRIAE GLEADVSQVNVKATTTEKLGFTGRAEGIAAQATVLIQKGDFL (SEQ ID NO:3).

[0057] In some embodiments, a heterologous nucleic acid may comprise a nucleotide sequence encoding an IspE polypeptide with 80% or more (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) amino acid sequence identity to the amino acid sequence:

[0058] MRILEKAPAKINLSLDVTRKRPDGYHEVEMIMTTIDLADRIELTELAEDEVRVSS HNRFVPDDQRNLAYQAAKLIKDRYNVKKGVSIMITKVIPVAAGLAGGSSDAAATLRGLNRLWN LNLSAETLAELGAEIGSDVSFCVYGGTALATGRGEKIKHISTPPHCWVILAKPTIGVSTAEVYRA LKLDGIEHPDVQGMIEAIEEKSFQKMCSRLGNVLESVTLDMHPEVAMIKNQMKRFGADAVLMS GSGPTVFGLVQYESKVQRIYNGLRGFCDQVYAVRMIGEQNALDHLG (SEQ ID NO: 4).

[0059] In some embodiments, a heterologous nucleic acid may comprise a nucleotide sequence encoding a GcpE polypeptide with 80% or more (e.g., at least 80%. at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%. or 100%) amino acid sequence identity to the amino acid sequence:

[0060] MQVSEITHRTKTRPVKVGPLTIGGNNEVVIQSMTTTKTHDVEATVAEINRLAEA GCQIVRVACPDERAANAIADIKKRISIPLVVDIHFDYKLALKAIEGGADKIRINPGNIGRREKVEA VVKAAKDKGIPIRIGVNAGSLEKRILEKYGYPTADGMVESALHHIKILEDLDFHDIIVSMKASDV NLAIEAYEKAAKAFDYPLHLGITESGTLFAGTVKSAAGLGAILSKGIGNTMRISLSADPVEEVKVARELEKSFGLASNAATLISCPTCGR1EIDLIS1ANEVEEY1SK1KAP1KVAVLGCAVNGPGEAREAD1 GIAGARGEGLLFRKGKIVRKVPEETMVEELKKEIDILAEEHYAKLEAEKAKLKEETQKADFL (SEQ ID NO:5).

[0061] In some embodiments, a heterologous nucleic acid may comprise a nucleotide sequence encoding an IspA polypeptide with 80% or more (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%. or 100%) amino acid sequence identity to the amino acid sequence:

[0062] MTNKLTSFLADRKKTIENQLSVYTEKLDMPDSLKKSMLYSLQAGGKRLRPLIVL AVLNAYGKSEKDGIPVGCAVEMIHTYSLIHDDLPCMDDDDLRRGKPTNHKVFGEATAVLAGD GLLTESFKLITSHVSDEVSAEKRLRLVNELISAAGTEGMVGGQVADMEAENRQVTLEELESIHER KTAKLLGFCVIAGAILADAPEEDIETLRTFSSHIGIGFQIRDDILDLEGSEEKIGKRVGSDTTNDKS TYPSLLSLEGAKHKLDVHIKEAKRLIGGLSLQKDLLYELCDLIAARDHL (SEQ ID NO:6).

[0063] In some cases, the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) amino acid sequence identity to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5. and SEQ ID NO:6. In some cases, the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) amino acid sequence identity to the amino acid sequences of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NO:5. and SEQ ID NO:6.

[0064] In some embodiments, a variant Listeria bacterium further comprises (e.g., in addition to the one or more heterologous nucleic acids comprising nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway) a mutation in an endogenous LytB gene such that the endogenous LytB gene is non -functional. In some cases, the mutation in the endogenous LytB gene is a deletion of all or a portion of the endogenous LytB gene. As described above, the L tB (i.e., IspH) enzyme encoded by the LytB gene catalyzes the conversion of (E)-4-hydroxy-3- methyl-but-2-enyl pyrophosphate (HMBPP) to dimethylallyl pyrophosphate (DMAPP) in the non- mevalonate pathway of isoprenoid synthesis. As such, a subject variant Listeria bacterium comprising a mutation in an endogenous LytB gene, such that the endogenous LytB gene is non-fimctional, may find use when increased production of HMBPP by the subject Listeria bacterium is desired.

[0065] When referring throughout the disclosure herein to heterologous nucleic acids in the context of a subject variant Listeria bacterium (e.g., see the previous paragraphs), the heterologous nucleotides can also be referred to as exogenous nucleic acids. As such, any disclosure of a "heterologous nucleic acid” or a variant Listeria bacterium genetically modified to comprise one or more heterologous nucleic acids can be considered a disclosure of an exogenous nucleic acid or a variant Listeria bacterium genetically modified to comprise one or more exogenous nucleic acids. As such, for example, a subject variant Listeria bacterium can be genetically modified to comprise one or more exogenous nucleic acids.

[0066] Mutations in additional genes may enhance the function of the non-mevalonate pathway under aerobic conditions in a subject Listeria bacterium. For example, a loss of function mutation in fur, which encodes a ferric uptake protein, may improve the aerobic function of the non-mevalonate pathway. In some embodiments, a variant Listeria bacterium of the present disclosure may comprise a loss of function mutation in a fur gene. In some embodiments, a variant Listeria bacterium of the present disclosure may comprise a loss of function mutation in a Imo 1694 gene. In some embodiments, a variant Listeria bacterium of the present disclosure may comprise a loss of function mutation in a Imo 1694 gene and a fur gene. In some cases, the loss of function mutation comprises a frameshift in the Imo 1694 geneand a frameshift mutation or pre-mature stop in the fur gene. In some cases, the loss of function mutation comprises a deletion of all or a portion of the Imo 1694 and / or fur gene. In some embodiments, a variant Listeria bacterium of the present disclosure may comprise a mutation in any one of a flgE gene, a ribF gene, a uracil-DNA glycosylase gene, a DNA-directed RNA polymerase subunit a gene, a UDP-N- acetylglucosamine 1-carboxyvinyltransferase gene, and a heptaprenyl diphosphate synthase component I gene. Additional embodiments of subject variant Listeria bacteria may include any combination of mutations that enhance the function of the non-mevalonate pathway under aerobic conditions discussed above.

[0067] In some embodiments, the nucleotide sequences encoding tire polypeptides of a non- mevalonate pathway are operably linked to a suitable promoter to facilitate expression of the nucleic acid comprising the nucleotide sequence and production of the polypeptides. For example. L. monocytogenes promoter / regulatory sequences which may be used to direct expression of nucleic acids comprising nucleotide sequences encoding the polypeptides of a non-mevalonate pathway include, but are not limited to, promoter sequences of the plcA gene which encodes PI-PLC, the Listeria mpl gene, which encodes a metalloprotease, and the Listeria inlA gene which encodes intemalin, a Listeria membrane protein. Heterologous regulatory elements such as promoters derived from phage, and promoters or signal sequences derived from other bacterial species, may be employed for the expression of a heterologous operon by the Listeria species. Another suitable promoter is the constitutive HyPer promoter; see, e.g., Reniere et al. (2016) PLoS Pathogens doi.org / 10.1371 / joumal.ppat.1005741.

[0068] In some embodiments, at least one of the one or more heterologous nucleic acids comprises a nucleotide sequence encoding an antigen. Tire heterologous antigen is, in certain embodiments, one that is capable of providing protection in a host organism against challenge by the infectious agent from which the heterologous antigen was derived, or which is capable of affecting tumor growth and metastasis in a manner which is of benefit to the host organism. Heterologous antigens therefore include those specified by infectious agents, wherein an immune response directed against the antigen serves to prevent or treat disease caused by the agent. Such heterologous antigens include, but are not limited to, viral, bacterial, fungal or parasite surface proteins and any other proteins, glycoproteins, lipoprotein, glycolipids, and the like. Heterologous antigens include cancer-associated antigens (e.g.. tumor antigens). Heterologous antigens also include those which provide benefit to a host organism which is at risk for acquiring or which is diagnosed as having a tumor that expresses the heterologous antigen(s). The host organism may be a mammal, such as a human.

[0069] By the temi "antigen" or “heterologous antigen”, as used herein, is meant a protein or peptide, a glycoprotein or glycopeptide, a lipoprotein or lipopeptide, or any other macromolecule which is not normally expressed in Listeria, which substantially corresponds to the same antigen in an infectious agent, a cancer cell, a tumor cell, or a tumor-related protein. The heterologous antigen isexpressed by a variant Listeria bacterium according to the present disclosure, and is processed and presented to cytotoxic T-cells upon infection of the host organism (e.g., a mammal) by the bacterium. Hie heterologous antigen expressed by a variant Listeria bacterium need not precisely match the corresponding unmodified antigen or protein in the tumor cell or infectious agent so long as it results in a T-cell response that recognizes the unmodified antigen or protein which is naturally expressed in the mammal. In other examples, the tumor cell antigen may be a mutant form of that which is naturally expressed in the host organism (e.g., a mammal), and the antigen expressed by the variant Listeria bacterium will conform to that tumor cell mutated antigen. By the term "tumor-related antigen" or “cancer-associated antigen”, as used herein, is meant an antigen which affects tumor grow th or metastasis in a host organism. The tumor-related antigen may be an antigen expressed by a tumor cell, or it may be an antigen which is expressed by a non-tumor cell, but which when so expressed, promotes the growth or metastasis of tumor cells. The types of tumor antigens and tumor-related antigens which may be introduced into Listeria by way of incorporating DNA encoding the same, include any known or heretofore unknown tumor antigen. In other examples, the “tumor-related antigen” or “cancer-associated antigen” has no effect on tumor growth or metastasis, but is used as a component of a Listeria vaccine because it is expressed specifically in the tissue (and tumor) from which the tumor is derived. In still other examples, the “tumor-related antigen” or “cancer-associated antigen” has no effect on tumor growth or metastasis, but is used as a component of a Listeria vaccine because it is selectively expressed in the tumor cell and not in any other normal tissues.

[0070] The heterologous antigen useful in vaccine development may be selected using knowledge available to the skilled artisan, and many antigenic proteins which are expressed by tumor cells or which affect tumor growth or metastasis or which are expressed by infectious agents are currently known. For example, viral antigens which may be considered as usefill as heterologous antigens include but are not limited to the nucleoprotein (NP) of influenza virus and the gag protein of human immunodeficiency virus (HIV). Other heterologous antigens include, but are not limited to, HIV env protein or its component parts gp!20 and gp41, HIV nef protein, and the HIV pol proteins, reverse transcriptase and protease. Still other heterologous antigens can be those related to hepatitis C virus (HCV), including but not limited to the El and E2 glycoproteins, as well as non-structural (NS) proteins, for example NS3. In addition, other viral antigens such as herpesvirus proteins may be useful. The heterologous antigens need not be limited to being of viral origin. Parasitic antigens, such as, for example, malarial antigens, are included, as are fungal antigens, bacterial antigens and tumor antigens.

[0071] As noted herein, a number of proteins expressed by tumor cells are also known and are of interest as heterologous antigens which may be inserted into the vaccine strain of the invention. These include, but are not limited to, the bcr / abl antigen in leukemia, human papilloma virus (HPV) E6 and E7 antigens of the oncogenic virus associated with cervical cancer, the MAGE1 and MZ2-E antigens in orassociated with melanoma, and the MVC-1 and HER-2 antigens in or associated with breast cancer. Suitable heterologous antigens include cancer-associated antigens such as, e.g., carcinoembryonic antigen (CEA); epithelial glycoprotein-2 (EGP-2); epithelial glycoprotein-40 (EGP-40); folate binding protein (FBP); fetal acetylcholine receptor; ganglioside antigen GD2; Her2 / neu; IL-13R-a2; kappa light chain; LeY; LI cell adhesion molecule; melanoma-associated antigen (MAGE); MAGE-A1; mesothelin; MUC1; NKG2D ligands; oncofetal antigen (h5T4); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); tumor-associate glycoprotein-72 (TAG-72); vascular endothelial grow th factor receptor-2 (VEGF-R2); and epidermal grow th factor receptor (EGFR) vIII polypeptide. Other coding sequences of interest include, but are not limited to, costimulatory molecules, immunoregulatory molecules, and the like.

[0072] Several approaches may be employed to express the heterologous antigen in Listeria species as wall be understood by one skilled in the art once armed with the present disclosure. In certain embodiments, genes encoding heterologous antigens are designed to either facilitate secretion of the heterologous antigen from the bacterium or to facilitate expression of the heterologous antigen on the Listeria cell surface. In certain embodiments, a fusion protein which includes the desired heterologous antigen and a secreted or cell surface protein of Listeria is employed. Listerial proteins which are suitable components of such fusion proteins include, but are not limited to, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase (PI-PLC). A fusion protein may be generated by ligating the genes w hich encode each of the components of the desired fusion protein, such that both genes are in frame with each other. Thus, expression of the ligated genes results in a protein comprising both the heterologous antigen and the Listerial protein. Expression of the ligated genes may be placed under the transcriptional control of a Listerial promoter / regulatory sequence such that expression of the gene is effected during growth and replication of the organism. Signal sequences for cell surface expression and / or secretion of the fused protein may also be added to genes encoding heterologous antigens in order to effect cell surface expression and / or secretion of the fused protein. When the heterologous antigen is used alone (i.e., in the absence of fused Listeria sequences), it may be advantageous to fuse thereto signal sequences for cell surface expression and / or secretion of the heterologous antigen. The procedures for accomplishing this are well known in the art of bacteriology and molecular biology.

[0073] In some instances, a subject variant Listeria bacterium of the present disclosure is attenuated. '‘Attenuation” and “attenuated” encompasses a Listeria bacterium that is modified to reduce virulence. The host can be a human or animal host, or an organ, tissue, or cell. The Listeria bacterium, to give a non-limiting example, can be attenuated to reduce binding to a host cell (e.g., human or nonhuman animal cell, such as a human or non-human mammalian cell), to reduce spread from one host cell to another host cell, to reduce extracellular growth, or to reduce intracellular growth in a host cell. Attenuation can be assessed by measuring, e.g.. an indicum or indicia of virulence, the LD50. the rate ofclearance from an organ, orthe competitive index (see, e.g., Auerb ch, et al. (2001) Infect. Immunity 69:5953-5957). Generally, an attenuation results in an increase in the LD50 (the lethal dose, 50%; the dose (number of bacteria) required to kill half the members of a tested population after a specified test duration) and / or an increase in the rate of clearance by at least 25%; more generally by at least 50%; most generally by at least 100% (2-fold); normally by at least 5-fold; more normally by at least 10-fold; most normally by at least 50-fold; often by at least 100-fold; more often by at least 500-fold; and most often by at least 1000-fold; usually by at least 5000-fold; more usually by at least 10,000-fold; and most usually by at least 50,000-fold; and most often by at least 100,000-fold. Attenuation can also be assessed by determining the number of colony-fonning units (CFUs).

[0074] In certain embodiments, attenuated Listeria according to the present disclosure are ones that exhibit a decreased virulence compared to a corresponding wild type strain in the Competitive Index Assay as described in Auerbach et aL, “Development of a Competitive Index Assay To Evaluate the Virulence of Listeria monocytogenes actA Mutants during Primary and Secondary Infection of Mice,” Infection and Immunity, September 2001, p. 5953-5957, Vol. 69, No. 9. In this assay, mice are inoculated with test and reference, e.g., wild-type, strains of bacteria. Following a period of time, e.g., 48 to 60 hours, the inoculated mice are sacrificed and one or more organs, e.g., liver, spleen, are evaluated for bacterial abundance. In these embodiments, a given bacterial strain is considered to be less virulent if its abundance in the spleen is at least about 50-fold, or more, such as 70-fold or more less than that observed with the corresponding wild-type strain, and / or its abundance in the liver is at least about 10- fold less, or more, such as 20-fold or more less than that observed with the corresponding wild-type strain.

[0075] In yet other embodiments, bacteria are considered to be less virulent if they show abortive replication in less than about 8 hours, such as less than about 6 hours, including less than about 4 hours, as determined using the assay described in Jones and Portnoy, Intracellular growth of bacteria. (1994b) Methods Enzymol. 236:463-467. In yet other embodiments, bacteria are considered to be attenuated or less virulent if, compared to wild-type, they fonn smaller plaques in the plaque assay employed in U.S. Patent No. 7,794,728 (the disclosure of which is herein incorporated by reference) where cells, such as murine L2 cells, are grown to confluency, e.g., in six-well tissue culture dishes, and then infected with bacteria. Subsequently. DME-agar containing gentamicin is added and plaques are grown for a period of time, e g., 3 days. Living cells are then visualized by adding an additional DME- agar overlay, e.g., containing neutral red (GIBCO BRL) and incubated overnight. In such an assay, the magnitude in reduction in plaque size observed with the attenuated mutant as compared to the wild-type is, in certain embodiments, 10%, including 15%, such as 25% or more.

[0076] Attenuated bacteria may include one or more different mutations which confer the attenuated phenotype, where mutations of interest include hly mutations and / or IplA mutations, e.g., asdescribed in U.S. Patent No. 7,794,728 (the disclosure of which is herein incorporated by reference); actA and / or internalin B (InlB) mutations, e.g., as reported in Dung et al., Clin. Cancer Res. (2012) 18:858-868); etc. Thus, in some embodiments, a variant Listeria bacterium of the present disclosure may further comprise a mutation in an actA gene and / or an inlB gene. In some cases, tire mutation comprises a deletion of all or a portion of the actA gene and / or the inlB gene.

[0077] A variant Listeria bacterium of the present disclosure may include one or more genetic modifications in addition to those described above, which one or more additional modifications provide for desirable qualities in the host cell, e.g., attenuation, enhanced immunogenicity, etc. Examples of such additional modifications include, but are not limited to, those described in PCT Published Application Nos.: WO 2014 / 106123; WO 2014 / 074635: WO 2009 / 143085; WO 2008027560 WO 2008066774; WO 2007117371; WO 2007103225: WO 2005071088; WO 2003102168; WO 2003 / 092600;WO / 2000 / 009733; and WO 1999 / 025376; the disclosures of which applications are herein incorporated by reference. A variant Listeria bacterium of the present disclosure may include, in addition to the modifications described above, an Lm-RIID (L. monocytogenes recombinase-induced intracellular death) mutation. See, e.g., USPN 9,511,129.

[0078] Additional embodiments of a variant Listeria bacterium of the present disclosure may comprise any combination of the mutations / modifications described above.

[0079] Variant Listeria bacteria as described herein may be generated using a variety of different protocols. As such, generation of the subject Listeria bacterium may be accomplished in a number of ways that are well known to those of skill in the art, including deletion mutagenesis, insertion mutagenesis, point mutations, mutagenesis which results in the generation of frameshift mutations, mutations which effect premature termination of a protein, and 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. Representative protocols of different ways to generate bacteria according to the present invention are provided in the Experimental Section, below.

[0080] Variant Listeria bacteria genetically modified to further comprise one or more heterologous nucleic acids as described herein may be generated using a variety of different protocols. The introduction of one or more heterologous nucleic acids into a variant Listeria bacterium may be accomplished, for example, by the creation of a recombinant variant Listeria bacterium in which the one or more heterologous nucleic acids are harbored on a vector, such as a plasmid for example, which plasmid is maintained and expressed in the variant Listeria bacterium, where expression of the heterologous nucleotide sequences is under the control of prokaryotic promoter / regulatory sequences. Alternatively, the one or more heterologous nucleic acids may be stably integrated into the variant Listeria bacterium chromosome (i.e., the bacterial genome) by employing, for example, transposonmutagenesis, homologous recombination, or integrase mediated site-specific integration (as described in USSN 10 / 136,860, the disclosure of which is herein incorporated by reference).

[0081] The Listeria cell (e.g., parental Listeria cell) that is used to generate a variant Listeria bacterium of the present disclosure can be any one of a number of different Listeria spp. Listeria spp of interest include, but are not limited to: L. fleischmannii, L. innocua. L. ivanovii. L. marthii, L. monocytogenes, L. rocourtiae, L. seeligeri, L. weihenstephanensis, and A. welshimeri. Thus, strains of Listeria other than L. monocytogenes may be a parental Listeria cell of the present disclosure. In certain cases, the Listeria strain is L. monocytogenes.COMPOSITIONS AND KITS

[0082] The present disclosure provides a composition comprising a subject variant Listeria bacterium. The present disclosure provides a composition comprising: a) a subject variant Listeria bacterium; and b) a multispecific antibody. A multispecific antibody is, in some instances, a bispecific T cell engaging (BiTE) antibody. A multispecific antibody can include a first antigen-binding site specific for a cancer-associated antigen and a second antigen-binding site specific for a T cell (e.g., a mucosal- associated invariant T (MAIT) cell, a y / 5 T cell, a CD8+cytotoxic T cell, a natural killer (NK) cell). In some embodiments, the T cell is a y / 6 T cell. A composition of the present disclosure can comprise, in addition to a variant Listeria bacterium and a multispecific antibody, one or more of: a salt (e.g., NaCl, MgCL. KC1, MgSCL, etc.), a buffering agent, and the like. In some instances, a composition of the present disclosure comprises, in addition to a variant Listeria bacterium and multispecific antibody of the present disclosure, saline.

[0083] Tire present disclosure also provides an immunogenic composition (also referred to herein as a “a vaccine composition”) comprising a variant Listeria bacterium of the present disclosure. In some embodiments, an immunogenic composition of the present disclosure can comprise: a) a subject variant Listeria bacterium; and b) an antigen or a nucleic acid encoding thereof. Suitable antigens include, but are not limited to, cancer-associated antigens, pathogen-associated antigens (e.g., viral antigens, pathogenic protozoan antigens, and the like). In certain embodiments, a subject immunogenic composition may additionally comprise pharmaceutically acceptable diluents or excipients. Suitable diluents include, for example, sterile, distilled water, saline, phosphate buffered solution, and the like. Suitable excipients are also well known to those skilled in the art and may be selected, for example, from A. Wade and P.J. Weller, eds., Handbook of Pharmaceutical Excipients (1994) The Pharmaceutical Press: London.

[0084] Hie present disclosure additionally provides kits comprising a unit dose of a subject immunogenic composition comprising a variant Listeria bacterium of the present disclosure. A unit dose of the variant Listeria can be in a range of from 104to 1010bacteria per dose. For example, in some cases.an “effective amount” of a variant Listeria of the present disclosure (a variant Listeria bacterium comprising a mutation in a purA gene encoding adenylosuccinate synthetase, wherein the variant Listeria bacterium is an adenosine auxotroph) is in a range of from 104to 5 x 104, from 5 x 104to 105, from 105to 5 x 105, from 5 x 105to 106. from 106to 5 x 106, from 5 x 106to 107, from 107to 5 x 107, from 5 x 107to 10s. from 10sto 109, or from 109to 1010, bacteria per unit dose. In some embodiments, the unit dose is an oral dose. An oral dose of a subject kit may be in any suitable form. Examples of suitable oral dosage forms include, without limitation, capsules, liquid solutions, suspensions, and elixirs. In some embodiments, the unit dose is injectable. An injectable dose of a subject kit may be in any suitable form (e.g., a sterile liquid for formulations).

[0085] In certain embodiments, a subject kit of the present disclosure further comprises a recombinant expression vector (e.g., a plasmid) comprising a nucleotide sequence encoding a heterologous polypeptide. In some embodiments, the heterologous peptide is an antigen. Suitable antigens include, but are not limited to, cancer-associated antigens, pathogen-associated antigens (e.g. viral antigens, pathogenic protozoan antigens, and the like). In some cases, the antigen is a cancer- associated antigen.METHODS OF INDUCING AN IMMUNE RESPONSE

[0086] The present disclosure provides methods of inducing an immune response in an individual, the methods comprising administering to the individual an effective amount of a subject immunogenic composition (i.e., “vaccine composition”).

[0087] Hie subject bacteria (variant Listeria bacterium comprising a mutation in a pur A gene encoding adenylosuccinate synthetase, wherein the variant Listeria bacterium is an adenosine auxotroph) find use as vaccines (also referred to herein as an “immunogenic composition”). The vaccines of the present disclosure are administered to a vertebrate by contacting the vertebrate with a sub-lethal dose of the attenuated Listeria vaccine, where contact typically includes administering the vaccine to the host. In some embodiments, the bacteria are provided in a pharmaceutically acceptable formulation. Administration can be oral, parenteral, intranasal, intramuscular, intradermal, intraperitoneal, intravascular, subcutaneous, direct vaccination of lymph nodes, administration by catheter or any one or more of a variety of well-known administration routes. In farm animals, for example, the vaccine may be administered orally by incorporation of the vaccine in feed or liquid (such as water). It may be supplied as a lyophilized powder, as a frozen formulation or as a component of a capsule, or any other convenient, pharmaceutically acceptable formulation that preserves the antigenicity of the vaccine. Any one of a number of well-known pharmaceutically acceptable diluents or excipients may be employed in the vaccines of the invention. Suitable diluents include, for example, sterile, distilled water, saline, phosphate buffered solution, and the like. The amount of the diluent may van' widely, as those skilled inthe art will recognize. Suitable excipients are also well known to those skilled in the art and may be selected, for example, from A. Wade and P.J. Weller, eds., Handbook of Pharmaceutical Excipients (1994) The Pharmaceutical Press: London. The dosage administered may be dependent upon the age, health and weight of the individual, the type of individual, and the existence of concurrent treatment, if any.

[0088] In some cases, a subject variant Listeria bacterium is administered at a dose in a range of 100-10,000 bacteria (e.g., 100-7,500, 100-5,000, 100-4,000, 100-3,000, 100-2,500, 100-2,000, 100-1,500, 100-1,000, 500-10,000, 500-7,500, 500-5,000, 500-4,000, 500-3,000, 500-2,500, 500-2,000, 500-1.500, 500-1,000, 1,000-10,000, 1,000-7,500, 1,000-5,000, 1,000-4,000, 1,000-3,000, 1,000-2,500. 1,000-2,000, 1,000-1,500. 1,500-10,000, 1,500-7,500, 1,500-5,000, 1,500-4,000, 1,500-3.000, 1,500-2.500, 1,500-2,000. 2,000-10,000, 2,000-7.500, 2,000-5,000. 2,000-4,000, 2.000-3,000, 2,000-2,500. 2,500-10,000, 2,500-7,500, 2,500-5,000, 2,500-4,000, or 2,500-3,000). In some cases, the dose is about 1,000 bacteria. In some cases, the dose is about 1,500 bacteria. In some cases, the dose is about 2,000 bacteria. In some cases, the dose is about 2,500 bacteria. In some cases, the dose is about 2,000 bacteria. In some cases, the dose is about 3,000 bacteria.

[0089] Tire vaccines can be employed in dosage forms such as capsules, liquid solutions, suspensions, or elixirs, for oral administration, or sterile liquid for formulations such as solutions or suspensions for parenteral, intranasal intramuscular, or intravascular use. In accordance with the invention, the vaccine may be employed, in combination with a pharmaceutically acceptable diluent, as a vaccine composition, useful in immunizing an individual against infection from a selected organism or virus or with respect to a tumor, etc. Immunizing an individual means providing the individual with at least some degree of therapeutic or prophylactic immunity against selected pathogens, cancerous cells, etc.

[0090] The individual may be any human and non-human animal susceptible to infection with the selected organism. The subject vaccines will find particular use with vertebrates such as mammals (including humans and non-human mammals); and with domestic animals. Domestic animals include domestic fowl, bovine, porcine, ovine, equine, caprine, canine, feline, Leporidate (such as rabbits), or other non-human animal.

[0091] The subject vaccines find use in methods for eliciting or boosting a cellular immune response, e.g., a y / 5 T cell response to a selected agent, e.g., pathogenic organism, tumor, etc., in a vertebrate, where such methods include administering an effective amount of the Listeria vaccine. Hie subject vaccines find use in methods for eliciting in a vertebrate an innate immune response that augments the antigen-specific immune response. Furthermore, the vaccines of the present invention may be used for treatment post-exposure or post diagnosis. In general, the use of vaccines for post-exposure treatment would be recognized by one skilled in the art. for example, in the treatment of rabies andtetanus. The same vaccine of the present invention may be used, for example, both for immunization and to boost immunity after exposure. Alternatively, a different vaccine of the present invention may be used for post-exposure treatment, for example, such as one that is specific for antigens expressed in later stages of exposure. As such, the subject vaccines find use as both prophylactic and therapeutic vaccines to induce immune responses that are specific for antigens that are relevant to various disease conditions.

[0092] In certain embodiments of the methods, a subject immunogenic composition comprises a variant Listeria bacterium that is genetically modified to comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway, wherein the variant Listeria bacterium grows aerobically. In some cases, the Listeria bacterium additionally comprises a loss of function mutation in an endogenous LytB gene. In said embodiments of the methods, the immune response is induced to a phosphoantigen produced by the variant Listeria bacterium. In some cases, the phosphoantigen is (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP).

[0093] In certain embodiments of the methods, a subject immunogenic composition comprises a variant Listeria bacterium that is genetically modified to comprise a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous polypeptide. In said embodiments of the methods, the immune response is induced to the heterologous polypeptide. In some cases, the heterologous polypeptide is an antigen as discussed herein. Suitable antigens include, but are not limited to, cancer-associated antigens, pathogen-associated antigens (e.g. viral antigens, pathogenic protozoan antigens, and the like). In some embodiments, the antigen is a cancer-associated antigen.

[0094] Additional embodiments of methods include administering to the individual an effective amount of an immunogenic composition comprising a variant Listeria bacterium of the present disclosure comprising any combination of the mutations / modifications described above.

[0095] In certain embodiments, said immune response comprises a T cell response. In some instances, the immune response comprises a mucosal -associated invariant T (MAIT) cell, a y / 5 T cell, a CD8+cytotoxic T cell, and / or a natural killer (NK) cell response. In some embodiments, said immune response comprises a y / 5 T cell response.

[0096] Tire subject vaccines find use in vaccination applications as described in PCT Published Application Nos.: WO 2014 / 106123; WO 2014 / 074635; WO 2009 / 143085; WO 2008027560 WO 2008066774; WO 20071 17371 ; WO 2007103225; WO 2005071088; WO 2003102168; WO 2003 / 092600; WO / 2000 / 009733; and WO 1999 / 025376; the disclosures of which applications are herein incorporated by reference.

[0097] Tire subject bacterial strains (variant Listeria bacterium comprising a mutation in a pur A gene encoding adenylosuccinate synthetase, wherein the variant Listeria bacterium is an adenosine auxotroph) also find use as immunopotentiating agents, i.e., as adjuvants. In such applications, thesubject attenuated bacteria may be administered in conjunction with an immunogen, e.g., a tumor antigen, modified tumor cell, etc., according to methods known in the art where live bacterial strains are employed as adjuvants. See, e.g., Berd et al., Vaccine 2001 Mar 21; 19( 17-19):2565-70.

[0098] In some embodiments, the bacterial strains are employed as adjuvants by chemically coupling to a sensitizing antigen. The sensitizing antigen can be any antigen of interest, where representative antigens of interest include, but are not limited to: viral agents, e.g.. Herpes simplex virus; malaria parasite; bacteria, e.g., staphylococcus aureus bacteria, diphtheria toxoid, tetanus toxoid, shistosomula; tumor cells, e.g. CAD2mammary adenocarcinomia tumor cells, and hormones such as thyroxine T , triiiodothyronine Ts, and cortisol. Tire coupling of the sensitizing antigen to the immunopotentiating agent can be accomplished by means of various chemical agents having two reactive sites such as, for example, bisdiazobenzidine, glutaraldehyde, di-iodoacetate. and diisocyanates, e.g., m-xylenediisocyanate and toluene-2,4-diisocyanate. Use of Listeria spp. as adjuvants is further described in U.S. Patent No. 4,816,253; the disclosure of which is herein incorporated by reference.Examples of Non-Limiting Aspects of the Disclosure

[0099] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[0100] Aspect 1. A variant Listeria bacterium comprising a mutation in a purA gene encoding adenylosuccinate synthetase, wherein the variant Listeria bacterium is an adenosine auxotroph.

[0101] Aspect 2. The variant Listeria bacterium of aspect 1, wherein the variant Listeria does not grow extracellularly in a mammal.

[0102] Aspect 3. Hie variant Listeria bacterium of aspect 1 or aspect 2, wherein the mutation is a deletion of all or a portion of the pur A gene.

[0103] Aspect 4. The variant Listeria bacterium of any one of aspects 1-3. wherein the variant Listeria bacterium requires from 100 pM to 500 pM adenosine supplementation to grow.

[0104] Aspect 5. The variant Listeria bacterium of any one of aspects 1-4, wherein the variant Listeria bacterium further comprises a deletion of all or a portion of an endogenous ribC gene and / or an endogenous ribF gene.

[0105] Aspect 6. The variant Listeria bacterium of any one of aspects 1-5, wherein the variant Listeria bacterium is genetically modified to comprise one or more heterologous nucleic acids.

[0106] Aspect 7. The variant Listeria bacterium of aspect 6, wherein the one or more heterologous nucleic acids are integrated into the bacterial genome.

[0107] Aspect 8. The variant Listeria bacterium of aspect 6 or 7, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway, wherein the variant Listeria bacterium grows aerobically.

[0108] Aspect 9. The variant Listeria bacterium of aspect 8, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding IspE, GcpE, and IspA polypeptides.

[0109] Aspect 10. Tire variant Listeria bacterium of aspect 8, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding Dxs, IspD, IspF, IspE, GcpE, and IspA polypeptides.

[0110] Aspect 11. The variant Listeria bacterium of any one of aspects 8-10, further comprising a loss of function mutation in an endogenous LytB gene.

[0111] Aspect 12. Tire variant Listeria bacterium of any one of aspects 8-11, wherein the variant Listeria bacterium further comprises one or more additional mutations that confers enhanced function of the non-mevalonate pathway under aerobic conditions.

[0112] Aspect 13. The variant Listeria bacterium of aspect 12. wherein tire one or more additional mutations that confers enhanced function of the non-mevalonate pathway under aerobic conditions comprises a mutation in an endogenous gene selected from lmol694,fur,flgE, ribF. uracil- DNA glycosylase, DNA-directed RNA polymerase subunit a, UDP-N-acetylglucosamine 1- carboxyvinyltransferase, and heptaprenyl diphosphate synthase component I.

[0113] Aspect 14. The variant Listeria bacterium of any one of aspects 6-13. wherein at least one of the one or more heterologous nucleic acids comprises a nucleotide sequence encoding an antigen polypeptide.

[0114] Aspect 15. Tire variant Listeria bacterium of aspect 14, wherein the antigen is a cancer- associated antigen.

[0115] Aspect 16. The variant Listeria bacterium of any one of aspects 1-15, wherein said variant Listeria bacterium is a variant Listeria monocytogenes bacterium.

[0116] Aspect 17. The variant Listeria bacterium of any one of aspects 1-16. wherein the variant Listeria bacterium further comprises one or more additional mutations that confers an attenuated phenotype on the bacterium and / or one or more additional mutations that provide a growth advantage.

[0117] Aspect 18. Tire variant Listeria bacterium of aspect 17, wherein the one or more additional mutations that confers an attenuated phenotype comprises a mutation in a gene selected from actA and inlB, and wherein the one or more additional mutations that provide a growth advantage comprises a mutation in an eetB gene.

[0118] Aspect 19. A composition comprising a variant Listeria bacterium of any one of aspects 1-18.

[0119] Aspect 20. The composition of aspect 19. further comprising a multispecific antibody that comprises: i) a first antigen-binding site specific for a cancer-associated antigen; and ii) a second antigen-binding site specific for a T cell.

[0120] Aspect 21. The composition of aspect 20, wherein the T cell is a mucosal-associated invariant T (MAIT) cell, a y / 8 T cell, a CD8+T cell, or a natural killer (NK) cell.

[0121] Aspect 22. An immunogenic composition comprising the variant Listeria bacterium of any one of aspects 1-18.

[0122] Aspect 23. A method of inducing an immune response in an individual, the method comprising administering to the individual an effective amount of an immunogenic composition according to aspect 22.

[0123] Aspect 24. The method of aspect 23, wherein the variant Listeria bacterium is genetically modified to comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway, wherein the variant Listeria bacterium grows aerobically, and wherein said immune response is induced to a phosphoantigen produced by the variant Listeria bacterium.

[0124] Aspect 25. The method of aspect 24, wherein the phosphoantigen is (E)-4-hydroxy-3- methyl-but-2-enyl pyrophosphate (HMBPP).

[0125] Aspect 26. Tire method of aspect 23, wherein the variant Listeria bacterium is genetically modified to comprise a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous polypeptide, and wherein said immune response is induced to the heterologous polypeptide.

[0126] Aspect 27. The method of aspect 26, wherein the heterologous polypeptide is an antigen.

[0127] Aspect 28. Tire method of aspect 27, wherein the antigen is a cancer-associated antigen.

[0128] Aspect 29. Tire method of any one of aspects 23-28, wherein said immune response comprises a gamma-delta T cell response.

[0129] Aspect 30. A kit comprising a unit dose of the immunogenic composition of aspect 22.

[0130] Aspect 31. The kit of aspect 30, wherein the unit dose is an oral dose.

[0131] Aspect 32. The kit of aspect 30, wherein the unit dose is injectable.

[0132] Aspect 33. Tire kit of any one of aspects 30-32, further comprising a recombinant expression vector comprising a nucleotide sequence encoding a heterologous polypeptide.

[0133] Aspect 34. The kit of aspect 33, wherein the heterologous polypeptide is an antigen.

[0134] Aspect 35. The kit of aspect 34, wherein the antigen is a cancer-associated antigen.EXAMPLES

[0135] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl. picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s): aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s): i.m., intramuscularly ): i.p., intraperitoneal(ly): s.c., subcutaneous(ly); and the like.Example 1

[0136] As a facultative intracellular pathogen, L. monocytogenes can thrive in extracellular environments during systemic infection, causing tremendous concern for the safe administration of this bacterium in humans. The in vivo extracellular growth niches of L. monocytogenes include, but are not limited to, blood and gallbladders. An engineered strain losing the ability to thrive in these niches would significantly improve the safe administration of L. monocytogenes . To accomplish this, nutrients that were limited in the extracellular environment were explored and it was found that the average concentration of purine bases and nucleosides in plasma and other extracellular fluids was low, generally in the range of 0.4-6 pM. More importantly, de novo nucleotide biosynthesis was identified to be the single most critical metabolic function required for bacterial growth in human blood. Based on these findings, it was hypothesized that purine auxotrophic L. monocytogenes would not grow in extracellular environments and would thus greatly improve the safety profile of L. monocytogenes therapeutics.

[0137] A L. monocytogenes auxotroph of adenosine, a purine nucleoside that is limited in blood and healthy cells but abundant in the tumor microenvironment, was generated. The adenosine auxotroph was constructed by generating an in-frame deletion of the gene encoding adenylosuccinate synthetase (purA) for adenyl species biosynthesis. The strain is referred to as tspurA hereafter. Tire tspurA mutants were adenosine auxotrophic as they did not replicate in the chemically defined medium without adenosine supplementation (Fig. 1). The minimal concentration of exogenous adenosine to support optimal growth of the auxotrophs was between 100 pM and 500 pM (Fig. 1), a concentration that does not normally exist in extracellular environments. Consistent with the hypothesis, the L. monocytogenes ApnrA mutants did not grow in extracellular conditions, as demonstrated in defibrinated sheep blood and human serum (Fig. 2).

[0138] Next, the entry and growth of L. monocytogenes strain in host cells was examined. In murine bone marrow-derived macrophages (BMMs), the majority of fpurA mutants, 70% relative to WT, escaped from phagosome and entered host cytosol (Fig. 3A), however, the cytosolic ApnrA mutants failed to grow (Fig. 3B). The restricted growth in the host cytosol may favor the induction of cell- mediated immunity (CMI) since the chance of cell death of APCs is reduced because of a lower bacterial burden per cell. Similar to the observations in the chemically defined media (Fig. 1), replication of the purA mutants in BMMs could be restored only if adenosine was supplemented at a concentration far higher than the physiologically relevant concentration in vivo (Fig. 3B).

[0139] The virulence, as well as the immunogenicity, of the purA mutants during in vivo infection in mice was then determined. Since the growth of AyntrA was restricted in blood and cells, it was reasonable to observe a severe attenuation of the strain during systemic infection of mice. The mutants presented a 2-logio defect compared to WT in bacterial burdens in the spleens and a 4-logio attenuation in the livers (Fig. 4A). The purA strain was compared with tsactA, the primary attenuating mutation contained in live, attenuated L. monocytogenes cancer vaccine strains that was investigated in previous preclinical and clinical trials. While tire two mutants behaved similarly with regard to CFUs in the livers and spleens, the purA strain was less detectable in the gallbladders (Fig. 4B), another in vivo extracellular growth niche of L. monocytogenes besides blood. Although attenuated in mouse spleens, the ApurA mutants grew more than 10-fold during the first 24 hours of infection (Fig. 4C), that likely? contributed to the establishment of adaptive immunity. Therefore, it was tested if the invented strain was able to induce robust CMI, especially when administered at a low dose which may be desired for safetyreasons. Tire ItspurA mutants administered at a very low dose (1000 bacteria) almost fully protected the mice from a second lethal dose challenge of WT L. monocytogenes, which was better protection than observed with the tsactA strain (Fig. 5A). Consistently, the purA mutants elicited significantly more antigen-specific CD8+cytotoxic T-cells than the Ar / cM mutant (Fig. 5B).

[0140] Another facet of this strain is that because of the high concentration of adenosine in tumor microenvironments, the purA strain may7survive in tumors. The purA mutation can also be combined with other genetic / metabolic manipulations (e.g., actA / inlB and ribC / ribF deletions, the suicidal L. monocytogenes strain, or the killed but metabolically active strain, to further improve the safety of therapeutic strains. The pur A strain can be combined with multi-specific antibodies that specifically drive T-cells including Gamma-Delta (y8) T cells or Mucosal-Associated Invariant T (MAIT) cells to tumors.

[0141] REFERENCES1. Radoshevich L. Cossart P. 2018. Listeria monocytogenes: towards a complete picture of its physiology and pathogenesis. Nat Rev Microbiol 16:32-46.2. Buchanan RL, Gorris LGM, Hayman MM, Jackson TC, Whiting RC. 2017. A review of Listeria monocytogenes: an update on outbreaks, virulence, dose-response, ecology, and risk assessments. Food Control 75: 1-13.3. Portnoy DA, Auerbuch V, Glomski IJ. 2002. Hie cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity. J Cell Biol 158:409- 414.4. Chavez-Arroyo A, Portnoy? DA. 2020. Why is Listeria monocytogenes such a potent inducer of CD8+ T-cells? Cell Microbiol 22:el3175.5. Flickinger JC, Rodeck U, Snook AE. 2018. Listeria monocytogenes as a vector for cancer immunotherapy: Current understanding and progress. Vaccines 6:48.6. Oladejo M. Paterson Y, Wood LM. 2021. Clinical experience and recent advances in the development of Listeria-based tumor immunotherapies. Front Immunol 12:642316.7. Sacco JJ, Evans M, Harrington KJ, Man S, Powell N, Shaw RJ, Jones TM. 2016. Systemic listeriosis following vaccination with the attenuated Listeria monocytogenes therapeutic vaccine, ADXS11-001. Hum Vaccines Immunother 12: 1085-1086.8. Zhang T, Abel S, Wiesch PA, Sasabe J, Davis BM. Higgins DE, Waldor MK. 2017. Deciphering the landscape of host barriers to Listeria monocytogenes infection. Proc Natl Acad Sci U S A 114:6334-6339.9. Louie A, Zhang T, Becattini S, Waldor MK, Portnoy DA. 2019. A multiorgan trafficking circuit provides purifying selection of Listeria monocytogenes virulence genes. mBio 10:e02948-19.10. Traut TW. 1994. Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 140: 1-22.11. Samant S, Lee H, Ghassemi M, Chen J, Cook JL, Mankin AS, Neyfakh AA. 2008. Nucleotide biosynthesis is critical for growth of bacteria in human blood. PLoS Pathog 4:e37.12. Hasko G, Antonioli L, Cronstein BN. 2018. Adenosine metabolism, immunity and joint health. Biochem Pharmacol 151:307-313.13. Sorrentino C. Morello S. 2017. Role of adenosine in tumor progression: focus on A2B receptor as potential therapeutic target. J Cancer Metastasis Treat 3: 127.14. Vaupel P, Mayer A. 2016. Hypoxia-driven adenosine accumulation: A crucial microenvironmental factor promoting tumor progression. Adv Exp Med Biol 876: 177-183.15. Brocksted DG, Giedlin MA, Leong ML, Bahjat KS, Gao Y, Luckett W, Liu W, Cook DN. Portnoy DA, Dubensky TW. 2004. Listeria-based cancer vaccines that segregate immunogenicity from toxicity. Proc Natl Acad Sci U S A 101: 13832-13837.16. Alvarez CL, Troncoso MF, Espelt M V. 2022. Extracellular ATP and adenosine in tumor microenvironment: roles in epithelial-mesenchymal transition, cell migration, and invasion. J Cell Physiol 237:389-400.17. Rivera-Lugo R. Light SH, Garelis NE, Portnoy DA. 2022. RibU is an essential determinant of Listeria pathogenesis that mediates acquisition of FMN and FAD during intracellular growth. Proc Natl Acad Sci U S A 119:e2122173119.18. Hanson WG, Benanti EL, Lemmens EE, Liu W, Skoble J, Leong ML, Rae CS, Fasso M, Brockstedt DG, Chen C, Portnoy DA, Dubensky TW, Lauer P. 2019. A potent and effective suicidal Listeria vaccine platform. Infect Immun 87:e00144-19.19. Brockstedt DG. Bahjat KS, Giedlin MA, Liu W, Leong M, Luckett W, Gao Y, Schnupf P. Kapadia D. Castro G, Lim JYH, Sampson-Johannes A. Herskovits AA, Stassinopoulos A, Bouwer HGA, Hearst JE, Portnoy DA, Cook DN, Dubensky TW. 2005. Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity. Nat Med 11:853-860.20. Elshiaty M. Schindler H, Christopoulos P. 2021. Principles and current clinical landscape of multispecific antibodies against cancer. Int J Mol Sci 22:5632.21. Zhang Y. Anaya-Sanchez A, Portnoy DA. 2022. para-aminobenzoic acid biosynthesis is required for Listeria monocytogenes growth and pathogenesis. Infect Immun 9:e0020722.

[0142] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMSWhat is claimed is:

1. A variant Listeria bacterium comprising a mutation in a pur A gene encoding adenylosuccinate synthetase, wherein the variant Listeria bacterium is an adenosine auxotroph.

2. The variant Listeria bacterium of claim 1, wherein the variant Listeria does not grow extracellularly in a mammal.

3. The variant Listeria bacterium of claim 1 or claim 2, wherein the mutation is a deletion of all or a portion of the pur A gene.

4. The variant Listeria bacterium of any one of claims 1-3, wherein the variant Listeria bacterium requires from 100 pM to 500 pM adenosine supplementation to grow.

5. The variant Listeria bacterium of any one of claims 1-4, wherein the variant Listeria bacterium further comprises a deletion of all or a portion of an endogenous ribC gene and / or an endogenous ribF gene.

6. Hie variant Listeria bacterium of any one of claims 1-5, wherein the variant Listeria bacterium is genetically modified to comprise one or more heterologous nucleic acids.

7. The variant Listeria bacterium of claim 6, wherein the one or more heterologous nucleic acids are integrated into the bacterial genome.

8. The variant Listeria bacterium of claim 6 or 7, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway, wherein the variant Listeria bacterium grows aerobically.

9. The variant Listeria bacterium of claim 8, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding IspE, GcpE, and IspA polypeptides.

10. The variant Listeria bacterium of claim 8, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding Dxs. IspD. IspF. IspE. GcpE, and IspA polypeptides.

11. The variant Listeria bacterium of any one of claims 8-10, further comprising a loss of function mutation in an endogenous LytB gene.

12. The variant Listeria bacterium of any one of claims 8-11. wherein the variant Listeria bacterium further comprises one or more additional mutations that confers enhanced function of the nonmevalonate pathway under aerobic conditions.

13. The variant Listeria bacterium of claim 12, wherein the one or more additional mutations that confers enhanced function of the non-mevalonate pathway under aerobic conditions comprises a mutation in an endogenous gene selected from lmol694, fur, flgE, ribF. uracil-DNA glycosylase. DNA- directed RNA polymerase subunit a, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, and heptaprenyl diphosphate synthase component I.

14. Hie variant Listeria bacterium of any one of claims 6-13, wherein at least one of the one or more heterologous nucleic acids comprises a nucleotide sequence encoding an antigen polypeptide.

15. The variant Listeria bacterium of claim 14, wherein the antigen is a cancer-associated antigen.

16. The variant Listeria bacterium of any one of claims 1-15, wherein said variant Listeria bacterium is a variant Listeria monocytogenes bacterium.

17. The variant Listeria bacterium of any one of claims 1-16, wherein the variant Listeria bacterium further comprises one or more additional mutations that confers an attenuated phenotype on the bacterium and / or one or more additional mutations that provide a grow th advantage.

18. The variant Listeria bacterium of claim 17, wherein the one or more additional mutations that confers an attenuated phenotype comprises a mutation in a gene selected from actA and inlB. and wherein the one or more additional mutations that provide a grow th advantage comprises a mutation in an eetB gene.

19. A composition comprising a variant Listeria bacterium of any one of claims 1-18.

20. The composition of claim 19, further comprising a multispecific antibody that comprises: i) a first antigen-binding site specific for a cancer-associated antigen; and ii) a second antigen-binding site specific for a T cell.

21. The composition of claim 20. wherein the T cell is a mucosal-associated invariant T (MAIT) cell, a y / 5 T cell, a CD8+T cell, or a natural killer (NK) cell.

22. An immunogenic composition comprising the variant Listeria bacterium of any one of claims 1-18.

23. A method of inducing an immune response in an individual, the method comprising administering to the individual an effective amount of an immunogenic composition according to claim 22.

24. Hie method of claim 23, wherein the variant Listeria bacterium is genetically modified to comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway, wherein the variant Listeria bacterium grows aerobically, and wherein said immune response is induced to a phosphoantigen produced by the variant Listeria bacterium.

25. Hie method of claim 24, wherein the phosphoantigen is (E)-4-hydroxy-3-methyl-but-2- enyl pyrophosphate (HMBPP).

26. The method of claim 23, wherein the variant Listeria bacterium is genetically modified to comprise a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous polypeptide, and wherein said immune response is induced to the heterologous polypeptide.

27. Hie method of claim 26, wherein the heterologous polypeptide is an antigen.

28. The method of claim 27, wherein the antigen is a cancer-associated antigen.

29. The method of any one of claims 23-28, wherein said immune response comprises a gamma-delta T cell response.

30. A kit comprising a unit dose of the immunogenic composition of claim 22.

31. The kit of claim 30, wherein the unit dose is an oral dose.

32. Tire kit of claim 30, wherein the unit dose is injectable.

33. The kit of any one of claims 30-32, further comprising a recombinant expression vector comprising a nucleotide sequence encoding a heterologous polypeptide.

34. The kit of claim 33, wherein the heterologous polypeptide is an antigen.

35. The kit of claim 34, wherein the antigen is a cancer-associated antigen.

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