Genetically modified listeria and methods of use thereof

Abstract

The present disclosure provides variant Listeria bacteria that 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 bacteria grow aerobically. The present disclosure provides compositions comprising a variant Listeria bacterium of the present disclosure and a multispecific antibody. Also provided are immunogenic compositions comprising a variant Listeria bacterium of the present disclosure. 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

GENETICALLY MODIFIED LISTERIA AND METHODS OF USE THEREOFCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 516,781 filed July 31, 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 and AI063302 awarded by the National Institutes of Health. The 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-488PRV SEQ LIST” created on July 28, 2023 and having a size of 9,151 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.INTRODUCTION

[0004] Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that is highly amenable for genetic manipulation and the cell biology of its infection has been extensively characterized. Upon phagocytosis by antigen-presenting cells, L. monocytogenes escapes from phagosomes and grows directly in the host cell cytosol where it secretes antigens and metabolites. 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. While the generation of robust antigen-specific CD8+ T-cell responses leading to efficient antitumor immunity have been observed in preclinical trials in mice, the same is not seen in clinical trials with humans.

[0005] y8 T-cells represent a unique T-cell population constituting between 1 to 5% of all T-cells in human blood and have important roles in generating an immune response against tumors and pathogens. Unlike aP T-cells, y8 T-cell recognition of target cells is independent of major histocompatibility complex-restricted antigen presentation and encompasses characteristics of both innate and adaptive immunity. Vy9V82 T-cells are a major subset of y8 T-cells in human peripheral blood and constitute 50 to 95% of all y8 T-cells in circulation. Upon encountering phosphoantigen,Vy9V82 T-cells undergo clonal expansion and activation that includes production of pro- inflammatory cytokines and adoption of a cytotoxic phenotype capable of killing cancer cells.

[0006] The most potent Vy9V62 T-cell phosphoantigen is the bacterial metabolite (E)-4-hydroxy- 3-methyl-but-2-enyl pyrophosphate (HMBPP). HMBPP is an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis, which is unique to bacteria and some parasites, but absent in mammals. Many pathogens including L. monocytogenes , Mycobacterium tuberculosis and Salmonella enterica use the non-mevalonate pathway to produce HMBPP, which induces expansion and activation of V' / 9V82 T-cells as an immune countermeasure. Although most organisms encode either the mevalonate or non-mevalonate pathway of isoprenoid biosynthesis, L. monocytogenes is one of the very few bacteria that encodes both pathways.SUMMARY

[0007] The present disclosure provides variant Listeria bacteria that 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 the presence or absence of a functional mevalonate pathway. The present disclosure provides compositions, including immunogenic compositions, comprising a variant Listeria bacterium of the present disclosure. 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.

[0008] The present disclosure provides variant Listeria bacteria that 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 the presence or absence of a functional mevalonate pathway. In some embodiments, the one or more heterologous nucleic acids comprise nucleotide sequences encoding LytB, IspE, GcpE, and IspA polypeptides. In some embodiments, the one or more heterologous nucleic acids comprise nucleotide sequences encoding Dxs, IspD, IspF, LytB, IspE, GcpE, and IspA polypeptides. In some cases, the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more identity to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. In some cases, the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more identity to the amino acid sequences of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDN0:6, and SEQ ID N0:7. 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 coding for Dxs, IspD, IspF, IspE, GcpE, and IspA polypeptides. In some cases, the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more identity to the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. In some cases, the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more identity to the amino acid sequences of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. In some embodiments, the one or more heterologous nucleic acids comprise nucleotide sequences encoding GcpE, LytB, and IspE polypeptides. In some cases, the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more identity to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0009] In some embodiments, a variant Listeria bacterium of the present disclosure comprises a loss of function mutation in a hmgR gene. In some cases, the loss of function mutation comprises a deletion of all or a portion of the hmgR gene. In some embodiments, a variant Listeria bacterium of the present disclosure comprises a loss of function mutation in a lmol694 gene and a fur gene. In some cases, the loss of function mutation comprises a frameshift in the lmol694 gene and 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 lmo!694 and / or fur gene. In some embodiments, a variant Listeria bacterium of the present disclosure comprises 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 -carboxy vinyltransferase gene, and a heptaprenyl diphosphate synthase component I gene. In some embodiments, a variant Listeria bacterium of the present disclosure is a conditionally obligate intracellular bacterium. In some cases, a variant Listeria bacterium of the present disclosure is a variant Listeria monocytogenes bacterium. In some embodiments, a variant Listeria bacterium of the present disclosure comprises a mutation in a ribC gene and / or a ribF gene. In some cases, the mutation comprises a deletion of all or a portion of the ribC gene and / or the ribF gene. In some embodiments, a variant Listeria bacterium of the present disclosure comprises a mutation in an actA gene and / or an MB gene. In some cases, the mutation comprises a deletion of all or a portion of the actA gene and / or the MB gene.

[0010] The present disclosure provides compositions comprising: (a) any variant Listeria bacterium of the present disclosure and (b) a multispecific antibody. In some embodiments, the multispecific antibody 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. In some cases, the T cell is a y / 5 T cell.

[0011] The present disclosure provides immunogenic compositions comprising any variant Listeria bacterium of the present disclosure and methods of using thereof. The present disclosure provides methods of inducing an immune response in an individual, the methods comprising administering to the individual an effective amount of any immunogenic composition of the present disclosure. In some embodiments, the immune response comprises a gamma-delta T cell response.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 depicts a schematic of the mevalonate and non- mevalonate pathways of isoprenoid biosynthesis. L. monocytogenes is one of the very few organisms that harbors genes for both pathways. The mevalonate pathway was made non-functional by deleting hmgR which codes for HMG-CoA reductase.

[0013] FIG. 2A-2C depict aerobic growth of suppressor strains of L. monocytogenes harboring a heterologous operon comprising non-mevalonate pathway genes. (A) Non-mevalonate pathway genes of B. subtilis were assembled as a synthetic operon, PH-7NMBS, and inserted in L. monocytogenes hmgR using pPL2x plasmid. (B) A subset of suppressors have lost three genes of the synthetic operon and contain only 4 genes in the insert (PH-4NMBS) (C) Growth of wildtype (WT), RhmgR, suppressors 4NMBS 1 and 7NMBS 2 was monitored in BHI medium at 37°C under aerobic conditions. Overnight cultures of all strains were sub-cultured to ODGOO of 0.05 and growth was monitored by measuring ODeoo periodically.

[0014] FIG. 3 depicts mutations acquired by L. monocytogenes to use the non-mevalonate pathway aerobically. Whole genome sequencing of L. monocytogenes strains that were able to grow aerobically using the non-mevalonate pathway was carried out to identify the suppressor mutations. Common loss of function mutations was identified in fur and lmol694.

[0015] FIG. 4 demonstrates L. monocytogenes with modified non-mevalonate pathway induces enhanced expansion of Vy9V82 T cells. Representative flow cytometry plot of Vy9+ V62+ CD3+ T-cells in human PBMC after stimulation with PBS only and LMW of L. monocytogenes 7NMBS 2. Human PBMC was stimulated for six days after addition of the indicated inducer and Vy9V82 T-cell expansion was analyzed by staining for surface markers and flow cytometry. The cells were pre-gated on CD3+ T-cells.

[0016] FIG. 5 depicts frequencies of Vy9+ V62+ T cells in human PBMC after stimulation for 6 days by the indicated undiluted dilutions of LMW extracts from / .. monocytogenes strains and purified HMBPP. After 6 days, samples were stained with fluorochrome-conjugated antibodies specific to CD3+ cells, Vy9+ TCR and V52+ TCR and Vy9V82 T-cell expansion was analyzed by flow cytometry.

[0017] FIG. 6 depicts a Dxs amino acid sequence. Highlighted amino acid(s) have been synthetically modified from the wildtype Bacillus subtilis amino acid sequence.

[0018] FIG. 7 depicts an IspD amino acid sequence. Highlighted amino acid(s) have been synthetically modified from the wildtype Bacillus subtilis amino acid sequence.

[0019] FIG. 8 depicts an IspF amino acid sequence. Highlighted amino acid(s) have been synthetically modified from the wildtype Bacillus subtilis amino acid sequence.

[0020] FIG. 9 depicts an IspH / LytB amino acid sequence. Highlighted amino acid(s) have been synthetically modified from the wildtype Bacillus subtilis amino acid sequence.

[0021] FIG. 10 depicts an IspE amino acid sequence. Highlighted amino acid(s) have been synthetically modified from the wildtype Bacillus subtilis amino acid sequence.

[0022] FIG. 11 depicts an IspG / GcpE amino acid sequence. Highlighted amino acid(s) have been synthetically modified from the wildtype Bacillus subtilis amino acid sequence.

[0023] FIG. 12 depicts an IspA amino acid sequence. Highlighted amino acid(s) have been synthetically modified from the wildtype Bacillus subtilis amino acid sequence.

[0024] FIG. 13A-13C The repaired non-meval onate pathway supports aerobic growth of L. monocytogenes. (FIG. 13A) Non-mevalonate pathway genes of B. subtilis were assembled as a synthetic operon, Ph-7NMBs, and inserted in / .. monocytogenes RhmgR using pPL2x plasmid. (FIG. 13B) A subset of suppressors have lost three genes of the synthetic operon and contain only 4 genes in the insert (P1I-4NMBS) (FIG. 13C) Growth of wildtype (WT), RhmgR. suppressors 4NMBS 1, 7NMBS 1 and 7NMBS 2 was monitored in BHI medium at 37°C under aerobic conditions. Overnight cultures of all strains were sub-cultured to ODeoo of 0.05 and growth was monitored by measuring ODeoo periodically. Standard deviation was calculated from three biological replicates.

[0025] FIG. 14 Suppressors have accumulated more intracellular iron. The sensitivity of WT, 4NMBS 1, 7NMBS 1, and 7NMBS 2 to the antibiotic streptonigrin was measured under aerobic conditions. Strains were grown to OD 0.4 and plated using 0.7% top agar onto BHI plates. 5ul of 2mg / ml streptonigrin was added to the disk and the plates were incubated overnight at 37°C.Diameter of the zone of inhibition for each strain is noted in mm. The diameter of the paper disk is 7mm.

[0026] FIG. 15 Growth of suppressors in cells of a host (e.g., human or animal host). Mice bone- marrow derived macrophages were infected with WT, 4NMBS 1 and 7NMBS1. At the indicated timepoints, macrophages were lysed and the intracellular bacteria enumerated by plating on BHI plates. Standard deviation was calculated from three technical replicates.

[0027] FIG. 16 L. monocytogenes with the repaired non-mevalonate pathway induces enhanced expansion of Vy9V82 T-cells. Representative flow cytometry plot of Vy9+V82+T-cells in human PBMC after stimulation with PBS only and LMW extract of / ., monocytogenes 7NMBS1. Human PBMC was stimulated for six days after addition of the indicated inducer and Vy9V82 T-cell expansion was analyzed by staining for surface markers and flow cytometry. The cells were pregated on CD3+T-cells.DEFINITIONS

[0028] “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.

[0029] The term “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.

[0030] 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 term 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, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0031] The terms "polypeptide," "peptide," and "protein", are used interchangeably herein, refer to a polymeric form 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 term 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.

[0032] As used herein the term “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 host cell may be present in a mixed population of genetically modified host cells.

[0033] “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 synthetic oligonucleotides, 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 form of an open reading frame uninterrupted by internal non-translated 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).

[0034] Thus, e.g., the term “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 the 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. Thisartificial 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.

[0035] Similarly, the term “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.

[0036] By “construct” or “vector” is meant a recombinant nucleic acid, generally recombinant DNA, 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.

[0037] The 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.

[0038] 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 by transient 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.

[0039] “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 innature. For example, a “transcriptional control region heterologous to a coding region” is a transcriptional control region that is not normally associated with the coding region in nature.

[0040] A “host cell,” as used herein, denotes a prokaryotic cell (e.g., a Listeria bacterium) that can be, or has been, used as a recipient for a nucleic acid (e.g., an expression vector), and includes the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector. For example, a subject prokaryotic host cell is a genetically modified prokaryotic host cell, by virtue of introduction into a suitable prokaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the prokaryotic host cell, or a recombinant nucleic acid that is not normally found in the prokaryotic host cell.

[0041] 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.nlm.nih.gov / BLAST. See, e.g., Altschul et al. (1990), J. Mol. 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 permits gaps in sequence alignments. 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).

[0042] 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.

[0043] 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 and lower 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.

[0044] 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.

[0045] 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 genetically modified Listeria bacterium” includes a plurality of such bacteria and reference to “the non-mevalonate pathway enzyme” includes reference to one or more non-meval onate pathway enzymes 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.

[0046] 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 (i.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 hereincan 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.

[0047] 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 of the 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.

[0048] 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).

[0049] It is understood that aspects and embodiments of the present disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0050] 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 sub-combination was individually and explicitly disclosed herein.

[0051] 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

[0052] The present disclosure provides variant Listeria bacteria that 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 the presence or absence of a functional mevalonate pathway. Also provided are methods of making and using the variant Listeria bacteria, e.g., as vectors, vaccines, and therapeutics. The present disclosure provides compositions comprising: (a) any variant Listeria bacterium of the present disclosure and (b) a multispecific antibody. The present disclosure provides immunogenic compositions comprising any variant Listeria bacterium of the present disclosure. The present disclosure provides methods of inducing an immune response in an individual, the methods comprising administering to the individual an effective amount of any immunogenic composition of the present disclosure.VARIANT LISTERIA BACTERIA

[0053] The present disclosure provides variant Listeria bacteria that 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 the presence or absence of a functional mevalonate pathway. A variant Listeria bacterium of the present disclosure is genetically modified with one or more heterologous nucleic acids, where at least one of the one or more heterologous nucleic acids comprises nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway. In some cases, a variant Listeria bacterium of the present disclosure comprises nucleic acids encoding polypeptides required for a mevalonate pathway for isoprenoid synthesis. In some cases, a variant Listeria bacterium of the present disclosure lacks one or more nucleic acids encoding one or more polypeptides required for isoprenoid synthesis via a mevalonate pathway and / or comprises one or more mutations in one or more nucleic acids encoding polypeptides required for a mevalonate pathway, such that the mevalonate pathway is non-functional.

[0054] 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 fromaccumulation 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 Farnesyl pyrophosphate (FPP). However, the native non- mevalonate pathway can only be used anaerobically in Listeria bacteria and therefore the growth of a Listeria bacterium reliant on the non-mevalonate pathway for isoprenoid synthesis is limited to anaerobic conditions.

[0055] Thus, the present disclosure provides variant Listeria bacteria that 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 the presence or in the absence of a functional mevalonate pathway.

[0056] The one or more heterologous nucleic acids comprises one or more nucleotide sequences coding for polypeptides required for isoprenoid synthesis through the non-mevalonate pathway. In some cases, all polypeptides required for isoprenoid synthesis through the non-mevalonate pathway are encoded on a single nucleic acid. In some cases, all polypeptides required for isoprenoid synthesis through the non-mevalonate pathway are encoded on a single 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 nucleic acids. In some embodiments, polypeptides required for isoprenoid synthesis through the non-mevalonate pathway include: Dxs, IspD, IspF, LytB, IspE, GcpE, and IspA polypeptides. In some embodiments, polypeptides required for isoprenoid synthesis through the non-mevalonate pathway include polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity tothe amino acid sequences set forth in: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, and SEQ ID NO:7. A polypeptide required for isoprenoid synthesis through the non-mevalonate pathway of the present disclosure may comprise an amino acid sequence with 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 set forth in any one of SEQ ID NOs: l-7

[0057] In some embodiments, the one or more heterologous nucleic acids comprising nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway comprise nucleotide sequences encoding LytB, IspE, GcpE, and IspA polypeptides. In some embodiments, the one or more heterologous nucleic acids comprise nucleotide sequences encoding Dxs, IspD, IspF, LytB, IspE, GcpE, and IspA polypeptides. In some embodiments, the one or more heterologous nucleic acids comprise nucleotide sequences encoding GcpE, LytB, and IspE polypeptides. 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, SEQ ID NO:6, and SEQ ID NO:7. 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 NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. 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. 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:5, SEQ ID NO:6, and SEQ ID NO:7. 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 IDN0:5, SEQ ID N0:6, and SEQ ID N0:7. In some cases, the one or more heterologous nucleic acidscomprise 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.

[0058] In some cases, a variant Listeria bacterium of the present disclosure comprises nucleic acids encoding polypeptides required for a mevalonate pathway for isoprenoid synthesis. In some cases, a variant Listeria bacterium of the present disclosure lacks one or more nucleic acids encoding one or more polypeptides required for a mevalonate pathway for isoprenoid synthesis. Polypeptides of the mevalonate pathway for isoprenoid biosynthesis include: i) an enzyme (e.g., acetoacetyl-CoA thiolase, e.g., encoded by atoB) that catalyzes the condensation of two molecules of acetyl-CoA to form acetylacetyl-CoA; ii) an enzyme (e.g., HMG-CoA synthase) that catalyzes the condensation of acetyl-CoA and acetyl acetyl -Co A to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA); iii) an enzyme (e.g., HMG-CoA reductase) that catalyzes the conversion of HMG- CoA to mevalonate; iv) an enzyme (e g., mevalonate kinase) that catalyzes the conversion of mevalonate to mevalonate 5-phosphate; v) an enzyme (e.g., phosphomevalonate kinase) that catalyzes the conversion of mevalonate 5-phosphate to mevalonate pyrophosphate; and vi) an enzyme (e.g., mevalonate pyrophosphate decarboxylase) that catalyzes the conversion of mevalonate 5-phosphate into isopentenyl pyrophosphate (IPP). In some cases, a variant Listeria bacterium of the present disclosure comprises a mutation in a lytB gene such that the lytB gene is non-functional. In some cases, the mevalonate pathway of the variant Listeria bacterium is intact, e.g., the variant Listeria bacterium does not have a loss of function mutation in any polypeptide of the mevalonate pathway for isoprenoid biosynthesis.

[0059] The hmgR gene encodes an HMG-CoA reductase that is essential for functioning of the mevalonate pathway of isoprenoid synthesis. HMG-CoA reductase catalyzes the conversion of 3- hydroxy-3-methylglutaryl-CoA (HMG-CoA) In some embodiments, a variant Listeria bacterium of the present disclosure comprises a mutation in a hmgR gene. In some cases, the mutation in a hmgR gene comprises a deletion of all or a portion of the hmgR gene. In some cases, a variant Listeria bacterium of the present disclosure does not have a mutation in a hmgR gene.

[0060] Mutations in other genes may enhance the function of the non-mevalonate pathway under aerobic conditions in a Listeria bacterium. As a functional pathway for isoprenoid synthesis is essential for growth, such mutations may also enhance the aerobic growth of a bacterium reliant on the non-mevalonate pathway for isoprenoid synthesis. For example, a loss of function mutation in fur, which encodes a ferric uptake protein, may improve the aerobic function of the non-mevalonatepathway. 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 lmol694 gene. In some embodiments, a variant Listeria bacterium of the present disclosure may comprise a loss of function mutation in a lmo!694 gene and a / z / r gene. In some cases, the loss of function mutation comprises a frameshift in the lmo!694 gene and 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 lmo!694 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.

[0061] In some embodiments, a variant Listeria bacterium of the present disclosure can be grown (cultured) in vitro in a culture medium supplemented with flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), it cannot grow in vivo (e g., inside a mammal) extracellularly. In some embodiments, a variant Listeria bacterium of the present disclosure 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). Genes required for FMN and FAD biosynthesis include ribC and ribF. RibC is a bifunctional enzyme that catalyzes the 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 comprises a mutation in a ribC gene and / or a ribF gene. In some cases, the mutation comprises a deletion of all or a portion of the ribC gene and / or the ribF gene.

[0062] In some instances, the Listeria host cell is attenuated. "Attenuation" and "attenuated" encompasses a Listeria host cell that is modified to reduce virulence. The host can be a human or animal host, or an organ, tissue, or cell. The Listeria host cell, to give a non-limiting example, can be attenuated to reduce binding to a host cell (e g., human or non-human 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. In other words, the host (a eukaryotic host) can be a human or animal host, or an organ, tissue, or cell (a eukaryotic cell), and a Listeria host cell (a bacterial host cell) can be attenuated to reduce binding to a cell (a eukaryotic cell) of the host (e.g., a human or non-human animal cell, such as a human or non-human mammalian cell), to reduce spread from one cell of the host to another cell of the host, to reduce extracellular growth, or to reduce intracellular growth in a cell of the host. Attenuation can beassessed by measuring, e.g., an indicum or indicia of virulence, the LD50, the rate of clearance from an organ, or the competitive index (see, e.g., Auerbuch, 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-forming units (CFUs).

[0063] 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.

[0064] 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 Enzymo 236:463-467. In yet other embodiments, bacteria are considered to be attenuated or less virulent if, compared to wild-type, they form 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 withthe attenuated mutant as compared to the wild-type is, in certain embodiments, 10%, including 15%, such as 25% or more.

[0065] Attenuated bacteria may include one or more different mutations which confer the attenuated phenotype, where mutations of interest include hly mutations and / or Ip I A mutations, e.g., as described 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 comprise a mutation in an actA gene and / or an inlB gene. In some cases, the mutation comprises a deletion of all or a portion of the actA gene and / or the inlB gene.

[0066] 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; WG / 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.

[0067] Additional embodiments of a variant Listeria bacterium of the present disclosure may comprise any combination of the mutations / modifications described above.

[0068] 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, / .. seeligeri, L. weihenstephanensis, and L. welshimeri. Thus, strains of Listeria other than L. monocytogenes may be host cells. In certain cases, the Listeria strain is L. monocytogenes.

[0069] Bacteria as described herein may be generated using a variety of different protocols. The introduction of one or more heterologous DNA molecules encoding polypeptides of a nonmevalonate pathway into a strain of Listeria may be accomplished, for example, by the creation of a recombinant Listeria in which DNA encoding the polypeptides is harbored on a vector, such as a plasmid for example, which plasmid is maintained and expressed in the Listeria species, where expression of the nucleotide sequences encoding the polypeptides is under the control ofprokaryotic promoter / regulatory sequences. Alternatively, DNA encoding the polypeptides of a non-mevalonate pathway may be stably integrated into the Listeria chromosome by employing, for example, transposon mutagenesis, homologous recombination, or integrase mediated site-specific integration (as described in USSN 10 / 136,860, the disclosure of which is herein incorporated by reference).

[0070] The nucleotide sequences encoding the polypeptides of a non-mevalonate pathway in some embodiments operably linked 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 internalin, a Listeria membrane protein. The 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 / journal.ppat.1005741.

[0071] In certain instances, a variant Listeria bacterium of the present disclosure expresses a heterologous antigen. The heterologous antigen is, in certain embodiments, one that is capable of providing protection in an animal 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 a host organism. Heterologous antigens which may be introduced into a Listeria strain of the subject disclosure by way of DNA encoding the same thus include any antigen which when expressed by Listeria serves to elicit a cellular immune response which is of benefit to the host in which the response is induced. 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 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 is maybe a mammal, such as a human.

[0072] By the term "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 tumor cell or a tumor-related protein. The heterologous antigen is expressed by a strain of Listeria according to the present disclosure, and is processed and presented to cytotoxic T-cells upon infection of mammalian cells by the strain. The heterologous antigen expressed by Listeria species 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 mammal, and the antigen expressed by the Listeria species will conform to that tumor cell mutated antigen. By the term "tumor-related antigen," as used herein, is meant an antigen which affects tumor growth 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” has no effect on tumor growth or metastasis, but is used as a component of the 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” has no effect on tumor growth or metastasis, but is used as a component of the Listeria vaccine because it is selectively expressed in the tumor cell and not in any other normal tissues.

[0073] 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 useful as heterologous antigens include but are not limited to the nucleoprotein (NP) of influenza virus and the gag protein of HIV. Other heterologous antigens include, but are not limited to, HIV env protein or its component parts gpl20 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 heterologousantigens 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.

[0074] 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, HPVE6 and E7 antigens of the oncogenic virus associated with cervical cancer, the MAGE1 and MZ2-E antigens in or associated 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 growth factor receptor-2 (VEGF-R2);and epidermal growth factor receptor (EGFR) vIII polypeptide. Other coding sequences of interest include, but are not limited to, costimulatory molecules, immunoregulatory molecules, and the like.

[0075] The introduction of DNA encoding a heterologous antigen into a strain of Listeria may be accomplished, for example, by the creation of a recombinant Listeria in which DNA encoding the heterologous antigen is harbored on a vector, such as a plasmid for example, which plasmid is maintained and expressed in the Listeria species, and in whose antigen expression is under the control of prokaryotic promoter / regulatory sequences. Alternatively, DNA encoding the heterologous antigen may be stably integrated into the Listeria chromosome by employing, for example, transposon mutagenesis, homologous recombination, or integrase mediated site-specific integration (as described in application serial no. 10 / 136,860, the disclosure of which is herein incorporated by reference).

[0076] Several approaches may be employed to express the heterologous antigen in Listeria species as will 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.

[0077] In certain embodiments, a fusion protein which includes the desired heterologous antigen and a secreted or cell surface protein of Listeria is employed. Listeria! proteins which are suitable components of such fusion proteins include, but are not limited to, listeriolysin O (LLO) andphosphatidylinositol-specific phospholipase (PI-PLC). A fusion protein may be generated by ligating the genes which 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.COMPOSITIONS

[0078] The present disclosure provides a composition comprising a variant Listeria bacterium of the present disclosure. The present disclosure provides a composition comprising: a) a variant Listeria bacterium of the present disclosure; 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 / 5 T cell. A composition of the present disclosure can comprise, in addition to a variant Listeria and a multispecific antibody, one or more of: a salt (e.g., NaCl, MgCh, KC1, MgSCU, 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.

[0079] The 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.

[0080] In some cases, a composition comprising a variant Listeria of the present disclosure (e.g., a variant Listeria bacterium comprising one or more heterologous nucleic acids comprising nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway) comprises a unit dose of the variant Listeria. 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 avariant Listeria of the present disclosure is in a range of from 104to 5 x 104, from 5 x 104to 1 C , 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 108, from 108to 109, or from 109to IO10, bacteria per unit dose. The present disclosure further provides a kit comprising a unit dose of a variant Listeria.METHODS OF INDUCING AN IMMUNE RESPONSE

[0081] The present disclosure provides methods of inducing an immune response in an individual, the methods comprising administering to the individual an effective amount of an immunogenic composition (i.e., “vaccine composition”) of the present disclosure.

[0082] The subject bacteria (variant Listeria bacterium comprising one or more heterologous nucleic acids comprising nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway) 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 vary widely, as those skilled in the 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 patient, the type of patient, and the existence of concurrent treatment, if any. The 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 combinationwith a pharmaceutically acceptable diluent, as a vaccine composition, useful in immunizing a patient against infection from a selected organism or virus or with respect to a tumor, etc. Immunizing a patient means providing the patient with at least some degree of therapeutic or prophylactic immunity against selected pathogens, cancerous cells, etc.

[0083] The subject vaccines find use in methods for eliciting or boosting a cellular immune response, e.g., a y / 8 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. The 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 and tetanus. 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.

[0084] The patient 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.

[0085] The 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 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.

[0086] The subject bacterial strains (variant Listeria bacterium comprising one or more nucleic acids comprising nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the non-mevalonate pathway) also find use as immunopotentiating agents, i.e., as adjuvants. In such applications, the subject 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 artwhere live bacterial strains are employed as adjuvants. See, e.g., Berd et al., Vaccine 2001 Mar 21;19(17-19):2565-70.

[0087] 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. CAD2 mammary adenocarcinomia tumor cells, and hormones such as thyroxine T4, triiiodothyronine T3, and cortisol. The 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.

[0088] The bacterial strains of this disclosure (e g., engineered L. monocytogenes strains) can be used to induce enhanced expansion and activation of y5 T-cells (e.g., in humans). These strains can be used as therapeutics in gamma delta-based cancer immunotherapy to target tumors (e.g., solid and hematological tumors). The genetic manipulations that enable L. monocytogenes to use the nonmevalonate pathway aerobically can also be combined with other genetic mutations, such as actAUnlB and ribC / ribF deletions that attenuate L. monocytogenes strains for therapeutic use (see, e.g., Brockstedt, D. G. et al. Proc. Natl. Acad. Sci. 101, 13832-13837 (2004); and Rivera-Lugo, et al., Proc. Natl. Acad. Sci. 119, e2122173119 (2022)).

[0089] The bacterial strains of this disclosure (e.g., engineered L. monocytogenes strains) can be used in combination therapy with existing methods to induce gamma delta T-cell expansion, e.g., in vivo stimulation using the bisphosphonate drug zoledronate (see, e.g., Yazdanifar et al., Cells vol. 9 at doi.org / 10.3390 / cells9051305 (2020)). The bacterial strains of this disclosure (e.g., engineered A. monocytogenes strains) can be used in combination with bispecific antibodies that can target the expanded gamma delta T-cells to tumor cells.Examples of Non-Limiting Aspects of the Disclosure

[0090] 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 (see Set A and Set B). 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:Set AAspect 1. A variant Listeria bacterium comprising 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 the presence or absence of a functional mevalonate pathway.Aspect 2. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding LytB, IspE, GcpE, and IspA polypeptides.Aspect 3. The variant Listeria bacterium of aspectl, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding Dxs, IspD, IspF, LytB, IspE, GcpE, and IspA polypeptides.Aspect 4. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more (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, SEQ ID NO: 6, and SEQ ID NO: 7.Aspect 5. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more (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 NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.Aspect 6. The variant Listeria bacterium of any one of aspects 1-5, further comprising a loss of function mutation in a hmgR gene.Aspect 7. The variant Listeria bacterium of aspect 6, wherein the loss of function mutation comprises a deletion of all or a portion of the hmgR gene.Aspect 8. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences coding for IspE, GcpE, and IspA polypeptides.Aspect 9. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding Dxs, IspD, IspF, IspE, GcpE, and IspA polypeptides.Aspect 10. The variant Listeria bacterium of aspect 1, wherein the heterologous operon comprises nucleotide sequences coding for polypeptides comprising amino acid sequences with 80% or more identity to the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.Aspect 11. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more (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 NO:5, SEQ ID NO:6, and SEQ ID NO:7.Aspect 12. The variant Listeria bacterium of any one of aspects 1-11, further comprising a loss of function mutation in a Imo 1694 gene and a fur gene.Aspect 13. The variant Listeria bacterium of aspect 12, wherein the loss of function mutation comprises a frameshift in the / w l 694 gene and a frameshift mutation or premature stop in the fur gene.Aspect 14. The variant Listeria bacterium of aspect 12, wherein the loss of function mutation comprises a deletion of all or a portion of the lmo\ 694 and / or fur gene.Aspect 15. The variant Listeria bacterium of any one of aspects 1-14, further comprising 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. Aspect 16. The variant Listeria bacterium of any one of aspects 1 -15, wherein the variant Listeria bacterium is a conditionally obligate intracellular bacterium.Aspect 17. The variant Listeria bacterium of any one of aspects 1-16, wherein said variant Listeria bacterium is a variant Listeria monocytogenes bacterium.Aspect 18. The variant Listeria bacterium of any one of aspects 1-17, further comprising a mutation in a ribC gene and / or a ribF gene.Aspect 19. The variant Listeria bacterium of aspect 18, wherein the mutation comprises a deletion of all or a portion of the ribC gene and / or the ribF gene.Aspect 20. The variant Listeria bacterium of any one of aspects 1-19, further comprising a mutation in an actA gene and / or an itilB gene.Aspect 21. The variant Listeria bacterium of aspect 20, wherein the mutation comprises a deletion of all or a portion of the actA gene and / or the inlB gene.Aspect 22. A composition comprising: a) a variant Listeria bacterium of any one of aspects 1-21; and b) a multispecific antibody.Aspect 23. The composition of aspect 22, wherein the multispecific antibody comprises: i) a first antigen-binding site specific for a cancer-associated antigen; and ii) a second antigenbinding site specific for a T cell.Aspect 24. The composition of aspect 23, wherein the T cell is a y / 8 T cell.Aspect 25. An immunogenic composition comprising the variant Listeria of any one of aspects 1-21.Aspect 26. 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 25.Aspect 27. The method of aspect 26, wherein said immune response comprises a gammadelta T cell response.SetBAspect 1. A variant Listeria bacterium comprising 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.Aspect 2. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding LytB, IspE, GcpE, and IspA polypeptides.Aspect 3. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprises nucleotide sequences encoding Dxs, IspD, IspF, LytB, IspE, GcpE, and IspA polypeptides.Aspect 4. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.Aspect 5. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences coding for polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity to the amino acid sequences of SEQ ID NO : 1, SEQ ID NO:2, SEQ ID NO 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.Aspect 6. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding IspE, GcpE, and IspA polypeptides. Aspect 7. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding Dxs, IspD, IspF, IspE, GcpE, and IspA polypeptides.Aspect 8. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding GcpE, LytB, and IspE polypeptides Aspect 9. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity to the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.Aspect 10. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.Aspect 11. The variant Listeria bacterium of aspect 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.Aspect 12. The variant Listeria bacterium of any one of aspects 1-11, further comprising a loss of function mutation in a hmgR gene.Aspect 13. The variant Listeria bacterium of aspect 12, wherein the loss of function mutation comprises a deletion of all or a portion of the hmgR gene.Aspect 14. The variant Listeria bacterium of any one of aspects 1-11, wherein the variant Listeria bacterium comprises an intact mevalonate pathway.Aspect 15. The variant Listeria bacterium of any one of aspects 1-11, wherein the variant Listeria bacterium does not have a loss of function mutation in a hmgR gene.Aspect 16. The variant Listeria bacterium of any one of aspects 1-15, further comprising a loss of function mutation in a fur gene.Aspect 17. The variant Listeria bacterium of aspect 16, further comprising a loss of function mutation in a lmol694 gene.Aspect 18. The variant Listeria bacterium of aspect 17, wherein the loss of function mutations comprise a frameshift in the lmo!694 gene and a frameshift mutation or premature stop in the fur gene.Aspect 19. The variant Listeria bacterium of aspect 17, wherein the loss of function mutations comprise a deletion of all or a portion of the lmol694 and / or fur gene.Aspect 20. The variant Listeria bacterium of any one of aspects 1-19, further comprising 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. Aspect 21. The variant Listeria bacterium of any one of aspects 1-20, wherein the variant Listeria bacterium is a conditionally obligate intracellular bacterium.Aspect 22. The variant Listeria bacterium of any one of aspects 1-21, wherein said variant Listeria bacterium is a variant Listeria monocytogenes bacterium.Aspect 23. The variant Listeria bacterium of any one of aspects 1-22, further comprising a mutation in a ribC gene and / or a ribF gene.Aspect 24. The variant Listeria bacterium of aspect 23, wherein the mutation comprises a deletion of all or a portion of the ribC gene and / or the ribF gene.Aspect 25. The variant Listeria bacterium of any one of aspects 1-24, further comprising a mutation in an actA gene and / or an inlB gene.Aspect 26. The variant Listeria bacterium of aspect 25, wherein the mutation comprises a deletion of all or a portion of the actA gene and / or the inlB gene.Aspect 27. A composition comprising: a) a variant Listeria bacterium of any one of aspects 1-26; and b) a multispecific antibody.Aspect 28. The composition of aspect 27, wherein the multispecific antibody comprises: i) a first antigen-binding site specific for a cancer-associated antigen; and ii) a second antigenbinding site specific for a T cell.Aspect 29. The composition of aspect 28, wherein the T cell is a y / 5 T cell.Aspect 30. An immunogenic composition comprising the variant Listeria of any one of aspects 1-26.Aspect 31. 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 30.Aspect 32. The method of aspect 31, wherein said immune response comprises a gammadelta T cell response.EXAMPLES

[0091] 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., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.Example 1Introduction

[0092] The most potent Vy9V82 T-cell phosphoantigen is the bacterial metabolite (E)-4-hydroxy- 3-methyl-but-2-enyl pyrophosphate (HMBPP). HMBPP is an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (Fig. 1), which is unique to bacteria and some parasites, but absent in mammals. Many pathogens including L. monocytogenes, Mycobacterium tuberculosis and Salmonella enterica use the non-mevalonate pathway to produce HMBPP, which induces expansion and activation of VY9V82 T-cells as an immune countermeasure. Although most organisms encode either the mevalonate or non-mevalonate pathway of isoprenoid biosynthesis, L. monocytogenes is one of the very few bacteria that encodes both pathways (Fig. 1). As demonstrated by a L. monocytogenes strain in which the mevalonate pathway was made non-functional by deletion of hmgR, the gene encoding a key enzyme of the pathway (Fig. 1), the non-mevalonate pathway canfunction, but only anaerobically. L. monocytogenes must have at least one functioning isoprenoid biosynthesis pathway to be viable and deletion of the mevalonate pathway hindered the ability of L. monocytogenes to grow aerobically unless mevalonate was supplemented in the medium. However, this strain can grow anaerobically using the non-mevalonate pathway without addition of mevalonate, showing that / .. monocytogenes can only use the non-mevalonate pathway anaerobically. Additionally, GcpE, the enzyme that produces HMBPP, is defective in L. monocytogenes. The following results address the functioning of the non-mevalonate pathway in / .. monocytogenes under aerobic conditions.Results

[0093] L. monocytogenes was engineered to harbor a functioning non-mevalonate pathway that enables it to grow aerobically exclusively using this pathway. It was reasoned that by harboring a functioning non-mevalonate pathway, L. monocytogenes strains can produce elevated levels of (E)- 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), the phosphoantigen of Vy9V82 T-cells. HMBPP is an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis, which is unique to bacteria and some parasites, but absent in mammals. To repair the non-mevalonate pathway and improve HMBPP production in A. monocytogenes, a synthetic operon consisting of seven genes from Bacillus subtilis, a Gram-positive bacterium that can use the non-mevalonate pathway aerobically, was introduced. This synthetic operon consisted of six genes of the non- mevalonate pathway and a downstream gene. The synthetic operon was constitutively expressed using a Phyper promoter, hereafter referred to as Ph-7NMns(Fig. 2A), and introduced in the L. monocytogenes RhmgR strain. Growth of the hmgR strain was used to monitor functionality of the non-mevalonate pathway, since L. monocytogenes needs one functioning isoprenoid biosynthesis pathway for survival. L. monocytogenes modified with the synthetic Ph-7NMBs operon did not grow aerobically. However, rare colonies, which turned out to harbor suppressor mutations, appeared after two days of growth and two colonies were selected for further study. One suppressor colony had lost dxs, ispl), and ispF genes of the insert, hereafter referred to as Ph-4NMBs (Fig. 2B), while another colony had all 7 genes of the synthetic operon insert. Serial aerobic passage of these two suppressor colonies in liquid media resulted in additional suppressor mutations that supported rapid and stable aerobic growth of L. monocytogenes using the recombinant non-mevalonate pathway (Fig. 2C). The doubling time of final suppressor strains 4NMBS 1 and 7NMBS 2 were 62 minutes, compared to the 41 minutes doubling time of wildtype, which has a functional mevalonate pathway. Whole genome sequencing of the suppressor strains identified two common mutations in the ferricuptake regulator (Fur) and lmo!694, and three mutations that were not shared between the two suppressors (Fig. 3). This indicates that the synthetic non-mevalonate operon plus suppressor mutations allows L. monocytogenes to have a functioning non-mevalonate pathway in the presence of oxygen and allows it to grow aerobically in the absence of the mevalonate pathway.

[0094] Vy9V82 T-cell expansion in vitro in human peripheral blood mononuclear cells (PBMC) upon stimulation with L. monocytogenes harboring the repaired or native non-mevalonate pathway was compared. Dilutions of low molecular weight (LMW) extracts of the repaired L. monocytogenes 4NMBS 1 and 7NMBS 2 strains, the wildtype strain, and strains with deletion of gcpE or lytB in a wildtype background were prepared using published methods. L. monocytogenes wildtype, EgcpE, and ElytB strains harbor the native non-mevalonate pathway and not the repaired one. Human PBMCs were stimulated with the LMW extracts of each strain for 6 days in the presence of IL -2 and Vy9V82 T-cell expansion was monitored by flow cytometry (Fig. 4). With undiluted extracts, all strains except EgcpE induced high V' 9V82 T-cell response similar to purified HMBPP (Fig. 5). At a 1 :10 dilution, 7NMBS2 strain induced enhanced expansion, as seen with HMBPP, yielding 47% of Vy9+ V82+ T-cells compared to 16% induced by wildtype L. monocytogenes (Fig. 4, Fig. 5). As expected, a EgcpE strain, which lacks the HMBPP-producing enzyme, had minimal Vy9V82 T-cell expansion, while a / lytB strain, which lacks the HMBPP- consuming enzyme, had increased Vy9V82 T-cell expansion compared to wildtype (Fig. 5). Remarkably, the 7NMBS 2 strain had more Vy9V82 T-cells expansion compared to a ElytB strain, which is considered a strong Vy9V82 T-cells inducer in previous studies.Example 2

[0095] This example includes some information from the above example, but also includes additional infonnation.Results

[0096] L. monocytogenes were engineered to harbor a functioning non-mevalonate pathway that enables it to grow aerobically exclusively using this pathway. It was reasoned that by harboring a functioning non-mevalonate pathway, L. monocytogenes strains can produce elevated levels of HMBPP, the ligand of Vy9V82 T-cells. To repair the non-mevalonate pathway and improve HMBPP production in L. monocytogenes, a synthetic operon consisting of seven genes from Bacillus subtilis, a Gram-positive bacterium that can use the non-mevalonate pathway aerobically, was introduced. This synthetic operon consisted of six genes of the non-mevalonate pathway and adownstream gene. The synthetic operon was constitutively expressed using a Phyper promoter, hereafter referred to as Ph-7NMBs (Fig- 13A), and introduced in the L. monocytogenes RhmgR strain. Growth of the RhmgR strain was used to monitor functionality of the non-mevalonate pathway, since L. monocytogenes needs one functioning isoprenoid biosynthesis pathway for survival. L. monocytogenes modified with the synthetic Ph-7NMBs operon did not grow aerobically. However, rare colonies, which turned out to harbor suppressor mutations, appeared after two days of growth and two colonies were selected for further study. One suppressor colony had lost dxs, ispD, and ispF genes of the Ph-7NMBs insert, hereafter referred to as Ph-4NMBs (Fig. 13B), while another colony retained all 7 genes of the synthetic operon insert. Serial aerobic passage of these two suppressor colonies in liquid media resulted in additional suppressor mutations that supported rapid and stable aerobic growth of L. monocytogenes using the recombinant non-mevalonate pathway (Fig. 13C). The doubling time of the three suppressor strains 4NMBS1, 7NMBS 1 and 7NMBS 2 in BHI medium during aerobic growth were found to be 58, 62 and 64 minutes respectively, compared to 42 minutes of wildtype L. monocytogenes which has a functional mevalonate pathway (Fig. 13C). Whole genome sequencing of the three different suppressor strains identified two common mutations in ferric uptake regulator (Fur) and lmol694 (putative oxidoreductase) and three mutations that were not shared between the two suppressors (Table 1). This indicates that the synthetic non-mevalonate operon plus suppressor mutations allows L. monocytogenes to have a functioning non-mevalonate pathway in the presence of oxygen and allows it to grow aerobically in the absence of the mevalonate pathway.

[0097] Table 1. Mutations acquired by L. monocytogenes to use the non-mevalonate pathway aerobically. Whole genome sequencing of L. monocytogenes strains that were able to grow aerobically using the non-mevalonate pathway was carried out to identify the suppressor mutations. Common loss of function mutations was identified in fur and lmol694.

[0098] Since all three independent suppressors (4NMBS 1, 7NMBS 1 and 7NMBS 2) had accumulated different mutations in a single gene, / ; / / ', it was examined if these mutations conferred a gain or loss of function of Fur. Fur is a global regulator of iron metabolism in bacteria and is well-known as a transcriptional repressor of genes involved in iron acquisition. Therefore, a loss of function of Fur results in more accumulation of iron in bacteria. Streptonigrin, which is an antibiotic whose bactericidal activity is directly proportional to the intracellular iron content of bacteria, was used to test if the suppressor mutations in fur leads to accumulation of iron. By performing a disk diffusion assay using streptonigrin, it was found that the zone of inhibition for all three suppressors were larger than that of wildtype (Fig. 14). Therefore, the suppressor mutations accumulated in fur were indeed loss of function mutations of the Fur repressor.

[0099] Next, it was examined if the aerobically growing suppressors with a repaired nonmevalonate pathway can grow inside cells of a host (human or animal host). For this, an in vitro infection was performed of murine bone-marrow derived macrophages with wildtype L. monocytogenes and the two fastest growing suppressors, 4NMBS1 and 7NMBS 1, and their intracellular growth was tracked. The two suppressors had a slight reduction in colony forming units (CFU) at 30 minutes post infection, but at later timepoints (5 and 8 hours post-infection), the suppressors were able to replicate and survive inside the macrophages using only the nonmevalonate pathway (Fig. 15). The doubling time of 4NMBS 1 and 7NMBS 1 strains in murine macrophages were 107 and 81 minutes respectively compared to 67 minutes of wildtype L. monocytogenes.

[0100] Vy9V52 T-cell expansion in vitro in human peripheral blood mononuclear cells (PBMC) from two healthy donors upon stimulation with L. monocytogenes harboring the repaired or native non-meval onate pathway was compared. Dilutions of low molecular weight (LMW) extracts of the repaired L. monocytogenes 4NMBS1, 7NMBS1 and 7NMBS 2 strains, the wildtype strain, and strains with deletion of gcpE or lytB in a wildtype background were prepared using published methods. L. monocytogenes wildtype, gcpE, and ElytB strains harbor the native non-mevalonate pathway and not the repaired one. Human PBMCs were stimulated with the LMW extracts of each strain for 6 days in the presence of lOOU / ml IL-2 and Vy9V52 T-cell expansion was monitored by flowcytometry (Fig. 16). With no dilution, all strains except EgcpE (which lacks the enzyme that produces HMBPP) induced a high Vy9V82 T-cell expansion similar to purified HMBPP (Fig. 5 and Table 2). At 1 : 10 dilution, while the suppressors still maintained a high Vy9V62 T-cell expansion, wildtype and ElytB (which accumulates HMBPP) had notably reduced expansion (Fig. 5 and Table 2). At 1 : 100 dilution, only the suppressor strains induced high V' 9V82 T-cell expansion (Fig. 5 and Table 2). At 1 : 1000 dilution, 4NMBS1 induced reduced expansion of Vy9V82 T-cells, while 7NMBS 1 and 2 still induced a strong expansion (Fig. 5 and Table 2). Overall, the suppressors were able to induce incredibly robust expansion of Vy9V82 T-cells compared to wildtype L. monocytogenes. Remarkably, the suppressors also induced more Vy9V82 T-cells expansion compared to a ElytB strain, which is considered a strong Vy9V82 T-cells inducer in previous studies.

[0101] Table 2. Frequencies in % of Vy9+V82+T-cells in healthy human PBMCs (donor 2) after stimulation by indicated dilutions of LMW extracts from Lm strains for 6 days. The cells were pregated on CD3+T-cells.

[0102] It was also tested if introduction of the Ph-7NMBs insert (Fig. 2A) in wildtype L. monocytogenes, which does not harbor any suppressor mutations, induced Vy9V82 T-cells response in human PBMCs. Using LMW extracts, wildtype with the Ph-7NMBs insert induced a stronger Vy9V82 T-cells expansion compared to wildtype without the B. subtilis non-mevalonate genes insert (Table 3). Interestingly, including just three genes of B. subtilis as a synthetic operon, gcpE, lytB and ispE, also induced a higher Vy9V82 T-cells expansion than wildtype L. monocytogenes, but the expansion was ~10-fold lower than the wildtype Ph-7NMBs strain (Table 3).

[0103] Table 3. Frequencies in % of Vy9' V52 T-cells in healthy human PBMCs after stimulation by indicated dilutions of LMW extracts from Lm strains for 6 days. The cells were pregated on CD3+T-cells.

[0104] 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 one or more heterologous nucleic acids comprising nucleotide sequences encoding polypeptides required for isoprenoid synthesis through the nonmevalonate pathway, wherein the variant Listeria bacterium grows aerobically.

2. The variant Listeria bacterium of claim 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding LytB, IspE, GcpE, and IspA polypeptides.

3. The variant Listeria bacterium of claim 1, wherein the one or more heterologous nucleic acids comprises nucleotide sequences encoding Dxs, IspD, IspF, LytB, IspE, GcpE, and IspA polypeptides.

4. The variant Listeria bacterium of claim 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

5. The variant Listeria bacterium of claim 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences coding for polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

6. The variant Listeria bacterium of claim 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding IspE, GcpE, and IspA polypeptides.

7. The variant Listeria bacterium of claim 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding Dxs, IspD, IspF, IspE, GcpE, and IspA polypeptides.

8. The variant Listeria bacterium of claim 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding GcpE, LytB, and IspE polypeptides.

9. The variant Listeria bacterium of claim 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity to the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

10. The variant Listeria bacterium of claim 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

11. The variant Listeria bacterium of claim 1, wherein the one or more heterologous nucleic acids comprise nucleotide sequences encoding polypeptides comprising amino acid sequences with 80% or more amino acid sequence identity to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

12. The variant Listeria bacterium of any one of claims 1-11, further comprising a loss of function mutation in a hmgR gene.

13. The variant Listeria bacterium of claim 12, wherein the loss of function mutation comprises a deletion of all or a portion of the hmgR gene.

14. The variant Listeria bacterium of any one of claims 1-11, wherein the variant Listeria bacterium comprises an intact mevalonate pathway.

15. The variant Listeria bacterium of any one of claims 1-11, wherein the variant Listeria bacterium does not have a loss of function mutation in a hmgR gene.

16. The variant Listeria bacterium of any one of claims 1-15, further comprising a loss of function mutation in a fur gene.

17. The variant Listeria bacterium of claim 16, further comprising a loss of function mutation in a lmol694 gene.

18. The variant Listeria bacterium of claim 17, wherein the loss of function mutations comprise a frameshift in the lmol694 gene and a frameshift mutation or pre-mature stop in the fur gene.

19. The variant Listeria bacterium of claim 17, wherein the loss of function mutations comprise a deletion of all or a portion of the Imo 1694 and / or fur gene.

20. The variant Listeria bacterium of any one of claims 1-19, further comprising 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 -carboxy vinyltransferase gene, and a heptaprenyl diphosphate synthase component I gene.

21. The variant Listeria bacterium of any one of claims 1-20, wherein the variant Listeria bacterium is a conditionally obligate intracellular bacterium.

22. The variant Listeria bacterium of any one of claims 1-21, wherein said variant Listeria bacterium is a variant Listeria monocytogenes bacterium.

23. The variant Listeria bacterium of any one of claims 1-22, further comprising a mutation in a ribC gene and / or a ribF gene.

24. The variant Listeria bacterium of claim 23, wherein the mutation comprises a deletion of all or a portion of the ribC gene and / or the ribF gene.

25. The variant Listeria bacterium of any one of claims 1-24, further comprising a mutation in an act A gene and / or an inlB gene.

26. The variant Listeria bacterium of claim 25, wherein the mutation comprises a deletion of all or a portion of the actA gene and / or the inlB gene.

27. A composition comprising: a) a variant Listeria bacterium of any one of claims 1-26; and b) a multispecific antibody.

28. The composition of claim 27, wherein the multispecific antibody 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.

29. The composition of claim 28, wherein the T cell is a y / 8 T cell.

30. An immunogenic composition comprising the variant Listeria of any one of claims 1-26.

31. 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 30.

32. The method of claim 31, wherein said immune response comprises a gamma-delta T cell response.

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