Listeria variants and methods of use thereof

Abstract

The present disclosure provides variant Listeria bacteria that lack functional ribC and ribF genes. Also provided are methods of making and using the variant Listeria bacteria, e.g., as vectors, vaccines, and therapeutics. The present disclosure provides variant Listeria bacteria that result in the production of mucosal-associated invariant T (MAIT) cell ligands in vivo. Also provided are methods of making and using the variant Listeria bacteria, e.g., to stimulate and increase the number of MAIT cells in an individual. TIFF2025502983000002.tif36169

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 304,235, filed January 28, 2022, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING U.S. GOVERNMENT SPONSORED RESEARCH This invention was made with Government support under Grant No. AI027655 and Grant No. AI063302 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Introduction Listeria monocytogenes is a gram-positive foodborne human and animal pathogen that is responsible for severe infections in immunocompromised individuals and pregnant women. Severe L. monocytogenes infections in humans are characterized by meningitis, meningoencephalitis, septicemia, and fetal death. L. monocytogenes is ubiquitous in nature and can be isolated from a wide variety of warm-blooded animals.

[0004] L. monocytogenes elicits a predominantly cell-mediated immune response when inoculated into animals. Therefore, L. monocytogenes is widely used as an experimental model to study many aspects of infection and immunity. Importantly, long-term cell-mediated immunity (CMI) is induced by infection of mice with sublethal doses of L. monocytogenes. In preclinical studies, attenuated strains of L. monocytogenes have shown tremendous potential as recombinant vaccine vectors. Attenuated recombinant strains have shown clinical efficacy as therapeutic vaccines for cancer immunotherapy.

[0005] Most known prokaryotes rely on a single bifunctional enzyme, encoded by the ribC (or ribF in some organisms) gene, called riboflavin kinase / FAD synthetase (FADS), to convert riboflavin (RF) first to flavin mononucleotide (FMN) and then to flavin adenine dinucleotide (FAD). Listeria has two separate enzymes encoded by the genes ribC and ribF. Summary of the Invention

[0006] overview The present disclosure provides variant Listeria bacteria that lack functional ribC and ribF genes. Also provided are methods of making and using the variant Listeria bacteria, e.g., as vectors, vaccines, and therapeutics. The present disclosure provides variant Listeria bacteria that result in the production of mucosal-associated invariant T (MAIT) cell ligands in vivo. Also provided are methods of making and using the variant Listeria bacteria, e.g., to stimulate and increase the number of MAIT cells in an individual. [Brief description of the drawings]

[0007] [Figure 1] Figures 1A-1D. Figure 1A shows the structures of riboflavin, FMN, and FAD. Figure 1B shows the broth growth curves of L. monocytogenes strains grown in rich medium. Figures 1C-1D show the colony forming units (CFU) of L. monocytogenes strains in the spleen (C) and liver (D) of mice 48 hours after intravenous infection with 1x105 CFU of each strain. [Diagram 2] Figures 2A-2B. Figure 2A shows broth growth curves of L. monocytogenes strains grown in chemically defined medium lacking flavins. Figure 2B shows images of culture supernatants of wild-type (left) and ΔribU+ribDEAHT strains (right) after 24 h of growth with shaking at 37 °C. [Diagram 3]Figures 3A-3D. Figure 3A shows the percentage of L. monocytogenes strains that colocalized with the autophagy receptor p62 in infected bone marrow-derived macrophages (BMMs). Figures 3B-C show intracellular growth curves of L. monocytogenes strains in BMMs. (B) shows growth curves of the indicated L. monocytogenes strains in wild-type BMMs. (C) shows growth curves of the indicated flavin-starved L. monocytogenes strains in wild-type BMMs lacking riboflavin (for 3 h) and supplemented with 1 μM riboflavin immediately prior to infection. Figure 3D shows generation times between 2 and 5 h for L. monocytogenes strains growing intracellularly in riboflavin-sufficient and riboflavin-deficient BMMs. [Figure 4] Figures 4A-4F. Figures 4A-C show intracellular growth curves of L. monocytogenes strains in BMM. (A) shows growth curves of the indicated L. monocytogenes strains in wild-type BMM. (B) shows growth curves of the indicated L. monocytogenes strains in wild-type BMM incubated in cell medium containing excess (10 μM) riboflavin over the course of infection. (C) shows growth curves of the indicated flavin-deficient L. monocytogenes in riboflavin-deficient wild-type BMM. Figures 4D-E show cell death of wild-type (D) or AIM2 KO (E) BMM infected with specific L. monocytogenes strains. Figure 4F shows intracellular growth curves of the indicated L. monocytogenes strains in AIM2 KO BMM. [Diagram 5]Figures 5A-5F. Figures 5A-C show broth growth curves of L. monocytogenes strains grown in chemically defined media containing different flavin sources. (A) shows the growth curve of wild-type L. monocytogenes grown in media containing riboflavin, FMN, or FAD as the sole flavin source. (B) shows the growth curve of ΔribU mutant L. monocytogenes strains grown in media containing riboflavin, FMN, or FAD as the sole flavin source. (C) shows the growth curve of the indicated L. monocytogenes strains grown in media containing riboflavin. Figure 5D shows the intracellular growth curves of L. monocytogenes strains in mouse BMM. Figures 5E-5F show the bacterial load in the spleen (C) and liver (D) 48 h postinfection of CD-1 mice infected intravenously with 1x105 CFU of the indicated L. monocytogenes strains. [Figure 6] Figures 6A-6C. Figure 6A shows in vitro growth of L. monocytogenes strains in defibrinated sheep blood. Figure 6B shows bacterial burden in the gallbladder 48 hours after infection of CD-1 mice infected intravenously with 1x105 CFU of the indicated L. monocytogenes strains. Figure 6C shows bacterial burden in the gastrointestinal tract of CD-1 mice infected orally with 1x108 CFU of the indicated L. monocytogenes strains. [Figure 7] FIG. 1 is a diagram of the riboflavin operon introduced into the L. monocytogenes chromosome with both pHyper (constitutive promoter) and pNative (inducible, native promoter from B. subtilis) shown. [Figure 8] 1 shows intracellular growth curves of L. monocytogenes strains in mouse BMMs. [Figure 9] Bacterial burden in the spleen and liver of B6 mice infected intravenously with 1x103 CFU of the indicated L. monocytogenes strains at 96 hours post-infection is shown. [Figure 10A] 10A-10D. Shows the effect of infection with an attenuated riboflavin-producing L. monocytogenes strain in an ActA minus background. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 11A] 11A-11B. Shows the effect of perforin-minus MAIT cells in riboflavin-producing L. monocytogenes. [Figure 11B] See legend to Figure 11A. [Figure 12] 1 shows intracellular growth curves of L. monocytogenes strains in wild-type mouse bone marrow-derived macrophages (BMMs). [Figure 13] 1 shows the bacterial burden in the spleen and liver of CD-1 mice infected intravenously with various L. monocytogenes strains 47 hours after infection. [Figure 14] 1 shows growth of L. monocytogenes ΔactAΔinlB, ΔactAΔinlB+ΔribCΔribF, and ΔactAΔinlB+ΔribCΔribF+eetB::tn strains in defibrinated sheep blood. [Figure 15] 1 shows the effect of vaccination with the ΔactA and ΔactAΔribCΔribF mutant strains of L. monocytogenes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description The present disclosure provides variant Listeria bacteria that lack functional ribC and ribF genes. Also provided are methods of making and using the variant Listeria bacteria, e.g., as vectors, vaccines, and therapeutics. The present disclosure provides variant Listeria bacteria that result in the production of mucosal-associated invariant T (MAIT) cell ligands in vivo. Also provided are methods of making and using the variant Listeria bacteria, e.g., to stimulate and increase the number of MAIT cells in an individual.

[0009] Before the present invention is further described, it should be understood that the invention is not limited to particular embodiments described, as such may, of course, vary. It should also 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 is limited only by the appended claims.

[0010] Where a range of values ​​is provided, it is to be understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value in that stated range, to the tenth of the unit of the lower limit, is encompassed within the invention, unless the context clearly dictates otherwise. 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.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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 described herein. All publications mentioned herein are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0012] It should 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, a reference to "a variant Listeria bacterium" includes a plurality of such variant Listeria bacteria, a reference to "the immunogenic composition" includes a reference to one or more immunogenic compositions 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. Thus, this statement is intended to serve as a predicate for the use of exclusive language, such as "solely," "only," and the like, or the use of "negative" limitations in connection with the recitation of claim elements.

[0013] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the invention are specifically embraced by the invention and are disclosed herein as if each and every combination were individually and expressly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0014] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0015] Variant Listeria spp. containing mutations in genes required for FMN and FAD biosynthesis The present disclosure provides a variant Listeria bacterium that contains mutations in genes required for flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) biosynthesis, and the variant Listeria is a facultative obligate intracellular bacterium in vivo (i.e., unable to grow extracellularly in a mammal). The variant Listeria can grow (culture) in vitro in a medium supplemented with FMN and FAD, but cannot grow extracellularly in vivo (e.g., in a mammal). The variant Listeria of the present disclosure requires supplementation of flavin mononucleotide and flavin adenine dinucleotide to grow, and thus is a facultative obligate intracellular bacterium when in vivo (e.g., when in a mammal or other animal host).

[0016] Genes required for FMN and FAD biosynthesis include ribC and ribF. The present disclosure provides variant Listeria bacteria lacking functional ribC and ribF genes. Variant Listeria bacteria can be generated by genetically modifying a parent Listeria cell such that the ribC and ribF genes are non-functional. A non-functional gene can be a completely deleted gene; a 5%-95% deleted gene (i.e., 5%-95% of the nucleotides are deleted compared to the corresponding wild-type gene); a gene having an insertion of one or more nucleotides (e.g., 1-10, 10-100, or more than 100 nucleotides) compared to the corresponding wild-type gene, where the insertion of the one or more nucleotides renders the gene non-functional, etc. In some cases, between 5% and 100% (e.g., between 5% and 10%, 10% and 20%, 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 100%) of the ribC genes and / or ribF genes are deleted to generate a variant Listeria of the present disclosure.

[0017] In some cases, the variant Listeria bacteria of the present disclosure are ΔribC / ΔribF bacteria, e.g., the ribC and ribF genes are deleted. 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.

[0018] The Listeria cell (e.g., parent Listeria cell) used to generate the variant Listeria bacteria of the present disclosure can be any one of several different Listeria species, and is usually a riboflavin-requiring strain. Listeria species of interest include, but are not limited to, L. fleischmannii, L. innocua, L. ivanovii, L. marthii, L. monocytogenes, L. rocourtiae, L. seeligeri, L. weihenstephanensis, and L. welshimeri. Thus, Listeria strains other than L. monocytogenes can also be host cells. In certain cases, the Listeria strain is L. monocytogenes.

[0019] In some cases, the Listeria host cell is attenuated. "Attenuation" and "attenuated" encompass Listeria host cells that have been modified to reduce virulence. The host may be a human or animal host, or an organ, tissue, or cell. By way of non-limiting example, the Listeria host cell may be attenuated to reduce binding to a host cell (e.g., a human or non-human animal cell, e.g., a human or non-human mammalian cell), reduce spread from one host cell to another, reduce extracellular proliferation, or reduce intracellular proliferation in a host cell. Attenuation may be assessed, for example, by measuring an index of virulence, LD50, clearance rate from an organ, or competitive index (see, e.g., Auerbuch, et al. (2001) Infect. Immunity 69:5953-5957). Typically, attenuation results in an increase in the LD50 (lethal dose, 50%, the dose (number of bacteria) required to kill half the members of a tested population after a particular test period) and / or an increase in the clearance rate by at least 25%, more typically at least 50%, most typically at least 100% (2-fold), typically at least 5-fold, more typically at least 10-fold, most typically at least 50-fold, often at least 100-fold, more often at least 500-fold, most often at least 1000-fold, usually at least 5000-fold, more typically at least 10,000-fold, most typically at least 50,000-fold, and most often at least 100,000-fold. Attenuation can also be assessed by measuring the number of colony forming units (CFU).

[0020] In certain embodiments, the attenuated Listeria of the present disclosure exhibits reduced virulence compared to the corresponding wild-type strain in a 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 bacterial strains. After a period of time, e.g., 48-60 hours, the inoculated mice are sacrificed and the abundance of bacteria in one or more organs, e.g., liver, spleen, is evaluated. In these embodiments, a given bacterial strain is considered to be less pathogenic if its abundance in the spleen is at least about 50-fold less, e.g., 70-fold less, than that observed in the corresponding wild-type strain, and / or if its abundance in the liver is at least about 10-fold less, e.g., 20-fold less, than that observed in the corresponding wild-type strain.

[0021] In yet another embodiment, the bacteria is considered to be less virulent if it shows failure to replicate for less than about 8 hours, for example, less than about 6 hours, including less than about 4 hours, as measured using the assay described in Jones and Portnoy, Intracellular growth of bacteria. (1994b) Methods Enzymol. 236:463-467. In yet another embodiment, the bacteria is considered to be attenuated or less virulent if it forms smaller plaques in a plaque assay as compared to wild type, as employed in U.S. Pat. No. 7,794,728, the disclosure of which is incorporated herein by reference. In the assay, cells, for example, mouse L2 cells, are grown to confluency, for example, in a 6-well tissue culture dish, and then infected with the bacteria. DME agar containing gentamicin is then added, and plaques are allowed to grow for a period of time, for example, 3 days. Live cells are then visualized, for example, by adding an additional DME agar overlay containing neutral red (GIBCO BRL), and incubated overnight. In such assays, the degree of reduction in plaque size observed for the attenuated mutant compared to the wild type is, in certain embodiments, 10%, including 15%, for example, 25% or more.

[0022] Attenuated bacteria may contain one or more different mutations that confer an attenuated phenotype, with mutations of interest including, for example, hly and / or lplA mutations as described in U.S. Pat. No. 7,794,728, the disclosure of which is incorporated herein by reference, and actA and / or internalin B (InlB) mutations as reported in Dung et al., Clin. Cancer Res. (2012) 18:858-868. Thus, in some cases, variant Listeria of the present disclosure contain mutations in actA (encoding the acting assembly-inducing protein ActA) and / or inlB (encoding internalin B), in addition to mutations that render the ribC and ribF genes non-functional (e.g., by deletion of all or part of the ribC and ribF genes). In some cases, a variant Listeria of the present disclosure includes a) a deletion of all or a portion of ribC, which renders the ribC gene non-functional; b) a deletion of all or a portion of ribF, which renders the ribF gene non-functional; c) a deletion of all or a portion of actA, which renders the actA gene non-functional; and d) a deletion of all or a portion of inlB, which renders the inlB gene non-functional.

[0023] The variant Listeria bacteria of the present disclosure may contain a deletion of all or a portion of the eetB gene in addition to the ribC / ribF modifications described above. Deleting all or a portion of the eetB gene may provide a growth advantage. In some cases, such variant strains exhibit an increased growth rate in broth compared to the growth rate in broth of Listeria bacteria containing ribC / ribF modifications and lacking the deletion of the eetB gene in vitro. In some cases, the variant Listeria of the present disclosure contains a) a deletion of all or a portion of ribC, which renders the ribC gene non-functional, b) a deletion of all or a portion of ribF, which renders the ribF gene non-functional, and c) a deletion of all or a portion of eetB, which renders the eetB gene non-functional. In some cases, a variant Listeria of the present disclosure includes a) a deletion of all or a portion of ribC, which renders the ribC gene non-functional; b) a deletion of all or a portion of ribF, which renders the ribF gene non-functional; c) a deletion of all or a portion of actA, which renders the actA gene non-functional; d) a deletion of all or a portion of inlB, which renders the inlB gene non-functional; and e) a deletion of all or a portion of eetB, which renders the eetB gene non-functional.

[0024] The variant Listeria bacteria of the present disclosure may contain one or more genetic modifications in addition to the ribC / ribF modifications described above, where the one or more further modifications provide desirable properties in a host cell, such as attenuation, improved immunogenicity, etc. Examples of such further modifications include, but are not limited to, those described in PCT published applications 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 are incorporated herein by reference.

[0025] In addition to the ribC / ribF modifications described above, variant Listeria bacteria of the present disclosure can include an Lm-RIID (L. monocytogenes recombinase-induced intracellular death) mutation. See, e.g., USPN 9,511,129.

[0026] The bacteria can be live or dead but metabolically active ("KBMA"). The KBMA vaccine strain is constructed by abolishing the ability of nucleotide excision repair through the deletion of DNA repair genes, e.g., uvrA and uvrB. The deletion of the genes renders the bacteria vulnerable to photochemical inactivation through combined treatment with psoralen and UVA. Due to the inability to repair the formed psoralen-induced DNA crosslinks, the KBMA bacterial strains are unable to replicate and are therefore functionally non-infectious. This property improves the safety profile compared to live attenuated strains. However, the number of crosslinks is very limited, preserving their metabolic activity, including antigen expression, and thus their immunocompetence. The KBMA vaccine strain is described in U.S. Pat. No. 7,833,775, the disclosure of which is incorporated herein by reference.

[0027] In certain examples, the variant Listeria bacteria of the present disclosure express a heterologous antigen, which in certain embodiments can protect an animal from challenge with the infectious agent from which the heterologous antigen is derived, or can affect tumor growth and metastasis, beneficial to the host organism. Thus, heterologous antigens that can be introduced into the Listeria strains of the present disclosure by DNA encoding the heterologous antigen include any antigen that, when expressed by Listeria, serves to induce a cellular immune response that is beneficial to the host to which the response is induced. Thus, heterologous antigens include those that are specified by an infectious agent, and an immune response to the antigen serves to prevent or treat a disease caused by the agent. Such heterologous antigens include, but are not limited to, surface proteins of viruses, bacteria, fungi, or parasites, and any other proteins, glycoproteins, lipoproteins, glycolipids, and the like. Heterologous antigens include tumor antigens. Heterologous antigens also include those that provide benefit to a host organism at risk of or diagnosed with a tumor expressing the heterologous antigen. The host organism may be a mammal, for example a human.

[0028] As used herein, the term "heterologous antigen" refers to a protein or peptide, glycoprotein or glycopeptide, lipoprotein or lipopeptide, or any other macromolecule not normally expressed in Listeria, which corresponds substantially to the same antigen in an infectious agent, tumor cell, or tumor-associated protein. The heterologous antigen is expressed by a Listeria strain according to the present disclosure, and is processed and presented to cytotoxic T cells after infection of a mammalian cell with the strain. The heterologous antigen expressed by the Listeria species does not have to exactly match the corresponding unmodified antigen or protein in the tumor cell or infectious agent, as long as it results in a T cell response that recognizes the unmodified antigen or protein naturally expressed in the mammal. In other examples, the tumor cell antigen may be a mutated version of one naturally expressed in a mammal, and the antigen expressed by the Listeria species matches the tumor cell mutated antigen. As used herein, the term "tumor-associated antigen" refers to an antigen that affects tumor growth or metastasis in a host organism. The tumor-associated antigen may be an antigen expressed by tumor cells or an antigen expressed by non-tumor cells, which when so expressed promotes tumor cell growth or metastasis. The types of tumor and tumor-associated antigens that can be introduced into Listeria by incorporating DNA encoding the tumor and tumor-associated antigens include any known or previously unknown tumor antigen. In another example, the "tumor-associated antigen" does not affect tumor growth or metastasis, but is specifically expressed in the tissue (and tumor) from which the tumor originates, and is therefore used as a component of a Listeria vaccine. In yet another example, the "tumor-associated antigen" does not affect tumor growth or metastasis, but is selectively expressed in the tumor cells and not in any other normal tissue, and is therefore used as a component of a Listeria vaccine.

[0029] Heterologous antigens useful in vaccine development may be selected using knowledge available to those skilled in the art, and many antigenic proteins are currently known that are expressed by tumor cells or that affect tumor growth or metastasis, or that are expressed by infectious agents. For example, viral antigens that may be considered 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, the env protein of HIV or its components gp120 and gp41, the nef protein of HIV, and the pol protein, reverse transcriptase, and protease of HIV. Still other heterologous antigens may be those associated with the Hepatitis C virus (HCV), including, but not limited to, the E1 and E2 glycoproteins, and nonstructural (NS) proteins, such as NS3. Additionally, other viral antigens, such as herpes virus proteins, may also be useful. The heterologous antigens need not be limited to being of viral origin. Included are parasitic antigens, such as malarial antigens, and also fungal antigens, bacterial antigens, and tumor antigens.

[0030] As described herein, several proteins expressed by tumor cells are also known and are of interest as heterologous antigens that can be inserted into the vaccine strains of the present invention. These include, but are not limited to, the bcr / abl antigens in leukemia, the HPVE6 and E7 antigens of oncogenic viruses 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 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, L1 cell adhesion molecule, melanoma-associated antigen (MAGE), MAGE-A1, mesothelin, MUC1, NKG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein-72 (TAG-72), vascular endothelial growth factor receptor-2 (VEGF-R2), and epidermal growth factor receptor (EGFR) vIII polypeptides, etc. Other coding sequences of interest include, but are not limited to, costimulatory molecules, immunomodulatory molecules, etc.

[0031] The bacteria described herein can be produced using a variety of different protocols. Thus, the production of the subject attenuated bacteria can be achieved in several ways well known to those skilled in the art, including deletion mutagenesis, insertion mutagenesis, point mutations, mutagenesis resulting in the production of frameshift mutations, mutations resulting in premature ends of proteins, and mutations of regulatory sequences that affect gene expression. Mutagenesis can be achieved using recombinant DNA technology, or using conventional mutagenesis techniques that use mutagenic chemicals or radiation, followed by selection of mutants. Representative protocols of the various methods of producing bacteria according to the present invention are provided in the experimental section below.

[0032] Introduction of DNA encoding a heterologous antigen into a Listeria strain may be accomplished, for example, by creation of a recombinant Listeria in which the DNA encoding the heterologous antigen is carried on a vector, such as a plasmid, which is maintained and expressed in the Listeria species, with the antigen expression being under the control of a prokaryotic promoter / regulatory sequence. Alternatively, the DNA encoding the heterologous antigen may be stably integrated into the Listeria chromosome, for example, by using transposon mutagenesis, homologous recombination, or integrase-mediated site-specific integration (as described in application Ser. No. 10 / 136,860, the disclosure of which is incorporated herein by reference).

[0033] As will be appreciated by those of skill in the art after being given the present disclosure, several approaches can be used to express heterologous antigens in Listeria spp. In certain embodiments, the gene encoding the heterologous antigen is designed to promote secretion of the heterologous antigen from the bacterium or to promote expression of the heterologous antigen on the Listeria cell surface.

[0034] In certain embodiments, a fusion protein is used that includes the desired heterologous antigen and a Listeria secreted or cell surface protein. Listeria proteins that are suitable components of such a fusion protein include, but are not limited to, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase (PI-PLC). Fusion proteins can be generated by ligating genes encoding 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 that includes both the heterologous antigen and the Listeria protein. Expression of the ligated genes can be placed under the transcriptional control of a Listeria promoter / regulatory sequence such that expression of the genes is effected during growth and replication of the organism. Signal sequences for cell surface expression and / or secretion of the fusion protein can also be added to the gene encoding the heterologous antigen to effect cell surface expression and / or secretion of the fusion protein. If the heterologous antigen is used alone (i.e., in the absence of fused Listeria sequences), it may be advantageous to fuse a signal sequence to it for cell surface expression and / or secretion of the heterologous antigen. Procedures for accomplishing this are well known in the art of bacteriology and molecular biology.

[0035] The DNA encoding the heterologous antigen to be expressed is, in some embodiments, preceded by an appropriate promoter to facilitate such expression. The appropriate promoter / regulatory and signal sequences used will depend on the type of Listeria protein desired in the fusion protein and will be readily apparent to one skilled in the art of Listeria molecular biology. For example, L. monocytogenes promoter / regulatory and / or signal sequences that can be used to direct expression of a fusion protein include, but are not limited to, sequences from the Listeria hly gene, which encodes LLO, the Listeria p60(iap) gene, and the Listeria actA gene, which encodes a surface protein required for L. monocytogenes actin assembly. Other promoter sequences of interest include the Listeria plcA gene, which encodes PI-PLC, the Listeria mpl gene, which encodes a metalloprotease, and the Listeria inlA gene, which encodes the Listeria membrane protein internalin. The heterologous regulatory elements, such as promoters from phages and promoters or signal sequences from other bacterial species, can be used to express heterologous antigens by the Listeria species. Another suitable promoter is the constitutive HyPer promoter, see, e.g., Renier et al. (2016) PLoS Pathogens doi.org / 10.1371 / journal.ppat.1005741.

[0036] In certain embodiments, the attenuated Listeria comprises a vector. The vector may comprise DNA encoding a heterologous antigen. In some cases, the vector is a plasmid capable of replicating in Listeria. The vector may encode a heterologous antigen, and the expression of the antigen is under the control of a eukaryotic promoter / regulatory sequence, e.g., in an expression cassette. Exemplary plasmids with suitable promoters of interest include, but are not limited to, pCMV beta, which comprises the immediate early promoter / enhancer region of human cytomegalovirus, and those which comprise the SV40 early promoter region or the mouse mammary tumor virus LTR promoter region.

[0037] Thus, in certain embodiments, the subject bacterium comprises at least one coding sequence of the heterologous polypeptide / protein described above. In some cases, the coding sequence is devoid of introns and is a continuous open reading frame with the same sequence as a cDNA sequence that can be produced, for example, from a chromosomal sequence. In some embodiments, the coding sequence is part of an expression cassette that provides expression of the coding sequence in the Listeria cell for which the vector is designed. As used herein, the term "expression cassette" refers to an expression module or expression construct that is composed of a recombinant DNA molecule that contains at least one desired coding sequence and the appropriate nucleic acid sequences, such as the promoter / regulatory / signal sequences identified above, required for the expression of the coding sequence in a particular host organism, i.e., the Listeria cell for which the vector is designed, in an operably linked manner, and the expression cassette may contain coding sequences for two or more different polypeptides, or multiple copies of the same coding sequence, as desired. Thus, the size of the coding product may vary greatly, and a wide range of different products may be encoded by the expression cassette present in the vector of this embodiment.

[0038] As noted above, the vector may contain at least one coding sequence, and in certain embodiments, the vector may contain two or more coding sequences, which may code for products that act together to provide a desired result. In general, the coding sequence may code for any of a number of different products and may be of a variety of different sizes, and the above discussion merely provides representative coding sequences of interest.

[0039] Compositions Comprising Variant Listeria spp. The present disclosure provides compositions comprising a variant Listeria of the present disclosure (variant Listeria comprising one or more mutations in one or more genes required for FMN and FAD biosynthesis, e.g., a ΔribCΔribF variant). The compositions of the present disclosure may further comprise, in addition to the variant Listeria of the present disclosure, a salt (e.g., NaCl, MgCl 2 , KCl, MgSO 4 etc.), buffering agent, etc. In some cases, the compositions of the present disclosure include, in addition to the variant Listeria of the present disclosure, saline. In some cases, the compositions of the present disclosure further include a multispecific antibody (e.g., a bispecific antibody). The multispecific antibody is optionally a bispecific T cell-inducing (BiTE) antibody. A multispecific antibody comprises a first antigen-binding site specific for a cancer-associated antigen and a second antigen-binding site specific for a T cell (e.g., mucosal-associated invariant T (MAIT) cell, γ / δ T cell, CD8 + The antibody may comprise a second antigen-binding site specific for cytotoxic T cells, natural killer (NK) cells.

[0040] The present disclosure provides an immunogenic composition (also referred to herein as a "vaccine composition") comprising a variant Listeria of the present disclosure. The immunogenic composition of the present disclosure may comprise a) a variant Listeria of the present disclosure, and b) an antigen. Suitable antigens include, but are not limited to, cancer-associated antigens, pathogen-associated antigens (e.g., viral antigens, pathogenic animal antigens, etc.).

[0041] In some cases, a composition comprising a variant Listeria of the present disclosure (a variant Listeria comprising one or more mutations in one or more genes required for FMN and FAD biosynthesis, e.g., a ΔribCΔribF variant) comprises a unit dose of the variant Listeria. The unit dose of the variant Listeria comprises 10 or more units per dose. 4 ~10 10 For example, in some cases, an "effective amount" of a variant Listeria of the present disclosure (a variant Listeria containing one or more mutations in one or more genes required for FMN and FAD biosynthesis, e.g., a ΔribCΔribF variant) may range from 10 to 100 micrograms per unit dose.4 ~5x10 4 , 5x10 4 ~10 5 , 10 5 ~5x10 5 , 5x10 5 ~10 6 , 10 6 ~5x10 6 , 5x10 6 ~10 7 , 10 7 ~5x10 7 , 5x10 7 ~10 8 , 10 8 ~10 9 , or 10 9 ~10 10 The disclosure further provides kits comprising unit doses of variant Listeria.

[0042] usefulness The above bacteria (variant Listeria containing one or more mutations in one or more genes required for FMN and FAD biosynthesis, e.g., ΔribCΔribF variants) are used in several different applications. Representative uses of the subject bacteria include, but are not limited to, (a) immunogens for generating antibodies against Listeria species, (b) adjuvant compositions in immunization protocols, (c) vectors for introducing macromolecules, e.g., nucleic acids or proteins, into the cytoplasm of target cells, and (d) vaccine compositions, e.g., for eliciting or enhancing a cellular immune response in a host. Each of these representative applications will now be further described individually below. The use of attenuated Listeria spp. is also described in U.S. Patent Nos. 8,679,476, 8,277,797, 8,192,991, 7,842,289, 7,794,728, 7,749,510, 7,488,487, 7,425,449, 6,599,502, 6,504,020, 6,287,556, 6,099,848, 6,004,815, 5,830,702, and 5,643,599, the disclosures of which are incorporated herein by reference.

[0043] vaccine The subject bacterium (a variant Listeria containing one or more mutations in one or more genes required for FMN and FAD biosynthesis, e.g., a ΔribCΔribF variant) is used as a vaccine (also referred to herein as an "immunogenic composition"). The vaccine of the present disclosure is administered to a vertebrate by contacting the vertebrate with a sublethal amount of an attenuated Listeria vaccine, which contact typically involves administering the vaccine to the host. In some embodiments, the bacterium is 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 routes of administration. For example, in livestock, the vaccine can be administered orally by incorporating the vaccine in feed or liquid (e.g., water). It can 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 maintains the antigenicity of the vaccine. Any one of several well-known pharma- ceutically acceptable diluents or excipients may be used in the vaccine of the present invention. Suitable diluents include, for example, sterile distilled water, physiological saline, phosphate buffer, and the like. The amount of diluent may vary widely, as recognized by those skilled in the art. Suitable excipients are also well-known to those skilled in the art and may be selected, for example, from A. Wade and PJ Weller, eds., Handbook of Pharmaceutical Excipients (1994) The Pharmaceutical Press: London. The dose administered may depend on the age, health, and weight of the patient, the type of patient, and the presence of concomitant therapy, if any. The vaccine may be used in a dosage form for oral administration, such as capsules, solutions, suspensions, or elixirs, or in a formulation for parenteral, intranasal, intramuscular, or intravascular use, such as sterile liquids for solutions or suspensions. According to the present invention, the vaccine may be used in combination with a pharma-ceutically acceptable diluent as a vaccine composition useful for immunizing a patient against infection with a selected organism or virus, or for tumors, etc.Immunizing a patient means providing the patient with at least some degree of therapeutic or prophylactic immunity against a selected pathogen, cancer cell, and the like.

[0044] The subject vaccines are used in methods for inducing or enhancing a cellular immune response, e.g., a helper T cell or a cytotoxic T cell response, in a vertebrate against a selected agent, e.g., a pathogenic organism, a tumor, etc., comprising administering an effective amount of the Listeria vaccine. The subject vaccines are used in methods for inducing an innate immune response in a vertebrate that enhances the antigen-specific immune response. Additionally, the vaccines of the invention can be used for post-exposure or post-diagnosis treatment. In general, the use of vaccines for post-exposure treatment will be recognized by those skilled in the art, e.g., in the treatment of rabies and tetanus. The same vaccine of the invention can be used for both immunization and post-exposure immune enhancement, for example. Alternatively, a different vaccine of the invention, e.g., specific for an antigen expressed at a later stage of exposure, may be used for post-exposure treatment. Thus, the subject vaccines prepared using the subject vectors are used as both prophylactic and therapeutic vaccines to induce immune responses specific for antigens associated with various disease states.

[0045] The patient may be any human or non-human animal susceptible to infection with the selected organism. The subject vaccines are particularly useful for vertebrate animals, such as mammals (including humans and non-human mammals), and for livestock, including poultry, cattle, pigs, sheep, horses, goats, dogs, cats, lagomorphs (e.g., rabbits), or other non-human animals.

[0046] The subject vaccines are used for vaccination applications as described in PCT Published Applications Nos. WO2014 / 106123, WO2014 / 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 are incorporated herein by reference.

[0047] Adjuvant Compositions The subject bacterial strains (variant Listeria containing one or more mutations in one or more genes required for FMN and FAD biosynthesis, e.g., ΔribCΔribF variants) are also used as immune enhancing agents, i.e., as adjuvants. In such applications, the subject attenuated bacteria can be administered in combination with an immunogen, e.g., tumor antigens, modified tumor cells, etc., according to methods known in the art in which live bacterial strains are used as adjuvants. See, e.g., Berd et al., Vaccin 2001 Mar 21;19(17-19):2565-70.

[0048] In some embodiments, the bacterial strains are used as adjuvants by chemically coupling to a sensitizing antigen, which can be any antigen of interest, with representative antigens of interest including viral agents such as herpes simplex virus, malaria parasites, bacteria such as staphylococcus aureus bacteria, diphtheria toxoid, tetanus toxoid, schistosoma, tumor cells such as CAD, and the like. 2 Breast adenocarcinoma tumor cells, as well as hormones such as thyroxine T 4 , Triiodothyronine T 3These include, but are not limited to, cyclosporine, cyclohexyl ester ...

[0049] delivery vehicle The subject bacteria (variant Listeria containing one or more mutations in one or more genes required for FMN and FAD biosynthesis, e.g., ΔribCΔribF variants) are also used as vectors or vehicles for the delivery of macromolecules to target cells, as described, for example, in PCT Publication No. WO 00 / 09733, the disclosures of which are incorporated herein by reference, and Dietrich et al., Nature Biotechnology (1998) 16:181-185. As described in these publications, a variety of different types of macromolecules can be delivered, including, but not limited to, nucleic acids, polypeptides / proteins, etc.

[0050] Variant Listeria spp. producing MAIT cell ligands The present disclosure provides variant Listeria that result in the production of the mucosal-associated invariant T (MAIT) cell ligand 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) and activates and stimulates MAIT cells. In some cases, the variant Listeria of the present disclosure that produces the MAIT cell ligand also produces riboflavin. The present disclosure provides variant Listeria that produces riboflavin (i.e., synthesizes riboflavin de novo) and activates and stimulates the proliferation of MAIT cells in vivo. The riboflavin-producing MAIT cell-stimulating variant Listeria of the present disclosure is also referred to herein as "ribDEAHT variant Listeria." The ribDEAHT variant Listeria of the present disclosure is genetically modified to include a riboflavin operon ("ribDEAHT operon") derived from a bacterium other than Listeria. Thus, the ribDEAHT variant Listeria of the present disclosure is genetically modified to include a heterologous ribDEAHT operon (i.e., a ribDEAHT operon derived from a bacterium other than Listeria). The heterologous ribDEAHT operon can be derived from any riboflavin-synthesizing bacterium. For example, the heterologous ribDEAHT operon can be derived from a Gram-positive bacterium, such as a Bacillus species, e.g., B. subtilis. As shown in FIG. 7, the heterologous ribDEAHT can include ribD, ribE, ribA, ribH, and ribT genes, which can be operably linked to a transcriptional regulator (e.g., a promoter), which can be constitutive or inducible.

[0051] In some cases, the variant Listeria of the present disclosure that produces the MAIT cell ligand 5-OP-RU (and can activate and stimulate proliferation of MAIT cells) does not produce riboflavin. An example of such a variant Listeria is one that is genetically modified with a heterologous ribD gene and a heterologous ribA gene. Such variant Listeria are referred to herein as "ribDA variant Listeria."

[0052] The ribDEAHT variant Listeria and ribDA variant Listeria of the present disclosure are collectively referred to herein as "5-OP-RU precursor-producing variant Listeria." The 5-OP-RU precursor-producing variant Listeria of the present disclosure produce the precursor 5-amino-6-(D-ribitylamino)uracil (5-A-RU), which reacts with an endogenous electrophile called methylglyoxal or glyoxal to produce 5-OP-RU.

[0053] In some cases, the 5-OP-RU precursor-producing variant Listeria of the present disclosure also has a mutation in the gloA gene (e.g., gloA - strain). See, e.g., Anaya-Sanchez et al. (2021) PLoS Pathogens 17:e1009819.

[0054] In some cases, the ribDEAHT variant Listeria of the present disclosure has a deletion in the ribU gene such that it does not produce a ribU protein. Thus, for example, in some cases, the ribDEAHT variant Listeria of the present disclosure is a ΔribU variant.

[0055] The Listeria host cell used to produce the ribDEAHT variant Listeria of the present disclosure or the ribDA variant Listeria of the present disclosure (i.e., the 5-OP-RU precursor-producing variant Listeria of the present disclosure) can be any one of several different Listeria species, and is usually a riboflavin-requiring strain. Listeria species of interest include, but are not limited to, L. fleischmannii, L. innocua, L. ivanovii, L. marsui, L. monocytogenes, L. leucourtiae, L. seeligeri, L. weihenstephanensis, and L. welshimeri. Thus, Listeria strains other than L. monocytogenes can also be host cells. In certain cases, the Listeria strain is L. monocytogenes.

[0056] In some cases, the Listeria host cell is attenuated. "Attenuation" and "attenuated" encompass Listeria host cells that have been modified to reduce virulence. The host may be a human or animal host, or an organ, tissue, or cell. By way of non-limiting example, the Listeria host cell may be attenuated to reduce binding to host cells, reduce spread from one host cell to another, reduce extracellular growth, or reduce intracellular growth in host cells. Attenuation may be assessed, for example, by measuring an index of virulence, LD50, clearance rate from an organ, or competitive index (see, for example, Auerbuch, et al. (2001) Infect. Immunity 69:5953-5957). Typically, attenuation results in an increase in the LD50 (lethal dose, 50%, the dose (number of bacteria) required to kill half of the members of a tested population after a particular test period) and / or an increase in the clearance rate by at least 25%, more typically at least 50%, most typically at least 100% (2-fold), typically at least 5-fold, more typically at least 10-fold, most typically at least 50-fold, often at least 100-fold, more typically at least 500-fold, most typically at least 1000-fold, more typically at least 5000-fold, more typically at least 10,000-fold, most typically at least 50,000-fold, and most typically at least 100,000-fold.

[0057] In certain embodiments, the attenuated Listeria of the present disclosure exhibits reduced virulence compared to the corresponding wild-type strain in a 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 bacterial strains. After a period of time, e.g., 48-60 hours, the inoculated mice are sacrificed and the abundance of bacteria in one or more organs, e.g., liver, spleen, is evaluated. In these embodiments, a given bacterial strain is considered to be less pathogenic if its abundance in the spleen is at least about 50-fold less, e.g., 70-fold less, than that observed in the corresponding wild-type strain, and / or if its abundance in the liver is at least about 10-fold less, e.g., 20-fold less, than that observed in the corresponding wild-type strain.

[0058] In yet another embodiment, the bacteria is considered to be less virulent if it shows failure to replicate for less than about 8 hours, for example, less than about 6 hours, including less than about 4 hours, as measured using the assay described in Jones and Portnoy, Intracellular growth of bacteria. (1994b) Methods Enzymol. 236:463-467. In yet another embodiment, the bacteria is considered to be attenuated or less virulent if it forms smaller plaques in a plaque assay as compared to wild type, as employed in U.S. Pat. No. 7,794,728, the disclosure of which is incorporated herein by reference. In the assay, cells, for example, mouse L2 cells, are grown to confluency, for example, in a 6-well tissue culture dish, and then infected with the bacteria. DME agar containing gentamicin is then added, and plaques are allowed to grow for a period of time, for example, 3 days. Live cells are then visualized, for example, by adding an additional DME agar overlay containing neutral red (GIBCO BRL), and incubated overnight. In such assays, the degree of reduction in plaque size observed for the attenuated mutant compared to the wild type is, in certain embodiments, 10%, including 15%, for example, 25% or more.

[0059] The attenuated bacteria may contain one or more different mutations that confer an attenuated phenotype, with mutations of interest including, for example, hly and / or lplA mutations as described in U.S. Patent No. 7,794,728, the disclosure of which is incorporated herein by reference, actA and / or internalin B (InlB) mutations as reported in Dung et al., Clin. Cancer Res. (2012) 18:858-868, etc. Thus, in some cases, the ribDEAHT variant Listeria of the present disclosure contains a mutation in actA and / or lnlB in addition to the heterologous ribDEAHT operon.

[0060] The variant Listeria bacteria of the present disclosure may contain one or more genetic modifications in addition to the heterologous ribDEAHT operon or heterologous ribDA gene, as described above, which further modify the one or more genes to provide desirable properties in a host cell, such as attenuation, improved immunogenicity, etc. Examples of such further modifications include, but are not limited to, those described in PCT published applications 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 are incorporated herein by reference.

[0061] The bacteria can be live or dead but metabolically active ("KBMA"). The KBMA vaccine strain is constructed by abolishing the ability of nucleotide excision repair through the deletion of DNA repair genes, e.g., uvrA and uvrB. The deletion of the genes renders the bacteria vulnerable to photochemical inactivation through combined treatment with psoralen and UVA. Due to the inability to repair the formed psoralen-induced DNA crosslinks, the KBMA bacterial strains are unable to replicate and are therefore functionally non-infectious. This property improves the safety profile compared to live attenuated strains. However, the number of crosslinks is very limited, preserving their metabolic activity, including antigen expression, and thus their immunocompetence. The KBMA vaccine strain is described in U.S. Pat. No. 7,833,775, the disclosure of which is incorporated herein by reference.

[0062] In certain examples, the ribDEAHT variant Listeria of the present disclosure or the ribDA variant Listeria of the present disclosure (5-OP-RU precursor-producing variant Listeria of the present disclosure) expresses a heterologous antigen. In certain embodiments, the heterologous antigen can protect an animal from challenge with the infectious agent from which the heterologous antigen is derived, or can affect tumor growth and metastasis, beneficial to the host organism. Thus, the heterologous antigen that can be introduced into the 5-OP-RU precursor-producing variant Listeria of the present disclosure by DNA encoding the heterologous antigen includes any antigen that, when expressed by Listeria, helps to induce a cellular immune response that is beneficial to the host to which the response is induced. Thus, heterologous antigens include those that are specified by infectious agents, and the immune response to the antigen plays a role in preventing or treating the disease caused by the agent. Such heterologous antigens include, but are not limited to, surface proteins of viruses, bacteria, fungi, or parasites, and any other proteins, glycoproteins, lipoproteins, glycolipids, etc. Heterologous antigens also include those that provide a benefit to a host organism at risk of developing or diagnosed as having a tumor that expresses the heterologous antigen. The host organism may be a mammal, such as a human.

[0063] As used herein, the term "heterologous antigen" refers to a protein or peptide, glycoprotein or glycopeptide, lipoprotein or lipopeptide, or any other macromolecule that is not normally expressed in Listeria, and corresponds substantially to the same antigen in an infectious agent, tumor cell, or tumor-associated protein. The heterologous antigen is expressed by the 5-OP-RU precursor-producing variant Listeria of the present disclosure, and is processed and presented to cytotoxic T cells after infection of mammalian cells with the strain. The heterologous antigen expressed by the 5-OP-RU precursor-producing variant Listeria of the present disclosure does not need to exactly match the corresponding unmodified antigen or protein in a tumor cell or infectious agent, as long as it produces a T cell response that recognizes the unmodified antigen or protein naturally expressed in a mammal. In another example, the tumor cell antigen may be a mutant of one naturally expressed in a mammal, and the antigen expressed by the Listeria species matches the tumor cell mutant antigen. As used herein, the term "tumor-associated antigen" refers to an antigen that affects tumor growth or metastasis in a host organism. The tumor-associated antigen may be an antigen expressed by tumor cells or an antigen expressed by non-tumor cells, which when so expressed promotes tumor cell growth or metastasis. The types of tumor and tumor-associated antigens that can be introduced into Listeria by incorporating DNA encoding the tumor and tumor-associated antigens include any known or previously unknown tumor antigen. In another example, the "tumor-associated antigen" does not affect tumor growth or metastasis, but is specifically expressed in the tissue (and tumor) from which the tumor originates, and is therefore used as a component of a Listeria vaccine. In yet another example, the "tumor-associated antigen" does not affect tumor growth or metastasis, but is selectively expressed in the tumor cells and not in any other normal tissue, and is therefore used as a component of a Listeria vaccine.

[0064] Heterologous antigens useful in vaccine development may be selected using knowledge available to those skilled in the art, and many antigenic proteins are currently known that are expressed by tumor cells or that affect tumor growth or metastasis, or that are expressed by infectious agents. For example, viral antigens that may be considered 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, the env protein of HIV or its components gp120 and gp41, the nef protein of HIV, and the pol protein, reverse transcriptase, and protease of HIV. Still other heterologous antigens may be those associated with the Hepatitis C virus (HCV), including, but not limited to, the E1 and E2 glycoproteins, and nonstructural (NS) proteins, such as NS3. Additionally, other viral antigens, such as herpes virus proteins, may also be useful. The heterologous antigens need not be limited to being of viral origin. Included are parasitic antigens, such as malarial antigens, and also fungal antigens, bacterial antigens, and tumor antigens.

[0065] As described herein, several proteins expressed by tumor cells are also known and are of interest as heterologous antigens that can be inserted into the vaccine strains of the present invention. These include, but are not limited to, the bcr / abl antigens in leukemia, the HPVE6 and E7 antigens of oncogenic viruses 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 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, L1 cell adhesion molecule, melanoma-associated antigen (MAGE), MAGE-A1, mesothelin, MUC1, NKG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein-72 (TAG-72), vascular endothelial growth factor receptor-2 (VEGF-R2), and epidermal growth factor receptor (EGFR) vIII polypeptides, etc. Other coding sequences of interest include, but are not limited to, costimulatory molecules, immunomodulatory molecules, etc.

[0066] In some cases, the 5-OP-RU precursor-producing variant Listeria of the present disclosure is genetically modified to produce a chimeric antigen receptor (CAR). The CAR can comprise an antigen-binding portion that binds to a cancer-associated antigen. The antigen-binding portion can be, for example, a single-chain Fv, a nanobody, etc.

[0067] Compositions Comprising ribDEAHT Variant Listeria or ribDA Variant Listeria The present disclosure provides compositions comprising variant Listeria of the present disclosure (ribDEAHT variant Listeria of the present disclosure, ribDA variant Listeria of the present disclosure). The compositions of the present disclosure may contain, in addition to the variant Listeria of the present disclosure, a salt (e.g., NaCl, MgCl 2 , KCl, MgSO 4etc.), buffering agent, etc. In some cases, the compositions of the present disclosure include, in addition to the variant Listeria of the present disclosure, saline. In some cases, the compositions of the present disclosure further include a multispecific antibody (e.g., a bispecific antibody). The multispecific antibody is optionally a bispecific T cell-inducing (BiTE) antibody. A multispecific antibody comprises a first antigen-binding site specific for a cancer-associated antigen and a second antigen-binding site specific for a T cell (e.g., mucosal-associated invariant T (MAIT) cell, γ / δ T cell, CD8 + The antibody may comprise a second antigen-binding site specific for a T cell (cytotoxic T lymphocyte).

[0068] The present disclosure provides an immunogenic composition (also referred to herein as a "vaccine composition") comprising a variant Listeria of the present disclosure. The immunogenic composition of the present disclosure may comprise a) a variant Listeria of the present disclosure, and b) an antigen. Suitable antigens include, but are not limited to, cancer-associated antigens, pathogen-associated antigens (e.g., viral antigens, pathogenic protozoan antigens, etc.).

[0069] usefulness The disclosed 5-OP-RU precursor producing variant Listeria is used to activate and / or stimulate the proliferation of MAIT cells. MAIT cells are innate-like T cells defined by their semi-invariant αβ T cell receptor (TCR) that recognizes small biosynthetic derivatives of riboflavin synthesis presented on the restriction molecule major histocompatibility complex (MHC)-related protein-1 (MR1). MAIT cell ligands include 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU), which are produced during riboflavin (vitamin B2) synthesis by a wide variety of bacteria, mycobacteria, and yeast. MAIT cells have an intrinsic effector memory phenotype, e.g., CD45RA - CD45RO + CD95 Hi CD62L Lo CD44 Hiand has the ability to secrete various inflammatory cytokines.

[0070] In some cases, a 5-OP-RU precursor-producing variant Listeria of the present disclosure is administered to an individual, and the administration increases the number of MAIT cells in the individual. In some cases, administering an effective amount of a 5-OP-RU precursor-producing variant Listeria of the present disclosure to an individual increases the number of MAIT cells in the individual by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or more than 50%. In some cases, administering an effective amount of a 5-OP-RU precursor-producing variant Listeria of the present disclosure to an individual activates MAIT cells in the individual.

[0071] In some cases, the 5-OP-RU precursor producing variant Listeria of the present disclosure is orally administered to an individual to modulate the immune response in the individual's intestine.

[0072] An "effective amount" of a 5-OP-RU precursor-producing variant Listeria of the present disclosure is an amount that, when administered to an individual in need thereof, results in a beneficial effect (e.g., a clinically beneficial effect) in the individual. In some cases, an effective amount of a 5-OP-RU precursor-producing variant Listeria of the present disclosure is an amount that, when administered to an individual in need thereof, increases the number of MAIT cells in the individual. In some cases, an effective amount of a 5-OP-RU precursor-producing variant Listeria of the present disclosure is an amount that, when administered to an individual with a tumor, reduces tumor volume and / or reduces the number of cancer cells in the individual.

[0073] In some cases, an "effective amount" of the 5-OP-RU precursor producing variant Listeria of the present disclosure is about 10 per dose. 4 ~10 10 For example, in some cases, an "effective amount" of a 5-OP-RU precursor-producing variant Listeria of the present disclosure is in the range of 10 4 ~5x10 4 , 5x104 ~10 5 , 10 5 ~5x10 5 , 5x10 5 ~10 6 , 10 6 ~5x10 6 , 5x10 6 ~10 7 , 10 7 ~5x10 7 , 5x10 7 ~10 8 , 10 8 ~10 9 , or 10 9 ~10 10 The range is 100 bacteria.

[0074] Examples of Non-Limiting Aspects of the Disclosure The above aspects including the embodiments of the subject matter may be useful alone or in combination with one or more other aspects or embodiments.Without limiting the above description, certain non-limiting aspects of the present disclosure are given below.As will be clear to those skilled in the art after reading this disclosure, each of the individually numbered aspects can be used or combined with any of the preceding or succeeding individually numbered aspects.This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly shown below.

[0075] Aspect 1. A variant Listeria bacterium comprising a mutation in a gene required for the biosynthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), wherein the variant Listeria bacterium does not grow extracellularly in a mammal.

[0076] Aspect 2. A variant Listeria bacterium according to aspect 1, wherein the genes required for FMN and FAD synthesis are the ribC and / or ribF genes.

[0077] Embodiment 3. The variant Listeria bacterium of embodiment 1 or embodiment 2, which is a conditionally obligate intracellular bacterium.

[0078] Aspect 4. A variant Listeria bacterium according to any one of aspects 1 to 3, which requires supplementation of flavin mononucleotide and flavin adenine dinucleotide for growth.

[0079] Aspect 5. The variant Listeria bacterium according to any one of Aspects 2 to 4, wherein the mutation comprises a deletion of all or a portion of the ribC gene and / or the ribF gene.

[0080] Embodiment 6. A variant Listeria bacterium according to any one of embodiments 5, wherein said variant is genetically modified to comprise a heterologous nucleic acid comprising a nucleotide sequence encoding at least one heterologous gene product.

[0081] The variant Listeria bacterium of embodiment 6, wherein the heterologous nucleic acid is integrated into the genome of the bacterium.

[0082] Embodiment 8. The variant Listeria bacterium of embodiment 6 or embodiment 7, wherein the at least one heterologous gene product comprises an antigen.

[0083] Aspect 9. The variant Listeria of aspect 8, wherein the antigen is a cancer-associated antigen.

[0084] Aspect 10. A variant Listeria bacterium according to any one of aspects 1 to 9, which is a variant Listeria monocytogenes bacterium.

[0085] Embodiment 11. A variant Listeria bacterium according to any one of embodiments 1 to 10, further comprising one or more additional mutations that confer an attenuated phenotype to the bacterium, and / or one or more additional mutations that confer a growth advantage.

[0086] Aspect 12. A variant Listeria bacterium according to aspect 11, wherein said one or more further mutations conferring an attenuated phenotype comprise a mutation in a gene selected from actA and inlB, and optionally said one or more further mutations conferring a growth advantage comprise a mutation in the eetB gene.

[0087] Aspect 13. a) a variant Listeria bacterium according to any one of aspects 1 to 12, and b) Multispecific antibodies A composition comprising:

[0088] Embodiment 14. The composition according to embodiment 13, wherein said 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.

[0089] Embodiment 15. The T cells are mucosa-associated invariant T (MAIT) cells, γ / δ T cells, CD8 + The composition of embodiment 14, wherein the cell is a T cell or a natural killer (NK) cell.

[0090] Embodiment 16. An immunogenic composition comprising a variant Listeria according to any one of embodiments 1 to 12.

[0091] A method for inducing an immune response in an individual, comprising administering to the individual an effective amount of an immunogenic composition according to aspect 16.

[0092] Aspect 18. The method of aspect 17, wherein said variant Listeria bacterium has been genetically modified to contain a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous polypeptide, and wherein said immune response is directed against said heterologous polypeptide.

[0093] The method of claim 18, wherein the heterologous polypeptide is an antigen.

[0094] The method of claim 18, wherein the antigen is a cancer associated antigen.

[0095] Embodiment 21 The method according to any one of embodiments 17 to 20, wherein said immune response comprises a gamma-delta T cell response.

[0096] Embodiment 22. A kit comprising a unit dose of the immunogenic composition according to embodiment 16.

[0097] Aspect 23. The kit of aspect 22, wherein the unit dose is an oral dose.

[0098] Aspect 24. The kit of aspect 22, wherein the unit dose is for injection.

[0099] Embodiment 25. The kit according to any one of embodiments 22 to 24, further comprising a recombinant expression vector comprising a nucleotide sequence encoding a heterologous antigen.

[0100] Aspect 26. A variant Listeria producing a 5-OP-RU precursor that results in the production of the mucosal-associated invariant T (MAIT) cell ligand 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU).

[0101] Aspect 27. The 5-OP-RU precursor producing variant Listeria of aspect 26, wherein said variant Listeria comprises a heterologous ribDEAHT operon, and wherein said variant Listeria synthesizes riboflavin and stimulates the proliferation of MAIT cells.

[0102] Aspect 28. A 5-OP-RU precursor producing variant Listeria according to aspect 26, wherein the variant Listeria comprises heterologous ribD and ribA genes.

[0103] Aspect 29. A 5-OP-RU precursor-producing variant Listeria of any one of aspects 26 to 28, wherein the variant has been genetically modified to contain a heterologous nucleic acid comprising a nucleotide sequence encoding at least one heterologous gene product.

[0104] Aspect 30. A 5-OP-RU precursor-producing variant Listeria bacterium according to aspect 29, wherein the heterologous nucleic acid is integrated into the genome of the bacterium.

[0105] Aspect 31. A 5-OP-RU precursor-producing variant Listeria bacterium according to aspect 29 or aspect 30, wherein the at least one heterologous gene product comprises an antigen.

[0106] Aspect 32. The variant Listeria of aspect 31, wherein the antigen is a cancer-associated antigen.

[0107] Embodiment 33 The variant Listeria bacterium of embodiment 29 or embodiment 30, wherein the at least one heterologous gene product comprises a chimeric antigen receptor.

[0108] Embodiment 34. A variant Listeria bacterium according to any one of embodiments 26 to 33, further comprising one or more additional mutations which confer an attenuated phenotype to the bacterium and / or one or more additional mutations which confer a growth advantage.

[0109] Aspect 35. A variant Listeria bacterium according to aspect 34, wherein the one or more further mutations that confer an attenuated phenotype comprise a mutation in a gene selected from actA and inlB, and the one or more further mutations that confer a growth advantage comprise a mutation in the eetB gene.

[0110] Embodiment 36. A composition comprising a variant Listeria according to any one of embodiments 26 to 35.

[0111] Aspect 37. A method for increasing the number and activation state of mucosal-associated invariant T (MAIT) cells in an individual, comprising administering to said individual an effective amount of a variant Listeria of any one of aspects 26 to 35.

[0112] Aspect 38. A method for treating cancer in an individual, comprising administering to said individual an effective amount of a variant Listeria according to any one of aspects 26 to 35. EXAMPLES

[0113] The following examples are presented 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 following experiments 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 error and deviation should be accounted for. Unless otherwise indicated, 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, such as bp, base pairs, kb, kilobases, pl, picoliters, s or sec, seconds, min, minutes, h or hr, hours, aa, amino acids, kb, kilobases, bp, base pairs, nt, nucleotides, im, intramuscular (into the muscle), ip, intraperitoneal (into the abdominal cavity), iv, intravenous (into a vein), sc, subcutaneous (under the skin), etc.

[0114] Example 1: Obligate intracellular pathogenic strains of L. monocytogenes Materials and Methods Bacterial cultures and strains L. monocytogenes strains (Table 1) were derived from the wild-type 10403S strain and were grown in filter-sterilized rich brain heart infusion (BHI) medium (BD, Sparks, MD, USA) containing 200 μg / mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Construction of ΔribU (lmo1945), ΔribC (lmo1329), ΔribF (lmo0728), and ΔribCΔribF strains was performed using allelic exchange with the temperature-sensitive plasmid pKSV7. During the generation process of ΔribC, ΔribF, and the double ribC / ribF mutant strains, bacteria were always grown in BHI medium containing 2.5 μM FMN (Sigma-Aldrich, St. Louis, MO, USA) and 2.5 μM FAD (Sigma-Aldrich, St. Louis, MO, USA) to avoid synthetic lethality. For all procedures using ΔribC, ΔribF, and ΔribCΔribF strains, bacteria were always grown in BHI medium containing 2.5 μM FMN and 2.5 μM FAD.

[0115] The generation of the ΔribU strain expressing the ribDEAHT operon or the complemented strain expressing ribU was performed by amplifying the ribDEAHT operon with its native promoter from Bacillus subtilis and the ribU gene with its native promoter from wild-type L. monocytogenes, respectively, and cloning them into the pPL2 integration vector. Similarly, the complementation of the ΔribCΔribF mutant was performed by amplifying the ribC gene with its native promoter from wild-type L. monocytogenes and cloning it into the pPL2 vector. They were integrated into the genome of L. monocytogenes via conjugation. Broth growth curves were performed with L. monocytogenes strains from overnight cultures grown at 37°C with shaking (200 rpm). Growth curves in eutrophic (BHI) and chemically defined synthetic media were started at an optical density (OD600) of 0.03. Growth curves were measured spectrophotometrically by optical density at a wavelength of 600 nm (OD600).

[0116] Tissue Culture and Growth Media Bone marrow-derived macrophages (BMMs) were prepared by harvesting bone marrow from 8-week-old female wild-type (Jackson laboratory) and AIM2 KO (University of Massachusetts Medical School) C57BL / 6J mice. BMMs were cultured in high glucose Gibco Dulbecco's Modified Eagle's Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) with 20% fetal bovine serum (FBS) (Avantor-Seradigm, Radnor, PA, USA), 10% M-CSF-producing 3T3 cell supernatant, 1% L-glutamine (Corning, Lowell, MA, USA), 1% sodium pyruvate (Corning, 315 Lowell, MA, USA), and 14 mM 2-mercaptoethanol (Gibco Thermo Fisher Scientific, Waltham, MA, USA) or using the modified DMEM recipe described in the experimental procedures.

[0117] Intracellular proliferation curve 16-18 hours before infection, 3x10 6 BMMs were seeded in 60 mm non-TC-treated dishes (MIDSCI, St. Louis, MO, USA) containing 14 x 12 mm glass cover slips (Thermo Fischer Scientific, Waltham, MA, USA) in each dish. L. monocytogenes strains were grown overnight at 30°C in a tilted position in 14 ml round polypropylene tubes (Thermo Fisher Scientific, Waltham, MA, USA). Bacteria were washed and diluted in sterile 1X phosphate-buffered saline (PBS) and used to infect BMMs at a multiplicity of infection (MOI) of 0.25. Thirty minutes after infection, cells were washed twice with 1X PBS. One hour after infection, 50 μg / mL gentamicin sulfate (Sigma Aldrich, St. Louis, MO, USA) was added to the cell medium to kill bacteria / prevent extracellular bacterial growth.

[0118] Intracellular growth curves in media lacking riboflavin To deplete intracellular flavins in L. monocytogenes, bacterial cultures were initiated in chemically defined medium containing 1 μM riboflavin 2 days prior to BMM infection and grown with shaking at 37°C. Bacteria were washed twice with 1X PBS 16–18 h prior to infection and then diluted in chemically defined medium lacking flavin and grown with shaking at 37°C.

[0119] Macrophages were washed twice with 1X PBS 3 hours prior to BMM infection, and cell medium was replaced with DMEM high glucose lacking riboflavin (Millipore Sigma, Burlington, MA, USA) containing 20% ​​dialyzed FBS using SnakeSkin dialysis tubing, 3.5K MWCO (Thermo Fisher Scientific, Waltham, MA, USA), and other components as described in the Tissue Culture and Growth Media section above. These riboflavin-starved L. monocytogenes were washed and diluted in sterile 1X PBS and used to infect BMM at an MOI of 0.25. These growth curves were performed without the addition of riboflavin unless otherwise stated in the legend.

[0120] Cell death (lactate dehydrogenase release) assay 16-18 hours before infection, 5x10 5 BMMs / well were seeded in 24-well plates in DMEM medium containing 100 ng / mL Pam3CSK4 (InvivoGen, San Diego, CA, USA). Prior to infecting BMMs, the cell medium was replaced with DMEM medium containing 5% FBS. L. monocytogenes strains were grown overnight on a slant at 30°C. For infection, bacteria were diluted in 1X PBS and BMMs were infected at an MOI of 4. Thirty minutes after infection, BMMs were washed twice with 1X PBS and DMEM medium containing 5% FBS and 50 μg / mL gentamicin was added to the wells.

[0121] Mouse intravenous infection Eight-week-old female CD-1 mice (Charles River Laboratories, Wilmington, MA, USA) were injected with 1x10 5 Mice were infected with 200 μL of PBS containing logarithmically growing bacteria. At 48 hours post-infection, mice were euthanized and the spleen, liver, and gallbladder were harvested, homogenized, and plated to determine the number of CFU per organ.

[0122] Blood Growth Curve Growth of L. monocytogenes strains in blood was measured using defibrinated sheep blood (HemoStat Laboratories, Dixon, CA, USA). Bacteria were grown logarithmically for 2.5 h, washed, and diluted in 3 mL of defibrinated sheep blood at a concentration of 1x10 6 The blood was resuspended at 100 / mL. The blood cultures were incubated with shaking at 37° C. Growth of L. monocytogenes in the blood was monitored for 3 days by diluting the blood in 1× PBS and plating to determine the number of CFU in whole blood.

[0123] Oral infection of mice Mice were given 5 mg / mL streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA) in drinking water 48 h before infection. 18–24 h before infection, mice were transferred to clean cages and food sources (mouse colony chow) were removed to initiate an overnight fast. On the day of infection, 1 × 10 8 1X PBS containing logarithmically growing bacteria was inoculated and smeared with 3 μL of butter. Each 8-week-old female CD-1 mouse (Charles River Laboratories, Wilmington, MA, USA) was then fed a piece of the inoculated bread. Streptomycin sulfate water was replaced with standard drinking water and diet was reinstated. Fecal samples were collected daily for 5 days post-infection, weighed, vortexed for 10 min at 4°C, and plated to determine the number of CFU per gram of fecal matter.

[0124] Phagosome escape assay BMMs were seeded in 24-well plates containing 12 mm glass cover slips (Thermo Fischer Scientific, Waltham, MA, USA) and cultured overnight. BMMs were treated with 250 ng / mL cytochalasin D (Sigma-Aldrich, St. Louis, MO, USA) and 30 min later infected with L. monocytogenes strains grown overnight at 30°C in a tilted position (MOI 15). 1 h 15 min post-infection, BMMs were washed twice with 1X PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 15 min. Immunofluorescence staining, microscopy, and image analysis were then performed. The primary antibodies used were rabbit anti-Listeria (BD Difco, Franklin Lakes, NJ, USA, Catalog No. 223021) at a dilution of 1:1000, and guinea pig anti-p62 (Fitzgerald, Acton, MA, USA, Catalog No. 20R-PP001) at a dilution of 1:200. The secondary antibodies used were Rhodamine Red-X goat anti-rabbit IgG (Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA, Catalog No. R6394) at a dilution of 1:2000, and AlexaFluor-647 goat anti-guinea pig IgG (Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA, Catalog No. A21450) at a dilution of 1:2000. At least 100 bacteria per condition were quantified for analysis.

[0125] result RibU is required for virulence in mice We isolated an L. monocytogenes mutant with a transposon insertion in the ribU gene, the only annotated riboflavin transporter in L. monocytogenes. To confirm that RibU is not essential for growth and to assess flavin acquisition and requirement during pathogenesis, we generated an L. monocytogenes strain with an in-frame deletion of ribU (ΔribU). Similar to the transposon mutant, the ΔribU strain had no detectable growth defect in rich medium compared to wild-type L. monocytogenes (Figure 1b). In contrast, the ΔribU strain had a 5-log virulence defect in mouse spleens compared to wild-type L. monocytogenes (Figure 1c), and no colony forming units (CFUs) could be recovered from the livers of infected mice (Figure 1d). Complementation of the ribU gene of the ΔribU mutant with its endogenous promoter (ΔribU+ribU) fully restored virulence in vivo (Fig. 1c, d).

[0126] To determine whether the virulence defect of the ΔribU strain was caused by riboflavin starvation in vivo, we engineered the ΔribU mutant to synthesize riboflavin by inserting the riboflavin operon ribDEAHT from the closely related Gram-positive bacterium B. subtilis into the L. monocytogenes chromosome. The strain grew in colorless, chemically defined, synthetic medium without supplementation of riboflavin, turning the medium yellow, the natural color of flavin (Fig. 2). Expression of the ribDEAHT operon rescued the virulence of the ΔribU strain to the level of wild-type L. monocytogenes in mouse spleen and liver (Fig. 1c, d). Based on these observations, we conclude that RibU is essential for pathogenesis of L. monocytogenes and that its function is linked to the acquisition of flavin, since de novo production of riboflavin in a RibU-minus strain completely bypassed the essentiality of RibU in vivo.

[0127] L. monocytogenes uses RibU to grow in macrophages To investigate why RibU is essential for the growth of L. monocytogenes in mice, infections were performed in vitro using bone marrow-derived macrophages (BMMs). At 2 h postinfection, the ΔribU mutant had a small but significant growth advantage over wild-type L. monocytogenes (Figure 4a), which was associated with increased phagosome escape (Figure 3a). However, during exponential growth (2-5 h postinfection), the ΔribU mutant had a clear defect in replication kinetics and showed a loss of CFUs at late infection stages (5-8 h postinfection) (Figure 4a). Complementation of the ΔribU strain with the ribU gene or the ribDEAHT operon completely restored the growth defect in BMMs (Figure 3b).

[0128] To examine whether the ΔribU strain has an intrinsic intracellular virulence defect that is not related to riboflavin, we incubated BMMs with excess riboflavin (10 μM) prior to infection, increasing the concentration of intracellular riboflavin. In this condition of riboflavin excess, the ΔribU mutant replicated to wild-type levels (Figure 4b). To assess whether the observed growth of the ΔribU strain during exponential growth phase in BMMs (Figure 4a) was due to residual flavin derived from the medium, riboflavin-deficient BMMs were infected with riboflavin-starved bacteria. In this experiment, the ΔribU mutant and wild-type L. monocytogenes strains were incubated in chemically defined medium lacking flavin for 16–18 h prior to infection. The BMM cell medium was replaced with medium lacking riboflavin, and macrophages were incubated for 3 h before being infected with riboflavin-starved bacteria. It was observed that riboflavin-starved wild-type L. monocytogenes was able to grow in riboflavin-deficient BMM. However, the riboflavin-starved ΔribU mutant was unable to replicate in riboflavin-deficient BMM (Figure 4c). The riboflavin-starved ΔribU mutant was able to grow in riboflavin-deficient BMM supplemented with 1 μM riboflavin immediately prior to infection (Figure 3c).

[0129] To examine whether infection with the ΔribU strain resulted in host cell death, we performed a lactate dehydrogenase (LDH) release assay. These results showed that ΔribU caused a significant increase in host cell death (Fig. 4d). To examine whether ΔribU induced AIM2-dependent pyroptosis, we performed an LDH release assay using AIM2 knockout (KO) BMMs and observed that the ΔribU mutant did not cause LDH release (Fig. 4e). As a control, L. monocytogenes (WT+LpflaA), which secretes flagellin that activates the NLRC4 inflammasome, still mediated cell death in infected AIM2 KO BMMs (Fig. 4e). The ΔribU mutant did not cause cell death in AIM2 KO BMMs. Indeed, at 8 h postinfection, there was no loss of CFU for the ΔribU mutant (Fig. 4f). These data suggested that the RibU-minus strain lyses to some extent in vivo and activates AIM2-dependent pyroptosis, which negatively impacts the virulence of the strain.

[0130] L. monocytogenes uses RibU to capture FMN and FAD from the host cell cytosol The doubling times of riboflavin-starved wild-type L. monocytogenes in riboflavin-deficient BMM were very similar to those of wild-type L. monocytogenes grown in BMM with riboflavin, 48.6 and 51.5 min, respectively (Figure 3d). Both these results and the fact that mammalian cells rapidly convert riboflavin to FMN and FAD upon uptake indicate that wild-type L. monocytogenes grows by uptake of FMN and / or FAD intracellularly and using RibU for their transport. To examine whether L. monocytogenes can uptake FMN and FAD to support growth, we used chemically defined media supplemented with riboflavin, FMN, or FAD as the only flavin and found that wild-type L. monocytogenes grew in media containing each of the three flavins (Figure 5a). In contrast, the ΔribU strain did not replicate in chemically defined medium containing FMN or FAD (Figure 5b) and had only a slight growth defect in medium containing riboflavin. These results suggest that RibU is involved in growth on FMN and FAD and that riboflavin can enter cells using RibU and / or another, as yet unidentified, riboflavin transporter.

[0131] To investigate whether L. monocytogenes utilizes RibU to capture FMN and FAD from the host cell cytosol, FMN- and FAD-auxotrophic strains of L. monocytogenes were generated by constructing strains lacking ribC, ribF, or both (ΔribCΔribF), i.e., enzymes involved in converting riboflavin to FMN and FAD. Because FMN and FAD are essential cofactors, construction of this strain was performed in rich medium containing excess FMN and FAD to avoid synthetic lethality. The ΔribCΔribF mutant was unable to replicate in chemically defined medium with riboflavin as the sole flavin source (Figure 5c).

[0132] If L. monocytogenes takes up FMN and FAD from the host cytosol, the ΔribCΔribF mutants should not be impaired in intracellular growth. Indeed, these strains replicated to the level of wild-type L. monocytogenes in BMM cells (Fig. 5d). To examine whether the ΔribC, ΔribF, and ΔribCΔribF mutant L. monocytogenes strains grow in vivo, mouse virulence assays were performed. The ΔribC, ΔribF, and ΔribCΔribF mutants remained virulent and grew to high levels in both the spleen and liver of mice, whereas the ΔribC and ΔribCΔribF strains had a statistically significant 2-log defect in the liver (Fig. 5e, f). Complementation of the ribCΔribF strain with the ribC gene by its endogenous promoter was able to restore most of the growth in the spleen and liver of infected mice (Fig. 5e, f). Thus, these results support a model in which L. monocytogenes uses RibU to import FMN and FAD from the host cell cytosol.

[0133] The ΔribCΔribF mutant cannot grow in blood, gallbladder, or gastrointestinal tract Wild-type L. monocytogenes can grow extracellularly in the gallbladder, blood, and gastrointestinal (GI) tract of mice. Upon infection, L. monocytogenes colonizes the lumen of the gallbladder, which is connected to the liver via the bile duct, and rapidly replicates extracellularly in the bile, establishing this organ as a bacterial reservoir. The ΔribCΔribF mutant was unable to colonize the gallbladder, but the ΔribC and ΔribF strains grew to the level of wild-type L. monocytogenes (Figure 6a). Complementation of the ΔribCΔribF mutant with the ribC gene via its endogenous promoter fully rescued growth of the ΔribCΔribF strain in the gallbladder.

[0134] To assess whether the ΔribCΔribF mutant can grow extracellularly in blood, we performed growth curves in defibrinated sheep blood and found that the ΔribCΔribF mutant did not replicate, with a 2-3 log loss of CFU observed by 24 hours post-inoculation (Figure 6b). To examine whether the ΔribCΔribF mutant can grow extracellularly within the lumen of the GI tract, mice were pretreated with streptomycin 2 days prior to infection and infected with 1x10 8 CFU / mouse. Fecal pellets were collected daily for 5 days and plated to assess bacterial load. The ΔribCΔribF mutant had a 7 log deficit in fecal pellet CFU compared to wild-type L. monocytogenes at 24 hours post-infection (Figure 6c). No CFU were recovered from ΔribCΔribF mutant-infected mice after day 1 post-infection (Figure 6c). Taken together, these observations suggest that the ΔribCΔribF strain is unable to grow extracellularly in the gallbladder, blood, or GI tract, and that this mutant is restricted to intracellular growth in vivo.

[0135] Figure 1A shows the structures of riboflavin (black), FMN (red), and FAD (purple). Figure 1B shows the broth growth curves of L. monocytogenes strains grown in rich medium. The average and standard deviation of three independent experiments are displayed. Figures 1C-1D show the structure of riboflavin (black), FMN (red), and FAD (purple). 5 The colony forming units (CFU) of L. monocytogenes strains in the spleen (C) and liver (D) of mice 48 hours after infection with each strain are shown. The black line represents the median CFU for each strain. The dashed line represents the limit of detection.

[0136] Figure 2A shows broth growth curves of L. monocytogenes strains grown in chemically defined medium lacking flavins. The mean and standard deviation of three independent experiments are displayed. In chemically defined medium lacking flavins, wild-type L. monocytogenes grows until it depletes its flavin pool. In contrast, the ΔribU+ribDEAHT strain grows to a higher density. Figure 2B shows images of culture supernatants of wild-type (left) and ΔribU+ribDEAHT strains (right) after 24 h of growth with shaking at 37 °C.

[0137] Figure 3A shows the percentage of L. monocytogenes strains that colocalized with the autophagy receptor p62 in infected bone marrow-derived macrophages (BMMs). In BMMs treated with cytochalasin D, bacteria that escape the phagosome are tagged with p62. Percent phagosome escape is calculated based on the percentage of p62 in total bacteria. + The generation time is calculated by counting the number of bacteria. Data represent the mean and standard error of the mean of two independent experiments. Figures 3B-3C show intracellular growth curves of L. monocytogenes strains in BMM. BMM were infected at an MOI of 0.1 and CFU were counted at the indicated times. (B) shows growth curves of the indicated L. monocytogenes strains in wild-type BMM. Data represent the mean and standard error of the mean of two independent experiments. (C) shows growth curves of the indicated flavin-starved L. monocytogenes strains in wild-type BMM without riboflavin (3 h) supplemented with 1 μM riboflavin immediately before infection. Data represent the mean and standard error of the mean of three independent experiments. Figure 3D shows generation times between 2 and 5 h for L. monocytogenes strains growing intracellularly in riboflavin-sufficient and riboflavin-deficient BMM. Negative values ​​indicate that the number of recoverable bacteria decreased over time.

[0138] Figures 4A-4C show intracellular growth curves of L. monocytogenes strains in BMMs. BMMs were infected at an MOI of 0.1 and CFUs were counted at the indicated times. (A) shows growth curves of the indicated L. monocytogenes strains in wild-type BMMs. Data represent the mean and standard error of the mean of two independent experiments. (B) shows growth curves of the indicated L. monocytogenes strains in wild-type BMMs incubated in cell medium containing excess (10 μM) riboflavin at the time of infection. Means and standard error of the mean of three independent experiments are shown. (C) shows growth curves of the indicated flavin-starved L. monocytogenes strains in riboflavin-deficient wild-type BMMs. Data represent the mean and standard error of the mean of three independent experiments. Figures 4D-E show cell death of wild-type (D) or AIM2 KO (E) BMMs infected with the specified L. monocytogenes strains. Data represent the mean and standard error of the mean of three technical replicates of at least two or four independent experiments, respectively. Figure 4F shows intracellular growth curves of the indicated L. monocytogenes strains in AIM2 KO BMMs. The mean and standard error of the mean of five independent experiments are shown.

[0139] Figures 5A-5C show broth growth curves of L. monocytogenes strains grown in chemically defined media containing different flavin sources. (A) shows growth curves of wild-type L. monocytogenes grown in media containing riboflavin, FMN, or FAD as the sole flavin source. Data show the mean and standard deviation of four independent experiments. (B) shows growth curves of ΔribU mutant L. monocytogenes strains grown in media containing riboflavin, FMN, or FAD as the sole flavin source. Wild-type L. monocytogenes grown in media containing riboflavin is used as a reference. Data show the mean and standard deviation of four independent experiments. (C) shows growth curves of the indicated L. monocytogenes strains grown in media containing riboflavin. Data show the mean and standard deviation of two independent experiments. Figure 5D shows intracellular growth curves of L. monocytogenes strains in mouse BMMs. BMMs were infected at an MOI of 0.1 and CFU were counted at the indicated times. The mean and standard error of the mean of two independent experiments are shown. Figures 5E-5F show the results of 1x10 5 5E and 5F show bacterial burden in the spleen (5E) and liver (5F) of CD-1 mice infected intravenously with the indicated CFU of L. monocytogenes strains 48 hours post-infection. Data represent a combination of at least two independent experiments. Black lines represent the median CFU of each strain.

[0140] Figure 6A shows the in vitro growth of L. monocytogenes strains in defibrinated sheep blood. The mean and standard error of the mean of three independent experiments are shown. Figure 6B shows the growth of L. monocytogenes strains in defibrinated sheep blood. 5 Figure 6C shows the bacterial burden in the gallbladder 48 hours after infection of CD-1 mice infected intravenously with the indicated CFU of L. monocytogenes strains. Data represent a combination of at least two independent experiments. Black lines represent the median CFU of each strain. Dashed lines represent the limit of detection. ...D shows the bacterial burden in the gallbladder 48 hours after infection of CD-1 mice infected intravenously with the indicated CFU of L. monocytogenes strains. 8Figure 1 shows the bacterial burden in the gastrointestinal tract of CD-1 mice orally infected with the indicated CFU of L. monocytogenes strains. Mice were pretreated orally with streptomycin prior to infection. Fecal samples were collected on days 1-5 and plated to determine CFU per gram of fecal stool. Data represent the mean and standard deviation of the mean from a combination of three independent experiments. The dashed line represents the limit of detection.

[0141] Example 2: Activation of MAIT cells using engineered L. monocytogenes strains To address the case in which L. monocytogenes evades the MAIT cell response, an L. monocytogenes strain that synthesizes riboflavin de novo was engineered by cloning the riboflavin biosynthetic operon (ribDEAHT) from the closely related bacterium, Bacillus subtilis, into the L. monocytogenes chromosome. Two background strains were created with two different promoters. The first one expresses the operon, i.e., pHyper ribDEAHT (Figure 7), from a constitutively active promoter (pHyper) in a wild-type L. monocytogenes background. The second strain expresses the riboflavin operon (Figure 7) from the native B. subtilis promoter (pNative), an FMN riboswitch that turns on / off translation of ribDEAHT depending on the flavin concentration within the bacterium in a riboflavin transporter (ribU) minus L. monocytogenes background, preventing the bacterium from uptake of flavin from the host cell, thus allowing the bacterium to produce riboflavin de novo. To determine whether the ribDEAHT strain was able to synthesize riboflavin de novo, broth growth curves were performed in chemically defined medium lacking flavin, showing that while wild-type L. monocytogenes could grow to a certain extent until it depleted its internal flavin pool, the ΔribU pNative ribDEAHT strain grew to a higher density (Figure 2a). Furthermore, there was a visual change in color from colorless to light yellow (the natural color of flavin) in the medium of the strain containing the ribDEAHT operon, suggesting that the ribDEAHT operon is functional and that riboflavin can be synthesized de novo in the engineered strain. Because expressing an exogenous operon in L. monocytogenes and synthesizing riboflavin may affect its virulence or fitness, we performed intracellular growth curves in bone marrow-derived macrophages. The strain expressing the ribDEAHT operon grew intracellularly similarly to wild-type L. monocytogenes, indicating that the production of riboflavin does not affect the intracellular fitness of L. monocytogenes in vitro (Figure 3b and Figure 8).These results suggested that the ribDEAHT strains were capable of producing riboflavin and were not attenuated with respect to their intracellular growth.

[0142] To determine whether L. monocytogenes may evade the MAIT cell response in vivo, mice were infected with wild-type L. monocytogenes or a riboflavin-producing (ribDEAHT) strain. By day 4 postinfection, pHyper ribDEAHT and ΔribU pNative ribDEAHT strains were found to be highly attenuated compared to wild-type L. monocytogenes in the spleens and livers of infected mice, suggesting that MAIT cells sense the riboflavin-producing strain and control bacterial load (Figure 9). To determine whether MAIT cells respond to the riboflavin-producing strain and are activated and / or accumulating in infected tissues, mice were infected with the riboflavin-producing strain in the ΔactA background and spleens and livers were harvested to examine the number of MAIT cells (Figure 10A). ActA is a virulence factor that allows L. monocytogenes to spread from cell to cell, and because strains lacking this factor are less virulent, using a strain in this background allows mice to be infected with a higher dose of bacteria, increasing the opportunity for MAIT cells to encounter cells infected with the ribDEAHT strain. The frequency of MAIT cells in perforin KO mice infected with ΔactA pHyper ribDEAHT was approximately 15% and 20% of total ab-T cells in the spleen and liver, respectively (Figure S6B), similar to the frequency observed in wild-type-infected mice (Figure S7C).

[0143] FIG. 7 is a diagram of the riboflavin operon from B. subtilis introduced into the L. monocytogenes chromosome with both pHyper (constitutive promoter) and pNative (inducible, the native promoter from B. subtilis) indicated.

[0144] Figure 8 shows intracellular growth curves of L. monocytogenes strains in mouse BMM. BMM were infected at an MOI of 0.1 and CFU were counted at the indicated times. The mean and standard error of the mean of two independent experiments are shown.

[0145] Figure 9 shows the 1x10 3 Figure 1 shows the bacterial burden in the spleen and liver 96 hours after infection of B6 mice infected intravenously with the indicated CFU of L. monocytogenes strains. Data represent a combination of at least three independent experiments. Black lines represent the median CFU of each strain.

[0146] Example 3 Although a five-fold increase in MAIT cells was observed in tissues infected with riboflavin-producing L. monocytogenes, the question was raised as to whether this response could be augmented by infecting mice with more bacteria. However, the strain used in the previous experiments was in a wild-type L. monocytogenes background and was found to be capable of infecting mice with a 50% lethal dose (LD) of 1000 cells in 100% of mice. 50 ) is approximately 5-fold the dose administered, so infection with higher doses would result in the death of mice. It was therefore decided to introduce the pNative and pHyper ribDEAHT operon construct into the AcA minus L. monocytogenes background (ΔactAΔribU pNative and pHyper ribDEAHT). ActA minus L. monocytogenes is highly attenuated in mice (approximately 1000-fold compared to wild type in BALB / C) due to its inability to spread from cell to cell, allowing mice to be infected with substantially more bacteria without killing them.

[0147] To confirm that the attenuation observed in the riboflavin-producing strain in a wild-type background was reproducible, first, a strain in an AcA-negative background was administered at a higher dose (1x10 7 CFU / mouse, 1x10 3We performed virulence experiments by infecting mice with 1000 riboflavin-producing strains (instead of 1000 CFU / mouse) of AcA minus strain. Indeed, at day 4 post-infection, the AcA minus riboflavin-producing strain was attenuated by 2 logs in the spleen and liver, as observed for the strain in the wild-type L. monocytogenes background (Figure 10A). Surprisingly, the MAIT cells (CD3 + TCR-β + MR1:5-OP-RU tetramer + The frequency of MAIT cells (αβ-T cells) was observed to be as high as 30% in the livers of infected mice (Figure 10B). The median frequency of MAIT cells in the spleens and livers of mice infected with ActA minus pHyper ribDEAHT strain was 15% and 20% of total αβ-T cells, respectively (Figure 10C). In contrast, the frequency of MAIT cells in the spleens and livers of mice infected with the ΔactA L. monocytogenes control was 0.5%-1% of total T cells, which was the same as in naive mice (Figure 10C).

[0148] Following infection, MAIT cells can persist in tissues long after the infection has subsided. The question arose as to whether this was the case following infection with riboflavin-producing L. monocytogenes. Mice were infected with AcA minus pHyper ribDEAHT and spleens and livers were harvested at days 2, 4, 7, 14, and 60 postinfection. From less than 1% of total αβ T cells at 2 days postinfection, the frequency of MAIT cells reached a maximum of 20% in the spleen at 4 days postinfection and in the liver at 14 days postinfection (Figure 10D). At 60 days postinfection, MAIT cells constituted an average of 5% and 10% of total αβ T cells in the spleen and liver, respectively (Figure 10D). The frequency of CD8+ T cells started at approximately 20% in both organs and reached a maximum of 40% in the spleen and 60% in the liver at 4 days postinfection (Figure 10D). In contrast, at 2 days postinfection, CD4+ T cells were at their highest frequency, 60% of αβ T cells in the spleen and 40% in the liver (Fig. S10D). At 4 days postinfection, their frequency was as low as 25% and 10% in the spleen and liver, respectively (Fig. S10D). At 60 days postinfection, CD4+ T cells ended up at lower frequencies in both organs than the initial frequency at the beginning of the experiment (Fig. S10D). These MAIT cell kinetic experiments showed that the frequency of MAIT cells peaked at 4 days postinfection in the spleen and 14 days in the liver and was maintained in these organs at a higher frequency for 60 days than that in naive mice (Fig. S10C) and in 2-day infected mice (Fig. S10D). These data suggest that MAIT cells respond strongly to attenuated riboflavin-producing L. monocytogenes strains by accumulating at high frequencies in infected organs and that they can persist in tissues at higher frequencies than those found in naive mice.

[0149] Figures 10A-10D. Infection with an attenuated riboflavin-producing L. monocytogenes strain in an ActA-negative background results in substantial and sustained accumulation of MAIT cells in infected tissues. (A) 1 x 10 7Bacterial burden in C57BL / 6 mice infected intravenously with the indicated L. monocytogenes strains at CFU. Four days post-infection, spleens (left) and livers (right) were harvested, homogenized, and plated to determine CFU per organ. These data represent a combination of three independent experiments: ΔactA, ΔactAΔribU pNat ribDEAHT, and ΔactA pHyp ribDEAHT (n=15 mice). Black lines represent median CFU for each strain. Dashed lines represent the limit of detection. Statistical significance of log-transformed CFU values ​​was determined using one-way ANOVA and Dunnett's post-hoc test using WT as control. ****P<0.0001. (B and C) Representative flow cytometry plots from hepatic MAIT cells (B) and summarized data from spleen and liver (C) showing the frequency of MAIT cells in naive, ΔactA, or ΔactA pHyper ribDEAHT infected C57BL / 6 mice 4 days after infection. The infecting dose was 1x10 7 CFU / mouse. MAIT cell percentages of viable, CD45 positive, TCRβ positive, MR1:5-OP-RU tetramer positive T cells are shown. In (C) Mean and SEM of three independent experiments: naïve (n=14 mice), ΔactA (n=10 mice), and ΔactA pHyp ribDEAHT (n=13 mice) are shown. Statistical significance was determined using one-way ANOVA and Dunnett's post-hoc test using (PBS) naïve mice as control. ****P<0.0001, ns, not significant, P>0.05. (D) 1x10 7 MAIT cells, CD4 CFU / mouse in mice infected with ΔactA pHyper ribDEAHT at 2, 4, 7, 14, and 60 days after infection + T cells and CD8 + MAIT cell kinetics experiments showing the frequency of T cells that were viable, CD45 positive, TCRβ positive, and subsequently MR1:5-OP-RU tetramer positive CD4 - / CD8 - Negative cells (MAIT cells), CD4 + / CD8 - (CD4 + T cells), and CD4 - / CD8+ (CD8 + The percentages of cells gated as T cells are shown. PBS, phosphate-buffered saline.

[0150] These data indicate that MAIT cells specifically restrict riboflavin-producing L. monocytogenes. The question arose as to which mechanism is involved. MAIT cells have two major effector functions that mediate pathogen control, producing cytokines that activate bystander cells or directly killing infected cells using the cytolytic effectors granzyme B and perforin. Because L. monocytogenes is an intracellular pathogen, we hypothesized that direct killing of infected cells is the response MAIT cells use to restrict riboflavin-producing L. monocytogenes. We infected mice lacking perforin (perforin KO), which should prevent all cytotoxic cells, including MAIT cells, from directly killing infected cells. We observed that riboflavin-producing L. monocytogenes had no virulence defect compared to wild-type L. monocytogenes (Figure 11A). To confirm that MAIT cells were still responding and accumulating in infected tissues and that the response at day 4 postinfection was not different in perforin KO mice, we infected these mice with an ActA minus riboflavin-producing L. monocytogenes strain and determined the frequency of MAIT cells in the organs. The frequency of MAIT cells in perforin KO mice was 15% and 20% of total αβ-T cells in the spleen and liver, respectively (Figure 11B), similar to the frequency observed in wild-type infected mice. These results suggested that the primary mechanism used by MAIT cells to restrict riboflavin-producing L. monocytogenes is direct killing of infected cells.

[0151] Figures 11A-11B. Perforin-minus MAIT cells restrict riboflavin-producing L. monocytogenes. 1 x 10 3Bacterial burden in perforin KO C57BL / 6 mice lacking the cytolytic effector perforin infected intravenously with the indicated L. monocytogenes strains at CFU. Four days post-infection, spleens (left) and livers (right) were harvested, homogenized, and plated to measure CFU per organ. These data represent a combination of two independent experiments: WT (n=10 mice), ΔribU pNat ribDEAHT (n=11 mice), and pHyper ribDEAHT (n=10 mice). Statistical significance of log-transformed CFU values ​​was determined using one-way ANOVA and Dunnett's post-hoc test using WT as control. ns, not significant, P>0.05. (B) Summary data showing the frequency of MAIT cells in the spleens and livers of naïve, ΔactA, or ΔactA pHyper ribDEAHT perforin KO C57BL / 6 mice at 4 days post-infection. The infectious dose was 1x10 7 CFU / mouse. MAIT cell percentages of viable, CD45 positive, TCRβ positive, MR1:5-OP-RU tetramer positive T cells are shown. Means and SEM of two independent experiments: ΔactA (n=8 mice), ΔactAΔribU pNat ribDEAHT (n=9 mice), and ΔactA pHyp ribDEAHT (n=9 mice) are shown. Statistical significance was determined using one-way ANOVA and Dunnett's post-hoc test using ΔactA mice as control. ***P<0.001, **P<0.01, *P<0.05, ns, not significant, P>0.05.

[0152] Example 4 : RibC / RibF strains carrying ActA and inlB mutations L. monocytogenes ΔribC, ΔribF, ΔactA, and ΔinlB strains were constructed and compared to L. monocytogenes ΔactA and ΔinlB strains and wild-type L. monocytogenes in macrophages in vitro. The data are shown in FIG.

[0153] FIG. 12 shows intracellular growth curves of L. monocytogenes strains in wild-type mouse BMMs. BMMs were infected at an MOI of 0.1 and CFUs were counted at the indicated times. The data show that the L. monocytogenes ΔribCΔribFΔactAΔinlB strain grew intracellularly to the levels of wild-type, ΔactAΔinlB, and ΔribCΔribF strains (no defects were detected). Combining the actA and inlB deletions with the L. monocytogenes ΔribCΔribF strain could make the ΔribCΔribF strain a safer vaccine vector or therapeutic without affecting its intracellular replication.

[0154] Figure 13 shows the 1x10 5 Bacterial burden in the spleen and liver of CD-1 mice infected intravenously with the indicated CFUs of L. monocytogenes strains 48 hours after infection. The data show that ΔribCΔribFΔactAΔinlB and ΔribCΔribFΔactAΔinlB+eetB::tn strains grow to CFU levels of ΔactAΔinlB (no defects detected). In this background, deletion of actA and inlB combined with a ΔribCΔribF strain and disruption of the eetB gene (making it non-functional) resulted in a strain that was as virulent in vivo in mice as the control ΔactAΔinlB strain.

[0155] To assess whether the ΔactAΔinlB+ΔribCΔribF and ΔactAΔinlB+ΔribCΔribF+eetB::tn mutants were able to grow extracellularly in blood, growth curves in defibrinated sheep blood were performed. Growth of the ΔactAΔinlB+ΔribCΔribF and ΔactAΔinlB+ΔribCΔribF+eetB::tn mutant strains of L. monocytogenes in blood was measured using defibrinated sheep blood (HemoStat Laboratories, Dixon, CA, USA). Bacteria were grown to log phase for 2.5 h, washed, and diluted in 3 mL of defibrinated sheep blood at a concentration of 1x10 6The blood was resuspended at 100 / mL. The blood cultures were incubated at 37° C. with shaking. Growth of L. monocytogenes in the blood was monitored for 4 days by diluting the blood in 1× PBS and plating to determine the number of CFU in whole blood. The data are shown in FIG.

[0156] As shown in Figure 14, 24 hours after inoculation of blood, both ΔactAΔinlB+ΔribCΔribF and ΔactAΔinlB+ΔribCΔribF+eetB::tn did not grow, and a 2-3 log loss of CFU was observed for both strains compared to ΔactAΔinlB, which replicated to high numbers. ΔactAΔinlB+ΔribCΔribF and ΔactAΔinlB+ΔribCΔribF+eetB::tn are highly attenuated in blood, as observed with the ΔribCΔribF mutant (Figure 6A).

[0157] Example 5 Vaccination To examine whether the ΔribCΔribF mutant strain could robustly induce adaptive immune responses and confer protection against future challenge with L. monocytogenes, mice were cultured at 1x10 5 After allowing the mice to rest for 30 days, they were immunized with 5x10 4 Mice were infected with wild-type L. monocytogenes to investigate the level of protection offered by the ΔactAΔribCΔribF strain. In general, if the strain used for vaccination is capable of generating a robust immune response against L. monocytogenes, the mice will be protected as they are able to mount a strong secondary immune response to antigen challenge. Depending on the efficacy of this experimental strain in activating the adaptive immune response, different levels of protection can be observed.

[0158] C57BL / 6 mice, 1x10 5 Mice were infected with logarithmic-phase ΔactA and ΔactAΔribCΔribF bacteria at 5x10 ng / mL and the infection was allowed to resolve for 30 days. Mice were then infected with 5x10 4Mice were challenged (infected) with logarithmic-phase wild-type L. monocytogenes to determine the level of protection offered by the ΔactAΔribCΔribF strain. Three days after challenge, spleens (left) and livers (right) were harvested and plated to count CFU per organ. ΔactA and ΔactAΔribCΔribF, n=10; PBS control (mice injected with 1X PBS as a control on day 0), n=5.

[0159] The data are shown in Figure 15. As shown in Figure 15, ΔactA and ΔactAΔribCΔribF provided similar levels of protection, suggesting that ΔactAΔribCΔribF may be a safe and effective vaccine platform or therapeutic for T cell / immune stimulation in vivo.

[0160] Although the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various modifications can be made and equivalents 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

1. A bacterium of the genus Listeria containing mutations in genes necessary for the biosynthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), wherein the bacterium does not proliferate extracellularly in mammals.

2. The bacterium of the genus Variantlisteria according to claim 1, wherein the gene necessary for the synthesis of FMN and FAD is the ribC gene and / or the ribF gene.

3. A bacterium of the genus Variantlisteria according to claim 1 or claim 2, which is a conditionally obligate intracellular bacterium.

4. A bacterium of the genus Variantlisteria according to claim 1, which requires the supplementation of flavin mononucleotide and flavin adenine dinucleotide in order to grow.

5. The variant of the genus Listeria according to claim 1, wherein the mutation comprises the deletion of all or part of the ribC gene and / or the ribF gene.

6. The variant of Listeria bacterium according to claim 5, wherein the variant is genetically modified to include a heterologous nucleic acid comprising a nucleotide sequence encoding at least one heterologous gene product.

7. The Variantlisteria bacterium according to claim 6, wherein the heterologous nucleic acid is incorporated into the genome of the bacterium.

8. The bacterium of the genus Variantlisteria according to claim 6 or 7, wherein the at least one heterologous gene product comprises an antigen.

9. The bacterium of the genus Variantlisteria according to claim 8, wherein the antigen is a cancer-associated antigen.

10. A bacterium of the genus Listeria according to claim 1, which is a bacterium of the genus Listeria monocytogenes.

11. The variant of the genus *Bacteria* according to claim 1, further comprising one or more further mutations that confer a weakened phenotype to the bacterium, and / or one or more further mutations that result in a growth advantage.

12. The variant of the genus Listeria according to claim 11, wherein one or more further mutations conferring the attenuated phenotype include mutations in genes selected from actA and inlB, and one or more further mutations resulting in the growth advantage include mutations in the eatB gene.

13. a) A bacterium of the genus Variantlisteria as described in claim 1, and b) Multispecific antibodies A composition containing the following:

14. The composition according to claim 13, wherein the multispecific antibody comprises i) a first antigen-binding site specific to a cancer-associated antigen, and ii) a second antigen-binding site specific to T cells.

15. The T cells include mucosa-associated invariant T (MAIT) cells, γ / δ T cells, CD8 ＋ The composition according to claim 14, wherein the composition is a T cell or a natural killer (NK) cell.

16. An immunogenic composition comprising the genus Variantlisteria as described in claim 1.

17. A method for inducing an immune response in an individual, comprising administering an effective amount of the immunogenic composition described in claim 16 to the individual.

18. The method according to claim 17, wherein the bacterium of the genus Variantlisteria is genetically modified to contain a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous polypeptide, and the immune response is induced against the heterologous polypeptide.

19. The method according to claim 18, wherein the heterologous polypeptide is an antigen.

20. The method according to claim 18, wherein the antigen is a cancer-related antigen.

21. The method according to any one of claims 17 to 20, wherein the immune response includes a gamma-delta T cell response.

22. A kit comprising a unit dose of the immunogenic composition described in claim 16.

23. The kit according to claim 22, wherein the unit dose is an oral dose.

24. The kit according to claim 22, wherein the unit dose is for injection.

25. The kit according to claim 22, further comprising a recombinant expression vector containing a nucleotide sequence encoding a heterologous antigen.

26. A variant of the genus Listeria that produces the 5-OP-RU precursor, leading to the production of the mucosa-associated invariant T (MAIT) cell ligand 5-(2-oxopropyrideneamino)-6-D-ribitylaminouracil (5-OP-RU).

27. The 5-OP-RU precursor-producing variantantlisteria species according to claim 26, wherein the variantantlisteria species contains a different ribDEAHT operon, and the variantantlisteria species synthesizes riboflavin and stimulates the proliferation of MAIT cells.

28. The genus Variantlisteria according to claim 26, wherein the Variantlisteria genus comprises heterologous ribD and ribA genes.

29. The 5-OP-RU precursor-producing variant of Listeria according to any one of claims 26 to 28, wherein the variant is genetically modified to include a heterologous nucleic acid comprising a nucleotide sequence encoding at least one heterologous gene product.

30. The 5-OP-RU precursor-producing variant of the genus Listeria according to claim 29, wherein the heterologous nucleic acid is incorporated into the genome of the bacterium.

31. The 5-OP-RU precursor-producing variant of the genus Listeria according to claim 29, wherein the at least one heterogeneous gene product comprises an antigen.

32. The bacterium of the genus Variantlisteria according to claim 31, wherein the antigen is a cancer-associated antigen.

33. The Variantlisteria bacterium according to claim 29, wherein the at least one heterologous gene product comprises a chimeric antigen receptor.

34. The variant of the genus Listeria according to claim 26, further comprising one or more further mutations that confer a weakened phenotype to the bacterium, and / or one or more further mutations that result in a growth advantage.

35. The variant of the genus Listeria according to claim 34, wherein one or more further mutations conferring the attenuated phenotype include mutations in genes selected from actA and inlB, and one or more further mutations resulting in the growth advantage include mutations in the eatB gene.

36. A composition comprising the genus Variantlisteria as described in claim 26.

37. A method for increasing the number and activation state of mucosal-associated invariant T cells (MAIT) in an individual, comprising administering to the individual an effective amount of a variant of the genus Listeria described in claim 26.

38. A method for treating cancer in an individual, comprising administering to the individual an effective amount of a Variantlisteria species as described in claim 26.

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