Live Salmonella typhi vectors engineered to express cancer protein antigens and methods of use thereof

Abstract

The present invention provides compositions and methods for inducing an immune response in a subject in need thereof, comprising administering to the subject an immunologically effective amount of a live Salmonella typhi vector, the Salmonella typhi vector engineered to express one or more cancer antigens. In some embodiments, the vector is designed to express the outer membrane folding protein BamA, or a fragment or variant thereof, and the lipid A deacylase PagL, or a fragment or variant thereof, and the Salmonella typhi vector, when administered to a subject, is capable of delivering antigens via outer membrane vesicles to dendritic cells in mucosal tissues or subcutaneously.

Description

[Technical Field]

[0001] Incorporation by Reference of Electronically Submitted Materials Incorporated herein by reference in its entirety is a computer-readable sequence listing identified as a 66,734 byte XML file entitled "Sequence_listing.xml," created on May 30, 2023.

[0002] The field of the invention relates generally to the fields of medicine, cancer, and molecular biology, and in particular to the technology of vaccine immunotherapy. [Background technology]

[0003] Cancer is the second leading cause of death in the United States (after heart disease), and colorectal cancer (CRC) ranked among the top three carcinogenic causes in terms of incidence and mortality in both men and women in the United States in 2019 (Non-Patent Documents 1 and 2). Traditional treatments for colon, rectal, and anal cancer typically involve surgical resection, radiation therapy, and / or chemotherapy, encompassing a growing number of diverse and continually improving compounds (Non-Patent Documents 3-5). Until recently, minimally invasive treatment strategies that attempted to utilize the host's immune system to eliminate tumor tissue seemed elusive. However, with the recent success of immune checkpoint inhibitors that activate tumor-specific T cells, which can circumvent immunosuppressive mechanisms in the tumor microenvironment and promote tumor elimination, interest in immunotherapeutic approaches to cancer treatment is now showing growing optimism (Non-Patent Document 6). In addition to using immune checkpoint inhibitors to attenuate T cell immunosuppression, efforts are also underway to enhance innate immunity through dendritic cell activation, which results in cross-presentation of tumor-associated antigens (TAA) to cytotoxic T cells and helps recruit T cells to enhance both T cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) responses (Non-Patent Document 7).

[0004] Salmonella is one of the most studied organisms for use as a mucosal live carrier vaccine to deliver foreign antigens to the immune system. Over the years, several attenuated vaccine strains of Salmonella derived from serovar Typhi have been developed (Non-Patent Documents 8-11). In clinical trials, some attenuated vaccine strains have induced a wide range of immune responses, including intestinal secretory IgA antibodies, serum IgG antibodies, and T cell-mediated immunity (Non-Patent Documents 8 and 11). The ability of orally administered live Salmonella to activate circulating human monocytes and dendritic cells and to initially activate CD8+ T cells through dendritic cell cross-presentation has recently been confirmed in humans (Non-Patent Documents 12 and 13).

[0005] Attempts to use Salmonella for therapeutic cancer intervention in humans have been conducted in clinical trials using both Salmonella Typhimurium and Salmonella Typhi serotypes with disappointing results. However, the effectiveness of using Salmonella strains in humans depends on the serotype used. Salmonella Typhimurium is adapted to the human host and is able to penetrate deep into human tissues after oral administration, whereas Salmonella Typhimurium is rapidly cleared after entering the bloodstream. 1 (Non-Patent Document 14). Therapeutic treatment with the attenuated Salmonella typhimurium strain VNP20009 has shown great promise in mouse models but has been unsuccessful despite administering the organism directly into the bloodstream for several hours (Non-Patent Documents 15 and 16). Clinical trials using the approved Salmonella typhi vaccine strain Ty21a, which was subsequently used as a carrier vaccine VXM01 expressing the angiogenic TAA VEGFR2, also achieved limited therapeutic efficacy. This is likely due to excessive attenuation of the already very weak parent vaccine strain Ty21a (Non-Patent Documents 17-19).

[0006] Carcinoembryonic antigen (CEA) is a surface glycoprotein of approximately 180 kDa expressed on various tumor tissues, including colorectal cancer, and is hypothesized to possess both cell adhesive and proangiogenic properties (Non-Patent Documents 20 and 21). It is a member of the immunoglobulin gene superfamily and consists of a variable N-terminal domain followed by three sets of constant Ig-like domains (designated A1B1, A2B2, and A3B3), each consisting of an "A" and "B" subdomain, with over 90% amino acid sequence identity (Non-Patent Documents 20 and 22). Multiple cytotoxic T cell epitopes have been mapped to the A3B3 domain of CEA (Non-Patent Documents 23-25). Clinical trials have clearly established the safety of successfully targeting CEA without inducing autoimmunity. Two recent studies have attempted to enhance tumor-specific adaptive immune responses by targeting dendritic cells with recombinant CEA, circumventing both immune tolerance and immunosuppression. Both studies reportedly succeeded in inducing clinically relevant CEA-specific T cell and antibody responses (Non-Patent Documents 26 and 27).

[0007] The human mucin gene MUC-1 is a membrane-expressed, glycosylated colon cancer-associated antigen that is also associated with other solid tumors. The extracellular protein core of this heterodimeric glycoprotein consists of a variable number of 20-residue proline- and threonine-rich repeat (VNTR) sequences, including up to 120 copies, noncovalently linked to a transmembrane region of approximately 20 kDa (Non-Patent Document 28). MUC-1 is normally present in a highly glycosylated form on most polarized mucosal epithelial tissues. However, overexpression in solid tumor tissues can result in significantly hypoglycosylated epitopes, exposing normally masked epitopes to immune surveillance (Non-Patent Documents 29-31).

[0008] Clinical trials have shown that patients who responded to MUC-1 experienced mild side effects, whereas non-responders were found to have elevated levels of circulating myeloid-derived suppressor cells, which may interfere with both humoral and cellular tumor-specific immunity (Non-Patent Documents 32 and 33). A poxvirus-based bivalent vaccine approach targeting CEA and MUC-1 was recently confirmed to be safe and immunogenic in a phase 1 dose-escalation clinical trial (Non-Patent Document 34). An immunosuppressive tumor microenvironment (TME) surrounds CRC solid tumors, significantly hindering the ability of the host's adaptive immune system to target and eliminate tumor tissue. This microenvironment is composed of various innate and adaptive immune cells, as well as cancer-associated fibroblasts, vascular tissue, and surrounding stromal tissue consisting of a rigid extracellular matrix that can eliminate infiltrating T cells (Non-Patent Documents 35 and 36). Tumor-derived cytokines, such as TGF-β, attenuate the antigen-presenting function of dendritic cells, inhibit T cell proliferation and effector function, and suppress immunosuppressive T cells. reg Tumor-associated macrophages induce the formation of M2 cells and inhibit the function of cytotoxic NK cells (Non-Patent Documents 37 and 38). In addition, tumor-associated macrophages are anergic (M2-polarized), exhibiting reduced phagocytic activity and reduced secretion of proinflammatory cytokines, which would otherwise facilitate tumor clearance by classically activated M1-polarized macrophages (Non-Patent Documents 39-41). In addition to the difficulty in accessing tumor tissue, tumor cells inherently possess genetic instability, which allows them to rapidly change their surface antigens and evade active immunosurveillance by immune effector cells. Therefore, more genetically stable surrounding stromal cell types represent promising alternative therapeutic targets for inducing tumor-specific adaptive cytotoxic responses that can activate innate immunity, disrupt the immunosuppressive environment of the TME, and kill tumor cells. NK cells are particularly suitable for this purpose due to their ability to kill tumor cells without the aid of T cells (Non-Patent Documents 35 and 39).

[0009] Nanomedicines can be defined as any therapeutic agent (nanostructure) with a physical dimension of less than 1000 nm (i.e., 1 mm) used to treat disease. In this regard, nanoparticles are typically less than about 100 nm (Non-Patent Document 42). Virus-like particles (VLPs) and small liposomes are typically less than about 200 nm (Non-Patent Documents 38 and 43), and outer membrane vesicles are typically in the 30-250 nm range (Non-Patent Documents 44 and 45). In comparison, viruses are typically less than 350 nm, bacteria range in size from 1 to 5 mm, and T cells are 7 to 20 mm. Recent data from animal models indicate that the inherent size and other associated physical properties of nanomedicines can themselves confer a direct therapeutic effect on tumors through a phenomenon called the "enhanced permeation and retention (EPR) effect," which relies on the leaky vasculature of tumors, independent of immunological targeting of tumor-associated antigens (Non-Patent Documents 42, 46, and 47). The blood vessels of rapidly growing tumors differ from normal vasculature in that they contain defective endothelial cells, irregular vascular arrangements, lack smooth muscle layers and innervation, and lack functional angiotensin II receptors. These leaky tumor vessels allow passive extravasation of small nanostructures from the blood into tumor tissue (Non-Patent Document 46). From within the tumor tissue, these nanostructures can then passively escape through the lymphatic system and reach local lymph nodes, where they may contact and activate antigen-presenting cells, including dendritic cells (Non-Patent Documents 46 and 47). As solid tumors grow, the vasculature becomes restricted, significantly reducing the EPR effect (Non-Patent Document 46). This observation may partly explain why the EPR effect has shown greater therapeutic efficacy in small animal models, perhaps even more so than in humans. These types of experiments are usually performed on animals with small, early-stage tumors with leaky blood vessels, whereas treatments for CRC patients usually involve larger, more advanced solid tumors, in which the EPR effect may be less effective (46).However, Islam et al. recently reported that the combined administration of tumor-targeting nanoparticles and vasodilators significantly improved the deposition and regression of very large solid tumors in a mouse animal model through the enhanced EPR effect (Non-Patent Document 48). Thus, nanomedicine offers the potential for treatment of "cold" tumors, in which the target tumor-associated antigen(s) has yet to be identified, by passively targeting and activating immune-suppressed myeloid cells in tumor tissue through the EPR effect.

[0010] In a recent experimental CRC mouse model, bacterial outer membrane vesicles were also observed to be able to deposit in mouse tumor tissues via the EPR effect, which transformed tissue macrophages from anergic M2 to inflammatory M1, leading to infiltration of activated T cells and subsequent tumor regression (Non-Patent Documents 49-51). Kim et al. used engineered OMVs derived from the nonpathogenic Gram-negative bacterium E. coli W3110, which lacked msbB to reduce endotoxin activity, to test the possibility of suppressing TLR4 activation and extending the half-life of the vesicles in circulation. These vesicles were systemically administered to mice bearing syngeneic subcutaneous tumors derived from CT26 or MC38 tumor cells (Non-Patent Document 49). Notably, DmsbB OMVs passively accumulated exclusively in tumor tissues, an effect also observed with OMVs isolated from Gram-positive bacteria Staphylococcus aureus and Lactobacillus acidophilus. The accumulated vesicles induced NK cell- and T cell-associated IFN-γ responses and completely eliminated established tumors without significant side effects. Interestingly, subcutaneous administration of host bacteria isolated from these vesicles failed to induce antitumor responses (Non-Patent Document 49). Qing et al. also reported therapeutic deposition of OMVs into tumor tissue, but used calcium phosphate-coated OMVs rather than genetically engineering the host E. coli strain to extend their systemic half-life (Non-Patent Document 50). Similarly, systemically administered vesicles accumulated in CT26-induced solid tumor tissue and induced a reversal of M1 / M2 polarization toward activated M1 macrophages, a significant increase in infiltrating CD8+ T cells, and the immunosuppressive Treg reported that this was accompanied by a decrease in tumor size, an increase in tumor apoptotic cells, and an overall improvement in survival (Non-Patent Document 50). As a further development of this OMV therapeutic strategy, Cheng et al. used systemically administered recombinant rOMVs engineered in Escherichia coli to target the Adpgk neoantigen present in MC38 colon cancer cells to retarget established solid tumors (Non-Patent Document 51). This treatment resulted in complete tumor regression in 60% of mice, and MC38 tumor tissue after subcutaneous immunization showed a significant increase in CD4+ and CD8+ T cells, activated neutrophils, and DCs, but not immunosuppressive T cells. reg The levels of β-glucan were reduced (Non-patent Document 51).

[0011] There is an urgent need for the development of new compositions and methods for treating cancer. The present invention fulfills this need and provides additional advantages.

[0012] This background information is provided for informational purposes only. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Siegel et al.,CA Cancer J Clin 2019;69(1):7-34 [Non-patent document 2] Miller et al., CA Cancer J Clin 2019;69(5):363-85 [Non-patent document 3] Libutti et al.,Philadelphia:Wolters Kluwer Health;2016 [Non-patent document 4] Libutti et al.,Philadelphia:Wolters Kluwer Health;2016

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[0014] It is to be understood that both the foregoing general description and the following detailed description of the embodiments are exemplary and explanatory and are not restrictive of the scope of the embodiments.

[0015] In one aspect, the invention provides a live Salmonella typhi vector engineered to express one or more cancer antigens, the outer membrane folding protein BamA or a fragment or variant thereof, and the lipid A deacylase PagL or a fragment or variant thereof, which, when administered to a subject, is capable of delivering the antigens to mucosal tissues via outer membrane vesicles.

[0016] In some embodiments, expression of one or more of BamA, PagL, and the antigen is inducible and under the control of an inducible promoter. In some embodiments, the promoter is sensitive to osmolality. In some embodiments, the osmotically controlled inducible promoter is the promoter of the outer membrane protein C (ompC) gene. In some embodiments, the antigen comprises an outer membrane protein, an antigenic fragment thereof, or a variant thereof.

[0017] In some embodiments, the cancer antigen is derived from colon cancer. In some embodiments, the present invention provides an attenuated live vector vaccine strain derived from Salmonella typhi that expresses a fusion protein comprising antigenic sequences from the proteins CEA and MUC1, wherein the live vector derived from Salmonella typhi exhibits enhanced delivery of the fusion protein to the immune system through increased formation of recombinant outer membrane vesicles (rOMVs). In some embodiments, the live vector derived from Salmonella typhi expresses a ClyA protein that is transported from the live vaccine via the rOMV. In some embodiments, extracellular transport of the fusion protein and ClyA from the live vaccine via the rOMV is increased.

[0018] In another aspect, the present invention provides a composition comprising isolated recombinant outer membrane vesicles from Salmonella typhi comprising one or more cancer antigens, where the Salmonella typhi has been engineered to express the antigens.

[0019] In another aspect, the present invention provides a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject an immunologically effective amount of a live Salmonella enterica Typhi vector engineered to express one or more cancer antigens, outer membrane folding protein BamA or a fragment or variant thereof, and lipid A deacylase PagL or a fragment or variant thereof, wherein the S. Typhi vector, when administered to a subject, is capable of delivering antigens to mucosal tissues via outer membrane vesicles.

[0020] In another aspect, the present invention provides a method for inducing an immune response in a subject in need thereof, the method comprising administering to the subject an immunologically effective amount of isolated recombinant outer membrane vesicles derived from a live Salmonella typhi vector comprising one or more cancer antigens, wherein the Salmonella typhi vector has been engineered to express one or more cancer antigens, the outer membrane folding protein BamA or a fragment or variant thereof, and the lipid A deacylase PagL or a fragment or variant thereof.

[0021] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: One or more cancer antigens, and b. providing a live Salmonella typhi vector engineered to express lipid A deacylase PagL or a fragment or variant thereof; The live Salmonella typhi vector, when administered to a subject, is capable of delivering the antigen to mucosal tissue via the outer membrane vesicles. In another aspect, the present invention provides a composition comprising an isolated recombinant outer membrane vesicle comprising one or more cancer antigens from a Salmonella typhi vector.

[0022] In another aspect, the present invention provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject: One or more cancer antigens, and b. administering a live Salmonella typhi vector engineered to express lipid A deacylase PagL or a fragment or variant thereof; The antigen is delivered to the mucosal tissue of the subject by outer membrane vesicles produced by the Salmonella typhi vector.

[0023] In another aspect, the present invention provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject: i. One or more cancer antigens, and b. lipid A deacylase PagL or a fragment or variant thereof; an immunologically effective amount of a live Salmonella typhi vector engineered to express ii. Administering an immunologically effective amount of isolated recombinant outer membrane vesicles derived from a live Salmonella typhi vector, wherein the Salmonella typhi vector has been engineered to express one or more cancer antigens, the outer membrane folding protein BamA or a fragment or variant thereof, and the lipid A deacylase PagL or a fragment or variant thereof.

[0024] In another aspect, the present invention provides a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject One or more cancer antigens, and b. lipid A deacylase PagL or a fragment or variant thereof The method includes administering an immunologically effective amount of a live Salmonella typhi vector engineered to express the antigen, which is delivered to the mucosal tissue of the subject after early detection of the target tumor by an unrelated diagnostic method. See, e.g., Cohen et al. 2018. Science, 359:926-930.

[0025] In another aspect, the present invention provides a method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a live Salmonella typhi vector described herein and / or an effective amount of an isolated recombinant outer membrane vesicle described herein.

[0026] Other objects, features, and advantages of the present invention will become apparent from the following detailed description, but it should be understood that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the above detailed description.

[0027] Those skilled in the art will understand that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. [Brief explanation of the drawings]

[0028] [Figure 1] Hemolytic activity of the isogenic attenuated Salmonella typhi CVD 910 live vector strain expressing chromosomally encoded ClyA delivered by overexpression of PagL. Samples from synchronized bacterial cultures of approximately 2 x 10 CFU were analyzed for hemolytic activity using sheep red blood cells, with five determinations per group. Lane 1: PBS; Lane 2: 910; Lane 3: 910ΔguaBA::clyA; Lane 4: 910ΔguaBA::clyA(pPagL). [Figure 2] FIG. 1 shows the transport of the antigen OmpAAb in OMVs from the CVD910 live vaccine strain. [Figure 3] Hemolytic activity of the isogenic, attenuated Salmonella typhi CVD 910 live vector strain expressing chromosomally encoded ClyA delivered by overexpression of PagL. Samples from synchronized bacterial cultures of approximately 2 x 10 CFU were analyzed for hemolytic activity using sheep red blood cells, with five replicates per group. Lane 1: PBS; Lane 2: 910; Lane 3: 910ΔguaBA::clyA; Lane 4: 910ΔguaBA::clyA(pPagLv1); Lane 5: 910ΔguaBA::clyA(pPagLv2); Lane 6: 910ΔguaBA::clyA(pPagLv3). [Figure 4]Hemolytic activity of an isogenic, attenuated Salmonella typhi CVD910 live vector strain expressing chromosomally encoded ClyA delivered by overexpression of BamA. Samples from synchronized bacterial cultures of approximately 2 x 10 CFU were analyzed for hemolytic activity using sheep red blood cells, with five replicates per group. Lane 1: PBS; Lane 2: 910; Lane 3: 910 (pSEC10); Lane 4: 910ΔguaBA::clyA; Lane 5: 910ΔguaBA::clyA (pAbBamAv1); Lane 6: 910ΔguaBA::clyA (pAbBamAv2). [Figure 5] A candidate bivalent Salmonella typhi-based colon immunotherapy vaccine is presented. Targeted colon antigen domains derived from carcinoembryonic antigen (CEA) and human mucin MUC-1 are expressed as surface-expressed fusion proteins and then presented to the site of immune induction via a novel inducible PagL-mediated outer membrane vesicle (OMV) delivery system. The plasmid is genetically stabilized using a novel single-stranded binding protein-based strategy. [Figure 6] Targeted colon cancer-associated antigens expressed in an attenuated Salmonella typhi-based bivalent carrier vaccine are shown. A. Carcinoembryonic antigen (CEA). B. MUC-1. [Figure 7] CRC cancer antigen(s) cassette is shown. [Figure 8] 1 shows engineered modifications to the lipid A structure that reduce TLR4 activation. 1 shows a schematic representation of the Salmonella lipid A structure with cleavage sites indicated for PagL and LpxE. [Figure 9]The genetic structure of CVD910ΔguaBA::PompC-bamAAbΔfliC::PompC-lpxEFn (pPagL-LOAM) is shown. The diagram illustrates the formation of a nested set of PfliC-PompC promoters by chromosomal insertion of PompC-lpxEFn, which replaces the fliC gene while retaining the original upstream PfliC promoter. This same strategy is used to form a nested set of PguaBA-PompC promoters by chromosomal insertion of PompC-bamAAb, which replaces the guaBA gene while retaining the original upstream PguaBA promoter. [Figure 10] In vitro characterization of CRC fusion protein expression in purified rOMVs. 0.5 μg of purified rOMVs was loaded per lane on an SDS-PAGE gel stained with Coomassie Brilliant Blue (Panel A) and further characterized by Western immunoblot analysis using purified rabbit A3B3-specific primary antibody and Alexa Fluor 680 goat anti-rabbit IgG (H+L) secondary antibody. [Figure 11] Figure 1 shows the IFN-γ response to A3B3-MUC1 in BALB / c mice immunized intramuscularly with 2 μg of rOMV on days 0 and 21. Spleen cells were harvested on day 35, pooled for each group, and immediately used for ELISpot analysis without cryopreservation. Each pooled sample was analyzed in quadruplicate and stimulated with 10 mg / mL of A3B3-MUC1 purified antigen for 40 hours prior to measuring spot counts. 250,000 splenocytes were analyzed per well. [Figure 12] IFN-γ responses to A3B3-MUC1 in C57BL / 6 mice immunized as described in Table 4 are shown. Spleen cells were harvested on day 21, pooled for each group, and used immediately for ELISpot analysis without cryopreservation. See text for experimental details. [Figure 13]Immunotherapeutic treatment of C57BL / 6 mice implanted with 300,000 MC38 colon cancer cells engineered to constitutively express MUC1 or CEAv2 antigens is shown. CEAv2 is a truncated form of CEA homologous to the domain expressed by our rOMVs (A and B). Cells were implanted subcutaneously on day 0, and mice were subsequently treated intravenously on days 3, 5, 7, and 9 (red arrows) with PBS or ΔfliC or lpxE rOMVs expressing CEA-MUC1-targeted tumor-associated fusion proteins at 0.75 mg per dose. Tumor progression, as measured by tumor volume, is plotted as the mean volume, with the standard error of the mean (SEM) shown in the bar graph. Statistical significance at day 28 was assessed by ANOVA with Geisser-Greenhouse correction and Tukey's multiple comparison analysis. In mice implanted with MC38-MUC1, p = 0.0483 for ΔfliC vs. lpxE, p = 0.0001 for ΔfliC vs. PBS, and p = 0.0004 for lpxE vs. PBS. In mice implanted with MC38-CEAv2, p = 0.2202 for ΔfliC vs. lpxE, p = 0.2122 for ΔfliC vs. PBS, and p = 0.0178 for lpxE vs. PBS (C and D). Cells were implanted subcutaneously and allowed to grow to mean tumor volumes of 115 mm3 for CEAv2 and 120 mm3 for MUC1. Mice were treated with 0.75 μg of PBS or rOMV per injection by intravenous injection on days 0, 2, 4, and 6 (red arrows). Tumor progression, plots, and statistical analysis are shown in panels A and B. No statistically significant differences were observed between CEAv2-lpxE and MUC1-lpxE, or between CEAv2-ΔfliC and MUC1-ΔfliC. (E) Kaplan-Meier survival curves for the experiments are plotted in panels C and D. No significant differences were observed between CEAv2-ΔfliC and MUC1-ΔfliC, or between MUC1-ΔfliC and MUC1-lpxE. [Figure 14]Figure 1 shows the immunogenicity and therapeutic efficacy of rOMVs re-engineered to express individual domains of the CRC fusion protein compared to unmodified rOMVs expressing the full-length A3B3-MUC1 fusion protein. A: Re-engineering strategy for constructing rOMVs expressing a single CRC domain. B: Anti-A3B3-MUC1 serum IgG antibody titers. C: ELISPOT assay measuring IFN-γ secretion from isolated splenocytes stimulated with A3B3-MUC1 fusion protein. D: Tumor volume in C57BL / 6 mice administered MC38-CEAv2 on day 0 and treated intravenously with 0.75 mg of rA3B3ΔfliC, rMUC1ΔfliC, rOMVΔfliC-CRC, or empty rOMVΔfliC-pagL vesicles on days 3, 5, 7, and 9. DETAILED DESCRIPTION OF THE INVENTION

[0029] Reference will now be made in detail to the presently preferred embodiments of the invention, which, together with the drawings and the following examples, serve to explain the principles of the invention. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, it being understood that other embodiments may be utilized and structural, biological, and chemical changes may be made without departing from the spirit and scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0030] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of one of ordinary skill in the art. Such techniques are fully explained in the literature. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2 ndedition(1989), Current Protocols in Molecular Biology(FMAusubel et al. eds.(1987)), the series Methods in Enzymology(Academic Press, Inc.), PCR:A Practical Approach(M.MacPherson et al.IRL Press at Oxford University Press(1991)), PCR 2:A Practical Approach(MJMacPherson,BDHames and GRTaylor (1995)), Antibodies, A Laboratory Manual (Harlow and Lane eds. (1988)), Using Antibodies, A Laboratory Manual (Harlow and Lane eds. (1999)), and Animal Cell Culture (RIFreshney ed. (1987)).

[0031] Definitions of common terms in molecular biology can be found, for example, in Benjamin Lewin, Genes VII, Oxford University Press, 2000 (ISBN 019879276X), Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Publishers, 1994 (ISBN 0632021829), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, Wiley, John & Sons, Inc., 1995 (ISBN 0471186341).

[0032] For purposes of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular include the plural, and vice versa. If any definition set forth below conflicts with the use of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims, unless a contrary meaning is clearly intended (e.g., in the document in which the term is originally used). The use of "or" means "and / or" unless otherwise stated. As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes multiple cells, including mixtures thereof. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. Furthermore, when a description of one or more embodiments uses the term "comprising," those skilled in the art will understand that in some specific instances, those one or more embodiments may alternatively be described using the language "consisting essentially of" and / or "consisting of."

[0033] As used herein, the term "about" means ±10% of the numerical value of the number with which it is being used.

[0034] Live vectors and recombinant outer membrane vesicles In some embodiments, the invention provides live Salmonella vectors, such as Salmonella typhi, engineered to express one or more cancer antigens, outer membrane folding protein BamA or a fragment or variant thereof, and lipid A deacylase PagL or a fragment or variant thereof, wherein the Salmonella vector, when administered to a subject, is capable of delivering antigens to mucosal tissues via outer membrane vesicles.

[0035] In some embodiments, the present invention provides live Salmonella typhi vectors engineered to express one or more cancer antigens and lipid A deacylase PagL or a fragment or variant thereof, wherein the Salmonella typhi vector, when administered to a subject, is capable of delivering antigens to mucosal tissues via outer membrane vesicles.

[0036] BamA is an approximately 90 kDa protein and is an essential component of the five-protein outer membrane β-barrel assembly machinery (BAM) complex, which catalyzes the insertion of β-barrel proteins into the outer membrane of Gram-negative bacteria. There are no particular limitations on the BamA that can be used in the present invention. BamA encompasses full-length BamA as well as biologically active fragments and variants of BamA. In some embodiments, BamA is derived from Acinetobacter baumannii. In some embodiments, a nucleotide sequence comprising BamA is optimized. In some embodiments, one or more codons (e.g., rare codons) are optimized for enhanced expression. In some embodiments, a putative ribosome binding site is optimized for enhanced expression. In some embodiments, the amino acid sequence of BamA is SEQ ID NO:8. In some embodiments, the nucleic acid sequence of BamA is SEQ ID NO:10.

[0037] The lipid A deacylase PagL that can be used in the present invention is not particularly limited. PagL encompasses full-length PagL as well as biologically active fragments and variants of PagL. In some embodiments, PagL is derived from Salmonella. In some embodiments, PagL is derived from Salmonella enterica serovar Typhimurium. In some embodiments, the nucleotide sequence comprising PagL is optimized. In some embodiments, one or more codons (e.g., rare codons) are optimized to enhance expression. In some embodiments, the putative ribosome binding site is optimized to enhance expression. In some embodiments, the nucleotide sequence of PagL comprises SEQ ID NO: 1, 3, or 5. In some embodiments, the amino acid sequence of PagL comprises SEQ ID NO: 2 or 4.

[0038] In some embodiments, the Salmonella typhi-derived live vector overexpresses one or more additional vesicle catalytic proteins, such as ClyA, which is involved in the natural induction of OMV formation in Salmonella typhi. ClyA includes full-length ClyA, as well as biologically active fragments and variants of ClyA.

[0039] ClyA is an endogenous protein of Salmonella typhi that, when overexpressed, can catalyze the formation of large outer membrane vesicles. This mechanism for vesicle formation raises the intriguing possibility of engineering ClyA to transport heterologous foreign antigens from live vectors via vesicles. These vesicles could also potentially carry immunomodulatory lipopolysaccharide (LPS) to improve the immunogenicity of otherwise poorly immunogenic antigens. The utility of ClyA in a strategy to enhance the immunogenicity of a foreign protective antigen (PA83) derived from anthrax toxin, i.e., to produce a live vector anthrax vaccine with demonstrated immunogenicity in both mouse and nonhuman primate animal models, has been confirmed (Galen et al. 2010. Infect. Immun. 78(1):337-47; Galen et al. 2009. J. Infect. Dis. 199(3):326-35). Similar to ClyA, overexpression of PagL has recently been reported to induce significant formation of outer membrane vesicles (Elhenawy et al.2016.mBio.7(4):e00940-16.doi:10.1128 / mBio.00940-16). Interestingly, the pagL gene is present in the mouse pathogen Salmonella typhimurium but absent in Salmonella typhi.

[0040] ClyA from Salmonella typhi was first described by Wallace et al., who also reported the crystal structure of the homologous HlyE hemolysin from Escherichia coli (Wallace et al. 2000. Cell 100:265-276). ClyA proteins can cause hemolysis in target cells. The present invention encompasses the use of both hemolytically active and hemolytically inactive forms of ClyA, with the hemolytically inactive variant being more preferred if the antigen transport and immunogenicity of the resulting protein can be maintained. In some embodiments, the nucleotide and amino acid sequences of ClyA correspond to SEQ ID NOs: 15 and 16, respectively. In some embodiments, ClyA is mutated to reduce its hemolytic activity while still retaining its transport function. In one embodiment, the ClyA mutant is ClyA I198N. In another embodiment, the ClyA mutant is ClyA C285W. In some embodiments, ClyA is mutated to reduce its hemolytic activity. In some embodiments, the ClyA mutant is selected from the group consisting of ClyA I198N, ClyA C285W, ClyA A199D, and ClyA E204K. In some embodiments, the ClyA is a fusion protein. In some embodiments, the ClyA comprises the following substitution mutations: I198N, A199D, and E204K. The sequence of the mutant is set forth in SEQ ID NO: 16.

[0041] In some embodiments, the S. typhi vector is engineered to reduce both TLR4- and TLR5-mediated reactogenicity. In some embodiments, the S. typhi vector has a deletion in the fliC gene, which is a TLR5 agonist. In some embodiments, the sequence of the fliC gene deleted from the chromosome of a candidate attenuated S. typhi vaccine strain, such as CVD910, or its derivatives, is SEQ ID NO:29.

[0042] In some embodiments, the Salmonella typhi vector comprises lpxE from Francisella novicida (lpxE Fn) in some embodiments. In some embodiments, the nucleotide sequence of IpxE is SEQ ID NO:26 and the amino acid sequence is SEQ ID NO:27. LpxE is a lipid A1-phosphatase that dephosphorylates lipid A to generate a less reactive monophosphoryl species (FIG. 8). By co-expressing PagL (which deacylates lipid A while promoting hypervesicle formation), rOMVs containing pentaacyl-monophosphoryl-lipid A with significantly reduced TLR4 and TLR5 activity can be produced. In some embodiments, a Salmonella typhi vector is engineered to insert a nucleic acid sequence encoding LpxE into the fliC locus of Salmonella typhi.

[0043] In some embodiments, the present invention provides compositions comprising isolated recombinant outer membrane vesicles from Salmonella typhi vectors of the invention comprising one or more cancer antigens, in some embodiments, the antigen comprises an outer membrane protein, an antigenic fragment thereof, or a variant thereof, and the Salmonella typhi has been engineered to express the antigen.

[0044] In some embodiments, the cancer is selected from colon cancer, colorectal cancer, leukemia (e.g., chronic lymphocytic leukemia or acute myeloid leukemia), lymphoma (e.g., non-Hodgkin's lymphoma), breast cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, lung cancer (e.g., small cell carcinoma or non-small cell lung cancer), and skin cancer (e.g., melanoma), uterine cancer, gallbladder cancer, adenocarcinoma, bile duct cancer, esophageal cancer, gastric cancer, glioblastoma, ovarian cancer, bladder cancer, and head and neck cancer. In some embodiments, the cancer is colon cancer.

[0045] In some embodiments, the cancer antigen is a neoantigen, carcinoembryonic antigen (CEA), human epithelial mucin MUC-1, cancer-testis antigen NY-ESO-1, HER2 / neu, SART-1, SART-2, KIAA0156, ART-1, ART-4, cyclophilin B, mutant elongation factor 2, malic enzyme, and alpha actinin-4, eIF4G, aldolase, annexin XI, Rip-1, NY-LU-12, fibromodulin, RHAMM / CD168, MDM2, hTERT, oncofetal antigen-immature laminin receptor protein (OFAiLRP), adipophilin, survivin, KW1-KW14, and tumor-derived IgV. The antibody comprises one or more antigens (or antigen fragments or derivatives thereof) selected from the group consisting of an HCDR3 region, a RHAMM-derived R3 peptide, B7.1, ICAM-1, LFA-3, epidermal growth factor receptor variant III (EGFRvIII), heat shock protein, IL-13 receptor alpha 2, alpha-fetoprotein, MART1, gp100, cancer-testis antigen MAGEA3, L-BLP25, p53, Wilms' tumor-1, KRAS, reactive telomerase, gastrin, prostate-specific antigen (PSA), tumor-associated antigen 5T4, cancer-testis antigen MAGE, PASD1, B-cell antigen CD20, and viral tumor protein E6 or E7.

[0046] In some embodiments, the cancer antigen comprises one or more antigenic fragments of CEA and / or MUC1. In some embodiments, the CEA antigen and the MUC1 antigen are part of a fusion protein. In some embodiments, the CEA antigen and the MUC1 antigen are part of the same fusion protein.

[0047] In some embodiments, the CEA antigen comprises domain A3B3. In some embodiments, the amino acid sequence of domain A3B3 comprises SEQ ID NO: 23. In some embodiments, the nucleotide sequence of domain A3B3 comprises SEQ ID NO: 30.

[0048] In some embodiments, the MUC1 antigen is a fragment comprising multiple repeat domains. In some embodiments, the amino acid sequence of the MUC1 fragment comprises SEQ ID NO: 24. In some embodiments, the nucleotide sequence of the MUC1 fragment comprises SEQ ID NO: 31.

[0049] In some embodiments, the cancer antigen is expressed in Salmonella typhi as a fusion protein. In some embodiments, the cancer antigen is fused to a polypeptide sequence comprising a surface-displayed protein. In some embodiments, the polypeptide sequence comprising a surface-displayed protein is selected from Lpp-OmpA, Lpp-OmpT, and ClyA. In some embodiments, the amino acid sequence of Lpp-OmpA comprises SEQ ID NO:21. In some embodiments, the nucleotide sequence of lpp-ompA comprises SEQ ID NO:28.

[0050] In some embodiments, the invention provides an isolated nucleic acid encoding an expression cassette for expression in a Salmonella typhi vector of the invention, wherein the expression cassette encodes a fusion protein comprising a surface-displayed protein and one or more antigens, in some embodiments, the surface-expressed protein is selected from Lpp-OmpA, Lpp-OmpT, and ClyA.

[0051] In some embodiments, the cancer antigen is a fusion protein comprising the CEA domain A3B3 and a MUC1 fragment fused to a surface-displayed protein such as Lpp-OmpA. In some embodiments, the cancer antigen comprises an Lpp-OmpA:A3B3:MUC1 fusion protein comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the cancer antigen comprises an lpp-ompA:a3b3:muc1 gene fusion encoded by SEQ ID NO: 11.

[0052] In some embodiments, the cancer antigen is a fusion protein comprising the CEA domain A3B3 fused to a surface-displayed protein such as Lpp-OmpA. In some embodiments, the cancer antigen comprises an Lpp-OmpA:A3B3 fusion protein comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the cancer antigen comprises an Lpp-OmpA:A3B3 gene fusion encoded by SEQ ID NO: 13.

[0053] In some embodiments, the cancer antigen is a fusion protein comprising a MUC1 fragment fused to a surface-displayed protein such as Lpp-OmpA. In some embodiments, the cancer antigen comprises an Lpp-OmpA:MUC1 fusion protein comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the cancer antigen comprises an lpp-ompA:muc1 gene fusion protein encoded by SEQ ID NO: 17.

[0054] In some embodiments, the cancer antigen is a ClyA comprising the amino acid sequence of SEQ ID NO: 20. I198N In some embodiments, the cancer antigen comprises a clyA:A3B3:MUC1 fusion protein. I198N In some embodiments, the ClyA:a3b3:muc1 fusion protein I198N The amino acid sequence comprises SEQ ID NO:25.

[0055] In some embodiments, the polypeptide components of the fusion protein are separated by one or more linker amino acid sequences. In some embodiments, the linker amino acid sequence is SEQ ID NO: 22. In preferred embodiments, the cancer antigen does not exhibit glycosylation.

[0056] In some embodiments, the present invention provides a nucleic acid comprising any of SEQ ID NOs: 11, 13, 17, or 19.

[0057] In some embodiments, the present invention provides compositions comprising a combination of isolated recombinant outer membrane vesicles from the engineered Salmonella typhi vectors described in the present invention.

[0058] In some embodiments, the present invention provides a genetically engineered, attenuated strain of Salmonella typhi as a live vaccine platform for delivery of one or more cancer antigens to protect against the development and / or progression of cancer, such as colon cancer. The one or more antigens can be expressed on the surface of the live vaccine after induction of synthesis in vivo and transported from the surface to the site of immune induction via a unique inducible OMV-mediated transport system, as described in more detail below.

[0059] In some embodiments, the live vaccine expresses the cancer antigens carcinoembryonic antigen (CEA) and human epithelial mucin MUC-1 and is useful as a vaccine for colon cancer.

[0060] There are no limitations on the Salmonella typhi strains that can be used as vaccines in the present invention. For example, this can include any specific strain genetically attenuated from the original clinical isolate Ty2. Any attenuated Salmonella typhi strain derived from Ty2 can be used as a live vector in the present invention. Exemplary attenuated Salmonella typhi strains include, but are not limited to, Salmonella typhi Ty21a, CVD908, Salmonella typhi CVD909, CVD908-htrA, CVD915, and CVD910. In some embodiments, the Salmonella typhi strain can carry one or more additional chromosomal mutations in essential genes expressed on a plasmid. In some embodiments, the plasmid also encodes a heterologous protein in accordance with the present invention, allowing for selection and genetic stabilization of the plasmid and preventing loss of Salmonella typhi. In some embodiments, the Salmonella typhi strain carries a mutation in the ssb gene encoded on a selection expression plasmid.

[0061] When heterologous antigens or other proteins are overexpressed using plasmids, plasmid stability can be an important factor in developing a high-quality attenuated Salmonella typhi vaccine. Plasmid-free bacterial cells tend to accumulate more rapidly than plasmid-containing cells. One reason for this increased rate of accumulation is that transcription and translation of plasmid genes imposes a metabolic burden that slows cell growth and gives plasmid-free cells a competitive advantage. Furthermore, foreign plasmid gene products can sometimes be toxic to host cells. Therefore, it is preferable for the plasmid to be under some form of selective pressure to ensure that the encoded antigen is expressed appropriately and efficiently, so that a strong and effective immune response can be achieved.

[0062] In some embodiments, the plasmid is selected in Salmonella typhi using a non-antibiotic selection system. For example, the plasmid can encode an essential gene that complements an otherwise lethal deletion / mutation at this locus from the live vector chromosome. Examples of non-antibiotic expression plasmids that can be used in the present invention are described herein. Further, plasmid systems that can be used in the present invention are described in, for example, U.S. Patent Application Publication No. 2007 / 0281348, U.S. Patent Nos. 7,141,408, 7,138,112, 7,125,720, 6,977,176, 6,969,513, 6,703,233, and 6,413,768, which are incorporated herein by reference.

[0063] In one embodiment, a non-antibiotic genetic stabilization and selection system for an expression plasmid is designed to encode single-stranded binding protein (SSB), an essential protein involved in DNA replication, recombination, and repair that can be deleted from the Salmonella typhi live vector chromosome (Lohman et al., Annu Rev Biochem. 1994;63:527-570; Chase et al., Annu Rev Biochem. 1986;55:103-136; Galen et al., Infect Immun. 2010 January;78(1):337-47). In some embodiments, a plasmid expression vector for Salmonella typhi encodes single-stranded binding protein (SSB). In some embodiments, the expression vector is pSEC10S.

[0064] Some embodiments of the present invention utilize expression plasmids that eliminate both the random segregation and catalytic limitations inherent in non-antibiotic plasmid selection systems. Segregation of these plasmids within live Salmonella typhi vectors is improved using the active partitioning system (parA) for Salmonella typhi CVD908-htrA (Galen et al., Infect. Immun. 67:6424-6433). In some embodiments, reliance on catalytic enzymes is circumvented by using a plasmid selection / post-segregational killing system based on the ssb gene.

[0065] A solution to the instability of multicopy plasmids and the foreign antigens they encode is to integrate the foreign gene cassette into the chromosome of the live vector. However, reducing copy number has both advantages and disadvantages. While reducing copy number certainly reduces the metabolic burden associated with both the multicopy plasmid itself and the encoded foreign protein(s), this reduction in foreign antigen synthesis ultimately results in reduced delivery of these antigens to the host immune system and possibly reduced immunogenicity. This explanation could explain why serum immune responses to chromosomally encoded antigens have been modest to date in clinical trials (Gonzalez et al., J Infect Dis. 1994;169:927-931; Khan et al., Vaccine 2007;25:4175-4182).

[0066] An antigenic or biologically active fragment is a polypeptide having an amino acid sequence that is entirely the same as part, but not all, of the amino acid sequence of one of the polypeptides. An antigenic fragment may be "free-standing" or may be contained within a larger polypeptide, that portion or region, most preferably forming a single contiguous region.

[0067] In some embodiments, antigenic or biologically active fragments include, for example, truncated polypeptides having the amino acid sequence of a polypeptide but with a deletion of a contiguous series of residues including the amino terminus, a deletion of a contiguous series of residues including the carboxyl terminus, or a deletion of two contiguous series of residues including the amino and carboxyl termini. In some embodiments, fragments are characterized by structural or functional attributes such as alpha-helix and alpha-helix-forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha-amphipathic regions, beta-amphipathic regions, flexible regions, surface-forming regions, and high antigenic index regions.

[0068] Fragments can be of any size. An antigenic fragment can induce an immune response in a subject or can be recognized by a specific antibody. In some embodiments, the fragment corresponds to an amino-terminal truncation mutant. In some embodiments, the number of amino-terminal amino acids missing from the fragment ranges from 1 to 100 amino acids. In some embodiments, it ranges from 1 to 75 amino acids, 1 to 50 amino acids, 1 to 40 amino acids, 1 to 30 amino acids, 1 to 25 amino acids, 1 to 20 amino acids, 1 to 15 amino acids, 1 to 10 amino acids, and 1 to 5 amino acids.

[0069] In some embodiments, the fragment corresponds to a carboxyl-terminal truncation mutant. In some embodiments, the number of carboxyl-terminal amino acids deleted from the fragment ranges from 1 to 100 amino acids. In some embodiments, it ranges from 1 to 75 amino acids, 1 to 50 amino acids, 1 to 40 amino acids, 1 to 30 amino acids, 1 to 25 amino acids, 1 to 20 amino acids, 1 to 15 amino acids, 1 to 10 amino acids, and 1 to 5 amino acids.

[0070] In some embodiments, the fragment corresponds to an internal fragment lacking both the amino- and carboxyl-terminal amino acids. In some embodiments, the fragment is 7 to 200 amino acid residues in length. In some embodiments, the fragment is 10 to 100, 15 to 85, 25 to 65, or 30 to 50 amino acid residues in length. In some embodiments, the fragment is 7, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 80, or 100 amino acids in length.

[0071] In some embodiments, the fragments are at least 50, 100, 150, 200, or at least 250 amino acids in length. Of course, larger antigenic fragments are also useful in accordance with the present invention, as are fragments corresponding to most, if not all, of the amino acid sequences of the polypeptides described herein.

[0072] In some embodiments, the present invention provides a polypeptide comprising any of SEQ ID NOs: 12, 14, 18, or 20.

[0073] In some embodiments, a polypeptide has an amino acid sequence at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide described herein, or an antigenic or biologically active fragment thereof. In some embodiments, a variant varies from the reference by conservative amino acid substitutions, i.e., replacing a residue with one of similar characteristics. Typical substitutions are between Ala, Val, Leu, and Ile; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; between basic residues Lys and Arg; or between aromatic residues Phe and Tyr. In some embodiments, a polypeptide is a variant in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.

[0074] In some embodiments, the polypeptide is encoded by a polynucleotide optimized for high-level expression in Salmonella using codons that are preferred in Salmonella. As used herein, a codon "optimized for high-level expression in Salmonella" refers to a codon that is relatively abundant in Salmonella compared to all other codons that correspond to the same amino acid. In some embodiments, at least 10% of the codons are optimized for high-level expression in Salmonella. In some embodiments, at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the codons are optimized for high-level expression in Salmonella.

[0075] In some embodiments, the cancer antigen is expressed on a plasmid in Salmonella typhi. In some embodiments, the plasmid has a non-antibiotic-based system for plasmid selection and gene stabilization. In some embodiments, the plasmid expresses a gene that is essential for growth of Salmonella typhi and is chromosomally mutated in Salmonella typhi. In some embodiments, the gene encodes a single-stranded binding protein (SSB).

[0076] In some embodiments, the cancer antigen is integrated into the chromosome of Salmonella typhi. It will be appreciated that insertion of the gene cassette(s) into, for example, the guaBA, htrA, ssb, and / or rpoS loci of Salmonella typhi can be accomplished using, for example, the lambda red recombination system (Datsenko et al., PNAS. 2000. 97(12):6640-5). In some embodiments, the cancer antigen is inserted into the guaBA locus of Salmonella typhi. In some embodiments, the cancer antigen is inserted into the rpoS locus of Salmonella typhi.

[0077] In some embodiments, immunogenic cassettes can be integrated into either the ΔguaBA or ΔrpoS locus of CVD910ssb, for example, to compare the immunogenicity of chromosomal integration with that of plasmid-based expression. In some embodiments, only the ΔguaBA and ΔrpoS open reading frames are deleted, while the original promoters for these sites remain intact. In some embodiments, the cassette insertion includes the PompC promoter from the low-copy expression plasmid, so that integration into ΔguaBA or ΔrpoS creates a nested promoter that controls inducible expression of a given cassette in two stages.

[0078] Pharmaceutical Composition In some embodiments, the present invention provides pharmaceutical compositions comprising the Salmonella typhi live vector vaccine of the present invention. Such compositions may be for use in vaccinating an individual, such as a human. Such pharmaceutical compositions may include a pharmaceutically effective carrier and, optionally, other therapeutic ingredients, such as various adjuvants known in the art. Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers or excipients, such as phosphate-buffered saline, water, emulsions, such as oil / water emulsions, and microemulsions.

[0079] In some embodiments, the composition comprises one or more live Salmonella typhi vectors of the invention. In some embodiments, the composition comprises a combination of live Salmonella typhi vectors.

[0080] In some embodiments, the present invention provides compositions comprising isolated recombinant outer membrane vesicles from live Salmonella typhi vectors of the present invention, wherein the recombinant outer membrane vesicles comprise one or more cancer antigens expressed from the Salmonella typhi vector.

[0081] In some embodiments, the present invention provides compositions comprising a combination of isolated recombinant outer membrane vesicles from the live Salmonella typhi vectors of the present disclosure.

[0082] In some embodiments, the present invention provides compositions comprising an immunologically effective amount of a live Salmonella typhi vector and / or isolated recombinant outer membrane vesicles comprising one or more cancer antigens, and further comprising an immunologically effective amount of one or more adjuvants.

[0083] In some embodiments, one or more adjuvants are administered in the same composition as the Salmonella typhi vector and / or isolated recombinant outer membrane vesicles, or in separate compositions. There are no particular limitations on the adjuvant. In some embodiments, the adjuvant is a Toll-like receptor agonist. Toll-like receptor agonists include, but are not limited to, imiquimod, resiquimod, MBS8, gretrimod (DSP-0509), LPS, flagellin, diacylated lipopeptides, triacylated lipopeptides, monophosphoryl lipid A (MPLA), bacillus Calmette-Guerin (BCG), GSK1795091, entrimod, polyinosinic-polycytidylic acid (poly-ICLC), lintatolimod, BO-112, motolimod, SBT6050, TQ-A3334, SHR2150, RO7119929, DSP-0509, BNT411, APR003, BDB001, BDC-1001, CV8102, TransCon The therapeutic agent may include one or more agents selected from TLR7 / 8, MBS-8, BDB-018, imiquimod (UGN-201), lefitolimod (MGN1703), tilsotolimod (IMO-2125), bidutolimod / CMP-001, SD-101, TAC-001, LHC165, NJH395, NKTR-262, and cabrotolimod (AST-008). See, for example, Rolfo et al. Applications and clinical trial landscape using toll-like receptor agonists to reduce the toll of cancer. npj Precis. Onc. 7, 26 (2023). This document is incorporated herein by reference.

[0084] In some embodiments, the adjuvant comprises isolated recombinant outer membrane vesicles. In some embodiments, the isolated recombinant outer membrane vesicles do not contain a cancer antigen (i.e., are empty vesicles). In some embodiments, the recombinant outer membrane vesicles are derived from a Salmonella typhi vector. In some embodiments, the cancer antigen is an antigenic fragment of CEA, MUC1, or both. In some embodiments, the cancer antigen comprises a fusion protein comprising lpp-ompA or lpp-ompT and antigenic fragments of CEA and MUC1.

[0085] The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the therapeutic ingredient(s) and not overly harmful to the recipient thereof. The therapeutic ingredient(s) will be provided in an amount and frequency necessary to achieve the desired immunological effect.

[0086] The mode of administration and dosage form will influence the therapeutic dose of the Salmonella typhi live vector or isolated recombinant outer membrane vesicles desired for vaccination applications. This application is not specifically limited to oral administration of the vaccine, but may also include parenteral or other mucosal routes, including sublingual administration, as appropriate. The bacterial live vector material or recombinant outer membrane vesicles will be delivered in an amount capable of eliciting an immune response effective to mount a patient's immune response to the expressed antigen.

[0087] The bacterial live vector vaccines or isolated recombinant outer membrane vesicles of the present invention can be usefully administered to a host animal together with any other suitable pharmacologically or physiologically active agent, such as an antigenic and / or other biologically active substance.

[0088] Live vectors or isolated recombinant outer membrane vesicles derived from attenuated Salmonella typhi expressing one or more antigens described herein can be prepared and / or formulated without undue experimentation for administration to mammals, including humans, as appropriate for a particular application. Pharmaceutical compositions can be prepared without undue experimentation in a manner known per se, for example, by conventional mixing, dissolving, dragee-making, levitating, emulsifying, encapsulating, entrapping, spray-drying, or lyophilizing processes, or any combination thereof.

[0089] In one embodiment, live vectors or isolated recombinant outer membrane vesicles derived from attenuated Salmonella typhi expressing one or more antigens are administered mucosally. Suitable routes of administration can include, for example, oral, lingual, sublingual, rectal, transmucosal, nasal, buccal, intrabuccal, intravaginal, or intestinal administration, intravesicular, intraurethral, ​​inhalation, intranasal, or intraocular injection, and optionally, depot or sustained-release formulations. Furthermore, the compositions can be administered in targeted drug delivery systems. Routes of administration can be combined.

[0090] Dosage rates and appropriate dosage forms for the bacterial live vector vaccine compositions or recombinant isolated outer membrane vesicles of the invention can be readily determined by one of ordinary skill in the art without undue trial and error through the use of conventional antibody titration techniques and conventional bioefficacy / biocompatibility protocols. Among other things, the dose rate and appropriate dosage form will depend on the particular antigen used, the desired therapeutic effect, and the desired duration of biological activity.

[0091] In some embodiments, attenuated live vectors or isolated recombinant outer membrane vesicles derived from Salmonella typhi expressing one or more antigens can also be prepared for nasal administration. As used herein, nasal administration includes administering a compound to the mucous membranes of the nasal passages or nasal cavity of a subject. Pharmaceutical compositions for nasal administration of live vectors or isolated recombinant outer membrane vesicles derived from Salmonella typhi include a therapeutically effective amount of live vectors or isolated recombinant outer membrane vesicles derived from Salmonella typhi prepared by well-known methods and administered, for example, as a nasal spray, nasal drops, suspension, gel, ointment, cream, or powder. Administration of live vectors or isolated recombinant outer membrane vesicles derived from Salmonella typhi can also be performed using a nasal tampon or nasal sponge.

[0092] The compositions may also suitably contain one or more preservatives, antioxidants, etc. Some examples of techniques for formulating and administering live vectors or isolated recombinant outer membrane vesicles derived from Salmonella typhi can be found in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishing Co., 21st addition, which is incorporated herein by reference.

[0093] In one embodiment, the pharmaceutical composition contains a live vector or isolated recombinant outer membrane vesicles derived from Salmonella typhi in an amount effective to achieve their intended purpose. In one embodiment, an effective amount refers to an amount sufficient to prevent or treat cancer. In one embodiment, treating refers to reducing the occurrence of, inhibiting the progression of, or ameliorating the symptoms of a disease, such as cancer, in the subject being treated. In one embodiment, preventing refers to administering prophylactically, for example, when, in the opinion of the attending physician, the subject's background, genetics, environment, occupational history, etc., creates a prediction or increased likelihood that the subject is at risk of having a disease, even if the subject does not yet have the disease or is asymptomatic of the disease at the time of diagnosis or administration.

[0094] Treatment method The present invention also includes a method of treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a live Salmonella typhi vector described herein and / or an effective amount of an isolated recombinant outer membrane vesicle described herein. The present invention also includes a method of inducing an immune response in a subject. The immune response may be directed against one or more cancer antigens expressed by the Salmonella typhi live vector.

[0095] In some embodiments, the present invention provides a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject an immunologically effective amount of a live Salmonella typhi vector engineered to express one or more cancer antigens, wherein the antigens are delivered to mucosal tissues of the subject by outer membrane vesicles produced by the Salmonella typhi vector.

[0096] In some embodiments, the present invention provides a method for inducing an immune response in a subject in need thereof, the method comprising administering to the subject an immunologically effective amount of isolated recombinant outer membrane vesicles comprising one or more cancer antigens.

[0097] In some embodiments, the present invention provides methods for treating or preventing cancer in a subject, comprising administering to the subject an immunologically effective amount of isolated recombinant outer membrane vesicles comprising one or more cancer antigens.

[0098] The route of administration of the recombinant outer membrane vesicles is not limited. In some embodiments, the recombinant outer membrane vesicles are administered intranasally, mucosally, orally, parenterally, intravenously, intramuscularly, intradermally, topically, or a combination thereof.

[0099] In one embodiment, the recombinant outer membrane vesicles used in the methods herein comprise one or more cancer antigens, including CEA or a fragment or variant thereof, MUC1 or a fragment or variant thereof, or a fusion protein comprising CEA or a fragment or variant thereof and MUC1 or a fragment or variant thereof. In some embodiments, the antigen is fused to a surface-displayed protein, such as Lpp-OmpA.

[0100] In some embodiments, the present invention provides a method for inducing an immune response in a subject in need thereof, the method comprising administering to the subject an immunologically effective amount of isolated recombinant outer membrane vesicles comprising one or more cancer antigens, wherein the recombinant outer membrane vesicles are administered to the subject by intravenous route, intramuscular route, or both, and the recombinant outer membrane vesicles are administered one or more times.

[0101] In one embodiment, the method comprises administering to a subject a combination of live Salmonella typhi vectors of the invention. In some embodiments, the combination of vectors is present in the same composition. In some embodiments, the vectors are present in separate compositions.

[0102] In one embodiment, the method comprises administering to a subject a combination of isolated recombinant outer membrane vesicles.

[0103] In some embodiments, the present invention provides a method for inducing an immune response in a subject in need thereof, comprising administering to the subject an immunologically effective amount of isolated recombinant outer membrane vesicles comprising one or more cancer antigens, wherein the recombinant outer membrane vesicles are administered to the subject by intravenous route, intramuscular route, or both, and the recombinant outer membrane vesicles are administered one or more times.

[0104] In some embodiments, the recombinant outer membrane vesicle comprises: One or more cancer antigens, b. the outer membrane folding protein BamA or a fragment or variant thereof, and c. Derived from a live Salmonella typhi vector engineered to express lipid A deacylase PagL, or a fragment or variant thereof.

[0105] In some embodiments, the subject is first administered the isolated recombinant outer membrane vesicles, followed at a later time by an immunologically effective amount of the outer membrane vesicles.

[0106] In some embodiments, a subject is first administered isolated recombinant outer membrane vesicles, followed by one to three subsequent administrations of an immunologically effective amount of outer membrane vesicles, the subsequent administrations being spaced apart in time.

[0107] In some embodiments, the outer membrane vesicles are administered a total of at least four times.

[0108] In some embodiments, the outer membrane vesicles are administered to the subject by an intravenous route.

[0109] In some embodiments, the outer membrane vesicles are administered at a dose of about 5 μg / kg to about 25 μg / kg.

[0110] In some embodiments, the outer membrane vesicles are administered at a dose of 10 μg / kg to about 25 μg / kg.

[0111] In some embodiments, the doses are spaced about 2 days to 12 weeks apart. In some embodiments, the doses are spaced at least 7 days apart. In some embodiments, the doses are spaced at least 14 days apart. In some embodiments, the doses are spaced at least 21 days apart.

[0112] In some embodiments, the subject receives one or more administrations of an immunologically effective amount of an adjuvant in combination with the outer membrane vesicles.

[0113] In some embodiments, the subject is administered the adjuvant in a composition separate from the composition comprising the outer membrane vesicles.

[0114] In some embodiments, the outer membrane vesicles and the adjuvant are administered in the same composition.

[0115] In some embodiments, a live Salmonella typhi vector or isolated recombinant outer membrane vesicle of the present invention is administered to a subject with cancer. In some embodiments, a live Salmonella typhi vector or isolated recombinant outer membrane vesicle of the present invention is administered to a subject at risk of developing cancer. In some embodiments, a live Salmonella typhi vector or isolated recombinant outer membrane vesicle is administered to a subject along with one or more additional therapies to treat cancer. In some embodiments, the one or more additional therapies are selected from chemotherapy, radiation therapy, surgery, and immunotherapy. In a preferred embodiment, the live Salmonella typhi vector or isolated recombinant outer membrane vesicle is co-administered to a subject as soon as possible after early detection of the target cancer. It is now well understood that early detection increases the likelihood of successful treatment of solid tumors before they progress to large solid masses, which can dramatically reduce the availability of target cancer antigens for any type of therapeutic treatment.

[0116] In some embodiments, the Salmonella vector and / or recombinant outer membrane vesicle is administered to a subject after early detection of cancer. This is advantageous because early detection and intervention can improve the subject's chances of curing cancer. In some embodiments, the method includes a diagnostic screening method for detecting cancer present in a subject. Such diagnostic screening methods may include imaging and / or screening tissue or blood samples obtained from the subject for the presence of cancer cells or cancer markers. Suitable early diagnostic methods are described, for example, in Cohen et al. Science, 359:926-930 (2018) and Lennon et al. Science 10.1126 / science.abb9601 (2020), the entire contents of which are incorporated herein by reference.

[0117] In some embodiments, cancer can be detected by performing a liquid biopsy, for example, by taking a blood sample and detecting cancer cells or markers in the sample. In some embodiments, a positive blood test for detecting cancer can be confirmed by a scan, such as a PET-CT scan, to identify a mass. In some embodiments, protein or nucleic acid markers are detected in the liquid sample to identify the cancer present in the subject. There is no necessary limit to the type of cancer that can be identified in the blood. Such cancers may include lung cancer, ovarian cancer, colon cancer, breast cancer, lymphoma, kidney cancer, thyroid cancer, uterine cancer, and appendix cancer.

[0118] In some embodiments, ovarian cancer can be detected in the blood by detecting the presence of TP53, CA19-9, CA125, CA15-3 markers, or combinations thereof.

[0119] In some embodiments, lung cancer can be detected in the blood by detecting the presence of KRAS, TP53, CA15-3, HGF, CEA, EGFR, PIK3CA markers, or combinations thereof.

[0120] In some embodiments, uterine cancer can be detected in the blood by detecting the presence of the TP53, CA19-9 markers, or a combination thereof.

[0121] In some embodiments, thyroid cancer can be detected in the blood by detecting the presence of the CEA marker.

[0122] In some embodiments, colon cancer can be detected in the blood by detecting the presence of the BRAF, TP53 markers, or a combination thereof.

[0123] In some embodiments, breast cancer can be detected in the blood by detecting the presence of the PIK3CA, TP53 markers, or a combination thereof.

[0124] In some embodiments, lymphoma can be detected in the blood by detecting the presence of HGF, NRAS markers, or a combination thereof.

[0125] In some embodiments, kidney can be detected in the blood by detecting the presence of the KRAS marker.

[0126] In some embodiments, appendix cancer can be detected in the blood by detecting the presence of the CEA marker.

[0127] Vaccine strategies are well known in the art, and therefore the vaccination strategies encompassed by the present invention are not intended to limit the invention in any way. In certain embodiments of the present invention, a Salmonella typhi live vector vaccine expressing one or more cancer antigens or isolated recombinant outer membrane vesicles is administered alone in a single application or in sequential applications spaced over time.

[0128] In other embodiments of the present invention, the Salmonella typhi live vector vaccine is administered as a component of a heterologous prime / boost regimen. A "heterologous prime / boost" strategy is a two-phase immunization regime involving sequential administration (priming and boosting phases) of the same antigen in two different vaccine formulations by the same or different routes. In certain embodiments of the present invention incorporating a heterologous prime / boost regimen, a mucosal prime / parenteral boost immunization strategy is used. For example, one or more Salmonella typhi live vector vaccines as taught herein can be administered orally or via another mucosal route, followed by a parenteral boost with a vaccine composition comprising isolated recombinant outer membrane vesicles from a Salmonella typhi vector containing one or more cancer antigens.

[0129] In another aspect, the present invention is directed to a method of inducing an immune response to an antigen in a subject in need thereof, the method comprising administering to the subject an immunologically effective amount of a live Salmonella typhi vector of the present invention as an initial administration, and subsequently administering a boost composition comprising a composition comprising isolated recombinant outer membrane vesicles from the Salmonella typhi vector comprising one or more cancer antigens.

[0130] In some embodiments, the isolated recombinant outer membrane vesicles of the invention are primed and boosted with a Salmonella typhi live vector vaccine of the invention, hi some embodiments, the boost is administered mucosally, e.g., orally, or parenterally.

[0131] In some embodiments, in the heterologous prime / boost setting, the subject: i. a live Salmonella typhi vector engineered to express one or more cancer antigens and lipid A deacylase PagL or a fragment or variant thereof; and ii. isolated recombinant outer membrane vesicles isolated from a live Salmonella typhi vector engineered to express one or more cancer antigens, lipid A deacylase PagL or a fragment or variant thereof, and outer membrane folding protein BamA or a fragment or variant thereof. In some embodiments, the live Salmonella typhi vector of part i is administered as a prime dose and the isolated recombinant outer membrane vesicles of part ii are administered as a boost dose. In some embodiments, the isolated recombinant outer membrane vesicles of part ii are administered as a prime dose and the live Salmonella typhi vector of part ii is administered as a boost dose.

[0132] As used herein, an "immune response" is a physiological response of a subject's immune system to an immune composition. The immune response may include an innate immune response, an adaptive immune response, or both. In one embodiment of the present invention, the immune response is a protective immune response. A protective immune response is one in which secondary exposure to the same or similar antigen confers immunological cellular memory in a subject, along with effects characterized by one or more of the following characteristics: a lag period that is shorter than the lag period that would result from exposure to the selected antigen in the absence of prior exposure to the immunizing composition; production of antibodies that last longer than the production of antibodies that would result from exposure to the selected antigen in the absence of prior exposure to the immunizing composition; a change in the type and quality of antibodies produced compared to the type and quality of antibodies produced upon exposure to the selected antigen in the absence of prior exposure to the immunizing composition; a shift in the class response with IgG antibodies appearing in higher concentrations and with greater persistence than IgM than occurs in response to exposure to the selected antigen in the absence of prior exposure to the immunizing composition; an increase in the average affinity (binding constant) of antibodies for the antigen compared to the average affinity of antibodies for the antigen that results from exposure to the selected antigen in the absence of prior exposure to the immunizing composition; and / or other characteristics known in the art for characterizing a secondary immune response.

[0133] In further embodiments, the method of inducing an immune response comprises administering to a subject a pharmaceutical formulation provided herein comprising one or more Salmonella live vectors or isolated recombinant outer membrane vesicles of the invention in an amount sufficient to induce an immune response in the subject (an immunologically effective amount). In some embodiments, the composition is administered intranasally.

[0134] In some embodiments, one or more Salmonella typhi live vector vaccines or isolated recombinant outer membrane vesicles of the invention are administered mucosally in an initial priming dose, optionally followed about 2 to about 10 weeks later by a second (or third, fourth, fifth, etc.) priming dose of live vector vaccine or isolated recombinant outer membrane vesicles. In some embodiments, a boosting composition is administered about 3 to about 12 weeks after the priming dose. In some embodiments, a boosting composition is administered about 3 to about 6 weeks after the priming dose. In some embodiments, the boosting composition is substantially the same type of composition administered as the priming composition (e.g., a homologous prime / boost regimen).

[0135] In carrying out a therapeutic and / or prophylactic immunization protocol, an immunologically effective amount of a live Salmonella typhi vector or isolated recombinant outer membrane vesicles is administered to a subject. As used herein, the term "immunologically effective amount" refers to the total amount of a live Salmonella typhi vector or isolated recombinant outer membrane vesicles sufficient to induce an enhanced immune response in a subject. When applied to an individual therapeutic agent administered alone, the term refers to that therapeutic agent alone. When applied to a combination, the term refers to the combined amount of components that result in a therapeutic effect, regardless of whether administered concomitantly, sequentially, or simultaneously.

[0136] The specific dosage will depend on the age, weight, sex and medical condition of the subject being treated, as well as the method of administration. Appropriate dosages can be readily determined by those skilled in the art.

[0137] There is no necessary limit to the dose of recombinant outer membrane vesicles that can be administered. The dose of recombinant outer membrane vesicles can be administered one or more times spaced apart over time.

[0138] In some embodiments, the dose is increased or decreased over time. In some embodiments, a dose of about 0.001 μg / kg to about 10 μg / kg of recombinant outer membrane vesicles is administered to the subject based on the subject's body weight (kg). In some embodiments, a dose of about 0.1 μg / kg to about 10 μg / kg of recombinant outer membrane vesicles is administered to the subject. In some embodiments, a dose of about 0.25 μg / kg to about 5.0 μg / kg of recombinant outer membrane vesicles is administered to the subject. In some embodiments, a dose of about 0.25 μg / kg to about 2.5 μg / kg of recombinant outer membrane vesicles is administered to the subject. In some embodiments, a dose of about 0.25 μg / kg to about 1.0 μg / kg of recombinant outer membrane vesicles is administered to the subject.

[0139] In some embodiments, the recombinant outer membrane vesicles are administered via a parenteral route. In some embodiments, the route of administration is intravenous. In some embodiments, the route of administration is intramuscular.

[0140] In some embodiments, a subject receives multiple doses of recombinant outer membrane vesicles (or multiple doses of live Salmonella typhi vector, or a combination of recombinant outer membrane vesicles and live Salmonella typhi vector) spaced apart. In some embodiments, a subject receives two, three, four, five, six, or more doses. In some embodiments, doses are administered at intervals of about 2 days to 12 weeks. In some embodiments, doses are administered at intervals of about 2 to 7 days. In some embodiments, a subject receives two to five doses, spaced apart by 2 to 7 days. In some embodiments, doses are administered at intervals of about 1 to 6 weeks. In some embodiments, doses are administered at intervals of about 1 week. In some embodiments, doses are administered at intervals of about 2 weeks. In some embodiments, doses are administered at intervals of about 3 weeks.

[0141] In some embodiments, a subject is initially administered a recombinant outer membrane vesicle parenterally, e.g., intravenously or intramuscularly, followed, e.g., about 1-4 weeks later, by a boosting dose of a live Salmonella typhi vector administered mucosally, e.g., intranasally.

[0142] In some embodiments, a subject is initially administered a live Salmonella typhi vector via a mucosal route, e.g., intranasally, followed, e.g., about 1-4 weeks later, by a boosting dose of recombinant outer membrane vesicles administered parenterally, e.g., intravenously or intramuscularly.

[0143] In some embodiments, a subject is administered an initial dose of recombinant outer membrane vesicles parenterally, for example, intravenously or intramuscularly, followed by a boosting dose of recombinant outer membrane vesicles parenterally, for example, intravenously or intramuscularly, for example, about 1 to 4 weeks later.

[0144] In some embodiments, a subject is administered an initial dose of a live S. Typhi vector via a mucosal route, e.g., intranasally, followed by, e.g., about 1-4 weeks later, a boosting dose of a live S. Typhi vector via a mucosal route, e.g., intranasally.

[0145] In some embodiments, a live vector derived from Salmonella typhi is about 1 x 10 9 In some embodiments, the attenuated live vector from Salmonella typhi can be administered intranasally at a dose of about 1 x 10 CFU. 8 CFU ~ approx. 1 x 10 10 CFU doses can be administered intranasally.

[0146] As used herein, the term "subject" refers to an animal, such as a mammal. For example, contemplated mammals include humans, primates, dogs, cats, sheep, cows, goats, pigs, horses, mice, rats, rabbits, and guinea pigs. The terms "subject," "patient," and "host" are used interchangeably.

[0147] In some embodiments, multivalent recombinant outer membrane vesicles are administered to a subject after an early diagnosis of colorectal cancer or to a subject at an early stage of progression of a solid tumor with sufficient vascular density to allow passive deposition of the recombinant outer membrane vesicles. Such patient populations may be identified using newly developed blood-based diagnostic assays (Lennon et al., Science, (2020), 369(6499). In some embodiments, subjects with more advanced stage III cancer who are recovering from surgery and have completed adjuvant systemic chemotherapy are treated with multiple doses of recombinant outer membrane vesicles alone or in combination with a VLP-CEA vaccine (or a live Salmonella typhi-derived vector as described herein) administered in a heterologous prime-boost format. In some embodiments, subjects receiving recombinant outer membrane vesicles are at high risk of developing colorectal cancer or other cancers in which MUC1 is often overexpressed (see, e.g., Crosby et al., Journal for Immunotherapy of cancer, (2020), 8(2); Kimura et al., Cancer Prev Res (Phila) 2013;6(1):18-26; Lohmueller et al., Sci Rep 2016;6:31740).

[0148] The live Salmonella typhi vector or isolated recombinant outer membrane vesicles of the present invention may be administered to warm-blooded animals of any age. The live Salmonella typhi vector can be administered as a single or multiple initial doses, followed by one or more booster doses. For example, a subject can receive a single dose followed by a booster dose at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years or more.

[0149] Adapting the teachings of the present invention to a particular problem is within the skill of one in the art in light of the teachings contained herein. Examples of compositions and methods of the present invention are illustrated in the following non-limiting examples. [Example]

[0150] Example 1. Development of a PagL-mediated antigen delivery platform. Because ClyA is a hemolysin with cytopathic properties that may reduce the clinical acceptability of candidate vaccine strains in which ClyA is overexpressed, we sought to develop a nonpathogenic alternative to induce OMV formation and transport based on PagL (Ludwig et al., Mol Microbiol 1999;31(2):557-67; Lai et al., Infect Immun 2000;68(7):4363-7). Therefore, we constructed a synthetic pagL gene and inserted it into our non-antibiotic, low-copy-number expression plasmid pSEC10, replacing the clyA gene to create pPagL. Similar to our previous experiments with inducible outer membrane vesicles, we wanted to monitor OMV transport by measuring the hemolytic activity associated with ClyA-mediated vesicle formation. Therefore, we integrated a cassette encoding ClyA into the guaBA locus of CVD910 and then introduced pPagL into the resulting strain, generating CVD910ΔguaBA::clyA(pPagL). Note that in this particular strain, ClyA acts as a surrogate hemolytic reporter for the chromosomally encoded antigen protein, and overexpression of plasmid-encoded PagL is expected to significantly improve rOMV transport. All strains were grown at 37°C to early logarithmic phase growth and showed approximately 2 × 10 binding to sheep erythrocytes. 7 For CFU of bacteria, OD 540Hemolytic activity was measured using the CVD910 strain. As expected, hemolytic activity was absent in the vaccine strain CVD910 (lane 2), as shown in Figure 1. Surprisingly, hemolytic activity of the chromosomally encoded ClyA was not detected in CVD910ΔguaBA::clyA (lane 3). This is due to the reduced copy number compared to the plasmid-encoded hemolytic activity observed in CVD910(pSEC10) [see Figure 3, lane 3]. However, significant hemolytic activity was observed when pPagL was introduced into 910ΔguaBA::clyA (lane 4), clearly demonstrating that overexpression of PagL induces superior transport of outer membrane proteins (i.e., ClyA in this example) through outer membrane vesicles.

[0151] Study Overview: Taken together, our results firmly establish the feasibility of developing an attenuated Salmonella typhi-based mucosal live vector vaccine capable of efficiently expressing and delivering properly folded foreign proteins to the surface of our live vector vaccine. These foreign antigens can be expressed from a chromosomally integrated gene cassette, enabling the construction of a live vector vaccine without the need for large, potentially unstable, multicopy expression plasmids for antigen delivery. We also designed a unique outer membrane vesicle antigen delivery platform and successfully completed a proof-of-principle study demonstrating the efficacy of the PagL-mediated antigen delivery system for delivery via recombinant rOMVs using ClyA as a model outer membrane protein.

[0152] Example 2. Development of a PagL-mediated antigen delivery platform. We constructed three synthetic alleles of pagL, designated pagL v1 (SEQ ID NOs: 1 and 2), pagL v2 (SEQ ID NOs: 3 and 4), and pagL v3 (SEQ ID NO: 5). These three versions differ in the 5'-terminal DNA sequence controlling the translation efficiency of each allele. This careful engineering approach was adopted because the optimal translation efficiency of pagL, which would ensure sufficient synthesis of biologically active PagL while avoiding potentially lethal overexpression of this protein, was unknown at the time of these experiments. The amino acid sequences of pagL v2 and v3 are identical. To this end, pagL v1 has an optimized ribosome binding site (RBS), an ATG start codon, and multiple optimized codons at the start of the gene to increase translation efficiency. pagL v2 is similar to v1 but contains a GTG start codon to slightly reduce translation efficiency. pagL v3 is essentially identical to the wild-type chromosomal sequence of the pagL gene naturally present in Salmonella typhimurium. Therefore, we expected the highest levels of PagL synthesis from v1, lower levels from v2, and the lowest levels from v3.

[0153] Each cassette was inserted as a BamHI-NheI fragment into our non-antibiotic, low-copy-number expression plasmid pSEC10, digested with BamHI and NheI, replacing the clyA gene to generate pPagL. The predicted sequence of pPagL v1 is shown in SEQ ID NO:6. Similar to our previous experiments with inducible recombinant outer membrane vesicles (rOMVs), we wanted to monitor OMV transport by measuring the hemolytic activity associated with ClyA-containing vesicles. Therefore, we integrated a ClyA-encoding cassette into the guaBA locus of CVD910 and then introduced pPagL into the resulting strain, generating CVD910ΔguaBA::clyA(pPagL). Note that in this particular strain, ClyA serves as a hemolytic reporter, surrogate for chromosomally encoded antigen proteins, and we expect that overexpression of plasmid-encoded PagL will significantly improve rOMV transport. All strains were grown at 37°C to early logarithmic phase growth, with a virulence of approximately 2 × 10 against sheep red blood cells. 7 For CFU of bacteria, OD 540 Hemolytic activity was measured using the CVD910DguaBA::clyA vector. As expected, hemolytic activity was absent in the vaccine strain CVD910 (lane 2), as shown in Figure 2. Surprisingly, due to the reduced copy number relative to the plasmid-encoded hemolytic activity observed in CVD910(pSEC10), the hemolytic activity of the chromosomally encoded ClyA was not detected in CVD910DguaBA::clyA (lane 3). However, significant hemolytic activity was observed when pPagL was introduced into 910DguaBA::clyA (lane 4), clearly demonstrating that overexpression of PagL induces superior transport of rOMVs (in this case, containing ClyA as a surrogate outer membrane protein).

[0154] Example 3. Development of an inducible vesicle delivery system. We engineered a novel osmotically inducible vaccine antigen delivery system in which foreign antigens expressed on the outer membrane surface of our attenuated Salmonella typhi candidate vaccine strain CVD910 can be efficiently transported from the surface of the vaccine strain via recombinant outer membrane vesicles carrying these foreign surface-expressed protein antigens. To validate this concept in the context of vaccine development, we further engineered our first prototype attenuated CVD910 candidate vaccine in which outer membrane proteins are efficiently expressed on the surface of the live strain. We introduced a low-copy-number expression plasmid encoding inducible overexpression of PagL, a novel outer membrane lipid A deacylase recently reported to catalyze hypervesicle formation when overexpressed in Salmonella. We hypothesized that overexpression of PagL might catalyze the formation of antigen-loaded rOMVs for efficient delivery to the site of immune induction, thereby inducing protection against disease (Elhenawy et al., mBio 2016;7(4):pii:e00940-16.doi:10.1128 / mBio.-16.). To improve the efficiency of outer membrane delivery of both antigen and PagL (to enhance rOMV-mediated antigen delivery and improve vaccine efficacy), we also integrated an inducible gene cassette encoding the outer membrane folding protein BamA into the chromosome of this prototype vaccine.

[0155] BamA is a ∼90 kDa protein that is an essential component of the five-protein outer membrane β-barrel assembly machinery (BAM) complex, which catalyzes the insertion of β-barrel proteins into the outer membrane of Gram-negative bacteria (Noinaj et al., Nature Reviews Microbiology 2017;15(4):197-204). However, the entire BamABCDE complex is not necessarily required for efficient insertion of selected outer membrane proteins. Indeed, several groups have reported that OmpA efficiently integrates into lipid bilayers containing only BamA (Gessmann et al., Proc Natl Acad Sci USA 2014;111(16):5878-83; Plummer et al., Biochemistry 2015;54(39):6009-11). Therefore, we hypothesized that overexpression of BamA in vaccine strains coexpressing PagL might also result in more efficient transport of outer membrane vesicles carrying surface-expressed foreign antigens. We chose to use the bamAAb allele from A. baumannii given a recent report that purified A. baumannii -derived AbBamA was protective against mice infected with MDR A. baumannii (Singh et al., Sci Rep 2017;7(1):12411).

[0156] Development of a PagL-Mediated Antigen Delivery Platform. After demonstrating antigen expression on the surface of our candidate vaccine strain CVD910, we initiated the development of an inducible outer membrane vesicle antigen transport system to deliver surface-expressed antigens to the site of immune induction after immunization. To achieve this, we focused on the use of PagL, a lipid A deacylase recently reported to catalyze hypervesicle formation when overexpressed in Salmonella (Elhenawy et al., mBio 2016;7(4):pii:e00940-16.doi:10.1128 / mBio.-16). Given that overexpression of PagL could theoretically induce hypervesicle formation of antigen-containing rOMVs, this strategy also presented a unique opportunity to use purified multivalent rOMVs as mucosal vaccines by themselves or in combination with the live carrier vaccine from which they were purified.

[0157] To explore this intriguing possibility, we first attempted to monitor OMV transport by phenotypically tagging vesicles with cytolysin A (ClyA), a novel endogenous Salmonella hemolysin first reported by Wai et al. to catalyze the formation of large outer membrane vesicles when overexpressed (Wai et al., Cell 2003;115(1):25-35). This simple hemolytic reporter phenotype allowed for rapid and quantitative assessment of OMV transport, efficiently guiding the optimization of expression cassettes and avoiding lethal overexpression of vesicle-forming proteins. We have also successfully utilized ClyA expression to transport foreign antigens from engineered carrier strains as fusion proteins encoded by low-copy-number expression plasmids (Galen et al., Infect Immun 2004;72(12):7096-106). However, to test PagL-mediated vesicle formation, we needed to reduce the expression of ClyA hemolysin so that ClyA was released to the surface at a level sufficient to detect hemolytic activity (i.e., phenotypically "tagging" the membrane surface), but not high enough to actually catalyze ClyA-mediated vesicle formation. We therefore integrated a ClyA-encoding cassette into the guaBA locus of CVD910 to generate the reporter strain CVD910ΔguaBA::clyA. We then constructed three synthetic pagL genes with different translation efficiencies. These genes varied in the distance between the consensus ribosome binding site (AGGAGG) and the start codon: one located 5 bases upstream of the optimal ATG start codon (pagL v1), one located 6 bases upstream of the less efficient GTG start codon (pagL v2), or one located 5 bases upstream of the GTG start codon (pagL v3). Since the ideal position of the RBS is 7 to 9 bases away from the ATG start codon, we predicted that the expression levels of these three isogenic alleles would decrease in the order pagLv1 > pagLv2 > pagLv3 (Ringquist et al., Mol Microbiol 1992;6(9):1219-29.). Each allele is involved in the osmoregulatory PompC The PagL gene was inserted into the low-copy-number expression plasmid pSEC10 so that it was positioned downstream of the ompC promoter, generating pPagL v1, pPagL v2, and pPagL v3, respectively. Inducible expression of PagL in the resulting expression plasmids is controlled at the transcriptional level by osmotic induction of the ompC promoter (Stokes et al., Infect Immun 2007;75(4):1827-34; Galen et al., Infect Immun 2010;78(1):337-47; Galen et al., Infect Immun 1999;67(12):6424-33). We hypothesized that increasing the expression level of the plasmid-encoded PagL, along with the efficiency of the RBS, would increase the transport of ClyA-tagged rOMVs and, consequently, their hemolytic activity.

[0158] To test this hypothesis, each plasmid was introduced into the reporter strain CVD910ΔguaBA::clyA. The strain was then grown to early logarithmic phase under inducing conditions at 37°C, and approximately 2 × 10 7 Using CFU of bacteria, hemolytic activity against sheep red blood cells was measured using OD 540 As shown in Figure 3, hemolytic activity was absent in the vaccine strain CVD910 (lane 2). As expected, hemolytic activity of the chromosomally encoded ClyA was not detected in CVD910ΔguaBA::clyA (lane 3) due to reduced expression levels from the chromosome. However, significant hemolytic activity was observed for 910ΔguaBA::clyA(pPagLv1), which decreased when the efficiency of the RBS was manipulated (lane 4 vs. lanes 5 and 6), supporting the hypothesis that overexpression of PagL induces superior transport of outer membrane proteins (i.e., ClyA in this case) through outer membrane vesicles.

[0159] Enhanced surface expression of OMPs by overexpression of AbBamA. Preliminary results suggested that the antigen was successfully expressed on the surface of CVD910 cells, but expression from non-permeabilized cells was reduced relative to the levels detected in permeabilized cells. We hypothesized that this difference may be due to the transport rate and / or proper insertion of the protein into the outer membrane, and that overexpression of transport proteins that affect translocation rate may enhance surface expression. We noted that both the antigen and PagL are β-barrel transmembrane proteins (Rutten et al., Proc Natl Acad Sci USA 2006;103(18):7071-6). The insertion of β-barrel proteins into the outer membrane of Gram-negative bacteria is mediated by the β-barrel assembly (BAM) complex, of which BamA (itself a β-barrel protein) constitutes an essential core component (Albrecht et al., Acta Crystallogr D Biol Crystallogr 2014;70(Pt 6):1779-89. Noinaj et al., Nature reviews Microbiology 2017;15(4):197-204). It has also been reported that BamA alone can promote outer membrane folding and membrane insertion of β-barrel proteins in vitro. Therefore, we hypothesized that overexpression of BamA could improve the surface expression of outer membrane proteins, including exemplary vaccine antigens. Potentially, enhanced transport of PagL to the outer membrane could also enhance rOMV formation and thus the delivery of foreign antigens to the site of immune induction.

[0160] To test this hypothesis, we designed a synthetic gene cassette encoding AbBamA. Interestingly, our original cassette, in which translation initiated at the ATG start codon, did not successfully insert into a low-copy expression plasmid. Therefore, to avoid potentially lethal overexpression of AbBamA, we inserted the nucleotide sequence 5 bases after the GTG start codon (bamA). Ab v1) or 4 bases (bamA Abv2) We engineered an upstream ribosome binding site to more tightly control the level of translation. Assuming that the ideal location of the RBS is 7–9 bases away from the start codon, we used the bamA Ab v1 is bamA Ab v2 (Ringquist et al., Mol Microbiol 1992;6(9):1219-29). As with the pagL allele, we predicted that P ompC We engineered the baniA allele under the transcriptional control of the promoter and inserted the resulting cassette into our low-copy expression plasmids, generating pAbBamAvl and pAbBamAv2. These plasmids were then introduced into CVD910ΔguaBA::clyA. Although ClyA does not possess a β-barrel structure, we also wanted to investigate any potential effects of AbBamA overexpression on OMV formation (Wallace et al., Cell 2000;100:265-76). As summarized in Figure 4, the hemolytic activity for plasmid-based expression of ClyA in CVD910(pSEC10) was significantly higher than that observed for chromosomally encoded ClyA in CVD910ΔguaBA::clyA (lane 3 vs. lane 4) due to the increased copy number of clyA in CVD910(pSEC10). Surprisingly, introduction of pAbBamAvl into CVD910ΔguaBA::clyA was able to enhance hemolytic activity to a level comparable to that of plasmid-based expression in CVD910(pSEC10), an effect that was consistent with that of bamA Ab It was reduced in strains that expressed the v2 allele less efficiently (lane 5 vs. lane 6). From these experiments, we conclude that AbBamA can enhance the formation of outer membrane vesicles phenotypically tagged with ClyA and transported from CVD910, and can also promote the transport of vesicles carrying antigens relevant for vaccine development or other foreign antigens.

[0161] Encouraged by successfully demonstrating surface expression of antigen in our candidate vaccine strain CVD910, as well as the ability of both PagL and AbBamA to enhance rOMV transport, we then tested the hypothesis that surface expression of antigen could be optimized by co-expression of both PagL and AbBamA in a single vaccine strain. Given that surface-expressed outer membrane proteins are not immediately transported via vesicles, we reasoned that increased surface expression would ultimately increase outer membrane vesicle formation, even though this was not clearly determined in these preliminary experiments. To accomplish this, we engineered an osmotically controlled PAGL encoding the 93.2 kDa AbBamA protein (SEQ ID NO: 8). ompC -bamA Ab v1 (SEQ ID NO: 7) was integrated into the guaBA locus of CVD910. The resulting strain was then transformed with the pAntigen expression plasmid, CVD910ΔguaBA::bamA Ab v1 (Antigen) was produced.

[0162] These results firmly establish the feasibility of developing an attenuated Salmonella typhi-based mucosal live carrier vaccine capable of efficiently expressing and delivering properly folded foreign outer membrane proteins to the surface of our carrier vaccine. To improve the clinical acceptability of our candidate live carrier vaccine, we formally eliminated any effect of antigen expression on the pathogenicity of our live carrier. We also designed a unique PagL-mediated outer membrane vesicle antigen delivery platform in which the efficiency of antigen surface expression is enhanced by overexpression of the outer membrane folding protein AbBamA. This innovative modification improves the surface expression of outer membrane proteins. We conclude that the enhanced surface expression catalyzed by AbBamA also enhances the surface expression of other surface-targeted foreign proteins, and that induction of PagL then catalyzes the efficient transport of recombinant OMVs potentially carrying a wide variety of foreign proteins from either prokaryotes or eukaryotes. This technology is not limited to the development of vaccines against human pathogens, but can also be used in veterinary medicine and other applications, such as the development of immunotherapeutic vaccines against solid tumors (Niethammer et al., BMC Cancer 2012;12:361; Schmitz-Winnenthal et al., Oncoimmunology 2015;4(4):e1001217; Schmitz-Winnenthal et al., Oncoimmunology 2018;7(4):e1303584).

[0163] Example 4. Development of a candidate bivalent Salmonella typhi-based colon cancer vaccine Using a previously attenuated and highly immunogenic Salmonella typhi-based carrier vaccine strain, we recently designed and functionally evaluated an osmotically inducible and highly efficient recombinant outer membrane vesicle (rOMV) antigen delivery system driven by overexpression of the lipid A deacylase PagL (Galen et al., J Infect Dis 2009;199(3):326-35; Galen et al., Vaccine 2014;32(35):4376-85; Galen et al., Infect Immun 2015;83(1):161-72; Elhenawy et al., mBio 2016;7(4):pii:e00940-16.doi:10.1128 / mBio.-16). PagL is encoded by a genetically stabilized low-copy-number expression plasmid in which an otherwise lethal chromosomal deletion of the single-stranded binding (SSB) protein is stabilized by trans-complementation (Figure 5) (Galen et al., Infect Immun 2010;78(1):337-47). We used a chimeric Lpp-OmpA surface-display peptide to deliver a bivalent fusion protein encoding domains derived from CEA and MUC-1 to the surface of our carrier vaccine (Figure 10) (Francisco et al., Proc Natl Acad Sci USA 1992;89(7):2713-7. Hui et al., Biotechnol Lett 2019;41(6-7):763-77). Subsequently, in vivo osmotic induction of our orally administered recombinant carrier vaccine results in presentation of our target colon-associated antigens via highly immunogenic and highly efficient rOMVs expressing the CEA-MUC1 fusion protein on their surface.

[0164] Example 5. Cancer vaccine cassette. Here, we describe the design of two master synthetic gene cassettes encoding fusion proteins intended to function as antigens for vaccination against colon cancer (Figure 6). These synthetic cassettes are intended to be synthesized flanked by the restriction enzymes BamHI at the 5' end and SpeI at the 3' end; therefore, in a preferred embodiment, the cassettes are intended to be inserted downstream of the PagL open reading frame of the proprietary expression plasmid pPagL. The resulting plasmid will then encode an operon consisting of PagL and this fusion gene, the transcription of which is regulated by the osmoregulated PAGL gene. ompC Upon osmotic induction of this vaccine plasmid, targeted cancer antigens are expressed on the surface of the Salmonella typhi-based carrier vaccine, followed by transport of these antigens through outer membrane vesicles induced by co-expression of PagL.

[0165] The two fusion proteins intended to be co-expressed with PagL each consist of a surface expression cassette operably linked to two additional cancer antigen cassettes, each separated by an engineered linker region [designated A(EAAAK)4A] (Figure 7). This linker region is designed to fold into a rigid alpha helix separating each cancer domain to allow proper folding after translation (Chen et al., Advanced Drug Delivery Reviews 2013;65(10):1357-69). The surface expression cassettes are linked to a modified and patented non-hemolytic version of the ClyA protein (referred to herein as clyA). I198NThese surface expression cassettes are then operably linked to a cassette encoding an oncofusion protein composed of two domains, one derived from the cancer antigen carcinoembryonic antigen (CEA) and the other from MUC1 (Figure 7). The CEA-derived domain, designated A3B3 (encoded by a3b3), encodes a 179-amino acid region derived from the sixth and seventh Ig domains of CEA (Oikawa et al., Biochem Biophys Res Commun 1987;142(2):511-8; Hefta et al., Cancer Res 1992;52(20):5647-55; Zaremba et al., Cancer Res 1997;57(20):4570-7; Nukaya et al., Int J Cancer 1999;80(1):92-7; Gu et al., Gastroenterology 2020;158(1):238-52). The MUC1 domain consists of 140 amino acids and represents the seven repeat regions of the human MUC1 protein (Engelmann et al., J Biol Chem 2001;276(30):27764-9; Soares et al., J Immunol 2001;166(11):6555-63; Scheikl-Gatard et al., J Transl Med 2017;15(1):154; Guan et al., Bioconjug Chem 1998;9(4):451-8). The DNA sequence of the LOA master gene cassette is set forth in SEQ ID NO:11, and the encoded fusion protein is set forth in SEQ ID NO:12. ClyA I198N The master gene cassette is set forth in SEQ ID NO:19 and the encoded fusion protein is set forth in SEQ ID NO:20.

[0166] Both of these master gene cassettes were designed so that cleavage of either cassette with the restriction enzymes XbaI and AvrII, followed by religation, would yield a truncated gene encoding a fusion protein containing only the surface expression domain, linker, A3B3 domain, and final linker (e.g., SEQ ID NOS: 13 and 14 for LOA). Similarly, cleavage of either cassette with Nhel and XbaI, followed by religation, would yield a truncated gene encoding a fusion protein containing only the surface expression domain and MUC1 domain separated by a single linker sequence (e.g., SEQ ID NOS: 17 and 18 for LOA). These master gene cassettes were designed in this manner to rapidly generate isogenic constructs that can be used for isogenic carrier vaccines to immunize mice and determine the contribution of A3B3, MUC1, or both to eliciting cancer-specific immune responses.

[0167] Example 6: Construction of a ΔfliC::lpxE strain As demonstrated by our data, we have now successfully constructed a hypervesicle-forming reagent strain derived from the attenuated Salmonella typhi strain CVD910, in which the foreign outer membrane protein-targeting vaccine antigens AbOmpA and AbBamA from A. baumannii are coexpressed on the surface and can now be transported from the reagent strain via rOMVs (through the action of PagL) for use as a parenteral vaccine. We demonstrated that the reactogenicity of these vesicles is reduced due to the enzymatic activity of PagL, which deacylates lipid A, reducing in vitro reactogenicity by approximately 10-fold. However, it has previously been reported that purified rOMVs can have variable but significant amounts of flagellin adsorbed to their surface, which is an agonist of TLR5. Because we intend to use purified rOMVs as a vaccine administered intramuscularly to humans, we must minimize the unacceptable reactogenicity induced by both TLR4 and TLR5 agonists while still maintaining optimal immunogenicity and protective efficacy (Liu, Q. et al., Sci. Rep. 6, 34776, doi:10.1038 / srep34776(2016)). Therefore, to further improve the clinical acceptability and purity of our rOMV-based vaccine, we cloned chromosomally encoded FliC (TLR5 agonist, GenBank locus #AE014613) into IpxE (IpxE) from Francisella novicida. Fn) (SEQ ID NO: 26) reduced both TLR4- and TLR5-mediated reactogenicity (Zhao, J. & Raetz, CR, Mol. Microbiol. 78, 820-836, doi:10.1111 / j.1365-2958.2010.07305.x (2010); Zhao, J. et al., mBio 10, doi:10.1128 / mBio.00886-19 (2019)). LpxE (SEQ ID NO: 27) is a lipid A 1-phosphatase that dephosphorylates lipid A to generate less reactive monophosphoryl species (Figure 8). Coexpression of PagL (which deacylates lipid A but promotes hypervesicle formation) produced rOMVs containing pentaacyl-monophosphate 1-lipid A with significantly reduced TLR4 and TLR5 activity. To engineer the strains, we used the well-established Lambda Red site-specific chromosomal recombination system, which has been used previously for all of the strain constructions reported herein (Datsenko et al., Proc. Natl. Acad. Sci. USA 97, 6640-6645 (2000)). A set of isogenic strains was constructed, as listed in Table 1. A low-copy-number plasmid, designated pPagL-LOAM, expressing both PagL and the Lpp-OmpA-A3B3-MUC1-targeted CRC fusion protein, was then introduced into these strains. The final strain, CVD910ΔguaBA::P ompC -bamA Ab ΔfliC::P ompC -lpxE Fn (pPagL-LOAM) is shown graphically in FIG. [Table 1]

[0168] Example 7: Expression of CRC fusion proteins in six novel strains. Vesicles were purified from liquid cultures of the isogenic strains listed in Table 1 by low-speed centrifugation and filtration of the supernatant through a 0.2 μm filter to remove bacteria and debris. They were then pelleted by high-speed ultracentrifugation. The pellet was resuspended in PBS. The rOMV concentration was determined rigorously using the 3-Deoxy-D-manno-Octulosonic Acid (KDO) assay described by REW Hanock (http: / / cmdr.ubc.ca / bobh / method / kdo-assay / ). 0.5 μg of each purified rOMV was then subjected to Coomassie Brilliant Blue staining and Western immunoblot analysis. As shown in Figure 10A, multiple bands were detected in the Coomassie-stained sample, as expected for outer membrane vesicles containing various proteins and lipoproteins found in the outer membrane of Salmonella typhi. Notably, a strong band migrating at approximately 50 kDa in lanes 2 and 3 of panel A is absent in lanes 4–7. This is consistent with the known molecular weight (approximately 50 kDa) of the flagellin FliC deleted from the strains in lanes 4–7. As shown in Figure 10B, the CRC fusion protein is clearly detected in lanes 3, 5, and 7, as expected, but is not observed as a single band. Given that the A3B3 domain of CEA contains two disulfide bridges in the expression domain of the fusion protein (Figure 10C), we hypothesized that the faster-moving species below the intense top band are additional species containing one or more of the predicted disulfide bridges. Even though the gel was run under reducing conditions with 200 mM dithiothreitol (DTT), it was clear that disulfide bridges still formed.

[0169] Example 8. Immunogenicity of rOMVs in BALB / c mice following a single parenteral administration. We designed a synthetic gene encoding a CRC-targeting fusion protein that fused the A3B3 Ig-like domain from CEA to a subdomain consisting of seven 20-residue repeats derived from the VNTR domain of MUC1. Because the domains used in this construction are normally expressed on the surface of gastrointestinal epithelial cells, we needed to genetically fuse the A3B3-MUC1 fusion to the Lpp-OmpA surface-localization peptide to ensure properly folded surface expression in S. typhi carrier strains (and the rOMVs that are subsequently transported) (Francisco et al., Proc Natl Acad Sci USA, 1992;89:2713-7). The lpp-ompA-a3b3-muc1 gene encoding this fusion protein was inserted into pPagL downstream of pagLv1 to generate the final expression plasmid, pPagL-LOAM. This plasmid was transformed into CVD910ΔguaBA::P ompC -bamA Ab ΔfliC::P ompC -lpxEFn (see Table 1) by electroporation into the CRC-targeted carrier strain CVD910ΔguaBA::P ompC -bamA Ab ΔfliC::P ompC -lpxE Fn (pPagL-LOAM) was constructed, and rOMV LpxE-CRC This enabled the delivery of CRC-targeted vesicles, designated rOMVs. We then isolated these vesicles and confirmed strong expression of A3B3-MUC1 in the isolated vesicles by Western immunoblot analysis using rabbit antisera against the A3B3-MUC1 fusion protein (data not shown). However, theoretically, a reduction in both TLR4 and TLR5 activity in these vesicles could severely impact the CRC-specific immune response induced by these vesicles. Therefore, we investigated the feasibility of using rOMVs to deliver CRC-targeted vesicles. LpxE-CRC A preliminary dose-response immunogenicity study was conducted to evaluate humoral immunity to

[0170] Fifty BALB / c mice (6–8 weeks old) were randomly divided into five groups and administered either empty, non-adjuvanted rOMVs as a negative control. LpxE 4.0 μg of vesicles were administered intramuscularly, or unadjuvanted rOMV LpxE-CRC Intramuscular administration of rOMVs was performed at increasing doses of 0.5, 1.0, 2.0, or 4.0 μg. We also administered PBS to five mice as an additional negative control. The rOMV concentration was rigorously determined using a 3-Deoxy-D-manno-Octulosonic Acid (KDO) assay. Serum was collected on days 0 and 21, and antigen-specific serum IgG in pooled serum was measured by ELISA. As shown in Table 2, robust serum IgG titers against A3B3-MUC1 were detected after a single intramuscular administration of rOMVs, and the titers appeared to peak at a dose of 2 μg. Therefore, we selected 2 μg for subsequent administration experiments. [Table 2]

[0171] Example 9: Immunogenicity of rOMVs in BALB / c mice following two parenteral doses at 3-week intervals. Despite reduced TLR activity, rOMV LpxE-CRC Although a strong humoral response was observed with the vesicles, we still wanted to test the immunogenicity of the entire panel of A3B3-MUC1-expressing rOMVs with graded reductions in TLR activity, as summarized in Table 3. In addition to testing CRC-specific serum IgG responses, we also tested T cell responses, assessed by an interferon-γ (IFN-γ) ELISpot assay. We hypothesized that rOMVs offer a unique opportunity for the treatment of colorectal cancer by disrupting the immunosuppressive tumor microenvironment through activation of the innate immune response, thereby eliciting a tumor-specific adaptive response of CD8+ cytotoxic T lymphocytes. [Table 3] Sixty BALB / c mice (6-8 weeks old) were randomly divided into six groups and immunized intramuscularly with 2.0 μg of unadjuvanted rOMV vesicles on days 0 and 21. We also administered PBS to an additional 10 mice as a negative control. Serum was collected on days 0, 20, and 35. Antigen-specific serum IgG was measured by ELISA, and IFN-γ responses were measured using harvested splenocytes and the MabTech Mouse IFN-γ ELISpotPLUS HRP Kit according to the manufacturer's instructions. Surprisingly, robust serum IgG titers against A3B3-MUC1 were again observed after intramuscular administration of 2 μg of rOMV, regardless of whether or not the mice were genetically engineered for TLR activity (Table 4). Despite the high background observed in the ELISpot assay, the IFN-γ response to A3B3-MUC1 was significantly higher than that observed with rOMV, as shown in Figure 11. DfliC-CRC The levels were clearly higher in animals receiving [Table 4]

[0172] Example 10. Immunogenicity of rOMVs in C57BL / 6 mice following two parenteral doses at one-week intervals. We plan to conduct a proof-of-concept therapeutic efficacy study to test tumor regression in a murine mouse model by eliciting CRC-specific immunity against our A3B3-MUC1 fusion protein. Therefore, we require a syngeneic tumor model in which both innate and adaptive immune responses are fully functional. We therefore target tumors induced in C57BL / 6 mice using the syngeneic MC38-CEA cell line purchased from Kerafast. This MC38-CEA model has been used in preclinical safety testing of a CEA vaccine antigen containing the immunodominant T cell epitope CEA(6D), which is also contained in our engineered fusion protein. Significant therapeutic effects were reported in the MC38-CEA mouse model and the drug successfully progressed to clinical trials (Crosby et al., Journal for immunotherapy of cancer, (2020), 8(2); Morse et al., J Clin Invest, (2010), 120:3234-41; Osada et al., Cancer Immunol Immunother 2012; 61(11):1941-51).

[0173] Recent data from animal models indicate that the inherent size and other associated physical properties of outer membrane vesicles alone can confer direct therapeutic effects on tumors, independent of immunological targeting of tumor-associated antigens, through the phenomenon of enhanced permeability and retention (EPR), which relies on leaky tumor vasculature (Irvine et al., Nat Rev Immunol, 2020, 20:321-34; Fang et al., Advanced drug delivery reviews, (2020), 157:142-60; Kelly et al., Expert review of vaccines, (2019), 18:269-80). This leaky tumor vasculature allows small nanostructures, such as OMVs, to passively extravasate from the blood into tumor tissue (Fang et al., Advanced drug delivery reviews, (2020), 157:142-60). From within tumor tissue, these OMVs are passively discharged through the lymphatic system to local lymph nodes, where they may encounter and activate antigen-presenting cells, including dendritic cells, and induce tumor-specific immunity (Fang et al., Advanced drug delivery reviews, (2020), 157:142-60; Kelly et al., Expert review of vaccines, (2019), 18:269-80).

[0174] Therefore, to take advantage of this property of our rOMVs, we wanted to validate the humoral and cellular immunity of rOMVs in C57BL / 6 mice immunized intravenously with our rOMVs. We also wanted to test the immunogenicity of a heterologous prime-boost strategy using both rOMVs and live carrier strains for vaccination. Thirty-five C57BL / 6 mice (6–8 weeks old) were randomly divided into six groups and primed on day 0 with either unadjuvanted rOMV vesicles or live carrier vaccine. We also administered PBS to an additional five mice as a negative control (see Table 5, "Priming Dose"). All mice were boosted on day 7 with live carrier vaccine or unadjuvanted rOMV vesicles (see Table 5, "Boost Dose"). Serum was collected on days 0, 6, and 20, and splenocytes were harvested on day 21. Antigen-specific serum IgG was measured by ELISA, and IFN-γ responses were measured again by ELISpot assay using freshly harvested splenocytes. Surprisingly, robust CRC-specific serum IgG responses were observed just 6 days after all parenteral priming doses. Responses increased significantly 14 days after all booster doses, regardless of route of administration or vaccine vehicle tested. [Table 5]

[0175] For the IFN-γ ELISpot assay, spleen cells were seeded onto mouse IFN-γ ELISpot plates (250,000 cells / well, six replicates per rOMV vaccine) and stimulated with either (1) A3B3-MUC1 (10 μg / mL), (2) concanavalin A (4 μg / mL, positive control), or (3) medium (negative control). Cells were incubated for 40 hours (37°C, 5% CO2), then washed and anti-IFN-γ (biotinylated) antibody was added. After a 1-hour incubation, the plate was washed, and streptavidin conjugated to horseradish peroxidase (SA-HRP) was added (1 hour), and the plate was washed again. Spot-forming cells (SFCs) were grown using tetramethylbenzidine (TMB) HRP substrate (7–8 minutes). The plate was scanned, and spots were counted using an ELISpot Immunospot® reader (Cellular Technology). Data were analyzed using Immunospot® version 7.0 software. Total SFC / well is reported as SFC / 106 splenocytes and the results are reported in Figure 12.

[0176] Both the humoral and cellular immune responses in this experiment were lower than those reported in Example 9, likely due to the shorter interval between prime and boost administration (21 days in Example 9 vs. 7 days in Example 10) and the shorter experimental period (35 days in Example 9 vs. 21 days in Example 10). The short interval in the prime-boost protocol used in Example 10 was adopted based on the work of Kim et al. and Cheng et al., who showed that therapeutic tumor regression was achieved in both groups of mice administered OMV IV at short intervals (Kim et al., Nature Communications, (2017), 8:626); Cheng et al., Nature Communications, (2021), 12:2041). Despite these modifications, all IFN-γ responses to A3B3-MUC1 reported in Figure 12 were statistically significant (p < 0.05 vs. PBS control group G) and closely matched the A3B3-MUC1-specific antibody responses (Table 5). The highest IFN-γ responses were induced in animals receiving homologous rOMV challenge (i.e., groups C and D immunized with 0.25 μg of unadjuvanted rOMV intravenously or group F with 2.0 μg intramuscularly). Heterologous prime-boost challenge with rOMV and live-attenuated carrier strain (groups A and B), or challenge with the same rOMV administered by different routes (intravenously and intramuscularly, group E), elicited the lowest CRC-specific antibody and cellular immune responses.

[0177] Taken together, the results of Examples 8–10 clearly established the feasibility of engineering an attenuated Salmonella typhi-based carrier strain capable of efficiently expressing and delivering the targeted colon-derived A3B3-MUC1 protein and inducing antigen-specific immune responses. We successfully designed a unique PagL-mediated outer membrane vesicle antigen delivery platform in which the efficiency of rOMV transport and isolation was enhanced by overexpression of the outer membrane folding protein BamA. We purified these rOMVs and demonstrated both humoral and cellular antigen-specific immunity against our CRC-targeted A3B3-MUC1 fusion protein. These data clearly demonstrate that intravenous administration of unadjuvanted rOMVs to C57BL / 6 mice induced excellent CRC-specific immunity, while heterologous prime-boost administration of rOMVs with a live carrier strain induced the lowest response. In a preferred embodiment, the inventors will further develop purified rOMVs intended for intravenous administration to humans as therapeutic immune intervention against primary tumor progression and prevention of liver metastasis. The inventors plan to utilize engineered rOMVs with reduced potent innate immune stimulation via TLR4 and TLR5 agonists to improve the safety and clinical acceptability of intravenously administered rOMVs by avoiding the induction of septic shock and cytokine storm responses in tumor patients.

[0178] Example 11. Therapeutic efficacy of rOMVs expressing a CEA-MUC1 fusion protein in a syngeneic C57BL / 6 mouse tumor challenge model. Despite re-engineering the rOMVs to reduce TLR4 and TLR5 activity, these modified vesicles maintained strong immunogenicity when tested in C57BL / 6 mice, demonstrating excellent serum IgG responses to our CEA-MUC1 fusion protein and excellent antigen-specific cellular responses as assessed by IFN-γ ELISPOT assays. Encouraged by these highly robust humoral and cellular responses, we conducted initial therapeutic efficacy studies using a syngeneic C57BL / 6 mouse model implanted with MC38 murine tumor cells expressing either CEA or MUC1. Mice (10 per group) were implanted subcutaneously with 300,000 tumor cells on day 1, followed by intravenous administration of 0.75 μg of vesicles on days 3, 5, 7, and 9 (based on quantification of LPS concentrations using the KDO assay). Tumor progression was monitored by calculating tumor volume up to day 28. As shown in Figures 13A and B, tumor volume was reduced in all groups receiving intravenous rOMV administration. ΔfliC-CRC When treated with rOMV, 50% achieved complete remission. lpxE-CRC In mice treated with rOMVl, 20% achieved complete remission. pxE-CRC Complete remission was observed in 50% of patients treated with rOMV ΔfliC-CRC When treated with rOMV, 30% achieved complete remission. Given the ability of our rOMV to significantly suppress the progression of new tumors, we next investigated their ability to act therapeutically against existing tumors. C57BL / 6 mice were re-injected with 300,000 tumor cells on day 1, and tumors grew to an average of 115-120 mm. 3The tumor volume was allowed to progress until it reached a volume of 100 μg. Mice were then treated intravenously with vesicles at 0.75 μg on days 0, 2, 4, and 6. Because tumor progression necessitated humane euthanasia, tumor volume was recorded only until day 18 (Figures 13C and D), and subsequent mortality was recorded until day 28 (Figure 13E). As in the previous experiment, a significant reduction in tumor volume was observed in all groups treated intravenously with vesicles. As shown in Figure 13C, after the first intravenous administration, the initial tumor volume began to shrink and remained significantly lower than the untreated PBS control group in all experimental groups until day 18 (Figure 13D). The survival data shown in Figure 13E demonstrate that tumor volume was significantly reduced in all groups treated with rOMV, regardless of the tumor cell line used for challenge. ΔfliC-CRC The group treated with rOMV showed a survival rate of over 90%. Although complete remissions were not observed in individual mice in this study, we believe these data support the currently mainstream strategy in oncology, which is that early treatment initiation (as modeled in the experiment summarized in Figures 13A and B) results in better therapeutic outcomes. Taken together, the data from these two challenge studies suggest that genetic modification of lipid A does not interfere with the therapeutic efficacy of our rOMV.

[0179] Example 12. Therapeutic efficacy of rOMVs expressing individual CRC target proteins and CRC fusion proteins in a syngeneic C57BL / 6 mouse tumor challenge model. We investigated the reasons for the inferior efficacy of MC38-CEAv2 challenge compared to MC38-MUC1 cell challenge. We therefore decided to create isogenic constructs in which either the A3B3 or MUC1 domain was deleted from the expression plasmid expressing the CRC fusion protein, as shown in Figure 14A. Here, we were only interested in evaluating the immunogenicity of individual domains and fusion proteins compared to the fusion protein. Therefore, we used rOMVs expressing the full-length A3B3-MUC1 fusion protein. ΔfliC-CRCWe focused on construct modifications only. rMUC1 expresses only the heptad repeat sequence of the human MUC1 protein by deleting the A3B3 domain. ΔfliC Similarly, rA3B3 expressing only the A3B3 domain of the human CEA protein was obtained by deleting the MUC1 domain (see Figure 13A). ΔfliC These re-engineered plasmids were then re-introduced into the carrier strain CVD911 by standard electroporation methods to express and purify the therapeutic candidate rOMVs. These re-engineered rOMVs were used to express and purify the unmodified rOMVs. ΔfliC-CRC We immunized mice as described in Table 5 above. Thirty-five C57BL / 6 mice (6-8 weeks old) were randomly divided into three groups and primed intravenously with 0.25 μg of unadjuvanted rOMV vesicles on day 0. We also administered PBS to five additional mice as a negative control. All mice were boosted intravenously with 0.25 μg of unadjuvanted rOMV vesicles or PBS on day 7. Serum was collected on days 0, 6, and 21, and splenocytes were harvested on day 21 for ELISPOT analysis. The results are reported in Figures 14B and 14C, respectively. These data suggest that MUC1 is strongly immunogenic, whereas A3B3 is less immunogenic, when evaluating tumor-specific serum IgG responses (Figure 14B). This difference in immunogenicity was less pronounced in tumor-specific T cell responses assessed by ELISPOT assay (Figure 14C). Having demonstrated that both independent domains induce humoral and cellular tumor-specific responses, we performed another syngeneic tumor challenge experiment. In this experiment, C57BL / 6 mice were implanted with 300,000 MC38-CEAv2 cells on day 0, followed by rA3B3 on days 3, 5, 7, and 9. ΔfliC , rMUC1 ΔfliC , rOMV ΔfliC-CRC , or empty rOMV ΔfliC-pagL0.75 μg of either rA3B3 or rA3B3 vesicles was administered intravenously. To attempt to distinguish antigen-specific protection against CEAv2, we challenged with MC38-CEAv2 cells alone. We found that when MUC1 was immunodominant, rA3B3 ΔfliC and rOMV ΔfliC-CRC The therapeutic effect of rMUC1 against CEAv2 challenge should be confirmed. ΔfliC We hypothesized that the effect would be reduced in rOMVs and no effect would be observed in empty vesicles. As shown in Figure 14D, we found that the effect of rOMVs ΔfliC-CRC The excellent therapeutic effect on MC38-CEAv2 conferred by rMUC1 ΔfliC Surprisingly, the therapeutic effect of rA3B3 on MC38-CEAv2 was reduced. ΔfliC Although the rA3B3 induced a superior therapeutic effect, the response was not predicted based on the humoral and cellular responses (Figure 14B and C). Most surprisingly, empty vesicles also conferred superior therapeutic effects upon challenge with MC38-CEAv2, despite not expressing tumor-specific antigen(s) on the surface of the empty rOMV. However, the EPR effect was not as pronounced as that of rA3B3. ΔfliC Vesicles and rMUC1 ΔfliC Although the vesicles should have been similarly effective, this was not the case. While we cannot clearly explain this discrepancy, we suspect that the surface charge of the rOMVs may be involved. While the original, unmodified surface charge allows empty vesicles to penetrate tumors, the introduction of either the A3B3 domain or the MUC1 domain alone may disrupt the surface charge, potentially reducing tumor penetration via the EPR effect. However, this does not affect the induction of tumor-specific immunity, which is at least somewhat effective compared to the PBS control. Based on this observation, it is clear that the A3B3-MUC1 fusion protein does not interfere with either the EPR effect or the induction of tumor-specific immunity.

[0180] Example 13. rOMVs activate Toll-like receptors. Toll-like receptors (TLRs), which act as a bridge between innate and adaptive immune responses, are a relevant target class in cancer therapy, both as monotherapy and in combination with immune checkpoint inhibitors (ICIs). TLR agonists have the potential to transform "cold tumors" into "hot tumors," making TLRs a promising target for cancer therapy in combination with immune checkpoint inhibitors. TLR agonists induce cytokine secretion, which activates cytotoxic T lymphocytes (CTLs), triggering immune responses that can mediate inflammation and reduce tumor burden. See, for example, Rolfo et al., "Applications and clinical trial landscape using toll-like receptor agonists to reduce the toll of cancer." npj Precis. Onc. 7, 26 (2023).

[0181] rOMV DfliC-CRC To demonstrate that rOMVs have the ability to activate TLRs, we performed a screen to test stimulation of Toll-like receptors (TLRs) by assessing NF-κB activation in HEK293 cells expressing a given TLR and harboring an NF-κB / AP-1-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. This reporter gene allows for monitoring signaling through TLRs based on NF-κB activation. ΔDfliC-CRC The activity of rOMV was tested against seven human TLRs (TLR2, 3, 4, 5, 7, 8 and 9) as potential agonists. ΔDfliC-CRC The rOMVs were evaluated at four concentrations and compared to a control ligand. These steps were performed in triplicate. As shown in Table 6, DfliC-CRCshowed a significant stimulatory effect on HEK-Blue hTLR2 and HEK-Blue hTLR4 at dilutions of 1 / 10000 or less, and a mild stimulatory effect on HEK-Blue hTLR3, HEK-Blue hTLR5, HEK-Blue hTLR7, HEK-Blue hTLR8, and HEK-Blue hTLR9 at a dilution of 1 / 10. [Table 6]

[0182] Example 14. Efficacy of rOMV vaccines administered by intravenous and intramuscular routes. To correlate the immunogenicity and route of administration of our OMV vaccine with antitumor efficacy, we administered rOMV to C57BL / 6 mice bearing MC38-MUC1 tumors. ΔfliC-CRC Briefly, seven groups of mice (10 mice per group) were inoculated with 300,000 MC38-MUC1 cells, and tumors were grown to an average volume of 100 mm. 3 Animals were randomized based on tumor size and injected with PBS (negative control) or 0.05 μg, 0.2 μg, and 0.75 μg of rOMV. ΔfliC-CRC administered intravenously (IV) for four doses every two days, or 0.2 μg, 0.75 μg, and 3 μg of rOMV ΔfliC-CRC was administered intramuscularly (IM) four times at 4-day intervals. Tumor progression was monitored up to day 18 after randomization by calculating tumor volume and tumor growth inhibition (TGI). As shown in Table 7, rOMV ΔfliC-CRC IM or IV administration of produced a dose-dependent antitumor effect as reflected by an increase in TGI in vaccinated mice. The tumor growth inhibition rate (TGI%) was calculated using the formula: TGI% = (1 - {Tt / T0 / Ct / C0} / 1 - {C0 / Ct}) x 100. Tt = mean tumor volume of treated animals at time point t TO = mean tumor volume of treated animals at time point 0 Ct = mean tumor volume of controls at time point t C0 = mean tumor volume of the control group at time point 0. [Table 7]

[0183] Throughout this disclosure, various publications, patents and published patent specifications are referenced by a specific citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into this disclosure in order to more fully describe the state of the art to which this invention pertains.

[0184] While the present teachings have been described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

Claims

1. A method for inducing an immune response in a subject in need thereof, comprising administering to the subject an immunologically effective amount of isolated recombinant outer membrane vesicles comprising one or more cancer antigens, wherein the recombinant outer membrane vesicles are administered to the subject by intravenous route, intramuscular route, or both, and the recombinant outer membrane vesicles are administered one or more times.

2. the recombinant outer membrane vesicles a. one or more cancer antigens; b. the outer membrane folding protein BamA or a fragment or variant thereof, and c) The method of claim 1, wherein the vector is obtained from a live Salmonella typhi vector engineered to express lipid A deacylase PagL or a fragment or variant thereof.

3. 3. The method of claim 1 or 2, wherein the subject is first administered the isolated recombinant outer membrane vesicles, and then at a later time point is administered an immunologically effective amount of the outer membrane vesicles.

4. The method of claim 1 or 2, wherein the subject is first administered the isolated recombinant outer membrane vesicles, and then administered an immunologically effective amount of the outer membrane vesicles at subsequent time points ranging from one to three times, with the subsequent administrations being spaced apart in time.

5. 5. The method of claim 4, wherein the outer membrane vesicles are administered a total of four times.

6. The method of any one of claims 1 to 5, wherein the outer membrane vesicles are administered to the subject by intravenous route.

7. The method of any one of claims 1 to 6, wherein the outer membrane vesicles are administered at a dose of about 5 μg / kg to about 25 μg / kg.

8. The method of any one of claims 1 to 6, wherein the outer membrane vesicles are administered at a dose of 10 μg / kg to about 25 μg / kg.

9. The method of any one of claims 1 to 8, wherein the administrations are performed at intervals of about 2 days to 12 weeks.

10. 10. The method of claim 9, wherein the administrations are spaced at least 7 days apart.

11. 10. The method of claim 9, wherein the administrations are spaced at least 14 days apart.

12. 10. The method of claim 9, wherein the administrations are spaced at least 21 days apart.

13. The method of any one of claims 1 to 9, wherein the subject is administered one or more immunologically effective amounts of an adjuvant in combination with the outer membrane vesicles.

14. 11. The method of claim 10, wherein the adjuvant is administered to the subject in a composition separate from a composition comprising the outer membrane vesicles.

15. 11. The method of claim 10, wherein the outer membrane vesicles and the adjuvant are administered in the same composition.

16. The method according to any one of claims 1 to 15, wherein the cancer antigen is an outer membrane protein.

17. The method of any one of claims 2 to 16, wherein the cancer antigen is encoded by a nucleic acid integrated into the chromosome of Salmonella typhi.

18. The method of any of claims 2 to 17, wherein the antigen is expressed from a plasmid.

19. The method of any one of claims 2 to 18, wherein the Salmonella typhi vector comprises a deletion in guaBA and htrA.

20. 20. The method of any of claims 2 to 19, wherein the antigen is inserted into a locus of Salmonella typhi selected from the group consisting of guaBA, rpoS, htrA, ssb, and combinations thereof.

21. The method of any of claims 2 to 20, wherein the antigen is inserted into the rpoS locus of Salmonella typhi.

22. 22. The method of any one of claims 2 to 21, wherein the Salmonella typhi overexpresses cytolysin A (ClyA) protein to promote the formation of outer membrane vesicles.

23. 23. The method of claim 22, wherein the ClyA is mutated to reduce the hemolytic activity of the ClyA.

24. 24. The method of claim 23, wherein the ClyA mutant is selected from the group consisting of ClyA I198N, ClyA A199D, ClyA E204K, ClyA C285W, and combinations thereof.

25. The method of any one of claims 22 to 24, wherein the ClyA is a fusion protein.

26. 24. The method of claim 23, wherein the ClyA comprises the substitution mutations I198N, A199D, and E204K.

27. The method of any one of claims 2 to 26, wherein the BamA is derived from Acinetobacter baumannii.

28. The method of any one of claims 2 to 27, wherein the amino acid sequence of BamA comprises SEQ ID NO:

8.

29. 29. The method of claim 28, wherein the bama gene encoding the BamA protein is integrated into the genome of Salmonella typhi.

30. 30. The method of claim 29, wherein the bamA is integrated into the guaBA locus of Salmonella typhi.

31. The method of any one of claims 27 to 30, wherein the bamA is expressed by an inducible promoter.

32. 32. The method of claim 31 , wherein the inducible promoter is osmoregulatory.

33. 33. The method of claim 32, wherein the osmoregulatory inducible promoter is the promoter of the outer membrane protein C (ompC) gene.

34. 34. The method of claim 33, wherein the promoter of the outer membrane protein C (ompC) gene comprises SEQ ID NO:

9.

35. The method according to any one of claims 2 to 34, wherein the pagL gene encoding PagL is integrated into the genome of Salmonella typhi.

36. The method of any one of claims 2 to 35, wherein pagL is expressed from a plasmid.

37. 37. The method of claim 36, wherein the plasmid expressing PagL is a low copy number expression plasmid.

38. The method of any one of claims 2 to 37, wherein the expression of pagL is controlled by an inducible promoter.

39. The method of any one of claims 36 to 38, wherein the plasmid has a non-antibiotic type plasmid selection system.

40. 40. The method of claim 39, wherein the plasmid expresses a gene that is essential for growth of Salmonella typhi and that is mutated on the chromosome in Salmonella typhi.

41. 41. The method of claim 40, wherein the gene encodes a single-stranded binding protein (SSB).

42. 42. The method of claim 41, wherein the inducible promoter is osmoregulatory.

43. 43. The method of claim 42, wherein the osmoregulatory inducible promoter is the promoter of the outer membrane protein C (ompC) gene.

44. The method of any one of claims 36 to 43, wherein the plasmid further encodes and expresses the antigen.

45. The method of any one of claims 2 to 44, wherein the amino acid sequence of PagL is selected from SEQ ID NO: 2 and SEQ ID NO:

4.

46. The method of any one of claims 22 to 45, wherein ClyA is expressed on a plasmid in Salmonella typhi.

47. 47. The method of claim 46, wherein the plasmid has a non-antibiotic type plasmid selection system.

48. 48. The method of claim 47, wherein the plasmid expresses a gene that is essential for growth of the Salmonella typhi and that is mutated on the chromosome in Salmonella typhi.

49. 49. The method of claim 48, wherein the gene encodes a single-stranded binding protein (SSB).

50. The method of any one of claims 1 to 49, wherein the cancer is a colon cancer antigen.

51. The method of any one of claims 2 to 50, wherein the Salmonella typhi vector comprises two cancer antigens.

52. 52. The method of any one of claims 1 to 51, wherein the cancer antigen is fused to a polypeptide to promote surface presentation of the antigen.

53. 53. The method of any one of claims 1 to 52, wherein the cancer antigen is fused to a chimeric Lpp-OmpA surface-displayed polypeptide.

54. 53. The method of any of claims 1-52, wherein the cancer antigen is fused to a ClyA surface-displayed polypeptide.

55. 55. The method of any of claims 50 to 54, wherein the colon cancer antigen is selected from CEA or an antigenic fragment thereof, MUC1 or an antigenic fragment thereof, and combinations thereof.

56. 56. The method of claim 55, wherein the CEA antigen and MUC1 are part of the same fusion protein.

57. 57. The method of claim 56, wherein the MUC1 antigen is a fragment comprising multiple repeat domains.

58. 58. The method of claim 57, wherein the amino acid sequence of the MUC1 antigen comprises SEQ ID NO:

24.

59. 59. The method of any one of claims 55 to 58, wherein the CEA antigen comprises the domain A3B3.

60. 54. The method of claim 53, wherein the amino acid sequence of Lpp-OmpA comprises SEQ ID NO:

21.

61. 61. The method of any one of claims 55 to 60, wherein the amino acid sequence of domain A3B3 comprises SEQ ID NO:

23.

62. 62. The method of any of claims 53 or 55-61, wherein the Lpp-OmpA:CEA:MUC1 fusion protein comprises the amino acid sequence of SEQ ID NO:

12.

63. 63. The method of any one of claims 50 to 62, wherein the cancer antigen is expressed on a plasmid, and the plasmid expresses a gene that is essential for growth of Salmonella typhi and that is mutated on the chromosome in Salmonella typhi.

64. 64. The method of claim 63, wherein the gene encodes a single-stranded binding protein (SSB).

65. 65. The method of claim 63 or 64, wherein the plasmid further expresses PagL.

66. The method of any of claims 50 to 65, wherein the cancer antigen and / or PagL is expressed under the control of an inducible promoter.

67. 67. The method of claim 66, wherein the inducible promoter is osmoregulatory.

68. 68. The method of claim 67, wherein the osmoregulatory inducible promoter is the promoter of the outer membrane protein C (ompC) gene.

69. 69. The method of claim 68, wherein the promoter of the outer membrane protein C (ompC) gene comprises SEQ ID NO:

9.

70. 69. The method of any one of claims 2 to 68, wherein the Salmonella typhi vector has a deletion in the fliC gene.

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