WO2025080765A1

WO2025080765A1 - Methods to deliver exogenous protein/peptide cargo to cell cytosol and uses thereof

Info

Publication number: WO2025080765A1
Application number: PCT/US2024/050658
Authority: WO
Inventors: Bobby Brooke HERRERA
Original assignee: Rutgers State University of New Jersey
Current assignee: Rutgers State University of New Jersey
Priority date: 2023-10-12
Filing date: 2024-10-10
Publication date: 2025-04-17
Anticipated expiration: 2026-04-12

Abstract

This disclosure provides compositions and methods for eliciting a T-cell mediated response to a pathogen or a tumor cell. Also provided are T-cell vaccines, T-cell adjuvants, and methods for performing T-cell diagnostics.

Description

METHODS TO DELIVER EXOGENOUS PROTEIN/PEPTIDE CARGO TO CELL CYTOSOL AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Application No. 63/589,709, filed on October 12, 2023, which is hereby incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on October 7, 2024, is named 070439.01840SeqList.xml and is 6,334 bytes in size.

FIELD OF THE INVENTION

[0003] The present invention relates to compositions and methods for eliciting a T-cell response to a pathogen or a tumor cell.

BACKGROUND OF THE INVENTION

[0004] T-cell responses are critical components of the immune system. T-cells are activated upon antigen recognition by antigen-presenting cells (APCs) that present alloantigens. This recognition process depends on the presence of MHC (major histocompatibility complex) proteins that bind antigens and carry them to the cell surface. There are two types of MHC proteins: 1. Class I MHC proteins that present foreign peptides to cytotoxic T-cells and 2. Class II MHC proteins that present foreign peptides to helper T-cells. Activated T-cells proliferate, differentiate and produce cytokines. Consequently, a T-cell response is implicated in various diseases including infectious diseases and cancers. Thus, targeting a T-cell response could serve as a therapeutic treatment for numerous diseases with associated T-cell responses. Additionally, induction of a T-cell response can be used 1. in combination with existing therapeutic agents to improve their efficacy and 2. for detecting cell-mediated immune responses for diagnostic purposes (e.g., detecting tuberculosis infection). However, there is currently no approved T-cell vaccine for inducing a T-cell response. SUMMARY OF THE INVENTION

[0005] In one aspect, this disclosure provides compositions and methods for eliciting a T- cell mediated response to a pathogen or a tumor cell.

[0006] In another aspect, this disclosure provides an immunogenic composition comprising an antigen fused to a cytotoxin, wherein the cytotoxin is a bacterial cytotoxin with the catalytic infectious domain removed, and wherein the antigen induces a T-cell response to a pathogen or a tumor cell.

[0007] In some embodiments, the bacterial cytotoxin is Bacillius thuringiensis or Bacillus shihchuchen.

[0008] In some embodiments, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0009] In some embodiments, the antigen fused is fused to the cytotoxin via the amino acid sequence of SEQ ID NO: 5.

[0010] In some embodiments, the pathogen is selected from the group comprising a bacteria, a virus, a parasite, and a fungus.

[0011] In some embodiments, the tumor cell is a cancer cell, and wherein the cancer is selected from the group comprising anal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, lung cancer, lymphoma, multiple myeloma, ovarian cancer, pancreatic cancer, renal transitional cell cancer, skin cancer, synovial sarcoma, and thyroid cancer.

[0012] In some embodiments, a pharmaceutical composition comprises the composition described herein and a pharmaceutically acceptable carrier.

[0013] In some embodiments, a kit comprises the composition described herein, wherein the kit comprises: a) an immunoassay to quantify cytokine levels from a sample of blood for detecting a T-cell-mediated immune response; and b) instructions for use.

[0014] In some embodiments, provided is a method of enhancing an existing therapeutic agent comprising administering the composition described herein to a subject in need thereof. [0015] In some embodiments, the existing therapeutic agent is selected from the group comprising a vaccine, an anti-viral, an antibody, and a small molecule.

[0016] In some embodiments, provided is a method of monitoring T-cells, comprising the following steps: a) mixing blood from a subject with the composition described herein; b) incubating the mixture of a) for at least 24 hours at about 37 °C; c) centrifuging the mixture of b) following the incubation period; d) collecting the supernatant from c); and e) quantifying cytokine levels with an immunoassay.

[0017] In some embodiments, the cytokine levels are quantified are quantified with an ELIspot, an ELISA assay, or flow cytometry.

[0018] In some embodiments, provided is a T-cell vaccine comprising the composition described herein, wherein the T-cell vaccine comprises an aqueous or liposomal formulation. In some embodiments, the T-cell vaccine further comprises an adjuvant.

[0019] In one aspect, this disclosure provides an isolated polynucleotide sequence encoding an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, said sequence is fused to an antigen. In some embodiments, said sequence is fused to the antigen via the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antigen is selected from the group comprising a bacterial antigen, a viral antigen, a parasitic antigen, a fungal antigen, a tumor-associated antigen (TAA), and a tumor-specific antigen (TSA).

[0020] In some embodiments, provided is a pharmaceutical composition comprising the antigen-fused amino acid sequence of the composition described herein and a pharmaceutically acceptable carrier.

[0021] In one aspect, provided is a method of eliciting a T-cell response in a subject comprising administering to the subject a composition comprising an antigen fused to a cytotoxin, wherein the antigen induces a T-cell response to a pathogen or a tumor cell.

[0022] In some embodiments, the T-cell response: a) enhances an existing therapeutic agent; b) serves as a T-cell vaccine; and/or enables T-cell diagnostics.

BRIEF DESCRIPTION OF THE DRAWINGS [0023] FIG. 1 depicts Coomassie stains of the indicated immunogenic compositions. L: ladder; FT, flow-through; Wl, wash 1; W2, wash 2; El, elution 1; and E2, elution 2.

[0024] FIG. 2 shows the resulting number of CD8+ T-cells in mice infected with yellow fever virus (YFV-17D) or dengue virus (DENV2). C, capsid; NS3, non-structural protein 3; P, protease; H, helicase; P/I P/I (phorbol 12-myristate 13-acetate (P)/ionomycin) (positive control). * = p < 0.05; One-Way ANOVA.

DETAILED DESCRIPTION OF THE INVENTION

[0025] This disclosure relates to an immunogenic composition comprising an antigen fused to a cytotoxin, wherein the antigen induces a T-cell response to a pathogen or a tumor cell. Also provided are methods of performing T-cell diagnostics using the immunogenic composition described herein.

Immunogenic Composition

[0026] In one aspect, provided is an immunogenic composition comprising an antigen fused to a cytotoxin, wherein the antigen induces a T-cell response to a pathogen or a tumor cell.

[0027] As used herein, the term “cytotoxin” refers to a substance that has a toxic effect on a cellular function. In some embodiments, the cytotoxin is a bacterial cytotoxin with the catalytic infectious domains removed. In some embodiments, the bacterial cytotoxin is Bacillius thuringiensis (Bt) or Bacillus shihchuchen. Bacillius thuringiensis is a soil- borne bacteria that produces a variety of insecticidal proteins. Bacillus shihchuchen is related to Bacillius thuringiensis (Cheng et al. Int. J. Mol. Sci. 2023. 24(11): 9636). Both Bacillius thuringiensis and Bacillus shihchuchen are Gram-positive bacteria.

[0028] In some embodiments, the cytotoxin comprises an amino acid sequence as shown in Table 1. In some embodiments, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Truncated SEQ ID NOs: 2 and 4 were generated based on domains believed to be involved in protein trafficking into the cell.

[0029] Table 1. Representative amino acid sequences of the cytotoxin fused to an antigen.

[0030] In some embodiments, the amino acid sequence of the cytotoxin is no greater than

78 amino acids in length such as 50 amino acids, 55 amino acids, 60 amino acids, 65 amino acids, 70 amino acids, 75 amino acids, 78 amino acids, or any number of amino acids in-between. In some embodiments, the amino acid sequence of the cytotoxin is no greater than 90 amino acids in length such as 70 amino acids, 75 amino acids, 80 amino acids, 85 amino acids, 90 amino acids, or any number of amino acids in-between. In some embodiments, the amino acid sequence of the cytotoxin is no greater than 104 amino acids in length such as 80 amino acids, 85 amino acids, 90 amino acids, 95 amino acids, 100 amino acids, 104 amino acids, or any number of amino acids in-between. In some embodiments, the amino acid sequence of the cytotoxin is no greater than 250 amino acids in length, such as about 100 amino acids, 150 amino acids, 200 amino acids, 250 amino acids, or any number of amino acids in-between. In some embodiments, the amino acid sequence of the cytotoxin is no greater than 350 amino acids in length, such as about 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, or any number of amino acids in-between. In some embodiments, the amino acid sequence of the cytotoxin is no greater than 450 amino acids in length, such as about 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or any number of amino acids inbetween.

[0031] Notably, the cytotoxin may or may not bind the target antigen, but it will elicit a T-cell mediated immune response. As used herein, the term “target antigen” refers to a molecular target for which it would be desirable to elicit a T-cell mediated immune response.

[0032] As used herein “antigen” refers to a substance that induces an immune response. The antigen can be a protein, peptide, polysaccharide, lipid, or a nucleic acid. In some embodiments, the antigen is an exogenous antigen. In some embodiments, the antigen is an endogenous antigen. In some embodiments, the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). As used herein, a “tumor-associated antigen (TAA)” is present on both tumor cells and healthy cells. As used herein, a “tumorspecific antigen (TSA)” is present only on tumor cells.

[0033] In some embodiments, the pathogen is selected from the group comprising a bacteria, a virus, a parasite, and a fungus. For example, the bacteria is selected from the group including, but not limited to Streptococcus pneumoniae, Bacillus anthracis, Clostridium botulinum, Clostridium difficile, Vibrio cholerae, Leptospira sp., Neisseria meningitidis, Clostridium tetani, and Mycobacterium tuberculosis. In some embodiments, the virus is selected from the group including, but not limited to varicella, coronavirus, influenza A, and rhinovirus. In some embodiments, the parasite is selected from the group including, but not limited to Toxoplasma gondii, hookworms, tapeworms, roundworms, and Giardia. In some embodiments, the fungus is selected from the group including, but not limited to Candidia, Aspergillus, Taloromyces, and Pneumocystis jirovecii.

[0034] In some embodiments, the tumor cell is a cancer cell. In some embodiments, cancer is selected from the group comprising anal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, lung cancer, lymphoma, multiple myeloma, ovarian cancer, pancreatic cancer, renal transitional cell cancer, skin cancer, synovial sarcoma, and thyroid cancer.

[0035] In some embodiments, the immunogenic composition is prepared according to known methods in the art. In some embodiments, the cytotoxin is fused to the antigen of interest via a protein linker. In some embodiments, the protein linker is GGSGGS (SEQ ID NO: 5).

Pharmaceutical Composition

[0036] In some embodiments, provided is a pharmaceutical composition comprising the immunogenic composition described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition is particularly suitable in the treatment or prophylaxis of the pathogens or tumor cells described herein. In some embodiments, the pharmaceutical composition is in the form of a tablet, capsule or parenteral. In some embodiments, the pharmaceutical composition is a liposome. In some embodiments, the pharmaceutical composition is administered to a subject in need thereof via various delivery modes including, but not limited to intramuscular, intravenous, intraperitoneal, intradermal injection, oral, sublingual, parenteral, and subcutaneous.

[0037] As used herein, the term “subject” refers to an animal, preferably a mammal such as a human.

[0038] In some embodiments, the pharmaceutical composition is formulated with a mixture of active and inactive ingredients. The active ingredients of the pharmaceutical composition can be in amount from about 0.5% to about 15% w/w. For example, the active ingredients can be in amount of about 0.5%, about 1%, about 2%, about 3 %, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or any amount in-between.

[0039] In some embodiments, the pharmaceutical composition further comprises an adjuvant. In some embodiments, the adjuvant is selected from the group comprising alum, calcium phosphate, poly-IC, Freund’s adjuvant, MF59, and AS0.

[0040] In some embodiments, the pharmaceutical composition can comprise lubricants, binders, diluents, and glidants as known in the art. [0041] In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils, or injectable organic esters. The choice of the pharmaceutically acceptable carrier depends on the route of administration of the composition. Furthermore, the pharmaceutically acceptable carrier can contain ingredients that stabilize, increase solubility or increase the uptake of the immunogenic composition as disclosed herein.

Kit

[0042] In some embodiments, provided is a kit that comprises the immunogenic composition described herein. In one embodiment, the kit is used for T-cell diagnostics, wherein the kit detects cytokine levels (indicative of a T-cell response) via an assay such as an immunoassay. In some embodiments, the kit comprises an immunogenic composition described herein, blood draw tubes, and antibodies for flow cytometry or intracellular cytokine staining.

[0043] In some embodiments, the kit comprises an immunogenic composition described herein, blood draw tubes, and an immunoassay (such as for detecting cytokine levels). In some embodiments, the kit comprises an immunogenic composition described herein, blood draw tubes, and an ELISpot (enzyme-linked immunosorbent spot) assay or an ELISA assay. For example, the kit can comprise kit comprises Vacutainer™ venous blood collections tubes, wherein the inner walls of the tubes are coated with a single or multiple (multiplex test) fusion immunogen(s). Blood from a subject is collected in the tubes, mixed, and incubated at 37 °C for about 24 to about 48 hours. Following the incubation period, blood is spun down and the resulting supernatant is analyzed via an anti-cytokine ELISA assay.

[0044] In some embodiments, the kit comprises an immunogenic composition described herein and an immunoassay to detect cytokine levels such as an ELIspot assay. In some embodiments, the kit comprises an immunogenic composition described herein, cells of interest, and an immunoassay to detect cytokine levels. For example, PBMCs (human peripheral blood mononuclear cells) can be treated with the immunogenic composition in a 96-well plate coated with the anti-cytokine antibodies. The cytokine levels can then be measured via an ELIspot assay. [0045] In some embodiments, the kit comprises an LFn (N-terminal domain of lethal factor) protein fused to a cytotoxin described herein that is used to treat cells. For example, PBMCs can be treated with an LFn fusion protein in a 96-well plate coated with the anti-cytokine antibodies. Resulting cytokine production can then be measured by ELIspot or ELISA assay.

[0046] The kit can further include instructions for use.

Methods of Use

[0047] Also provided are methods of using the immunogenic composition described herein. The immunogenic composition can be used as a T-cell vaccine, a T-cell adjuvant, and for T-cell diagnostics.

I. T-cell Vaccine

[0048] In some embodiments, the immunogenic composition described herein is used in a T-cell vaccine to elicit a T-cell response in a subject.

[0049] In some embodiments, the T-cell vaccine comprises an aqueous formulation. In some embodiments, the T-cell vaccine is a liposomal vaccine. In some embodiments, the T-cell vaccine further comprises an adjuvant. In some embodiments, the adjuvant is selected from the group comprising alum, calcium phosphate, poly-IC, Freund’s adjuvant, MF59, and AS0. In some embodiments, the adjuvant is alum.

[0050] In some embodiments, the T-cell vaccine is administered to a subject in need thereof intramuscularly, intravenously, intraperitoneally, intradermally, orally, sublingually, parenterally, or subcutaneously.

[0051] In some embodiments, the T-cell vaccine is administered at a dose of about 0.1 mg/kg of body weight to about 100 mg/kg of body weight of the subject in need thereof. For example, the T-cell vaccine can be administered to a subject in need thereof at a dose of about 0.1 mg/kg, about 1.0 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg of body weight, or any dose in-between. In other embodiments, the T-cell vaccine is administered in an amount of about 50 mg to about 1000 mg to the subject in need thereof. For example, the T-cell vaccine can be administered to a subject in need thereof in amount of about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, or any amount in-between.

[0052] In some embodiments, the T-cell vaccine is administered to a subject in need thereof in an amount of about 10 pg to about 1 gram. For example, the T-cell vaccine can be administered to a subject in need thereof in amount of about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 200 pg, about 300 pg, about 400 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, about 1000 pg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 800 mg, about 900 mg, about 1 gram (1000 mg), or any amount in-between.

[0053] In some embodiments, the T-cell vaccine is prepared using standard methods known in the art. For example, the T-cell vaccine can be produced in bacterial fermenters in a bioreactor along with purification processes. The bacterial fermenters can be E. coll.

II. T-cell Adjuvant

[0054] In some embodiments, the immunogenic composition described herein is used to enhance an existing therapeutic agent and thus function as a T-cell adjuvant.

[0055] In some embodiments, the immunogenic composition described herein is coadministered with an existing therapeutic agent. In some embodiments, the immunogenic composition described herein is administered to a subject prior to administration of an existing therapeutic agent. In some embodiments, the immunogenic composition described herein is administered to a subject following administration of an existing therapeutic agent. In some embodiments, the immunogenic composition described herein is administered to a subject within 48 hours of diagnosis, which includes administering either within 24 hours of diagnosis or at the time of diagnosis. In some embodiments, the immunogenic composition described herein is administered to a subject within 48 hours of infection, which includes administering within 24 hours of infection.

[0056] In some embodiments, the existing therapeutic agent is selected from the group comprising a vaccine, an anti-viral, an antibody, and a small molecule. For example, the vaccine can include, but is not limited to the influenza vaccine, the COVID-19 vaccine, the Hepatitis A vaccine, Hepatitis B vaccine, smallpox vaccine, Respiratory Syncytial Virus vaccine, Mpox vaccine, and Rabies vaccine. For example, the anti-viral can include, but is not limited to ritonavir (NORVIR®), oseltamivir (Tamiflu®), penciclovir (DENAVIR®), nirmatrelvir/ritonavir (PAXLOVID™), and acyclovir (Zovirax®). For example, the antibody can include, but is not limited to teclistamab (TECVAYLI®), cemiplimab (LIBTAYO®), nivolumab (OPDIVO®), and ofatumumab (KESIMPTA®). For example, the small molecule can include, but is not limited to a small molecule agonist, a small molecule antagonist, and antibiotics.

[0057] In some embodiments, the immunogenic composition as a T-cell adjuvant, enhances the response to the existing therapeutic by at least 50% such as by about 50%, about 60%, about 70%, about 80%, or about 90%. As used herein the term “enhances the response” or “enhanced response” refers to a measurable improvement in at least one symptom of a disease or condition.

[0058] In some embodiments, the T-cell adjuvant is administered at a dose of about 0.1 mg/kg of body weight to about 100 mg/kg of body weight of the subject in need thereof. For example, the T-cell vaccine can be administered to a subject in need thereof at a dose of about 0.1 mg/kg, about 1.0 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg of body weight, or any dose in-between. In other embodiments, the T-cell adjuvant is administered in an amount of about 50 mg to about 1000 mg to the subject in need thereof. For example, the T-cell adjuvant can be administered to a subject in need thereof in amount of about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, or any amount in-between.

[0059] In some embodiments, the T-cell adjuvant is administered to a subject in need thereof in an amount of about 10 pg to about 1 gram. For example, the T-cell adjuvant can be administered to a subject in need thereof in amount of about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 200 pg, about 300 pg, about 400 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, about 1000 pg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 800 mg, about 900 mg, about 1 gram (1,000 mg), or any amount in-between.

III. T-cell Diagnostics

[0060] In some embodiments, provided is a method of using the immunogenic composition for T-cell monitoring or diagnostics. For example, a sample (e.g., blood) can be combined with the immunogenic composition, incubated with said composition for a given period of time, and then cytokine production can be measured using an immunoassay such as flow cytometry and ELISpot assays. Increased cytokine levels correlate with an activated T-cell response.

[0061] In some embodiments, the immunogenic composition is combined with blood from a subject. After about 24-48 hours of incubation at 37 °C, the blood is spun down and the supernatant is analyzed for cytokine levels. The cytokine levels are quantified via ELISpot assays, ELISA assays, flow cytometry, and/or intracellular cytokine staining.

Definitions

[0062] As used herein and in the appended claims, the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.

[0063] As used herein, the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0064] As used herein, the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

[0065] As used herein, the terms “and/or” or “/” means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0066] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise. All publications mentioned herein are incorporated herein by reference in their entireties. EXAMPLES

[0067] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments or examples disclosed, but it is intended to cover modifications that are within the spirit and scope of the present invention as defined by the appended claims.

Example 1. Preparation of Immunogenic Compositions

[0068] The immunogenic compositions were prepared by fusing the N-terminus of the amino acid sequence of SEQ ID NO: 4 with gene fragments of YFV (Yellow fever virus) NS3P (non- structural protein 3 protease), YFV NS3H (non- structural protein 3 helicase), DENV (Dengue virus) NS3P, and DENV NS3H. The YSV and DENV gene fragments were fused to the N-terminus of SEQ ID NO: 4 via a GGS2 linker (GGSGGS; SEQ ID NO: 5). The constructs were cloned into an expression vector, transformed into E. Coli, and expressed and purified (FIG. 1) using standard methods known in the art.

Example 2. Eliciting an Immune Response with Immunogenic Compositions in Mice with Yellow Fever Virus or Dengue Virus

[0069] The immunogenicity of the compositions in Example 1 were tested in Ifnar ¹' mice infected with yellow fever virus (YFV-17D) or dengue virus (DENV2). The viruses were obtained from BEI Resources and mice were infected by intraperitoneal injection. Mice that did not receive the immunogenic compositions served as a negative control. Mice that were administered P/I (phorbol 12-myristate 13-acetate (P)/ionomycin) served as a positive control.

[0070] Following 7 days post-infection, blood was collected from the mice and CD8+ T- cells were isolated using the Dynabeads™ FlowComp™ Mouse CD8 Kit (Thermo Fisher Scientific, catalog number 11462D). As shown in FIG. 2, mice infected with either YFV- 17D or DENV2 exhibited an increase in the percentage of CD8+ IFN-y T-cells to homologous and heterologous fusion proteins compared to the no immunogen stimulation. Thus, mice infected with YFV-17D or DENV2 demonstrated robust T-cell responses against YFV and DENV2 structural and non-structural proteins.

[0071] Sequence Listing [0072] SEQ ID NO: 1 - Bacillus mycoides (WP_227590919.1)

MKIKKEIVKVLSMSFLLTTLPVTFSTTITHAAGPYGGGSENEIEIDYKRDEENKKKKEEEK

RKVYEEELKEIVKRVVQIDVKEENVVRREETEKLVKDLPSEILEMYDKVGGKIKIVEGSL

AEYPDLKGRKAINDNGKEVSLDQYYAYSIEGKAPEVFIRASEDYEESIHKRTSVYNEIGK

SLVRDVLKTEVLMDSSFLQAVNQMRLDKDTEAEFFSQNLRTYKGSFDETYVKEHIKDF

QDIFAKAFAYSRIPEFKEFFNTRSGPPFYIKKRTKQVVSYFFICFNKKLIKTVKLSLGKLLR

TFLYI

[0073] SEQ ID NO: 2 - Smaller modified fragment

GKSLVRDVLKTEVLMDSSFLQAVNQMRLDKDTEAEFFSQNLRTYKGSFDETYVKEHIK DFQDIFAKAFAYSRIPEFKE

[0074] SEQ ID NO: 3 - Enterococcus sp. 5H (WP_2171496993.1)

MSKKKNLITIIAFIALSAVYPLKVHAINDFISGDNDENIDRVIEIFEPFRDVLDDRQEAFLIT

FEQTIENERTMNNMLRLTGGDFSLIPRGASIESQIRMERFADELKLMDALFLLRESRNHT

GFYLYSLVSSNDFYIPEAYYYKVNNPNILDNTKVEHLREFYRYGFLSDFLIGSATGNEPQ

VNGQFKVRIKVPERTPLVQVNNAIDNLIINRNMGLEIESIRRITDRGREVLRVEANLISKEE

VDKKIQKVKNNIDTIVADKIGDKYKLIAFDLSGKGASLISEQAEQMVSRFIDVIPLPILKE

GISFMGEKGANIVLTDKKLGYVREALPVDILPSQEEKVFNEANRSAGRYNPYKRTIVING

HARGIVGNHMDPERLLHEFGHVVDNMRGQGYSDDNLSNSAEFKVIFNEERDKLTEYGK

TNIQEFFAEAFRLTYSTNLQKREHIKESAPKSYEFITKIR

[0075] SEQ ID NO: 4 - Brevibacillus laterosporus strain ZQ2

GKAIARDTLDESTCVNEAFLDALHQAKNAADEDANALLFSHQLPAYEGKYDTAYVREH

IDEFREVFAQAFAYYYEPSYQPVLKAYVPEMFRYMNDINEQEINLS

[0076] SEQ ID NO: 5 - Protein linker

GGSGGS

Claims

1. An immunogenic composition comprising an antigen fused to a cytotoxin, wherein the cytotoxin is a bacterial cytotoxin with the catalytic infectious domain removed, and the antigen induces a T-cell response to a pathogen or a tumor cell.

2. The composition of claim 1, wherein the bacterial cytotoxin is Bacillius thuringiensis or Bacillus shihchuchen.

3. The composition of claim 1, wherein the cytotoxin comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

4. The composition of claim 1, wherein the antigen fused is fused to the cytotoxin via the amino acid sequence of SEQ ID NO: 5.

5. The composition of claim 1, wherein the pathogen is selected from the group comprising a bacteria, a virus, a parasite, and a fungus.

6. The composition of claim 1, wherein the tumor cell is a cancer cell, and wherein the cancer is selected from the group comprising anal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, lung cancer, lymphoma, multiple myeloma, ovarian cancer, pancreatic cancer, renal transitional cell cancer, skin cancer, synovial sarcoma, and thyroid cancer.

7. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

8. A kit comprising the composition of claim 1, wherein the kit comprises: a) an immunoassay to quantify cytokine levels from a sample of blood for detecting a T-cell-mediated immune response; and b) instructions for use.

9. A method of enhancing an existing therapeutic agent comprising administering the composition of claim 1 to a subject in need thereof.

10. The method of claim 9, wherein the existing therapeutic agent is selected from the group comprising a vaccine, an anti-viral, an antibody, and a small molecule.

11. A method of monitoring T-cells, comprising the following steps: a) mixing blood from a subject with the composition of claim 1; b) incubating the mixture of a) for at least 24 hours at about 37 °C; c) centrifuging the mixture of b) following the incubation period; d) collecting the supernatant from c); and e) quantifying cytokine levels with an immunoassay.

12. The method of claim 11, wherein the cytokine levels are quantified with an ELIspot assay, an ELISA assay, or flow cytometry.

13. A T-cell vaccine comprising the composition of claim 1, wherein the T-cell vaccine comprises an aqueous or liposomal formulation.

14. The T-cell vaccine of claim 13, wherein the T-cell vaccine further comprises an adjuvant.

15. An isolated polynucleotide sequence encoding an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

16. The amino acid sequence of claim 15, wherein said sequence is fused to an antigen.

17. The amino acid sequence of claim 15, wherein said sequence is fused to the antigen via the amino acid sequence of SEQ ID NO: 5.

18. The amino acid sequence of claim 17, wherein the antigen is selected from the group comprising a bacterial antigen, a viral antigen, a parasitic antigen, a fungal antigen, a tumor-associated antigen (TAA), and a tumor-specific antigen (TSA).

19. A pharmaceutical composition comprising the antigen-fused amino acid sequence of claim 17 and a pharmaceutically acceptable carrier.

20. A method of eliciting a T-cell response in a subject comprising administering to the subject a composition comprising an antigen fused to a cytotoxin, wherein the antigen induces a T-cell response to a pathogen or a tumor cell.

21. The method of claim 20, wherein the cytotoxin is a bacterial cytotoxin.

22. The method of claim 21, wherein the bacterial cytotoxin is Bacillius thuringiensis or Bacillus shihchuchen.

23. The method of claim 20, wherein the cytotoxin comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

24. The method of claim 20, wherein the pathogen is selected from the group comprising a bacteria, a virus, a parasite, and a fungus.

25. The method of claim 20, wherein the tumor cell is a cancer cell, and wherein the cancer is selected from the group comprising anal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, lung cancer, lymphoma, multiple myeloma, ovarian cancer, pancreatic cancer, renal transitional cell cancer, skin cancer, synovial sarcoma, and thyroid cancer.

26. The method of claim 20, wherein the T-cell response: a) enhances an existing therapeutic agent; b) serves as a T-cell vaccine; and/or enables T-cell diagnostics.