Multivalent tuberculosis vaccine

Abstract

A multivalent tuberculosis vaccine comprises one or both Mycobacterium tuberculosis transmembrane copper transporters CtpV (SEQ ID NO: 1) and MctB (SEQ ID NO: 2) reconstituted into proteoliposomes at a protein-to-lipid ratio of 1:15 to 1:40 (w / w). The proteoliposomes, optionally combined in a 1:1 to 3:1 ratio and formulated with CpG ODN 2395, 7909, or 1018, are encapsulated in ZIF-8 or multivariate Mn-ZIF (15-50 % Mn) to yield shelf- stable nanoparticles. Intranasal or pulmonary administration elicits robust Thl-biased immunity, polyfunctional CD4+ T-cells, and IgG2a-dominant antibodies, achieving >0.3 login CPU reduction in lungs versus saline and superior Th1 / Th2 balance versus BCG in murine aerosol challenge models. A metalloimmunological adjuvant comprising CDA-loaded Mn-ZIF synergistically activates cGAS-STING, enhancing dendritic cell maturation and protective efficacy when combined with tuberculosis antigens. Kits and methods for vaccination and manufacture are provided.

Description

FJ ref. UTD-P0001US / Client ref. 25003MULTIVALENT TUBERCULOSIS VACCINEPRIORITY PARAGRAPH

[0001] The present Application claims priority to US Provisional Application 63 / 718,732 filed 11 / 10 / 2024 which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under GM128704 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] A sequence listing is being submitted electronically with this application. The sequence listing is incorporated herein by reference. The sequence listing is contained in the XML file named "UTDP0001" which is 9,714 bytes (as measured in Microsoft Windows®) and was created on November 7, 2025.FIELD

[0004] Certain embodiments of the invention are directed generally to the field of medicine and infectious diseases, more particularly to vaccines and treatments for tuberculosis.BACKGROUND

[0005] Mycobacterium tuberculosis Mtb) infects around one-fourth of the world’s population. It is the second deadliest infectious disease affecting humans behind COVID-19. Despite the disease being a pervasive issue in many high-population developing countries, the only vaccine that has been distributed on a wide scale has been Bacillus Calmette-Guerin (BCG), a / / vc-attenuated vaccine form of Mycobacterium bovis. While BCG has lowered the occurrence of disseminated tuberculosis (TB) in children, vaccine efficacies have been reported to vary as widely as 0-80% in different populations around the world against the more common pulmonary adult form of the disease.1With the emergence of drug-resistant Mtb strains, there is a need for a radically new approach to developing alternative biotechnologies for developing TB vaccines.FJ ref. UTD-P0001US / Client ref. 25003

[0006] Research for whole cell inactivated or live attenuated vaccines often requires a biosafety level 3 facility. Moreover, such formulations carry a high risk of reverting back to virulence due to mutations introduced via continuous subculturing, creating an everlasting biosafety hazard that will always accompany the mass production of these vaccines. The inevitable switch to safer subunit vaccines has yet to show promise. This has not been for a lack of effort, as hundreds of papers have explored antigen-based approaches, though they have mostly been soluble proteins and often those secreted from Mtb.2-6There is a need to expand the library of TB antigens that can be recognized by the immune system, especially those present on the surface of Mtb, as their pathogen-associated molecular patterns (PAMPs) can trigger the pattern recognition receptors to invoke an immune response.

[0007] There remains a need for additional vaccines and therapies for treating or ameliorating Mycobacterium tuberculosis (Mtb) infection.SUMMARY

[0008] Certain embodiments provide a solution to the problems described above through the recombinant expression and purification of two novel transmembrane proteins expressed on the surface of Mtb and their reconstitution into artificial lipid bilayers, mimicking their presentation in the pathogen. The lipid bilayer can be in the form of proteoliposomes, forming size-controlled nanoparticles. The incorporation of both proteins onto / into the same proteoliposome has been demonstrated. This model of a liposomal vaccine also offers the advantages of PAMP-like presentation, enhanced cellular uptake, and adjuvant properties.

[0009] Certain embodiments are directed to a tuberculosis vaccine composition comprising CtpV, MctB, or CtpV and MctB in a liposome carrier forming a proteoliposome. The vaccine composition can further include one or more adjuvants. In certain aspects the adjuvant can be a CpG oligonucleotide adjuvant. The CpG oligonucleotide adjuvant can be selected from CpG ODN 2395, CpG 7909, or CpG 1018. In certain aspects the proteoliposome is coated with or encapsulated in a metal-organic framework (MOF). The MOF can be ZIF-8 or a similar MOF. In certain aspects CtpV and MctB are present in separate proteoliposomes. In other aspects CtpV and MctB are present in the same proteoliposomes. CtpV and MctB can be present in a ratio of at least, at most, or about 5: 1, 4: 1, 3: 1, 2:1, 1 : 1, 1 :2, 1 :3, 1 :4, or 1 :5. The proteoliposomes can haveFJ ref. UTD-P0001US / Client ref. 25003 a protein to lipid ratio of at least, at most, or about 1 : 15, 1 :25, or 1 :40. Tn certain aspects the proteoliposomes are formulated for intranasal or pulmonary administration.

[0010] Certain embodiments are directed to methods of treating tuberculosis comprising administering an effective amount of a vaccine composition described herein.

[0011] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0012] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0013] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0014] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0015] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0016] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and / or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps), but may include other elements (orFJ ref. UTD-P0001US / Client ref. 25003 components or features or steps) not expressly listed or inherent to the chemical composition and / or method.

[0017] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0018] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0019] Definitions

[0020] The following definitions are provided to facilitate a clear understanding of the terms used throughout this patent application. These definitions are intended to be illustrative rather than limiting and are applied consistently unless otherwise specified in the context of the claims or detailed description. The terms defined encompass their ordinary meanings in the art, as well as any specific interpretations provided, to support the invention's disclosure and scope.

[0021] “Adjuvant” refers to any substance that is added to a vaccine composition to enhance, direct, or modulate the immune response to the antigen(s) contained therein. Certain embodiments include adjuvants that include, but are not limited to, CpG oligonucleotides, aluminum salts, oil-in-water emulsions, saponins, and metal-organic frameworks (MOFs) such as ZIF-8 or Mn-ZIF.

[0022] “CtpV” refers to the Cation transporter protein V of Mycobacterium tuberculosis, having the amino acid sequence set forth in SEQ ID NO: 1, or a biologically functional equivalent thereof, including fragments, variants, or fusion proteins that retain immunogenicity.FJ ref. UTD-P0001US / Client ref. 25003

[0023] “MctB” refers to the Mycobacterial copper transporter protein B of Mycobacterium tuberculosis, having the amino acid sequence set forth in SEQ ID NO:2, or a biologically functional equivalent thereof, including fragments, variants, or fusion proteins that retain immunogenicity.

[0024] “Antigen” means any substance capable of inducing a specific immune response, including production of antibodies and / or activation of T cells. In the context of this invention, the antigens are the transmembrane proteins CtpV and / or MctB derived from Mycobacterium tuberculosis (Mtb).

[0025] “Effective amount” refers to an amount of a vaccine composition sufficient to elicit a detectable immune response, reduce bacterial load, prevent infection, ameliorate symptoms, or treat tuberculosis in a subject.

[0026] “Lipid nanoparticle” or “liposome” refers to a spherical vesicle having at least one lipid bilayer, including small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), and multilam ellar vesicles (MLVs). The term encompasses both empty liposomes and those containing embedded or reconstituted proteins.

[0027] “Metal-organic framework (MOF)” refers to a class of crystalline compounds consisting of metal ions or clusters coordinated to organic ligands to form one-, two-, or three- dimensional porous structures. Exemplary MOFs include zeolitic imidazolate frameworks (ZIFs) such as ZIF-8, ZIF-67, ZIF-7, and multivariate MOFs such as Mn-ZIF.

[0028] “Proteoliposome” refers to a liposome in which one or more membrane proteins, such as CtpV and / or MctB, are reconstituted into the lipid bilayer in a functional orientation, mimicking their native presentation on the surface of Mycobacterium tuberculosis.

[0029] “Subject” means a human or non-human animal selected for treatment or therapy, including but not limited to mammals such as mice, non-human primates, and humans.

[0030] “Tuberculosis” or “TB” refers to the infectious disease caused by Mycobacterium tuberculosis, including latent TB infection (LTBI), active pulmonary TB, and extrapulmonary TB.

[0031] “Vaccine composition” refers to a formulation comprising at least one antigen (CtpV, MctB, or both) in a proteoliposome carrier, optionally with one or more adjuvants, stabilizers, preservatives, or delivery vehicles, suitable for administration to elicit protective immunity against Mycobacterium tuberculosis.FJ ref. UTD-P0001US / Client ref. 25003

[0032] “ZTF-8” refers to zeolitic imidazolate framework-8, a porous MOF composed of zinc ions coordinated with 2-methylimidazole linkers, used herein for encapsulation or coating of proteoliposomes to enhance stability and shelf-life.

[0033] A “STING agonist” is a small-molecule activator of the Stimulator of Interferon Genes (STING) pathway, specifically a cyclic dinucleotide (CDN) or CDN analogue that binds directly to the STING protein (TMEM173) in the cytosol of mammalian cells, inducing STING dimerization, phosphorylation of TBK1 and IRF3, nuclear translocation of phosphorylated IRF3, and subsequent transcription of type-I interferons (IFN-a / p) and proinflammatory cytokines (TNF-a, IL-6, IL-12, etc.). STING agonists include CDA (cyclic di-adenosine monophosphate, also known as c-di-AMP or 3 '3 '-c-di-AMP), cGAMP (cyclic guanosine monophosphateadenosine monophosphate, specifically the natural 2'3 '-cGAMP produced by cGAS), 3'3'- cGAMP (synthetic bacterial-type cyclic di-GMP-AMP) These STING agonists are post- synthetically loaded into multivariate Mn-ZIF (15-50 % Mn doping) or co-encapsulated with proteoliposomes inside ZIF-8 or Mn-ZIF.

[0034] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the 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 this detailed description.DESCRIPTION OF THE DRAWINGS

[0035] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0036] FIG. 1. Utilization of membrane proteins as TB antigens. Transmembrane copper transporter proteins found on the outer (MctB) and inner (CtpV) membranes were recombinantly expressed in E. coli. The proteins were purified as detergent micelle complexes. CtpV micelles were tested as antigens against the plasma of TB-positive patients, and their immune response in the form of TB-specific IgG and IgM was analyzed using a Luminex assay. CtpV and MctBFJ ref. UTD-P0001US / Client ref. 25003 were reconstituted into proteoliposomes, individually and in combination (Combo), whose lipid bilayers mimic the native environment of transmembrane proteins. Combo was encapsulated in ZIF-8 (Combo@Z) to create a shelf-stable formulation. The immune profile of CtpV, MctB, Combo, and Combo@Z, along with saline and BCG as controls, were evaluated in vivo in a mouse model using ELISA and flow cytometry.

[0037] FIG. 2A-2D. Purification and characterization of proteoliposomes. Size-exclusion chromatogram of (A) CtpV and (B) MctB, and corresponding analysis by (C) SDS-PAGE gel electrophoresis (theoretical masses: CtpV - 82.1 KDa, MctB - 34.5 KDa). (D) DLS of MctB, CtpV, and Combo proteoliposomes. (E) Gel electrophoresis of all three proteoliposomes. The incorporation efficiency of the proteins was determined using gel band densitometry.

[0038] FIG. 3A-3C. Luminex data showing CtpV, PPD, and ESAT6CFP10-specific IgG titers. Plasma IgG levels were measured using customized Luminex assay over three dilutions and the area under the curve (AUC) calculated. Each column represents an individual patient. Titers for (A) CtpV, (B) purified protein derivative (PPD), and (C) ESAT-6 and CFP-10 were determined in latent (n = 18) and active TB (n=19) plasma samples as well endemic negative controls (n = 8). Dashed lines represent the median values for latent TB, active TB, endemic negative controls, and saline background.

[0039] FIG. 4A-4I. In vivo study of individual and combination of antigens. (A) Experimental timeline for study. Cytokines (B) TNF-a, (C) IL-2, (D) IFN-y, and (E) IL-17A were measured on days 21, 28, 35, and 42. Antibody titers of (F) total IgG, (G) IgGl, and (H) IgG2a were measured on day 28, and the IgG2a / IgGl ratio was used to derive the (I) Thl / Th2 ratio. Statistical analysis was performed using student’s T-test with a confidence interval of 95%.

[0040] FIG. 5A-5F. Flow cytometry analysis of (A-B) spleen, (C-D) lymph nodes, and (E-F) lungs of mice after being sacrificed on day 42. Statistical analysis was performed using student’s T-test with a confidence interval of 95%.

[0041] FIG. 6A-6C. (A) Synthetic scheme for encapsulation of Combo inside ZIF-8 (C@Z). Characterization of C@Z using (B) PXRD and (C) SEM (scale bar = 1 pm).

[0042] FIG. 7A-7M. Comparison of C@Z’s performance in vivo with Combo and BCG. Cytokines (A) TNF-a, (B) IL -2, (C) IFN-y, and (D) IL-17A were measured on days 21, 28, 35, and 42. Antibody titers of (E) total IgG, (F) IgGl, and (G) IgG2a were measured on day 28. Flow cytometry analysis of (H, K) spleen, (I, L) lymph nodes, and (J, M) lungs of mice afterFJ ref. UTD-P0001US / Client ref. 25003 being sacrificed on day 42. Statistical analysis was performed using student’s T-test with a confidence interval of 95%.

[0043] FIG. 8A-8G. Proteoliposomes incorporation efficiency determination by band densitometry in SDS-PAGE.

[0044] FIG. 9A-9C. CtpV IgM titers are detectable in plasma from latent and active TB patients. (A - C) Plasma IgM levels were measured using customized Luminex assay over three dilutions and the area under the curve (AUC) calculated. Each column represents an individual patient. Titers for (A) CtpV, (B) purified protein derivative (PPD), and (C) ESAT-6 & CFP-10 were determined in latent (n = 18) and active TB (n=19) plasma samples as well endemic negative controls (n = 8). Dashed lines represent the median values for latent TB, active TB, endemic negative controls, and saline background.

[0045] FIG. 10. Lipid / MOF precursor ratio optimization for encapsulation.

[0046] FIG. 11. Gating strategy for flow cytometry data.

[0047] FIG. 12A-12D. Synthesis of Mn-ZIF and delivery of Mn, Zn, and CDA for immune activation in-vitro and in-vivo. (A) Synthetic overview of Mn-ZIF and CDA@Mn-ZIF. Zn2+, Mn2+, a reducing agent TCEP, and 2-methylimidazole (HMIM) were mixed in water and left static at RT to afford Mn-ZIF. CDA was incorporated post-synthetically by incubating with Mn- ZIF suspended in water and methanol at RT on a rotisserie to make CDA@Mn-ZIF. (B) Uptake of the multivariate MOF was measured to demonstrate the delivery and release of Mn, Zn, and CDA into cells. (C) The immune- stimulatory effect of Mn and CDA delivery to trigger the cGAS-STING pathway. First, Mn and Zn can bind to cGAS to trigger cGAMP production that activates STING. Additionally, Mn and CDA can directly activate STING, causing it to phosphorylate TANK -binding kinase 1 (TBK-1), which phosphorylates and dimerizes interferon regulatory factor 3 (IRF-3). Phosphorylated IRF-3 dimers translocate to the nucleus, turning on gene expression of pro-inflammatory cytokines. (D) CDA@Mn-ZIF was combined with the Mtb fusion protein CysVac2 and used a vaccine in a pre-clinical mouse Mtb challenge experiment.

[0048] FIG. 13A-13E. Characterization of Mn-ZIF. (A) PXRD diffractogram of different Mn ratios starting with ZIF-8 control with 0% Mn up to 50% Mn. SEM micrographs of (B) ZIF-8 control, (C) 15% Mn-ZIF, (D) 30% Mn-ZIF, and (E) 50% Mn-ZIF.

[0049] FIG. 14A-14D. In vitro cytotoxicity measured by resazurin assay. Viability of (A) RAW 264.7 cells, (B) 4T1 cells, and (C) HEK 293 cells were measured after treatment withFJ ref. UTD-P0001US / Client ref. 25003 different doses of 0% Mn-ZTF, 15% Mn-ZIF, 30% Mn-ZIF, and 50% Mn-ZTF and plotted. (D) Calculated IC50 values of each sample on the three cell lines. Statistical analysis was performed using Two-way ANOVA with Tukey’s multiple comparison test, confidence interval of 95%. Data is presented as mean ± standard deviation.

[0050] FIG. 15A-15F. Characterization of CDA@Mn-ZIF, association, and uptake. (A) Scheme of post-synthetic loading of CD A onto Mn-ZIF. (B) CDA loading after incubating each concentration of CDA with 1 mg / mL of Mn-ZIF. (C) Surface charge of Mn-ZIF before and after loading with CDA measured by ^-potential. (D) Epifluorescent micrographs of CDA@Mn-ZIF loaded with rhodamine in RAW 264.7 cells stained with Hoechst and lysotracker green after 6 h. (E) Representative histogram of RAW 264.7 cell uptake of CF encapsulated in Mn-ZIF at 4 h and 8 h. (F) Average uptake at 4 h and 8 h of CF, Mn-ZIF(CF), CDA@Mn-ZIF(CF) calculated by geometric mean of FITC intensity where n = 5. Statistical analysis was performed using Two- way ANOVA with Tukey’s multiple comparison test, confidence interval of 95%. Data is presented as mean ± standard deviation.

[0051] FIG. 16A-16D. Immune activating potential of 50% Mn-ZIF and CDA@Mn-ZIF. (A) BMDC activation measured by flow cytometry. Activation was defined as CDl lc+ cells double positive for CD80+ and CD86+. (B) Western blot of cGAS-STING downstream proteins with molecular weights in kDa annotated on the left. (C) TNF-a and (D) IL-6 produced by BMDCs after 24 h incubation with each sample measured by ELISA on the cell supernatant. Statistical analysis was performed with Welches t-test, confidence interval of 95%. Data is presented as mean ± standard deviation.

[0052] FIG. 17A-17G. Adjuvant effect of CDA@Mn-ZIF in-vivo in a pre-clinical Tb mouse model. (A) Timeline of experiments including vaccination, blood draw, challenge, and sacrifice for lung CFU counting. Percentage of CD4+CD44hlCD62L10T cells secreting (B) IL- 17, (C) IFN- y, (D) IL-2, and (E) TNF from PBMCs after in vitro stimulation with CysVac2. PBMC was taken approximately 2 weeks after the final vaccination. (F) Example flow cytometry gating strategy for phenotyping of PBMCs. (G) Mean Logic colony forming units (CFU) in the lungs of C57BL / 6 mice inoculated with CDA@Mn-ZIF formulated with either 10, 3, or 1 pg of CysVac2 via the I.M. route, 3-times at 2-week intervals. Mice were rested and infected with low-dose aerosol (50-100 CFU) of Mtb Erdman KOI, and CFU was determined at day 30 post-infection.FJ ref. UTD-P0001US / Client ref. 25003N=5 mice per group. Statistical analysis was performed using a one-way ANOVA. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. Data is presented as mean ± standard deviation.DESCRIPTION

[0053] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment, and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0054] Tuberculosis (TB) is one of the deadliest infectious diseases, which continues to be a major health risk in many parts of the world. Even today, the century-old BCG vaccine is the only formulation on the market.I. Tuberculosis Vaccine Formulations

[0055] Certain embodiments describe the purification and testing of Cation transporter protein V (CtpV) and / or Mycobacterial copper transporter B (MctB) antigens presented in a lipid bilayer as a Mycobacterium tuberculosis vaccine. CtpV was tested as an antigen against the plasma of tuberculosis patients. CtpV and MctB were reconstituted into proteoliposomes, individually and in combination, to create a stable nanoparticle vaccine platform. Since tuberculosis vaccines often need to be shipped to areas with fluctuating power supply, the proteoliposomes can be encapsulated in ZIF-8 to create a shelf-stable formulation. In vivo studies were conducted to test the immunogenicity of the formulations with and without the ZIF-8 coating.A. Antigens

[0056] The two transmembrane proteins are the Cation transporter protein V (CtpV) and the Mycobacterial copper transporter protein B (MctB). CtpV is localized on the inner membrane, while MctB resides on the outer membrane / mycomembrane oiMtb.7 8Both proteins are putative copper ion exporters, overexpressed in response to increased cytoplasmic copper concentrationsFJ ref. UTD-P0001US / Client ref. 25003 resulting from host nutritional immunity responses.9 10The host immune system employs copper as an anti-invading agent, leveraging the generation of toxic reactive oxygen species through the redox cycling of free copper ions, ultimately leading to bacterial cell death. Upon infection, the host alveolar macrophages modulate molecular strategies and express proteins that facilitate the transport of Cu(I) ions inside the phagosome compartment, where Mtb replicates, to overload the compartment with copper and promote toxic reactive oxygen species (ROS) production via oxygen-dependent copper redox cycling.11This process primarily includes the trafficking of the host-encoded transmembrane Cu(I) pump ATP7A to vesicles that fuse with phagosomes, stimulated by IFN-y which is produced after infection.10 12

[0057] Cation transporter protein V (CtpV) has a full length amino acid sequence of MRVCVTGFNVDAVRAVAIEETVSQVTGVHAVHAYPRTASVVIWYSPELGDTAAVLSAI TKAQHVPAELVPARAPHSAGVRGVGVVRKITGGIRRMLSRPPGVDKPLKASRCGGRPR GPVRGSASWPGEQNRRERRTWLPRVWLALPLGLLALGSSMFFGAYPWAGWLAFAATL PVQFVAGWPILRGAVQQARALTSNMDTLIALGTLTAFVYSTYQLFAGGPLFFDTSALIIA FVVLGRHLEARATGKASEAISKLLELGAKEATLLVDGQELLVPVDQVQVGDLVRVRPG EKIPVDGEVTDGRAAVDESMLTGESVPVEKTAGDRVAGATVNLDGLLTVRATAVGAD TALAQIVRLVEQAQGDKAPVQRLADRVSAVFVPAVIGVAVATFAGWTLIAANPVAGMT AAVAVLIIACPCALGLATPTAIMVGTGRGAELGILVKGGEVLEASKKIDTVVFDKTGTLT RARMRVTDVIAGQRRQPDQVLRLAAAVESGSEHPIGAAIVAAAHERGLAIPAANAFTAV AGHGVRAQVNGGPVVVGRRKLVDEQHLVLPDHLAAAAVEQEERGRTAVFVGQDGQV VGVLAVADTVKDDAADVVGRLHAMGLQVAMITGDNARTAAAIAKQVGIEKVLAEVLP QDKVAEVRRLQDQGRVVAMVGDGVNDAPALVQADLGIAIGTGTDVAIEASDITLMSGR LDGVVRAIELSRQTLRTIYQNLGWAFGYNTAAIPLAALGALNPVVAGAAMGFSSVSVVT NSLRLRRFGRDGRTA (SEQ ID NO: 1). Certain embodiments include fragments comprising any segment of consecutive amino acids from (starting at the amino or the carboxy terminus) 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760 to 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,FJ ref. UTD-P0001US / Client ref. 25003430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770 amino acids of SEQ ID NO: 1, including all values and ranges there between.

[0058] Mycobacterial copper transporter protein B (MctB) MISLRQHAVSLAAVFLALAMGVVLGSGFF SDTLLS SLRSEKRDLYTQIDRLTDQRDALR EKLSAADNFDIQVGSRIVHDALVGKSVVIFRTPDAHDDDIAAVSKIVGQAGGAVTATVS LTQEFVEANSAEKLRSVVNSSILPAGSQLSTKLVDQGSQAGDLLGIALLSNADPAAPTVE QAQRDTVLAALRETGFITYQPRDRIGTANATVVVTGGALSTDAGNQGVSVARFAAALA PRGSGTLLAGRDGSANRPAAVAVTRADADMAAEISTVDDIDAEPGRITVILALHDLING GHVGHYGTGHGAMSVTVSQ (SEQ ID NO:2). Certain aspects include any segment of consecutive amino acids from (starting at either amino or carboxy terminus) 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 314 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 314 amino acids ofSEQ ID NO:2, including all values and ranges there between.

[0059] In certain embodiments, modifications and / or changes may be made in the amino acid sequence of the described polypeptides, and thus the present invention contemplates variation in sequences of the polypeptides, and nucleic acids coding therefor, where they are nonetheless able to retain substantial activity with respect to the therapeutic, preventative, and curative aspects of the present invention.

[0060] In certain aspects a polypeptide can contain an amino-terminal (N-terminal) tag and / or a carboxy-terminal (C-terminal) tag. These tags are amino acid sequences that are added to recombinant proteins to aid in their purification, detection, and / or study. Amino-terminal (N- terminal) tags are attached to the beginning (N-terminus) of the protein sequence. Carboxy- terminal (C-terminal) tags are attached to the end (C -terminus) of the protein sequence. Protein tags include, but are not limited to His-tag (Polyhistidine tag), GST-tag (Glutathione S- transferase), FLAG-tag (DYKDDDDK SEQ ID NO:3), Myc-tag (EQKLISEEDL. SEQ ID NO:4), HA-tag (Hemagglutinin tag, YPYDVPDYA SEQ ID NO:5), Strep-tag (WSHPQFEK SEQ ID NO: 6), MBP-tag (Maltose Binding Protein), Thioredoxin (Trx) tag, V5 tag (GKPIPNPLLGLDST SEQ ID NO: 7), SUMO tag (Small Ubiquitin-like Modifier), T7-tag (MASMTGGQQMG SEQ ID NO: 8), or AviTag (GLNDIFEAQKIEWHE SEQ ID NO: 9)FJ ref. UTD-P0001US / Client ref. 25003

[0061] The decision to use an N-terminal or C-terminal tag depends on the protein's structure, function, and the experimental objectives. It is often an empirical process to determine which configuration is best for a particular protein. In certain embodiments, both types of tags can often be removed by proteolytic cleavage if they interfere with the protein's function, although this step adds complexity to the purification process.

[0062] A biological functional equivalent of a polypeptide or fragment / segment thereof can include one or more amino acid substitution without appreciable loss of antigenicity. So-called “conservative” changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges on the ability to carry out a designed function. It is thus contemplated by the inventors that various changes may be made in the sequence of proteins, polypeptides, or fragments / segments disclosed herein, while still fulfilling the goals of the present invention.

[0063] In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a “biologically functional equivalent” protein and / or polypeptide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalents are thus defined herein as those proteins, polypeptides, or fragments / segments where amino acids may be substituted but function is maintained. In certain aspects, a polypeptide or fragment thereof is 80, 85, 90, 92, 94, 96, 98, or 100% identical to the wildtype form of the polypeptide, e g., SEQ ID NO: 1 or SEQ ID NO:2 or fragments thereof. In certain aspects, polypeptide(s) 80, 85, 90, 92, 94, 96, 98, or 100% identical to SEQ ID NO: 1 or 2 are used or nucleic acids encoding the same.

[0064] In general, the shorter the length of the molecule, the fewer changes that can be made within the molecule while retaining function. Longer domains may have an intermediate number of changes. The full-length protein will have the most tolerance for a larger number of changes. However, it must be appreciated that certain molecules or domains that are highly dependent upon their structure may tolerate little or no modification. Function of a polypeptide can be determined by using various assays known to detect the activity of the polypeptide of interest.

[0065] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and / or the like. An analysis of the size, shape and / or type of the amino acid side-chainFJ ref. UTD-P0001US / Client ref. 25003 substituents reveals that arginine, lysine, and / or histidine are all positively charged residues; that alanine, glycine, and / or serine are all a similar size; and / or that phenylalanine, tryptophan, and / or tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine, and / or histidine; alanine, glycine, and / or serine; and / or phenylalanine, tryptophan, and / or tyrosine are defined herein as biologically functional equivalents.

[0066] To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and / or charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and / or arginine (-4.5).

[0067] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index and / or score and / or still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those that are within ±1 are particularly preferred, and / or those within ±0.5 are even more particularly preferred.

[0068] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and / or those within ±0.5 are even more particularly preferred.FJ ref. UTD-P0001US / Client ref. 25003B. Proteoliposomes

[0069] Proteoliposomes are artificial vesicles composed of a lipid bilayer containing embedded or reconstituted proteins, particularly membrane proteins. The lipid bilayer can be in the form of a lipid nanoparticle that is a vehicle comprising one or more lipids. The term “lipid nanoparticle” also refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm). The one or more lipids can be cationic lipids, non-cationic lipids, or PEG- modified lipids. The lipid nanoparticles can be formulated to deliver one or more agents. Suitable lipids include, but is not limited to phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. The lipid nanoparticle can form spontaneously when phospholipids are placed in water due to the amphipathic nature of phospholipids, which have a hydrophilic head and hydrophobic tails. When membrane proteins are incorporated into these liposomes, they become proteoliposomes. These proteins can include integral membrane proteins that span or are embedded within the lipid bilayer, or peripheral membrane proteins that are loosely bound to the surface of the lipid bilayer or to integral proteins.

[0070] Proteoliposomes can be manufactured by solubilizing or purifying membrane proteins and reconstituting the proteins in a lipid particle. The proteins can be mixed with lipids in the presence of detergents which are later removed, allowing the lipids to form vesicles around the proteins. After formation, proteoliposomes can be purified and / or isolated by removing any remaining detergent or non-incorporated proteins.

[0071] In certain aspects the lipid nanoparticles (e.g., proteoliposomes) of the invention can be 125 nm to 225 nm. Lipid nanoparticles can be unilamellar consisting of a single phospholipid bilayer. Unilamellar lipid nanoparticles can be further divided into: small unilamellar vesicles (SUVs) - usually 20-100 nm in diameter, large unilamellar vesicles (LUVs) - typically greater than 100 nm, and multilamellar vesicles (MLVs) composed of several concentric bilayers. In certain aspects the liposomes can be SUVs.

[0072] The primary components of lipid nanoparticles are phospholipids, which naturally form bilayers in aqueous environments due to their amphiphilic nature. Key lipids include, but are not limited to phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylglycerol (PG), Cholesterol, or E. col polar lipids (e.g., PE, PG, and Cardiolipin (CL)). Any lipid may be used in the composition described herein so long as it is aFJ ref. UTD-P0001US / Client ref. 25003 membrane-forming lipid. Although any lipid as defined above may be used, particularly suitable lipids may include those with at least one fatty acid chain containing at least 4 carbons, and typically about 4 to 28 carbons. The fatty acid chain may contain any number of saturated and / or unsaturated bonds. The lipid may be a natural lipid or a synthetic lipid. Non-limiting examples of lipids may include phospholipids, sphingolipids, sphingomyelin, cerebrosides, gangliosides, ether lipids, sterols, cardiolipin, cationic lipids and lipids modified with poly (ethylene glycol) and other polymers. Synthetic lipids may include, without limitation, the following fatty acid constituents: lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these fatty acids. In some embodiments, the lipid or lipids of the lipid nanoparticle are amphiphilic lipids, meaning that they possess both hydrophilic and hydrophobic (lipophilic) properties.

[0073] Lipids suitable for use in the composition of the present disclosure include, but are not limited to phospholipids, cationic lipids, cholesterol and / or cholesterol derivatives, or a combination thereof. It is to be understood that the terms “phospholipids”, “cationic lipids” or “cholesterol derivatives”, are not necessarily mutually exclusive of each other.

[0074] Broadly defined, a “phospholipid” is a member of a group of lipid compounds that yield on hydrolysis phosphoric acid, an alcohol, fatty acid, and nitrogenous base. Phospholipids that are preferably used in the preparation of the composition of the present disclosure are those with at least one head group selected from the group consisting of phosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine and phosphoinositol. More preferred are lipids which are about 94-100% phosphatidylcholine. In some embodiments, the phospholipid used in the preparation of the composition of the present disclosure is phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidyl serine (PS), Phosphatidylglycerol (PG), Cholesterol, E. coli polar lipids (e.g., PE, PG, and Cardiolipin (CL)), di oleoyl phosphatidylcholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), Dioleoyl Phosphatidylethanolamine (DOPE), l,2-dipalmitoyl-sn-glycero-3 -succinate (DGS), or a combination thereof. Another common phospholipid is sphingomyelin. Sphingomyelin contains sphingosine, an amino alcohol with a long unsaturated hydrocarbon chain. A fatty acyl side chain is linked to the amino group of sphingosine by an amide bond, to form ceramide. The hydroxyl group of sphingosine is esterified to phosphocholine. Like phosphoglycerides, sphingomyelin is amphipathic. Lecithin, which also may be used, is a natural mixture of phospholipids typicallyFJ ref. UTD-P0001US / Client ref. 25003 derived from chicken eggs, sheep's wool, soybean and other vegetable sources. All of these and other phospholipids may be used in the practice of the disclosure. Phospholipids can be purchased, for example, from Avanti lipids (Alabastar, AL, USA), Lipoid LLC (Newark, NJ, USA) and Lipoid GmbH (Germany), among various other suppliers.

[0075] Anionic phospholipids include Phosphatidylserine (PS), Phosphatidylinositol (PI), Phosphatidylinositol phosphates (PIPs), Phosphatidylglycerol (PG), Cardiolipin, Phosphatidic Acid (PA), and the like.

[0076] Cationic phospholipids include DOTAP (l,2-dioleoyl-3-trimethylammonium- propane), DC-cholesterol (3P-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol), DODMA (l,2-dioleyloxy-3-dimethylaminopropane), DOTMA (N-[l-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride), DDAB (Dimethyldioctadecylammonium bromide), Ethyl PC (Phosphatidylcholine with an ethyl group), and the like.

[0077] Zwitterionic phospholipids are those that contain both a positive and a negative charge within their head group, making them electrically neutral overall, and include Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Sphingomyelin, and the like.

[0078] Cholesterol and / or cholesterol derivatives may be used in the composition of the present disclosure. When unesterified cholesterol is used in the composition, the cholesterol is usually used in an amount equivalent to about 10% of the amount of phospholipid. If a compound other than cholesterol is used to stabilize the composition, one skilled in the art can readily determine the amount needed in the composition. Cholesterol derivatives suitable for use in the present disclosure include cholesterol P-D-glucoside, cholesterol 3-sulfate sodium salt, positively charged cholesterol such as DC-cholesterol and other cholesterol like molecules such as Campesterol, Ergosterol, Betulin, Lupeol, P-Sitosterol, a,P-Amyrin and bile acids.

[0079] In some embodiments, positively charged lipids (or cationic lipids) are used in the composition of the present disclosure. Exemplary cationic lipids suitable for use in the compositions of the present disclosure include but are not limited to, l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), dioctadecylamidoglycylspermine-4trifluoroacetic acid (DOGS), dioleyldimethylammonium chloride (DODAC), dimethyldioctadecylammonium bromide (DDAB), l,2-distearoyl-3-dimethylammonium-propane (DAP), N-(4-carboxybenzyl)-FJ ref. UTD-P0001US / Client ref. 25003N,N-dimethyl-2,3-bis(oleoyloxy)propan-l -aminium (DOB AQ), 1 ,2-dipalmitoyl-sn-glycero-3- succinate (DGS), N-palmitoyl homocysteine ammonium salt (PHC), l,2-dioleyloxy-3- dimethylaminopropane (DODMA), Dimethyldioctadecylammonium Bromide Salt (DDAB), 1,2- dilauroyl-sn-glycero-3-ethylphosphocholine chloride salt (EPC), N4-Cholesteryl-Spermine HC1 Salt (GL67), l,2-dioleoyloxypropyl-3-dimethyl-hydroxy ethylammonium bromide (DORI), N-(3- aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-l-propanammonium bromide (GAP-DLRIE), 2,3-dioleyloxy-N-[2[sperminecarboxaminino]ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), l,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and SAINT 2.

[0080] SUVs are typically produced by methods like sonication or extrusion of larger vesicles through polycarbonate filters with defined pore sizes. Their small size means they have a high surface area to volume ratio, which can affect drug loading efficiency and release kinetics.C. Coating or Cage

[0081] In certain embodiments a proteoliposome can be coated with a metal-organic framework (MOF) or similar material. A MOF is a class of compounds consisting of metal ions or clusters coordinated to organic ligands to form one-, two-, or three-dimensional structures. These structures often exhibit high porosity and can be designed to have specific pore sizes, shapes, and chemical functionalities. A particular MOF can be a zeolitic imidazolate framework- 8 (ZIF-8) that can be used to coat proteoliposomes described herein. ZIF-8 is a type of MOF known for its zeolite-like properties due to its structure, which consists of tetrahedrally coordinated transition metal ions connected by imidazolate linkers. ZIF-8 primarily consists of zinc ions (Zn2+) linked by 2-methylimidazolate (2-mim) ligands, forming a porous, crystalline structure. ZIF-8 is recognized for its high thermal and chemical stability, large surface area, and tunable pore sizes, making it suitable for applications like gas storage, separation, and drug delivery. Its structure allows for modifications, where functional groups can be attached to the ligands to alter properties or interactions. ZIF-8 can be used to encapsulate or coat particles or surfaces, providing stability, controlled release mechanisms (like in drug delivery), or enhancing material properties such as thermostability or catalytic activity.

[0082] Zeolitic Imidazolate Frameworks (ZIFs) are a subclass of Metal-Organic Frameworks (MOFs) known for their zeolite-like structures and remarkable thermal and chemical stability.FJ ref. UTD-P0001US / Client ref. 25003Members of the ZIF family include ZTF-8 (composed of zinc ions (Zn2+) connected by 2- methylimidazolate linkers); ZIF-67 (similar to ZIF-8 but uses cobalt ions (Co2+) instead of zinc); ZIF-7 (contains zinc ions and benzimidazolate linkers); ZIF-90 (uses zinc ions with imidazolate- 2-carboxyaldehyde linkers); ZIF-20 (made with zinc ions and imidazolate linkers); ZIF-11 (zinc- based, with 4,5-dichloroimidazolate linkers); ZIF-4 (zinc-based ZIF, with 2-imidazolate linkers); ZIF-68 (uses zinc with a combination of imidazolate and other functionalized imidazolate linkers); ZIF-93 (includes indium ions (In3+) and imidazolate linkers); ZIF-95 (indium-based, with different linker configurations).

[0083] Each member of the ZIF family can be tailored for specific applications by changing the metal ions or the functional groups on the imidazolate linkers. Substitutes for ZIF-8 coating include, but are not limited to (i) other MOFs such as ZIF-L, ZIF-67, ZIF-7, ZIF-90, ZIF-20, ZIF-11, ZIF-4, ZIF-68, ZIF-93, ZIF-95, MAF-7, UiO-66 (composed of zirconium and terephthalic acid) or MIL-101 (with chromium or iron metal centers) which offer similar benefits but might differ in stability or pore size, (ii) polymer-based coatings for applications where flexibility or specific mechanical properties are needed, polymers like polylactic acid (PLA) or polyethylene glycol (PEG) coatings can provide controlled release or biocompatibility, though they might lack the porosity or specific chemical interactions of ZIF-8, (iii) inorganic alternatives include silica or zeolite coatings, (iv) functionalized carbon materials, (v) hybrid materials that combine MOFs with polymers or other inorganic materials to create hybrid coatings that leverage the benefits of both.II. Vaccine Formulation and Administration

[0084] In light of the current specification, the determination of an appropriate treatment regimen (e.g., dosage, frequency of administration, systemic vs. local, etc.) is within the skill of the art. For administration, the components described herein will be formulated in a unit dosage form (solution, suspension, emulsion, etc.) in association with a pharmaceutically acceptable carrier. Such vehicles are usually nontoxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and Hank's solution. A preferred vehicle is 5% (w / w) human albumin in saline. The vehicle may contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.FJ ref. UTD-P0001US / Client ref. 25003

[0085] The compositions described herein, as well as their biological equivalents, can be administered independently or in combination by any suitable route. Examples of parenteral administration include intravenous, intraarterial, intramuscular, intraperitoneal, and the like. The routes of administration described herein are merely an example and in no way limiting.

[0086] The dose of the therapeutic compositions administered to an animal, particularly in a human, in accordance with embodiments of the invention, should be sufficient to result in a desired response in the subject over a reasonable time frame. It is known that the dosage of therapeutic compositions depends upon a variety of factors, including the efficacy of the composition employed, the age, species, condition or disease state, and the body weight of the subject.

[0087] Moreover, dose and dosage regimen will depend mainly on the type of biological damage to the host, the type of subject, the history of the subject, and the type of composition being administered. The size of the dose will be determined by the route, timing and frequency of administration as well as the existence, nature and extent of any adverse side effects that might accompany the administration of a particular therapeutic composition and the desired physiological effect. It is also known that various conditions or disease states, in particular, chronic conditions or disease states, may require prolonged treatment involving multiple administrations.

[0088] Therefore, the amount of the therapeutic composition must be effective to achieve an enhanced therapeutic index. If multiple doses are employed, the frequency of administration will depend, for example, on the type of subject. One skilled in the art can ascertain upon routine experimentation the appropriate route and frequency of administration in a given subject that are most effective in any particular case. Suitable doses and dosage regimens can be determined by conventionally known range-finding techniques. Generally, treatment is initiated with smaller dosages, which are less than the optimal dose of the compound. Thereafter, the dosage is increased by small increments until the optimal effect under the circumstances is obtained.

[0089] The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optional adjuvants and other therapeutic ingredients.FJ ref. UTD-P0001US / Client ref. 25003

[0090] For use in therapy, an effective amount of the vaccine composition as described herein can be administered to a subject by any mode that delivers the vaccine to the desired location or surface. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to oral, parenteral, intramuscular, intranasal, intradermal, sublingual, intratracheal, inhalation, ocular, vaginal, and rectal. Preferred routes are by injection or by inhalation.

[0091] For oral administration, the vaccine composition can be formulated readily by combining the vaccine with pharmaceutically acceptable carriers well known in the art. Such carriers enable the vaccine of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.

[0092] For administration by inhalation, the vaccine according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the vaccine and a suitable powder base such as lactose or starch.

[0093] Nasal delivery of a vaccine composition of the present invention is also contemplated. Nasal delivery allows the passage of a vaccine to the nasal mucosa. Intranasal drug delivery can offer several advantages including rapid absorption and bypassing the gastrointestinal tract. Various methods and technologies can be used for intranasal delivery, including, but not limited to nasal sprays, pump sprays, metered dose sprays, nasal drops, nasal inhalers, dry powder inhalers (DPIs), etc. Formulations for nasal delivery include those with dextran or cyclodextran. For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the vaccine composition of the present invention solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when aFJ ref. UTD-P0001US / Client ref. 25003 liquid in the chamber is compressed. The chamber is compressed to administer the composition of the present invention.

[0094] Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation.

[0095] The vaccine, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain agents such as suspending, stabilizing and / or dispersing agents.

[0096] Pharmaceutical formulations for parenteral administration include aqueous solutions of the vaccine in water-soluble form. Additionally, suspensions may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0097] Suitable buffering agents include: acetic acid and a salt (1-2% w / v); citric acid and a salt (1-3% w / v); boric acid and a salt (0.5-2.5% w / v); and phosphoric acid and a salt (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02% w / v).

[0098] The compositions for use in embodiments of the invention generally include carriers. These carriers may be any of those conventionally used and are limited only by the route of administration and chemical and physical considerations, such as solubility and reactivity with the immunizing agent(s). In addition, the composition may be formulated as polymeric compositions, inclusion complexes, such as cyclodextrin inclusion complexes, liposomes, microspheres, microcapsules, and the like, without limitation.

[0099] The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers, or diluents, are well known and readily available. It is preferred that theFJ ref. UTD-P0001US / Client ref. 25003 pharmaceutically acceptable carrier be one which is chemically inert with respect to the composition and one that has no detrimental side effects or toxicity under the conditions of use.

[0100] The choice of excipient will be determined, in part, by the composition, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the composition(s) used in the embodiments of the invention.

[0101] Vaccine adjuvants are substances added to vaccines to enhance the body's immune response to the antigen, which is the part of the vaccine that specifically provokes an immune response. Adjuvants help to make vaccines more effective by boosting the immune response, which can lead to longer-lasting immunity; reducing the amount of antigen needed, which can be crucial for vaccines where the antigen is scarce or expensive to produce; or improving the effectiveness, especially in populations where the immune system might be less responsive, like the elderly or infants.

[0102] Types of adjuvants include: (i) Aluminum Salts (Alum) which is one of the most widely used adjuvants, known for its safety and efficacy. It primarily induces a humoral (antibody-mediated) immune response, (ii) Oil-Based Emulsions such as MF59 and AS03, which are often used in flu vaccines to enhance immune responses, particularly in older adults, (iii) Saponins, such as QS-21, which stimulates both humoral and cellular immune responses, (iv) Bacterial derivatives like monophosphoryl lipid A (MPL), derived from bacteria, which can stimulate Toll-like receptors and enhance cellular immunity, (v) Virosomes which are virus-like particles that can carry antigens and mimic natural infection, improving immune recognition, (vi) Nucleic acid adjuvants such as CpG oligodeoxynucleotides, which stimulate Toll-like receptor 9, enhancing the Thl type of immune response. CpG Oligodeoxynucleotides (ODNs) are synthetic oligodeoxynucleotides containing unmethylated CpG motifs, which mimic bacterial DNA and are potent stimulators of TLR9. Examples include CpG ODN 2395, CpG 7909 (also known as ProMune) and CpG 1018 (used in hepatitis B vaccine HEPLISAV-B).

[0103] In certain aspects, bacterial lipids can be used as adjuvants, such as Monophosphoryl Lipid A (MPL), Lipid A, Trehalose Dibehenate (TDB), Mycobacterial Cell Wall Components, Lipoteichoic Acid (LTA), Ornithine-containing Lipids, Lipid A Analogues, Lipopolysaccharide (LPS) from Photosynthetic Bacteria, and the like. These lipids and their derivatives are utilized in various vaccine formulations to enhance the immune response by acting as pattern recognitionFJ ref. UTD-P0001US / Client ref. 25003 receptor agonists, particularly through Toll-like receptors, thereby promoting a more robust and appropriate immune reaction to the vaccine antigen.III. Examples

[0104] The following examples as well as the figures are included to demonstrate embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMPLE 1TESTING THE ANTIGENIC POTENTIAL OF NOVEL TRANSMEMBRANE PROTEINS TO DEVELOP A THERMOSTABLE TUBE CULOSIS MOF-LIPOSOMAL VACCINE

[0105] Described here is the immunogenic profile of a new multi-antigen proteoliposome formulation comprised of CtpV and MctB. It is shown that the approach generates robust immunological responses. Further, CtpV was studied by observing the antibody response in plasma of TB patients and compared to commonly studied antigen mixes such as purified protein derivative (PPD) and the Mtb virulence proteins ESAT-6 and CFP-10. Both antigens were studied in a mouse model; mice were vaccinated with CtpV, MctB, and the combination proteoliposome (Combo) - all benchmarked against BCG-vaccinated mice - to assess their T-cell activation profile as well as cytokine and antibody response. To enhance the immunogenicity of the antigens, the proteoliposomes were delivered with class A CpG as an adjuvant that has shown much promise in clinical studies and is approved for human use.13,14Intranasal delivery was selected as the route of administration to reduce the use of needles and improve patient compliance. Additionally, recent publications have shown how intranasal administration of a vaccine formulation can be more effective in lowering bacterial burden in mice when compared to the routine subcutaneous or intramuscular mode of administration.15

[0106] There are potential challenges associated with translating proteoliposome-based vaccines to clinical use. These biomaterials are delicate, prone to degradation and aggregation,FJ ref. UTD-P0001US / Client ref. 25003 and require strict cold chain shipping using ultra-low temperature freezers, similar to the process for delivering mRNA-based COVID vaccines. To address these challenges, the proteoliposomes are coated with ZIF-8, a biofriendly metal organic framework, to improve thermal stability of the formulation. The ZIF-8 coating degrades when exposed to mucosal surfaces without causing bioaccumulation.16 17ZIF-8 has been reported to protect biomaterials like proteins,18 19liposomes,20,21and nucleic acids19,22from higher temperatures and denaturing agents. Subsequent in vivo studies were carried out to ascertain that encapsulation of the biomaterials in ZIF-8 does not compromise the antigens’ immunogenicity (FIG. 1).A. Results and Discussion

[0107] Purification and reconstitution of proteins. Protocols are developed for the recombinant expression of CtpV and MctB in E. coli BL21(DE3)-Gold cells using codon- optimized constructs in pET-52(+) vectors encoding for the sequence of each protein which included a C-terminus Hisio-tag for affinity chromatography purification. The solubilization of each protein was maximized using the biocompatible detergent n-tetradecyl-phosphocholine (Fos-choline-14), which forms stable micellar-protein complexes with the protein targets. Protein-detergent micelles were purified by immobilized metal affinity chromatography (IMAC), followed by size exclusion chromatography (SEC). The chromatogram for purified CtpV (FIG. 2A) and MctB (FIG. 2B) revealed monodisperse peaks, yielding 6 and 8 mg / L of culture media, respectively. Protein purity (95-99%) was determined by sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (FIG. 2C).

[0108] To create a native-like environment for the proteins to remain stable in the vaccine formulation, a methodology was established to reconstitute CtpV and MctB - both individually and concurrently - into artificial lipid bilayers in the form of proteoliposomes. Small unilamellar vesicles (SUVs) were generated by extrusion and subsequently destabilized by detergent addition. A concentrated protein solution was added to the destabilized SUVs (1 :25 protein-to- lipid ratio), followed by detergent removal by Bio-Beads resin and separation of the proteoliposomes by ultracentrifugation. Characterization of the obtained proteoliposomes was performed by dynamic light scattering (FIG. 2D). CtpV proteoliposomes were determined to be 212 ± 80 nm in diameter (PDI: 0.133), MctB proteoliposomes were 202 ± 73 nm (PDI: 0.125), and Combo proteoliposomes were 155 ± 80 nm (PDI: 0.133). Liposomal incorporation andFJ ref. UTD-P0001US / Client ref. 25003 reconstitution efficiencies for both target proteins were determined to be >90% by SDS-PAGE gel band densitometry analysis of the supernatant and proteoliposome pellet (FIG. 2E).

[0109] From these data, it has been determined that multiple mycobacterial metal transporters have been successfully reconstituted concomitantly in proteoliposomes for the first time. Subsequent sections, describe the experiments carried out using the aforementioned proteoliposomes for in vivo profiling.

[0110] Response against CtpV in plasma from TB patients. The reconstituted CtpV was used to assess if immune responses were detectable in TB patients. Owing to a limited supply of plasma from patients, similar analyses could not be carried out for MctB. A customized multiplex assay was used to determine levels of antigen-specific IgM and IgG. Across samples from patients diagnosed with latent or active TB, CtpV-reactive IgG (FIG. 3) and IgM were detected, as were antibodies reactive to the controls: a mixture of Mtb proteins (PPD) (FIG. 3B) and a mix of highly / Wi-specific ESAT6 and CFP10 (FIG. 3C). These results are interesting as CtpV is present on the inner membrane and is not a surface-exposed antigen, and the fact that patients exposed to Mtb have existing antibodies against this antigen suggests that transmembrane proteins are exploitable antigens in vaccine formulation. It is contemplated that MctB will produce a similar - if not more robust - antibody response when tested against patient plasma. It is also interesting to note that if an individual patient has a high titer for one antigen, it does not necessarily correlate to have higher titers for other antigens. Such an observation further elucidates the need for including a variety of antigens - possibly even a mix of soluble and membrane proteins - to provide a robust induction of humoral immunity.

[0111] Immunogenicity of antigens benchmarked against BCG in a mouse model. A breadth of immune responses could potentially be leveraged by vaccines to protect against Mtb. It is well established that the immune response to Mtb is cell-mediated.23Thl cells promote cell-mediated immune responses and are required for the host’s defenses against intracellular infections.24An in vivo study was set up in C57BL / 6 mice (female, 4-6 weeks of age, n = 5-10 for each group) to observe if the selected protein antigens, individually or in combination, can provide a Thl-biased response along with elevated antibody titers. Class A CpG - an adjuvant known to provide a Thl-biased response - was delivered along with these proteoliposomes intranasally.25The vaccines were administered in triplicate, at one-week intervals. Negative and positive controls for benchmarking were single-dose saline and single-dose BCG-vaccinated mice, respectively.FJ ref. UTD-P0001US / Client ref. 25003CpG was not added externally to the BCG group as the bacteria carries its own CpG, which can stimulate cells. Serum was collected submandibularly on days 7, 14, 21, and 28 after the last vaccine dose from each group, after which the mice were sacrificed (FIG. 4A).

[0112] The serum across four weeks was used to quantify and compare TNF-a (FIG. 4B), IL-2 (FIG. 4C), IFN-y (FIG. 4D), and IL-17A levels (FIG. 4E) at baseline. IFN-y and TNF-a are important Th-1 cytokines that synergize to activate the microbicidal mechanism of macrophages. TNF-a attracts infiltrating macrophages and lymphocytes to the site of infection,26and we observe a significant increase in serum TNF-a levels in all proteoliposome groups, the highest being of the Combo group, whose levels were on par with that of the BCG-vaccinated group. This trend is also observed with the IFN-y data; remarkably, the Combo groups outperform BCG across all four weeks. This is an exciting result as IFN-y activates macrophage phagocytosis to kill intracellular bacteria,26and there are reports of a correlation between low IFN-y response to latency antigens and decreased protection against pulmonary TB in adults27IL-2 is also a Thl- type cytokine that promotes the proliferation and maturation of T-cells.28Elevated levels of IL-2 have also been reported to maintain cellular immunity and granuloma formation.29The initial advantage of using Combo is observed on day 21, with serum IL-2 levels in the group being on par with BCG. Still, the BCG group’s elevated IL-2 levels persist longer than the Combo. For IL-17A, although elevated significantly from baseline, serum cytokine levels of the proteoliposome groups did not match up to that of BCG, possibly indicating that these antigens may not mount a robust enough Th 17 biased response, which has been known to improve vaccine efficacy alongside a Thl biased response synergistically.30

[0113] Although not as extensively investigated as a cell-mediated response, it has been hypothesized that protective antibodies could play a role in preventing the initial acquisition of infection with Mtb23Some reports suggest opsonization as one of the mechanisms of protection; in the presence of antibodies, innate immune cells like macrophages and neutrophils show increased internalization and killing of Mtb.31,32Since the cytokine response in all study groups was the highest on day 28, the serum from that day was used to evaluate bulk levels of total IgG (FIG. 4F), IgGl (FIG. 4G), and IgG2a (FIG. 4H) titers. Although the variation of total IgG and IgGl across groups is statistically insignificant (except when compared to saline), the increase of IgG2a levels in the Combo group skew the IgG2a / IgGl ratio. This value determines the Thl / Th2FJ ref. UTD-P0001US / Client ref. 25003(FIG. 41), confirming our other findings indicating Thl -biasing of the immune response for the Combo group surpassing that of BCG.

[0114] The general consensus in the field points to T cells playing the primary role in protective immunity against Mlh. especially CD4+ T cells. A report by Singh et al. suggests that CD44+ T cells play a role in defending against TB by activating and migrating lymphocytes to the site of infection.33To quantify the cellular response from these vaccinations, the mice were sacrificed on day 42 (four weeks after the last vaccine dose). The spleen (FIG. 5A-5B), lymph nodes (FIG. 5C-5D), and lungs (FIG. 5E-5F) from the sacrificed mice were studied for their effector CD4+ (left column) and CD8+ (right column) T-cell populations using flow cytometry. Across all organs and both cell types, there was a statistically significant enhancement in T-cell activation for groups vaccinated with CtpV, MctB observed, or a combination of both. The Combo and BCG groups were observed to have comparable CD4+ and CD8+ T cell activation in the spleens and lymph nodes, but Combo’s T cell activation in the lungs was slightly higher than that of BCG. Considering that these proteins have been purified and studied in vivo as potential vaccine antigens for the first time, these results are very promising. It is noteworthy to mention that many papers show that subunit vaccines - even with adjuvants included - are unable to outperform BCG in a mouse model.3’5,6’34'36This is even noted in the animal studies published for GamTBVac - a subunit vaccine candidate currently in Phase 3 clinical trials.37

[0115] Protecting fragile proteoliposomes in a thermally stable MOF coating to preserve their immunogenicity. Since transmembrane proteins are found in nature in a lipid bilayer environment with aqueous systems present on either side of the protein, it is only possible to study their therapeutic potential in an artificial system that mimics a cell membrane. Hence, the target antigens require to be reconstituted into proteoliposomes (small unilamellar liposomes with proteins embedded in their lipid bilayer), as they are not soluble in aqueous media without detergents, which form micellar-protein complexes. However, it is well-known that lipid-based vaccine formulations are fragile, prone to aggregation, and can only be stored and shipped through the expensive cold chain. Lipid nanoparticles - another lipid-based nanoparticle made of phospholipid building blocks - have famously been utilized to develop the Pfizer and Modema vaccines for COVID-19. As a result, a significant fraction of COVID vaccine doses went to waste because the thawed vaccines cannot be refrozen.FJ ref. UTD-P0001US / Client ref. 25003

[0116] Using metal-organic frameworks (MOFs) - specifically ZIF-8 - has been demonstrated to grow a protective shell around proteoliposomes to protect them from thermal and transportation stressors. Herbert et al. demonstrated the difference in thermal stability between pristine and ZIF-8-encapsulated proteoliposomes containing the transmembrane Cu(I) P-type ATPase pump CopA (a homolog of CtpV), where the ZIF-8 coating protected CopA’s enzymatic activity to >80% at RT and >60% at 55°C. ZIF-8-encapsulated proteoliposomes where shipped across the United States and subsequently left at room temperature (RT) for two months, only to find no change in size and catalytic activity.21This allows the utilization of these novel antigens in an easily accessible, shelf-stable formulation. Moreover, an extensive biocompatibility study has previously been published that describes administering up to 1 mg ZIF-8 intranasally, which had no side effects on mice.38

[0117] Using previously published conditions for liposome encapsulation,20the combo proteoliposome (chosen due to the best performance in the previous experiment) in ZIF-8 (C@Z) were encapsulated (FIG. 6A). Although previous work established suitable encapsulation conditions, it did not optimize the synthesis to maximize encapsulation in minimal MOF. This is rectified and screened for the maximum amount of lipid that can be loaded onto ZIF-8. The C@Z formulation was characterized using powder x-ray diffraction (PXRD) to ascertain its sodalite topology (FIG. 6B). The formulation was also observed using scanning electron microscopy (SEM) to ensure the presence of nano-sized particles (FIG. 6C).

[0118] The same in vivo experiments were conducted as discussed in FIGs 4 and 5, but this time with C@Z; the data was benchmarked against Combo and BCG to observe whether encapsulation in ZIF-8 compromises the immunogenicity of these delicate protein antigens. Cytokine levels in serum were compared between the three groups (FIG. 7A-7D), along with total IgG, IgGl, and IgG2a (FIG. 7E-7G). The differences in performance of C@Z for all cytokines across weeks were statistically insignificant. The same trend was observed for all antibody titers. The Thl / Th2 ratio of the C@Z group was calculated to be 1.84, which is greater than the ratio for BCG (FIG. 41). The effector T-cell subpopulations were quantified for activated CD4+ (column 3) and activated CD8a+ (column 4) cells in the spleens (FIG. 7H and 7K), lymph nodes (FIG. 71 and 7L), and lungs (FIG. 7J and 7M). While the group’s activated CD4+ cell counts for C@Z are equivalent to those of Combo and BCG, we observe a slight reduction in CD8+ activation levels for the C@Z group.FJ ref. UTD-P0001US / Client ref. 25003

[0119] Two novel transmembrane proteins expressed on the surface of M. tuberculosis have been characterized for their potential as antigens for vaccine development - a rather sparsely investigated area of research. It is contemplated that a protein that presents itself on the surface of a bacterium is highly likely to be recognized by the immune system as a PAMP and the results support this hypothesis.

[0120] The compositions described herein provide an alternative to soluble and secreted antigensB. Materials and Methods

[0121] Chemicals and Biologicals. Synthetic DNA plasmid constructs were purchased from Genscript Inc. E. coli BL21 (DE3) GOLD competent cells were purchased from Agilent Technologies. Zinc acetate dihydrate, 2-methylimidazole, tween-20, sodium hydroxide, glycerol, P-mercaptoethanol (molecular biology grade), deoxyribonuclease I from bovine pancreas and bovine serum albumin (BSA) were purchased from Millipore Sigma. RBC lysis buffer, Zombie UV fixable viability kit, cell staining buffer, PE / cyanine7 anti-mouse CD4 antibody, PE antimouse CD8 antibody, Alexa Fluor 594 anti-mouse CD3 antibody, Alexa Fluor 700 anti-mouse CD44 antibody, Pacific Blue anti-mouse CD62L antibody, ELISA max standard set mouse TNF- a, ELISA max standard set mouse IL-2, ELISA max standard set mouse IFN y, ELISA max standard set mouse IL- 17, purified goat anti -mouse IgG (minimal x-reactivity), purified antimouse IgGl, purified anti-mouse IgG2a, biotin goat anti-mouse IgG (minimal x-reactivity), biotin anti-mouse IgGl, biotin anti-mouse IgG2a, and TMB substrate set were purchased from Biolegend. E. coli polar lipids and L- a-phosphatidylcholine were purchased from Avanti Polar Lipids. Terrific broth media, ampicillin sodium salt, isopropyl thiogalactopyranoside, magnesium chloride hexahydrate, imidazole, dithiothreitol, EDTA-free protease inhibitor cocktail tablets, MOPS 3-(4-morpholino) propane sulfonic acid, tris(2-carboxyethyl) phosphine (TCEP), sodium chloride, sodium pyruvate, chloroform, nitric acid (trace metal grade), hydrochloric acid, oleic acid, and glacial acetic acid were purchased from Thermo Fisher Scientific. n-Tetradecyl-phosphocholine (Fos-choline-14) and n-dodecyl-B-D-maltoside (DDM) were purchased from Anatrace. Middlebrook 7H10 agar base and Middlebrook 7H9 broth base were purchased from Remel. Catalase, Bovine was purchased from MP Biomedicals. Anhydrous D-(+)-glucose was purchased from Alfa Aesar. Polycarbonate membranes, HyClone phosphateFJ ref. UTD-P0001US / Client ref. 25003 buffered saline solution (PBS), HyClone RPMI 1640 medium were purchased from Cytiva. Chel ex-resin, Bio-Beads and 4-15% Tris-Glycine Mini -PROTEAN gels were purchased from Bio Rad. FB Essence was purchased from Avantor. Isoflurane solution was purchased from Covetrus.

[0122] Instruments. Ultrapure water was fdtered in lab with the ELGA PURELAB flex 2 system. Bacterial cells were harvested, and cell debris was separated using Themo Scientific Sorvall LYNX 6000 centrifuge. Bacterial cells were lysed using M110P Microfluidizer Processor. Membranes were isolated using Ultracentrifuge Beckman Optima XPN80. Chromatographic purifications were carried out in AKTA Pure FPLC system, using HisTrap FF 5mL, HiPrep 26 / 10 desalting, Superdex 200 10 / 300, and HiLoad 16-600 Superdex 200pg columns from Cytiva. Protein solutions were concentrated in Thermo scientific Sorvall ST8 centrifuge using 100,000 MWCO and 50,000 MWCO concentrators from Sartori ous VIVASPIN 20. Absorbance at 280 nm of the protein solutions was measured using ThermoScientific NanoDrop One. Liposomes were extruded using Avanti Mini Extruder. DLS measurements were carried out using Malvern Analytical Zetasizer Nano ZS. PXRD diffractograms were obtained from Rigaku SmartLab X-ray diffractometer. SEM micrographs were taken on Zeiss Supra 40. Absorbance spectra were obtained on Biotek Synergy H4 Hybrid microplate reader. Proteoliposomes were pelleted using Sorvall MX- 120 micro-ultracentrifuge. ZIF-encapsulated materials, cells, and BCG cultures were pelleted down using Beckman Coulter Allegra X-14R and Sorvall Legend Micro 17 centrifuges. BCG culture plates were grown in Thermo Scientific Heratherm incubator. Overnight precultures were growth in Thermo Scientific Shaker MAXQ 4450. Large scale bacterial cultures were grown in Infers HT multitron standard incubator. Cell counting was carried out on a Thermo Countess II. Flow cytometry data were acquired on a BD LSRFortessa.

[0123] BCG Culturing. To make agar plates, Middlebrook 7H10 agar (19 g), glycerol (5 mL), sodium pyruvate (4.4 g), and DI water (900 mL) were autoclaved. Once cooled to 60 °C, enrichment containing oleic acid (50 mg), BSA (5 g), dextrose (2 g), beef catalase (4 mg), sodium chloride (850 mg), and DI water (100 mL) was added to the agar via syringe-filter, and the solution was poured onto plates. To make media, Middlebrook 7H9 broth (4.7 g), glycerol (2 mL), sodium pyruvate (4.4 g), and DI water (900 mL) were autoclaved. Once cooled to 60°C, enrichment containing BSA (5 g), dextrose (2 g), beef catalase (3 mg), and DI water (100 mL)FJ ref. UTD-P0001US / Client ref. 25003 was added to the media via a syringe filter. Bacterial stocks received from the Lu Lab were used as is. Agar plates were streaked using the stock and stored in the incubator at 37 °C for a few days until single colonies appeared. 3-4 single colonies were transferred to a T75 flask (with a filter cap to allow airflow) containing 10 mL media and stored in the incubator at 37 °C in static conditions. Every 4 d, absorbance at 600 nm was recorded till optical density reached a range of 0.4-0.7. 1 mL of bacteria culture was transferred to a 1.5 mL and pelleted down by centrifuging at 5000 xg for 10 mins at RT. 1 mL of ODooo = 1.0 bacterial culture contains 5 * 108CFU / mL. This equation was used to determine how much water the bacterial pellet should be resuspended in to obtain a 2.5 x 107CFU / mL stock.

[0124] Animals and ethics. Female C57BL / 6 mice (4-6 weeks old) were purchased from Charles Rivers Laboratories. All in vivo experiments were carried out under protocol #19-06, which was approved by the University of Texas at Dallas Institutional Animal Care and Use Committee (IACUC).

[0125] Expression and purification of CtpV. Synthetic DNA encoding for CtpV (Uniprot accession number: P9WPS3) was cloned into the pET-52b (+) vector, featuring a C-terminal Hisio-tag and a N-terminal STREP -tag II. The expression plasmid was transformed into E. coli BL21 (DE3) GOLD competent cells. Overnight preculture was cultivated in Terrific Broth (TB) media supplemented with 50 pg / mL ampicillin and 1% glycerol (v / v). Cells were inoculated in fresh TB media and grown at 37°C with agitation. Upon reaching OD6oo= 2, the cells were induced for protein expression by adding isopropyl thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, followed by incubation at 37°C with agitation for 4 hours.

[0126] Cells were harvested through centrifugation (4°C, 14,000 x g, 20 min) and subsequently resuspended in lysis buffer (20 mM Tris / HCl pH 8, 5mM MgCL, 150 mM NaCl, 30 pg / mL deoxyribonuclease I from bovine pancreas, and EDTA-free protease inhibitor cocktail tablet). Cell lysis was performed using an ice-cold microfluidizer at 20000 PSI by circulating the cell suspension through a Z-shaped diamond chamber three times. The cell lysate was centrifuged to remove cell debris (20 min, 4°C, 20,000 x g). Membranes were isolated via ultracentrifugation (1 h, 4°C, 205,100 x g), followed by resuspension in buffer (20 mM Tris / HCl, pH 8, 500 mM NaCl, EDTA-free protease inhibitor cocktail). Membrane suspension was aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C until protein purification.FJ ref. UTD-P0001US / Client ref. 25003

[0127] Protein extraction was performed by mixing the membrane suspension (8 mL) with 35 mL of extraction buffer (20 mM Tris / HCl, pH 8, 500 mM NaCl, 25 mM imidazole, 1 mM dithiothreitol (DTT), 1% (w / v) n-Tetradecyl-phosphocholine (Fos-choline-14) and EDTA-free protease inhibitor cocktail). The suspension was stirred at 4°C for 2 h, followed by ultracentrifugation to remove residual membranes and precipitates (1 h, 4°C, 205,100 x g). The supernatant containing detergent-solubilized CtpV was loaded onto 2x HisTrap FF 5 mL columns connected in series. The columns were pre-equilibrated in wash buffer (20 mM Tris / HCl, pH 8, 500 mM NaCl, 35 mM imidazole, 1 mM DTT, 0.05% (w / v) Fos-choline-14). The columns were washed with 40 CV buffer and subjected to a gradient elution of 0 to 100% elution buffer (20 mM Tris / HCl, pH 8, 500 mM NaCl, 500 mM imidazole, 1 mM DTT, 0.05% (w / v) Fos-choline-14) over 12 CV. Protein elution occurred at 6CV (50% elution buffer) within the elution gradient.

[0128] Subsequent to elution, buffer exchange was performed using a HiPrep 26 / 10 desalting column to desalting buffer (20 mM MOPS / NaOH, pH 7, 500 mM NaCl, 1 mM DTT, 0.05% (w / v) Fos-choline-14). The protein solution was concentrated using a 100,000 MWCO concentrator by centrifugation (4°C, 2100 ' gl to a final protein concentration of 2.0-3.0 mg / mL. Aggregated protein and impurities were removed by size exclusion chromatography on a Superdex 200 10 / 300 column using a desalting buffer. Protein purity was confirmed by SDS- PAGE (4-15% Tris-Glycine Mini-PROTEAN gel), and concentration was quantified by measuring Abs2so (£=65400 M"1cm-1).

[0129] Reconstitution of CtpV in proteoliposome small unilamellar vesicles (SUVs)^ Chloroform solutions of E. coli polar lipids (25 mg / mL) and L- a-phosphatidylcholine (25 mg / mL), mixed in 3: 1 ratio (w / w), were dried under nitrogen flow to obtain a thin lipid film. Lipids were dried under a vacuum overnight to remove chloroform completely. The dried lipid film was hydrated with a ImM DTT solution in MiliQ water. A concentrated Chel ex-treated buffer stock was added to the lipid suspension to a final lipid concentration of 25 mg / mL of lipids, 20 mM MOPS / NaOH, pH 7, 50 mM NaCl, and 1 mM DTT. After three freeze-thaw cycles in liquid nitrogen, small unilamellar vesicles (SUVs) were obtained through 11 extrusions utilizing polycarbonate membranes (sequentially decreasing pore size of 1 pm, 400 nm, and 200 nm) and a 1 mL gas-tight-syringe system. A Fos-choline-14 stock solution was added to theFJ ref. UTD-P0001US / Client ref. 25003SUVs to a final concentration of 0.02% (w / v), and the mixture was tilted for 1 hour at room temperature to destabilize the vesicles.

[0130] A CtpV solution concentrated to ~ 2.0 mg / mL was added to the detergent- destabilized SUVs solution to a final ratio of proteimlipid equal to 1 :25 (w / w), followed by tilting of the mixture for 1 hour at 4°C. Fos-choline-14 was removed from the SUVs with the addition of Bio-Beads (previously activated by washing with methanol, ethanol, and water consecutively, and overnight vacuum drying) to a final concentration of 60 mg / mL. The BioBeads slurry in the proteoliposomes suspension was continuously tilted at 4°C, and the BioBeads resin was exchanged after 1, 12, 14, and 16 hours.

[0131] Proteoliposomes were separated by ultracentrifugation (4°C, 45 min, 160,000 x g). Pelleted SUVs were collected and resuspended in transport buffer (20 mM MOPS / NaOH, pH 7, 50 mM NaCl, 1 mM DTT) to a final lipid concentration of 25 mg / mL and CtpV concentration of 1 mg / mL. Protein incorporation and concentration were assessed via SDS-PAGE 4-15% Tris- Glycine Mini-PROTEAN gel band densitometric analysis using purified CtpV as a standard. The proteoliposomes were aliquoted, frozen in liquid nitrogen, and stored at -80°C.

[0132] Expression and purification of MctB. The synthetic DNA encoding for MctB (UniProt accession number: P9WJ83) was cloned into the pET-52b (+) vector, including a C- terminal Hisio-tag and an N-terminal STREP -tag II. The expression plasmid was transformed into E. coli BL21 (DE3) GOLD competent cells. Overnight preculture was grown in Terrific Broth (TB) media containing 50 pg / mL ampicillin and 1% glycerol (v / v). Cells were inoculated in fresh TB media and grown at 37°C with agitation until ODeoo= 2 was reached. Protein expression was induced by adding IPTG to a final concentration of 1 mM. Cell cultures were incubated at 30°C, under agitation for 4 hours.

[0133] Harvested cells underwent lysis, and the membrane fraction was isolated and stored as described for recombinant CtpV expression. Protein extraction followed a similar protocol to that of CtpV membranes, mixing 4 mL of the membranes with 40 mL extraction buffer, the suspension stirred at room temperature for 10 hours, followed by ultracentrifugation. The supernatant containing detergent-solubilized MctB was loaded onto a HisTrap FF 5 mL column pre-equilibrated in wash buffer (20 mM Tris / HCl, pH 8, 500 mM NaCl, 1 mM DTT, 0.05% (w / v) Fos-choline-14). The column was washed with a 20 CV buffer. A gradient of 0 to 100% elution buffer (20 mM Tris / HCl, pH 8, 500 mM NaCl, 500 mM imidazole, 1 mM DTT, 0.05%FJ ref. UTD-P0001US / Client ref. 25003(w / v) fos-choline-14) was applied over 1 CV, followed by 3CV of 100% elution buffer. Protein was eluted at 1.5 CV of the 100% elution buffer.

[0134] The protein solution was immediately injected into a HiLoad 16-600 superdex 200pg column to separate the protein from aggregates and imidazole and exchange the buffer (20 mM Tris / HCl, pH 8, 500 mM NaCl, 1 mM DTT, 0.05% (w / v) n-dodecyl-B-D-maltoside (DDM)). Protein purity was verified by SDS-PAGE (4-15% Tris-Glycine Mini-PROTEAN gels), and concentration was determined by measuring Abs280 (e=4470 cm'1).

[0135] Reconstitution of MctB in proteoliposome SUVs. Preparation of MctB proteoliposomes mirrored the method used for CtpV proteoliposomes, utilizing DDM to destabilize the SUVs and concentrating the MctB solution to ~1.5 mg / mL.

[0136] Reconstitution of CtpV and MctB concurrently into proteoliposome SUVs. To generate proteoliposomes containing simultaneously both proteins, liposomes were prepared using the previously described method. The concentrated CtpV solution was added first to the SUVs (previously destabilized with Fos-choline-14), on ice, and tilted for 1 h, followed by the addition of concentrated MctB in the same manner. Detergent removal and separation of the pelleted SUVs followed the procedure used for single protein proteoliposome reconstitutions. SVUs pellets were resuspended in transport buffer to a final concentration of 25 mg / mL lipids and 1 mg / mL of each protein. CtpV and MctB incorporation and concentration in the proteoliposomes were determined with by SDS-PAGE 4-15% Tris-Glycine Mini-PROTEAN gel band densitometry analysis.

[0137] Human IgG data methods. Adults were recruited from the Texas / Mexico border (2006-2010).39Latent TB (n = 18) was defined by a positive IFN-y release assay (IGRA) TSPOT or QuantiFERON with no history of prior TB diagnosis or treatment and no clinical signs and symptoms of active TB disease. Active TB (n = 19) was defined by sputum acid-fast bacilli smear and culture in combination with clinical signs and symptoms of disease. Endemic controls (n = 8) were defined by negative TSPOT or QuantiFERON with no history, clinical signs and symptoms, or exposure to TB. To limit confounding variables, groups for latent and active TB were matched by age and sex, tested negative for HIV and type 2 diabetes as defined by WHO criteria, and received <8 days TB treatment.40’41Written informed consent was obtained from study participants and approved by institutional IRBs.FJ ref. UTD-P0001US / Client ref. 25003

[0138] Sample collection. Blood samples were collected by venipuncture in sodium heparin tubes; plasma was isolated by centrifugation, aliquoted, stored at -80°C, and heat-inactivated (30 min, 55°C) prior to use.

[0139] Antigens. H37Rv purified protein derivative (PPD) (Statens Serum Institute), ESAT-6 (BEI), CFP-10 (BEI) were used as Mtb antigens in addition to CtpV.

[0140] Quantification of antigen-specific IgG and subclasses. Customized Luminex assays were used to measure antigen-specific IgG and subclass levels as previously described.42'44Carboxylated microspheres (Bio-Rad, MCI 00 series) were coupled to protein antigens using an NHS-ester reaction (Thermo Fisher, Cat A32269) following the manufacturer’s instructions. Serial dilutions of IgG purified from each individual patient plasma samples (1 :100, 1 : 1000, and 1 : 10000 for IgG and 1 : 100, 1 :300, and 1 :900 for IgM) were added to antigen-coupled beads (18 h, 4°C) and washed. PE-conjugated antibodies detecting total IgG (IDC-10, Southern Biotech) and IgM (SA-DA4, Southern Biotech) were added (2 h, RT), washed with PBS 0.05% Tween- 20, and re-suspended in PBS to acquire fluorescence intensity on Magpix (Luminex). The relative level of antigen-specific antibodies was defined as the area under the curve (AUC) calculated from the serial dilutions for each individual sample.

[0141] Synthesis of C@Z. IM zinc acetate and 3M 2-m ethylimidazole (HMIM) stocks were made in sterile water (filtered and autoclaved). 150 pL of the Combo proteoliposome was added to a 1.5 mb tube, followed by 50 pL of CpG stock (2 mg / mL), 567 pL of water, 213 pL of HMIM stock, 20 pL of zinc acetate stock. The zinc acetate is added last. The reaction mixture is vortexed and allowed to incubate at RT for 3 h. Formed C@Z was washed twice by centrifuging at 4000 x g for 15 mins at RT, and the supernatant was exchanged with water. After washing, C@Z was resuspended in water and brought up to 300 pL.

[0142] Vaccination, blood draws, and sacrifice. Female C57BL / 6 (n = 5) were intranasally vaccinated with saline, CtpV + CpG, MctB + CpG, Combo + CpG, C@Z, and BCG, respectively. The dosing and schedule shown in the table below were followed. The vaccine was administered intranasally by pipetting droplets into the nostrils of anesthetized mice using a 20 pL pipette and waiting between drops to ensure the mice inhaled the administered vaccine. For CtpV, MctB, and CpG, the stocks were all 2 mg / mL each. For Combo, the stock was 2 mg / mL per protein (4 mg / mL total protein concentration).FJ ref. UTD-P0001US / Client ref. 25003

[0143] Submandibular blood was collected from all mice on days 21, 28, 35, and 42. On day 42, the mice were sacrificed via CO2 asphyxiation. Lungs, spleen, and axillary and superficial cervical lymph nodes were collected and kept in PBS on ice until further processing.

[0144] Serum isolation from blood and cytokine and antibody ELISAs. Blood drawn from mice was allowed to sit for ~30 minutes at RT and then centrifuged at 1400 x g for 10 mins. The serum in the top layer was collected into separate tubes. Serum was stored at -80°C until further use. For TNF a, IL-2, IFN y, and IL-17 quantification, 5 pL of serum was used per sample (diluted to 100 pL), and the assay was performed using ELISA kits following the manufacturer’s instructions. For total IgG, TgGl, and IgG2a titer quantification, 0.5 pL of serum was used per sample (diluted to 100 pL and serially diluted from there). For all antibody titer assays, 96-well Nunc plates were coated with anti-mouse IgG / IgGl / IgG2a and sealed and stored for incubation overnight at 4°C. The next day, the plates were washed (4x with 300 pL of wash buffer - 0.1 M PBS 0.05% v / v tween-20) and subsequently blocked (200 pL of 1% BSA in wash buffer) for 1.5 h at RT. Plates were then washed, and serially diluted serum was added to the wells and incubated for 2 h at RT. After that, plates were washed again, and biotin anti-mouse IgG / IgGl / IgG2a was added to the wells and incubated for 1.5 h at RT. Post incubation, plates were washed, and then HRP-avidin was added. The plates were incubated for 30 mins at RT. After a final wash, TMB substrate was added and incubated in the dark till a color gradient of blue was observed. The reaction was then stopped using 100 pL 2N H2SO4, and the plates were read at 450 nm and 570 nm. All antibodies, HRP-avidin, and TMB substrate were diluted according to the manufacturer’s instructions.

[0145] Single-cell suspension preparation and staining for flow cytometry. Collagen digestion buffer was prepared by adding collagenase D (200 mg) and DNAse I (3000 units) to 25 mL PBS. Each extracted lung was incubated in 1 mL of digestion buffer for 45 mins at 37°C.FJ ref. UTD-P0001US / Client ref. 25003Subsequently, the lungs were removed from the buffer. The lungs, spleen, and lymph nodes were homogenized with a mortar and cell strainer (70 pm for lungs and 100 pm for spleens and lymph nodes) whilst adding 5 mL cell media (RPMI containing 10% FB Essence and 0.4% 0- mercaptoethanol) per sample. Cells were pelleted down by centrifuging them at 500 * g for 5 mins. RBC lysis buffer (3 mL) was added to each cell pellet; then, the pellets were resuspended and incubated on ice. After 10 mins, 7 mL cell media was used to neutralize the lysis buffer, the cells were pelleted down, and the supernatant was replaced with 1 mL media. At this stage, cells were counted for all samples. Three million cells of each sample were added to a 96-well plate. Zombie UV was added to the cells and incubated on ice in the dark for 30 mins. Then, the antibody cocktail containing PE Cy7 CD4, PE CD8a, AF594 CD3, AF700 CD44, and PacBlue CD62L was added to the cells and allowed to incubate in the same conditions for 60 more min. All antibodies were diluted according to the manufacturer’s instructions, which are available on BioLegend’s website. Cells were pelleted down and washed with cell staining buffer twice. Lastly, cells were resuspended in 200 pL cell staining buffer and analyzed via flow cytometry. Gating strategy used for analyzing cell populations has been included in the supplementary document.EXAMPLE 2MN AND ZN-DOPED MULTIVALENT METAL-ORGANIC FRAMEWORK AS A METALLOIMMUNOLOGICAL ADJUVANT TO PROMOTE PROTECTION AGAINST TUBERCULOSIS INFECTION

[0146] Described herein is a first-in-class vaccine adjuvant delivery system, Mn-ZIF, it was developed by incorporating manganese (Mn) into the zinc-containing zeolitic-imidazolate framework-8 (ZIF-8). The mixed metal approach, allowed for tunable Mn doping, was made possible by including a mild reducing agent into the reaction mixture. This approach allowed up to 50% Mn, with the remaining 50% Zn within the ZIF. This multivariate approach exhibited significantly decreased cytotoxicity compared to ZIF-8. The porous structure of Mn-ZIF enabled the co-delivery of the STING agonist cyclic di-adenosine monophosphate (CDA) through postsynthetic loading, forming CDA@Mn-ZlF. The composite demonstrated enhanced cellular uptake and synergistic activation of the cGAS-STING pathway, producing proinflammatory cytokines and activating antigen-presenting cells (APCs). In a preclinical Mycobacterium tuberculosis (Mtb) model, CDA@Mn-ZIF formulated with the CysVac2 fusion protein elicited aFJ ref. UTD-P0001US / Client ref. 25003 potent antigen-specific T-cell response and significantly reduced the mycobacterial burden in the lungs of infected mice. These findings highlight the potential of CDA@Mn-ZIF as a promising adjuvant for subunit vaccines, offering a novel approach to enhancing vaccine efficacy and protection against infectious diseases such as tuberculosis.A. Results and Discussion

[0147] An aqueous synthesis incorporating Mn2+and Zn2+was developed to make the reaction green and biofriendly. This method allows for scalability, more direct use in vitro and in vivo, and future application in the growing field of biomimetic encapsulation, where an expanded library of MOFs is desirable. (Kumari, Matter 2023, 6, 2570) Various synthetic conditions were tested to achieve a wide range of Mn incorporation in nano- and micrometer sizes of ZIF. Mn2+oxidizes in water to form various manganese oxides, and we realized quickly that a mild reducing agent is needed to keep the Mn2+stable as stocks and during the reaction. Three reducing agents, sodium ascorbate, sodium citrate, and tris(2-carboxyethyl)phosphine (TCEP), were tested. We found that TCEP did not interfere with the formation of the MOF while allowing for the maximum Mn loading possible and was, hence, the reducing agent of choice for future experiments. TCEP-assisted Mn-doped ZIF (Mn-ZIF) was prepared by adding DI water, TCEP, manganese(II) acetate tetrahydrate, zinc(II) acetate dihydrate, and 2-methylimidazole (HMIM) — in that order — and left to react statically at room temperature for 20 min. We used inductively coupled plasma mass spectrometry (ICP-MS) to determine the amount of Mn doped into ZIF. The percent doping was calculated based on the mole percent of Mn from the total moles of Mn and Zn in a sample (Equation El). We found that the concentration of Mn was highly tunable; 15%, 30%, and 50% were chosen as representative samples whose crystallinity was retained when examined using powder x-ray diffraction (PXRD) (FIG. 13 A). The PXRD diffractograms confirmed that the Mn-ZIF formulas and ZIF-8 synthesized with TCEP (0% Mn) were isostructural to simulated sodalite ZIF-8. The diffraction results demonstrate that TCEP does not interfere with the sodalite topology formation of ZIF-8 or Mn-ZIF. A loss in crystallinity is observed in the diffraction patterns as Mn doping is increased, and past 50%, the material became amorphous with no distinct peaks in the PXRD diffractograms. The PXRD of a representative sample that was calculated to have 70% Mn is not shown. The morphology of the various Mn-loaded samples was then assessed by scanning electron microscopy (SEM). A less common rounded cubic structure can be seen for each sample compared to the typical rhombicFJ ref. UTD-P0001US / Client ref. 25003 dodecahedron of sodalite ZIF-8. SEM further shows a size between 400-600 nm for each formulation, but the size slightly increases as more Mn is incorporated. X-ray photoelectron spectroscopy (XPS) survey and Ols spectra were collected for 50% Mn-ZIF and ZIF-8. Oxygen was detected in both samples, most likely due to three sources: surface metal oxides, metal hydroxides, and residual water with respective bond energies of 529-530 eV, 530-532 eV, and 533-534 eV. (Munoz-Gil, Nanomaterials 2019, 9, 1369; Yang, Sci. Rep. 2015, 5, 17473) A similar oxidation character was observed with Mn-ZIF having a slightly shifted peak to higher energies, suggesting more water and hydroxide bonding than oxide bonding, as seen in ZIF-8. From this, we can conclude that most Mn is incorporated into the lattice and not just trapped as metal oxides. Further, the survey spectrum for Mn-ZIF shows the signature Mn peaks along with Zn peaks seen in the ZIF-8 spectrum, supporting the mixed metal results of ICP-MS.

[0148] Before proceeding to immune activation experiments, it is important to understand the cytotoxicity of our material so we can determine the right dosage for in vitro and in vivo applications. Cell viability assays were used to determine the half-maximal inhibitory concentration (IC50) of Mn-ZIF with different doping percentages compared to control ZIF-8. Three cell lines were used: RAW 264.7, 4T1, and HEK-293. These represent various cell types and tissues: murine immune cells, murine breast cancer epithelial cells, and human kidney epithelial cells, respectively. Three viability assays were tested, including tetrazolium-based lactate dehydrogenase (LDH), MTT assay, and resazurin assay. Tetrazolium-based dyes that utilize redox-active formazan rings were incompatible with Mn as they interfered with the assay’s color development; consequently, the LDH and MTT assays produced spurious results. An example is where after lysing cells in the presence of Mn-ZIF or Mn2+salt, LDH still had a low signal, indicating living cells similar to media when they should produce a signal similar to the lysed control group. Assays using tetrazolium dyes have been previously reported to have issues measuring redox-active compounds. (Ettlinger, Chem. Soc. Rev. 2022, 51, 464; Scarcello, PLOS ONE 2020, 15, e0231634) In contrast, the resazurin-based dye appears inert to Mn. Resazurin assays are also redox-based; however, extensive control experiments were performed to confirm that Mn-ZIF did not react with the dye. Further, as a fluorescent assay, it avoids interference from the MOF’s absorbance. Across all three cell lines, viability increased as Mn substitution increased. While the difference observed in RAW 264.7 was small but not statistically significant (FIG. 14A), cell viability of 50% Mn-ZIF was considerably higher thanFJ ref. UTD-P0001US / Client ref. 25003 that of ZIF-8 in 4T1 and HEK-293 cells. 4T1 showed an 84% increase in tolerance (60.3 ± 3.1 pg / mL vs. 111.3 ± 0.8 pg / mL), and HEK-293 was even higher, with an 87% increase (81.8 ± 9.6 pg / mL vs. 153.1 ± 0.4 pg / mL) when comparing ZIF-8 to 50% Mn-ZIF (FIG. 14B-C). These results have been summarized in FIG. 14D. We hypothesize that the increased viability could be attributed to Mn and Zn being metabolized using different biological processes, and the reduced amount of each metal likely reduces the overall toxicity in some cell lines compared to a pure Zn MOF. (Jomova, Chem.-Biol. Interact. 2022, 367, 110173) The IC50 values are promising as only a small amount of Mn - in the pM range based on endogenous levels (Killilea, Free Radical Biol. Med. 2022, 182, 182) - is needed to induce cGAS-STING activation, allowing for flexible dosing with Mn-ZIF. (Zhao, Cell Rep. 2020, 32, 108053). With cytotoxicity data for Mn-ZIF, we next sought to exploit it as a delivery method for Mn2+to activate cGAS-STING. The 50% Mn-ZIF formulation was used for all future experiments because it had the highest biocompatibility of all formulations tested. Additionally, we looked to boost STING activation by also incorporating an agonist. Mn co-delivered with a CDN can work synergistically to activate STING and create a more robust immune response. (Sun, Nat. Nanotechnol. 2021, 16, 1260) As a model, we incorporated the known STING agonist CD A, a molecule belonging to the CDN family. (Cheng, Front. Microbiol. 2022, 13) An additional benefit of loading CDA onto Mn-ZIF is nanoparticle- mediated delivery, which can enhance uptake compared to just CDA - a negatively charged small molecule. First, CDA was post-synthetically loaded onto the surface of Mn-ZIF for 24 h on a rotisserie at room temperature (FIG. 15A). Owing to the previously reported higher affinity of CDA to metals in methanol over other solvents, we chose to try that approach first. (Sun, Nat. Nanotechnol. 2021, 16, 1260) As determined by UV-Vis spectroscopy, over 96% of the CDA in the solution was absorbed into the Mn-ZIF (FIG. 15B). Liquid chromatography (LC) supported the nanodrop result with greater than 94% absorption. Loading was also tested in water to eliminate the need for methanol, and we were excited to find that a comparable 93% was absorbed by Mn-ZIF, as determined by LC. The highest percent loading was observed when adding 30 pg / mL concentration of CDA to 1 mg / mL of Mn-ZIF, and when increasing CDA concentrations were used, the loading percentage decreased. We thus used the most efficient loading route for future experiments, giving us about 28 pg of CDA per one mg of Mn-ZIF. C,- potential measurements show that the surface of Mn-ZIF became more negatively charged owing to the anionic phosphodi esters of the loaded CDA (FIG. 15C). Additionally, the CDA-loadedFJ ref. UTD-P0001US / Client ref. 25003Mn-ZIF (CDA@Mn-ZIF) retained 94% of the loaded CDA after 24 h, confirming good stability and CDA retention of the composite. Cytotoxicity was performed on the CDA@Mn-ZIF in the same way as in FIG. 14 and IC50 was determined to be 61.0 ± 6.3 pg / mL, which was slightly lower than ZIF but not significantly different.

[0149] Mn-ZIF was meant to serve as an adjuvant delivery vehicle for the CDA and was also designed to protect CDA in vitro. Cells contain enzymes known as phosphodiesterases that can hydrolyze the phosphodiester bond of CDNs, converting them to linear dinucleotides that will not activate STING. An experiment was adapted from literature using snake venom phosphodiesterase (SVPD) to degrade CDA to linear phosphadenylyl-adenosine (pApA). (Zhou, Anal. Chem. 2014, 86, 2412) The change in structure generated a large shift in elution time, so the degradation could be observed by LC, as seen in the pApA and CDA traces. CDA@Mn-ZIF was treated with SVPD or buffer as a control, heated to deactivate the enzyme, and finally treated with dilute acetic acid to decompose the MOF and release the CDA into supernatant. After running on LC, traces overlapping with intact CDA could be seen for the SVPD-treated and untreated samples. This suggests the Mn-ZIF could preserve the CDA from degradation even with a surface loading process.

[0150] To demonstrate the uptake of Mn-ZIF and co-delivery of Mn and CDA, a fluorescent dye was incorporated into the pore structure via coprecipitation during synthesis before loading CDA. 5,6-carboxyfluorescein (CF) and rhodamine B (Rh) were chosen because of their high quantum yield. CF and Rh were incorporated into the crystalline structure during the Mn-ZIF reaction by conducting the MOF synthesis in an aqueous solution of concentrated CF and Rh to form fluorescent nanoparticles (Mn-ZIF(CF)) and (Mn-ZIF (Rh)). Mn-ZIF(CF) and Mn-ZIF (Rh) were then coated with CDA post-synthetically with the same method to create CDA@Mn- ZIF(CF) and CDA@Mn-ZIF(Rh) and can be seen in the green or red channels of an epifluorescence microscope respectively. Both Mn-ZIF(Rh) and CDA@Mn-ZIF(Rh) were used to conduct uptake studies on RAW 264.7 cells. Particle-cell association of CDA@Mn-ZIF(Rh) was visualized on an epifluorescence microscope at 6 h and is represented in FIG. 15D. The CDA@Mn-ZIF(Rh) signal can be seen around the nucleus in the red channel, associating with the green lysotracker channel and has a Pearson colocalization coefficient of 0.72. This suggests CDA@Mn-ZIF can effectively associate with the cell, potentially through the lysosome. Since 2- D epifluorescence can only confirm association, a quantitative uptake assessment was performedFJ ref. UTD-P0001US / Client ref. 25003 using flow cytometry on RAW 264.7 cells. Mn-ZIF was used as a non-fluorescent control to account for scattering as a potential source of background signal. Pure CF dye was used to show the uptake of a representative, negatively charged small molecule. Samples were washed with both PBS and flow cytometry buffer, which contains the chelating agent EDTA, to remove material only bound to the surface so that signal is from dye internalized in the cell and not bound to the surface. Four- and eight-hour time points were assessed for uptake by incubating cells with Mn-ZIF, CF, Mn-ZIF(CF), and CDA@Mn-ZIF(CF) at each time. A representative histogram of 8 h is shown in FIG. 15E, and the geometric mean of the CF intensity using the FITC channel is represented in FIG. 15F. Free CF’s uptake was very limited but significantly increased once encapsulated within Mn-ZIF. The enhanced CDA@Mn-ZIF(CF) uptake compared to Mn-ZIF(CF) was surprising, given that its surface charge was more negative than Mn-ZIF (CF) alone. We hypothesize that this result could be attributed to protein corona formation from the bovine serum albumin used in the cell media, which has previously been reported to enhance the uptake of negatively charged particles. (Shahabi, ACS Appl. Mater. Interfaces 2015, 7, 13821; Augustine, Mater. Today Commun. 2020, 25, 101692). Taken together, these results suggest that Mn-ZIF is a good delivery vehicle for both therapeutic metals like manganese and adjuvanting small molecules like CDA.

[0151] Following uptake studies of Mn-ZIF, we checked if APCs could be activated in vitro by delivering a combination of Mn and CDA. Activating APCs is extremely important for host defense against pathogens and cancer, and stimulation of cGAS-STING is one way to achieve activation. (Ou, Front. Immunol. 2021, 12) cGAS-STING responses can be measured in vitro by surface marker staining and by quantifying the pro-inflammatory cytokines produced by the cells. BMDCs were isolated and used as target APCs. MnCE, ZIF-8, CDA, Mn-ZIF, and CDA@Mn-ZIF were normalized to achieve 10 pg / mL Mn and 1.7 pg / mL CDA then tested on BMDCs. Successful dendritic cell (DC) activation was measured by the percent of CDl lc+cells double positive for CD80+and CD86+, the surface proteins that allow APCs to present and activate T-cells. When added to BMDC cultures, Mn-ZIF provided greater activation than the mole-equivalent dose of MnCE and lipopolysaccharides (LPS), a positive control known to activate BMDCs. Excitingly, CDA@Mn-ZIF outperformed all the groups and offered greater DC activation than Mn-ZIF alone, suggesting a strong synergistic effect (FIG. 16A). ELISA was used to measure cytokines released from the BMDCs into the cell supernatants. Mn-ZIF andFJ ref. UTD-P0001US / Client ref. 25003CDA@Mn-ZIF elicited a proinflammatory response, quantified using cytokines TNF-a (FIG. 16C) and IL-6 (FIG. 16D). Cytokine production from equal doses of ZIF-8 and CDA was below the limit of detection, and MnCL alone only produced IL-6. While cytokine production was significantly higher in the LPS control, it is important to note this level is undesirably high; excess IL-6 produced from LPS can lead to toxicity, as seen in sepsis. (Smiechowicz, J. Clin. Med. 2022, 11) This demonstrates that Mn-ZIF can generate an immune response alone and synergistically increase the potency of CDA. To confirm that the BMDC activation and cytokine production resulted from STING, we used a western blot to see the activation of two proteins downstream of STING — TBK-1 and IRF-3. TBK-1 will phosphorylate (P-TBK-1) upon STING activation and induce IRF-3 phosphorylation (P-IRF-3), further leading to proinflammatory gene expression generating cytokines. (Motwani, Nat. Rev. Genet. 2019, 20, 657) Western blot confirmed that Mn-ZIF and CDA@Mn-ZIF could trigger phosphorylation of both TBK-1 and IRF-3 in RAW 264.7 cells, while only P-TBK-1 was seen for MnCL and the other controls did not induce phosphorylation of either protein (FIG. 16B and SI 1 A). The western blot bands were quantified and normalized to 0-actin and results are not shown. This result links the cytokine production observed to Mn-ZIF and CDA@Mn-ZIF’s ability to activate STING. IFN-0, a type-1 interferon, was measured and a modest increase in IFN- production was observed from CDA@Mn-ZIF. IFN-y was also measured but no sample generated significant production. In addition to cytokine production, we observed increased radical oxygen species (ROS) generation inside RAW macrophages for the ZIF-8 and Mn-ZIF containing samples using an intercellular ROS detection kit. ROS have been implicated in promoting lymphocyte activation by playing a role in antigen process and presentation, and increased ROS is indicative of activated immune cells. (Bassoy, Front. Immunol. 2021, 12).

[0152] We next wanted to determine if CDA@Mn-ZIF could be formulated with a protein and be used as a vaccine. We looked to test CDA@Mn-ZIF in a Mtb model for two reasons: cGAS-STING is important for intracellular bacterial recognition, particularly in Mtb, where the bacteria go so far to inhibit this pathway by overexpressing phosphodiesteraces. (Chai, Cell. Mol. Immunol. 2020, 17, 901; Dey, Nat. Chem. Biol. 2017, 13, 210). Additionally, adjuvants that target cGAS-STING to induce protective immunity with subunit vaccines are relatively underexplored but a CDN-based STING agonist Mtb subunit vaccine has shown promising results as an intranasal vaccine in pre-clinical animal models. (long, I. Immunol. 2022, 208, 407)FJ ref. UTD-P0001US / Client ref. 25003To test CDA@Mn-ZTF as an adjuvant in an Mtb model, we looked to combine it with a well- characterized subunit protein. The CysVac2 fusion protein is made of the Mtb antigens Ag85B and CysD (Counoupas, npj Vaccines 2016, 1, 16012). It has been shown to induce protective immunity in mouse models when formulated with AdvaxCpG — a commercially available adjuvant made from polysaccharide and CpG oligonucleotide — and administered parenterally (Counoupas, Sci. Rep. 2017, 7, 8582) or intrapulmonary. (Stewart, Vaccines 2024, 12) Therefore, as a well-characterized vaccine fusion protein, CysVac2 was a good candidate to test the adjuvant activity of the CDA@Mn-ZIF. An intramuscular (I.M) vaccination route was chosen as this is currently the standard used in humans for new subunit TB vaccines. Mice were inoculated three times at two-week intervals with either 10, 3, or 1 pg of CysVac2 and 125 pg CDA@Mn-ZIF via the I.M. route. A full timeline of the experiments can be seen in FIG. 17A. A cohort of mice chosen as the positive control were vaccinated with BCG — the commercial standard for vaccination against TB. Two weeks after the final vaccination, peripheral blood mononuclear cells (PBMCs) were isolated and immunophenotyped for activated T-cells, as it is well established that the immune response to TB is cell -mediated. (Jacobs, Tuberculosis 2016, 101, 102) Thl cells promote cell-mediated immune responses and are required for the host’s defenses against intracellular infections. (Chung, Vaccine 2022, 40, 574) The CDA@Mn-ZIF, CysVac2 combo (CMZ / CysVac2) resulted in dose-dependent production of IL-17, IFN-y, IL -2, and TNF in the PBMCs after restimulation with antigen (FIG. 17 B-F). As expected, adjuvant alone could not produce activated T-cells but interestingly, BCG vaccinated mice also did not show a CysVac2 T-cell response, given that Ag85B is produced by BCG. Seeing that CMZ / CysVac2 generated a potent antigen-specific T-cell response in the blood, we looked to test the vaccine's efficacy in a Mtb challenge experiment. After aerosol infection with a low dose of Mtb, there was a significant reduction of colony-forming units (CFUs) in the lungs compared to saline-treated mice (FIG. 17G). The data suggest strong protective immunity induced from CDA@Mn-ZIF resulted in a significant reduction, approximately 0.3 Logw CFU reduction compared to the saline treated group, in mycobacterial burden regardless of the CysVac2 concentration. The significant reduction in bacterial burden in the CMZ / CysVac2 group is exciting, especially considering that this fusion protein contains only two known antigens commonly investigated for TB vaccine development. Since BCG is a whole-cell vaccine, enumerable epitopes and antigenic targets on its surface often result in better outcomes in mouseFJ ref. UTD-P0001US / Client ref. 25003 models; however, these results have not translated well into human models. (Ma, eBioMedicine 2017, 22, 143; Teng, Hum. Vaccines Immunother. 2015, 11, 1456; Troy, Tuberculosis 2020, 123, 101949; Stylianou, Infect. Immun. 2018, 86; Riccomi, Front. Immunol. 2019, 10; Ahmed, Vaccine 2017, 35, 4983) Consequently, BCG serves as a positive control more than a benchmark, particularly in mice. Interestingly, the dose-dependent response in PBMC cytokine expression did not translate to a dose-associated relationship in mycobacterial burden. There are multiple variables that could contribute to a lack of dose dependence in the challenge including the different T-cell profiles in the blood may not be reflected in the lung throughout the infection period. Additionally, there are many immune factors including immunoglobulin levels and cells other than T-cells involved during infection of a highly virulent strain, which could mask the dose dependence. Prior work using CysVac2 combined with commercially available adjuvants has shown similar levels of CFU reduction, suggesting CDA@Mn-ZIF is a promising adjuvant to promote protection against TB infection. (Counoupas, Sci. Rep. 2017, 7, 8582)B. Materials and Methods

[0153] Materials: TCEP-HC1 tris(2-carboxyethyl) phosphine hydrochloride was purchased from Goldbio. Zinc(II) acetate dihydrate, 2-methylimidazole, and manganese(II) acetate tetrahydrate, L-glutamine solution, sodium hydroxide, 5(6)-carboxyfluorescin and fluorometric intracellular ROS kit (deep red) were purchased from Millipore Sigma. Deep Blue Cell Viability kit, FITC anti-mouse CDl lc antibody, Alexa Fluor® 594 anti-mouse CD80 antibody, and Pacific Blue™ anti-mouse CD86 antibody were purchased from Biolegend. CDA was purchased from Invivogen. Nitric acid (trace metal grade) was purchased from Thermo Fisher Scientific. HyClone phosphate buffered saline solution, HyClone Dulbecco's modified eagle's medium (DMEM), HyClone RPMI 1640 medium, and penicillin-streptomycin were purchased from Cytiva. FB Essence was purchased from Avantor. TBK1, Phospho-TBKl, IRF-3, Phospho-IRF3, beta-actin, HRP-linked anti-rabbit IgG and SignalFire ECL reagent were purchased from Cell Signaling Technology

[0154] Instruments: SEM micrographs were captured on a Zeiss Supra 40. PXRD spectra were collected using Rigaku SmartLab X-ray Diffractometer. DLS measurements for size and zeta potential were carried out using Malvern Analytical Zetasizer Nano ZS. Fluorescence intensity measurements on 96-well plates were carried out using Biotek Synergy H4 Hybrid microplate reader. Epifluorescence images were taken on EVOS FL digital inverted fluorescenceFJ ref. UTD-P0001US / Client ref. 25003 microscope. Cell counting was carried out using Thermo Countess II. Zinc and manganese quantification was done using Agilent 7900 ICP-MS. CDA quantification was done using Thermo Fisher NanoDrop and Agilent 1100 series LC. X-ray photoelectron spectra was recorded using PHI VersaProbe II Scanning XPS Microprobe. Flow cytometry was performed on the BD Fortessa.

[0155] Cells and animals: RAW 264.7 cells were received as a gift from Dr. Rockford Draper (Department of Biological Sciences, UT Dallas). 4T1 cells were received as gifts from Dr. Laurentiu Pop (Department of Radiation Oncology, UT Southwestern). HEK 293 cells were received as a gift from Mateusz Durbacz (Department of Molecular Biology and Hamon Center for Regenerative Science and Medicine, UT Southwestern). Female C57BL / 6 mice (8-10 weeks) were purchased from Charles River Laboratories and were housed under the protocol #19-06, which was approved by IACUC. C57BL / 6 mice (8-10-week-old female, ABR Bioresources, Moss Vale, NSW, Australia) were used for the in pre-clinical assessment of the vaccine formulation. Mice were acclimated for one week prior to commencement of vaccination. All procedures were approved by the Animal Welfare Committee of the Local Sydney Health District under protocol # 2023 / 002.

[0156] Protein: CysVac2 was prepared by Sydney Analytical (University of Sydney, Camperdown, NSW, Australia) from transfected ClearColi BL21(DE3) bacteria (Gene Target Solutions Pty Ltd, Dural, NSW, Australia). The protein was purified using size exclusion and LPS removed to ensure limited reactivity. The LPS concentration was determined by Pierce Chromogenic Endotoxin Kit (ThermoFisher Scientific) and by assessing functional activity ina RAW-Blue® Assay (gift from Dr. Nicholas Shields, University of Sydney), which were used to monitor the NF-kB and AP-1 responses upon pattern recognition receptor stimulation. The RAW-Blue® assay showed no activity in the CysVac2 preparation.

[0157] Mn-ZIF synthesis: Four stocks were prepared in MilliQ water, a 1 M solution of zinc (II) acetate dihydrate, 3 M 2-methylimidazole (HMIM), 0.5 M TCEP-HC1, and 1 M manganese (II) acetate tetrahydrate with lOmM TCEP. For a 15 ml reaction of 50% Mn-ZIF reactants were added in the following order: (1) 550 pl H2O, (2) 450 pl TCEP, (3) 800 pl Mn, (4) 400 pl Zn, and (5) 12.8 ml HMIM. After adding HMIM, the reaction was vortexed and left static at RT for 20 mins, then centrifuged at 4,300 * G for 15 m. The supernatant was discarded, the pellets were washed twice with water and subsequently dried under a high vacuum overnight.FJ ref. UTD-P0001US / Client ref. 25003

[0158] Sample preparation for in vitro and in vivo experiments: 10 mg of ZIF-8 or 15 / 30 / 50% Mn-ZIF was weighed on an analytical balance and suspended in a glass vial with endotoxin-free, sterile water to make a 10 mg / ml stock. The stock solution was vortexed and sonicated to ensure proper dispersion.

[0159] In vitro cytotoxicity: Biolegend’s resazurin-based “Deep Blue Cell Viability” kit was used for cytotoxicity and IC50 calculations. 25,000 cells per well were seeded in a 96-well plate overnight in 50 pl of media and incubated at 37° C and 5% humidity. The following morning, 50 pl of 2* desired final concentration of the sample was added to n = 4 wells. The plate was placed back in the incubator for 24 h. 30 min before the 24 h time point, 10 pl of lyse buffer was added to appropriate wells as a negative control. At 24 h, 10 pl of the resazurin reagent was added to each well and mixed before being returned to the incubator to incubate for 4 h before being read at an excitation of 530 nm and an emission of 590 nm on a microplate reader. % viability was normalized to media only, and lysed cell control, and data are presented as average ± standard deviation (n = 4 with outlier analysis done in GraphPad Prism with Grubbs’ method). IC50 was determined using the linear regression function in Microsoft Excel.

[0160] CDA loading on Mn-ZIF: To prepare CDA@Mn-ZIF, 1 mg of Mn-ZIF was mixed with 30 pg CDA in methanol or water. This sample was placed on a rotisserie at RT for 24 h. After the 24 h period, the CDA@Mn-ZIF was centrifuged at 17,000 * G for 10 m, and the supernatant was collected for concentration determination by LC and NanoDrop UV-VIS. CDA@Mn-ZIF was washed once with water and then dried overnight in a high vacuum chamber for storage or directly resuspended in DI water for use.

[0161] Determination of CDA concentration by LC: A standard curve of CDA was first prepared by diluting a 1 mg / mL stock to 25 pg / mL and making half serial dilutions down to 1.5625 pg / mL. These standards were run through polymeric reverse phase (PLRP) column on the LC with UV detector set to 260 nm wavelength. Water and acetonitrile with 0.1% formic acid were used as eluting solvents. 5 pL of each sample was injected on the following gradient: 20% acetonitrile for 2 minutes, an increasing gradient from 20% to 60% for 30 minutes, hold at 60% for 3 minutes, and a decrease down to 20% over 2 minutes. Unknown samples were run under the same conditions, and concentration was determined by peak area integration on GraphPad.

[0162] Association and uptake experiments: 50% Mn-ZIF was prepared with 5,6- carboxyfluorescein (CF) and rhodamine B (Rh) encapsulated by replacing the water in theFJ ref. UTD-P0001US / Client ref. 25003 reaction with saturated solutions of each dye. For association by epi, 30,000 RAW 264.7 cells were seeded in an 8-well chamber slide overnight. 60 pg / mL Mn-ZIF (Rh) was added and incubated for 6 h then samples were washed with PBS and imaged. For uptake by flow, 200,000 cells per well were plated in a 96-well u-bottom plate and incubated overnight. Samples were added and allowed to incubate for 4 h and 8 h, then the cells were washed multiple times with PBS and then FACS buffer to remove material that was not taken in. Samples were run on a BD FortessaLSR flow cytometer.

[0163] Bone marrow dendritic cell cultures: Femurs and tibias were isolated from naive C57BL / 6 mice and placed in PBS. Surgical scissors were used to snip both ends of the bone, and a 25 G needle was used to flush the marrow out using RPMI over a 70 pm single cell suspension filter. Cells were centrifuged at 500 * G for 5 m, and the supernatant was removed. RBC lysis buffer was used to remove red blood cells, after which the cells were counted and suspended in 10 mL RPMI with 20 ng / mL of GM-CSF in a T-75 at a concentration of 2 x lO6cells / ml. 10 mb of RPMI with GM-CSF was added three days later, and media was refreshed on days 6 and 9.

[0164] BMDC activation and cytokine production: Non-adherent cells were collected on day 10 from the previously described cultures and seeded at a concentration of 200,000 cells per well in a 96-well plate with the final volume being 200 pL with samples. Final concentration of each sample was 1.7 pg / ml of CDA, 36 pg / ml of MnCk • 4 H2O, and 60 pg / ml of ZIF-8, 60 pg / ml Mn-ZIF loaded with 1.7 pg / ml CDA. Samples were incubated for 20 h, at which point the plate was spun, and supernatants were taken and frozen for cytokine ELISA. BMDCs were then washed with PBS and followed a staining protocol. First, samples were incubated with Zombie UV (biolegend) in PBS, followed by adding a staining cocktail of CDl lc FITC, CD80 AF647, and CD86 Pacific Blue (biolegend) and left on the ice. Samples were washed twice with cell staining buffer, resuspended, and ran on flow. TNF-a (biolegend), IL-6 (biolegend), IFN-y (biolegend), and IFN-P (R&D Systems) ELISAs were run according to manufactures protocol and absorbance was read on a Synergy H4 plate reader (Biotek).

[0165] Western blot:WasN 2.647 cells were seeded in a 6-well plate at l * 106cells per well overnight. Media was then removed and replaced with 2ml of media with a final concentration of each sample as follows: 1.7 pg / ml of CDA, 36 pg / ml of MnCL • 4 H2O, and 60 pg / ml of ZIF-8, 60 pg / ml Mn-ZIF and 60 pg / ml CDA@Mn-ZIF (1.7 pg / ml CDA). After 18 hours of incubation, supernatants with samples were removed and 200 pl of RIP A lyse buffer was added to each well,FJ ref. UTD-P0001US / Client ref. 25003 and cells were scraped and transferred to 15 ml centrifuged tubes. A Qsonica QI 25 probe sonicator then sonicated each sample at 50% intensity for three 15s cycles on ice. Samples were centrifuged at 14,0007g to pellet debris, and the supernatant was transferred to 500 pl tubes. Protein concentrations were normalized by Bradford assay and then run for 45 min on a 4 - 15% gradient gel at 200 V. The gel was transferred by semi-dry method to a nitrocellulose membrane and blocked with 3% milk buffer for 1 hour at RT. The blot was incubated overnight with 1 / 1000 dilutions of P-TBK-1 and P-IRF-3 antibodies in 5% BSA in TEST with rocking at 4°C. After 16 hours the blot was washed three times with TBST for 5 min each and incubated with 1 / 3000 dilution of secondary antibody for 1 h before adding substrate and reading on a Biorad gel imager (ChemiDoc Touch). The blot was stripped with stripping buffer and re-probed for TBK-1 and IRF-3, then P-actin as a loading control.

[0166] Vaccination and Infection: Mice (5 per group) were vaccinated in both hind quadricep muscles with 25 pL of the CDA@Mn-ZIF formulation mixed with either 10, 3 or 1 pg of CysVac2 protein 3-times at 2-week intervals. As a positive experimental control, mice were vaccinated subcutaneously with 5x104 CFU of BCG Danish (AJ Vaccines). Mice were then rested for 4 weeks during which (2 weeks after the final inoculation) they were bled to obtain peripheral blood mononuclear cells (PBMC) for immune assessment. At the end of 4 weeks, mice were infected with a low dose aerosol of Mtb, strain H37Rv (TMCC 107) and rested for another 4 weeks.

[0167] Intracellular cytokine assay: PBMC were obtained from blood taken from each mouse. Approximately 100 pL of blood was collected from a tail vein into EDTA tubes (source), diluted in endotoxin-free PBS and then PBMC obtained by centrifugation over a Histopaque- 1083 (source) gradient. PBMCs were washed, counted, and resuspended to IxlO6cells / mL in completed-RPMI-1640 (10% FBS, pen / strep, L-glut). Cells were cultured with a final concentration of lOpg / mL of CysVac2 protein for 5 hours, after which GolgiPlug was added according to manufacturer’s protocol and incubated for a further 11 hours. Cells were collected and the prepared for flow cytometry analysis using the following fluorochrome-conjugated mAb: IL17-Pacific Blue, IL2-PE, TNF-PerCP-Cy5.5, CD4-Alexaflour700, IFN-y-PECy7, CD62L- BV650, CD44-FITC, Live Dead Blue. Data was collected using a LSR Fortessa (BD Biosciences), running FACSDiva Software. Approximately 106cells per mouse were collected. FlowJo® software (BD Biosciences) was used to analyze the data.FJ ref. UTD-P0001US / Client ref. 25003

[0168] Determination of Colony Forming Units (CFU): The number of CFU in the lungs of mice was determined by plating 10-fold serial dilution of organ homogenates on 7H10 + OADC agar (Bacto). Cultures were incubated at 37°C for 14-21 days, after which colonies were counted. References(1) Ahmed, Immunol. Rev. 2021, 301, 98(2) Coppola, npj Vaccines 2021, 6, 81(3) Ma, eBioMedicine 2017, 22, 143(4) Olsen, Infect. Immun. 2001, 69, 2773(5) Ahmed, Vaccine 2017, 35, 4983(6) Teng, Hum. Vaccines Immunother. 2015, 11, 1456(7) Siroy, J. Biol. Chem. 2008, 283, 17827(8) Ward, J. Bacteriol. 2008, 190, 2939(9) Darwin, J. Biol. Chem. 2015, 290, 18962(10) Hood, Nat. Rev. Microbiol. 2012, 10, 525(11) Ahmad, Macrophage 2022(12) White, J. Biol. Chem. 2009, 284, 33949(13) Zhao, Signal Transduct. Target. Ther. 2023, 8, 283(14) Montamat, Allergen-Specific Immunotherapy 2021(15) Forsyth, mBio 2020(16) Wijesundara, Chem. Rev. 2024, 124, 3013(17) Kumari, Matter 2023, 6, 2570(18) Ehrman, Chem. Sci. 2024, 15, 2731(19) Wijesundara, Chem. Sci. 2022, 13, 13803(20) Kumari, Proc. Natl. Acad. Sci. U. S. A. 2023, 120, e2218247120(21) Herbert, Nat. Commun. 2021, 12, 2202(22) Brohlin, ACS Appl. Nano Mater. 2022, 5, 13697(23) Jacobs, Antibodies and tuberculosis 2016(24) Chung, Induction of Thl and Th2 2022(25) Jurk, Immunobiology 2004, 209, 141(26) Wang, PLOS ONE 2015, 10, e0122560(27) Leyten, Microbes Infect. 2006, 8, 2052FJ ref. UTD-P0001US / Client ref. 25003(28) Smith, Science 1988, 240, 1 169(29) Yamamoto, Polyfunctional CD4+ T-cell induction 2009(30) Counoupas, npj Vaccines 2020, 5, 105(31) Brown, Lipoarabinomannan-reactive human secretory 2003(32) de Valliere, Enhancement of innate and cell-mediated 2005(33) Singh, J. Control. Release 2022, 349, 796(34) Troy, Pulmonary mucosal immunity 2020(35) Stylianou, Identification and Evaluation 2018(36) Riccomi, Parenteral Vaccination 2019(37) Tkachuk, Multi-subunit BCG booster 2017(38) Kumari, Chem. Sci. 2023, 14, 5774(39) Restrepo, Cross-sectional assessment 2011(40) Grace, Antibody Subclass and Glycosylation 2021(41) Burel, IgG glycosylation associates 2024(42) Brown, High-throughput, multiplexed IgG 2012(43) Bates, BNT162b2-induced neutralizing 2022(44) Adhikari, Diverging Maternal and Cord 2024

Claims

FJ ref. UTD-P0001US / Client ref. 25003CLAIMS1. A tuberculosis vaccine composition comprising all or a portion of mycobacterial cation transporter protein V (CtpV), all or a portion of mycobacterial copper transporter protein B (MctB), or all or a portion of CtpV and all or a portion of MctB in a liposome carrier forming a proteoliposome.

2. The vaccine composition of claim 1, further comprising an adjuvant.

3. The vaccine composition of claim 2, wherein the adjuvant is a CpG oligonucleotide adjuvant.

4. The vaccine composition of claim 3, wherein the CpG oligonucleotide adjuvant is CpG ODN 2395, CpG 7909, or CpG 1018.

5. The vaccine composition of claim 1, wherein the proteoliposome is coated with or encapsulated in a metal-organic framework (MOF).

6. The vaccine composition of claim 5, wherein the MOF encapsulates a STING agonist selected from cyclic di-adenosine monophosphate (CDA), cGAMP, or 3'3'-cGAMP.

7. The vaccine composition of claim 6, wherein the MOF is a multivariate Mn-ZIF comprising 15-50% Mn doping.

8. The vaccine composition of claim 6, wherein the MOF is ZIF-8.

9. The vaccine composition of claim 1, wherein CtpV and MctB are present in separate proteoliposomes.

10. The vaccine composition of claim 1, wherein CtpV and MctB are present in the same proteoliposomes.

11. The vaccine composition of claim 1, wherein CtpV and MctB are present in a ratio of at least or about 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1 :2, 1 :3, 1 :4, or 1 :5.

12. The vaccine composition of claim 1, wherein the proteoliposomes have a protein to lipid ratio of at least or about 1 : 15, 1 :25, or 1 :40.

13. The vaccine composition of claim 1, wherein the proteoliposomes are formulated for intranasal, pulmonary, intramuscular, or subcutaneous administration.

14. The vaccine composition of claim 1, wherein CtpV comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1 and retains copper transporter activity.

15. The vaccine composition of claim 1, wherein MctB comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2 and retains copper transporter activity.FJ ref. UTD-P0001US / Client ref. 2500316. The vaccine composition of claim 1, wherein the proteoliposome comprises both CtpV and MctB in a molar ratio of 1 : 1 to 3 : 1 (CtpV :MctB).

17. The vaccine composition of claim 1, wherein the proteoliposome is a small unilamellar vesicle (SUV) having a diameter of 125-225 nm as measured by dynamic light scattering (DLS).

18. The vaccine composition of claim 1, wherein the proteoliposome is stabilized by encapsulation within a pharmaceutically acceptable biodegradable polymer or a hybrid polymerinorganic composite configured to enhance thermostability and / or control release of antigen.

19. A method of treating tuberculosis comprising administering an effective amount of a vaccine composition of claim 1.

20. A proteoliposome comprising: (a) a lipid bilayer comprising one or more phospholipids selected from E. coli polar lipids, DOPC, DPPC, DOPE, cholesterol, or combinations thereof; and (b) at least one transmembrane protein selected from CtpV (SEQ ID NO: 1) and MctB (SEQ ID NO: 2) reconstituted in the lipid bilayer at a protein-to-lipid ratio of about 1 :15 to about 1 :40 (w / w).

21. A metal-organic framework (MOF)-encapsulated vaccine composition comprising the proteoliposome of claim 13 encapsulated in ZIF-8, wherein the proteoliposome further comprises CpG ODN 2395, CpG 7909, or CpG 1018.

22. A method of inducing an immune response against Mycobacterium tuberculosis in a subject comprising administering an effective amount of the composition of any one of claims 1 - 18 to the subject by an intranasal, pulmonary, intramuscular, or subcutaneous route of administration.

23. A method of preventing or reducing Mycobacterium tuberculosis bacterial load in the lungs of a subject comprising administering the composition of any one of claims 1-18, , 20, or 21 via intranasal or pulmonary route, wherein the bacterial load is reduced by at least 0.3 logio CFU compared to saline control.

24. A method of eliciting a Thl-biased immune response in a subject comprising administering the composition of claim 1, wherein the Thl / Th2 ratio (IgG2a / IgGl) is at least 2- fold higher than BCG.

25. A method of eliciting polyfunctional CD4+T-cells co-expressing IFN-y, TNF-a, and IL -2 in spleen, lymph nodes, and lungs comprising administering the composition of claim 1-18.FJ ref. UTD-P0001US / Client ref. 2500326. A kit comprising: (a) a first container comprising lyophilized proteoliposomes containing CtpV and / or MctB; (b) a second container comprising ZIF-8 precursors (zinc acetate and 2- methylimidazole); and (c) instructions for encapsulating the proteoliposomes in ZIF-8 to form a shelf-stable vaccine.

27. A shelf-stable tuberculosis vaccine produced by the process of: (a) reconstituting CtpV and MctB into SUVs at a proteindipid ratio of 1 :25; (b) mixing the proteoliposomes with CpG and ZIF-8 precursors; and (c) incubating for 3 hours at room temperature to form Combo@Z.

28. A multivariate metalloimmunological adjuvant comprising Mn-ZIF having 30-50% Mn doping, wherein the adjuvant activates the cGAS-STING pathway and increases CD80 CD86 dendritic cell activation by at least 3 -fold compared to ZIF-8 alone.

29. The multivariate metalloimmunological adjuvant of claim 28, wherein the adjuvant is used as an adjuvant for the vaccine composition of claim 1 or for a composition comprising a subunit antigen.

30. The adjuvant of claim 28, further comprising a cyclic dinucleotide (CDN) selected from CDA, cGAMP, or 2'3'-cGAMP loaded post-synthetically at 20-30 pg CDA per mg Mn-ZIF.

31. A vaccine composition comprising: (a) an antigen selected from Mycobacterium tuberculosis antigens CysVac2, Ag85B, ESAT-6, CFP-10, or other protein subunit antigens, including the proteoliposome of claim 13; and (b) the adjuvant of claim 27 or 28, wherein the composition reduces lung CFU by at least 0.3 logio in an aerosol Mtb challenge model when administered intramuscularly at 1-10 pg antigen dose.

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