Compositions and methods for vaccination against mycobacterium tuberculosis

Abstract

Provided herein are compositions including epitopes for HBHA, Rv3351c, and ESAT6, and optionally one or both of Rv1490 and HUPB; nucleic acid(s) encoding the same, and combinations thereof. The epitopes are present on one or more polypeptides, optionally one or more fusion protein. In some forms, the epitope(s) for HBHA are present on a HBHA polypeptide. In some forms, the epitope(s) for Rv3351c and the epitope(s) for ESAT6 are present on a Rv3351c-ESAT6 fusion protein. In some forms, the one or more of the epitopes are package in or on a particle, optionally nanoparticles. Exemplary nanoparticles are formed of wax, PLGA, etc. In some forms, the composition further includes a hydrogel-based vaccine delivery platform. In some forms, the composition includes an adjuvant such as glucopyranosyl lipid A (GLA) and / or CpG. Also provided are methods of using the compositions for increasing an immune response.

Description

[0001] ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0002] COMPOSITIONS AND METHODS FOR VACCINATION AGAINST MYCOBACTERIUM TUBERCULOSIS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of and priority to U. S. S. N. 63 / 720,084 filed November 13, 2024, and which is specifically incorporated herein by reference in its entirety.

[0005] REFERENCE TO THE SEQUENCE LISTING

[0006] The Sequence Listing submitted as an XML file named “UGA_2025-062-02_PCT_ST26”, created November 13, 2025, and having a size of 30,603 bytes is hereby incorporated by reference pursuant to 37 C. E. R. § 1.834(c)(1).

[0007] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0008] This invention was made with government support under AI169204-01 awarded by National Institutes of Health. The government has certain rights in the invention.

[0009] FIELD OF THE INVENTION

[0010] The disclosed invention is generally in the field of immunogenic compositions and vaccines and specifically in the area of building immunity against tuberculosis.

[0011] BACKGROUND OF THE INVENTION

[0012] The current tuberculosis (TB) vaccine is Bacille Calmette-Guerin (BCG). It is only given to infants and small children and provides limited protection in adolescents / adults. Although safe, the efficacy of the vaccine is uncertain with different studies finding between 0 and 80% efficacy.

[0013] TB is both preventable and curable but is still a deadly infection killing more than a 1 million people with other 10 million infected in 2022. In 2022, the largest number of new TB cases occurred in WHO’s South-East Asian Region (46%), followed by the African Region (23%) and the Western Pacific (18%).

[0014] Therefore, there remains a need for improved TB vaccines.

[0015] Thus, provided are compositions and methods of use thereof for vaccination against TB. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0016] Throughout this specification the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0017] 45801771.1 1 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0018] BRIEF SUMMARY OF THE INVENTION

[0019] Provided herein are compositions including epitopes for HBHA, Rv3351c, ESAT6, Rvl490, HUPB, and combinations thereof; nucleic acid(s) encoding the same, and combinations thereof. The epitopes are present on one or more polypeptides. In some forms, one or more of the polypeptides is a fusion protein.

[0020] For example, in some forms, the epitope(s) for HBHA are present on a HBHA polypeptide optionally including the amino acid sequence of SEQ ID NO: 1 or a variant thereof with at least 70% sequence identity thereto.

[0021] In some forms, the epitope(s) for Rv3351 are present on a Rv3351 polypeptide optionally including the amino acid sequence of SEQ ID NOS:2, 3, or 29, or a variant thereof with at least 70% sequence identity thereto.

[0022] In some forms, the epitope(s) for ESAT6 are present on a ESAT6 polypeptide optionally including the amino acid sequence of SEQ ID NOS:4 or 5, or a variant thereof with at least 70% sequence identity thereto.

[0023] In some forms, the epitope(s) for Rvl490 are present on a Rvl490 polypeptide optionally including the amino acid sequence of SEQ ID NOS:7 or 6, or a variant thereof with at least 70% sequence identity thereto.

[0024] In some forms, the epitope(s) for HUPB are present on a HUPB polypeptide optionally including the amino acid sequence of SEQ ID NOS:8 or 9, or a variant thereof with at least 70% sequence identity thereto.

[0025] In some forms, the epitope(s) for Rv3351c and the epitope(s) for ESAT6 are present on a Rv3351c-ESAT6 fusion protein optionally including the amino acid sequence of SEQ ID NO:2 or a fragment or a variant thereof with at least 70% sequence identity thereto fused to the amino acid sequence of SEQ ID NO:4 or fragment or a variant thereof with at least 70% sequence identity thereto. In some forms, the fusion protein includes SEQ ID NOS:9 or 10, or a fragment or a variant thereof including at least 70% sequence identity thereto.

[0026] The nucleic acid can be, for example, RNA, optionally mRNA, or DNA, optionally a vector optionally a viral vector.

[0027] In some forms, the one or more of the epitopes are packaged in or on (e.g., adhered to) a particle, optional nanoparticles. Exemplary, non-limiting nanoparticles are wax nanoparticles, PLGA nanoparticles, or a combination thereof.

[0028] In some forms, the composition further includes a hydrogel-based vaccine delivery platform optionally VacSIM™.

[0029] 45801771.1 2 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0030] In some forms, the composition further includes an adjuvant such as glucopyranosyl lipid A (GLA) and / or CpG.

[0031] fhe composition can include a pharmaceutically acceptable carrier. In some forms, the composition is in a formulation suitable for subcutaneous and / or nasal delivery.

[0032] In some forms, the composition is in an effective amount to induce an immune response against one or more of HBHA, Rv3351c, ESAT6, Rvl490, and / or HUPB, when administered to a subject. Thus, any and all of the disclosed proteins and fusion proteins, and nucleic acids encoding the same, can be used alone or in any combination as antigens of the disclosure.

[0033] Thus, also provided are methods of inducing or increasing an immune response in a subject in need thereof including administering the subject a provided composition. Typically, the immune response is against one, two, three, four, or all five of HBHA, Rv3351c, ESAT6, Rvl490, and HUPB. In some forms, the composition is administered in an effective amount to increase immunity against one or more mycobacterial strains in the subject. Such strains include, for example, Mycobacterium tuberculosis (Mtb), M. bovis, M. africanum, M. canettii, M. orygis, M. microti, M. caprae, M. pinnipedii, M. mungi, and M. suricattae.

[0034] In some forms, the composition is administered in an effective amount to reduce or prevent one or more symptoms of TB. In some forms, the subject does not have TB. In some forms, the subject has TB. In some forms, the subject has been exposed to someone with TB.

[0035] In some forms, the composition is administered in combination with a BCG (Bacille Calmette-Guerin) vaccine. The BCG vaccine can be administered before or after the provided compositions. For example, in some forms, the BCG vaccine is administered to the subject first and composition is administered to the subject one or more times day(s), month(s), week(s), and / or year(s) later. Optionally, the administration is the same or similar to the regimen of Figure 7.

[0036] Routes of administration are also provided and include, for example, intranasal and subcutaneous routes. The subject can be, for example, an infant, child, adult optionally elderly adult. The subject can be mammal, preferably human. In some forms, the subject is alternatively or additional a non-human animal that can carry or otherwise contract Mycobacterium tuberculosis (Mtb), M. bovis, M. africanum, M. canettii, M. orygis, M. microti, M. caprae, M. pinnipedii, M. mungi, and M. suricattae.

[0037] Also provided are fusion proteins including the amino acid sequence of SEQ ID NO:2 or a fragment or a variant thereof with at least 70% sequence identity thereto fused to the amino acid sequence of SEQ ID NO:4 or fragment or a variant thereof with at least 70% sequence identity thereto. An exemplary fusion protein includes the amino acid sequence of SEQ ID NOS: 10 or 11,

[0038] 45801771.1 3 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0039] or a fragment or a variant thereof including at least 70% sequence identity thereto. Nucleic acids encoding the fusion protein are also provided, as are immunogenic compositions thereof.

[0040] Also provided are methods of purifying insoluble proteins as provided herein, such as the provided fusion proteins.

[0041] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings illustrate several forms of the disclosed methods and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0044] Figures 1A and IB are scanning electron micrographs of conalbumin- (1A) and Rv3351c-ESAT6-loaded (IB) 25-35 kDa PLGA NPs.

[0045] Figures 2A and 2B are graphs of percentage release of proteins from PLGA NPs. Figure 2A shows release of model protein conalbumin (CA) from 5-10 (circles) and 25-35 kDa (squares) PLGA NPs in pH 5.2 acetate buffer at 37 °C. Figure 2B shows release of Rv3351c-ESAT6 fusion antigen from 25-35 kDa PLGA in simulated lung fluid at 37 °C (n>3). Protein release was quantified using the bicinchoninic acid (BCA) assay.

[0046] Figures 3A-3D are scanning electron micrographs of carnauba wax nanoparticles after sonication (3A), centrifugation at 20,000 x g (3B), ultracentrifugation at 40,000 x g (3C), and combination with Rv3351c-ESAT6 fusion protein (3D).

[0047] Figures 4A and 4B are bar graphs showing stability of wax nanoparticles containing Rv3351c-ESAT6 upon storage at 4 °C up to 14 days as monitored by dynamic light scattering in terms of hydrodynamic size (4A) and polydispersity index (4B). All time points days 3-14 are not significant (p > 0.05) compared to day 0 measurements as determined using a one-way ANOVA with Dunnett’s multiple comparisons.

[0048] Figures 5A and 5B are bar graphs showing stability of PLGA nanoparticles containing Rv3351c-ESAT6 upon storage at 4 °C up to 14 days as monitored by dynamic light scattering in terms of hydrodynamic size (5 A) and polydispersity index (5B). *p < 0.05 and *p < 0.01 compared

[0049] 45801771.1 4 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0050] to day 0 measurements as determined using a one-way ANOVA with Dunnett’s multiple comparisons.

[0051] Figure 6 is a bar graph showing stability of PLGA nanoparticles containing Rv3351c-ESAT6 upon storage at 4 °C up to 14 days in terms of antigen retained within the nanoparticles (left bars) and antigen found released into the supernatant (right bars). Less than 3% of the antigen within the nanoparticles was released into the supernatant up to 14 days after preparation. All time points days 3-14 are not significant (p > 0.05) compared to day 0 measurements as determined using a one-way ANOVA with Dunnett’s multiple comparisons.

[0052] Figure 7 is a vaccine strategy and timeline utilized in the experiments described below. Figure 8 is a bar graph showing the # of lung granulomas at week 4 post infection (p.i.) Figure 9 is a bar graph showing the # of lung granulomas at week 8 p.i.

[0053] Figure 10 is a bar graph showing the lung area affect at week 8 p.i.

[0054] Figure 11 is a bar graph showing the loglO of Mtb count in the lung (CFU) at week 8 p.i. Figure 12 is a bar graph showing the loglO of Mtb count in the spleen (CFU) at week 8 p.i.

[0055] Figure 13 is a line graph showing the presence of anti-Rv3351 / ESAT6 IgG antibodies following subcutaneous delivery (SQ) two weeks after second booster.

[0056] Figure 14 is a line graph showing the presence of anti-Rv3351 / ESAT6 IgG antibodies following intranasal delivery (IN) two weeks after second booster.

[0057] Figure 15 is a series of images showing lung histopathology at 4 weeks p.i.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular forms and the Example included therein and to the Figures and their previous and following description.

[0060] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms only and is not intended to be limiting.

[0061] I. Definitions

[0062] As used herein, the term “nucleic acid(s)” refers to any nucleic acid containing molecule, including, but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4- acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5 -(carboxyhydroxylmethyl) uracil, 5 -fluorouracil, 5 -bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine,

[0063] 45801771.1 5 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0064] 1 -methylpseudouracil, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5 -methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine. In accordance with standard nomenclature, nucleic acid sequences are denominated by either a three letter, or single letter code as indicated as follows: adenine (Ade, A), thymine (Thy, T), guanine (Gua, G) cytosine (Cyt, C), uracil (Ura, U).

[0065] As used herein, the term “polynucleotide” refers to a chain of nucleotides of any length, regardless of modification (e.g., methylation).

[0066] As used herein, the term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide, RNA (e.g., including but not limited to, mRNA, tRNA and rRNA) or precursor. The polypeptide, RNA, or precursor can be encoded by a full-length coding sequence or by any portion thereof. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The term “gene” encompasses both cDNA and genomic forms of a gene, which may be made of DNA, or RNA. A genomic form or clone of a gene may contain the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0067] As used herein, the term “nucleic acid molecule encoding,” refers to the order or sequence of nucleotides along a strand of nucleotides. The order of these nucleotides can determine the order of amino acids along the polypeptide (protein) chain. The nucleotide sequence can thus code for the amino acid sequence.

[0068] As used herein, “heterologous” means derived from a different species.

[0069] As used herein, a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors described herein can be expression vectors.

[0070] 45801771.1 6 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0071] As used herein, the term “polypeptide” refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

[0072] As used herein, a “variant,” “mutant,” or “mutated” polynucleotide contains at least one polynucleotide sequence alteration as compared to the polynucleotide sequence of the corresponding wild-type or parent polynucleotide. Mutations may be natural, deliberate, or accidental. Mutations include substitutions, deletions, and insertions.

[0073] As used herein, “identity,” as known in the art, is a relationship between two or more polynucleotide or polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between the polynucleotide or polypeptide as determined by the match between strings of such sequences. “Identity” can also mean the degree of sequence relatedness of a polynucleotide or polypeptide compared to the full-length of a reference polynucleotide or polypeptide. “Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988).

[0074] As used herein, “operably linked” refers to a juxtaposition wherein the components are configured so as to perform their usual function. For example, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence, and an organelle localization sequence operably linked to protein will assist the linked protein to be localized at the specific organelle.

[0075] As used herein, the terms “subject,” “individual,” and “patient” refer to any individual who is the target of treatment using the disclosed compositions. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The subjects can be symptomatic or asymptomatic. The term does not denote a particular age or sex. Thus, adult and newborn subjects,

[0076] 45801771.1 7 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0077] whether male or female, are intended to be covered. A subject can include a control subject or a test subject.

[0078] As used herein, “treat” means to prevent, reduce, decrease, or ameliorate one or more symptoms, characteristics or comorbidities of an age-related disease, disorder or condition; to reverse the progression of one or more symptoms, characteristics or comorbidities of an age related disorder; to halt the progression of one or more symptoms, characteristics or comorbidities of an age-related disorder; to prevent the occurrence of one or more symptoms, characteristics or comorbidities of an age-related disorder; to inhibit the rate of development of one or more symptoms, characteristics or comorbidities or combinations thereof.

[0079] As used herein, the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being affected.

[0080] As used herein, an “adjuvant” is a substance that increases the ability of an antigen to stimulate the immune system.

[0081] As used herein, the term “carrier” or “excipient” refers to an organic or inorganic ingredient, natural or synthetic inactive ingredient in a formulation, with which one or more active ingredients are combined.

[0082] As used herein, the term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.

[0083] A used herein, the term “pharmaceutically-acceptable carrier” means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.

[0084] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0085] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

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[0087] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0088] These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific form or combination of forms of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0089] Disclosed are mixtures formed by performing or preparing to perform the disclosed method. For example, disclosed are mixtures comprising the disclosed compositions.

[0090] Whenever the method involves mixing or bringing into contact compositions or components or reagents, performing the method creates a number of different mixtures. For example, if the method includes 3 mixing steps, after each one of these steps a unique mixture is formed if the steps are performed separately. In addition, a mixture is formed at the completion of all of the steps regardless of how the steps were performed. The present disclosure contemplates these mixtures, obtained by the performance of the disclosed methods as well as mixtures containing any disclosed reagent, composition, or component, for example, disclosed herein.

[0091] 45801771.1 9 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0092] It is to be understood that the disclosed compounds, compositions, and methods are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms and forms only and is not intended to be limiting.

[0093] Every compound provided herein is intended to be and should be considered to be specifically disclosed herein. Further, every subgroup that can be identified herein is intended to be and should be considered to be specifically disclosed herein. As a result, it is specifically contemplated that any compound, or subgroup of compounds can be either specifically included for or excluded from use or included in or excluded from a list of compounds.

[0094] IL Compositions

[0095] A. Antigens

[0096] 1. Antigen Sequences

[0097] Any and all of the disclosed polypeptides and fusion proteins, and nucleic acids encoding the same, can be used alone or in any combination as antigens of the disclosure. Typically the antigens include one or more epitopes. An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, and / or T cells.

[0098] Thus, the disclosed compositions typically include epitopes of one, two, three, four, or all five of epithelial heparin-binding hemagglutinin adhesin (HBHA), inducer of lipid rafts protein Rv3351c, pore forming protein early secretory antigenic target 6 (ESAT6), Uncharacterized protein Rvl490 (Rvl490), and DNA-binding protein HupB (HUPB, also referred to as MDP1) as polypeptide(s), or nucleic acids encoding the same. In some forms, the disclosed compositions include epitopes of at least two, or preferably all three of HBHA, Rv3351c, and ESAT6, and optionally one or both of Rvl490 and HUPB as polypeptide(s), or nucleic acids encoding the same. The epitopes are typically part of one or more polypeptides.

[0099] The polypeptides can be in the foim of separate, individual polypeptides or fusion proteins that include two, three, four, or five of the individual polypeptides and / or include heterologous sequences such single (secretion) sequence(s), linker(s), tag(s), and / or reporter(s). Any one or more of these features can be cleaved / removed before use of the polypeptide or fusions protein as an antigen(s). Functional fragments and variants thereof are also provided. Thus, polypeptides provided herein with signal sequences, purification tags, reports, etc., are also expressly provided without these features.

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[0101] For example, consensus sequences for HBHA, Rv3351c, ESAT6, Rvl490, and HUPB are known in the art and include, but are not limited to,

[0102] HBHA homologs in Mtb, M. bovis, and M. orygis

[0103] > Mycobacterium tuberculosis H37RvlRvO475lhbhA MAENSNIDDIKAPLLAALGAADLALATVNELITNLRERAEETRTDTRSRVEESRARLTKLQEDLPE QLTELREKFTAEELRKAAEGYLEAATSRYNELVERGEAALERLRSQQSFEEVSARAEGYVDQAVEL TQEALGTVASQTRAVGERAAKLVGIELPKKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVTQ

[0104] K (SEQ ID NO:1, UniProt Accession No. P9WIP9 ■ HBHA_MYCTU);

[0105] > Mycobacterium bovis AF2122-97IMb0485lhbhA MAENSNIDDIKAPLLAALGAADLALATVNELITNLRERAEETRTDTRSRVEESRARLTKLQEDLPE QLTELREKFTAEELRKAAEGYLEAATSRYNELVERGEAALERLRSQQSFEEVSARAEGYVDQAVEL TQEALGTVASQTRAVGERAAKLVGIELPKKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVTQ

[0106] K (SEQ ID NO: 1)

[0107] > Mycobacterium orygis 51145IRJtmp_000499lhbhA MAENSNIDDIKAPLLAALGAADLALATVNELITNLRERAEETRTDTRSRVEESRARLTKLQEDLPE QLTELREKFTAEELRKAAEGYLEAATSRYNELVERGEAALERLRSQQSFEEVSARAEGYVDQAVEL TQEALGTVASQTRAVGERAAKLVGIELPKKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVTQ

[0108] K (SEQ ID NO: 1)

[0109] Rv3351c homologs in Mtb, M. bovis, and M. orygis

[0110] > Mycobacterium tuberculosis H37RvlRv3351clRv3351c VLASCPARSGAAVADAIKSAVGVQPSGVEHKTLRRMDLVRYLAGGHTTYPPEGFVAGSDVIGTTNP AAAQAIVAAIGTWPPAAGRASALIDSLGGAVGDMDPEGSAFPWCRQSAWQWYVNTPSDGQVATAN KWLSDAHHAVQHFSVGGYVNYLEANAAASQYFGANLSRLTTVRRKYDPDRIMYSGLDFSTRQVAER LLPALGFRVRFGVLVIRCALCTDTVKRLGTLPNLTWSRLKVNVAVTQEQAGVMDLPALPVRRTPRR

[0111] (SEQ ID NOG), MLASCPARSGAAVADAIKSAVGVQPSGVEHKTLRRMDLVRYLAGGHTTYPPEGFVAGSDVIGTTNP AAAQAIVAAIGTWPPAAGRASALIDSLGGAVGDMDPEGSAFPWCRQSAWQWYVNTPSDGQVATAN KWLSDAHHAVQHFSVGGYVNYLEANAAASQYFGANLSRLTTVRRKYDPDRIMYSGLDFSTRQVAER LLPALGFRVRFGVLVIRCALCTDTVKRLGTLPNLTWSRLKVNVAVTQEQAGVMDLPALPVRRTPRR

[0112] (SEQ ID NOG, UniProt Accession No. 050380 ■ 050380_MYCTU);

[0113] > Mycobacterium bovis AF2122-97IMb3386clMb3386c MLASCPARSGAAVADAIKSAVGVQPSGVEHKTLRRMDLVRYLAGGHTTYPPEGFVAGSDVIGTTNP AAAQAIVAAIGTWPPAAGRASALIDSLGGAVGDMDPEGSAFPWCRQSAWQWYVNTPSDGQVATAN KWLSDAHHAVQHFSVGGYVNYLEANAAASQYFGANLSRLTTVRRKYDPDRIMYSGLDFSTRQVAER

[0114] 45801771.1 11 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0115] LLPALGFRVRFGVLVIRCALCTDTVKRLGTLPNLTWSRLKVNVAVTQEQAGVMDLPALPVRRTPRR

[0116] (SEQ ID NO: 2)

[0117] > WP_003917774.1 MULTISPECIES: BBE domain-containing protein [Mycobacterium] includes M. orygis 1112400015 MDLVRYLAGGHTTYPPEGFVAGSDVIGTTNPAAAQAIVAAIGTWPPAAGRASALIDSLGGAVGDMD PEGSAFPWCRQSAWQWYVNTPSDGQVATANKWLSDAHHAVQHFSVGGYVNYLEANAAASQYFGAN LSRLTTVRRKYDPDRIMYSGLDFSTRQVAERLLPALGFRVRFGVLVIRCALCTDTVKRLGTLPNLT WSRLKVNVAVTQEQAGVMDLPALPVRRTPRR (SEQ ID NO:29)

[0118] ESAT-6 homologs in Mtb, M. bovis, and M. orygis

[0119] > Mycobacterium tuberculosis II37RvlRv3875lesxA MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELN NALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO:4, UniProt Accession No. P9WNK7 ■ ESXA_MYCTU),

[0120] > M. bovis accession: AAC44033.1 MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDTATELNN ALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO:5)

[0121] > Mycobacterium orygis 51145IRJtmp_003991lesxA MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELN NALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO:4)

[0122] Rvl490 homologs in Mtb, M. bovis, and M. orygis

[0123] > Mycobacterium tuberculosis H37RvlRvl490IRvl490 VSQCFAVKGIGGADQATLGSAEILVKYAQLADKRARVYVLVSTWLWWGIWHVYFVEAVFPNAILW LHYYAASYEFGFVRRGLGGELIRMLTGDHFFAGAYTVLWTSITVWLIALAVWWLILSTGNRSERR IMLALLVPVLPFAFSYAIYNPHPELFGMTALVAFSIFLTRAHTSRTRVILSTLYGLTMAVLALIHE AIPLEFALGAVLAIIVLSKNATGATRRICTALAIGPGTVSVLLLAWGRRDIADQLCAHIPHGMVE NPWAVATTPQRVLDYIFGRVESHADYHDWVCEHVTPWFNLDWITSAKLVAVVGFRALFGAFLLGLL FFVATTSMIRYVSAVPVRTFFAELRGNLALPVLASALLVPLFITAVDWTRWWVMITLDVAIVYILY AIDRPEIEQPPSRRNVQVFVCWLVLAVIPTGSANNIGR (SEQ ID NO:6) MSQCFAVKGIGGADQATLGSAEILVKYAQLADKRARVYVLVSTWLWWGIWHVYFVEAVFPNAILW LHYYAASYEFGFVRRGLGGELIRMLTGDHFFAGAYTVLWTSITVWLIALAVWWLILSTGNRSERR IMLALLVPVLPFAFSYAIYNPHPELFGMTALVAFSIFLTRAHTSRTRVILSTLYGLTMAVLALIHE AIPLEFALGAVLAIIVLSKNATGATRRICTALAIGPGTVSVLLLAWGRRDIADQLCAHIPHGMVE NPWAVATTPQRVLDYIFGRVESHADYHDWVCEHVTPWFNLDWITSAKLVAVVGFRALFGAFLLGLL FFVATTSMIRYVSAVPVRTFFAELRGNLALPVLASALLVPLFITAVDWTRWWVMITLDVAIVYILY

[0124] 45801771.1 12 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0125] AIDRPEIEQPPSRRNVQVFVCWLVLAVIPTGSANNIGR (SEQ ID NO:7, UniProt Accession No. P9WLX1 ■ Y1490_MYCTU),

[0126] > Mycobacterium bovis AF2122-97IMbl527IMbl527 MSQCFAVKGIGGADQATLGSAEILVKYAQLADKRARVYVLVSTWLWWGIWHVYFVEAVFPNAILW LHYYAASYEFGFVRRGLGGELIRMLTGDHFFAGAYTVLWTSITVWLIALAVWWLILSTGNRSERR IMLALLVPVLPFAFSYAIYNPHPELFGMTALVAFSIFLTRAHTSRTRVILSTLYGLTMAVLALIHE AIPLEFALGAVLAIIVLSKNATGATRRICTALAIGPGTVSVLLLAWGRRDIADQLCAHIPHGMVE NPWAVATTPQRVLDYIFGRVESHADYHDWVCEHVTPWFNLDWITSAKLVAVVGFRALFGAFLLGLL FFVATTSMIRYVSAVPVRTFFAELRGNLALPVLASALLVPLFITAVDWTRWWVMITLDVAIVYILY AIDRPEIEQPPSRRNVQVFVCWLVLAVIPTGSANNIGR (SEQ ID NO:7) > Mycobacterium orygis WP_031708495 MSQCFAVKGIGGADQATLGSAEILVKYAQLADKRARVYVLVSTWLWWGIWHVYFVEAVFPNAILW LHYYAASYEFGFVRRGLGGELIRMLTGDHFFAGAYTVLWTSITVWLIALAVWWLILSTGNRSERR IMLALLVPVLPFAFSYAIYNPHPELFGMTALVAFSIFLTRAHTSRTRVILSTLYGLTMAVLALIHE AIPLEFALGAVLAIIVLSKNATGATRRICTALAIGPGTVSVLLLAWGRRDIADQLCAHIPHGMVE NPWAVATTPQRVLDYIFGRVESHADYHDWVCEHVTPWFNLDWITSAKLVAVVGFRALFGAFLLGLL FFVATTSMIRYVSAVPVRTFFAELRGNLALPVLASALLVPLFITAVDWTRWWVMITLDVAIVYILY AIDRPEIEQPPSRRNVQVFVCWLVLAVIPTGSANNIGR (SEQ ID NO:7)

[0127] and

[0128] HUPB homologs in Mtb, M. bovis, and M. orygis

[0129] > Mycobacterium tuberculosis H37RvlRv2986clhupB MNKAELIDVLTQKLGSDRRQATAAVENWDTIVRAVHKGDSVTITGFGVFEQRRRAARVARNPRTG ETVKVKPTSVPAFRPGAQFKAWSGAQRLPAEGPAVKRGVGASAAKKVAKKAPAKKATKAAKKAAT KAPARKAATKAPAKKAATKAPAKKAVKATKSPAKKVTKAVKKTAVKASVRKAATKAPAKKAAAKRP ATKAPAKKATARRGRK (SEQ ID NO:8) MGMNKAELIDVLTQKLGSDRRQATAAVENWDTIVRAVHKGDSVTITGFGVFEQRRRAARVARNPR TGETVKVKPTSVPAFRPGAQFKAWSGAQRLPAEGPAVKRGVGASAAKKVAKKAP AKKATKAAKKA ATKAPARKAATKAPAKKAATKAPAKKAVKATKSPAKKVTKAVKKTAVKASVRKAATKAPAKKAAAK RPATKAPAKKATARRGRK (SEQ ID NO:9, UniProt Accession No. P9WMK7 ■ DBH_MYCTU)

[0130] > Mycobacterium bovis AF2122-97IMb3010clhupB MNKAELIDVLTQKLGSDRRQATAAVENWDTIVRAVHKGDSVTITGFGVFEQRRRAARVARNPRTG ETVKVKPTSVPAFRPGAQFKAWSGAQRLPAEGPAVKRGVGASAAKKVAKKAPAKKATKAAKKAAT

[0131] 45801771.1 13 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0132] KAPARKAATKAPAKKAATKAPAKKAVKATKSPAKKVTKAVKKTAVKASVRKAATKAPAKKAAAKRP ATKAPAKKATARRGRK (SEQ ID NO:8)

[0133] > Mycobacterium orygis 51145IRJtmp_003081lhupB MNKAELIDVLTQKLGSDRRQATAAVENWDTIVRAVHKGDSVTITGFGVFEQRRRAARVARNPRTG ETVKVKPTSVPAFRPGAQFKAWSGAQRLPAEGPAVKRGVGASAAKKVAKKAPAKKATKAAKKAAT KAPARKAATKAPAKKAATKAPAKKAVKATKSPAKKVTKAVKKTAVKASVRKAATKAPAKKAAAKRP ATKAPAKKATARRGRK (SEQ ID NO:8)

[0134] Variants of HBHA, Rv3351c, ESAT6, Rvl490, and HUPB can have, for example, at least 60, 65, 70, 75, 80, 85, 90, or 95 precent sequence identity to any one of SEQ ID NOS:l-9 and 29.

[0135] Fusion proteins can be formed of any combination of two or more of the proteins, or functional fragments or variants thereof. In some forms, the fusion proteins further includes additional sequences, e.g., a signal sequence and / or a purification tag, optionally appended to the N-terminal and C-terminal ends of the fusion protein, optionally respectively. In some forms, the proteins or fragments or variants thereof are separated by an optional linker sequence.

[0136] In some forms, at least ESAT6 is fused to one or more of Rc3351c, HBHA, Rvl490, and / or HUPB; and / or an additional feature that improves expression, solubility, and / or isolation in or from an expression system.

[0137] In some forms, an Rv3351c - ESAT6 fusion protein includes SEQ ID NO:4 fused to SEQ ID NO:2, optionally to the C-terminal end of SEQ ID NO:2, optionally including an intervening linker sequence.

[0138] An exemplary Rv3351c - ESAT6 fusion protein includes or is MLASCPARSGAAVADAIKSAVGVQPSGVEHKTLRRMDLVRYLAGGHTTYPPEGFVAGSDVIGTTNP AAAQAIVAAIGTWPPAAGRASALIDSLGGAVGDMDPEGSAFPWCRQSAWQWYVNTPSDGQVATAN KWLSDAHHAVQHFSVGGYVNYLEANAAASQYFGANLSRLTTVRRKYDPDRIMYSGLDFSTRQVAER LLPALGFRVRFGVLVIRCALCTDTVKRLGTLPNLTWSRLKVNVAVTQEQAGVMDLPALPVRRTPRR GDGACGGMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWD ATATELNNALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO: 10); or MASMTGGQQMGRDPNSSSVDKLMLASCPARSGAAVADAIKSAVGVQPSGVEHKTLRRMDLVRYLAG GHTTYPPEGFVAGSDVIGTTNPAAAQAIVAAIGTWPPAAGRASALIDSLGGAVGDMDPEGSAFPWC RQSAWQWYVNTPSDGQVATANKWLSDAHHAVQHFSVGGYVNYLEANAAASQYFGANLSRLTTVRR KYDPDRIMYSGLDFSTRQVAERLLPALGFRVRFGVLVIRCALCTDTVKRLGTLPNLTWSRLKVNVA VTQEQAGVMDLPALPVRRTPRRGDGACGGMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLT KLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTI SEAGQAMASTEGNVTGMFALEHHHHHH

[0139] (SEQ ID NO: 11)

[0140] 45801771.1 14 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0141] Also provided are variant Rv3351c - ESAT6 fusion proteins having at least 60, 65, 70, 75, 80, 85, 90, or 95 percent sequence identity to SEQ ID NOS: 10 and / or 11.

[0142] Although expressly provided as protein sequences from Mtb, M. bovis, and M. orygis the corresponding protein sequences in related species including, but not limited to, M. africanum, M. canettii, M. microti, M. leprae, M caprae, M pinnipedii, M microti, M mungi, M suricattae, M avium avium, M avium ho ninissuis, M intracellulare and M. kansasii, are known in the art and can be used in substitution or addition to the expressly provided protein sequences.

[0143] 2. Peptide Linkers

[0144] The fusion proteins can include one or more peptide linkers to e.g., separate various domains of the fusion protein.

[0145] Exemplary flexible linkers include, but are not limited to, Gly-Ser, Gly-Ser-Gly, Ala-Ser, Gly-Leu-Phe, Gly-Ser-Gly-Gly (SEQ ID NO: 12), Gly-Ser-Gly-Ser (SEQ ID NO: 13), Gly-Gly-Gly-Ser (SEQ ID NO: 14), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 15), Gly-Gly-Gly-Gly-Ser (SEQ ID NO:16), (Gly4-Ser)2(SEQ ID NO:17), (Gly4-Ser)4(SEQ ID NO:18), (Gly-Gly-Gly-Gly-Ser)3(SEQ ID NO: 19).

[0146] 3. Purification Tags and Reporters

[0147] The fusion protein can optionally include additional sequences or moieties, including, but not limited to signal sequences, purification tags, solubility enhancers, and / or reporters.

[0148] In some forms the purification tag is a polypeptide. Polypeptide purification tags are known in the art and include, but are not limited to His tags which typically include six or more, typically consecutive, histidine residues; green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein and protein A. More specific examples include FLAG tags including the sequence DYKDDDDK (SEQ ID NO:20); haemagglutinin (HA) tags including the sequence YPYDVP (SEQ ID NO:21); or MYC tags including the sequence ILKKATAYIL (SEQ ID NO:22) or EQKLISEEDL (SEQ ID NO:23). The fusion protein utilized in the experiments below includes 6 consecutive histidines at the C-terminus. Methods of using purification tags to facilitate protein purification are known in the art and include, for example, a chromatography step wherein the tag reversibly binds to a chromatography resin.

[0149] Although many proteins with therapeutic or commercial uses can be produced by recombinant organisms, the yield and quality of the expressed protein are variable due to many factors. For example, heterologous protein expression by genetically engineered organisms can be affected by the size and source of the protein to be expressed, the presence of an affinity tag linked to the protein to be expressed, codon biasing, the strain of the microorganism, the culture conditions

[0150] 45801771.1 15 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0151] of microorganism, and the in vivo degradation of the expressed protein. Some of these problems can be mitigated by fusing the protein of interest to an expression or solubility enhancing amino acid sequence. Exemplary expression or solubility enhancing amino acid sequences include maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and a small ubiquitin-related modifier (SUMO).

[0152] In some forms, the compositions disclosed herein include expression or solubility enhancing amino acid sequence. In some forms, the expression or solubility enhancing amino acid sequence is cleaved prior administration of the composition to a subject in need thereof. The expression or solubility enhancing amino acid sequence can be cleaved in the recombinant expression system, or after the expressed protein in purified. In some forms, the expression or solubility enhancing is a ULP1 or SUMO sequence. Recombinant protein expression systems that incorporate the SUMO protein (" SUMO fusion systems") have been shown to increase efficiency and reduce defective expression of recombinant proteins in E. coli., see for example Malakhov, et al., J. Struct. Funct. Genomics, 5: 75-86 (2004), U. S. Patent No. 7,060,461, and U. S. Patent No. 6,872,551. SUMO fusion systems enhance expression and solubility of certain proteins, including severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease, nucleocapsid, and membrane proteins (Zuo et al., J. Struct. Funct. Genomics, 6:103-111 (2005)).

[0153] A reporter protein typically provides for some phenotypic change or enzymatic property. Examples of such proteins are provided in K. Weising et al. Ann. Rev. Genetics, 22, 421 (1988), and include, but are not limited to, carcinoembryonic antigen, secreted alkaline phosphatase, and the beta subunit of chorionic gonadotropin, glucuronidase (GUS), luciferase (e.g., Gaussia Luciferase (GLuc), Nanoluciferase (NLuc), and fluorescent proteins such as green fluorescent protein (GFP), enhanced green fluorescent protein (eGEP), turbo red fluorescent protein (TurboREP), etc.

[0154] The reporter can serve as a measure or monitor of in vivo viral activity. For example, these reporters are released by cells infected with live virus into the blood, and can be measured peripherally to determine viral activity (Phuong, et al., Cancer Res., 63:2462-2469 (2003); Peng, et al., Nat. Med., 8:527-531 (2002); Shashkova, et al., Cancer Gene Ther., 15:61-72 (2008);

[0155] Hiramatsu, et al., Cancer Science. 100, 1389-1396 (2005)).

[0156] Purifications tags, solubility enhancers, and reporters can be inserted anywhere in the fusions preferably where they do not disturb the ability of the fusion protein to induce an immune response against Mtb. Preferred locations include the N-terminus and / or C-terminus, but internal locations relative to other domains of the fusion protein are also contemplated.

[0157] 45801771.1 16 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0158] B. Isolated Nucleic Acids

[0159] All of the accession numbers provided herein are specifically incorporated by reference herein in their entireties. Where an amino acid (e.g., polypeptide) is expressly provided or provided in an accession number, all nucleic acid sequences including but not limited to, gene, cDNA, and mRNA sequences and their complements, and codon variations thereof encoding the amino acid sequence, both as single strands and double strands, and in any form of nucleic acid, including, but not limited to DNA and RNA, and analogs and variations thereof including but limited to modified bases, sugars, and linkages (e.g., peptide nucleic acids (PNA)), are all expressly provided.

[0160] Isolated nucleic acid sequences encoding the chimeric fusion proteins and individual domains and fragments thereof, and vectors and other expression constructs encoding the foregoing are also disclosed herein. As used herein, “isolated nucleic acid’’ refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome. The term “isolated” as used herein with respect to nucleic acids also includes the combination with any non-naturally-occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.

[0161] An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.

[0162] The nucleic acid sequences encoding the disclosed proteins and polypeptides can be or include, for example, engineered genomic sequences and fragments of naturally occurring genomic sequence, mRNA sequence wherein the exons have been deleted, and other nucleic acid sequences. Nucleic acids encoding the chimeric fusion proteins and domains thereof may be optimized for expression in the expression host of choice. Codons may be substituted with alternative codons

[0163] 45801771.1 17 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0164] encoding the same amino acid to account for differences in codon usage different host organisms. In this manner, the nucleic acids may be synthesized using expression host-preferred codons. Nucleic acids can be in sense or antisense orientation, or can be complementary to a reference sequence encoding the chimeric fusion protein or domain(s) thereof. Nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of the nucleic acid. Common modifications are discussed in more detail below.

[0165] Nucleic acids encoding polypeptides can be administered to subjects in need thereof.

[0166] Nucleic delivery involves introduction of “foreign” nucleic acids into a cell and ultimately, into a live animal. Compositions and methods for delivering nucleic acids to a subject are known in the art (see Understanding Gene Therapy, Lemoine, N. R., ed., BIOS Scientific Publishers, Oxford, 2008).

[0167] 1. Vectors and Host Cells

[0168] Vectors encoding chimeric fusion proteins and domains thereof are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a “vector” is a replicon, such as a plasmid, phage, vims or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Vectors can be expression vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0169] Nucleic acids in vectors can be operably linked to one or more expression control sequences. Tor example, the control sequence can be incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.

[0170] Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA

[0171] 45801771.1 18 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0172] polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.

[0173] Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculo viruses, tobacco mosaic virus, herpes viruses, cytomegalo vims, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI). Clontech (Palo Alto. CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0174] An expression vector can include a tag sequence. Tag sequences are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein and protein A.

[0175] Vectors containing nucleic acids to be expressed can be transferred into host cells. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Host cells (e.g., a prokaryotic cell or a eukaryotic cell such as insect cells or mammalian cells (e.g., CHO cells) can be used to, for example, produce the fusion proteins described herein.

[0176] The vectors can be used to express fusion protein nucleic acids in cells. An exemplary vector includes, but is not limited to, an adenoviral vector. One approach includes nucleic acid transfer into primary cells in culture followed by autologous transplantation of the ex vivo transformed cells into the host, either systemically or into a particular organ or tissue. Ex vivo methods can include, for example, the steps of harvesting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the encoded polypeptides. These methods are known in the art of molecular biology. The transduction step can be accomplished by any standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Cells that have

[0177] 45801771.1 19 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0178] been successfully transduced then can be selected, for example, for expression of the coding sequence or of a drug resistance gene. The cells then can be lethally irradiated (if desired) and injected or implanted into the subject. In one form, expression vectors containing nucleic acids encoding fusion proteins are transfected into cells that are administered to a subject in need thereof.

[0179] In vivo nucleic acid therapy can be accomplished by direct transfer of a functionally active RNA or DNA into mammalian somatic tissue or organ in vivo. Nucleic acids may also be administered in vivo by viral means. Nucleic acid molecules encoding polypeptides or fusion proteins may be packaged into retrovirus vectors using packaging cell lines that produce replication-defective retroviruses, as is well-known in the art. Other virus vectors may also be used, including recombinant adenoviruses and vaccinia virus, which can be rendered non-replicating. In addition to naked DNA or RNA (e.g., mRNA), or viral vectors, or engineered bacteria may be used as vectors.

[0180] Nucleic acids may also be delivered by other carriers, including liposomes, polymeric micro- and nanoparticles, including but not limited to polymeric, liposomal, and protein nanoparticles, and polycations.

[0181] In addition to virus- and carrier-mediated gene transfer in vivo, physical means well-known in the art can be used for direct transfer of DNA, including administration of plasmid DNA and particle-bombardment mediated gene transfer.

[0182] 2. Oligonucleotide Composition

[0183] The disclosed nucleic acids nucleic acids can be DNA or RNA nucleotides which typically include a heterocyclic base (nucleic acid base), a sugar moiety attached to the heterocyclic base, and a phosphate moiety which esterifies a hydroxyl function of the sugar moiety. The principal naturally-occurring nucleotides include uracil, thymine, cytosine, adenine and guanine as the heterocyclic bases, and ribose or deoxyribose sugar linked by phosphodiester bonds.

[0184] In some forms, the oligonucleotides are composed of nucleotide analogs that have been chemically modified to improve stability, half-life, or specificity or affinity for a target receptor, relative to a DNA or RNA counterpart. The chemical modifications include chemical modification of nucleobases, sugar moieties, nucleotide linkages, or combinations thereof. As used herein ‘modified nucleotide” or “chemically modified nucleotide” defines a nucleotide that has a chemical modification of one or more of the heterocyclic base, sugar moiety or phosphate moiety constituents. In some forms, the charge of the modified nucleotide is reduced compared to DNA or RNA oligonucleotides of the same nucleobase sequence. For example, the oligonucleotide can have low negative charge, no charge, or positive charge.

[0185] 45801771.1 20 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0186] Typically, nucleoside analogs support bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequence-specific fashion between the oligonucleotide analog molecule and bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). In some forms, the analogs have a substantially uncharged, phosphorus containing backbone.

[0187] C. Adjuvants

[0188] Immunologic adjuvants stimulate the immune system's response to a target antigen, but do not provide immunity themselves. Adjuvants can act in various ways in presenting an antigen to the immune system. Adjuvants can act as a depot for the antigen, presenting the antigen over a longer period of time, thus maximizing the immune response before the body clears the antigen. Examples of depot type adjuvants are oil emulsions. An adjuvant can also act as an irritant, which engages and amplifies the body's immune response.

[0189] The adjuvant may be without limitation AS03, AddaSO3, MF59, CpG, glucopyranosyl lipid A (GLA), alum (e.g., aluminum hydroxide, aluminum phosphate); saponins purified from the bark of the Q. saponaria tree such as Quil A (a mixture of more than 25 different saponin molecules), or subcombinations or individual molecules thereof such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy) phosphazene (PCPP polymer; Virus Research Institute, USA), Ht3 ligand, Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium), non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Montanide IMS (e.g., IMS 1312, water-based nanoparticles combined with a soluble immunostimulant, Seppic).

[0190] Adjuvants may be TLR ligands. Adjuvants that act through TLR3 include without limitation double-stranded RNA. Adjuvants that act through TLR4 include without limitation derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland). Adjuvants that act through TLR5 include without limitation flagellin.

[0191] Adjuvants that act through TLR7 and / or TLR8 include single-stranded RNA, oligoribonucleotides

[0192] 45801771.1 21 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0193] (ORN), synthetic low molecular weight compounds such as imidazoquinolinamines (e.g., imiquimod (R-837), resiquimod (R-848)). Adjuvants acting through TLR9 include DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODN), such as CpG ODN. Another adjuvant class is phosphorothioate containing molecules such as phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbone linkages.

[0194] The adjuvant can also be oil emulsions (e.g., Freund’s adjuvant); saponin formulations; virosomes and viral-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineralcontaining compositions (e.g., mineral salts, such as aluminum salts and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and / or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester formulations; polyphosphazene; muramyl peptides; imidazoquinolone compounds; and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0195] Adjuvant GLA-LS is a vaccine adjuvant that contains a synthetic TLR4 agonist, glucopyranosyl lipid A (GLA), suspended in a liposome emulsion (LS).

[0196] Adjuvant GLA-SE is a vaccine adjuvant that contains a synthetic TLR4 agonist, glucopyranosyl lipid A (GLA), suspended in a squalene oil-in-water emulsion (SE).

[0197] Adjuvants may also include immunomodulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-gamma), macrophage colony stimulating factor, and tumor necrosis factor.

[0198] Immunostimulatory complexes called ISCOMs are particulate antigen delivery systems having antigen, cholesterol, phospholipid and saponin (Quil A or other saponin) with potent immunostimulatory activity. ISCOMATRIX® is a particulate adjuvant having cholesterol, phospholipids and saponins (Quil A) but without containing antigen. See, e.g., U. S. Patent No. 9,149,520, Sun, et al., Volume 27, Issue 33, 16 luly 2009, Pages 4388-4401, and Morelli, et al., J Med Microbiol. 2012 Jul;61(Pt 7):935-43. doi: 10.1099 / jmm.0.040857-0. Epub 2012 Mar 22. This adjuvant has principally the same structure as ISCOMs, consisting of perforated cage-like particles of approximately 40 nm in diameter. The antigens can be formulated with ISCOMATRIX® to produce vaccines capable of antigen presentation and immunostimulants similar to ISCOMs-type formulations, but with a wider range of applicability, since its use is not limited to hydrophobic membrane proteins. Modifications of ISCOMs formulations and ISCOMATRIX® have also been developed to achieve a better association of some antigens, such as described in WO 98 / 36772.

[0199] ISCOMs and ISCOMATRIX® combine the advantages of a particulate delivery system with the in situ presence of an adjuvant (Quil A) and consequently have been found to be more

[0200] 45801771.1 22 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0201] immunogenic than other colloidal systems such as liposomes and protein micelles. Formulations of ISCOMs and ISCOMATRIX® retained the adjuvant activity of the Quil A, while increasing its stability, reducing its hemolytic activity, and producing less toxicity. They also generate a similar immune response to the one obtained by immunizing with simple mixtures of antigen and saponin, but allow for the use of substantially smaller amounts of antigen. Several ISCOMs-type vaccine formulations or containing ISCOMATRIX® have been approved for veterinary use, for example against equine influenza virus.

[0202] Other liposomal systems mainly composed of saponins from Q. saponaria and sterols (primarily cholesterol) have been described, one of which is referred to as ASO1B. See, e.g., WO 96 / 33739, being also formulated as emulsions such as described in US 2005 / 0220814. See, also, U. S. Published Application No. 2011 / 0206758.

[0203] Iscomatrix-like adjuvants such as ISCOMATRIX® are thought to function via canonical inflammasome activation and subsequent release of pro-inflammatory cytokines such as IL-18 and IL-ip (Wilson, et al., Journal of immunology. 2014; 192(7): 3259-68. doi:

[0204] 10.4049 / jimmunol.l302011. PubMed PMID: 24610009). This mechanism is thought to be mediated at least in-part by endosomal degradation and the release of NRLP3-activating cathepsin proteases into the cytosol.

[0205] D. Delivery Vehicles

[0206] The disclosed compounds can be administered and / or taken up into the cells of a subject with or without the aid of a delivery vehicle. Appropriate delivery vehicles for the disclosed compositions are known in the art and can be selected to suit the particular composition.

[0207] In some forms, the antigens, or nucleic acids encoding the same are delivered to a subject in need thereof using nanoparticles, hydrogel, or a combination thereof. In some forms, the compositions are formulated for extended release of antigen.

[0208] 1. Nanoparticles

[0209] In some forms, the nucleic acid or protein is incorporated into or encapsulated by or absorbed onto a nanoparticle or microparticle (e.g., polymeric, liposomal, protein, etc.), micelle, synthetic lipoprotein particle, or carbon nanolube. For example, the compositions can be incorporated onto or into a vehicle such as polymeric or protein microparticles or nanoparticles which provide protection and / or controlled release of the compound. In some forms, release of the compound(s) is controlled by diffusion of the compound out of the microparticles or nanoparticles and / or degradation of the particles by hydrolysis and / or enzymatic degradation.

[0210] The compositions can include a biodegradable or bioerodible material in which the active agent, such an antigen, is embedded or encapsulated. Any of the active agents including, but not

[0211] 45801771.1 23 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0212] limited to, therapeutic, nutritional, diagnostic, prophylactic agents, etc., can be, but need not necessarily be, delivered to the target cells using a particle-based delivery vehicle. Thus, the active agent can be encapsulated and / or entrapped and / or dispersed in a particle(s). Compositions can include a plurality of particles having an active agent encapsulated and / or entrapped and / or dispersed therein and / or adhered thereon, in a pharmaceutically-acceptable carrier. In some forms, the formulations is for suitable for or otherwise designed for subcutaneous delivery, intravenous delivery, infusion, inhalation, internal delivery, or a combination thereof.

[0213] Nanoparticles can improve antigen stability, promote uptake by antigen-presenting cells, and preferentially target lymphatic tissues. Tissue targeting, antigen release rates, and penetration of epithelial barriers can be tuned by altering the physicochemical properties of the nanoparticles.

[0214] Any one or more of the disclosed active agents, e.g., protein and fusion protein antigens and / or adjuvants can be associated with nanoparticles. The nanoparticle formulation of the each of the active agents can be the same or different. In some forms, some of the antigens are associated with nanoparticles and some are not. Studies presented in the examples below show that Rv3351c-ESAT6 and HBHA can each can be associated with the nanoparticles.

[0215] fhe particles can be capable of controlled release of the active agent. The particles can be microparticle(s) and / or nanoparticle(s). The particles can include one or more polymers. One or more of the polymers can be a synthetic polymer. The particle or particles can be formed by, for example, single emulsion technique or double emulsion technique or nanoprecipitation.

[0216] In some forms, some of the compositions are packaged in particles and some are not. For example, one or more antigens can be incorporated into the same or separate particles while a coadministered other antigen(s) and / or adjuvant is not. Different compositions can be packaged in the same particles or different particles. For example, two or more active agents can be mixed and packaged together. In some forms, the different compositions are packaged separately into separate particles wherein the particles are similarly or identically composed and / or manufactured. In some forms, the different compositions are packaged separately into separate particles wherein the particles are differentially composed and / or manufactured.

[0217] The delivery vehicles can be nanoscale compositions, for example, 0.5 mu up to, but not including, about 1 micron. In some forms, and for some uses, the particles can be smaller, or larger. Thus, the particles can be microparticles, supraparticles, etc. For example, particle compositions can be between about 1 micron to about 1000 microns. Such compositions can be referred to as microparticulate compositions.

[0218] Nanoparticles generally refers to particles in the range of less than 0.5 nm up to, but not including 1,000 nm. In some forms, the nanoparticles have a diameter between 500 nm to less than

[0219] 45801771.1 24 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0220] 0.5 nm, or between 50 and 500 nm, or between 50 and 300 nm. Cellular internalization of polymeric particles can highly dependent upon their size, with nanoparticulate polymeric particles being internalized by cells with much higher efficiency than micoparticulate polymeric particles. For example, Desai, et al. have demonstrated that about 2.5 times more nanoparticles that are 100 nm in diameter are taken up by cultured Caco-2 cells as compared to microparticles having a diameter on 1 pM (Desai, et al., Pharm. Res., 14:1568-73 (1997)). Nanoparticles also have a greater ability to diffuse deeper into tissues in vivo.

[0221] In some forms, particularly those in which the particles need not be internalized by cells, the particles can be microparticles. Microparticle generally refers to a particle having a diameter, from about 1 micron to about 100 microns. The particles can also be from about 1 to about 50 microns, or from about 1 to about 30 microns, or from about 1 micron to about 10 microns. The microparticles can have any shape. Microparticles having a spherical shape may be referred to as “microspheres.”

[0222] Supraparticles are particles having a diameter above about 100 pm in size. For example, supraparticle may have a diameter of about 100 pm to about 1,000 pm in size.

[0223] The particles can have a mean particle size. Mean particle size generally refers to the statistical mean particle size (diameter) of the particles in the composition. Two populations can be said to have a substantially equivalent mean particle size when the statistical mean particle size of the first population of particles is within 20% of the statistical mean particle size of the second population of particles; more preferably within 15%, most preferably within 10%.

[0224] The weight average molecular weight can vary for a given polymer but is generally from about 1000 Daltons to 1,000,000 Daltons, 1000 Daltons to 500,000 Dalton, 1000 Daltons to 250,000 Daltons, 1000 Daltons to 100,000 Daltons, 5,000 Daltons to 100,000 Daltons, 5,000 Daltons to 75,000 Daltons, 5,000 Daltons to 50,000 Daltons, or 5,000 Daltons to 25,000 Daltons.

[0225] In some forms, the nanoparticles include at least 50, 60, or 70% antigen encapsulation; 200-400 nm nanoparticle size, low poly dispersity index (<0.2), sustained antigen release, stability on storage at 4 deg C, or any combination thereof.

[0226] Particles are can be formed of one or more polymers. Exemplary polymers are discussed below. Copolymers such as random, block, or graft copolymers, or blends of the polymers listed below can also be used.

[0227] Functional groups on the polymer can be capped to alter the properties of the polymer and / or modify (e.g., decrease or increase) the reactivity of the functional group. For example, the carboxyl termini of carboxylic acid contain polymers, such as lactide- and glycolide-containing polymers,

[0228] 45801771.1 25 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0229] may optionally be capped, e.g., by esterification, and the hydroxyl termini may optionally be capped, e.g. by etherification or esterification.

[0230] Copolymers of PEG or derivatives thereof with any of the polymers described below may be used to make the polymeric particles. In certain forms, the PEG or derivatives may be located in the interior positions of the copolymer. Alternatively, the PEG or derivatives may locate near or at the terminal positions of the copolymer. For example, one or more of the polymers above can be terminated with a block of polyethylene glycol. In some forms, the core polymer is a blend of pegylated polymer and non-pegylated polymer, wherein the base polymer is the same (e.g., PLGA and PLGA-PEG) or different (e.g., PLGA-PEG and PLA). In certain forms, the microparticles or nanoparticles are formed under conditions that allow regions of PEG to phase separate or otherwise locate to the surface of the particles. The surface-localized PEG regions alone may perform the function of, or include, the surface- altering agent. In particular forms, the particles are prepared from one or more polymers terminated with blocks of polyethylene glycol as the surface- altering material.

[0231] Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives. Polymers which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide may also be suitable as materials for drag containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybut rate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0232] Other examples of preferred biodegradable polymers include synthetic polymers that degrade by hydrolysis such as poly(hydroxy acids), such as polymers and copolymers of lactic acid and glycolic acid, other degradable polyesters, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), poly(lactide-co-caprolactone), and poly(amine-co-ester) polymers, such as those described in Zhou, et al., Nature Materials, 11 (l):82-90 (2011), Tietjen, el al. Nature Communications, 8:191 (2017) doi:10.1038 / s41467-017-00297-x, and WO 2013 / 082529, U. S. Published Application No.

[0233] 2014 / 0342003, and PCT / US2015 / 061375.

[0234] Natural polymers include alginate and other polysaccharides, collagen, albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.

[0235] 45801771.1 26 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0236] Exemplary polymers also include, but are not limited to, cyclodextrin-containing polymers, in particular cationic cyclodextrin-containing polymers, such as those described in U. S. Patent No.

[0237] 6,509.323,

[0238] In some forms, non-biodegradable polymers can be used, especially hydrophobic polymers. Examples of preferred non-biodegradable polymers include ethylene vinyl acetate, poly(meth) acrylic acid, copolymers of maleic anhydride with other unsaturated polymerizable monomers, poly(butadiene maleic anhydride), polyamides, copolymers and mixtures thereof, and dextran, cellulose and derivatives thereof.

[0239] Other suitable biodegradable and non-biodegradable polymers include, but are not limited to, polyanhydrides, polyamides, polycarbonates, poly alkylenes, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as polyethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate) and ethylene vinyl acetate polymer (EVA), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters such as poly( vinyl acetate), polyethylene, polypropylene, poly(vinyl acetate), poly vinyl chloride, polystyrene, polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polyvinylpyrrolidone, polymers of acrylic and methacrylic esters, polysiloxanes, polyurethanes and copolymers thereof, modified celluloses, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, and polyacrylates such as poly(methyl methacrylate), poly(ethylmethacrylate), Poly(2-hydroxyethyl methacrylate) (pHEMA), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate). These materials may be used alone, as physical mixtures (blends), or as co-polymers.

[0240] The polymer may be a bioadhesive polymer that is hydrophilic or hydrophobic. Hydrophilic polymers include CARBOPOL™ (a high molecular weight, crosslinked, acrylic acid-based polymers such as those manufactured by NOVEON™), polycarbophil, cellulose esters, and dextran, polymers of acrylic acids, include, but are not limited to, poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as "polyacrylic acids").

[0241] 45801771.1 27 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0242] Release rate controlling polymers may be included in the polymer matrix or in the coating on the formulation. Examples of rate controlling polymers that may be used are hydroxypropylmethylcellulose (HPMC) with viscosities of either 5, 50, 100 or 4,000 cps or blends of the different viscosities, ethylcellulose, methylmethacrylates, such as EUDRAGIT® RS 100, EUDRAGIT® RL100, EUDRAGIT® NE 30D (supplied by Rohm America). Gastrosoluble polymers, such as EUDRAGIT® E100 or enteric polymers such as EUDRAGIT® L100-55D, L100 and SI 00 may be blended with rate controlling polymers to achieve pH dependent release kinetics. Other hydrophilic polymers such as alginate, polyethylene oxide, carboxymethylcellulose, and hydroxyethylcellulose may be used as rate controlling polymers.

[0243] These polymers can be obtained from sources such as Sigma Chemical Co., St. Louis, MO; Polysciences, Warrenton, PA; Aldrich, Milwaukee, WI; Fluka, Ronkonkoma, NY; and BioRad, Richmond, CA, or can be synthesized from monomers obtained from these or other suppliers using standard techniques.

[0244] In certain forms, the hydrophobic polymer is an aliphatic polyester. In preferred forms, the hydrophobic polymer is polyhydroxyester such as poly(lactic acid), poly(glycolic acid), or poly (lactic acid-co-gly colic acid).

[0245] Other polymers include, but are not limited to, polyalkyl cyanoacralate, polyamino acids such as poly-L-lysine (PLL), poly(valeric acid), and poly-L-glutamic acid, hydroxypropyl methacrylate (HPMA), polyorthoesters, poly(ester amides), poly(ester ethers), polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poly(butyric acid), trimethylene carbonate, and polyphosphazenes.

[0246] The particles can be designed to release molecules to be encapsulated or attached over a period of days to months. Nonlimiting factors that affect the release profiles of molecules from particles include pH, temperature, polymer composition, formulation parameters, molecule-polymer interactions, particle size, and particle porosity. Aliphatic polyesters differ in hydrophobicity and that in turn affects the degradation rate. The hydrophobic poly (lactic acid) (PLA), more hydrophilic poly (glycolic acid) PGA and their copolymers, poly (lactide-co-glycolide) (PLGA) have different release rates. The degradation rate of these polymers, and often the corresponding drug release rate, can vary from days (PGA) to months (PLA) and is easily manipulated by varying the ratio of PLA to PGA. In some examples, the release rate of molecules from PLGA is dependent on the molecular weight of the PLGA polymer, the weight ratio of lactic acid to glycolic acid, as well as the polymer end-cap (carboxylic acid or ester). Rates of molecule release from PLGA and polymer degradation are tunable by varying the ratio of lactic acid to glycolic acid (L: G). For example, some forms may utilize ratios of 50:50 (L: G), 65:35 (L: G), 75:25 45801771.1 28 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0247] (L: G), 85:15 (L: G), or 100:0 (L: G). Release rates of molecules are also tuned by the chemistry of the terminal end of PLGA. Nonlimiting examples of terminal end groups on PLGA include carboxylic acid, esters, and hydroxyl groups.

[0248] In some preferred forms, the particles can contain one more of the following polyesters: homopolymers including glycolic acid units, referred to herein as “PGA”, and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D, L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D, L-lactide, collectively referred to herein as “PLA”, and caprolactone units, such as poly(8-caprolactone), collectively referred to herein as " POL"; and copolymers including lactic acid and glycolic acid units, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) characterized by the ratio of lactic acid:glycolic acid, collectively referred to herein as “PLGA”; and polyacrylates, and derivatives thereof. Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as " PEGylated polymers". In certain forms, the PEG region can be covalently associated with polymer to yield " PEGylated polymers" by a cleavable linker. For example, particles can also contain one or more polymer conjugates containing end-to-end linkages between the polymer and a targeting moiety or a detectable label. For example, a modified polymer can be a PLGA-PEG-peptide block polymer.

[0249] The in vivo stability / release of the particles can be adjusted during the production by using polymers such as poly(lactide-co-glycolide) copolymerized with polyethylene glycol (PEG). If PEG is exposed on the external surface, it may increase the time these materials circulate due to the hydrophilicity of PEG.

[0250] In some forms, the particles are wax particles (see, Hart, et al., Nanoparticle-Fusion Protein Complexes Protect against Mycobacterium tuberculosis Infection, Mol Ther. 2018 Mar 7;26(3):822-833. doi: 10.1016 / j.ymthe.2017.12.016. Epub 2017 Dec 22, which is specifically incorporated by reference herein in its entirety) or polylactic-co-glycolic acid (PLGA) (see, Shepherd, S. D., O’Buckley, S. C., Harrington, J. M. et al. A moldable sustained release bupivacaine formulation for tailored treatment of postoperative dental pain. Sci Rep 8, 12172 (2018).

[0251] An exemplary wax particles can be produced via the emulsification of yellow carnauba (YC) palm wax with sodium myristate (NaMA) (see, e.g., Arias M. A., Loxley A., Eatmon C., Van Roey G., Fairhurst D., Mitchnick M., Dash P., Cole T., Wegmann F., Sattentau Q., Shattock R. Carnauba wax nanoparticles enhance strong systemic and mucosal cellular and humoral immune responses to HIV-gpl40 antigen. Vaccine. 2011;29:1258-1269. doi: 10.1016 / j.vaccine.2010.11.084, which is specifically incorporated by reference herein in its entirety). YC wax and its derivatives are classified by the European Food Standards Agency as non-toxic, and their use is licensed in a wide

[0252] 45801771.1 29 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0253] range of edible and cosmetic products. These NPs have an average diameter of —400 nm (ranging from 200 to 800 nm) and are anionic, with a zeta potential of approximately -75 mV, thus imparting a high colloidal stability on YC-NaMA in suspension. Proteins can be absorbed on the particles by incubating the particles and protein, e.g., 1 hr at room temperature, and the mixture can then be centrifuged, e.g., at 100,000 x g for 30 min, to collect the loaded particles.

[0254] 2. Hydrogels

[0255] In some forms, the compositions additionally or alternatively include a Hydrogel-based vaccine delivery platform. The platform can include a self-assembling gel matrix that is biocompatible and biodegradable to deliver vaccines such that the vaccine antigens are released over time. The delivery platform reduces the immediate pro-inflammatory response in the host and enhances immunogenicity.

[0256] In some forms the hydrogel provides improved focal and sustained delivery of antigen at the site of vaccine administration; enhancing antigen presenting cell activation and antigen uptake and processing. In some forms, one or more immunogenic agents is administered in a composition including a slurry matrix component that is a liquid at room temperature, non-physiological pH, and / or low salt concentrations and a gel at physiological salt concentrations physiological pH, and / or physiological temperatures. Gelling may be induced by the physiological body temperature of a vertebrate, such as a mammal or bird. Such a temperature may be, for example, at least about 25° Celsius (C), at least about 30° Celsius, at least about 32° Celsius, at least about 35° Celsius, at least about 37° Celsius, at least about 39° Celsius, or at least about 40°. Gelling may be induced by the physiological salt concentrations. In some forms, gelling may in the presence of millimolar concentrations of salt, for example by a salt concentration of greater than about 0.05 molar (M). Gelling may be induced by the physiological pH of a vertebrate, such as a mammal or bird.

[0257] Thus, the vaccine composition gels or polymerizes after administration to a subject, localizing the vaccine antigens to a single site where innate antigen presenting cells can home and begin taking up vaccine antigens. Ideally, the slurry matrix is a biocompatible material will not induce undesirable reactions in the body as a result of contact with bodily fluids or tissue, such as tissue death, tumor formation, allergic reaction, foreign body reaction (rejection), inflammatory reaction, antibody response, or blood clotting, for example. A slurry matrix may also be referred to herein or elsewhere in the art as a “biomedical polymer hydrogel,” a “biomedical hydrogel,” “biomedical polymer,” a “polymer hydrogel,” a “biocompatible polymer hydrogel,” a “biocompatible hydrogel,” a “biocompatible polymer,” or a “hydrogel.” As used herein, a “hydrogel” is a 3-dimensional network of cross-linked, hydrophilic macromolecules capable of being swelled and incorporating about 20 percent to about 95 percent water by weight. A hydrogel

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[0259] is a gel in which the liquid constituent is water. As used herein, a gel is a solid, jelly-like material that can have properties ranging from soft and weak to hard and tough. A gel is a substantially dilute cross-linked system, which exhibits no flow when in the steady-state. By weight, gels are mostly liquid, yet they behave like solids due to a three-dimensional cross-linked network within the liquid. It is the crosslinks within the fluid that give a gel its structure (hardness). In this way gels are a dispersion of molecules of a liquid within a solid in which the solid is the continuous phase and the liquid is the discontinuous phase. Hydrogel is a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (they can contain over 99.9% water) natural or synthetic polymers. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content.

[0260] Any of a wide variety of biomedical polymer hydrogels available for use in medical technologies may be used with the methods and compositions described herein. In some forms, the slurry matrix is naturally occurring, such as for example, fibrin, collagen, elastin, agarose, methylcellulose, hyaluronan, and other naturally derived polymers. In some forms, the slurry matrix is MATRIGEL, or a derivative thereof. MATRIGEL is a basal membrane extract from mouse cells and includes laminin, collagen IV, entactin, nidogen, and proteoglycans. The invasion of tumor cells into MATRIGEL has been used to study the involvement of extracellular matrix receptors and matrix degrading enzymes in tumor progression and invasion and MATRIGEL has been used also as an in vitro and in vivo angiogenesis model (MATRIGEL plug assay) to study the activity of angiogenic and anti-angiogenic cytokines and other substances. MATRIGEL is commercially available as BD MATRIGEL™ Matrix. See the world wide web at bdbiosciences.com / cellculture / ecm / ecmtypes / index.jsp.

[0261] In some forms, a slurry matrix is synthetic, such as for example, a synthetic peptide hydrogel or a self-assembling peptide (sapeptide) scaffold. The sapeptide scaffolds are formed through the spontaneous assembly of ionic self-complementary beta-sheet oligopeptides under physiological conditions, producing a hydrogel material. These short peptides (typically about 8, about 12, about 16, about 24, or about 32 amino acid residues with internally-repeating sequences) self-assemble in aqueous salt solution into three-dimensional matrices. The peptides are characterized as being amphiphilic, having alternating hydrophobic and hydrophilic amino acid residues; greater than 12 amino acids, and preferably at least 16 amino acids; complementary and structurally compatible. Complementary refers to the ability of the peptides to interact through ionized pairs and / or hydrogen bonds which form between their hydrophilic side-chains, and structurally compatible refers to the ability of complementary peptides to maintain a constant

[0262] 45801771.1 31 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0263] distance between their peptide backbones. Peptides having these properties participate in intermolecular interactions which result in the formation and stabilization of beta-sheets at the secondary structure level and interwoven filaments at the tertiary structure level. Examples include, but are not limited to, peptide family members, RADA (SEQ ID NO:28), referred to as “RADA”; RADARADARADARADA (SEQ ID NO:24), referred to as “RAD16-I” and “(RADA)(4)”;

[0264] RARAD DARARADADA (SEQ ID NO:25), referred to as “RAD1641” and “RARADADA)(2)”; FKFEFKFE (SEQ ID NO:26), referred to as “KFE-8” and “(FKFE)(2)”; and KLDLKLDLKLDL (SEQ ID NO:27), referred to as “KLD-12” and “(KLDL)(3)”.

[0265] See, for example, U. S. Pat. No. 5,670,483; Holmes et al., 2000, Proc Natl Acad Sci USA; 97(12):6728-33; Yokoi et al., 2005, Proc Natl Acad Sci USA; 102(24):8414-9; Liu et al., 2012, Nanoscale; 4(8):2720-7, and BD PURAMATRIX™ Peptide Hydrogel, Guidelines for Use, Catalog Number 354250 (SPC-354250-G rev 2.0; BD Biosciences, Bedford, Mass.). In some aspects, a peptide hydrogel includes the peptide scaffold self-assembling building blocks of arginine-alanine-aspartate-alanine (RADA (SEQ ID NO:28)). In some aspects, a peptide hydrogel includes RADARADARADARADA (SEQ ID NO:24), or a derivative thereof. In some aspects, the peptide hydrogel includes PURAMATRIX, or a derivative thereof. Sec, for example, U. S. Pat. No.

[0266] 5,670,483; Holmes et al., 2000, Proc Natl Acad Sci USA; 97(12):6728-33; Yokoi et al., 2005, Proc Natl Acad Sci USA; 102(24):8414-9; Liu et al., 2012, Nanoscale; 4(8):2720-7, and BD PURAMATRIX™ Peptide Hydrogel, Guidelines for Use, Catalog Number 354250 (SPC-354250-G rev 2.0; BD Biosciences, Bedford, Mass.), each of which are incorporated herein in their entireties.

[0267] In some forms, a biomedical polymer hydrogel may be a polyethylene glycol (PEG) hydrogel that polymerizes spontaneously in vivo.

[0268] In some forms, a slurry matrix, in addition to gelling at vertebrate or mammalian body temperature and / or physiological salt concentrations, is a bioresorbable synthetic polymer that degrades and dissolves with time. Such compounds are naturally degraded in the body by hydrolysis and absorbed as water-soluble monomers. Examples include, polylactic acid, polylactide (PLA), poly (L-lactic acid), poly-D-lactide, polyglycolic acid (PGA), polyglycolide and its copolymers (poly(lactic-co-glycolic acid) with lactic acid, homo- and copolymers of lactic acid and glycolic acid, poly (DL-lactic acid / glycinc) copolymers, poly (DL-lacticco-glycolic acid) (PLGA), poly (DL-lacticco-glycolic acid) (PLGA), porous poly(DL-lactic-co-glycolic acid) foams, poly(amino acids) poly [ox(l -oxo- 1,2-ethanediyl)] ((C2H2O2)n: Biovek), poly(glycolide-co-caprolactone), poly(glycolide-co-trimethylene carbonate), polydioxanone (PDO, PDS), poly-p-dioxanone, caprolactone (also referred to as 2-oxepanone), epsilon-caprolactone, 6-hexanolactone, hexano-6-lactone, l-oxa-2-oxocycloheptane polyglactin 910. polyanhydrides, and polyorthoester 45801771.1 32 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0269] films formed from poly (D, L-lactic-co-glycolic acid, 88:12) (PLGA) or from a 50 / 50 (w / w) blend of PLGA and poly (L-lactic acid) (PLLA). See, for example, Schakenraad and Dijkstra, 1991, Clin Mater; 7(3):253-69; Mooney et al., 1997, J Biomed Mater Res; 37(3):413-20: and Lu et al., 2000, Biomaterials; 21 (18): 1837 -45.

[0270] For example, in some forms, the disclosed antigens are provided and / or administered to a subject in need thereof in an immunogenic or vaccine composition including the antigens optionally encapsulated or adhered into or onto nanoparticles, and a peptide hydrogel that is a liquid at room temperature and a gel at physiological pH, physiological salt concentrations, and / or physiological temperatures optionally wherein the peptide hydrogel includes a self-assembling peptide selected from the group consisting of RADA (SEQ ID NO:28), referred to as “RADA”;

[0271] RAD ARAD ARAD ARADA (SEQ ID NO:24), referred to as “RAD16-I” and “(RADA)(4)”;

[0272] RARADADARARADADA (SEQ ID NO:25), referred to as “RAD1641” and “RARADADA)(2)”; FKFEFKFE (SEQ ID NO:26), referred to as “KFE-8” and “(FKFE)(2)”; KLDLKLDLKLDL (SEQ ID NO:27), referred to as “KLD-12” and “(KLDL)(3)”; and combinations thereof, and optionally further including an adjuvant, optionally such as a TLR agonist such as a CpG oligodeoxynucleotide (ODN). Exemplary hydrogel forms are, e.g. 1.0% W / V of the (RADA)(4) peptide in sterile water buffered to pH 3.5 with HC1 optionally, but preferably for subcutaneous injection formulations and 0.5% W / V optionally, but preferably, for intranasal formulations.

[0273] See, e.g., Grenfell RFQ Shollenberger LM, Samli EF, Harn DA. 2015. Vaccine SelfAssembling Immune Matrix Is a New Delivery Platform That Enhances Immune Responses to Recombinant HBsAg in Mice. Clin Vaccine Immunol 22:doi.org / 10.1128 / CVI.00714-14, Samli, “VacSIM™: a Self-assembling immune matrix that enhances vaccine efficacy,” dissertation dated 2015, and U. S. Patent Nos. 9,566,338, 11,524,072, and 12,186,390, each of which is specifically incorporated by reference herein in its entirety. In some forms, the composition includes VacSIM™.

[0274] 3. Nucleic Acid Delivery

[0275] If the compound is a nucleic acid or vector, the delivery vehicle can be or include a viral vector, for example a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). The viral vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g.. Pastan et al., (1988) Proc. Natl. Acad. Sci. U. S. A. 85:4486; Miller et al., (1986) Mol. Cell. Biol. 6:2895). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding the compound. The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are

[0276] 45801771.1 33 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0277] widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948 (1994)), adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492-1500 (1994)), lentiviral vectors (Naidini et al., Science 272:263-267 (1996)), pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol. 24:738-747 (1996)).

[0278] Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood ^>1A12- H3 (1996)). For example in some forms, the composition is delivered via a liposome. Commercially available liposome preparations such as LIPOFECTIN, LIPOFECT AMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECT AM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to procedures standard in the art are well known. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.). This disclosed compositions and methods can be used in conjunction with any of these or other commonly used gene transfer methods.

[0279] E. Formulations

[0280] The disclosed compounds can be formulated in a pharmaceutical composition.

[0281] Pharmaceutical compositions can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), enteral, transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, pulmonary, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0282] The compositions can be administered locally or systemically.

[0283] 1. Formulations for Parenteral Administration

[0284] Compounds and pharmaceutical compositions thereof can be administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of the active agent(s) and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions include diluents sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally, additives such as detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to as POLYSORBATE® 20 or 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol,

[0285] 45801771.1 34 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0286] polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.

[0287] Recombinantly modified virus can also be introduced into a host with a physiologically acceptable carrier and / or adjuvant. Useful carriers are well known in the art, and include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

[0288] 2. Formulations for Mucosal and Pulmonary

[0289] Administration

[0290] Active agent(s) and compositions thereof can be formulated for pulmonary or mucosal administration. The administration can include delivery of the composition to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. In a particular form, the composition is formulated for and delivered to the subject sublingually.

[0291] In one form, the compounds are formulated for pulmonary delivery, such as intranasal administration or oral inhalation. The respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream. The lungs are branching structures ultimately ending with the alveoli where the exchange of gases occurs. The alveolar surface area is the largest in the respiratory system and is where drug absorption occurs. The alveoli are covered by a thin epithelium without cilia or a mucus blanket and secrete surfactant phospholipids. The respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchiole, which then lead to the ultimate respiratory zone, the alveoli, or deep lung. The deep lung, or alveoli, is the primary target of inhaled therapeutic aerosols for systemic drug delivery.

[0292] Pulmonary administration of therapeutic compositions composed of low molecular weight drugs has been observed, for example, beta- androgenic antagonists to treat asthma. Other therapeutic agents that are active in the lungs have been administered systemically and targeted via pulmonary absorption. Nasal delivery is considered to be a promising technique for administration of therapeutics for the following reasons: the nose has a large surface area available for drug absorption due to the coverage of the epithelial surface by numerous microvilli, the subepithelial layer is highly vascularized, the venous blood from the nose passes directly into the systemic circulation and therefore avoids the loss of drug by first-pass metabolism in the liver, it offers lower

[0293] 45801771.1 35 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0294] doses, more rapid atainment of therapeutic blood levels, quicker onset of pharmacological activity, fewer side effects, high total blood flow per cm3, porous endothelial basement membrane, and it is easily accessible.

[0295] The term aerosol as used herein refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant. Aerosols can be produced using standard techniques, such as ultrasonication or high-pressure treatment.

[0296] Carriers for pulmonary formulations can be divided into those for dry powder formulations and for administration as solutions. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. For administration via the upper respiratory tract, the formulation can be formulated into a solution, e.g., water or isotonic saline, buffered or un-buffered, or as a suspension, for intranasal administration as drops or as a spray. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2. One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and / or upper respiratory administration.

[0297] Preferably, the aqueous solution is water, physiologically acceptable aqueous solutions containing salts and / or buffers, such as phosphate buffered saline (PBS), or any other aqueous solution acceptable for administration to an animal or human. Such solutions are well known to a person skilled in the art and include, but are not limited to, distilled water, de-ionized water, pure or ultrapure water, saline, phosphate-buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer’s solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0298] In another form, solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ethyl ether, and propanol may be used for the formulations. The solvent is selected based on its ability to readily aerosolize the formulation. The solvent should not detrimentally react with the compounds. An appropriate solvent should be used that dissolves the compounds or forms a suspension of the compounds. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as freons, can be added as desired to increase the volatility of the solution or suspension.

[0299] 45801771.1 36 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0300] In some forms, compositions may contain polymers, surfactants or other excipients well known to those of the art. In this context, “minor amounts” means no excipients are present that might affect or mediate uptake of the compounds in the lungs and that the excipients that are present are present in amount that do not adversely affect uptake of compounds in the lungs.

[0301] Dry lipid powders can be directly dispersed in ethanol because of their hydrophobic character. For lipids stored in organic solvents such as chloroform, the desired quantity of solution is placed in a vial, and the chloroform is evaporated under a stream of nitrogen to form a dry thin film on the surface of a glass vial. The film swells easily when reconstituted with ethanol. To fully disperse the lipid molecules in the organic solvent, the suspension is sonicated. Nonaqueous suspensions of lipids can also be prepared in absolute ethanol using a reusable PARI LC Jet-r-nebulizer (PARI Respiratory Equipment, Monterey, CA).

[0302] Dry powder formulations (“DPFs”) with large particle size have improved flowability characteristics, such as less aggregation, easier aerosolization, and potentially less phagocytosis. Dry powder aerosols for inhalation therapy are generally produced with mean diameters primarily in the range of less than 5 microns, although a preferred range is between one and ten microns in aerodynamic diameter. Large “carrier” particles (containing no drug) have been co-delivered with therapeutic aerosols to aid in achieving efficient aerosolization among other possible benefits.

[0303] Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art. The preferred methods of manufacture are by spray drying and freeze drying, which entails using a solution containing the surfactant, spraying to form droplets of the desired size, and removing the solvent.

[0304] The particles may be fabricated with the appropriate material, surface roughness, diameter and tap density for localized delivery to selected regions of the respiratory tract such as the deep lung or upper airways. For example, higher density or larger particles may be used for upper airway delivery. Similarly, a mixture of different sized particles, provided with the same or different active agents may be administered to target different regions of the lung in one

[0305] 45801771.1 37 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0306] F. Immunogenic Compositions and Vaccines

[0307] Immunogenic compositions and vaccines are also provided. Typically, an immunogenic composition includes an adjuvant, an antigen (which may be e.g., polypeptide, a fusion protein, a nucleic acid encoding the same, or a virus having or encoding the polypeptide or fusion protein), or a combination thereof. The combination of an adjuvant and an antigen can be referred to as a vaccine. When administered to a subject in combination, the adjuvant and antigen can be administered in separate pharmaceutical compositions, or they can be administered together in the same pharmaceutical composition. The disclosed chimeric fusion proteins and nucleic acids encoding the same (e.g., mRNA) can serve as the antigen component of an immunogenic composition or vaccine formulation. In some forms of the antigen provided as a recombinant protein, the chimeric fusion protein is a monomeric form. In preferred forms of antigen provided as a recombinant protein, the chimeric fusion protein is a multimeric, preferably trimeric, form.

[0308] Additionally, the composition can include an adjuvant. Thus, in some forms, the composition includes both an antigen and an adjuvant. Two or more different antigens, one or more different adjuvants, or combinations thereof, can be used or combined. In particular forms, the formulation is an adjuvant + protein or a nanoparticle-based vaccines with or without adjuvant and using mRNA or DNA as the means of delivering antigen.

[0309] Thus, immunogenic compositions and vaccines including an effective amount antigen(s), or a nucleic acid or virus having or encoding the same, to induce an immune response thereto are provided alone and in combination with an effective amount of adjuvant to optionally further increase the immune response. The immunogenic compositions and vaccines can be used in methods of treating and preventing Mtb infections.

[0310] III. Methods of Use

[0311] Methods of inducing an immune response in a subject (e.g., a human) by administering to the subject a therapeutically effective amount of a disclosed immunogenic or vaccine composition are provided.

[0312] The genus Mycobacterium comprises more than 170 species, most of which are environmental organisms.

[0313] TB is an infectious disease usually caused in humans primarily by Mycobacterium tuberculosis Mtb) bacteria. The Mycobacterium tuberculosis complex (MTBC) includes nine other known TB-causing mycobacteria: M. bovis, M. africanum, M. canettii, M. orygis, M. microti. M caprae, M pinnipedii, M mungi, M suricattae, and the yet-unnamed dassie bacillus and chimpanzee bacillus.

[0314] 45801771.1 38 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0315] Mycobacterium africanum is not widespread, but it is a significant cause of human TB in parts of Africa. Mycobacterium bovis and M. orygis are the primary causes of TB in domestic and wild animals and can infect humans primarily through consumption of contaminated milk and cheese The introduction of pasteurized milk has almost eliminated this as a public health problem in developed countries, but it remains a problem underdeveloped countries. M. canettii is rare and seems to be limited to the Horn of Africa, although a few cases have been seen in African emigrants. M. microti is also rare and is seen almost only in immunodeficient people, although its prevalence may be significantly underestimated.

[0316] Thus, the immune response can be directed against any one or more of Mtb, M. bovis, M. africanum, M. canettii, M. orygis, M. microti, M. leprae, M caprae, M pinnipedii, M microti, M mungi, M suricattae, or to-be defined tuberculous mycobacteria.

[0317] In some forms, the subject is infected with any one or more of Mtb, M. bovis, M. africanum, M. canettii, M. orygis, M. microti, M. caprae, M. pinnipedii, M. mungi, or M. suricattae. In some forms, the subject has TB or a lung disease that resembles TB. In some forms, the subject is not infected with Mtb, M. bovis, M. africanum, M. canettii, M. orygis, M. microti, M. leprae, M. caprae, M. pinnipedii, M. mungi, or M. suricattae. Although the protein sequences provided above are from Mtb, it will be appreciated that these proteins or their corresponding proteins from the foregoing organisms can be used in the preparation of the disclosed immunogenic and vaccine formations, and their associate methods of use.

[0318] In some forms, the subject does not have TB. In some forms, the subject has been exposed to a subject with TB. In some forms, the subject has TB.

[0319] Tuberculosis generally affects the lungs, but it can also affect other parts of the body. Most infections show no symptoms, in which case it is known as subclinical, incipient, or latent TB. It is unknown what percentage of subclinical or incipient cases can convert to active disease, but approximately 10% of latent infections do progress to active disease that, if left untreated, kill about half of those affected. Typical symptoms of active TB are chronic cough with blood-containing mucus, fever, night sweats, and weight loss. Infection of other organs can cause a wide range of symptoms.

[0320] In some forms, the disclosed methods reduce or prevent one or more symptoms of TB. The immune response can be induced, increased, or enhanced by the composition compared to a control (e.g., absence of the composition or presence of another composition). The composition can include an effective amount of a antigen(s) protein, a nucleic acid encoding the same such as a viral vector or mRNA, or any combination hereof.

[0321] 45801771.1 39 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0322] Adjuvant can optionally be delivered together or separately. In some forms the components, particular nucleic acids encoding the antigen(s) (e.g., mRNA or DNA) are delivered using nanoparticles, e.g., polymeric or liposomal nanoparticles and / or a hydrogel-based vaccine delivery platform such as VacSIM™. The immune response is typically against the antigen(s), and thus, preferably increases immunity against one or more, preferably two or more bacterial polypeptides.

[0323] In some forms, the disclosed compositions increase a B cell response. In some forms, a disclosed composition is administered to a subject in need thereof in an effective amount to induce an antigen-specific antibody response (e.g., IgG, IgG2a, IgGl, or a combination thereof), increase a response in germinal centers, increase plasmablast frequency, increase inflammatory cytokine expression, or a combination thereof.

[0324] In some forms, the administration of the composition alternatively or additionally induces a B-memory cell response in subjects administered the composition compared to a control. A B-memory cell response is intended to mean an increased frequency of peripheral blood B lymphocytes capable of differentiation into antibody-secreting plasma cells upon antigen encounter.

[0325] In some forms, the compositions can induce an effector cell response such as a CD4 or CD8 T-cell immune response, against at least one of the component antigen(s) or antigenic compositions compared to the effector cell response obtained under control conditions (e.g., absence of the composition or presence of another composition). The term “improved effector cell response’’ refers to a higher effector cell response such as a CD8 or CD4 response obtained in a subject after administration of a disclosed composition than that obtained under control conditions.

[0326] The described compositions may be administered as part of prophylactic vaccines or immunogenic compositions which confer resistance in a subject to subsequent exposure to infectious agents, or as part of therapeutic vaccines, which can be used to initiate or enhance a subject’s immune response to a pre-existing antigen.

[0327] The desired outcome of a prophylactic or therapeutic immune response may vary according to the disease or condition to be treated, or according to principles well known in the art. For example, an immune response against an infectious agent may completely prevent colonization and replication of an infectious agent, affecting “sterile immunity’’ and the absence of any disease symptoms. However, a vaccine against infectious agents may also be considered effective if it reduces the number, severity or duration of symptoms; if it reduces the number of individuals in a population with symptoms; or reduces the transmission of an infectious agent. Similarly, immune responses may completely treat a disease, may alleviate symptoms, or may be one facet in an overall therapeutic intervention against a disease.

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[0329] The disclosed compositions may be used in methods of inducing protective immunity against an infectious agent, disease, or condition by administering to a subject (e.g., a human) a therapeutically effective amount of the compositions. “Protective immunity” or “protective immune response” refers to immunity or eliciting an immune response against an infectious agent, which is exhibited by a subject (e.g., a human), that prevents or ameliorates an infection or reduces at least one symptom thereof.

[0330] In some forms, the methods include inducing the production of neutralizing antibodies or inhibitory antibodies in a subject (e.g., a human) by administering any of the disclosed compositions to the subject. In some forms, a disclosed composition is administered to a subject in need thereof in an effective amount to increase an antigen- specific antibody response (e.g., IgA, IgD, IgE, IgM, IgG, IgG2a, IgGl, or a combination thereof). The antibody response is important for preventing many infections and may also contribute to resolution of infection.

[0331] Upon administration of an immunogenic or vaccine composition as described herein, e.g., via injection, aerosol, droplet, oral, topical or other route, the immune system of the host responds to the composition by producing antibodies specific for the antigen(s). As a result of the vaccination the host becomes at least partially or completely immune to TB.

[0332] The host to which the compositions can be administered can be any mammal susceptible to infection by a Mycobacterium tuberculosis complex species such as Mtb and capable of generating a protective immune response to antigens of the bacteria. Thus, suitable hosts include humans, nonhuman primates, bovine, equine, swine, ovine, caprine, lagamorph, rodents, such as mice or cotton rats, etc. Furthermore, TB is spread through contact with infected animals or their bodily fluids, such as saliva, mucus, or milk. It can also be transmitted through contaminated soil or

[0333] food. Accordingly, the disclosure provides methods for creating vaccines for a variety of human and veterinary uses.

[0334] Mtb is the most host-specific; it produces progressive disease more often in humans and nonhuman primates but it can infect other animals including pigs, cattle, and elephants.

[0335] M. bovis can cause progressive disease in many mammalian species, including humans. M. caprae. an organism closely related to M bovis, has been isolated from humans, goats, cattle, and several wildlife species in Europe.

[0336] M. orygis causes tuberculosis in oryx, rhinos, dairy cattle, rhesus monkeys, and humans. For example, bovine TB is a chronic infectious disease caused primarily by M. bovis and is characterized by the formation of tubercles in any organ or tissue. This disease affects various animal species and humans: therefore, it represents a significant veterinary and public health problem (Milian-Suazo, et al., Animals (Basel). 2022 Dec 1; 12(23):3377. doi:

[0337] 45801771.1 41 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0338] 10.3390 / anil2233377. PMID: 36496897; PMCID: PMC9735741.). Bovine TB control is based on the tuberculin test and the disposal of reactor animals. In underdeveloped countries, this strategy has not been successful due to the lack of economic resources to compensate producers for their slaughtered animals. Thus, in some forms, the disclosed compositions are administered to bovine to control bovine TB and reduce its transmission to humans.

[0339] The compositions can be administered to a subject susceptible to or otherwise at risk of mycobacterial infection in an "immunogenically effective dose" which is sufficient to induce or enhance the individual's immune response capabilities against the bacteria. The compositions can be administered to a subject via injection, aerosol delivery, nasal spray, nasal droplets, oral inoculation, or topical application.

[0340] In particular forms, the composition includes an immunogenically effective amount of the compounds (i.e., antigen(s) alone (or nucleic acid encoding the same) or in combination with adjuvant).

[0341] In all subjects, the precise amount of the composition administered and the timing and repetition of administration will be determined by various factors, including the nature of the composition, the patient's state of health and weight, the mode of administration, the nature of the formulation, etc. For polypeptide compositions, generally dosage levels of 0.001 to 20 mg / kg of body weight daily are administered to mammals. Generally, for intravenous injection or infusion, dosage may be lower.

[0342] Any of the compositions can be administered as part of vaccine regime including 1, 2, 3, 4, 5, or more administrations of the disclosed compositions, 1, 2, 3, 4, 5, 5, 6, or 7 days, weeks, or months apart. In some forms, the vaccine regime includes a prime and boost, or a prime, a first boost, and a second boost. In a specific, non-limiting form, the regime is a prime-boost regime, 3 or 4 weeks apart.

[0343] The prime and boost can be the same or different vaccine formulations. For example, in some forms, the prime is BCG (Bacille Calmette-Guerin), a live, attenuated vaccine and the boost is an immunogenic composition or vaccine disclosed herein. The term “BCG" refers to a strain of live, attenuated strain of derived from M. bovis. BCG is currently the only commercially available vaccine against TB infection and disease.

[0344] BCG was developed by attenuation of M. bovis at the Institute Pasteur over 100 years ago and during this process, the virulent strain lost several important gene segments, one segment encoding virulence associated antigen ESAT-6. This original mutation is referred to as RD1. The BCG vaccine was in the subsequent 30-40 years distributed to various laboratories and production facilities worldwide. This gave rise to various vaccine substrains often named after the location of

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[0346] the laboratory in which it is produced (BCG Danish, Prague, Tokyo etc). As many of these strains initially were propagated by continuous cultures, a large number of additional deletions of the original genome have been observed and with different distribution in different substrains. In total, at least 12 major alterations / deletions (RD1-11 and the SigK mutation) are reported among the BCG vaccine strains that have been analysed. Some of these deletions contain immunologically important antigens with vaccine potential and some deletions are immunologically silent.

[0347] The RD1 deletion is an example of a region that contains immune dominant antigens of great importance for vaccines. In the present context, BCG strains may be divided into two main groups; early BCG strains and late BCG strains. Early and late BCG strains are not to be confused with early and late antigens.

[0348] The early BCG strains lack the RD1 region. The early BCG strains include BCG Russia, BCG Japan, BCG Moreau, BCG Sweden and BCG Birkhaug.

[0349] The late BCG strains lack the RD1 and RD2 regions, and have a mutation in sigK. The late BCG strains include BCG Tice, BCG Frappier, BCG Pasteur, BCG Danish, BCG Glaxo, BCG Prague, BCG China as well as the genetically modified BCG strain, VPM1002.

[0350] I’he late and early BCG strains do not possess identical genotypes and phenotypes. Thus, in the late BCG strains some antigens encoded by the RD2 region have been deleted and other antigens are poorly expressed or non-secreted. Consequently, for these missing, poorly expressed or non-secreted antigens, no immune response will be induced upon vaccination with the late BCG strains.

[0351] The majority of the teenage and adult population in countries plagued by TB are vaccinated with BCG. Particularly because of the excellent protection induced by BCG in young children, the use of BCG likely will never be eliminated in these endemic areas. Thus, a prevalent strategy for enhancing protection against TB, is to boost the immune response raised as a reaction to the initial BCG vaccination. This may be accomplished by administrating the disclosed TB antigen(s), e.g., as a booster vaccine.

[0352] The immune responses can be characterized by a variety of methods. These include taking samples of nasal washes or sera for analysis of tuberculosis specific antibodies, which can be detected by tests including, but not limited to, enzyme-linked immunosorbent assay, luciferaseimmunoprecipitation assay, and flow cytometry. In addition, immune responses can be detected by assay of cytokines in nasal washes or sera, ELISPOT of immune cells from either source, quantitative RT-PCR or microarray analysis of nasal wash or serum samples, and restimulation of immune cells from nasal washes or serum by re-exposure to antigen in vitro and analysis for the production or display of cytokines, surface markers, or other immune correlates measures by flow

[0353] 45801771.1 43 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0354] cytometry or for cytotoxic activity against indicator target cells displaying antigens. In some forms, testing including use of one or more of the exemplary assays of the experiments provided below.

[0355] Due to some patients having been previously exposed to tuberculosis, tests may be performed before and after treatment with a disclosed composition, wherein an increased or induced immune response is evident by an increase or improvement after treatment relative to before treatment.

[0356] In some forms, the subjects, which may be neonates, infants, children, adolescents, adults including or excluding the elderly, or any combination thereof, are given multiple doses of the composition to elicit sufficient levels of immunity. For neonates and infants, administration may begin within the first month of life, and continue at intervals throughout childhood, such as at two months, four months, six months, one year and two years, as necessary to maintain sufficient levels of protection against tuberculosis. In other forms, adults who are particularly susceptible to repeated or serious infection, such as, for example, health care workers, day care workers, family members of young children, the elderly, individuals with compromised cardiopulmonary function, etc. are given multiple administrations to establish and / or maintain protective immune responses. Levels of induced immunity can be monitored by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted and / or administrations repeated as necessary to maintain desired levels of protection.

[0357] Further, different compositions may be indicated for administration to different recipient groups.

[0358] Any of the disclosed compositions can be combined with other conventional vaccination compositions and methods. See, e.g., WO 2024 / 103167, WO 2023 / 159121, WO 2020 / 249756, WO 2013 / 158061, and WO 2015 / 161853. In such forms, the different compositions can be in the same or different admixtures and administered simultaneously or present in separate preparations and administered separately.

[0359] IV. Methods of Isolating Insoluble Recombinant Proteins

[0360] During preparation of Rv3351c-ESAT6 fusion protein, it was observed that fusion protein was highly insoluble. To increase yield, an isolation protocol was developed. Thus, methods of isolating / purifying insoluble recombinant proteins are provided. The methods can include any one or more of the steps provided below. A specific form is provided in the Examples below, and is also full disclosed for use alone or in combination / substitution with any one or more of the steps provided below.

[0361] 1. Harvest cells expressing the protein e.g., by centrifugation.

[0362] 2. After centrifugation, decant supernatant.

[0363] 45801771.1 44 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0364] 3. Resuspend cell pellet e.g., at room temperature in Lysis Buffer.

[0365] 4. Add EDTA-free Protease Inhibitor.

[0366] 5. Add PMSP and mix.

[0367] 6. Add lysozyme and mix.

[0368] 7. Add tris(2-carboxyethyl)phosphine [TCEP] and mix.

[0369] 8. Sonicate cell pellet suspension, preferably on ice.

[0370] 9. Add DNAse I and mix.

[0371] 10. Insoluble and soluble components are separated, e.g. by centrifugation. Pellet will be prepared for recombinant protein isolation from the inclusion bodies (IB).

[0372] Solubilizing proteins from inclusion bodies (IB)

[0373] 11. Suspend the inclusion body (IB) pellet in IB buffer (IBB) and mix.

[0374] 12. Add PMSF and mix.

[0375] 13. Add lysozyme and mix.

[0376] 14. Prepare 12 volumes (1 volume = amount of lysis buffer used in step 3) of IBB diluted to 1:10 in dH20. Add 6 volumes 0.1X IBB to the IB suspension and mix.

[0377] 15. Centrifuge the suspension, remove supernatant.

[0378] 16. Resuspend the pellet in 0. IX IBB.

[0379] 17. Repeat step 15.

[0380] 18. Resuspend IB pellet as in step 15. *The washed IB pellet can be stored at -80C. 19. Resuspend the IB pellet in urea Tris-HCl CHAPS, and solubilize overnight in a 50 ml conical on an orbital shaker at room temperature.

[0381] 20. Centrifuge and supernatant to new tube (contains the recombinant protein).

[0382] 21. Collect soluble protein, e.g., by affinity chromatography.

[0383] 22. Release the bound protein and dialyze in accordance the release protocol.

[0384] 23. Suspend precipitated protein in, e. g., DMS O.

[0385] V. Kits

[0386] The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method.

[0387] The disclosed invention can be further understood by the following numbered paragraphs: 1. A composition including epitopes for HBHA, Rv3351c, and ESAT6, a nucleic acid(s) encoding the same, or a combination thereof.

[0388] 45801771.1 45 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0389] 2. The composition of paragraph 1, wherein the epitopes are present on one or more polypeptides.

[0390] 3. 1'he composition of paragraph 2, wherein one or more of the polypeptides is a fusion protein.

[0391] 4. The composition of any one of paragraphs 1-3, wherein the epitope(s) for HBHA are present on a HBHA polypeptide optionally including the amino acid sequence of SEQ ID NO:1 or a variant thereof with at least 70% sequence identity thereto.

[0392] 5. The composition of any one of paragraphs 1-4, where in the epitope(s) for Rv3351c and the epitope(s) for ESAT6 are present on a Rv3351c-ESAT6 fusion protein optionally including the amino acid sequence of SEQ ID NO:2 or a fragment or a variant thereof with at least 70% sequence identity thereto fused to the amino acid sequence of SEQ ID NO:4 or fragment or a variant thereof with at least 70% sequence identity thereto.

[0393] 6. The composition of paragraph 5, wherein the fusion protein includes SEQ ID NOS: 10 or 11, or a fragment or a variant thereof including at least 70% sequence identity thereto.

[0394] 7. The composition of any one of paragraphs 1-6, wherein the nucleic acid is RNA, optionally mRNA, or DNA, optionally a vector optionally a viral vector.

[0395] 8. The composition of any one of paragraphs 1-7, wherein the one or more of the epitopes are package in or on a particle, optionally nanoparticles.

[0396] 9. The composition of paragraph 8, wherein the nanoparticles are wax nanoparticles, PLGA nanoparticles, or a combination thereof.

[0397] 10. The composition of any one of paragraphs 1-9 further including a hydrogel-based vaccine delivery platform optionally VacSIM™.

[0398] 11. The composition of any one of paragraphs 1-10 further including an adjuvant.

[0399] 12. The composition of paragraph 11, wherein the adjuvant is glucopyranosyl lipid A (GLA) and / or CpG.

[0400] 13. The composition of any one of paragraphs 1-12 further including a pharmaceutically acceptable carrier.

[0401] 14. The composition of any one of paragraphs 1-13 in a formulation suitable for subcutaneous and / or nasal delivery.

[0402] 15. The composition of any one of paragraphs 1-14 in an effective amount to induce an immune response against one or more of HBHA, Rv3351c, and ESAT6 when administered to a subject.

[0403] 16. A method of inducing or increasing an immune response in a subject in need thereof including administering the subject the composition of any one of paragraphs 1-15.

[0404] 45801771.1 46 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0405] 17. The method of paragraph 16, wherein the composition is administered in an effective amount to increase immunity against one or more mycobacterial strains in the subject.

[0406] 18. fhe method of paragraph 17, wherein the one or more mycobacterial strains includes Mycobacterium tuberculosis (Mtb), M. bovis, M. africanum, M. canettii, M. orygis, M. microti, M. caprae, M. pinnipedii, M. surricattae, or M. mungi.

[0407] 19. The method of any one of paragraphs 16-18, the composition is administered in an effective amount to reduce or prevent one or more symptoms of tuberculosis.

[0408] 20. The method of any one of paragraphs 16-19, wherein the subject does not have tuberculosis.

[0409] 21. The method of any one of paragraphs 16-19, wherein the subject has tuberculosis.

[0410] 22. The method of any one of paragraphs 16-21, wherein the subject has been exposed to someone with tuberculosis.

[0411] 23. The method of any one of paragraphs 16-22, wherein the composition is administered in combination with a BCG (Bacille Calmette-Guerin) vaccine.

[0412] 24. The method of paragraph 23, wherein the BCG vaccine is administered to the subject first and composition is administered to the subject one or more times day(s), month(s), week(s), and / or year(s) later, optionally wherein the administration is the same or similar to the regimen of Figure 7.

[0413] 25. The method of any one of paragraphs 16-24, wherein the composition is administered by an intranasal route.

[0414] 26. The method of any one of paragraphs 16-24, wherein the composition is administered by a subcutaneous route.

[0415] 27. The method of any one of paragraphs 16-26, wherein the subject is an infant, child, adult optionally elderly adult; optionally wherein the subject is a human.

[0416] 28. The method of any one of paragraphs 16-27 further including separately administrating the subject an adjuvant.

[0417] 29. A fusion protein including the amino acid sequence of SEQ ID NO:2 or a fragment or a variant thereof with at least 70% sequence identity thereto fused to the amino acid sequence of SEQ ID NO:4 or fragment or a variant thereof with at least 70% sequence identity thereto.

[0418] 30. The fusion protein of paragraph 29 including SEQ ID NOS: 10 or 11, or a fragment or a variant thereof including at least 70% sequence identity thereto.

[0419] 31. A nucleic acid encoding the fusion protein of paragraphs 29 or 30.

[0420] 32. An immunogenic composition including the fusion protein of paragraphs 29 or 30 or the nucleic acid of paragraph 31.

[0421] 45801771.1 47 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0422] 33. A method of purifying insoluble proteins as provided herein.

[0423] 34. A compound, composition, method, or process as described herein including, but not limited to, in the text, drawings, and combinations thereof.

[0424] The invention can also be further understood by the following additional numbered paragraphs:

[0425] 1. A composition including epitopes for epithelial heparin-binding hemagglutinin adhesin (HBHA), inducer of lipid rafts protein Rv3351c, and pore forming protein early secretory antigenic target 6 (ESAT6), and optionally one or both of Uncharacterized protein Rvl490 (Rvl490) and DNA-binding protein HupB (HUPB), a nucleic acid(s) encoding the same, or a combination thereof.

[0426] 2. The composition of paragraph 1, wherein the epitopes are present on one or more polypeptides.

[0427] 3. The composition of paragraph 2, wherein one or more of the polypeptides include one or more fusion proteins.

[0428] 4. The composition of any one of paragraphs 1-3, wherein

[0429] the epitope(s) for HBHA are present on a HBHA polypeptide including the amino acid sequence of SEQ ID NO:1 or a variant thereof with at least 70% sequence identity thereto;

[0430] the epitope(s) for Rv3351 are present on a Rv3351 polypeptide including the amino acid sequence of SEQ ID NOS:2, 3, or 29, or a variant thereof with at least 70% sequence identity thereto;

[0431] the epitope(s) for ESAT6 are present on a ESAT6 polypeptide including the amino acid sequence of SEQ ID NOS:4 or 5, or a variant thereof with at least 70% sequence identity thereto;

[0432] the epitope(s) for Rvl490 are present on a Rvl490 polypeptide including the amino acid sequence of SEQ ID NOS:7 or 6, or a variant thereof with at least 70% sequence identity thereto;

[0433] the epitope(s) for HUPB are present on a HUPB polypeptide including the amino acid sequence of SEQ ID NOS:8 or 9, or a variant thereof with at least 70% sequence identity thereto; or a combination thereof.

[0434] 5. The composition of any one of paragraphs 1-4, where in the epitope(s) for Rv3351c and the epitope(s) for ESAT6 are present on a Rv3351c-ESAT6 fusion protein optionally including the amino acid sequence of SEQ ID NO:2 or a fragment or a variant thereof with at least 70% sequence identity thereto fused to the amino acid sequence of SEQ ID NO:4 or fragment or a variant thereof with at least 70% sequence identity thereto.

[0435] 6. The composition of paragraph 5, wherein the fusion protein includes SEQ ID NOS:10 or 11, or a fragment or a variant thereof including at least 70% sequence identity thereto.

[0436] 45801771.1 48 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0437] 7. The composition of any one of paragraphs 1-6, wherein the nucleic acid is RNA, optionally mRNA, or DNA, optionally a vector optionally a viral vector.

[0438] 8. fhe composition of any one of paragraphs 1-7, wherein the one or more of the epitopes are package in or on a particle, optionally nanoparticles.

[0439] 9. The composition of paragraph 8, wherein the nanoparticles are wax nanoparticles, PLGA nanoparticles, or a combination thereof.

[0440] 10. The composition of any one of paragraphs 1-9 further including a hydrogel-based vaccine delivery platform.

[0441] 11. The composition of paragraph 10, wherein the hydrogel-based vaccine delivery platform includes a peptide hydrogel that is a liquid at room temperature and a gel at physiological pH, physiological salt concentrations, and / or physiological temperatures.

[0442] 12. The composition of paragraph 11, wherein the peptide hydrogel includes a selfassembling peptide selected from the group consisting of RAD ARAD ARAD ARADA (SEQ ID NO:24), RADA (SEQ ID NO:28), RARADADARARADADA (SEQ ID NO:25), FKFEFKFE (SEQ ID NO:26), KLDLKLDLKLDL (SEQ ID NO:27), and combinations thereof.

[0443] 13. The composition of any one of paragraphs 1-12 further including an adjuvant.

[0444] 14. The composition of paragraph 12, wherein the adjuvant is CpG and / or glucopyranosyl lipid A (GLA).

[0445] 15. fhe composition of any one of paragraphs 1-14 further including a pharmaceutically acceptable carrier and / or in a formulation suitable for subcutaneous and / or nasal delivery.

[0446] 16. The composition of any one of paragraphs 1-15 in an effective amount to induce an immune response against one or more of HBHA, Rv3351c, ESAT6, Rvl490, and HUPB when administered to a subject.

[0447] 17. The composition of any one of paragraphs 1-15, wherein the epitope(s) for HBHA are present on a HBHA polypeptide including the amino acid sequence of SEQ ID NO: 1, the epitope(s) for Rv3351c and the epitope(s) for ESAT6 are present on a Rv3351c-ESAT6 fusion protein including the amino acid sequence of SEQ ID NO:2 fused to the amino acid sequence of SEQ ID NO:4, and wherein the HBHA polypeptide and Rv3351c-ESAT6 fusion protein are encapsulated or adhered to nanoparticles, optionally wax or PLGA nanoparticles.

[0448] 18. The composition of paragraph 17 including the hydrogel-based vaccine delivery platform including a peptide hydrogel that is a liquid at room temperature and a gel at physiological pH, physiological salt concentrations, and / or physiological temperatures, wherein the peptide hydrogel includes a self-assembling peptide selected from the group consisting of

[0449] RAD ARAD ARAD ARAD A (SEQ ID NO:24).

[0450] 45801771.1 49 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0451] 19. The composition of paragraph 18, including an adjuvant.

[0452] 20. The composition of paragraph 19, wherein the adjuvant is CpG.

[0453] 21. A method of inducing or increasing an immune response in a subject in need thereof including administering the subject the composition of any one of paragraphs 1-20.

[0454] 22. The method of paragraph 21, wherein the composition is administered in an effective amount to increase immunity against one or more mycobacterial strains in the subject.

[0455] 23. The method of paragraph 22, wherein the one or more mycobacterial strains includes Mycobacterium tuberculosis (Mtb), M. bovis, M. africanum, M. canettii, M. microti, M. orygis, M. caprae, M. pinnipedii, M. mungi, M. suricattae or subsequent additions to the Mtb complex.

[0456] 24. The method of any one of paragraphs 21-23, the composition is administered in an effective amount to reduce or prevent one or more symptoms of TB.

[0457] 25. The method of any one of paragraphs 21-24, wherein the subject does not have TB.

[0458] 26. The method of any one of paragraphs 21-24, wherein the subject has TB.

[0459] 27. The method of any one of paragraphs 21-24, wherein the subject has been exposed to someone with TB.

[0460] 28. fhe method of any one of paragraphs 21-27, wherein the composition is administered in combination with a BCG (Bacille Calmette-Guerin) vaccine.

[0461] 29. The method of paragraph 28, wherein the BCG vaccine is administered to the subject first and the composition is administered to the subject one or more times day(s), month(s), week(s), and / or year(s) later.

[0462] 30. The method of any one of paragraphs 21-29, wherein the composition is administered by an intranasal route.

[0463] 31. The method of any one of paragraphs 21-29, wherein the composition is administered by a subcutaneous route.

[0464] 32. The method of any one of paragraphs 21-31, wherein the subject is an infant, child, adult optionally elderly adult; optionally wherein the subject is selected from humans, non-human primates, pigs, cattle, sheep, deer, mink, dogs, cats, elephants, and birds.

[0465] 33. The method of any one of paragraphs 21-32 further including separately administrating the subject an adjuvant.

[0466] 34. The method of paragraph 30, wherein the epitope(s) for HBHA are present on a HBHA polypeptide including the amino acid sequence of SEQ ID NO:1, the epitope(s) for Rv3351c and the epitope(s) for ESAT6 are present on a Rv3351c-ESAT6 fusion protein including the amino acid sequence of SEQ ID NO:2 fused to the amino acid sequence of SEQ ID NO:4, and wherein the

[0467] 45801771.1 50 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0468] HBHA polypeptide and Rv3351c-ESAT6 fusion protein are encapsulated or adhered to nanoparticles, optionally wax or PLGA nanoparticles.

[0469] 35. 1'he method of paragraph 34 including the hydrogel- based vaccine delivery platform including a peptide hydrogel that is a liquid at room temperature and a gel at physiological pH, physiological salt concentrations, and / or physiological temperatures, wherein the peptide hydrogel includes a self-assembling peptide selected from the group consisting of RAD ARAD ARAD ARADA (SEQ ID NO: 24).

[0470] 36. The method of paragraph 35, including an adjuvant.

[0471] 37. The method of paragraph 36, wherein the adjuvant is CpG.

[0472] Examples

[0473] Example 1: Vaccines Against Tuberculosis

[0474] Experiments were designed to develop a mucosal booster vaccine including three Mycobacterium tuberculosis Mtb) vaccine antigens. An important reason to develop mucosal vaccines is the increasing evidence that mucosal immune responses are important for protection against diseases which initially interact with these surfaces. Epithelial cells including the respiratory epithelium and particularly alveolar epithelial cells (AECs) are primary targets for mucosal vaccines. See, e.g., Pavlicek RL, Fine-Coulson K, Gupta T, Quinn FD, Posey JE, Willby M, Castro-Garza J, Karls RK. Rv3351c, a Mycobacterium tuberculosis gene that affects bacterial growth and alveolar epithelial cell viability. Can J Microbiol. 2015 Dec:61(12):938-47. doi:

[0475] 10.1139 / cjm-2015-0528. Three of the Mtb secreted proteins that are involved in infecting and trafficking within AECs are the focus of this study: i) HBHA; epithelial cell heparin-binding hemagglutinin adhesin, ii) Rv3351c; an inducer of lipid rafts, and iii) ESAT6; pore forming protein early secretory antigenic target 6.

[0476] Results show that Mtb strains deleted for hbhA, Rv3351c or esxA (ESAT6 gene) replicate less effectively in AECs and in lungs of aerosol-infected mice, and the respective mutant strains disseminate less effectively from lungs. Humans with active tuberculosis (TB) generate antibodies against HBHA, Rv3351c, and ESAT6, demonstrating that these proteins are presented to the immune system during infection, and Mtb HBHA, Rv3351c, or ESAT6 administered subcutaneously or intranasally have been shown to induce such immune responses in mice.

[0477] Although the entire immune response repertoire required for protection against Mtb infection is not known, recent data in animal models indicates that vaccine-induced CD4+ cells of the T helper 17 (Thl7) cell subtype, which naturally traffic to the airways, can accelerate the recruitment of protective Thl cells through the production ol Il'Ny and IL-17. Cytotoxic CD8+ T cells are

[0478] 45801771.1 51 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0479] important for antigen-specific release of granulysin, perforin, and granzyme B which kill infected host cells.

[0480] Materials and Methods

[0481] A vaccine was designed that combines these three epithelial cell-targeting Mtb proteins as a booster mucosal vaccine with Bacille Calmette-Guerin (BCG) as a prime drive robust protective mucosal, cellular, and humoral responses in the lungs compared to subcutaneous vaccination with BCG alone.

[0482] Compositions

[0483] HBHA plus the Rv3351c-ESAT6 fusion protein was adsorbed onto two different nanoparticle (NP) types: wax (see, Hart, et al., Nanopaiticle-Eusion Protein Complexes Protect against Mycobacterium tuberculosis Infection, Mol Ther. 2018 Mar 7;26(3):822-833. doi:

[0484] 10.1016 / j.ymthe.2017.12.016. Epub 2017 Dec 22, which is specifically incorporated by reference herein in its entirety) or polylactic-co-glycolic acid (see, Shepherd, S. D., O’Buckley, S. C., Harrington, EM. et al. A moldable sustained release bupivacaine formulation for tailored treatment of postoperative dental pain. Sci Rep 8, 12172 (2018). doi.org / 10.1038 / s41598-018-29696-w, which is specifically incorporated by reference herein in its entirety). Native HBHA is purified by running sonicates of BCG Pasteur cell pellet over a cationic exchange column at neutral pH and eluting with a salt gradient. Fractions are analyzed, and finally dialyzed to remove impurities.

[0485] MAENSNIDDIKAPLLAALGAADLALATVNELITNLRERAEETRTDTRSRVEESRARLTKLQEDLPE QLTELREKFTAEELRKAAEGYLEAATSRYNELVERGEAALERLRSQQSFEEVSARAEGYVDQAVEL TQEALGTVASQTRAVGERAAKLVGIELPKKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVTQ

[0486] K (SEQ ID NO:1)

[0487] The Rv3351c-ESAT6 fusion protein is a recombinant protein purified from E. coli.

[0488] MASMTGGQQMGRDPNSSSVDKLMLASCPARSGAAVADAIKSAVGVQPSGVEHKTLRRMDLVRYLAG GHTTYPPEGFVAGSDVIGTTNPAAAQAIVAAIGTWPPAAGRASALIDSLGGAVGDMDPEGSAFPWC RQSAVVQWYVNTPSDGQVATANKWLSDAHHAVQHFSVGGYVNYLEANAAASQYFGANLSRLTTVRR KYDPDRIMYSGLDFSTRQVAERLLPALGFRVRFGVLVIRCALCTDTVKRLGTLPNLTWSRLKVNVA VTQEQAGVMDLPALPVRRTPRRGDGACGGMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLT KLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTI SEAGQAMASTEGNVTGMFALEHHHHHH

[0489] (SEQ ID NO:11)

[0490] The NP -protein complexes were combined in VacSIM® plus one of two vaccine adjuvants, CpG or GLA-SE, both well-studied with Mtb and VacSIM®. VacSIM® is an immune matrix that when mixed with target antigens plus adjuvants has been shown to enhance immunity and protection for vaccines against a number of pathogens (synthetic oligopeptides; biocompatible and

[0491] 45801771.1 52 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0492] biodegradable; ex vivo, a 1.0% VacSIM solution is liquid at acidic pH, resulting in the flexibility to mix virtually any antigen, organism, and adjuvant, at physiological conditions, VacSIM selfassembles into nanofibers, forming matrix depot, slow- release of Ag, leading to a robust adaptive immunity and memory responses, VacSIM, + CpG drives response toward Thl).

[0493] 25-35 kDa PLGA (50:50 lactide: glycolide) PLGA25 was used to encapsulate the Rv3351c-ESAT6 fusion protein. VacSIM (1.0% W / V of the (RADA)4 peptide in sterile water buffered to approximately pH 3.5 with HC1) is the preferred formulation for subcutaneous vaccines. VacSIM (0.5% W / V of the (RADA)4 peptide in sterile water buffered to approximately pH 3.5 with HO) is the preferred formulation used for intranasal vaccines.

[0494] Protocol for purification of Rv3351c-ESAT6-6His

[0495] E.coli Strain: ClearColi BL21(DE3) transformed with plasmid pRK278 (pET23b-Rv3351c-ESAT6-6His)

[0496] Cell Extract Purification

[0497] 1. Harvest cells [cultured in LB broth + carbenicillin (0.1 mg / ml) at 37C to OD600 = 0.6, and induced 6 hours with 1 mM IPTG] by centrifugation [6,000 x g, 15 min, 4C] in sterile, preweighed [record bottle weight | 250 ml polypropylene centrifugation bottle(s) |max volume 166 ml per bottle],

[0498] 2. After centrifugation, decant supernatant (saving 50 - 100 uL for SDS-PAGE analysis) and allow pellet to drain by inverting bottle on a paper towel for approx. 1 min, removing as much liquid as possible. Weigh 250 ml bottle(s) to determine the wet weight of the pellet. *Pellet may be stored at -80C at this point.

[0499] 3. Resuspend cell pellet at room temperature in 5 ml Lysis Buffer [300 mM NaCl, 100 mM Sodium Phosphate, 10 mM Tris-HCl, pH8.0, 0.5% Triton X100] per gram of wet cell paste by pipetting or gentle vortexing. *If pellet was stored at -80C, thaw pellets on ice, vortex until homogenous, then suspend in the above buffer. Combine multiple suspended pellets in a single 250 mL bottle.

[0500] 4. Add 1 cOmplete Mini, EDTA-free Protease Inhibitor Cocktail tablet (Roche) per cell pellet preparation.

[0501] 5. Add PMSF to [0.5 mM]finai and mix by pipetting or gentle vortexing.

[0502] 6. Add freshly-prepared lysozyme (10 mg / mL stock dissolved in dH2O) to [1 mg / mL]finaiand mix by pipetting or gentle vortexing.

[0503] 7. Add tris(2-carboxyethyl)phosphine [TCEP] to [1 mM]tmai and mix by pipetting or gentle vortexing.

[0504] 45801771.1 53 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0505] 8. Sonicate cell pellet suspension on ice with the following settings: 50% amplitude, 15 second pulse, 2 minute pause, for 8 cycles with a flat-tip sonicator probe [Branson],

[0506] 9. Add DNAse 1 to [5 ug / mL]finaiand mix by vortexing. *Sample viscosity should decrease significantly after incubation.

[0507] 10. Insoluble and soluble components are separated by centrifugation [16,000 x g, 20 min, 4C], Decant supernatant. Save 50 - 100 uL for SDS-PAGE gel analysis. *Pellet will be prepared for recombinant protein isolation from the inclusion bodies (IB).

[0508] Solubilizing proteins from inclusion bodies (IB)

[0509] 11. Suspend the inclusion body (IB) pellet in IB buffer (IBB) [300mM NaCl, lOOmM Sodium Phosphate, 10 mM Tris-HCL, pH8.0, 0.5% CHAPS] using same volume as in step 3 (i.e. 5 ml buffer / gram). Pipette and vortex to obtain uniform suspension.

[0510] 12. Add PMSF to [0.5 mM]fjnaiand mix by vortexing.

[0511] 13. Add freshly-prepared lysozyme to [1 mg / mL]finai. Vortex to mix and incubate at room temperature 5 min.

[0512] 14. Prepare 12 volumes (1 volume = amount of lysis buffer used in step 3) of IBB diluted to 1:10 in dH20. Add 6 volumes 0.1X IBB to the IB suspension and vortex for 1 min.

[0513] 15. Centrifuge the suspension at 5,000 x g for 15 min at 4C to sediment the IBs. Remove supernatant with a pipette. Save 50 - 100 uL of supernatant for SDS-PAGE analysis.

[0514] 16. Resuspend pellet in 0.1X IBB using 10 ml per gram of original cell paste, for example: 35 ml of 1 / 10 diluted buffer for an original cell pellet of 3.5 g) and centrifuge (5,000 x g for 15 min at 4C) and removing supernatant.

[0515] 17. Repeat step 15.

[0516] 18. Resuspend IB pellet as in step 15, but this time centrifuge at 16,000 x g, 15 min, 4C and remove supernatant saving 50 - 100 uLof supernatant for SDS-PAGE analysis.

[0517] *The washed IB pellet can be stored at -80C.

[0518] 19. Resuspend the IB pellet in 8M urea 10 mM Tris-HCl 0.05% CHAPS, (pH 8.0) (1 ml / 50 ml of starting culture) and solubilize overnight in a 50 ml conical on an orbital shaker at room temperature.

[0519] 20. Centrifuge at 16,000 x g for 15 min at 4C. Transfer supernatant to new tube (contains the fusion protein).

[0520] Ni-NTA His-Bind column prep and purification

[0521] 21. Prepare a gravity-flow column by mixing 50% Ni-NTA His-Bind slurry by inversion and transfer 2.5 mL to a conical tube (2.5 ml of slurry = 1.25 ml bed volume) or use a EconoFit Nuvia IMAC Ni -charged FPLC column (BioRad). If pouring column,

[0522] 45801771.1 54 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0523] 22. Wash the 2.5 mL of slurry with 3 volumes (7 mL) of dH20. Wash = add dH20 to slurry, mix by inversion, allowing slurry to settle, and remove liquid with a pipette

[0524] 23. Equilibrate the 2.5 mL of resin with 3 volumes (7 mL) of binding buffer (BB) [8M urea, 50 mM NaCl, 10 mM Tris-HCl, 0.05% CHAPS, pH 8.0], [Equilibrate = add binding buffer to slurry, mix by inversion, allowing slurry to settle / separate and removing the binding buffer with a pipette],

[0525] 21. Suspend resin in 2.5 ml BB

[0526] 22. Add 2 mL of the buffer-equilibrated resin to 15 mL of solubilized inclusion body supernatant and mix gently by inversion or slow-shaking platform for 15 minutes at room temperature. After incubation, load supernatant-resin onto a column, remove bottom cap, collect flow through and reload onto the column. Save 2nd flow through for SDS-PAGE analysis. [Alternatively, the FPLC column is equilibrated on an FPLC with lines previously equilibrated with BB.

[0527] 23. Wash column with 8 column volumes of wash buffer 1 (8 M urea, 50 mM NaCl, lOmM Tris-HCl, 0.05% CHAPS, pH 8.0, 10 mM imidazole). Save all column chromatography fractions for SDS-PAGE analysis.

[0528] 24. Wash column with 8 mL wash buffer 2 (8 M urea, 50 mM NaCl, 10 mM Tris-HCl 0.05% CHAPS, pH 8.0, 60 mM imidazole).

[0529] 25. Elute protein from column with elution buffer (8 M urea, 50 mM NaCl, 10 mM Tris-HCl, 0.05% CHAPS, pH 7.5, 500 mM imidazole) collecting 0.5 mL fractions.

[0530] 26. Run each fraction on SDS-PAGE gels and stain with Coomassie blue to assess protein purification.

[0531] 27. Pool eluted fractions containing significant recombinant protein. Save 50 pl for SDS-PAGE.

[0532] 28. Load the remaining pooled eluate into a 10,000 MWCO Slide-A-Lyzer cassette using a needle and syringe (see Slide-A-Lyzer instructions for where to inject the protein). Choose a Slide-A-Lyzer size that can hold the entire sample with extra volume to all for increased volume during dialysis. Attach to a Slide-A-Lyzer float to the top of the cassette (keeping the side where protein was injected on top) and immediately float in a beaker containing a stir bar and the first dialysis below buffer below. Dialyze at 4C for a minimum of 1.5 hr per buffer*:

[0533] 4 M urea, 100 mM KC1, 50 mM glycine, 10 mM Tris, 0.05% CHAPS, pH 7.5

[0534] 2 M urea, 100 mM KC1, 50 mM glycine, 10 mM Tris, 0.05% CHAPS, pH 7.5

[0535] 1 M urea, 100 mM KC1, 50 mM glycine, 10 mM Tris, 0.05% CHAPS, pH 7.5

[0536] 0.5 M urea, 100 mM KC1, 50 mM glycine, 10 mM Tris, 0.05% CHAPS, pH 7.5

[0537] 45801771.1 55 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0538] 0.1 M urea, 100 mM KC1, 50 mM glycine, 10 mM Tris, 0.05% CHAPS, pH 7.5

[0539] 0 M urea, 100 mM KC1, 50 mM glycine, 10 mM Tris, pH 7.5

[0540] 0 M M urea, 100 mM KC1, 50 mM glycine, 10 mM Tris, pH 7.5

[0541] * After each dialysis step, carefully hold the cassette upright, and pour the used buffer down the drain. Then pour in the next dialysis buffer and float the Slide- A-Lyzer in it.

[0542] 29. After final dialysis step, the protein will precipitate out of solution. Aseptically transfer the precipitate to a 50 ml conical tube, by cutting open dialysis cassette with sterile razor blade.

[0543] 30. Suspend the protein in DMSO.

[0544] 31. Quantify the protein by BCA assay.

[0545] 32. Examine protein on SDS-PAGE gel and western blot using antibody to C-terminal 6-His tag.

[0546] Protocol for preparation NPs for encapsulation of antigens Carnauba Wax Nanoparticles

[0547] Wax nanoparticles (NPs) were prepared via hot homogenization using the surfactant sodium myristate. To remove excess surfactant, we utilized serial centrifugation. Briefly, the wax nanoparticle solution was centrifuged at 20,000 x g for 20 min, with the supernatant collected and subsequently ultracentrifuged at 40,000 x g for 20 min. It was found that a 1:1 ratio of carnauba wax:sodium myristate to provide the most reproducible particles with porous morphology, increasing the potential surface area for protein adsorption. The nanoparticles were characterized using scanning electron microscopy (SEM) and dynamic light scattering (DLS) for morphology and size metrics, respectively. The resulting porous particles were free of debris after serial centrifugation and exhibited a hydrodynamic size of 346346 ± 20 nm (PDI = 0.44 ± 0.05). The addition of conalbumin (CA; 78kDa; isoelectric point of 6.1), a model protein, to the wax NPs upon stirring for 1 h at room temperature exhibited only a slight increase in size and negligible change in particle surface charge. Due to the low water solubility of the Rv335 Ic-ES AT6 fusion antigen, wax NPs in water were combined with fusion antigen in DMSO and dialyzed for 24 h (10 kDa MWCO) into ultrapure water to allow the antigen to precipitate onto the surface of the wax particles and the final formulation to be free of organic solvent. Wax NPs containing the Rv3351c-ESAT6 fusion antigen exhibited a reduced hydrodynamic size of 202 ± 8 nm (PDI = 0.45 ± 0.04) and comparable morphology to the blank NPs. The size of the wax NPs containing the fusion antigen was stable over 14 d at 4 °C storage.

[0548] PLGA Nanoparticles

[0549] PLGA NPs were prepared from 5-10 kDa or 25-35 kDa PLGA via solvent displacement. Prior to use of the fusion antigen, the model protein CA was utilized for PLGA NP optimization.

[0550] 45801771.1 56 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0551] PLGA NPs were characterized for morphology and size using SEM and DLS, respectively.

[0552] Morphologically, 25 kDa PLGA produced spherical, individual particles, whereas 5-10 kDa demonstrated NP aggregates. Protein encapsulation and release rates at both pH 5.2 and pH 7.4 were monitored using the BCA assay. Both molecular weights of PLGA were found to encapsulate CA at high efficiencies (-90%), with high reproducibility across 6 separate synthesis batches. At pH 7.4, both NP systems released 30-35% of the encapsulated CA over 56 d. At pH 5.2, mimicking endosomal pH, the 5-10 kDa PLGA NPs released only 5.0 ± 1.5% CA, whereas the 25-35 kDa NPs released 20.6 ± 2.3% CA over 56 d. Of note, the net surface charge of the NPs was highly driven by the charge of the protein, with resulting net charges in line with the isoelectric point of CA.

[0553] Remaining particles were degraded at 56 d, and the protein content was quantified. Indeed, the 25-35 kDa PLGA particles that released 497 ± 24 pg over 56 days at pH 7.4 still contained 558 ± 54 pg, and those that released 294 ± 34 pg over 56 days at pH 5.2 still contained 709 ± 54 pg, indicating that in vitro release into buffer was still ongoing after 8 weeks.

[0554] Due to increased release at pH 5.2 and superior morphology, the 25-35 kDa PLGA was selected for transition to use with the Rv3351c-ESAT6 fusion antigen. The necessity for DMSO incorporation to the PLGA synthesis due to the water insolubility of the fusion antigen led to a hydrodynamic size of 316 ± 10 nm (PDI = 0.30 ± 0.04) in water, which aligned with the results for PLGA NPs containing CA. Particles maintained spherical morphology. The encapsulation efficiency was slightly decreased to 82.2 ± 3.5% but was reproducible across 8 separate syntheses. The particle size of the PLGA NPs containing the fusion antigen demonstrated only minor decreases in size and polydispersity after 7 to 14 d at 4°C storage, with no changes indicating NP aggregation or degradation. Importantly, the Rv3351c-ESAT6 fusion antigen was not prematurely released during storage, with >97% fusion antigen remaining within the NPs after 3. 7, 10, or 14 d of storage at 4 °C. The NP charge remained at -6.3 to -6.5 mV at both pH 5.2 and pH 7.4, but slight differences in the release rates in buffer were still observed, with slightly faster release at pH 7.4 than pH 5.2. Of note, the release of the hydrophobic fusion antigen was at a slower rate than was found with CA, with less than 10% of the antigen released after 140 days at either pH. Release was then assessed into Gamble’s simulated lung fluid containing dipahnitoylphosphatidylcholine (DPPC), a prominent phospholipid in lung surfactant. The incorporation of surfactant was expected to improve the physiological similarities for the in vitro release study. In simulated lung fluid, sustained antigen release was observed over >200 days, with 42.4 ± 13.4% released after 234 days.

[0555] Protocol for vaccine formulations

[0556] Booster vaccines were formulated using either wax NPs or PLGA NPs for the delivery of the Rv3351c-ESAT6 fusion antigen. Briefly, Rv3351c-ESAT6-loaded NPs were combined with

[0557] 45801771.1 57 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0558] HBHA and adjuvant in dilute saline or within a biocompatible nanofiber gel-matrix (VacSIM®). VacSIM® comprises a liquid solution of the (RADA)4 synthetic oligopeptide that self-assembles into a hydrogel depot when administered based on the salt and temperature of physiological conditions. It was determined that a VacSIM® composition of 0.5 wt% and 1.0 wt% were suitable for intranasal and subcutaneous delivery, respectively. For the initial subcutaneous evaluation in vivo, 10 formulations were prepared, with 5 wax-based formulations and 5 PLGA-based formulations. Formulations included either VacSIM®, CpG adjuvant, GLA-LS adjuvant, or a combination of VacSIM® and adjuvant. The same 10 formulations were evaluated for intranasal delivery. For both studies, a total composition of 15 pg antigen was delivered in each dose, with 7.5 pg of HBHA and 7.5 pg of Rv3351c-ESAT6.

[0559] The three vaccine formulations that exhibited the greatest protective effect in vivo were selected to move forward, all of which were delivered intranasally and included PLGA NPs. Three total antigen contents were evaluated, including 5 pg, 15 pg, and 30 pg. Preparations without antigen (i.c., adjuvant only) were also prepared for comparison.

[0560] Table of Formulations:

[0561] Rv3351 C-ESAT6 fusion + native HBHA from BCG Danish

[0562] WAX nanoparticles

[0563] SQV IN1 -VacSIM

[0564] SQ2 / IN2- CpG 2395

[0565] SQ3 / IN3- GLA-SE

[0566] SQ4 / IN4 - VacSIM CpG

[0567] SQ5 / ! N5~ VacSIM GLA-SE

[0568] PG LA B ano pa rti cl e s

[0569] SQ6 / IN6- VacSIM

[0570] SQ7 / IN7-CpG

[0571] SQ8 / INS - GLA-SE

[0572] SQ9 f IN 9 - VacS M + CpG

[0573] SQ10 / IN 10 -VacSIM GLA-SE

[0574] SQ = subcutaneous administration; IN = intranasal administration

[0575] Vaccination Protocol

[0576] BCG vaccinated mice were given 2 boosters of sub-cutaneous or intranasal with the 3 vaccines with 2 different nanoparticles, 2 different adjuvants and with or without VacSIM™ matrix compound. See Table of Formulations above and Figure 7.

[0577] Results

[0578] Results are presented in Figures 1-15. Figure 1-6 show nanoparticle characterizations. Figures 7-15 show intranasal vaccines IN4, IN6, and IN9 are the best at reducing lung burden in the infected mice. Intranasal immunization was more protective than subcutaneous (lung bacillary

[0579] 45801771.1 58 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0580] load). Immunizations protected against Mtb infection-associated lung pathology. The humoral response was strong.

[0581] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0582] References

[0583] 1. Pavlicek RL, Fine-Coulson K, Gupta T, Quinn FD, Posey IE, Willby M, Castro-Garza I, Karls RK. Rv3351c. a Mycobacterium tuberculosis gene that affects bacterial growth and alveolar epithelial cell viability. Can J Microbiol. 2015 Dec;61(12):938-47. doi: 10.1139 / cjm-2015-0528.

[0584] 2. Patarroyo, et al., Vaccine. 2008 Aug 12;26(34):4387-95. doi:

[0585] 10.1016 / j. vaccine.2008.05.092. Epub 2008 lun 26. PMID: 18585422;

[0586] 3. Aoki, et al., I Biol Chem. 2004 Sep 17;279(38):39798-806. doi: 10.1074 / jbc. M402677200. Epub 2004 lul 2. PMID: 15234978,

[0587] 4. Milian-Suazo, et al., Animals (Basel). 2022 Dec 1;12(23):3377. doi: 10.3390 / anil2233377. PMID: 36496897; PMCID: PMC9735741.

[0588] 5. Hart, et al., Nanoparticle-Fusion Protein Complexes Protect against Mycobacterium tuberculosis Infection, Mol Ther. 2018 Mar 7;26(3):822-833. doi: 10.1016 / j.ymthe.2017.12.016. Epub 2017 Dec 22,

[0589] 6. Shepherd, S. D., O’Buckley, S. C., Harrington, J. M. et al. A moldable sustained release bupivacaine formulation for tailored treatment of postoperative dental pain. Sci Rep 8, 12172 (2018). doi.org / 10.1038 / s41598-018-29696-w

[0590] 7. WO 2024 / 103167A1

[0591] 8. WO 2023 / 159121A2

[0592] 9. WO 2020 / 249756A1

[0593] 10. WO 2013 / 158061A1

[0594] 11. WO 2015 / 161853A1

[0595] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0596] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps.

[0597] 45801771.1 59 ATTORNEY DOCKET NO. UGA 2025-062-02 PCT

[0598] “Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0599] Unless the context clearly indicates otherwise, use of the word “can” indicates an option or capability of the object or condition referred to. Generally, use of “can” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of the word “may” indicates an option or capability of the object or condition referred to. Generally, use of “may” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of “may” herein does not refer to an unknown or doubtful feature of an object or condition.

[0600] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Nothing herein is to be constmed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0601] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0602] 45801771.1 60

Claims

ATTORNEY DOCKET NO. UGA 2025-062-02 PCTCLAIMSWe claim:

1. A composition comprising epitopes for epithelial heparin-binding hemagglutinin adhesin (HBHA), inducer of lipid rafts protein Rv3351c, and pore forming protein early secretory antigenic target 6 (ESAT6), and optionally one or both of Uncharacterized protein Rvl490 (Rvl490) and DNA-binding protein HupB (HUPB), a nucleic acid(s) encoding the same, or a combination thereof.

2. The composition of claim 1, wherein the epitopes are present on one or more polypeptides.

3. The composition of claim 2, wherein one or more of the polypeptides comprise one or more fusion proteins.

4. The composition of any one of claims 1-3, whereinthe epitope(s) for HBHA are present on a HBHA polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a variant thereof with at least 70% sequence identity thereto;the epitope(s) for Rv3351 are present on a Rv3351 polypeptide comprising the amino acid sequence of SEQ ID NOS:2, 3, or 29, or a variant thereof with at least 70% sequence identity thereto;the epitope(s) for ESAT6 are present on a ESAT6 polypeptide comprising the amino acid sequence of SEQ ID NOS:4 or 5, or a variant thereof with at least 70% sequence identity thereto;the epitope(s) for Rvl490 are present on a Rvl490 polypeptide comprising the amino acid sequence of SEQ ID NOS:7 or 6, or a variant thereof with at least 70% sequence identity thereto;the epitope(s) for HUPB are present on a HUPB polypeptide comprising the amino acid sequence of SEQ ID NOS:8 or 9, or a variant thereof with at least 70% sequence identity thereto; or a combination thereof.

5. The composition of any one of claims 1 -4, where in the epitope(s) for Rv3351c and the epitope(s) for ESAT6 are present on a Rv3351c-ESAT6 fusion protein optionally comprising the amino acid sequence of SEQ ID NO:2 or a fragment or a variant thereof with at least 70% sequence identity thereto fused to the amino acid sequence of SEQ ID NO:4 or fragment or a variant thereof with at least 70% sequence identity thereto.

6. The composition of claim 5, wherein the fusion protein comprises SEQ ID NOS: 10 or 11, or a fragment or a variant thereof comprising at least 70% sequence identity thereto.

7. The composition of any one of claims 1-6, wherein the nucleic acid is RNA, optionally mRNA, or DNA, optionally a vector optionally a viral vector.45801771.1 61ATTORNEY DOCKET NO. UGA 2025-062-02 PCT8. The composition of any one of claims 1-7, wherein the one or more of the epitopes are package in or on a particle, optionally nanoparticles.

9. fhe composition of claim 8, wherein the nanoparticles are wax nanoparticles, PLGA nanoparticles, or a combination thereof.

10. The composition of any one of claims 1-9 further comprising a hydrogel-based vaccine delivery platform.

11. The composition of claim 10, wherein the hydrogel-based vaccine delivery platform comprises a peptide hydrogel that is a liquid at room temperature and a gel at physiological pH, physiological salt concentrations, and / or physiological temperatures.

12. The composition of claim 11, wherein the peptide hydrogel includes a selfassembling peptide selected from the group consisting of RAD ARAD ARAD ARADA (SEQ ID NO:24), RADA (SEQ ID NO:28), RARADADARARADADA (SEQ ID NO:25), FKFEFKFE (SEQ ID NO:26), KLDLKLDLKLDL (SEQ ID NO:27), and combinations thereof.

13. The composition of any one of claims 1-12 further comprising an adjuvant.

14. The composition of claim 12, wherein the adjuvant is CpG and / or glucopyranosyl lipid A (GLA).

15. The composition of any one of claims 1-14 further comprising a pharmaceutically acceptable carrier and / or in a formulation suitable for subcutaneous and / or nasal delivery.

16. The composition of any one of claims 1-15 in an effective amount to induce an immune response against one or more of HBHA, Rv3351c, ESAT6, Rvl490, and HUPB when administered to a subject.

17. The composition of any one of claims 1-15, wherein the epitope(s) for HBHA are present on a HBHA polypeptide comprising the amino acid sequence of SEQ ID NO: 1, the epitope(s) for Rv3351c and the epitope(s) for ESAT6 are present on a Rv3351c-ESAT6 fusion protein comprising the amino acid sequence of SEQ ID NO:2 fused to the amino acid sequence of SEQ ID NO:4, and wherein the HBHA polypeptide and Rv3351c-ESAT6 fusion protein are encapsulated or adhered to nanoparticles, optionally wax or PLGA nanoparticles.

18. The composition of claim 17 comprising the hydrogel-based vaccine delivery platform comprising a peptide hydrogel that is a liquid at room temperature and a gel at physiological pH, physiological salt concentrations, and / or physiological temperatures, wherein the peptide hydrogel includes a self-assembling peptide selected from the group consisting of RAD ARAD ARAD ARADA (SEQ ID NO:24).

19. The composition of claim 18, comprising an adjuvant.

20. The composition of claim 19, wherein the adjuvant is CpG.45801771.1 62ATTORNEY DOCKET NO. UGA 2025-062-02 PCT21. A method of inducing or increasing an immune response in a subject in need thereof comprising administering the subject the composition of any one of claims 1-20.

22. fhe method of claim 21, wherein the composition is administered in an effective amount to increase immunity against one or more mycobacterial strains in the subject.

23. The method of claim 22, wherein the one or more mycobacterial strains comprises Mycobacterium tuberculosis (Mtb), M. bovis, M. africanum, M. canetlii, M. microti, M. orygis, M. caprae, M. pinnipedii, M. mungi, M. suricattae or subsequent additions to the Mtb complex.

24. The method of any one of claims 21-23, the composition is administered in an effective amount to reduce or prevent one or more symptoms of TB.

25. The method of any one of claims 21-24, wherein the subject does not have TB.

26. The method of any one of claims 21-24, wherein the subject has TB.

27. The method of any one of claims 21-24, wherein the subject has been exposed to someone with TB.

28. The method of any one of claims 21-27, wherein the composition is administered in combination with a BCG (Bacille Calmette-Guerin) vaccine.

29. fhe method of claim 28, wherein the BCG vaccine is administered to the subject first and the composition is administered to the subject one or more times day(s), month(s), week(s), and / or year(s) later.

30. The method of any one of claims 21-29, wherein the composition is administered by an intranasal route.

31. The method of any one of claims 21-29, wherein the composition is administered by a subcutaneous route.

32. The method of any one of claims 21-31, wherein the subject is an infant, child, adult optionally elderly adult: optionally wherein the subject is selected from humans, non-human primates, pigs, cattle, sheep, deer, mink, dogs, cats, elephants, and birds.

33. The method of any one of claims 21-32 further comprising separately administrating the subject an adjuvant.

34. The method of claim 30, wherein the epitope(s) for HBHA are present on a HBHA polypeptide comprising the amino acid sequence of SEQ ID NO: 1, the epitope(s) for Rv3351c and the epitope(s) for ESAT6 are present on a Rv3351c-ESAT6 fusion protein comprising the amino acid sequence of SEQ ID NO:2 fused to the amino acid sequence of SEQ ID NO:4, and wherein the HBHA polypeptide and Rv3351c-ESAT6 fusion protein are encapsulated or adhered to nanoparticles, optionally wax or PLGA nanoparticles.45801771.1 63ATTORNEY DOCKET NO. UGA 2025-062-02 PCT35. The method of claim 34 comprising the hydrogel-based vaccine delivery platform comprising a peptide hydrogel that is a liquid at room temperature and a gel at physiological pH, physiological salt concentrations, and / or physiological temperatures, wherein the peptide hydrogel includes a self-assembling peptide selected from the group consisting of RADARADARADARADA (SEQ ID NO: 24).

36. The method of claim 35, comprising an adjuvant.

37. The method of claim 36, wherein the adjuvant is CpG.45801771.1 64

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