CROSS REFERENCE TO RELATED APPLICATIONS

[0601J This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/244,484 filed September 15, 2.021, and U.S. Provisional Patent Application No. 63/355,329 filed June 24, 2022, both of which are incorporated herein by reference in their entirety for all purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nudeotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 56,714 Byte (XML) file named “CSU-40979-60T’ created on September 14, 2022.

GOVERNMENT SUPPORT

[0003] Uns invention was made with government support under R21 A1158056 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

[0004] The present disclosure provides materials and methods related to engineered bacteria for use in vaccines. In particular, the present disclosure provides novel compositions and methods for generating vaccine compositions comprising bacteria (e.g., Mycobacterium bovis BCG) engineered to express immunogenic polypeptides and fusion proteins to treat and/or prevent infection from a pathogenic organism (e.g. , coronavirus).

BACKGROUND

[0005] The COVID- 19 pandemic and rapid global spread of its causative agent, the coronavirus SARS-CoV-2, have created an unprecedented public health challenge. Among the priorities listed in the NIAID Strategic Plan for COVID- 19 research is the need to pursue multiple strategies to develop a COVID-19 vaccine. Vaccine candidates should be efficacious across ah patent age groups, including in the elderly. Mycobacterium bovis BCG (a live attenuated vaccine and the only approved vaccine for TB prevention) has recently received attention for its potential to mitigate through non-specific immunity the prevalence and severity' of the symptoms of COVID-19. Accordingly, randomized controlled clinical trials are underway across the world, including in the United States, to assess the potential benefit of BCG vaccination. BCG vaccination has been known since the 1960s to non-specifically improve immunity against a number of viral pathogens, resulting in reduced morbidity' and mortality' in neonates, children and the elderly. Additionally, BCG vaccines have been shown to produce long-lasting (e.g., >10 years) CD4+ and CD8+ T cell responses after one inoculation, exhibit natural adjuvant properties and a remarkable safety profile (>5 billion doses given to date), and it is relatively easy and inexpensive to mass-produce (e.g.. does not typically require cold-chain distribution processes). For these reasons, BCG has been extensively used a vaccine platform to recombinant! y express a variety of antigens from different bacterial, parasite and viral pathogens with reports of promising candidates making it to clinical trials.

SUMMARY

Embodiments of the present disclosure include a recombinant bacterial cell engineered to express an exogenous polypeptide from a coronavirus. In some embodiments, the cell is Mycobacterium bovis BCG. In some embodiments, the exogenous polypeptide is immunogenic. In some embodiments, the exogenous polypeptide is expressed in the cytosol of the bacterial cell, or exported and/or secreted by the bacterial cell.

[0007] In some embodiments, the exogenous polypeptide expressed in the cytosol of the bacterial cell comprises or is derived from a nucleocapsid protein (N) from a coronavirus, or an immunogenic fragment thereof. In some embodiments, the exogenous polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the exogenous polypeptide does not comprise a signal peptide. In some embodiments, the bacterial cell comprises an exogenous polynucleotide encoding N operably linked to a heterologous promoter. In some embodiments, the heterologous promoter comprises or is derived from a mycolyltransferase Ag85B (fbpB) gene promoter from Mycobacterium tuberculosis . In some embodiments, the heterologous promoter is at least 80% identical to SEQ ID NO: 19.

[0908j In some embodiments, the exogenous polypeptide exported and/or secreted by the bacterial cell comprises an S 1/S2 subunit polypeptide from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from mycolyltransferase Ag85B (FbpB) from Mycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3. In some embodiments, the bacterial cell comprises a polynucleotide encoding the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB operably linked to a heterologous promoter. In some embodiments, the heterologous promoter comprises or is derived from a mycolyltransferase Ag85B (fbpB) gene promoter from Mycobacterium tuberculosis . In some embodiments, the polynucleotide encoding the heterologous promoter is at least 80% identical to SEQ ID NO: 19.

[0009] In some embodiments, the exogenous polypeptide exported and/or secreted by the bacterial cell comprises an S2’ subunit polypeptide from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from mycolyltransferase Ag85B (FbpB) from Mycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5. In some embodiments, the bacterial cell comprises a polynucleotide encoding the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB operably linked to a heterologous promoter. In some embodiments, the heterologous promoter comprises or is derived from a mycolyltransferase Ag85B (fbpB) gene promoter from Mycobacterium tuberculosis. In some embodiments, the polynucleotide encoding the heterologous promoter is at least 80*% identical to SEQ ID NO: 19.

[0010] In some embodiments, the exogenous polypeptide expressed in the cytosol, or exported and/or secreted by the bacterial cell comprises membrane protein (M) from a coronavirus, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from mycolyltransferase Ag85B (FbpB) from Mycobacterium tuberculosis, or a fragment thereof In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7. In some embodiments, the bacterial cell comprises a polynucleotide encoding the exogenous polypeptide comprising the M protein fused to the FbpB signal peptide operably linked to a heterologous promoter. In some embodiments, the heterologous promoter comprises or is derived from a mycolyltransferase Ag85B (fbpB) gene promoter from Mycobacterium tuberculosis . In some embodiments, the polynucleotide encoding the heterologous promoter is at least 80% identical to SEQ ID NO: 19.

[0011] In some embodiments, the exogenous polypeptide exported and/or secreted by the bacterial cell comprises a receptor binding domain (RBD) polypeptide from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from mycolyltransferase Ag85B (FbpB) from Mycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the RBD poly peptide fused to the FbpB comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, the RBD polypeptide comprises at least one of an N331 D and/or an N343D amino acid substitution. In some embodiments, the bacterial cell comprises a polynucleotide encoding the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB operably linked to a heterologous promoter. In some embodiments, the heterologous promoter comprises or is derived from a mycolyltransferase Ag85B (fbpB) gene promoter from Mycobacterium tuberculosis. In some embodiments, the polynucleotide encoding the heterologous promoter is at least 80% identical to SEQ ID NO: 19.

[0012] In some embodiments, the exogenous polypeptide exported and/or secreted by the bacterial cell comprises or is derived from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from beta-lactamase C (BlaC) from Mycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17. In some embodiments, the S protein polypeptide comprises at least one of an K986P and/or an K987P ammo acid substitution. In some embodiments, the bacterial cell comprises a polynucleotide encoding the S protein polypeptide operably linked to a homologous promoter. In some embodiments, the homologous promoter comprises or is derived from an hsp60 (groEL2) gene promoter Mycobacterium. bovis BCG.

[0013] In some embodiments, the exogenous polypeptide is a coronavirus antigen. In some embodiments, the coronavirus is selected from the group consisting of: SARS-CoV-2, SARS- CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, AND HKU1.

[0014] Embodiments if the present disclosure also include a vaccine composition comprising any of the engineered bacterial cells described herein, or any combination thereof. [0015] In some embodiments, the composition further comprises a pharmaceutically acceptable adjuvant and/or excipient.

[0016] In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁴ to about 1x10 ¹² CFU.

[0017] In some embodiments, the composition is formulated as lyophilized powder. [0018] Embodiments of the present disclosure also include a method of inducing an immune response in a subject. In accordance with these embodiments, the method includes administering a composition comprising any of the recombinant bacterial cells described herein to the subject.

[0019] In some embodiments, administering the composition to the subject immunizes the subject against a coronavirus infection and/or a tuberculous infection.

[0020] In some embodiments, the composition is administered percutaneously, subcutaneously, trans dermally, intramuscularly, via inhalation, or via ingestion.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1: Viral loads in the cranial lobe of the lung three days post-challenge in the various immunization groups. Asterisks denote statistically significant differences in viral load between the saline control and immunization groups per Dunnett’s multiple comparison test (P < 0.05).

[0022] FIG. 2: Representative lung sections probed with an anti-SARS-CoV-2 nucleocapsid protein antibody (eosin staining indicates positivity). Images shown are 2..5x magnification.

DETAILED DESCRIPTION

[0023] Emergence of novel human coronaviruses from animal reservoirs has likely been ongoing throughout the history of humanity . Only recently has this garnered more attention because of technological advances to detect new viruses and the devastating potential of rapid spread achieved by global movement of people. Despite persistent efforts, there is no vaccine platform available that can be quickly and efficiently adapted to target the growing number of human and animal coronavirus strains. Coronavirus infection in populations is characterized by a variety of virologic and serologic states that may not be associated with clinical disease. For example, virus shedding can vary in duration and may be episodic or persistent. Seropositivity does not predict virus shedding and may not persist once virus is cleared, so reinfection is possible. The outcome is that coronaviruses have adapted to continually churn within susceptible populations and under the right conditions can cause epidemic outbreaks.

[0024] Because the viral spike protein is responsible for binding the host cell receptor and because neutralizing antibodies against spike have been demonstrated, most coronavirus vaccines have targeted the spike protein as the key immunogen. Unfortunately, spike proteins, including the host receptor binding domain (RBD) also induces antibodies that can enhance infection and accelerate disease. This is termed antibody-dependent enhancement (ADE) and is primarily mediated by IgG binding to Fey receptors. Additionally, there are three structural proteins available for antibody recognition on the surface of SARS-CoV-2 particles: S, membrane (M) and envelope (E). E is expressed at low levels and mutations in E of SARS (a closely related human coronavirus) do not affect viral replication. Since immune-escape is possible, E is unlikely to be a useful immunogen. On the other hand, M is highly expressed, is essential for virus assembly and has been shown to suppress the type 1 interferon innate immune response. In support of coronavirus M protein as a vaccine immunogen, previous studies have identified eleven Thl epitope-containing peptides and two linear antibody binding peptides in the M protein. Additionally, the nucleocapsid protein (N) is an abundant RNA binding protein that is critical for viral genome packing: thus, it is also a potential useful immunogen.

[0625] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

1. Definitions

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0027] The terms “comprise(s),” “include(s),” "‘having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility' of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0028] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. [0029] “Correlated to” as used herein refers to compared to.

[0030] As used herein, the term “animal” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, pigs, rodents (e.g, mice, rats, etc.), flies, and the like.

[0031 ] As used herein, the term “non-human animals” refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcmes, caprines, equines, canines, felines, aves, etc.

[0032] As used herein, the term “microorganism” refers to an organism or microbe of microscopic, submicroscopic, or ultrami croscopic size that typically consists of a single cell. Examples of microorganisms include bacteria, yeast, viruses, parasites, fungi, certain algae, and protozoa. In some aspects, the microorganism is engineered (“engineered microorganism”) to produce one or more therapeutic molecules or proteins of interest. In certain aspects, the microorganism is engineered to take up and catabolize certain metabolites or other compounds from its environment. In certain aspects, the microorganism is engineered to synthesize certain beneficial metabolites or other compounds (synthetic or naturally occurring) and release them into its environment. In certain embodiments, the engineered microorganism is an engineered bacterium.

[0033] As used herein, the term “recombinant bacterial cell” or “recombinant bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, a recombinant bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.

[0034] As used herein, the term “nucleic acid molecule” 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, aziridinylcytosme, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1 -methylguanine, 1 -methylinosine, 2,2-dimethyIguanine, 2-methyIadenine, 2-methyIguanine, 3-methyIcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5 -methyl aminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,

5 ’ -methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methyithio-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 -methyl uracil, N- uracil-5-oxyacetic acid methylester, uracil -5 -oxy acetic acid, pseudouracil, queosine, 2- thiocytosine, and 2,6-diaminopurine.

[0035] The term “gene” refers to a nucleic acid (e.g. , DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragment are retained. 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 or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5’ of the coding region and present on the mRNA are referred to as 5’ non-translated sequences. Sequences located 3’ or downstream of the coding region and present on the mRNA are referred to as 3’ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene.

[0036] As used herein, the term “heterologous gene” refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc.). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature (e.g. , genes expressed in loci where the gene is not normally expressed).

[0037] As used herein, a “non-native” or “exogenous” nucleic acid sequence refers to a nucleic acid sequence not normally present in a bacterium, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype. In some embodiments, the non-native or exogenous nucleic acid sequence is a synthetic, non-naturally occurring sequence. The non- native or exogenous nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in a gene cassette. In some embodiments, “non-native” or “exogenous” refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non-native or exogenous nucleic acid sequence may be present on a plasmid or chromosome. In addition, multiple copies of any regulatory region, promoter, gene, and/or gene cassette may be present in the bacterium, wherein one or more copies of the regulatory' region, promoter, gene, and/or gene cassette may be mutated or otherwise altered as described herein. In some embodiments, genetically engineered bacteria are engineered to comprise multiple copies of the same regulatory region, promoter, gene, and/or gene cassette in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions. In some embodiments, the genetically engineered bacteria of the disclosure comprise an exogenous polypeptide encoding an immunogenic polypeptide or antigenic polypeptide that is operably linked to an inducible, heterologous promoter that is not associated with said gene in nature.

[0038] As used herein, “operably linked” refers a nucleic acid sequence encoding an immunogenic polypeptide or antigenic polypeptide that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence. A regulatory region is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5’ and 3’ untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

|0039] As used herein, “promoter” refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5’ of the sequence that they regulate. Promoters may be derived in their entirety' from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may' regulate expression of a coding sequence or gene in response to a particular stimulus, e.g., in a cell- or tissue- specific manner, in response to different environmental or physiological conditions, or in response to specific compounds. Prokaryotic promoters are typically classified into two classes: inducible and constitutive. In some embodiments, promoters of the present disclosure are “heterologous” in that they are not associated with a given gene or polynucleotide in nature. In some embodiments, promoters of the present disclosure are “non-heterologous” or endogenous in that they are associated with a given gene or polynucleotide in nature. In some embodiments, a polynucleotide can be exogenous to a given bacteria] cell, but can also be operably linked to its endogenous promoter, which is also heterologous to the bacterial ceil.

[0640] The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids as nucleic acids such as DNA and RNA found in the state they exist in nature. For example, a given DNA sequence (e.g. , a gene) is found on the host cell chromosome in proximity to neighboring genes: RNA sequences, such as a specific inRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (l.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).

[0041] As used herein, the term “purified” or “to purify” refers to the removal of components (e.g., contaminants) from a sample. For example, antibodies are purified byremoval of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non- immunoglobulin proteins and/or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.

[0042] As used herein, the term “subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g, cow; pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-hurnan primate (e.g., a monkey, such as a cynomolgus or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. In one embodiment, the subject is a human. The subject or patient may be undergoing various forms of treatment.

[0043] As used herein, the term “treat,'’ “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and/or injury, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the seventy’ of a disease prior to affliction refers to administration of a treatment to a subject that is not at the time of administration afflicted with the disease. “Preventing” also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease,

[0044] Unless otherwise defined herein, scientific and technical terms used in connection wdth the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology', immunology', microbiology', genetics and protein and nucleic acid chemistry' and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

2. Compositions and Methods

|0045] Embodiments of the present disclosure include a recombinant bacterial cell engineered to express an exogenous polypeptide from a coronavirus. In some embodiments, the cell is Mycobacterium bovis BCG, including any variants and derivatives thereof. As would be understood by one of ordinary skill in the art, BCG-based vaccines have been developed to protect against tuberculosis (TB); BCG strains are live, attenuated strains of Mycobacterium bovis that were developed by repeated subculture and have been shown to be effective for treating and preventing TB infections. Therefore, in some embodiments, the recombinant bacterial cells engineered to express an exogenous polypeptide from a coronavirus, as described in the present disclosure, can be used to prevent and/or treat both COVID- 19 and TB infections. [0046] In accordance with these embodiments, the present disclosure includes a recombinant bacterial cell engineered to express an exogenous, immunogenic polypeptide from a coronavirus. In some embodiments, the immunogenic, exogenous polypeptide from a coronavirus is engineered to be expressed in the cytosol of the bacterial cell, or exported and/or secreted by the bacterial cell. The immunogenic polypeptide can be from any coronavirus, including but not limited to, SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-NL63, HCoV- 229E, HCoV-OC43, AND HKU1. In some embodiments, the coronavirus is SARS-CoV-2. Additionally, the immunogenic polypeptide can be encoded by any of the genes present in a coronavirus genome, including but not limited to, genes for the structural proteins: surface (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Additionally, the structural genes encode nine accessory proteins, encoded by ORF3a, ORF3d, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF 14, and ORF 10 genes, which can also be included as an immunogenic, exogenous polypeptide of the present disclosure.

[0047] In some embodiments, the exogenous polypeptide expressed in the cytosol of the bacterial cell comprises or is derived from a nucleocapsid protein (N) from a coronavirus, or an immunogenic fragment thereof. In some embodiments, the exogenous polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 1. In some embodiments, the exogenous polypeptide comprises an ammo acid sequence that is at least 75% identical to SEQ ID NO: 1 . In some embodiments, the exogenous polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the exogenous polypeptide comprises an ammo acid sequence that is at least 85% identical to SEQ ID NO: 1. In some embodiments, the exogenous polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 . In some embodiments, the exogenous polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the exogenous polypeptide comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 1. In some embodiments, the exogenous polypeptide comprises an ammo acid sequence that is at least 97% identical to SEQ ID NO: 1, In some embodiments, the exogenous polypeptide comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1. In some embodiments, the exogenous polypeptide comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 1. In some embodiments, the exogenous polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 1.

[0048] In some embodiments, the exogenous polypeptide does not comprise a. signal peptide. In some embodiments, the bacterial cell comprises an exogenous polynucleotide encoding N operably linked to a heterologous promoter. In some embodiments, the heterologous promoter comprises or is derived from a mycolyltransferase Ag85B (fbpB) gene promoter fromMycobacterium tuberculosis . In some embodiments, the heterologous promoter is at least 70% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 75% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 80% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 85% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 90% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 95% identical to SEQ ID NO: 19.

[0049] In some embodiments, the exogenous polypeptide exported and/or secreted by the bacterial cell comprises an S1/S2 subunit polypeptide from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from mycolyltransferase Ag85B (FbpB) fromMycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 70% identical to SEQ ID NO: 3. In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 75% identical to SEQ ID NO: 3. In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 80% identical to SEQ ID NO: 3, In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 85% identical to SEQ ID NO: 3. In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3. In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 3. In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 3. In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 98% identical to SEQ ID NO: 3. In some embodiments, the exogenous polypeptide comprising the S 1/S2 subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 3. In some embodiments, the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB comprises an amino acid sequence that is 100% identical to SEQ ID NO: 3.

[0050] In some embodiments, the bacterial cell comprises a polynucleotide encoding the exogenous polypeptide comprising the S1/S2 subunit polypeptide fused to the FbpB operably linked to a heterologous promoter. In some embodiments, the heterologous promoter comprises or is derived from a mycolyltransferase Ag85B (fbpB) gene promoter from Mycobacterium tuberculosis . In some embodiments, the heterologous promoter is at least 70% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 75% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 80% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 85% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 90% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 95% identical to SEQ ID NO: 19.

[0051] In some embodiments, the exogenous polypeptide exported and/or secreted by the bacterial cell comprises an S2’ subunit polypeptide from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from mycolyltransferase Ag85B (FbpB) from Mycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 5. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 5. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 85% identical to SEQ ID NO: 5. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 90% identical to SEQ ID NO: 5. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 96% identical to SEQ ID NO: 5. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 5. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 5. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 99% identical to SEQ ID NO: 5. In some embodiments, the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB comprises an ammo acid sequence that is 100% identical to SEQ ID NO: 5.

[0052] In some embodiments, the bacterial cell comprises a polynucleotide encoding the exogenous polypeptide comprising the S2’ subunit polypeptide fused to the FbpB operably linked to a heterologous promoter. In some embodiments, the heterologous promoter comprises or is derived from a mycolyltransferase Ag85B (fbpB) gene promoter from Mycobacterium tuberculosis . In some embodiments, the heterologous promoter is at least 70% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 75% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 80% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 85% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 90% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 95% identical to SEQ ID NO: 19.

[0053] In some embodiments, the exogenous polypeptide expressed in the cytosol, or exported and/or secreted by the bacterial cell comprises membrane protein (M) from a coronavirus, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from mycolyltransferase Ag85B (FbpB) from Mycobacterium tuberculosis, or a fragment thereof In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 7. In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an ammo acid sequence that is at least 75% identical to SEQ ID NO: 7. In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 7. In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 7. In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7. In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 7. In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is at least 97% identical to SEQ) ID NO: 7. In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 7. In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 7. In some embodiments, the exogenous polypeptide comprising the M protein is fused to the FbpB signal peptide and comprises an amino acid sequence that is 100% identical to SEQ ID NO: 7.

[0054] In some embodiments, the bacterial cell comprises a polynucleotide encoding the exogenous polypeptide comprising the M protein fused to the FbpB signal peptide operably- linked to a heterologous promoter. In some embodiments, the heterologous promoter comprises or is derived from a mycolyltransferase Ag85B (fbpB) gene promoter from Mycobacterium tuberculosis . In some embodiments, the polynucleotide encoding the heterologous promoter is at least 80% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 70% identical to SEQ) ID NO: 19. In some embodiments, the heterologous promoter is at least 75% identical to SEQ) ID NO: 19. In some embodiments, the heterologous promoter is at least 80% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 85% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 90% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 95% identical to SEQ ID NO: 19.

[0055] In some embodiments, the exogenous polypeptide exported and/or secreted by the bacterial cell comprises a receptor binding domain (RBD) polypeptide from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from mycolyltransferase Ag85B (FbpB) from Mycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 9. In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an ammo acid sequence that is at ieast 75% identical to SEQ ID NO: 9. In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 9. In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 95% identical to SEQ ID NO: 9, In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 9. In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 97% identical to SEQ ID NO: 9. In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 98% identical to SEQ ID NO: 9. In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 9, In some embodiments, the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB comprises an ammo acid sequence that is at least 100% identical to SEQ ID NO: 9.

[0056] In some embodiments, the RBD polypeptide comprises at ieast one of an N331D and/or an N343D amino acid substitution. In some embodiments, the RBD polypeptide comprises an N331D amino acid substitution. In some embodiments, the RBD polypeptide comprises an N343D amino acid substitution. In some embodiments, the RBD polypeptide comprises both an N331D and an N343D amino acid substitution. In some embodiments, the bacterial cell comprises a polynucleotide encoding the exogenous polypeptide comprising the RBD polypeptide fused to the FbpB operably linked to a heterologous promoter. In some embodiments, the heterologous promoter comprises or is derived from a mycolyltransferase Ag85B (fbpB) gene promoter from Mycobacterium tuberculosis . In some embodiments, the heterologous promoter is at least 70% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 75% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 80% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 85% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 90% identical to SEQ ID NO: 19. In some embodiments, the heterologous promoter is at least 95% identical to SEQ ID NO: 19. [0057] In some embodiments, the exogenous polypeptide exported anchor secreted by the bacterial cell comprises or is derived from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from beta-lactamase C (BlaC) from Mycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 11. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 11. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 11. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 11. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 11. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 11. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 1 1 . In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 11. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 11. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is 100% identical to SEQ ID NO: 11 .

[0058] In some embodiments, the exogenous polypeptide exported and/or secreted by the bacterial cell comprises or is derived from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from beta-lactamase C (BlaC) from Mycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 13. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 13. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 13. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 13. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an ammo acid sequence that is at least 95% identical to SEQ ID NO: 13. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 13. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 13. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an ammo acid sequence that is at least 98% identical to SEQ ID NO: 13. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 13. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is 100% identical to SEQ ID NO: 13.

[0059] In some embodiments, the exogenous polypeptide exported and/or secreted by the bacterial cell comprises or is derived from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from beta-lactamase C (BlaC) from Mycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 15. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 15, In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an ammo acid sequence that is at least 80% identical to SEQ ID NO: 15. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 15. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 15. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 15. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 15. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 15. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 15. In some embodiments, the exogenous polypeptide comprising' the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is 100% identical to SEQ ID NO: 15.

[0060] In some embodiments, the exogenous polypeptide exported and/or secreted by the bacterial cell comprises or is derived from a coronavirus Spike (S) protein, or an immunogenic fragment thereof, fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises or is derived from beta-lactamase C (BlaC) from Mycobacterium tuberculosis, or a fragment thereof. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 17. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 17. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 17. In some embodiments, the exogenous polypeptide comprising' the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 17. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an ammo acid sequence that is at least 90% identical to SEQ ID NO: 17. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 17. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an ammo acid sequence that is at least 97% identical to SEQ ID NO: 17. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is al least 98% identical to SEQ ID NO: 17. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 17. In some embodiments, the exogenous polypeptide comprising the S protein polypeptide is fused to the BlaC signal peptide and comprises an amino acid sequence that is 100% identical to SEQ ID NO: 17.

[0061] In some embodiments, the S protein polypeptide comprises at least one of an K986P and/or an K987P ammo acid substitution. In some embodiments, the S protein polypeptide comprises an K986P amino acid substitution. In some embodiments, the S protein polypeptide comprises an K987P amino acid substitution. In some embodiments, the S protein polypeptide comprises both an K986P and an K987P amino acid substitution. In some embodiments, the bacterial cell comprises a polynucleotide encoding the S protein polypeptide operably linked to a homologous promoter. In some embodiments, the homologous promoter comprises or is derived from an hsp60 (groEL2) gene promoter from Mycobacterium bovis BCG.

Embodiments if the present disclosure also include a vaccine composition comprising any of the engineered bacterial cells described herein, or any combination thereof. In some embodiments, the composition further comprises a pharmaceutically acceptable adjuvant and/or excipient. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁴ to about 1x10 ¹² CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁵ to about 1x10 ¹² CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10° to about 1x10 ¹² CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁷ to about 1x10 ¹² CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁸ to about 1x10 ¹² CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁹ to about 1x10 ¹² CFU. In some embodiments, the engineered, bacterial cells are present in the composition in an amount ranging from about 1x10 ⁵⁰ to about 1x10 ⁵² CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ¹¹ to about 1x10 ¹² CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁴ to about 1x10 ⁵¹ CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁴ to about 1x10 ¹⁰ CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁴ to about 1x10 ⁹ CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁴ to about 1x10 ⁸ CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁴ to about 1x10 ⁵ CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁴ to about 1x10 ⁶ CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁴ to about 1x10 ⁵ CFU. In some embodiments, the engineered, bacterial cells are present in the composition in an amount ranging from about 1x10 ⁵ to about 1x10 ¹⁰ CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁶ to about 1x10 ⁹ CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁷ to about 1x10 ¹¹ CFU. In some embodiments, the engineered bacterial cells are present in the composition in an amount ranging from about 1x10 ⁸ to about 1x10 ¹⁰ CFU.

[0063] In some embodiments, the composition is formulated as lyophilized powder. In some embodiments, the composition is formulated as lyophilized powder and includes a pharmaceutically acceptable adj uvant and/or excipient.

[0064] Embodiments of the present disclosure also include a method of inducing an immune response in a subject. In accordance with these embodiments, the method includes administering a composition comprising any of the recombinant bacterial cells described herein to the subject. In some embodiments, administering the composition to the subject immunizes the subject against a coronavirus infection and/or a tuberculous infection. In some embodiments, the composition is administered percutaneously, subcutaneously, transdermally, intramuscularly, via inhalation, or via ingestion.

[0065] In accordance with these embodiments, the present disclosure includes pharmaceutical compositions comprising the genetically engineered bacteria and/or recombinant immunogenic polypeptides of the present disclosure, which can be used to treat, manage, ameliorate, and/or prevent diseases associated with infections from pathogenic organisms, and/or symptom(s) associated with diseases associated with infections from pathogenic organisms. Pharmaceutical compositions also include one or more genetically engineered bacteria, and/or one or more recombinant immunogenic polypeptides alone or m combination with prophylactic agents, therapeutic agents, and/or and pharmaceutically acceptable carriers or excipients.

[0066] In some embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic modifications described herein (e.g,, to express one or more immunogenic polypeptides). In some embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria that are each engineered to comprise the genetic modifications described herein.

[0067] The pharmaceutical compositions described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g, “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enter! cally coated or un coated. Appropriate formulation depends on the route of administration.

[0068] The genetically engineered bacteria and/or recombinant immunogenic polypeptides described herein may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, immediate-release, pulsatile-release, delay ed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 10 ⁴ to 10 ¹² bacteria, e.g., approximately 10 ⁵ bacteria, approximately 10 ^b bacteria, approximately 10 ⁷ bacteria, approximately 10 ⁸ bacteria, approximately 10 ⁹ bacteria, approximately 10 ¹⁰ bacteria, approximately 10 ¹¹ bacteria, or approximately 10 ¹² bacteria, or more. The compositions, which may comprise any combinations of the genetically engineered bacteria and/or recombinant polypeptides described herein, can be administered once or more daily, weekly, or monthly.

[0069] In some embodiments, the pharmaceutical compositions comprising the recombinant bacterial cells engineered to express an exogenous, immunogenic coronavirus polypeptide of the present disclosure, are administered to a subject consistent with medical guidelines for administering a BCG vaccine to treat or prevent a TB infection. For example, in some embodiments, the pharmaceutical compositions of the present disclosure can be administered at a dose of about 1 to 8 x 10 ⁸ CFU, which is roughly equivalent to about 50 mg (wet weight), as lyophilized powder.

[0070] The genetically engineered bacteria and/or recombinant immunogenic polypeptides may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the genetically engineered bacteria of the invention may be formulated in a solution of sodium bicarbonate, e.g, I molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example). The genetically engineered bacteria may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0071] The genetically engineered bacteria and/or recombinant immunogenic poly peptides disclosed herein may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate,

[0072] As would be recognized by one of ordinary skill in the art based on the present disclosure, embodiments of the pharmaceutical compositions comprising the genetically engineered bacteria and/or recombinant immunogenic polypeptides of the present disclosure can be packaged in a kit. The kit can comprise any of the genetically engineered bacteria and/or recombinant immunogenic polypeptides of the present disclosure, as well as any pharmaceutically acceptable adjuvants, earners, or excipients. In some embodiments, the kit includes a device for administering the pharmaceutical compositions (e.g, syringes, needles, patches, pumps, jet injectors, pen injectors, piston syringes, needle-free injectors, mechanically operated injectors, and injectors with computerized or electronic elements), as well as instructions and dosage information for administering the pharmaceutical compositions to a subject. In some embodiments, the composition is administered percutaneously, subcutaneously, trans dermally, intramuscularly, via inhalation, or via ingestion.

3. Examples

[0073] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.

[0074] lire present disclosure has multiple aspects, illustrated by the following non-limiting examples. Example 1

[0075] As described further herein, various recombinant BCG constructs were generated that express different SARS-CoV-2 antigens/antigen fusions, under control of different promoters and, in some cases, signal peptides. These include the soluble domain of the membrane protein (M), the full-size nudeocapsid protein (N), and various forms or segments of the Spike protein (e.g, full-size Spike protein devoid of its signal peptide and C-terminal transmembrane domain, Spike proteins with N- and C-terminal truncations, and Spike proteins comprising certain amino acid substitutions (e.g, K986P and/or K987P)); Spike immunogenic peptides S2’ (KPSKRSFIEDLLFNK (SEQ ID NO: 21)) and S1/S2 (QTQTNSPRRARSVASQS (SEQ ID NO: 22)) and RBD domain (wild-type or devoid of its glycosylation (Asn>Asp) or cysteine residues (Cys>Ser)), Table 1 below includes the various features of the constructs of the present disclosure.

[0076] Table 1 : Recombinant BCG constructs.

Example 2

[0077] Experiments were conducted to evaluate the immunogenicity and protective efficacy of recombinant BCG-based SARS-CoV -2 vaccines of the present disclosure. The golden Syrian hamster model of SARS-CoV-2 infection that is routinely used to test COVID-19 vaccines cannot be used in the case of BCG Pasteur due to the hypersusceptibility of this model to BCG Pasteur infection, which results in extensive pneumonia in vaccinated animals. Therefore, a recently developed mouse model of SARS-CoV-2 infection (see MAIO model; Lei st et al, 2020) was used to test the efficacy of several rBCG constructs in 10-12 weeks-old BALB/c mice.

[0078] Mice were immunized with (r)BCG (e.g., 10 ⁵ CFUs per mouse) through the intravenous (IV) route. Six weeks post-immunization, mice were challenged with SARS-CoV- 2 MAIO and viral loads in the cranial lung (PFU), histopathology, immunohistochemistry of lung sections (N antigen) and weight loss were analyzed 3 days post challenge. This study included the following treatment groups: Saline (mock immunization); SolaVAX in CpG 1018 adjuvant (Ragan et al., 2021) (inactivated SARS-CoV-2 virus; positive control for protection); intramuscular immunization with a boost after 3 weeks; BCG Pasteur 1173 P2 (WT parent strain not expressing any SARS-CoV-2 antigen); BCG Pasteur: :ESXl ^Mm3i (BCG Pasteur expressing the ESX-1 secretion system from M. mannum) (Groschel et al., 2017); BCGD/e«Z).-FbpB-S1/S2 (S1/S2 Spike peptide fused to full-size FbpB; secreted); BCGDZe«Z).‘FbpB-S2’ (S2’ Spike peptide fused to full-size FbpB; secreted); BCGDleuD):N (intracellularly expressed full-size N protein); BCGDZewD:Msol (intracellularly expressed soluble domain of the M protein); BCGD/eaD:RBD WT (secreted RBD domain); BCGD/e?zD:RBD (Cys>Ser) (secreted RBD domain with cysteines mutated to serines); and BCGDZeuD: Spike [K986P; K987P; full-size Spike devoid of its native signal peptide and C- terminal transmembrane domain] (potentially exported although not released in culture filtrates).

[0079] PCR-based and immunoblot analysis of rBCG strains recovered from the lungs and spleens of mice 6 weeks post-immunization showed that ah still contained a plasmid and produced each of the SARS-CoV-2 antigens. The rBCG constructs are thus stable in vivo.

Evidence of protection based on lung viral loads three days post-challenge w as achieved with BCG Pasteur, BCG Pasteur: :ESXl ^Mmar and three rBCG constructs (Spike, S1/S2, N), with the construct expressing the Spike protein showing the greatest efficacy (albeit less than that conferred by the SolaVAX control) (FIG. 1 ). The mice that received the rBCG construct expressing the Spike protein also displayed less N-protein immunohistochemical staining in the lung compared to those that recei ved the WT BCG Pasteur and saline controls (FIG. 2).

Example 3

In accordance with the above experiments, the recombinant constructs of the present disclosure can be tested in other models, including models designed to evaluate whether the constructs of the present disclosure protect against tuberculosis infections. For example, the above efficacy studies can be repeated with rBCG expressing the Spike protein in another murine model of SARS-CoV -2 infection in w-hich WT BCG (not expressing any S ARS-CoV- 2 antigen) given IV (10 ^fe CFU/mouse) has been shown to display protective efficacy (Hilligan et al., 2021 ). This model includes 7-9 weeks-old C57BL/6 mice challenged with the more virulent SARS-CoV -2 isolate alpha B.l.1.7. These experiments will help determine whether rBCG, given as one single dose, may induce longer-term protection than an inactivated virus such as SolaVAX (given as two doses three weeks apart). The experimental design for this experiment will be similar to the one used in the experiment described above and include immunizing C57BL/6 mice IV with 10 ⁵ to 10 ⁶ CFUs of WT BCG or rBCG constructs. Mice will be challenged with SARS-CoV-2 alpha B. l.1.7 six and fifteen weeks post-immunization, and viral loads in the cranial lung (PFU), lung histopathology, lung immunochemistry (SARS- CoV-2 N protein), immune responses and weight loss will be analyzed 3 days post-challenge. Tins study will include the following treatment groups: Saline (mock immunization); SolaVAX in CpG 1018 adjuvant; IM immunization with a boost after 3 weeks; BCG Pasteur 1173 P2 (WT parent strain not expressing any SARS-CoV-2 antigen); BCGDleuD): Spike [K986P; K987P; full-size Spike devoid of its native signal peptide and C -terminal transmembrane domain] (potentially exported although not released in culture filtrates); BCGDleuD :FbpB- RBD (Asn>Asp) (secreted RBD domain with Asn glycosylation sites mutated to Asp), [0081] These studies will also help evaluate whether the rBCG constructs of the present disclosure still protect against tuberculosis, making them potential dual vaccines against the two most deadly infectious diseases globally. To tins end, the same treatments groups described above will be used to immunize C57BL/6 mice IV (10 ⁵ to 10 ⁶ CFU/mouse). At about one hundred days post-immunization, animals will be challenged with virulent M. tuberculosis and M. tuberculosis loads in the lungs and spleens will be assessed 30 days post-challenge to assess and compare TB protective efficacy between treatment groups.

[0082] Sequences. The various embodiments of the present disclosure described herein may include one or more of the sequences referenced below, which can be found in the corresponding sequence listing. As described further herein, open reading frames are in bolded font, and restriction sites used for cloning are underlined. SARS-CoV-2 sequences are in italicized font, and promoter sequences are in lowercase. Signal peptides, where present, are underlined in their corresponding amino acid sequences.

[0083] Table 2: Nucleic acid and amino acid sequences.