Immunogenic composition comprising malonate semialdehyde decarboxylase (MSAD), and vaccine composition for preventing or treating nontuberculous mycobacteria infection, comprising same

Abstract

The present invention relates to an immunogenic composition comprising a malonate semialdehyde decarboxylase (MSAD) protein or a polynucleotide encoding same, and a vaccine composition and a pharmaceutical composition for preventing or treating nontuberculous mycobacteria infection, which comprise same. The vaccine composition and the pharmaceutical composition, which comprise the MSAD protein or the polynucleotide encoding same, of the present invention, have excellent preventive and therapeutic efficacy against nontuberculous mycobacteria infection. In addition, the vaccine composition and the pharmaceutical composition, which comprise the MSAD protein or the polynucleotide encoding same, of the present invention, have a greater effect of preventing and treating nontuberculous mycobacteria infection when administered intranasally, and have a greater effect of preventing and treating nontuberculous mycobacteria infection when used in combination with an immune adjuvant.

Description

Immunogenic composition containing MSAD (MALONATE SEMIALDEHYDE DECARBOXYLASE) and vaccine composition containing the same for preventing or treating nontuberculous mycobacterial infection

[0001] The present invention relates to an immunogenic composition comprising MSAD (Malonate Semialdehyde Decarboxylase) protein or a polynucleotide encoding the same, and a vaccine composition for preventing or treating nontuberculous mycobacterial infection comprising the same.

[0002] Non-tuberculous mycobacteria (NTM) are frequently found in the environment, such as soil, water, sewage systems, and medical equipment, and can cause infection in a variety of hosts, including humans.

[0003] As the number of patients with NTM infections increases worldwide, the most frequently occurring pathogens causing them are Mycobacterium avium complex (MAC) and M. abscessus.

[0004] MAC is a complex composed mainly of M. intracellulare and M. avium, and accounts for approximately 50% of the causative agents of NTM infections in Korea. M. abscessus is a rapidly growing acid-fast bacterium that was isolated from M. chelonae and recently named a new subspecies (M. abscessussubsp.abscessus). M. abscessus is reported to be involved in infectious diseases at a higher frequency than the previously known M. chelonae or M. fortuitum, and accounts for approximately 11-22% of the causative agents of NTM infections in Korea.

[0005] Infections caused by M. abscessus and MAC primarily cause pulmonary infections, especially in immunosuppressed individuals such as patients with cystic fibrosis and the elderly. M. abscessus is known to be highly antibiotic-resistant even among Mycobacteria. It has innate resistance to antibiotics such as β-lactamases, aminoglycoside phosphotransferases, and aminoglycosidase acetyltransferases, as well as clarithromycin and first-generation antituberculosis drugs (isoniazid, rifampin, pyrazinamide, and ethambutol). Along with M. abscessus, MAC is the most common NTM pathogen causing human infections. Like M. abscessus, MAC can cause pulmonary infections in patients with chronic lung disease and immunocompromised individuals. Similar to M. abscessus, it also exhibits multidrug resistance, which is accompanied by long-term treatment times, high costs, and drug side effects.

[0006] Infections caused by M. abscessus and MAC require long-term drug treatment, frequently cause side effects, and are difficult to treat with antibiotics due to their multidrug resistance characteristics, so there is a high tendency to rely on surgical treatment.

[0007] That is, infections caused by M. abscessus and MAC are difficult to treat due to their characteristic of multi-drug resistance to antibiotics, and require long-term treatment and surgical therapy, making infections caused by them an important public health problem worldwide.

[0008] Therefore, there is an urgent need to develop new drugs for infections caused by NTM, such as M. abscessus and MAC, due to difficulties in existing prevention and treatment.

[0009] Against this backdrop, the inventors of the present application conducted in-depth research to develop innovative vaccines and therapeutic agents for the prevention and treatment of nontuberculous mycobacterial infections.

[0010] The inventors of the present application, through genetic comparative analysis studies, noticed that the MSAD (Malonate Semialdehyde Decarboxylase) gene was deleted in highly pathogenic strains of Mycobacteria, such as M. tuberculosis, M. leprae, and M. ulcerans, and from this, they presumed that the MSAD protein would contribute to reducing the pathogenicity of the pathogen. Based on this, they attempted to confirm the possibility of MSAD as a vaccine and therapeutic agent for nontuberculous mycobacterial infections.

[0011] In particular, protein-based therapeutics can be useful as vaccines and therapeutics with minimal side effects because they target specific antigens of different diseases and induce immune responses by utilizing antigen-specific properties, and act only on specific pathogens or cells without affecting normal cells.

[0012] The inventors of the present application have conducted in-depth research using this protein-based therapeutic approach and have discovered that the MSAD protein can act as an effective vaccine and therapeutic agent against nontuberculous mycobacterial infections.

[0013] Furthermore, the inventors studied the route of administration to maximize the efficacy of the MSAD protein and discovered that the nasal administration technique could further enhance its effectiveness.

[0014] Meanwhile, intranasal administration is a technique that stimulates mucosal immunity by administering antigens into the nasal cavity, thereby inducing a strong local immune response. In addition to mucosal immune responses, systemic immune responses can also be activated through mucosal-associated lymphoid tissue. Unlike conventional vaccine and therapeutic administration methods that primarily rely on injections, intranasal drug administration is non-invasive, reducing pain and fear of injections. This makes it very easy to administer to children and the elderly. Patients can easily administer the drug themselves, reducing visits to medical institutions and the burden on medical personnel. Furthermore, compared to injections, intranasal drug administration has the advantage of fewer systemic side effects, and local side effects are relatively mild.

[0015] [Prior Art Literature]

[0016] [Patent Document]

[0017] (Patent Document 0001) Korean Patent Publication No. 10-2023-0085439

[0018] One aspect of the present invention provides an immunogenic composition comprising MSAD (Malonate Semialdehyde Decarboxylase) protein; or a polynucleotide encoding the same.

[0019] Another aspect provides a method of inducing an immune response comprising administering to a subject in need thereof an MSAD protein; or a polynucleotide encoding the same.

[0020] Another aspect provides a use of the MSAD protein; or a polynucleotide encoding the same, for inducing an immune response.

[0021] Another aspect provides the use of an MSAD protein for use in the manufacture of an immunogenic composition; or a polynucleotide encoding the same.

[0022] Another aspect is to provide a vaccine composition for preventing or treating nontuberculous mycobacterial infections comprising the immunogenic composition.

[0023] Another aspect provides a method for preventing or treating a nontuberculous mycobacterial infection, comprising administering the immunogenic composition to a subject in need thereof.

[0024] Another aspect provides a use of the immunogenic composition for the prevention or treatment of nontuberculous mycobacterial infections.

[0025] Another aspect provides a use of the immunogenic composition for the manufacture of a vaccine composition for the prevention or treatment of nontuberculous mycobacterial infections.

[0026] Another aspect provides a pharmaceutical composition for the prevention or treatment of nontuberculous mycobacterial infections, comprising the MSAD protein or a polynucleotide encoding the same.

[0027] Another aspect provides a method for preventing or treating a nontuberculous mycobacterial infection, comprising administering to a subject in need thereof an MSAD protein; or a polynucleotide encoding the same.

[0028] Another aspect provides the use of the MSAD protein; or a polynucleotide encoding the same, for use in the prevention or treatment of nontuberculous mycobacterial infections.

[0029] Another aspect provides the use of the MSAD protein; or a polynucleotide encoding the same, for use in the manufacture of a pharmaceutical composition for the prevention or treatment of nontuberculous mycobacterial infections.

[0030] Another aspect is to provide a health functional food for preventing or improving nontuberculous mycobacterial infections, comprising MSAD protein or a polynucleotide encoding the same.

[0031] Another aspect provides the use of MSAD protein; or a polynucleotide encoding the same, for use in the manufacture of a health functional food for preventing or improving nontuberculous mycobacterial infections.

[0032] Another aspect is to provide a feed composition for preventing or improving nontuberculous mycobacterial infections, comprising MSAD protein or a polynucleotide encoding the same.

[0033] Another aspect provides the use of MSAD protein; or a polynucleotide encoding the same, for use in the manufacture of a feed composition for preventing or ameliorating nontuberculous mycobacterial infections.

[0034] One aspect of the present invention provides an immunogenic composition comprising MSAD (Malonate Semialdehyde Decarboxylase) protein; or a polynucleotide encoding the same.

[0035] Another aspect provides a method for inducing an immune response comprising administering to a subject in need thereof an MSAD protein; or a polynucleotide encoding the same.

[0036] Another aspect provides for the use of the MSAD protein; or a polynucleotide encoding the same, for inducing an immune response.

[0037] Another aspect provides the use of an MSAD protein; or a polynucleotide encoding the same, for use in the manufacture of an immunogenic composition.

[0038] As used herein, the term “immunogen” means a substance that induces or is capable of inducing an immune response within an individual.

[0039] In one specific example, the immunogenic composition may comprise MSAD protein or a polynucleotide encoding the same as an active ingredient.

[0040] As used herein, the term "comprising as an active ingredient" in relation to an immunogenic composition means that the immunogenic composition includes an effective amount capable of inducing, stimulating or enhancing a targeted immune response.

[0041] As used herein, the terms "polynucleotide" and "nucleic acid" refer to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in double-stranded or single-stranded form. Unless otherwise specified, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.

[0042] The above polynucleotide may be used without limitation as long as it encodes the MSAD protein of the present invention, and includes all of DNA sequences, genomic DNA (gDNA) sequences, complementary DNA (cDNA) sequences, RNA sequences, and messenger RNA (mRNA). The above polynucleotide may be isolated from nature or produced by genetic engineering methods known in the art.

[0043] In one specific example, the polynucleotide may be a deoxyribonucleic acid (DNA) polynucleotide or a ribonucleic acid (RNA) polynucleotide.

[0044] In one specific example, the polynucleotide may be a deoxyribonucleic acid polynucleotide and / or may be included in a recombinant vector.

[0045] In one specific example, the recombinant vector may be an expression vector of the MSAD protein.

[0046] In one specific example, the immunogenic composition may comprise an MSAD protein or an expression vector thereof, and the method for inducing an immune response may comprise a step of administering the MSAD protein or an expression vector thereof to a subject in need thereof.

[0047] As used herein, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can integrate into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably herein. For the purposes of the present invention, the use of plasmid vectors is preferred. A typical plasmid vector useful for this purpose has a structure that includes (a) an origin of replication that allows efficient replication, such that hundreds of plasmid vectors can be contained per host cell, (b) an antibiotic resistance gene that allows selection of host cells transformed with the plasmid vector, and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site does not exist, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adaptor or linker according to conventional methods.

[0048] In the present invention, the vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like. Suitable recombinant vectors may include expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, and may be produced in various ways depending on the purpose. In the present invention, the vector may be any means used to deliver a polynucleotide encoding the MSAD protein of the present invention to a host cell, and a preferred vector may be a viral vector such as a retrovirus, a herpes virus, an adenovirus, and an adeno-associated virus.

[0049] In the present invention, the polynucleotide encoding the MSAD protein can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct DNA introduction. Expression within a target tissue can be achieved by targeting the recombinant vector to specific cells using a viral vector, a receptor ligand, or the like, using a tissue-specific promoter, or a combination of both.

[0050] In one specific example, the polynucleotide may be messenger RNA (mRNA) and may be formulated into a lipid nanoparticle (LNP).

[0051] As used herein, the term "lipid nanoparticle" (LNP) refers to nanometer-sized particles composed of lipids used to deliver bioactive substances such as genes, nucleic acids, drugs, or vaccines into cells. Lipid nanoparticles are composed of a bilayer or monolayer of lipid molecules that are spontaneously formed and have a structure capable of encapsulating nucleic acids or drugs within them. Lipid nanoparticles are generally composed of lipid components such as (i) ionizable cationic lipids capable of neutralizing the negative charge of mRNA, (ii) phospholipids and cholesterol that maintain the lipid bilayer structure of the lipid nanoparticle and enable cell membrane penetration, and (iii) polyethylene glycol (PEG) that enhances the stability of lipid nanoparticles in the body. Lipid nanoparticles can protect mRNA from degradation by enzymes in the body and neutralize the negative charge of mRNA, thereby increasing cell membrane penetration efficiency. Once inside the cell, lipid nanoparticles separate from and degrade mRNA, and the mRNA can participate in protein expression processes in the cytoplasm.

[0052] In one specific example, the immunogenic composition may be against non-tuberculous mycobacteria (NTM).

[0053] The above immunogenic composition may comprise, as an active ingredient, the MSAD protein or a polynucleotide encoding the same to an extent capable of inducing, stimulating or enhancing an immune response against nontuberculous mycobacteria.

[0054] The method for inducing an immune response may comprise a step of administering to a subject in need thereof an MSAD protein or a polynucleotide encoding the same, or an expression vector thereof, to an extent capable of inducing, stimulating, or enhancing an immune response against nontuberculous mycobacteria.

[0055] In one specific example, the nontuberculous mycobacteria are Mycobacterium avium complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium prarintracellulare, Mycobacterium intracellularesubsp.yongonense, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium marseillense, Mycobacterium arosiense, Mycobacterium bouchedurhonense, Mycobacterium timonense, Mycobacterium vulneris, Mycobacterium indicus pranii, Mycobacterium abscessuscomplex, Mycobacterium abscessus, Mycobacterium abscessussubsp.abscessus, Mycobacterium abscessussubsp.massiliense, Mycobacterium abscessussubsp. boletii.Bolletii), Mycobacterium smegmatis, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium paragordonae, Mycobacterium osloensis, Mycobacterium phlei, Mycobacterium terrae, Mycobacterium chelonae, Mycobacterium mucogenicum, Mycobacterium peregrinum, Mycobacterium simiae The spores may be at least one selected from the group consisting of Mycobacterium simiae, Mycobacterium wolinskyi, Mycobacterium ulcerans, and Mycobacterium marinum.

[0056] In one specific example, the MSAD protein may be derived from a nontuberculous mycobacterium.

[0057] The above MSAD protein may be derived from Mycobacterium abscessus or Mycobacterium avium complex.

[0058] The above MSAD protein may be derived from Mycobacterium abscessus or Mycobacterium intracellulare.

[0059] In one specific example, the MSAD protein may be MSAD-1 protein, MSAD-2 protein, or a combination thereof.

[0060] In one specific example, the MSAD protein may be a protein consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, homology or identity to a protein consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0061] In one specific example, the polynucleotide encoding the MSAD protein may be a polynucleotide consisting of a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, homology or identity to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In one specific example, the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 may be a deoxyribonucleic acid polynucleotide and may be included in a recombinant vector.

[0062] In one specific example, the polynucleotide encoding the MSAD protein may be a polynucleotide consisting of a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, homology or identity to a polynucleotide having a sequence complementary to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In one specific example, the polynucleotide having a sequence complementary to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 may be a ribonucleic acid polynucleotide or messenger RNA (mRNA), and may be formulated into a lipid nanoparticle (LNP).

[0063] As used herein, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences or nucleotide sequences are related, which may be expressed as a percentage. The terms homology and identity may be used interchangeably.

[0064] In one specific example, the polynucleotide of SEQ ID NO: 5 or SEQ ID NO: 6 may be a mammalian optimized sequence of a polynucleotide encoding an MSAD protein.

[0065] As used herein, the term “mammalian optimized sequence” refers to a gene sequence encoding a specific protein that is optimized so that the specific protein can be efficiently expressed in mammalian cells. Mammalian optimized sequences are generally designed by the following methods: (i) codon optimization: optimizing the gene sequence encoding a specific protein by selecting codons preferred in mammals; (ii) GC content control: regulating the GC content of the gene sequence so that it is suitable for expression in mammals; (iii) RNA structure optimization: optimizing the mRNA sequence by considering the secondary structure of mRNA, which can affect the protein translation process; (iv) transcription and translation control element optimization: optimizing promoter, enhancer, UTR (untranslated region) sequences, etc. that function well in mammalian cells so that the former can be effectively transcribed and translated.

[0066] Using mammalian-optimized sequences allows genes encoding specific proteins to be expressed at higher levels or to produce proteins more stably in mammalian cells. In particular, since the goal of vaccines is to sufficiently produce the target antigen protein to induce an immune response, using mammalian-optimized sequences allows for maximum protein expression in mammalian cells. In vaccine development, these mammalian-optimized sequences can enhance the efficacy of various vaccines, including DNA vaccines, mRNA vaccines, recombinant protein vaccines, and viral vector vaccines, and achieve higher yields during production.

[0067]

[0068] Another aspect provides a vaccine composition for preventing or treating nontuberculous mycobacterial infections comprising the immunogenic composition according to the present invention.

[0069] Another aspect provides a method for preventing or treating a nontuberculous mycobacterial infection, comprising administering the immunogenic composition to a subject in need thereof.

[0070] Another aspect provides the use of the immunogenic composition for the prevention or treatment of nontuberculous mycobacterial infections.

[0071] Another aspect provides the use of the immunogenic composition for the manufacture of a vaccine composition for the prevention or treatment of nontuberculous mycobacterial infections.

[0072] In one specific example, the vaccine composition may include the immunogenic composition according to the present invention as an active ingredient.

[0073] In this specification, the term "comprising as an active ingredient" in relation to a vaccine composition means including an effective amount capable of exhibiting the preventive or therapeutic effect of the vaccine composition on nontuberculous mycobacterial infections.

[0074] In this specification, the term “prevention” means any action that inhibits or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0075] In one specific embodiment, the vaccine composition may further comprise an immune adjuvant.

[0076] In one specific example, the method for preventing or treating nontuberculous mycobacterial infection may further include a step of administering an immune adjuvant to a subject in need thereof.

[0077] As used herein, the term "immune adjuvant" refers to a substance that, while not inherently capable of eliciting a specific antigen-antibody immune response, can stimulate an immune response to an antigen, such as a vaccine, thereby inducing enhanced immunity. For example, when an immune adjuvant is used in a purified antigen vaccine that exhibits a low immune response, a high immune response can be induced even with a low concentration of the antigen. The term "immune adjuvant" may be used interchangeably with immune adjuvant, immune enhancer, immune booster, or immune stimulator.

[0078] In one specific example, the immune adjuvant may be at least one selected from the group consisting of AS01 (liposome mixed with monophosphoryl lipid A and saponin QS-21), IC31 (oligo nucleotide and cationic peptide), CFA01 (cationic liposome), Alum (aluminum salts), HBsAg (Hepatitis B surface antigen), and a polypeptide having an amino acid sequence of SEQ ID NO: 9 (GRLVFQ).

[0079] In one specific example, the vaccine composition may be administered via one or more routes selected from the group consisting of oral, transdermal, intramuscular, intraperitoneal, inhalational, subcutaneous, intravenous, and nasal routes. Additionally, the vaccine composition may be administered via a non-invasive route.

[0080]

[0081] Another aspect provides a pharmaceutical composition for the prevention or treatment of nontuberculous mycobacterial infections, comprising the MSAD protein; or a polynucleotide encoding the same.

[0082] Another aspect provides a method for preventing or treating a nontuberculous mycobacterial infection, comprising administering to a subject in need thereof an MSAD protein according to the present invention; or a polynucleotide encoding the same.

[0083] Another aspect provides the use of the MSAD protein; or a polynucleotide encoding the same, for use in the prevention or treatment of nontuberculous mycobacterial infections.

[0084] Another aspect provides the use of the MSAD protein; or a polynucleotide encoding the same, for use in the manufacture of a pharmaceutical composition for the prevention or treatment of nontuberculous mycobacterial infections.

[0085] In one specific example, the pharmaceutical composition may comprise MSAD protein or a polynucleotide encoding the same as an active ingredient.

[0086] In this specification, the term "comprising as an active ingredient" in relation to a pharmaceutical composition means including an effective amount capable of exhibiting the preventive or therapeutic effect of the pharmaceutical composition on nontuberculous mycobacterial infections.

[0087] In one specific example, the polynucleotide may be a deoxyribonucleic acid (DNA) polynucleotide or a ribonucleic acid (RNA) polynucleotide.

[0088] The above polynucleotide may be used without limitation as long as it encodes the MSAD protein of the present invention, and includes all of DNA sequences, genomic DNA (gDNA) sequences, complementary DNA (cDNA) sequences, RNA sequences, and messenger RNA (mRNA). The above polynucleotide may be isolated from nature or produced by genetic engineering methods known in the art.

[0089] In one specific example, the polynucleotide is a deoxyribonucleic acid polynucleotide and may be included in a recombinant vector.

[0090] In one specific example, the recombinant vector may be an expression vector of the MSAD protein.

[0091] In one specific example, the pharmaceutical composition may comprise an MSAD protein or an expression vector thereof, and the method for preventing or treating a nontuberculous mycobacterial infection may comprise a step of administering the MSAD protein or an expression vector thereof to a subject in need thereof.

[0092] In one specific example, the polynucleotide may be messenger RNA (mRNA) and may be formulated into a lipid nanoparticle (LNP).

[0093] In one specific example, the nontuberculous mycobacteria are Mycobacterium avium complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium prarintracellulare, Mycobacterium intracellularesubsp.yongonense, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium marseillense, Mycobacterium arosiense, Mycobacterium bouchedurhonense, Mycobacterium timonense, Mycobacterium vulneris, Mycobacterium indicus pranii, Mycobacterium abscessuscomplex, Mycobacterium abscessus, Mycobacterium abscessussubsp.abscessus, Mycobacterium abscessussubsp.massiliense, Mycobacterium abscessussubsp. boletii.Bolletii), Mycobacterium smegmatis, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium paragordonae, Mycobacterium osloensis, Mycobacterium phlei, Mycobacterium terrae, Mycobacterium chelonae, Mycobacterium mucogenicum, Mycobacterium peregrinum, Mycobacterium simiae The spores may be at least one selected from the group consisting of Mycobacterium simiae, Mycobacterium wolinskyi, Mycobacterium ulcerans, and Mycobacterium marinum.

[0094] In one specific example, the MSAD protein may be derived from a nontuberculous mycobacterium.

[0095] The above MSAD protein may be derived from Mycobacterium abscessus or Mycobacterium avium complex.

[0096] The above MSAD protein may be derived from Mycobacterium abscessus or Mycobacterium intracellulare.

[0097] In one specific example, the MSAD protein may be MSAD-1 protein, MSAD-2 protein, or a combination thereof.

[0098] In one specific example, the MSAD protein may be a protein consisting of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, homology or identity to a protein consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0099] In one specific example, the polynucleotide encoding the MSAD protein may be a polynucleotide consisting of a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, homology or identity to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In one specific example, the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 may be a deoxyribonucleic acid polynucleotide and may be included in a recombinant vector.

[0100] In one specific example, the polynucleotide encoding the MSAD protein may be a polynucleotide consisting of a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100%, homology or identity to a polynucleotide having a sequence complementary to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In one specific example, the polynucleotide having a sequence complementary to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 may be a ribonucleic acid polynucleotide or messenger RNA (mRNA), and may be formulated into a lipid nanoparticle (LNP).

[0101] In one specific example, the polynucleotide of SEQ ID NO: 5 or SEQ ID NO: 6 may be a mammalian optimized sequence of a polynucleotide encoding an MSAD protein.

[0102] Using mammalian-optimized sequences allows genes encoding specific proteins to be expressed at higher levels or to produce proteins more stably in mammalian cells. In particular, since the goal of vaccines is to sufficiently produce the target antigen protein to induce an immune response, using mammalian-optimized sequences allows for maximum protein expression in mammalian cells. In vaccine development, these mammalian-optimized sequences can enhance the efficacy of various vaccines, including DNA vaccines, mRNA vaccines, recombinant protein vaccines, and viral vector vaccines, and achieve higher yields during production.

[0103] In one specific embodiment, the pharmaceutical composition may further comprise an immune adjuvant.

[0104] In one specific example, the method for preventing or treating nontuberculous mycobacterial infection may further include a step of administering an immune adjuvant to a subject in need thereof.

[0105] In one specific example, the immune adjuvant may be at least one selected from the group consisting of AS01 (liposome mixed with monophosphoryl lipid A and saponin QS-21), IC31 (oligo nucleotide and cationic peptide), CFA01 (cationic liposome), Alum (aluminum salts), HBsAg (Hepatitis B surface antigen), and a polypeptide having an amino acid sequence of SEQ ID NO: 9 (GRLVFQ).

[0106] In one specific example, the pharmaceutical composition may be administered by one or more routes selected from the group consisting of oral, transdermal, intramuscular, intraperitoneal, inhalational, subcutaneous, intravenous, and nasal routes. Additionally, the pharmaceutical composition may be administered by a non-invasive route.

[0107]

[0108] The vaccine composition or pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable salt.

[0109] As used herein, the term “pharmaceutically acceptable” means a substance that can be effectively used for a desired purpose without causing excessive toxicity, irritation, or allergic reactions, within the scope of pharmaceutical judgment.

[0110] As used herein, the term "pharmaceutically acceptable salt" means a salt according to one aspect of the present invention which is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound (e.g., protein, polynucleotide). Salts of the parent compound can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base, for example, sodium, calcium, magnesium or potassium, or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. These reactions are typically carried out in water or in an organic solvent or in a mixture of the two. Generally, when practical, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile can be used. The pharmaceutically acceptable salts include both addition salts of acids or bases and stereochemically isomeric forms thereof, and may be, for example, addition salts of organic or inorganic acids. The above salt includes any salt that maintains the activity of the parent compound in the subject of administration and does not cause undesirable effects, and is not particularly limited thereto.

[0111] These salts include inorganic and organic salts, for example, acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium idet, carbonic acid, chlorobenzoic acid, citric acid, idetic acid, toluenesulfonic acid, fumaric acid, gluceptic acid, esilinic acid, pamoic acid, gluconic acid, methylnitric acid, malonic acid, hydrochloric acid, hydroiodoic acid, hydroxynaphtholic acid, isethionic acid, lactobionic acid, mandelic acid, mucic acid, It can be naphthylic acid, muconic acid, p-nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, salicylic acid, sulfamic acid, sulfanilinic acid, methanesulfonic acid. In addition, the salt form includes salts of alkali and alkaline earth metals such as ammonium salt, lithium salt, sodium salt, potassium salt, magnesium salt, and calcium salt, salts with organic bases such as benzathine, N-methyl-D-glucamine, and hydrabamine salts, and salts with amino acids such as arginine and lysine. In addition, the salt form can be converted into a free form by treating with a suitable base or acid.

[0112] The vaccine composition or pharmaceutical composition according to the present invention may be administered as a separate drug, or may be administered in combination with an antibiotic or other drug for the prevention, treatment, or symptom relief of nontuberculous mycobacterial infections. The antibiotic refers to a conventional antibiotic for the prevention or treatment of nontuberculous mycobacterial infections.

[0113] As used herein, the term “combination administration” may be achieved by administering the individual components of the immuno / therapeutic regimen simultaneously, sequentially, in reverse order, or separately.

[0114] The terms "subject" and "patient" are used interchangeably herein. The subject may be an animal. In some embodiments, the subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate). In some embodiments, the subject is a cynomolgus monkey. In some embodiments, the subject is a human.

[0115] The above vaccine composition or pharmaceutical composition is administered in an immunologically or therapeutically effective amount.

[0116] As used herein, the term “immunologically effective amount” or “therapeutically effective amount” refers to a drug effective to achieve a desired immunological, prophylactic, or therapeutic result, such as the MSAD protein according to the present invention, a polynucleotide encoding the same, or an immunogenic composition. In some cases, the desired result is treatment of a disease or disorder in a subject. The immunologically or therapeutically effective amount level may be determined based on factors including the type and severity of the patient’s disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration, and the excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The compositions of the present disclosure may be administered as individual drugs or in combination with other drugs, may be administered sequentially or simultaneously with conventional drugs, and may be administered in single or multiple doses. That is, the total effective amount of the compositions of the present disclosure may be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. Taking all of the above factors into consideration, it is important to administer the amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0117] As used herein, terms such as "treating," "treatment," "to treat," "palliating," or "to palliate" refer to therapeutic measures aimed at curing, slowing, alleviating symptoms, and / or arresting the progression of a diagnosed pathological condition or disorder. Therefore, those requiring treatment include those who have already been diagnosed with or are suspected of having a disorder.

[0118] The vaccine composition or pharmaceutical composition according to the present invention may additionally include a pharmaceutically acceptable carrier and may be formulated together with the carrier.

[0119] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound (e.g., protein, polynucleotide). In a composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0120] A vaccine composition or pharmaceutical composition according to one embodiment of the present invention; or the composition comprising a pharmaceutically acceptable carrier, may be applied in any dosage form comprising the same as an active ingredient, and may be prepared as an oral or parenteral dosage form, and may be formulated in a unit dosage form for ease of administration and uniformity of dosage. In the present invention, the pharmaceutical dosage form includes a form suitable for oral, rectal, nasal, intranasal, topical (including buccal and sublingual), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration, or a form suitable for administration by inhalation or insufflation.

[0121] Oral administration dosage forms containing the composition of the present invention as an active ingredient may be formulated, for example, as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs.

[0122] The composition of the present invention may be formulated as a parenteral dosage form containing the active ingredient, such as a subcutaneous injection, intravenous injection, or intramuscular injection; a suppository injection; or a spray, such as an aerosol, that can be inhaled through the respiratory tract. To formulate the composition of the present invention as an injectable dosage form, the composition of the present invention may be mixed with a stabilizer or buffer in water to prepare a solution or suspension, which may then be formulated into a unit dosage form in an ampoule or vial.

[0123] The dosage of the pharmaceutical composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The daily dosage is preferably 0.01 μg to 1000 mg per kg of body weight per day when administered parenterally, more preferably 1 μg to 500 mg. However, since the dosage may increase or decrease depending on the route of administration, severity of obesity, sex, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0124]

[0125] Another aspect provides a health functional food for preventing or improving nontuberculous mycobacterial infections, comprising MSAD protein or a polynucleotide encoding the same.

[0126] Another aspect provides the use of MSAD protein; or a polynucleotide encoding the same, for use in the manufacture of a health functional food for preventing or ameliorating nontuberculous mycobacterial infections.

[0127] In the health functional food of the present invention according to one specific example, “MSAD protein”, “polynucleotide”, “nontuberculous mycobacteria”, “nontuberculous mycobacterial infection”, “prevention” and “improvement” are as described above.

[0128] According to one specific example, the health functional food of the present invention may include a salt that is acceptable from a food science perspective.

[0129] As used herein, the term "food-acceptable salt" means a formulation of a compound (e.g., a protein, a polynucleotide) that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. For example, the food-acceptable salt can be obtained by reacting the compound with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, and the like. In addition, it can be obtained by reacting the compound with a base to form a salt such as an alkali metal salt such as an ammonium salt, a sodium or potassium salt, an alkaline earth metal salt such as a calcium or magnesium salt, a salt of an organic base such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, etc., but is not limited thereto.

[0130] The health functional food herein may be formulated into any one form selected from the group consisting of powders, tablets, capsules, pills, granules, and liquids, using conventional methods known in the art, but is not limited thereto. Various forms may be manufactured using methods known in the art.

[0131] Additionally, it can be prepared in the form of a composition by mixing with a known substance or active ingredient known to have preventive, improving or therapeutic activity against nontuberculous mycobacterial infections.

[0132] In addition, the health functional food of the present invention may contain conventional food additives, and the suitability as the "food additive" is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sorghum pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

[0133] In addition to the above, the health functional food of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may include fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination.

[0134]

[0135] Another aspect provides a feed composition for preventing or improving nontuberculous mycobacterial infections, comprising MSAD protein or a polynucleotide encoding the same.

[0136] Another aspect provides the use of MSAD protein; or a polynucleotide encoding the same, for use in the manufacture of a feed composition for preventing or ameliorating nontuberculous mycobacterial infections.

[0137] In the feed composition of the present invention according to one specific example, “MSAD protein”, “polynucleotide”, “nontuberculous mycobacteria”, “nontuberculous mycobacterial infection”, “prevention” and “improvement” are as described above.

[0138] In this specification, “feed composition” may mean any natural or artificial diet, meal, etc. or a component of said meal, especially for or suitable for eating, ingesting and digesting by an animal.

[0139] The above feed composition may include, but is not particularly limited to, nutrients such as energy, protein, lipids, vitamins, and minerals required by the individual consuming the feed. The individual refers to the subject of breeding, and includes, without limitation, any living organism capable of consuming the feed of the present invention, including companion animals and livestock.

[0140] The type of the above feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant-based feed such as grains, roots, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal-based feed such as proteins, inorganic substances, oils, minerals, oils, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more types.

[0141] The feed composition according to the present invention can be manufactured by adding the MSAD protein of the present invention or a polynucleotide encoding the same in an appropriate effective concentration range according to various feed manufacturing methods known in the art.

[0142] The feed composition according to the present invention can be applied without limitation to any subject for the purpose of preventing or ameliorating nontuberculous mycobacterial infections. For example, it can be applied to any subject, including non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds, and fish.

[0143] The vaccine composition and pharmaceutical composition comprising the MSAD protein of the present invention or a polynucleotide encoding it have excellent preventive and therapeutic efficacy against nontuberculous mycobacterial infections. Furthermore, the vaccine composition and pharmaceutical composition comprising the MSAD protein of the present invention or a polynucleotide encoding it have enhanced preventive and therapeutic efficacy against nontuberculous mycobacterial infections when administered intranasally, and have enhanced preventive and therapeutic efficacy against nontuberculous mycobacterial infections when used in combination with an immune adjuvant.

[0144] Figure 1 is a graph comparing the expression levels of inflammatory cytokines TNF-α and IL-6 according to time periods ((A) 24 hours, (B) 48 hours) after treatment with MSAD protein at various concentrations in mouse macrophage cell line J774A.1.

[0145] Figure 2 is a schematic diagram of the immunization schedule to confirm the protective effect through intranasal administration (IN) of MSAD protein before and after infection in an animal model of M. abscessus respiratory infection (Cy, Cyclophosphamide; M. ab_R, M. abscessus_Rough; M. ab_S, M. abscessus_Smooth; M. mas, M. massiliense).

[0146] Figure 3 is a graph of CFU in each organ confirmed in a model of nasal administration of MSAD protein before and after infection ((A) M. abscessus_Rough, (B) M. abscessus_Smooth, (C) M. massiliense).

[0147] Figure 4 is a photograph confirming the ability to alleviate inflammation in the MSAD nasal administration group through lung tissue H&E staining.

[0148] Figure 5 is a graph comparing serum IgG1 antibody levels according to time after infection in a model of nasal administration of MSAD protein before and after infection.

[0149] Figure 6 is a graph comparing serum IgG2a antibody levels according to time after infection in a model of nasal administration of MSAD protein before and after infection.

[0150] Figure 7 is a graph comparing the immune activity of spleen cells and lung cells in a model of nasal administration of MSAD protein before and after infection, using IFN-γ ELISA using culture fluids of spleen cells and lung cells cultured for 3 days in the presence or absence of MSAD antigen.

[0151] Figure 8 is a graph comparing the immune activity of spleen cells and lung cells in a model of nasal administration of MSAD protein before and after infection, using IFN-γ ELISPOT using culture fluids of spleen cells and lung cells cultured for 3 days in the presence or absence of MSAD antigen.

[0152] Figure 9 is a graph comparing the immune activity of spleen cells and lung cells in a model of nasal administration of MSAD protein before and after infection, using the IFN-γ secreting CD4 cell ratio or CD8 T cell ratio confirmed using flow cytometry analysis of spleen cells.

[0153] Figure 10 is a schematic diagram of the experimental schedule for verifying the protective effect against M. abscessus respiratory infection by intranasal administration of MSAD protein before and after infection.

[0154] Figure 11 is a graph showing the post-infection weight change rate of a model administered intranasally with MSAD protein before and after infection, as a result of an experiment verifying the ability to protect against M. abscessus respiratory infection by intranasal administration of MSAD protein before and after infection.

[0155] Figure 12 is a graph comparing the immune activity of lung cells in a model of nasal administration of MSAD protein before and after infection, using IFN-γ ELISPOT, as a result of an experiment to verify the protective effect against M. abscessus respiratory infection by nasal administration of MSAD protein before and after infection.

[0156] Figure 13 is a graph of CFU in lung tissue of a model of nasal administration of MSAD protein before and after infection, as a result of an experiment to verify the protective effect against M. abscessus respiratory infection due to nasal administration of MSAD protein before and after infection.

[0157] Figure 14 is a schematic diagram of an experimental schedule to confirm the protective effect against M. abscessus respiratory infection according to the nasal administration dose of MSAD protein before and after infection in an animal model of M. abscessus respiratory infection.

[0158] Figure 15 is a graph showing the weight change rate after infection of the MSAD protein nasal administration model before and after infection according to the dose of MSAD protein administered intranasally, as a result of an experiment on the protective effect against M. abscessus respiratory infection according to the dose of MSAD protein administered intranasally before and after infection in an animal model of M. abscessus respiratory infection.

[0159] Figure 16 is a photograph showing the results of an experiment to determine the protective effect against M. abscessus respiratory infection according to the nasal administration dose of MSAD protein before and after infection in an animal model of M. abscessus respiratory infection, confirming the ability to alleviate inflammation findings according to the MSAD protein administration dose in the MSAD nasal administration group through lung tissue H&E staining.

[0160] Figure 17 is a graph comparing antibody titers (IgG1, IgG2a, total IgG) against MSAD antigen in mouse serum as a result of an experiment on the protective effect against M. abscessus respiratory infection according to the nasal administration dose of MSAD protein before and after infection in an animal model of M. abscessus respiratory infection.

[0161] Figure 18 is a graph of CFU in the lung tissue of an MSAD protein nasal administration model before and after infection, as a result of an experiment on the protective effect against M. abscessus respiratory infection according to the dose of MSAD protein nasal administration before and after infection in an M. abscessus respiratory infection animal model.

[0162] Figure 19 is a schematic diagram of an experimental schedule comparing survival rates according to MSAD protein administration methods before and after infection in a lethal dose M. abscessus respiratory infection model.

[0163] Figure 20 is a graph comparing the survival rate by day after M. abscessus infection according to the MSAD protein administration method before and after infection in a lethal dose M. abscessus respiratory infection model.

[0164] Figure 21 is a graph comparing the survival rates of each group on the 8th day after infection according to the pre- and post-infection MSAD protein administration method in a lethal dose M. abscessus respiratory infection model.

[0165] Figure 22 is a photograph confirming the ability to alleviate lung tissue inflammation findings according to nasal administration of MSAD protein before and after infection in a lethal dose M. abscessus respiratory infection model.

[0166] Figure 23 is a schematic diagram of an experimental schedule to confirm the therapeutic vaccine potential of MSAD protein in an animal model of M. abscessus respiratory infection.

[0167] Figure 24 is a graph showing the results of an experiment to confirm the possibility of a therapeutic vaccine for nasal administration of MSAD protein in a M. abscessus respiratory infection model, showing the change in mouse weight ratio for 7 days after M. abscessus respiratory infection.

[0168] Figure 25 is a graph showing the results of an experiment to confirm the possibility of a therapeutic vaccine for nasal administration of MSAD protein in a M. abscessus respiratory infection model, showing M. abscessus CFU in lung tissue 3 days and 7 days after M. abscessus respiratory infection.

[0169] Figure 26 is a schematic diagram of an experimental schedule to confirm the therapeutic vaccine efficacy of MSAD protein and MSAD protein + sHB in an IAV + M. abscessus respiratory infection model.

[0170] Figure 27 is a graph showing the change in mouse weight ratio 7 days after M. abscessus respiratory infection, as a result of a mouse experiment to confirm the therapeutic vaccine efficacy of nasal administration of MSAD protein and MSAD protein + sHB in an IAV + M. abscessus respiratory infection model.

[0171] Figure 28 is a graph of M. abscessus CFU in lung tissue 14 days after M. abscessus respiratory infection, as a result of a mouse experiment to confirm the therapeutic vaccine efficacy of nasal administration of MSAD protein and MSAD protein + sHB in an IAV + M. abscessus respiratory infection model.

[0172] Figure 29 is a photograph showing the results of a mouse experiment to confirm the therapeutic vaccine efficacy of nasal administration of MSAD protein and MSAD protein + sHB in an IAV + M. abscessus respiratory infection model, confirming the ability to alleviate inflammation findings in lung tissue 14 days after M. abscessus respiratory infection.

[0173] Figure 30 is a graph comparing the proportion of CD4+ or CD8+ T cells secreting IFN-γ, as confirmed by flow cytometry analysis of lung cells, as a result of a mouse experiment to confirm the therapeutic vaccine efficacy of nasal administration of MSAD protein and MSAD protein+sHB in an IAV+M. abscessus respiratory infection model.

[0174] Figure 31 is a graph comparing SFU for comparing the proportion of IFN-γ secreting cells among lung cells using the IFN-γ ELISPOT experiment, as a result of a mouse experiment to confirm the therapeutic vaccine efficacy of nasal administration of MSAD protein and MSAD protein + sHB in an IAV + M. abscessus respiratory infection model.

[0175] Figure 32 is a schematic diagram of an experimental schedule to determine the protective ability of an individual against M. abscessus respiratory infection according to the MSAD protein immunization administration route.

[0176] Figure 33 is a graph showing the results of an experiment to confirm the protective effect against M. abscessus respiratory infection according to the MSAD protein immunization administration route, and showing the change in mouse weight ratio for 7 days after M. abscessus respiratory infection.

[0177] Figure 34 is a graph comparing antibody titers against MSAD antigens in mouse serum 7 days after M. abscessus respiratory infection, showing the results of an experiment to confirm the protective effect against M. abscessus respiratory infection according to the MSAD protein immunization administration route.

[0178] Figure 35 is a graph comparing M. abscessus CFU in mouse lung tissue 7 days after M. abscessus respiratory infection, showing the results of an experiment to confirm the protective effect against M. abscessus respiratory infection according to the MSAD protein immunization administration route.

[0179] Figure 36 is a schematic diagram of the experimental schedule to determine the protective effect of MSAD protein intranasal administration or MSAD+PA subcutaneous administration immunization in an IAV +M. abscessus respiratory infection mouse model.

[0180] Figure 37 is a graph comparing antibody titers against MSAD antigens in mouse serum and BALF 7 days after M. abscessus respiratory infection, as a result of an experiment to confirm the protective effect of MSAD protein nasal administration or MSAD protein + PA subcutaneous administration immunization in an IAV + M. abscessus respiratory infection mouse model.

[0181] Figure 38 is a graph comparing M. abscessus CFU in mouse lung tissue 7 days after M. abscessus respiratory infection, as a result of an experiment to confirm the protective effect of MSAD protein nasal administration or MSAD protein + PA subcutaneous administration immunization in an IAV + M. abscessus respiratory infection mouse model.

[0182] Figure 39 is a schematic diagram of an experimental schedule to determine the preventive effect against M. abscessus respiratory infection in individuals following intranasal administration of MSAD protein or intranasal administration of MSAD protein + sHB prior to lethal M. abscessus respiratory infection.

[0183] Figure 40 is a graph comparing the survival rate against M. abscessus respiratory infection in mice following immunization with intranasal administration of MSAD protein or intranasal administration of MSAD protein + sHB prior to lethal M. abscessus respiratory infection.

[0184] Figure 41 shows the results of a statistical significance test between the survival rate graphs of the M. abscessus respiratory infection control group (PBS group) and the M. abscessus respiratory infection group after nasal administration of MSAD protein + sHB immunization (nasal administration of MSAD protein + sHB group).

[0185] Figure 42 is a schematic diagram of an experimental schedule for confirming the protective effect of MSAD protein in M. intracellulare infection.

[0186] Figure 43 is a graph showing the results of an experiment to confirm the protective effect of MSAD protein against the M. intracellulare respiratory infection model, showing the change in mouse weight ratio over 7 days after M. intracellulare respiratory infection.

[0187] Figure 44 is a graph showing the results of an experiment to confirm the protective effect of MSAD protein on the M. intracellulare respiratory infection model, showing the M. intracellulare CFU in lung tissue on day 7 of M. intracellulare respiratory infection.

[0188] Figure 45 is a graph comparing the antibody titer against MSAD antigen in lung tissue in serum 7 days after M. intracellulare respiratory infection, which is the result of an experiment to confirm the protective effect of MSAD protein on the M. intracellulare respiratory infection model.

[0189] Figure 46 is a schematic diagram of an experimental schedule for confirming the ability to protect against NTM infection through subcutaneous immunization with MSAD protein derived from M. yongonense.

[0190] Figures 47 to 49 are the results of an experiment to confirm the protective ability of M. yongonense-derived MSAD protein against an NTM infection model, and are graphs of CFU in lung tissues of the M. abscessus-infected group and the M. intracellulare-infected group (Figure 47), spleen weight (Figure 48), and the intracellular ratio of IFN-γ-secreting CD8 T cells using flow cytometry (Figure 49) in the M. yongonense-derived MSAD subcutaneously and one week after NTM respiratory infection.

[0191] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0192] Terms or words used in the specification and claims of the present invention are not to be construed as limited to their usual or dictionary meanings, and should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0193] Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0194] Throughout the specification of the present invention, “A and / or B” means A or B, or A and B.

[0195]

[0196] The research results of this patented invention are the result of research (Project ID: RS-2025-00553721) conducted with support from the National Research Foundation of Korea and funding from the Ministry of Science and ICT.

[0197]

[0198] Example 1. Expression, purification, and removal of bacterial endotoxin from MSAD (Malonate Semialdehyde Decarboxylase) protein.

[0199] In the following experimental examples, MSAD (Malonate Semialdehyde Decarboxylase) protein was expressed and purified, and bacterial endotoxin was removed.

[0200] Specifically, the MSAD protein was isolated from M. abscessus ATCC19977 T Sequence of MAB_0160 protein (Protein ID: CAM60260.1) (SEQ ID NO: 1) and M. intracellularesubsp.yongonense05-1390 T The sequence (SEQ ID NO: 2) of OEM_24710 protein (Protein ID: AGP64006.1) was introduced into the pET-28a vector and then transformed into Escherichia coli Rosetta2, and expression and purification processes were performed to secure the sequence. The sequences (SEQ ID NOs: 1 and 2) of proteins derived from M. abscessus and M. intracellularesubsp. yongonense and the sequences (SEQ ID NOs: 7 and 8) of proteins expressed with each Hist-tag attached are shown in Table 1 below.

[0201] Protein sequence in MSAD-derived strain E. coli expressed and purified protein sequence M. abscessusMPLVRIDLTADRSPAEQRAIADGVHEGLVAVLKIPARDRFQIITAHDAGDIIAEDAGLGFRRSRSVVIVHIFTQTGRTTETKQRIFAELADRLGAVGVTGADLFVGISENGPQDWSFGFGHAQYVTGELAVPAAGA (SEQ ID NO: 1)MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMPLVRIDLTADRSPAEQRAIADGVHEGLVAVLKIPARDRFQIITAHDAGDIIAEDAGLGFRRSRSVVIVHIFTQTGRTTETKQRIFAELADRLGAVGVTGADLFVGISENGPQDWSFGFGHAQYVTGELAVPAAGAKLAAALEHHHHHH (SEQ ID NO: 7)M. intracellularesubsp.yongonenseMPLLYIDLIEGRSPSEVRALLDAIHETVVEAFGVPERDRYQVVRTHPAHEVIALDTGLGIDRSARQVILHVVSRRRPRELKQKFYELLASRLADRCGLDPADLIVSVTENNDEDWSFGHGRAQFLTGELK (SEQ ID NO: 2)MGSSHHHHHHSSGLVPRGSHMASMPLLYIDLIEGRSPSEVRALLDAIHETVVEAFGVPERDRYQVVRTHPAHEVIALDTGLGIDRSARQVILHVVSRRRPRELKQKFYELLASRLADRCGLDPADLIVSVTENNDEDWSFGHGRAQFLTGELKKLAAALEHHHHHH (SEQ ID NO: 8)

[0202] The pET-28a vectors in which each MSAD protein sequence was cloned were transformed into E. coli and cultured under appropriate temperature and culture conditions for target protein overexpression. First, MSAD derived from M. abscessus was cultured in 1 L of LB broth containing 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 100 μg / mL kanamycin at 26°C for 6 h. MSAD derived from M. yongonense was cultured in 1 L of LB broth containing 1 mM IPTG and 100 μg / mL kanamycin at 15°C for 16 h.

[0203] Each culture was centrifuged at 4500 rpm for 10 minutes to obtain bacteria, and then 1 mg / mL lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor were added, and the bacteria were disrupted using sonication. The homogenate was centrifuged at 20,000 xg at 4°C for 30 minutes to isolate the supernatant containing the soluble protein. Since the expressed protein was labeled with a 6xHis tag, the protein was purified using the affinity chromatography technique of the AKTA go system that utilizes the property of binding to a Ni-NTA column. Afterwards, E. After removing the endotoxin generated during protein purification from E. coli, the amount of endotoxin was quantified and confirmed using a water-soluble extract of horseshoe crab amoebocytes (LAL, Limulus Amebocyte Lysate).

[0204]

[0205] Experimental Example 1. Confirmation of the ability of MSAD protein to induce inflammation in macrophages.

[0206] J774A.1, a mouse macrophage cell line, was cultured in opti-MEM, a minimal medium, at 37°C for 1 hour to minimize nutrition. Then, MSAD protein derived from M. abscessus was diluted according to concentration in RPMI (2% FBS RPMI) supplemented with 2% fetal bovine serum (FBS), and the cells were cultured at 37°C for 24 or 48 hours. The culture medium was obtained and an enzyme-linked immunosorbent assay (ELISA) was performed to compare the expression levels of inflammatory cytokines TNF-α and IL-6.

[0207] Figure 1 is a graph comparing the expression levels of inflammatory cytokines TNF-a and IL-6 according to time periods ((A) 24 hours, (B) 48 hours) after treatment with MSAD protein at various concentrations in mouse macrophage cell line J774A.1.

[0208] As shown in Figure 1, it was confirmed that the expression levels of TNF-α and IL-6 increased in a concentration-dependent manner depending on the MSAD protein treatment concentration, and it was confirmed that the expression levels of TNF-α and IL-6 increased as the treatment time increased. This confirmed that the MSAD protein can induce immunity against macrophages, which are one of the antigen-presenting cells.

[0209]

[0210] Experimental Example 2. Confirmation of protective effect through intranasal administration (IN) of MSAD protein before and after infection in a M. abscessus respiratory infection model.

[0211] M. abscessus is an opportunistic pathogen, so respiratory infections do not readily occur in healthy individuals. Therefore, to create a state of vulnerability to infection, cyclophosphamide was injected intraperitoneally (IP) into mice to induce neutropenia by suppressing neutrophil production.

[0212] Figure 2 is a schematic diagram of the immunization schedule to confirm the protective effect through intranasal administration (IN) of MSAD protein before and after infection in an animal model of M. abscessus respiratory infection (Cy, Cyclophosphamide; M. ab_R, M. abscessus_Rough; M. ab_S, M. abscessus_Smooth; M. mas, M. massiliense).

[0213] Specifically, the immunization schedule shown in Fig. 2 is explained. 12 days (D-12) before infection and 1 day (D-1) before infection, 150 mg / kg cylophosphamide was administered intraperitoneally (IP) to BALB / c mice to induce neutropenia. In addition, 5 μg of M. abscessus-derived MSAD protein was administered intranasally (IN) to mice after respiratory anesthesia using isoflurane 1 week before infection. When infected with M. abscessus (D0), ~10 7 CFU (Colony Forming Unit) was administered intranasally, and 5 μg of MSAD protein was administered intranasally to each mouse 1 day (D+1) and 8 days (D+8) after infection.

[0214] M. abscessus used for infection were classified according to the type of colony, and two strains derived from rough colonies and smooth colonies (M. abscessus_Rough, M. abscessus_Smooth, respectively), and M. massiliense, a subspecies of M. abscessus, were used for infection.

[0215]

[0216] Seven days after infection (D+7), mice were necropsied and the number of bacteria in lung tissue was confirmed using a CFU assay.

[0217] Figure 3 is a graph of CFU in each organ confirmed in a model of nasal administration of MSAD protein before and after infection ((A) M. abscessus_Rough, (B) M. abscessus_Smooth, (C) M. massiliense).

[0218] As shown in Figure 3, a tendency toward a decrease in the number of bacteria in lung tissue was confirmed in the MSAD-administered group compared to the non-protein-administered group (PBS) for all three strains.

[0219]

[0220] At 14 days post-infection (D+14), lung tissues of mice were fixed in 4% formaldehyde, embedded in paraffin, and stained with Hematoxylin & Eosin (H&E) to confirm inflammation in the lung tissues.

[0221] Figure 4 is a photograph confirming the ability to alleviate inflammation in the MSAD nasal administration group through lung tissue H&E staining.

[0222] As shown in Figure 4, when compared among the groups infected with only the fungus, M. abscessus_Rough showed the highest level of inflammation. In the M. abscessus_Rough infection group, which showed the highest level of inflammation, nasal administration of MSAD protein significantly alleviated the inflammation.

[0223]

[0224] The antibody titers for MSAD protein were compared using serum from mice obtained 7 days (D+7) and 14 days (D+14) after infection. The IgG ELISA experiment comparing antibody titers was conducted as follows. 100 μL of an antigen suspension at a concentration of 5 μg / mL was added to each well of a 96-well plate dedicated to enzyme-linked immunosorbent assay (ELISA), and coating was performed at 4°C for one day. After washing with phosphate-buffered saline (PBS-T), each well was blocked with ELISA diluent for 1 hour at room temperature. After washing once or twice, serum diluted to an appropriate concentration was loaded into each well, and the wells were left at room temperature for 2 hours to induce binding between antigen and antibody. After washing, antibodies detecting the appropriate subclass were attached, and antibody titers were detected using HRP and TMB.

[0225] Figure 5 is a graph comparing serum IgG1 antibody levels according to time after infection in a model of nasal administration of MSAD protein before and after infection.

[0226] Figure 6 is a graph comparing serum IgG2a antibody levels according to time after infection in a model of nasal administration of MSAD protein before and after infection.

[0227] As shown in Figures 5 and 6, when the antibody titer for the MSAD protein used for mouse immunization was checked, it was confirmed that the IgG1 and IgG2a antibody titers were higher in the MSAD protein administration group compared to the non-administered PBS group. In addition, a tendency for the titer to be higher in the serum obtained 2 weeks (14 days) after infection was also confirmed compared to the serum obtained 1 week (7 days) after infection. This confirmed that the MSAD protein administration was performed correctly and that it was possible to induce an MSAD-specific immune response. In addition, the antibody titer was higher 2 weeks after infection than 1 week after infection, confirming that it was necessary to pass more than 3 weeks after the first MSAD protein immunization to increase to a statistically significant level.

[0228] In addition, the finding that antibodies to the MSAD protein increased in the serum of the blood flowing throughout the body confirmed the possibility that immunity following nasal administration of the MSAD protein was not limited to the respiratory or lung tissue but led to the induction of systemic immunity.

[0229]

[0230] Splenocytes and lung cells were obtained from mice autopsied 14 days after infection, and the immune activity of each tissue was compared. The activity of IFN-γ, a key cytokine of the Th1 immune response known to be closely related to bacterial clearance, was determined using IFN-γ ELISPOT, ELISA of cell culture fluid, or flow cytometry.

[0231] Figure 7 is a graph comparing the immune activity of spleen cells and lung cells in a model of nasal administration of MSAD protein before and after infection, using IFN-γ ELISA using culture fluids of spleen cells and lung cells cultured for 3 days in the presence or absence of MSAD antigen.

[0232] Figure 8 is a graph comparing the immune activity of spleen cells and lung cells in a model of nasal administration of MSAD protein before and after infection, using IFN-γ ELISPOT using culture fluids of spleen cells and lung cells cultured for 3 days in the presence or absence of MSAD antigen.

[0233] Figure 9 is a graph comparing the immune activity of spleen cells and lung cells in a model of nasal administration of MSAD protein before and after infection, using the IFN-γ secreting CD4 cell ratio or CD8 T cell ratio confirmed using flow cytometry analysis of spleen cells.

[0234] As shown in Figure 7, when the cell culture fluids cultured for 3 days with or without MSAD antigen from spleen cells and lung cells were examined using IFN-γ ELISA, it was confirmed that the amount of IFN-γ expression increased specifically in the MSAD antigen in the MSAD immune group.

[0235] This trend was maintained in IFN-r ELISPOT, an experimental technique that can compare the proportion of cells secreting IFN-γ for each cell, as shown in Figure 8.

[0236] Through this, it was confirmed that the nasal administration of MSAD protein sufficiently induced immunity not only in lung tissue, which is located near the main route of administration, but also in the spleen, which is a major immune-related organ.

[0237] In the case of splenocytes used for flow cytometry, the analysis was performed without antigen stimulation, and as shown in Figure 9, immune activity was confirmed through an increase in the proportion of CD4+ or CD8+ T cells secreting IFN-γ in the MSAD protein nasal administration group without re-stimulation of the MSAD protein used for immunization.

[0238]

[0239] To verify the protective effect of intranasal administration of MSAD protein before and after infection against M. abscessus respiratory infection, repeated experiments were conducted. The experimental schedule is shown in Figure 10.

[0240] Figure 10 is a schematic diagram of the experimental schedule for verifying the protective effect against M. abscessus respiratory infection by intranasal administration of MSAD protein before and after infection.

[0241] Compared to the previous experimental set schedule (see Fig. 2), the Cyclophosphamide administration schedule was conducted on D-4 and D-1, and there was a difference in that the mouse autopsy was performed 11 days after the infection date (D+11).

[0242]

[0243] The change in mouse weight in each group was observed from the date of infection until the time of sacrifice. The results are shown in Figure 11.

[0244] Figure 11 is a graph showing the post-infection weight change rate of a model administered intranasally with MSAD protein before and after infection, as a result of an experiment verifying the ability to protect against M. abscessus respiratory infection by intranasal administration of MSAD protein before and after infection.

[0245] As shown in Figure 11, no difference in weight trend was confirmed between the non-administered group and the MSAD protein nasal administration group.

[0246] Immune activity was compared in the presence or absence of MSAD antigen using IFN-γ ELISPOT in lung cells from mice sacrificed on day 11 post-infection. The results are shown in Figure 12.

[0247] Figure 12 is a graph comparing the immune activity of lung cells in a model of nasal administration of MSAD protein before and after infection, using IFN-γ ELISPOT, as a result of an experiment to verify the protective effect against M. abscessus respiratory infection by nasal administration of MSAD protein before and after infection.

[0248] As shown in Figure 12, when MSAD antigen re-stimulation was performed, it was confirmed that the number of IFN-γ secreting cells increased in the MSAD protein nasal administration group compared to the non-administration group.

[0249] The number of bacteria in the lung tissue of mice sacrificed on the 11th day after infection was determined using a CFU assay. The results are shown in Figure 13.

[0250] Figure 13 is a graph of CFU in lung tissue of a model of nasal administration of MSAD protein before and after infection, as a result of an experiment to verify the protective effect against M. abscessus respiratory infection due to nasal administration of MSAD protein before and after infection.

[0251] As shown in Figure 13, when MSAD antigen re-stimulation was performed, it was confirmed that the CFU in the lung tissue of the MSAD protein nasal administration group decreased compared to the non-administration group.

[0252]

[0253] To determine the difference in efficacy according to the nasal administration dose of MSAD protein, the efficacy was determined by setting the MSAD administration doses to 1 μg and 5 μg according to the same schedule as in Figure 10. The experimental schedule is as shown in Figure 14.

[0254] Figure 14 is a schematic diagram of an experimental schedule to confirm the protective effect against M. abscessus respiratory infection according to the nasal administration dose of MSAD protein before and after infection in an animal model of M. abscessus respiratory infection.

[0255] Compared to the schedule in Figure 10, there is a difference in that the time of mouse autopsy is 14 days after the infection date (D+14).

[0256]

[0257] Weight changes in the MSAD protein nasal administration groups were tracked after M. abscessus infection. The results are shown in Figure 15.

[0258] Figure 15 is a graph showing the weight change rate after infection of the MSAD protein nasal administration model before and after infection according to the dose of MSAD protein administered intranasally, as a result of an experiment on the protective effect against M. abscessus respiratory infection according to the dose of MSAD protein administered intranasally before and after infection in an animal model of M. abscessus respiratory infection.

[0259] As shown in Figure 15, all MSAD protein-administered groups were found to have higher weight ratios compared to the infected group. This figure is similar to that of the non-infected group, and the 1 μg MSAD protein-administered group showed a slightly higher figure than the 5 μg MSAD protein-administered group.

[0260]

[0261] Inflammatory findings in lung tissue were observed in the MSAD protein nasal administration groups. The results are shown in Figure 16.

[0262] Figure 16 is a photograph showing the results of an experiment to determine the protective effect against M. abscessus respiratory infection according to the nasal administration dose of MSAD protein before and after infection in an animal model of M. abscessus respiratory infection, confirming the ability to alleviate inflammation findings according to the MSAD protein administration dose in the MSAD nasal administration group through lung tissue H&E staining.

[0263] As shown in Figure 16, the MSAD protein administration groups showed alleviated inflammation compared to the infected group, and in particular, the 1 μg administration group showed further alleviation of inflammation.

[0264]

[0265] The IgG levels in mouse serum against MSAD protein were compared between groups administered MSAD protein intranasally. The results are shown in Figure 17.

[0266] Figure 17 is a graph comparing antibody titers (IgG1, IgG2a, total IgG) against MSAD antigen in mouse serum as a result of an experiment on the protective effect against M. abscessus respiratory infection according to the nasal administration dose of MSAD protein before and after infection in an animal model of M. abscessus respiratory infection.

[0267] As shown in Figure 17, the antibody titer increased significantly in the 5 μg administration group, whereas this did not occur in the 1 μg administration group.

[0268]

[0269] The number of bacteria in the lung tissue of the MSAD protein nasal administration groups was confirmed using a CFU assay. The results are shown in Figure 18.

[0270] Figure 18 is a graph of CFU in the lung tissue of an MSAD protein nasal administration model before and after infection, as a result of an experiment on the protective effect against M. abscessus respiratory infection according to the dose of MSAD protein nasal administration before and after infection in an M. abscessus respiratory infection animal model.

[0271] As shown in Figure 18, the number of CFU in lung tissue decreased in the infected groups administered 1 μg and 5 μg of MSAD protein compared to the control group, and the numbers showed similar patterns. However, further research is needed to determine whether administration of 1 μg of MSAD can induce a systemic immune response.

[0272]

[0273] Experimental Example 3. Survival Rates According to MSAD Protein Administration Methods Before and After Infection in a Lethal Dose M. abscessus Respiratory Infection Model

[0274] To determine the difference in survival by administering MSAD protein before and after infection with M. abscessus, a mouse death model due to respiratory infection with M. abscessus was created. For this purpose, 150 mg / kg cyclophosphamide was intraperitoneally injected (IP) into BALB / c mice 4 days before and 1 day after infection with M. abscessus (D-4, D-1), and then ~10 7 Conditions for infection with CFU by intranasal injection (IN) were used.

[0275] Figure 19 is a schematic diagram of an experimental schedule comparing survival rates according to MSAD protein administration methods before and after infection in a lethal dose M. abscessus respiratory infection model.

[0276] As shown in Figure 19, MSAD protein was administered intranasally (IN) or subcutaneously (SC) 7 days before infection (D-7) and 1 day after infection (D+1). The survival rate of mice was then determined based on the date of infection.

[0277]

[0278] Ten mice were infected in each group, and no deaths were observed until the third day of infection. However, deaths occurred in each group on the eighth day of infection. The survival rates are shown in Figures 20 and 21.

[0279] Figure 20 is a graph comparing the survival rate by day after M. abscessus infection according to the MSAD protein administration method before and after infection in a lethal dose M. abscessus respiratory infection model.

[0280] Figure 21 is a graph comparing the survival rates of each group on the 8th day after infection according to the pre- and post-infection MSAD protein administration method in a lethal dose M. abscessus respiratory infection model.

[0281] As shown in Figures 20 and 21, the survival rate was significantly increased in subjects administered MSAD protein intranasally compared to subjects administered MSAD protein subcutaneously.

[0282]

[0283] In an experiment conducted again under identical conditions, lung tissues of mice were fixed with 4% formaldehyde, embedded in paraffin, and stained with H&E at 3 days (D+3), at which point no deaths occurred, in two groups: the non-administered PBS group and the MSAD protein nasal administration group. The lung tissues were examined for lung inflammation by fixing them with 4% formaldehyde, embedding them in paraffin, and staining with H&E. The results are shown in Figure 22.

[0284] Figure 22 is a photograph confirming the ability to alleviate lung tissue inflammation findings according to nasal administration of MSAD protein before and after infection in a lethal dose M. abscessus respiratory infection model.

[0285] As shown in Figure 22, a significant alleviation of lung tissue inflammation was observed in the MSAD protein nasal administration group compared to the PBS group, which may have led to an increase in survival rate.

[0286]

[0287] Experimental Example 4. Confirmation of the possibility of a therapeutic vaccine through nasal administration of MSAD protein after respiratory infection with M. abscessus.

[0288] To suppress natural clearance of M. abscessus respiratory infection in BALB / c mice, 150 mg / kg of cyclophosphamide was injected intraperitoneally one day before infection (D-1). M. abscessus~10 6Mice were infected with CFU via the intranasal route, and 5 μg of MSAD protein was administered intranasally one day later (D+1). The number of M. abscessus CFU in mouse lung tissue was determined on D+3 and D+7 after infection. The experimental schedule is shown in Figure 23.

[0289] Figure 23 is a schematic diagram of an experimental schedule to confirm the therapeutic vaccine potential of MSAD protein in an animal model of M. abscessus respiratory infection.

[0290]

[0291] The weight change rate of mice was observed for 7 days after respiratory infection with M. abscessus. The results are shown in Fig. 24.

[0292] Figure 24 is a graph showing the results of an experiment to confirm the possibility of a therapeutic vaccine for nasal administration of MSAD protein in a M. abscessus respiratory infection model, showing the change in mouse weight ratio for 7 days after M. abscessus respiratory infection.

[0293] As shown in Figure 24, there was no significant difference in the weight change rate between the PBS and MSAD protein intranasal administration groups after infection. The safety of MSAD intranasal administration was indirectly confirmed by the lack of effect on mouse weight.

[0294]

[0295] The number of M. abscessus bacteria in lung tissue was determined using a CFU assay 3 and 7 days after respiratory infection. The results are shown in Figure 25.

[0296] Figure 25 is a graph showing the results of an experiment to confirm the possibility of a therapeutic vaccine for nasal administration of MSAD protein in a M. abscessus respiratory infection model, showing M. abscessus CFU in lung tissue 3 days and 7 days after M. abscessus respiratory infection.

[0297] As shown in Figure 25, the MSAD protein administration vaccine group after M. abscessus infection showed no tendency for CFU in lung tissue to decrease until the third day of infection, but a decrease in CFU was confirmed on the seventh day. This confirmed that nasal administration of MSAD protein had therapeutic effect against M. abscessus respiratory infection from 6 days after MSAD protein administration.

[0298]

[0299] In a model where C57BL / 6 female mice of a different strain were infected with 100 PFU (Plaque Forming Units) of Influenza A virus strain PR8 (IAV) and then challenged with M. abscessus, the number of M. abscessus-infected bacteria further increased. In this model, the therapeutic efficacy of combined administration of MSAD protein and sHB, the surface antigen (HbsAg) of Hepatitis B virus, after M. abscessus infection was confirmed. The experimental schedule is as shown in Fig. 26.

[0300] Figure 26 is a schematic diagram of an experimental schedule to confirm the therapeutic vaccine efficacy of MSAD protein and MSAD protein + sHB in an IAV + M. abscessus respiratory infection model.

[0301]

[0302] The change in mouse weight over a 7-day period following respiratory infection with M. abscessus was observed. The results are shown in Fig. 27.

[0303] Figure 27 is a graph showing the change in mouse weight ratio 7 days after M. abscessus respiratory infection, as a result of a mouse experiment to confirm the therapeutic vaccine efficacy of nasal administration of MSAD protein and MSAD protein + sHB in an IAV + M. abscessus respiratory infection model.

[0304] As shown in Figure 27, on the 1st day after M. abscessus respiratory infection, the weight change rate was confirmed to increase in the MSAD protein only administration group compared to the PBS control group and MSAD protein + sHB administration group on the 3rd day after infection after nasal administration of MSAD protein or MSAD protein + sHB. The PBS group and MSAD protein + sHB administration group were confirmed to show a trend of weight recovery between the 5th and 6th days after infection, but the weight change rate of the PBS group and MSAD protein + sHB administration group was confirmed to be similar.

[0305]

[0306] Fourteen days after M. abscessus respiratory infection, the number of M. abscessus bacteria in lung tissue was determined. The results are shown in Figure 28.

[0307] Figure 28 is a graph of M. abscessus CFU in lung tissue 14 days after M. abscessus respiratory infection, as a result of a mouse experiment to confirm the therapeutic vaccine efficacy of nasal administration of MSAD protein and MSAD protein + sHB in an IAV + M. abscessus respiratory infection model.

[0308] As shown in Figure 28, it was confirmed that the number of bacteria in lung tissue was significantly reduced in both the MSAD protein administration group and the MSAD protein + sHB administration group compared to the PBS control group.

[0309]

[0310] Fourteen days after M. abscessus respiratory infection, inflammatory findings were observed in lung tissue. The results are shown in Figure 29.

[0311] Figure 29 is a photograph showing the results of a mouse experiment to confirm the therapeutic vaccine efficacy of nasal administration of MSAD protein and MSAD protein + sHB in an IAV + M. abscessus respiratory infection model, confirming the ability to alleviate inflammation findings in lung tissue 14 days after M. abscessus respiratory infection.

[0312] As shown in Figure 29, in the experimental set, the lung tissue inflammation findings due to M. abscessus respiratory infection were not significantly aggravated, but it was confirmed that the MSAD protein + sHB administration group showed significantly alleviated lung tissue inflammation findings compared to the PBS control group or the MSAD protein alone administration group.

[0313]

[0314] When this is comprehensively confirmed, it can be judged that the efficacy of the MSAD protein + sHB administration group is not only due to the efficacy of the MSAD protein, but also shows additional activity.

[0315]

[0316] Additionally, 14 days after M. abscessus respiratory infection, the proportion of CD4+ or CD8+ T cells secreting IFN-γ in lung cells and the proportion of IFN-γ-secreting cells among total lung cells were determined using flow cytometry and IFN-γ ELISPOT, respectively. The results are shown in Figures 30 and 31, respectively.

[0317] Figure 30 is a graph comparing the proportion of CD4+ or CD8+ T cells secreting IFN-γ, as confirmed by flow cytometry analysis of lung cells, as a result of a mouse experiment to confirm the therapeutic vaccine efficacy of nasal administration of MSAD protein and MSAD protein+sHB in an IAV+M. abscessus respiratory infection model.

[0318] Figure 31 is a graph comparing SFU for comparing the proportion of IFN-γ secreting cells among lung cells using the IFN-γ ELISPOT experiment, as a result of a mouse experiment to confirm the therapeutic vaccine efficacy of nasal administration of MSAD protein and MSAD protein + sHB in an IAV + M. abscessus respiratory infection model.

[0319] In Fig. 31, Mab lysate means M. abscessus_Rough whole cell lysate stimulation, and w / o simulation means non-stimulation.

[0320] As shown in Figures 30 and 31, the MSAD protein + sHB administration group was confirmed to show significantly higher proportions of CD4+ or CD8+ T cells secreting IFN-γ; and the proportion of cells secreting IFN-γ among total lung cells. This suggests that the MSAD protein + sHB nasal administration method may activate immunity at an earlier stage than when the IFN-γ that had increased through the existing pre- and post-infection MSAD protein nasal administration method was fully activated.

[0321]

[0322] Experimental Example 5. Confirmation of the preventive efficacy of MSAD protein immunization before respiratory infection with M. abscessus.

[0323] BLAB / c female mice were immunized twice with 5 μg of MSAD protein at 40 μl intranasal route and 100 μl subcutaneous route at 2-week intervals before infection with M. abscessus. Two weeks after the last immunization, ~10 7 All groups, except the Unchallenge group, were infected with M. abscessus via the nasal route at CFU levels. The experimental schedule is shown in Figure 32.

[0324] Figure 32 is a schematic diagram of an experimental schedule to determine the protective ability of an individual against M. abscessus respiratory infection according to the MSAD protein immunization administration route.

[0325]

[0326] The weight change rate of mice was observed for 7 days after MSAD protein immunization and M. abscessus respiratory infection. The results are shown in Figure 33.

[0327] Figure 33 is a graph showing the results of an experiment to confirm the protective effect against M. abscessus respiratory infection according to the MSAD protein immunization administration route, and showing the change in mouse weight ratio for 7 days after M. abscessus respiratory infection.

[0328] As shown in Figure 33, at the 7th day of infection, which was the time of mouse autopsy, all groups except the MSAD protein nasal administration group had similar weight ratios, and the MSAD protein nasal administration group had a weight ratio that was about 3% higher than the other groups.

[0329]

[0330] Antibody titers against MSAD antigen were compared using serum from mice obtained 7 days after infection (D+7). The results are shown in Figure 34.

[0331] Figure 34 is a graph comparing antibody titers against MSAD antigens in mouse serum 7 days after M. abscessus respiratory infection, showing the results of an experiment to confirm the protective effect against M. abscessus respiratory infection according to the MSAD protein immunization administration route.

[0332] As shown in Figure 34, both the intranasal and subcutaneous MSAD protein administration groups showed increased antibody titers compared to the infection-only group, confirming normal mouse immunization. Among these, the subcutaneous MSAD protein administration group showed significantly higher antibody titers than the intranasal MSAD protein administration group.

[0333]

[0334] At 7 days post-infection (D+7), the number of bacteria in mouse lung tissue was compared using a CFU assay. The results are shown in Figure 35.

[0335] Figure 35 is a graph comparing M. abscessus CFU in mouse lung tissue 7 days after M. abscessus respiratory infection, showing the results of an experiment to confirm the protective effect against M. abscessus respiratory infection according to the MSAD protein immunization administration route.

[0336] As shown in Figure 35, only the intranasal MSAD protein administration group showed a decrease in CFU compared to the infection only group, confirming the protective effect of MSAD protein nasal administration immunization before M. abscessus infection. It was confirmed that subcutaneous MSAD protein immunization did not show protective effect.

[0337]

[0338] C57BL / 6 female mice were challenged with a small dose of 100 PFU (Plaque Forming Units) of Influenza A virus strain PR8 (IAV) for 1 week after respiratory infection. 6 The preventive efficacy of MSAD protein immunity was confirmed using a model in which mice developed higher bacterial infection rates when infected with M. abscessus at CFU levels in the respiratory tract.

[0339] When MSAD protein was administered intranasally alone, it had a protective effect against respiratory infection with M. abscessus, but when administered subcutaneously, the protective effect was weak. Therefore, the MSAD+PA subcutaneous vaccine method, which added aluminum, which has been used as an immune adjuvant in many literatures, and Poly 6 (P6, SEQ ID NO: 9, GRLVFQ), a peptide derived from Hepatitis B virus that plays a role in enhancing Th1 immune responses, was confirmed to have a protective effect against M. abscessus. In PA, P represents the HBV-derived peptide Poly 6 (SEQ ID NO: 9, GRLVFQ), and A represents an aluminum salt, which were applied as immune adjuvants. The experimental schedule is as shown in Fig. 36.

[0340] Figure 36 is a schematic diagram of the experimental schedule to determine the protective effect of MSAD protein intranasal administration or MSAD+PA subcutaneous administration immunization in an IAV +M. abscessus respiratory infection mouse model.

[0341]

[0342] Antibody titers against the MSAD antigen were compared using serum and BALF from mice obtained 7 days after infection (D+7). The results are shown in Figure 37.

[0343] Figure 37 is a graph comparing antibody titers against MSAD antigens in mouse serum and BALF 7 days after M. abscessus respiratory infection, as a result of an experiment to confirm the protective effect of MSAD protein nasal administration or MSAD protein + PA subcutaneous administration immunization in an IAV + M. abscessus respiratory infection mouse model.

[0344] As shown in Figure 37, the MSAD protein + PA subcutaneous administration group showed the highest serum IgG (IgG1, IgG2, total IgG) expression levels against MSAD protein, similar to the subcutaneous administration of MSAD protein alone. In the case of nasal administration of MSAD protein, the broncheoalveolar fluid (BALF) of mice showed a higher expression level of IgA antibodies against MSAD protein than the other groups.

[0345]

[0346] The number of bacteria in mouse lung tissue obtained 7 days after infection (D+7) was compared using a CFU assay. The results are shown in Figure 38.

[0347] Figure 38 is a graph comparing M. abscessus CFU in mouse lung tissue 7 days after M. abscessus respiratory infection, as a result of an experiment to confirm the protective effect of MSAD protein nasal administration or MSAD protein + PA subcutaneous administration immunization in an IAV + M. abscessus respiratory infection mouse model.

[0348] As shown in Figure 38, the preventive efficacy of MSAD protein intranasal administration against M. abscessus was also confirmed in the IAV + M. abscessus respiratory infection model of C57BL / 6 mice, and in relation to the subcutaneous administration immunization method using MSAD protein, it was confirmed that the number of bacteria in lung tissue was significantly reduced compared to the PBS control group when MSAD protein + PA subcutaneous administration immunization was administered with PA.

[0349] Through this, MSAD protein was also confirmed to have potential as a subcutaneous immunotherapy through the use of appropriate immune adjuvants.

[0350]

[0351] In order to evaluate the preventive effect against M. abscessus infection, the MSAD protein + sHB nasal administration method, which was confirmed in the previous evaluation of the therapeutic efficacy of MSAD protein, was administered IP of Cyclophosphamide to C57BL / 6 female mice and 10 7 The mortality rates of mice were compared through respiratory infection with a high dose of M. abscessus (CFU). The experimental schedule is shown in Figure 39.

[0352] Figure 39 is a schematic diagram of an experimental schedule to determine the preventive effect against M. abscessus respiratory infection in individuals following intranasal administration of MSAD protein or intranasal administration of MSAD protein + sHB prior to lethal M. abscessus respiratory infection.

[0353]

[0354] Survival rates against M. abscessus respiratory infection were compared in mice immunized with either intranasal MSAD protein or intranasal MSAD protein plus sHB prior to lethal M. abscessus respiratory infection. The results are shown in Figure 40.

[0355] Figure 40 is a graph comparing the survival rate against M. abscessus respiratory infection in mice following immunization with intranasal administration of MSAD protein or intranasal administration of MSAD protein + sHB prior to lethal M. abscessus respiratory infection.

[0356] As shown in Figure 40, in the mouse survival rate comparison experiment, deaths were confirmed in the PBS group and the MSAD protein nasal immunization group that were only infected with M. abscessus, whereas no deaths were found in the MSAD protein + sHB nasal immunization group, as in the non-infected group.

[0357]

[0358] A statistical significance test was conducted for this. The results are shown in Figure 41.

[0359] Figure 41 shows the results of a statistical significance test between the survival rate graphs of the M. abscessus respiratory infection control group (PBS group) and the M. abscessus respiratory infection group after nasal administration of MSAD protein + sHB immunization (nasal administration of MSAD protein + sHB group).

[0360] As shown in Figure 41, when a statistical significance test was performed on the survival rate between the PBS control group and the MSAD protein + sHB nasal administration group, statistical significance was found in both the Log-rank test and the Gehan-Breslow-Wilcoxon test.

[0361]

[0362] However, unlike previous results showing preventive efficacy, the MSAD protein nasal administration group did not show any increase in survival in this study. This may be because it was administered over a shorter period of time than the existing immunization schedule, which may not have induced a sufficient immune response. Given that the MSAD protein + sHB nasal administration method showed high survival even under this short immunization schedule, it may be considered to be superior to the MSAD protein nasal administration method.

[0363]

[0364] Experimental Example 6. Confirmation of protective efficacy through intranasal (IN) and subcutaneous (SC) administration of MSAD protein before infection in a non-tuberculous mycobacteria (NTM) infection model.

[0365] The protective effect of MSAD protein against Mycobacterium intracellulare (M. intracellulare) was confirmed. MSAD-1 protein derived from M. intracellularesubsp. yongonense was used, and infection was carried out with M. intracellularesubsp. paraintracellulare. The experimental schedule is as shown in Figure 42.

[0366] Figure 42 is a schematic diagram of an experimental schedule for confirming the protective effect of MSAD protein in M. intracellulare infection.

[0367] The experimental groups included PBS, MSAD protein nasal administration, MSAD protein subcutaneous administration, and MSAD protein + PA subcutaneous administration. In PA, P represents the HBV-derived peptide Poly 6 (SEQ ID NO: 9, GRLVFQ), and A represents aluminum salt, which were used as immune adjuvants.

[0368] Five micrograms of MSAD protein were administered per mouse, with 30 μl intranasally and 100 μl subcutaneously after inhalation anesthesia. The same immunization schedule was repeated two weeks later, resulting in a total of two immunizations. Two weeks after the second immunization, ~10 M. intracellulararesubsp.paraintracellulare were detected. 7 CFU (Colony Forming Unit) was administered intranasally and sacrificed after 7 days.

[0369]

[0370] The weight change rate of mice was observed for 7 days after M. intracellulare respiratory infection. The results are shown in Figure 43.

[0371] Figure 43 is a graph showing the results of an experiment to confirm the protective effect of MSAD protein against the M. intracellulare respiratory infection model, showing the change in mouse weight ratio over 7 days after M. intracellulare respiratory infection.

[0372] When comparing the weight change rates of mice in each administration group after M. intracellulare infection, there was no significant difference in the weight change rates, as with M. abscessus. This indirectly confirmed the safety of MSAD, as administration of MSAD protein did not affect mouse weight.

[0373]

[0374] Seven days after respiratory infection with M. intracellulare (D+7), mice were necropsied, and the number of M. intracellulare bacteria in lung tissue was determined using a CFU assay. The results are shown in Figure 44.

[0375] Figure 44 is a graph showing the results of an experiment to confirm the protective effect of MSAD protein on the M. intracellulare respiratory infection model, showing the M. intracellulare CFU in lung tissue on day 7 of M. intracellulare respiratory infection.

[0376] As shown in Figure 44, a statistically significant decrease in the number of bacteria in lung tissue was observed in the groups administered MSAD protein compared to the non-administered group (PBS).

[0377]

[0378] Antibody titers against the M. intracellularesubsp.yongonense MSAD antigen were compared using mouse serum. The results are shown in Figure 45.

[0379] Figure 45 is a graph comparing the antibody titer against MSAD antigen in lung tissue in serum 7 days after M. intracellulare respiratory infection, which is the result of an experiment to confirm the protective effect of MSAD protein on the M. intracellulare respiratory infection model.

[0380] As shown in Figure 45, when the antibody titer for the M. intracellularesubsp.yongonense MSAD antigen used for mouse immunization was checked, it was confirmed that the IgG1 and IgG2a antibody titers were higher in the MSAD protein administration group compared to the non-administered PBS group.

[0381] In particular, in the M. intracellulare infection experiment, it was confirmed that the CFU of the subcutaneous administration immunization group was low and the antibody titer was high. In the case of MSAD intranasal administration, when comparing the anti-MSAD IgG1 antibody titer with the non-administered group alone, it was confirmed that it was statistically significantly higher, confirming the protective effect of MSAD protein in the M. intracellulare respiratory infection model after intranasal and subcutaneous administration.

[0382]

[0383] The protective efficacy of subcutaneous (SC) immunization with MSAD protein (MSAD_Yon) derived from M. intracellulares subsp. yongonense was reaffirmed, and its protective efficacy against Mycobacterium abscessus in addition to M. intracellulare subsp. paraintracellulare was also confirmed. The experimental schedule is shown in Fig. 46.

[0384] Figure 46 is a schematic diagram of an experimental schedule for confirming the ability to protect against NTM infection through subcutaneous immunization with MSAD protein derived from M. yongonense.

[0385] Mice were subcutaneously administered 5 μg of MSAD protein twice at 2-week intervals, followed by 1×102 immunizations 2 weeks after the last immunization. 7 Infection was performed by intranasal administration of CFU of M. abscessus or M. intracellulare.

[0386] One week after subcutaneous immunization with M. yongonense-derived MSAD and NTM respiratory infection, the amount of bacteria in lung tissue and spleen weight were measured in the M. abscessus-infected and M. intracellulare-infected groups. In addition, the proportion of lung cells containing IFN-γ-secreting cytotoxic T cells (CD8+IFN-γ+), a key cell type responsible for suppressing bacterial infection, was determined using flow cytometry. The results are presented in Figures 47 to 49.

[0387] Figures 47 to 49 are the results of an experiment to confirm the protective ability of M. yongonense-derived MSAD protein against an NTM infection model, and are graphs of CFU in lung tissues of the M. abscessus-infected group and the M. intracellulare-infected group (Figure 47), spleen weight (Figure 48), and the intracellular ratio of IFN-γ-secreting CD8 T cells using flow cytometry (Figure 49) in the M. yongonense-derived MSAD subcutaneously and one week after NTM respiratory infection.

[0388] As shown in Figures 47 to 49, the subcutaneous immunization with MSAD protein derived from M. yongonense showed protective effects against M. abscessus infection in addition to M. intracellulare, as confirmed by comparison of the amount of bacteria retained in lung tissue (Figure 47). In particular, under the M. abscessus infection condition, the group administered MSAD protein derived from M. yongonense showed a statistically significant decrease in the spleen weight increased by M. abscessus infection (Figure 48), and the proportion of IFN-γ secreting cytotoxic T cells (IFN-γ+ CD8+) among lung cells was confirmed to be significantly increased (Figure 49).

[0389]

[0390] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the experimental examples and embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

[0391] Attach electronic file

Claims

An immunogenic composition comprising MSAD (Malonate Semialdehyde Decarboxylase) protein or a polynucleotide encoding the same as an active ingredient. An immunogenic composition according to claim 1, wherein the polynucleotide is a deoxyribonucleic acid (DNA) polynucleotide or a ribonucleic acid (RNA) polynucleotide. In claim 1, the immunogenic composition is for non-tuberculous mycobacteria. In claim 3, the nontuberculous mycobacteria are Mycobacterium avium complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium prarintracellulare, Mycobacterium intracellularesubsp.yongonense, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium marseillense, Mycobacterium arosiense, Mycobacterium bouchedurhonense, Mycobacterium timonense, Mycobacterium vulneris, Mycobacterium indicus pranii, Mycobacterium abscessuscomplex, Mycobacterium abscessus, Mycobacterium abscessussubsp.abscessus, Mycobacterium abscessussubsp.massiliense, Mycobacterium abscessussubsp. boletii.Bolletii), Mycobacterium smegmatis, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium paragordonae, Mycobacterium osloensis, Mycobacterium phlei, Mycobacterium terrae, Mycobacterium chelonae, Mycobacterium mucogenicum, Mycobacterium peregrinum, Mycobacterium simiae An immunogenic composition comprising at least one selected from the group consisting of Mycobacterium simiae, Mycobacterium wolinskyi, Mycobacterium ulcerans and Mycobacterium marinum. An immunogenic composition according to claim 1, wherein the MSAD protein is derived from nontuberculous mycobacteria. A vaccine composition for the prevention or treatment of nontuberculous mycobacterial infection, comprising the immunogenic composition of any one of claims 1 to 5 as an active ingredient. A vaccine composition according to claim 6, further comprising an immune adjuvant. In claim 7, the immune adjuvant is a vaccine composition comprising at least one selected from the group consisting of AS01 (liposome mixed with monophosphoryl lipid A and saponin QS-21), IC31 (oligo nucleotide and cationic peptide), CFA01 (cationic liposome), Alum (Aluminium salts), HBsAg (Hepatitis B surface antigen), and a polypeptide having an amino acid sequence of SEQ ID NO:

9. In claim 6, the vaccine composition is administered by at least one route selected from the group consisting of oral, transdermal, intramuscular, intraperitoneal, inhalation, subcutaneous, intravenous, and nasal routes. A pharmaceutical composition for the prevention or treatment of nontuberculous mycobacterial infections, comprising MSAD protein or a polynucleotide encoding the same as an active ingredient. A pharmaceutical composition according to claim 10, wherein the polynucleotide is a deoxyribonucleic acid (DNA) polynucleotide or a ribonucleic acid (RNA) polynucleotide. A pharmaceutical composition according to claim 10, further comprising an immune adjuvant. A pharmaceutical composition according to claim 12, wherein the immune adjuvant is at least one selected from the group consisting of AS01 (liposome mixed with monophosphoryl lipid A and saponin QS-21), IC31 (oligo nucleotide and cationic peptide), CFA01 (cationic liposome), Alum (Aluminium salts), HBsAg (Hepatitis B surface antigen), and a polypeptide having an amino acid sequence of SEQ ID NO:

9. A pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is administered by at least one route selected from the group consisting of oral, transdermal, intramuscular, intraperitoneal, inhalation, subcutaneous, intravenous, and nasal routes. MSAD protein; or a health functional food for preventing or improving nontuberculous mycobacterial infection, comprising a polynucleotide encoding the same.

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