Vaccine composition and methods for inducing immunity against mycobacterium tuberculosis

Abstract

The present invention is a development of a vaccine formulation or a vaccine composition for inducing antigen-specific cells to generate immunity for resistance against Mycobacterium tuberculosis, comprising Bacillus Calmette-Guérin (BCG), ESAT-6 protein (ESAT-6) encapsulated with a nanoparticle-type carrier, and a STING ligand, wherein the BCG, the ESAT- 6 protein encapsulated with the nanoparticle-type carrier, and the STING ligand are physically and / or chemically combined, with a mass ratio of ESAT-6 to the STING ligand being in a range of 4-10 : 1, and wherein the vaccine composition according to the present invention is administered by a prime-pool strategy for preventing pulmonary tuberculosis infection.

Description

[0001] VACCINE COMPOSITION AND METHODS FOR INDUCING IMMUNITY AGAINST MYCOBACTERIUM TUBERCULOSIS

[0002] Technical Field

[0003] In the fields of medical science and pharmacology in the part relating to a vaccine composition for inducing immunity against Mycobacterium tuberculosis and methods for inducing immunity against Mycobacterium tuberculosis using the vaccine composition.

[0004] Background of Related Art

[0005] Tuberculosis remains one of the leading public health problems of many countries worldwide, including Thailand. According to a report by the World Health Organization, approximately one in four people worldwide are currently infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Tuberculosis causes approximately 1.3 million deaths worldwide per year 1 (World Health Organization. Global tuberculosis report 2023. World Health Organization; 2023). In Thailand, there are approximately 111,000 tuberculosis cases and about 13,700 deaths per year. The incidence rate is approximately 1.3 times higher than the global average. Thailand is still ranked among the 14 countries worldwide with a high tuberculosis burden, including drug -resistant tuberculosis, particularly multi drugresistant tuberculosis and extensively drug -resistant tuberculosis, and it has been reported that approximately 98 percent of patients infected with extensively drug-resistant tuberculosis die, on average, within 16 days after diagnosis. All of these data indicate a growing tuberculosis burden driven by drug-resistant tuberculosis. In addition, the vaccine used for preventing tuberculosis currently has only one type, namely the Bacillus Calmette-Guerin (BCG) vaccine, which is a live vaccine derived from the attenuation of Mycobacterium bovis, a tuberculosis-causing pathogen in cattle, which is very similar to Mycobacterium tuberculosis, thereby rendering BCG highly safe and suitable for administration to newborn infants. Although the BCG vaccine can help prevent severe tuberculosis in children, it does not provide sufficient efficacy in preventing pulmonary tuberculosis in adults 2 (Kaufmann SH. The contribution of immunology to the rational design of novel antibacterial vaccines. Nat Rev Microbiol. 2007;5(7):491-504. doi:10.1038 / nrmicrol688).

[0006] The reason why the BCG vaccine cannot effectively prevent pulmonary tuberculosis in adults is not fully understood; however, recent phase II clinical trial data from Africa showed that intradermal BCG revaccination in adults increased protection against tuberculosis by 45.4% compared with the control group over a 2-year follow-up period 3 (Nemes E, Geldenhuys H, Rozot V, et al. Prevention of M. tuberculosis infection with H4:IC31 vaccine or BCGrevaccination. NEngl JMed. 2018;379(2): 138-149. doi:10.1056 / NEJMoal714021). Therefore, it has been hypothesized that the limited efficacy of the BCG vaccine in preventing pulmonary tuberculosis in adults may be partly due to waning immunity over time following vaccination at birth.

[0007] In addition, previous studies indicate that the BCG vaccine is unable to prevent pulmonary tuberculosis in adults, and this limitation may arise from the fact that BCG predominantly stimulates the generation of Mtb- specific Thl CD4+T cells, but has limited capacity to induce Mtb-specific CD8+T cells 4 (Grode L, Seiler P, Baumann S, et al. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guerin mutants that secrete listeriolysin. J Clin Invest. 2005;115(9):2472-2479. doi:10.1172 / JCI24617), which play an important role in host resistance to tuberculosis 5 (Nieuwenhuizen NE, Kulkarni PS, Shaligram U, et al. The recombinant bacille Calmette -Guerin vaccine VPM1002: ready for clinical efficacy testing. Front Immunol. 2017;8:1147. doi:10.3389 / fimmu.2017.01147). This is because BCG lacks expression of several virulence-associated proteins, including proteins used by Mycobacterium tuberculosis to perforate the phagosomal membrane. Although the loss of virulence-associated proteins confers a high safety profile to BCG, allowing administration to newborn infants, this also prevents efficient delivery of antigen into the cytoplasm of antigen-presenting cells (APCs). Consequently, major histocompatibility complex class I antigen presentation is reduced, resulting in impaired induction of Mtb-specific CD8+T cells and limited protection against pulmonary tuberculosis in adults.

[0008] Furthermore, the inability of BCG to perforate the phagosomal membrane prevents activation of cytosolic inflammasomes in APCs, thereby impairing the production of interleukin-1 beta (IL- 1 P) 6 (Barclay WR, Anacker RL, Brehmer W, Leif W, Ribi E. Protection of monkeys against airborne tuberculosis by aerosol vaccination with bacillus Calmette -Guerin. Am Rev Respir Dis. 1973; 107(3):351 -358. doi:10.1164 / arrd,1973.107.3.351) and consequently limits the induction of Mtb-specific Thl 7 T cells 7 (Dijkman K, Sombroek CC, Vervenne RAW, et al. Prevention of tuberculosis infection and disease by local BCG in repeatedly exposed rhesus macaques. Nat Med. 2019;25(2):255-262. doi : 10.1038 / s41591 -018-0319-9), which also play an important role in host resistance to tuberculosis 8 (Perdomo C, Zedler U, Kuhl AA, et al. Mucosal BCG vaccination induces protective lung-resident memory T cell populations against tuberculosis. mBio. 2016;7(6):e01686-16. doi: 10.1128 / mBio.01686-16). In addition, the inability of BCG to perforate the phagosomal membrane prevents delivery of mycobacterial DNAto the cytoplasm to activate stimulator of interferon genes (STING) in APCs, thereby limiting activationof autophagy in APCs 9 (Hart P, Xue Y, Liu Y, et al. Nanop article -fusion protein complexes protect against Mycobacterium tuberculosis infection. Mol Ther. 2018;26(3):822-833. doi:10.1016 / j.ymthe.2017.12.016), which is an important process that enhances antigen presentation, including MHC class I antigen presentation, MHC class II antigen presentation, and antigen cross-presentation 10 (Miinz C. Autophagy proteins in antigen processing for presentation on MHC molecules. Immunol Rev. 2016;272(l): 17-27. doi:10.1111 / imr.l2422).

[0009] Because lung-localized immunity plays a critical role in preventing pulmonary tuberculosis, the generation of Mtb-specific T cells in the lung represents an important target for tuberculosis vaccine development. However, the currently used BCG vaccine is administered only once to newborn infants via the intradermal route, thereby making the generation of Mtb-specific T cells in the lung, or the recruitment of such cells from other anatomical sites to the lung, highly inefficient. Past studies indicate that parenteral administration of the BCG vaccine is insufficient to induce robust Mtb-specific T cell responses in the lung or to effectively recruit such cells from peripheral sites to the lung 11-13 (Barclay WR, Anacker RL, Brehmer W, Leif W, Ribi E. Protection of monkeys against airborne tuberculosis by aerosol vaccination with bacillus Calmette-Guerin. Am Rev Respir Dis. 1973; 107(3):351 -358; Dijkman K, Sombroek CC, Vervenne RAW, et al. Prevention of tuberculosis infection and disease by local BCG in repeatedly exposed rhesus macaques. Nat Med. 2019;25(2):255-262; Perdomo C, Zedler U, Kuhl AA, et al. Mucosal BCG vaccination induces protective lung-resident memory T cell populations against tuberculosis. mBio. 2016;7(6):e01686-16)

[0010] In view of the above-mentioned problems, efforts have been directed toward the development and investigation of new pharmaceutical compositions or vaccines capable of effectively preventing tuberculosis, in particular pulmonary tuberculosis in adults, which represents a major source of person-to-person transmission.

[0011] Summary of the Invention

[0012] A delivery system for inducing immunity against a pathogenic microorganism comprises an ESAT-6 protein (ESAT-6) encapsulated within a nanoparticle-type carrier and a STING ligand, wherein the ESAT-6 protein encapsulated within the nanoparticle-type carrier is physically or chemically combined with the STING ligand, and wherein a mass ratio of ESAT-6 to the STING ligand is in a range of 4-10:1.

[0013] A vaccine composition for inducing antigen-specific immune responses against Mycobacterium tuberculosis comprises Bacillus Calmette-Guerin (BCG), ESAT-6 proteinencapsulated within a nanoparticle-type carrier, and a STING ligand, wherein the BCG, the ESAT-6 protein encapsulated within the nanoparticle-type carrier, and the STING ligand are physically and / or chemically combined, and wherein a mass ratio of ESAT-6 to the STING ligand is in a range of 4-10:1.

[0014] The nanoparticle-type carrier is selected from the group consisting of chitosan, chitosan derivatives, polymer-based substances, polyamino acid-based substances, and surfactants. Preferably, the chitosan derivative is trimethyl chitosan (TMC).

[0015] In one embodiment, the vaccine composition further comprises a pharmaceutically acceptable excipient selected from one or more of a solvent, an adjuvant, a buffering agent, an additive, and a stabilizer.

[0016] In another embodiment, the vaccine composition according to the invention induces, in the lung, one or more of Mtb-specific CD8+T cells, Mtb-specific CD4+Thl T cells, Mtb-specific CD4+Thl7 T cells, Mtb-specific IgA, and Mtb-specific IgG.

[0017] A method for inducing immunity conferring resistance against Mycobacterium tuberculosis using the vaccine composition according to the invention comprises a first administration of the vaccine composition via a parenteral route and a subsequent administration of the vaccine composition via a mucosal route, wherein the subsequent administration is performed 2-8 weeks after the first administration.

[0018] The present invention provides an effective vaccine composition conferring resistance against Mycobacterium tuberculosis for preventing tuberculosis in both children and adults, wherein the vaccine comprises a protein capable of perforating a phagosomal membrane and encapsulated within TMC nanoparticles, thereby enabling delivery of BCG antigens into the cytoplasm of APCs, promoting antigen presentation via MHC class I molecules, and stimulating antigen-specific cells to secrete cytokines that induce the generation of Mtb-specific CD4+Thl cells, CD4+Thl7 cells, and CD8+T cells, as well as inducing lung-localized immunity to prevent tuberculosis infection.

[0019] Brief Description of the Drawings

[0020] Fig. 1 is a graph showing the delivery of ESAT-6 protein encapsulated within TMC nanoparticles (ESAT-6 / TMC nanoparticles) and Bacillus Calmette-Guerin (BCG) into human dendritic cells.Fig. 2 is a graph showing the effect of ESAT-6 / TMC nanoparticles on increasing the delivery of BCG antigens into the cytoplasm of human dendritic cells.

[0021] Fig. 3 is an image showing the effect of ESAT-6 / TMC nanoparticles on increasing the delivery of BCG antigens into the cytoplasm of human dendritic cells.

[0022] Fig. 4 is a graph showing the effect of ESAT-6 / TMC nanoparticles on increasing perforation of the phagosomal membrane of BCG.

[0023] Fig. 5 is an image showing the effect of ESAT-6 / TMC nanoparticles on increasing perforation of the phagosomal membrane of BCG.

[0024] Fig. 6 is a graph showing the cytotoxicity of ESAT-6 / TMC nanoparticles toward human dendritic cells.

[0025] Fig. 7 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating maturation of human dendritic cells, as measured by CD80 surface expression, compared with administration of BCG alone.

[0026] Fig. 8 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating maturation of human dendritic cells, as measured by CD83 surface expression, compared with administration of BCG alone.

[0027] Fig. 9 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating maturation of human dendritic cells, as measured by CD86 surface expression, compared with administration of BCG alone.

[0028] Fig. 10 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating maturation of human dendritic cells, as measured by HLA-DR surface expression, compared with administration of BCG alone.

[0029] Fig. 11 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating production of interleukin- 1 beta (IL-ip) in human dendritic cells, compared with administration of BCG alone.

[0030] Fig. 12 is a graph showing no significant difference in the stimulation of interleukin- 12 (IL-12) production in human dendritic cells between administration of a STING ligand together with ESAT-6 / TMC nanoparticles and BCG and administration of BCG alone.Fig. 13 is a graph showing no significant difference in the stimulation of interleukin-6 (IL-6) production in human dendritic cells between administration of a STING ligand together with ESAT-6 / TMC nanoparticles and BCG and administration of BCG alone.

[0031] Fig. 14 is a graph showing no significant difference in the stimulation of transforming growth factor-beta (TGF-P) production in human dendritic cells between administration of a STING ligand together with ESAT-6 / TMC nanoparticles and BCG and administration of BCG alone.

[0032] Fig. 15 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating the generation of Mtb-specific CD4+T cells in the lungs of mice, compared with administration of BCG alone.

[0033] Fig. 16 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating the generation of Mtb-specific CD4+Thl T cells in the lungs of mice, compared with administration of BCG alone.

[0034] Fig. 17 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating the generation of Mtb-specific CD4+Thl7 T cells in the lungs of mice, compared with administration of BCG alone.

[0035] Fig. 18 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating the generation of Mtb CD4+T cells in the airways of the lungs of mice, compared with administration of BCG alone.

[0036] Fig. 19 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating the generation of Mtb-specific CD4+T cells producing interferon-gamma (IFN-y) in the airways of the lungs of mice, compared with administration of BCG alone.

[0037] Fig. 20 is a graph showing no significant difference in the stimulation of Mtb-specific CD8+T cells in the airways of the lungs of mice between administration of a STING ligand together with ESAT-6 / TMC nanoparticles and BCG and administration of BCG alone.

[0038] Fig. 21 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating the generation of Mtb-specific CD8+T cells producing interferon-gamma (IFN-y) in the airways of the lungs of mice, compared with administration of BCG alone.Fig. 22 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating production of interferon-gamma (IFN-y) in lung cells of mice, compared with administration of BCG alone.

[0039] Fig. 23 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating production of interleukin- 17A (IL- 17 A) in lung cells of mice, compared with administration of BCG alone.

[0040] Fig. 24 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating production of interleukin -17F (IL-17F) in lung cells of mice, compared with administration of BCG alone.

[0041] Fig. 25 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating production of interleukin-22 (IL-22) in lung cells of mice, compared with administration of BCG alone.

[0042] Fig. 26 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating production of tumor necrosis factor-alpha (TNF-a) in lung cells of mice, compared with administration of BCG alone.

[0043] Fig. 27 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating production of interleukin-6 (IL-6) in lung cells of mice, compared with administration of BCG alone.

[0044] Fig. 28 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on stimulating the generation of Mtb-specific IgA and IgG antibodies in the airways of the lungs of mice, compared with administration of BCG alone.

[0045] Fig. 29 is a graph showing the effect of a STING ligand, when administered together with ESAT-6 / TMC nanoparticles and BCG, on preventing Mycobacterium tuberculosis infection in the lungs of mice, compared with administration of BCG alone.

[0046] Detailed Description

[0047] In the following description:

[0048] The term “BCG” means Mycobacterium species, in particular but not limited to Mycobacterium bovis bacillus Calmette-Guerin, wherein such BCG may be in the form of a BCG vaccine for preventing tuberculosis used in medical practice or laboratories, including any BCG vaccine manufactured and marketed in commerce, whether derived from attenuatedMycobacterium bovis or modified from any strain. For example, the Thai BCG vaccine produced by the Queen Saovabha Memorial Institute is the Tokyo strain, whereas European BCG vaccines include the Danish strain, and the like.

[0049] The term “prime-pull strategy” means administering a prime vaccine via a parenteral route, such as subcutaneous injection, intradermal injection, intraperitoneal injection, or intramuscular injection, followed by administering a booster vaccine via a mucosal route, such as intranasal administration, aerosol administration, intratracheal administration, intrabronchial administration, or oral administration, and the like.

[0050] The term “Mtb-specific” means a characteristic of an adaptive immune response of T cells and / or antibodies to antigens derived from part of, most of, or all of Mycobacterium tuberculosis, wherein such Mycobacterium tuberculosis antigens may be proteins / peptides, DNA, lipids, and the like.

[0051] For example, Mtb-specific CD8+T cells, Mtb-specific CD4+Thl T cells, and Mtb-specific CD4+Thl7 T cells refer to T cells that respond to one or more proteins / peptides derived from Mycobacterium tuberculosis, including CD8+T cells, T helper 1 cells, and T helper 17 cells, respectively. Mtb-specific IgA and Mtb-specific IgG refer to IgA and IgG antibodies, respectively, that recognize antigens derived from part of, most of, or all of Mycobacterium tuberculosis, wherein such Mycobacterium tuberculosis antigens may be proteins / peptides, DNA, lipids, and the like.

[0052] The present invention relates to a delivery system for inducing immunity against a pathogenic microorganism, a vaccine composition, and methods for inducing antigen -specific immune responses conferring resistance against Mycobacterium tuberculosis.

[0053] Delivery system for inducing immunity against a pathogenic microorganism

[0054] A delivery system for inducing immunity against a pathogenic microorganism comprises: (a) ESAT-6 protein encapsulated within a nanoparticle-type carrier; and

[0055] (b) a STING ligand,

[0056] wherein the ESAT-6 protein encapsulated within the nanoparticle-type carrier is physically or chemically combined with the STING ligand, and wherein a mass ratio of the ESAT-6 protein to the STING ligand is in a range of 4-10:1.In one embodiment, the nanoparticle-type carrier is selected from the group consisting of chitosan, chitosan derivatives, polymer-group substances, poly amino acid-group substances, and surfactants, wherein examples of each group are provided in the following section entitled “Vaccine composition.”

[0057] A drug delivery system facilitates effective function of a vaccine or pharmaceutical composition in inducing immunity, including enhancing antigen presentation, enhancing activation of immune cells involved in immune induction, improving stability and persistence of the vaccine, and facilitating cellular uptake by cells involved in immune induction.

[0058] Further details of components of the delivery system according to the present invention are as follows.

[0059] (a) ESAT-6 protein encapsulated within a nanoparticle-type carrier

[0060] ESAT-6 is a protein capable of perforating intracellular organelle membranes and / or cellular membranes, wherein the ESAT-6 protein may be derived from Mycobacterium species, synthesized, or produced by culturing bacteria, yeast, plants, insect cells, or animal cells, including variants comprising amino acid modifications.

[0061] (b) STING ligand

[0062] A STING ligand is a molecule capable of binding to STING protein, thereby stimulating innate immunity and / or autophagy to enhance antigen presentation. The STING ligand may be a cyclic dinucleotide (CDN) derived from bacterial production or chemical synthesis.

[0063] Vaccine composition

[0064] A vaccine composition for inducing antigen-specific immunity conferring resistance against Mycobacterium tuberculosis comprises:

[0065] (a) BCG;

[0066] (b) ESAT-6 protein encapsulated within a nanoparticle-type carrier; and

[0067] (c) a STING ligand,

[0068] wherein the BCG, the ESAT-6 protein encapsulated within the nanoparticle-type carrier, and the STING ligand are physically and / or chemically combined, and wherein a mass ratio of the ESAT-6 protein to the STING ligand is in a range of 4-10: 1.In one embodiment, the nanoparticle-type carrier is selected from the group consisting of chitosan, chitosan derivatives, polymer-group substances, poly amino acid-group substances, and surfactants.

[0069] The chitosan derivatives include trimethyl chitosan (TMC), carboxymethyl chitosan (CMC), N,O-carboxymethyl chitosan (NOCC), and the like.

[0070] The polymer-group substances include synthetic polymers comprising amine groups, such as poly(diallyldimethylammonium chloride) (PDDA), linear polyethyleneimine (1-PEI), branched polyethyleneimine (b-PEI), poly(2-aminoethyl methacrylate) (PAEM), and the like.

[0071] The polyamino acid-group substances include poly-L-lysine, poly-L-arginine, poly-L-histidine, and the like.

[0072] The surfactants include cationic surfactants, such as cetyltrimethylammonium bromide (CTAB) and N-palmitoyl-D-erythro-sphingosyl-l-O-carbamoyl-spermine triacetate salt (CCS), and the like.

[0073] In addition, the nanoparticle-type carrier may include dextran derivatives comprising amine groups and lipid-group substances, wherein examples of the dextran derivatives include diethylaminoethyl-dextran, and examples of the lipid-group substances include ammonium-containing lipids, such as dioctadecyldimethylammonium bromide (DODAB), dimethylaminoethane-carbamoyl-cholesterol (DC-Chol), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA).

[0074] In one embodiment, the vaccine composition further comprises a pharmaceutically acceptable excipient selected from one or more of a solvent (including saline and N -methyl -2-pyrrolidone (NMP)), an adjuvant (including CpG oligodeoxynucleotides (CpG ODNs) and monophosphoryl lipid A (MPL)), a buffering agent (including phosphate -buffered saline (PBS) and HEPES), an additive (including trehalose and polyethylene glycol (PEG)), and a stabilizer (including sugars, dextran, and sodium glutamate), alone or in any combination thereof.

[0075] Method for preparing the vaccine composition

[0076] (a) Method for preparing BCG

[0077] BCG may be prepared by culturing BCG microorganisms in a liquid medium suitable for mycobacterial growth until the exponential phase is reached. The amount of BCG may bedetermined by measuring optical density at 600 nm (ODeoo), and viable BCG may be quantified by colony-forming unit (CFU) analysis. The BCG may be harvested and washed with saline prior to formulation.

[0078] Alternatively, commercially manufactured BCG may be reconstituted with saline prior to formulation.

[0079] Alternatively, a live attenuated BCG microorganism, or a commercially manufactured and marketed BCG, may be reconstituted with saline prior to formulating such BCG into the vaccine composition according to the present invention.

[0080] (b) Method for encapsulating or loading of ESAT-6 into ESAT-6 / TMC nanoparticles TMC-type nanoparticles are prepared by dissolving 10-20 g of chitosan powder in 500 mL of N-methyl-2-pyrrolidone (NMP) in a 1-L reactor. Thereafter, 200 mL of sodium hydroxide (NaOH) solution (9% w / v) is added and the mixture is stirred at 25-38 °C for 30 minutes. Subsequently, 5-60 mL of methyl iodide is added, and the reaction proceeds under continuous stirring for 48 hours. The mixture is precipitated with ethanol, the precipitate is dissolved in 500 mL of 20% (w / v) sodium chloride (NaCl), and the solution is stirred at room temperature for 18 hours. The solution is dialyzed for 2-3 days and freeze-dried for 24 hours.

[0081] Then, an ESAT-6 protein solution (100-300 pg) is mixed with tripolyphosphate (TPP) solution (5 mg / mL; 50 pL) in HEPES buffer (5 mM, pH 7.4) containing Tween-80 (1% v / v). ESAT-6 / TMC nanoparticles are prepared by combining the ESAT-6 mixture with TMC solution (DQ 15-25%; 1 mg / mL; 100 pL) at a TMC:TPP mass ratio of 3-7:1 and mixing at 25-38 °C.

[0082] The ESAT-6 / TMC nanoparticles are collected by centrifugation at 10,000 x g for 10 minutes, reconstituted in 300 pL PBS, homogenized by sonication at 21% amplitude for 10 seconds, and stored at 4-8 °C prior to use.

[0083] (c) Method for preparing STING ligand

[0084] A STING ligand may be prepared by dissolving a commercially available STING ligand in saline prior to combination with ESAT-6 / TMC nanoparticles and BCG.

[0085] When all components are prepared, BCG, ESAT-6 / TMC nanoparticles, and the STING ligand are physically or chemically combined.

[0086] In one embodiment, ESAT-6 / TMC nanoparticles are mixed with the STING ligand in a buffer adjusted to pH 7.0-7.4 (preferably PBS or 0.9% saline), with a mass ratio of ESAT-6protein to STING ligand of 4-10:1 to provide a total volume of 50 pL. Thereafter, BCG (5.0 x 105to 1.0 x io6CFU) in 50 pL saline is added to provide a total volume of 100 pL.

[0087] In a second embodiment, ESAT-6 / TMC nanoparticles are mixed with the STING ligand in PBS or saline, with a mass ratio between the ESAT-6 protein and the STING ligand (pg: pg) of 4-10:1, and BCG in an amount of 5.0 x io5to 1.0 x io6CFU in saline is added to provide a total volume of at least 20 pL.

[0088] In another embodiment, BCG, ESAT-6 / TMC nanoparticles, and the STING ligand are mixed under conditions at a temperature in a range of 2-8 °C and a pH in a range of 7.0-7.4 in a suitable solvent, with a mass ratio between the ESAT-6 protein and the STING ligand of 4-10:1 together with BCG in an amount of 5.0 x 105to 1.0 x 106CFU, to provide a total volume in a range of 20-100 pL.

[0089] A person having ordinary skill in the art can improve, modify, amend, and add components, ratios / proportions, types of solvents, types of adjuvants, types of buffering agents, types of additives, and / or types of stabilizers, including temperature conditions, pH, dosage, and methods of use, to render the medicine / vaccine suitable for administration via a parenteral route, a mucosal route, or other routes, and suitable for characteristics of a user (including sex, age range, body weight, underlying diseases, drug allergies, and the like), without departing from the scope of the appended claims.

[0090] Methods for inducing antigen-specific immune responses conferring resistance against Mycobacterium tuberculosis

[0091] A method for inducing immunity conferring resistance against Mycobacterium tuberculosis using a vaccine composition comprising BCG, ESAT-6 / TMC nanoparticles, and a STING ligand comprises:

[0092] (a) a first administration of the pharmaceutical composition via a parenteral route; and (b) a subsequent administration of the pharmaceutical composition via a mucosal route, wherein the subsequent administration is 2-8 weeks apart from the first administration. Alternatively, the parenteral route is selected from subcutaneous injection, intradermal injection, intraperitoneal injection, and intramuscular injection, and the mucosal route is selected from intranasal administration, aerosol administration, intratracheal administration, intrabronchial administration, and oral administration.In one embodiment, the parenteral administration is carried out by mixing 20 pg of ESAT-6 / TMC nanoparticles with 5 pg of the STING ligand in saline to achieve a total volume of 50 pL. Subsequently, BCG in an amount of 5.0 x io5CFU in 50 pL of saline is added to achieve a final total volume of 100 pL, thereby providing a vaccine formulation suitable for administration via a parenteral route, preferably subcutaneous injection, with an interval of 2-8 weeks. Alternatively, a second or subsequent administration may be adjusted to be more than 8 weeks apart, depending on the immune response characteristics of a subject.

[0093] In another embodiment, the mucosal administration is carried out by mixing 20 pg of ESAT-6 / TMC nanoparticles with 5 pg of the STING ligand in saline and 5.0 x 105CFU of BCG in saline to achieve a total volume of 20-40 pL. The composition is then administered via a mucosal route, preferably intranasally.

[0094] The tuberculosis immunity -inducing vaccine composition comprising BCG, ESAT-6 / TMC, and a STING ligand can provide inhibition against Mycobacterium tuberculosis by exerting effects through three important mechanisms as follows:

[0095] (1) Delivery of BCG antigens into the cytoplasm of APCs via ESAT-6 / TMC nanoparticles, thereby enhancing the generation of Mtb-specific CD8+T cells in the lung, wherein the APCs include dendritic cells and macrophages.

[0096] (2) Stimulation of the generation of Mtb-specific CD4+Thl T cells and Mtb-specific CD4+Thl7 T cells in the lung.

[0097] (3) Stimulation of the generation of Mtb-specific IgA and Mtb-specific IgG in the lung, thereby contributing to prevention of pulmonary tuberculosis.

[0098] Example 1: Delivery of BCG antigens into the cytoplasm of dendritic cells via ESAT-6 / TMC nanoparticles

[0099] In this example, a test was performed to evaluate the capability of the carrier system according to the present invention to enhance delivery of BCG antigens into the cytoplasm of human dendritic cells by fluorescently labeling BCG with Alexa Fluor 647. Thereafter, Alexa Fluor 647-labeled BCG alone, TMC nanoparticles + Alexa Fluor 647-labeled BCG, ESAT-6 / TMC nanoparticles + Alexa Fluor 647-labeled BCG, or soluble ESAT-6 + Alexa Fluor 647-labeled BCG were administered to human dendritic cells for 4 hours. The cells were then washed with PBS and fixed at 0, 24, and 48 hours after washing, and subsequently stained with a red fluorescently labeled antibody against ESAT-6 protein and a green fluorescently labeled antibodyagainst the Ag85 complex (a BCG antigen), or a green fluorescently labeled antibody against galectin-3 (a marker of phagosomal membrane perforation), and nuclei were stained blue with Hoechst. The capability of ESAT-6 / TMC nanoparticles was assessed for: (1) delivery of ESAT-6 into human dendritic cells (Fig. 1 A); (2) delivery of BCG into human dendritic cells (Fig. IB); (3) delivery of the Ag85 complex (a BCG antigen) into the cytoplasm of human dendritic cells (Fig. 2 and Fig. 3); and (4) perforation of the phagosomal membrane of BCG in human dendritic cells (Fig. 4 and Fig. 5).

[0100] Fig. 1 A and Fig. IB show that ESAT-6 / TMC nanoparticles most effectively deliver ESAT-6 into human dendritic cells and that delivery of BCG into human dendritic cells is not significantly different from administration of BCG alone.

[0101] Fig. 2 and Fig. 3 demonstrate that ESAT-6 / TMC nanoparticles significantly enhance delivery of the Ag85 complex (a BCG antigen) into the cytoplasm of human dendritic cells compared with administration of BCG alone.

[0102] Fig. 4 and Fig. 5 show that ESAT-6 / TMC nanoparticles most effectively promote perforation of the phagosomal membrane of BCG in human dendritic cells compared with administration of BCG alone.

[0103] Accordingly, ESAT-6 / TMC nanoparticles, when administered together with BCG, enhance delivery of BCG antigens into the cytoplasm of antigen-presenting cells (APCs), thereby promoting induction of Mtb-specific CD8+T cells.

[0104] Example 2: Cytotoxicity test of ESAT-6 / TMC nanoparticles in human dendritic cells Human dendritic cells were treated with medium alone, soluble ESAT-6 alone, BCG alone, soluble ESAT-6 + BCG, TMC alone, TMC + BCG, ESAT-6 / TMC nanoparticles alone, or ESAT-6 / TMC nanoparticles + BCG for 48 hours. Cytotoxicity was then measured by MTS analysis.

[0105] As shown in Fig. 6, ESAT-6 / TMC nanoparticles did not induce cytotoxicity relative to medium alone, which was used as a negative control.

[0106] Example 3: Effect of STING ligand combined with ESAT-6 / TMC nanoparticles and BCG on dendritic cell maturation and cytokine production

[0107] In this example, a test was performed to evaluate the capability of the vaccine composition according to the present invention, compared with MPL or CpG, to stimulate dendritic cellmaturation for antigen presentation and to induce production of cytokines associated with Thl and Thl7 differentiation. Human dendritic cells were treated for 48 hours with medium alone, ESAT-6 / TMC nanoparticles alone, ESAT-6 / TMC nanoparticles + BCG, ESAT-6 / TMC nanoparticles + BCG + MPL, ESAT-6 / TMC nanoparticles + BCG + CpG, ESAT-6 / TMC nanoparticles + BCG + STING ligand, BCG alone, MPL alone, CpG alone, or STING ligand alone. Dendritic cell maturation was assessed by staining for CD80, CD83, CD86, and HLA-DR surface expression and analyzing by flow cytometry, with medium alone as a negative control (Fig. 7-10). Culture supernatants were analyzed by ELISA for interleukin- 1 beta (IL- 1 P), interleukin- 12 (IL-12), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-P), with medium alone as a negative control (Fig. 11-14).

[0108] Fig. 7-10 show that the vaccine composition according to the present invention most effectively induces maturation of APCs, particularly dendritic cells, resulting in significantly increased expression of CD83, CD86, and HLA-DR compared with BCG alone, and increased CD80 expression approaching statistical significance relative to BCG alone.

[0109] Fig. 11-14 show that the vaccine composition according to the present invention most effectively induces IL-ip production compared with BCG alone, wherein IL-ip is a cytokine important for Thl7 differentiation. In contrast, production of IL-12 (important for Thl differentiation) and production of IL-6 and TGF-P (important for Thl7 differentiation) were already robustly induced by BCG alone.

[0110] Example 4: Induction of Mtb-specific CD4+Thl, CD4+Thl7, and CD8+T Cells in the lungs of mice by administration of the vaccine composition using a prime-pull strategy The vaccine composition according to the present invention was administered to mice using a prime-pull strategy, comprising a first administration via a parenteral route (including subcutaneous, intradermal, intraperitoneal, or intramuscular injection) followed by a booster administration via a mucosal route (including intranasal, aerosol, intratracheal, intrabronchial, or oral administration).

[0111] In this test, BCG was administered by subcutaneous injection for 4 weeks to establish an immune baseline equivalent to intradermal BCG vaccination at birth in humans. For the primepull strategy, the vaccine composition according to the present invention was administered as a prime by subcutaneous injection for 4 weeks, followed by intranasal administration, and the mice were maintained for 6 weeks, in comparison with the following control groups: PBS alone (negative control), BCG alone, ESAT-6 / TMC + BCG, ESAT-6 / TMC + BCG + MPL, ESAT-6 / TMC + BCG + CpG, ESAT-6 / TMC alone, MPL alone, CpG alone, and STING ligand alone, wherein BCG alone administered once subcutaneously served as a benchmark control.

[0112] Thereafter, at the scheduled time point, a fluorescently labeled anti-CD45 antibody was injected via the tail vein and allowed to circulate for 1 minute to label intravascular leukocytes, thereby enabling discrimination of leukocytes residing in lung parenchyma (CD45‘) from leukocytes within pulmonary blood vessels (CD45+). The mice were then euthanized, and bronchoalveolar lavage (BAL) cells and lung cells were collected. The cells were stimulated with purified protein derivative (PPD) for 6 hours and stained with fluorescently labeled antibodies against CD3, CD4, CD8, CD44, CD69, CD103, interferon-gamma (IFN-y), and interleukin-17A (IL-17A), followed by flow cytometric analysis to quantify Mtb-specific CD4+Thl and Thl7 T cells in lung parenchyma (Fig. 15-17) and Mtb-specific IFN-y-producing CD4+T cells and CD8+T cells in the airways of the lungs (Fig. 18-21).

[0113] In addition, lung cells were incubated with PPD for 72 hours, and culture supernatants were analyzed for IFN-y, IL-17A, IL-17F, IL-22, TNF-a, and IL-6 using a cytokine bead multiplex array (Fig. 22-27).

[0114] Fig. 15-17 show that the vaccine composition according to the present invention administered using the prime-pull strategy significantly increases the generation of Mtb-specific CD4+Thl and Thl7 T cells in lung parenchyma compared with BCG alone, as determined by flow cytometry.

[0115] Fig. 18-21 further show that the vaccine composition according to the present invention administered using the prime-pull strategy most effectively induces Mtb-specific IFN-y-producing CD4+and CD8+T cells in the airways of the lungs, with significant increases compared with BCG alone. In contrast, BCG alone did not induce Mtb-specific CD8+T cells producing IFN-y in the airways of the lungs.

[0116] Fig. 22-27 demonstrate that the vaccine composition according to the present invention administered using the prime-pull strategy significantly increases production of IFN-y, IL-17A, IL-17F, IL-22, TNF-a, and IL-6 in lung cells following PPD stimulation compared with BCG alone, as determined by cytokine bead multiplex analysis.

[0117] Accordingly, the vaccine composition according to the present invention administered using the prime-pull strategy induces robust generation of Mtb-specific CD4+Thl, CD4+Thl7, and CD8+T cells in the lungs of mice.Example 5: Induction of Mtb-specific IgA and IgG in the airways of the lungs of mice by administration of the vaccine composition using a prime-pull strategy

[0118] BCG in combination with ESAT-6 / TMC nanoparticles and a STING ligand was administered to mice using a prime-pull strategy to evaluate induction of Mtb-specific IgA and IgG in the airways of the lungs.

[0119] BCG was administered by subcutaneous injection for 4 weeks to establish an immune baseline equivalent to intradermal BCG vaccination at birth in humans. Thereafter, the vaccine composition according to the present invention was administered using a prime-pull strategy, comprising subcutaneous priming for 4 weeks followed by intranasal boosting, and the mice were maintained for 6 weeks, in comparison with the following control groups: PBS alone (negative control), BCG alone, ESAT-6 / TMC + BCG, ESAT-6 / TMC + BCG + MPL, ESAT-6 / TMC + BCG + CpG, ESAT-6 / TMC alone, MPL alone, CpG alone, and STING ligand alone, wherein BCG alone administered once subcutaneously served as a benchmark control Thereafter, at the scheduled time point, an anti-CD45 antibody was injected via the tail vein and allowed to circulate for 1 minute to label intravascular leukocytes. The mice were euthanized, and bronchoalveolar lavage (BAL) fluid was collected for quantification of Mtb-specific IgA and IgG in the airways of the lungs by ELISA (Fig. 28A and Fig. 28B).

[0120] Fig. 28A and Fig. 28B show that the vaccine composition according to the present invention administered using the prime-pull strategy significantly increases Mtb-specific IgA and IgG levels in the airways of the lungs compared with BCG alone, as determined by ELISA.

[0121] Accordingly, the vaccine composition comprising BCG, ESAT-6 / TMC nanoparticles, and a STING ligand, when administered using a prime-pull strategy, induces mucosal and systemic immunity and is suitable for development as a vaccine for preventing pulmonary tuberculosis.

[0122] Example 6: Reduction of Mycobacterium tuberculosis burden in mouse lungs by administration of the vaccine composition using a prime-pull strategy

[0123] BCG was administered by subcutaneous injection for a period of 4 weeks to establish an immune baseline equivalent to intradermal BCG vaccination typically administered at birth in humans. Subsequently, the vaccine composition according to the present invention (comprising BCG in combination with ESAT-6 / TMC nanoparticles and a STING ligand) was administered using a prime-pull strategy. In this strategy, priming was performed by subcutaneous injection for4 weeks, followed by intranasal boosting, and the mice were maintained for an additional 6 weeks.

[0124] Efficacy was evaluated in comparison with the following groups: BCG alone; ESAT-6 / TMC + BCG; ESAT-6 / TMC alone; STING ligand alone; ESAT-6 / TMC + STING ligand; and BCG + STING ligand, wherein a single subcutaneous administration of BCG alone served as the benchmark control. At the scheduled time point, the mice were challenged intranasally with Mycobacterium tuberculosis strain H37Rv at a dose of 100-500 CFU and maintained for 6 weeks. Following the observation period, the mice were euthanized, and the lungs were harvested and homogenized in phosphate-buffered saline. Viable mycobacterial burden in the lungs was determined by serial dilution and cultivation on solid media for colony -forming unit (CFU) analysis (Fig. 29).

[0125] Fig. 29 illustrates that the vaccine composition according to the present invention administered using the prime-pull strategy significantly inhibits growth of Mycobacterium tuberculosis in the lungs of mice compared with BCG alone, as determined by CFU analysis.

[0126] Best Mode of the Invention

[0127] As disclosed in the detailed description of embodiments.

Claims

Claims1. A delivery system for inducing immunity against a pathogenic microorganism, comprising:(a) ESAT-6 protein (ESAT-6) encapsulated within a nanoparticle-type carrier; and (b) a STING ligand,wherein the ESAT-6 protein encapsulated within the nanoparticle-type carrier is physically or chemically combined with the STING ligand, and wherein a mass ratio of ESAT-6 to the STING ligand is in a range of 4-10:1.

2. The delivery system according to claim 1, wherein the nanoparticle -type carrier is selected from the group consisting of chitosan, chitosan derivatives, polymer-group substances, polyamino acid-group substances, and surfactants.

3. A vaccine composition for inducing antigen-specific immunity conferring resistance against Mycobacterium tuberculosis, comprising:(a) BCG;(b) ESAT-6 protein (ESAT-6) encapsulated within a nanoparticle-type carrier; and (c) a STING ligand,wherein the BCG, the ESAT-6 protein encapsulated within the nanoparticle-type carrier, and the STING ligand are physically and / or chemically combined, and wherein a mass ratio of the ESAT-6 protein to the STING ligand is in a range of 4-10: 1.

4. The vaccine composition according to claim 3, further comprising a pharmaceutically acceptable excipient selected from one or more of a solvent, an adjuvant, a buffering agent, an additive, and a stabilizer, alone or in any combination thereof.

5. The vaccine composition according to claim 3 or 4, wherein the nanoparticle-type carrier is selected from the group consisting of chitosan, chitosan derivatives, polymer-group substances, polyamino acid-group substances, and surfactants.

6. The vaccine composition according to claim 3 or 4, wherein the nanoparticle-type carrier comprises trimethyl chitosan (TMC).

7. The vaccine composition according to claim 3 or 4, wherein the antigen-specific cells comprise at least one of dendritic cells and macrophages.

8. The vaccine composition according to any one of claims 3-7, wherein the vaccine composition induces, in the lung, one or more of Mtb-specific CD8+T cells, Mtb-specific CD4+Thl T cells, Mtb-specific CD4+Thl7 T cells, Mtb-specific IgA, and Mtb-specific IgG.

9. A method for inducing immunity conferring resistance against Mycobacterium tuberculosis using the vaccine composition according to any one of claims 3-8, the method comprising:(a) a first administration of the vaccine composition via a parenteral route; and (b) a subsequent administration of the vaccine composition via a mucosal route, wherein the subsequent administration is performed 2-8 weeks after the first administration.

10. The method according to claim 9, wherein the parenteral route is subcutaneous injection and the mucosal route is intranasal administration.

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