KR102040665B1 - Pharmaceutical composition containing attenuated streptococcus pneumoniae and uses thereof - Google Patents

Abstract

The present application relates to pharmaceutical compositions comprising attenuated pneumococcal strains and uses for the prophylaxis or treatment of inflammatory diseases, respiratory viral infections or bacterial infectious diseases.

Description

Pharmaceutical composition comprising attenuated pneumococcal strain and use thereof {PHARMACEUTICAL COMPOSITION CONTAINING ATTENUATED STREPTOCOCCUS PNEUMONIAE AND USES THEREOF}

The present application relates to pharmaceutical compositions comprising attenuated pneumococcal strains and uses for the prophylaxis or treatment of inflammatory diseases, respiratory viral infections or bacterial infectious diseases.

It is known that nasal or oral administration of antigen induces regulatory T (Treg) cells, which induces mucosal immune tolerance in target organs (Faria and Weiner, 2005). Induction of mucosal immune tolerance in mice has been reported to inhibit various autoimmune diseases including atherosclerosis (Maron et al, 2002) (Faria and Weiner, 2005). Thus, mucosal immune tolerance principles can be applied to animals and humans. Stimulation of mucous membranes with multiple administrations of antigens induces T cells to secrete anti-inflammatory cytokines, such as IL-10 or TGF-β1, to defend tissues and induce mucosal immune tolerance (Faria and Weiner, 2005; Weiner, 2001 ). Certain types of Treg cells are preferentially induced to maintain immunotolerance of mucosal surfaces, but the mechanism of induction is not yet known.

However, current mucosal vaccines are not effective enough to induce a sufficient mucosal immune response without an immunostimulant. Mucosal adjuvants are used when antigen tolerance alone is low in immunotolerance induction efficiency, for example, the cholera toxin B subunit (CTB) (Faria and Weiner, 2005). However, the immune-increasing effects of cholera toxin after the inoculation of the throat are recognized and mediated by bacteria (Kim et al., 2016a), indicating that the microbial system plays an essential role. Indicates that it is preferable. In other words, the concept of mucosal vaccines without immunostimulants has not yet been demonstrated.

In addition, it has not yet been reported whether mucosal vaccines can be effective in a variety of diseases other than those directly related to the antigens used in vaccine manufacture, and currently used immunotherapy is only defended against antigens used for vaccination. In particular, the 23-valent polysaccharide vaccine or the 13-valent conjugate vaccine are used to prevent pneumococcal disease, but the 23-valent polysaccharide vaccine has no memory response, and the 13-valent conjugate vaccine can only protect 13 serotypes out of 90 or more serotypes. (Kalin, 1998).

1.Aaberge IS, Eng J, Lermark G, Lovik M. Virulence of Streptococcus pneumoniae in mice: a standardized method for preparation and frozen storage of the experimental bacterial inoculum. Microb Pathog. 1995 Feb; 18 (2): 141-52. 2.Avery OT, Macleod CM, McCarty M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types: Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J Exp Med. 1944 Feb 1; 79 (2): 137-58. Bosch AA, Biesbroek G, Trzcinski K, Sanders EA, Bogaert D. Viral and bacterial interactions in the upper respiratory tract. PLoS Pathog 2013; 9: e1003057. Briles DE, Forman C, Crain M. Mouse antibody to phosphocholine can protect mice from infection with mouse-virulent human isolates of Streptococcus pneumoniae. Infect Immun. 1992 May; 60 (5): 1957-62. 5. Choi SY, Tran TD, Briles DE, Rhee DK. Inactivated pep27 mutant as an effective mucosal vaccine against a secondary lethal pneumococcal challenge in mice. Clin Exp Vaccine Res. 2013 Jan; 2 (1): 58-65. 6. Cohen JM, Wilson R, Shah P, Baxendale HE, Brown JS. Lack of cross-protection against invasive pneumonia caused by heterologous strains following murine Streptococcus pneumoniae nasopharyngeal colonisation despite whole cell ELISAs showing significant cross-reactive IgG. Vaccine. 2013 May 1; 31 (19): 2328-32. 7. Faria AM, Weiner HL. Oral tolerance. Immunol Rev. 2005 Aug; 206: 232-59. 8.Geng S, Zhang H, Zhou X, He Y, Zhang X, Xie X, Li C, He Z, Yu Q, Zhong Y, Lowrie DB, Zheng G, Wang B. Diabetes tolerogenic vaccines targeting antigen-specific inflammation. Hum Vaccin Immunother. 2015; 11 (2): 522-30. 9. Jawhara S, Poulain D. Saccharomyces boulardii decreases inflammation and intestinal colonization by Candida albicans in a mouse model of chemically-induced colitis. Med Mycol 2007, 45 (8): 691-700. 10. Kalin M. Pneumococcal serotypes and their clinical relevance. Thorax, 1998; 53: 159-62. 11.Kim D, Kim YG, Seo SU, Kim DJ, Kamada N, Prescott D, Philpott DJ, Rosenstiel P, Inohara N, Nunez G. Nod2-mediated recognition of the microbiota is critical for mucosal adjuvant activity of cholera toxin. Nat Med. 2016a May; 22 (5): 524-30. 12.Kim GL, Choi SY, Seon SH, Lee S, Park SS, Song JY, Briles DE, Rhee DK. Pneumococcal pep27 mutant immunization stimulates cytokine secretion and confers long-term immunity with a wide range of protection, including against non-typeable strains. Vaccine. 2016b Dec 12; 34 (51): 6481-6492. 13.Maron R, Sukhova G, Faria AM, Hoffmann E, Mach F, Libby P, Weiner HL. Mucosal administration of heat shock protein-65 decreases atherosclerosis and inflammation in aortic arch of low-density lipoprotein receptor-deficient mice. Circulation. 2002; 106: 1708-1715. 14. McDaniel LS, Scott G, Kearney JF, Briles DE. Monoclonal antibodies against protease-sensitive pneumococcal antigens can protect mice from fatal infection with Streptococcus pneumoniae. J Exp Med. 1984 Aug 1; 160 (2): 386-97. 15. Metzger DW, Furuya Y, Salmon SL, Roberts S, Sun K. Limited efficacy of antibacterial vaccination against secondary serotype 3 pneumococcal pneumonia following influenza infection. J Infect Dis 2015; 212: 445-52. 16. Mina MJ, Klugman KP. Pathogen replication, host inflammation, and disease in the upper respiratory tract. Infect Immun 2013; 81: 625-8. 17. Pigny F, Lassus A, Terrettaz J, Tranquart F, Corthesy B, Bioley G. Intranasal vaccination with Salmonella-derived serodominant secreted effector protein B associated with gas-filled microbubbles partially protects against gut infection in mice. J Infect Dis. 2016 Aug 1; 214 (3): 438-46. 18. Rizzo A, Losacco A, Carratelli CR, Domenico MD, Bevilacqua N. Lactobacillus plantarum reduces Streptococcus pyogenes virulence by modulating the IL-17, IL-23 and Toll-like receptor 2/4 expressions in human epithelial cells. Int Immunopharmacol. 2013 Oct; 17 (2): 453-61. 19.Saint-Lu N, Tourdot S, Razafindratsita A, Mascarell L, Berjont N, Chabre H, Louise A, Van Overtvelt L, Moingeon P. Targeting the allergen to oral dendritic cells with mucoadhesive chitosan particles enhances tolerance induction. Allergy. 2009 Jul; 64 (7): 1003-13. 20. Schnoeller C, Roux X, Sawant D, Raze D, Olszewska W, Locht C, Openshaw PJ. Attenuated Bordetella pertussis vaccine protects against respiratory syncytial virus disease via an IL-17-dependent mechanism. Am J Respir Crit Care Med. 2014 Jan 15; 189 (2): 194-202. 21.Shak JR, Vidal JE, Klugman KP. Influence of bacterial interactions on pneumococcal colonization of the nasopharynx. Trends Microbiol 2013; 21: 129-35. Shim BS, Choi JA, Song HH, Park SM, Cheon IS, Jang JE, Woo SJ, Cho CH, Song MS, Kim H, Song KJ, Lee JM, Kim SW, Song DS, Choi YK, Kim JO, Nguyen HH, Kim DW, Bahk YY, Yun CH, Song MK. Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus. J Microbiol. 2013 Feb; 51 (1): 130-5. 23. Sun JB, Czerkinsky C, Holmgren J. Mucosally induced immunological tolerance, regulatory T cells and the adjuvant effect by cholera toxin B subunit. Scand J Immunol. 2010 Jan; 71 (1): 1-11. 24. Takagi H, Hiroi T, Yang L, Takamura K, Ishimitsu R, Kawauchi H, Takaiwa F. Efficient induction of oral tolerance by fusing cholera toxin B subunit with allergen-specific T-cell epitopes accumulated in rice seed. Vaccine. 2008 Nov 11; 26 (48): 6027-30. 25. Weiner HL. Oral tolerance: immune mechanisms and the generation of Th3-type TGF-beta-secreting regulatory cells. Microbes Infect. 2001 Sep; 3 (11): 947-54. 26. Weiner HL. The mucosal milieu creates tolerogenic dendritic cells and T (R) 1 and T (H) 3 regulatory cells. Nat. Immunol. 2001; 2: 671. 27. Wirtz S, Neufert C, Weigmann B, Neurath MF: Chemically induced mouse models of intestinal inflammation. Nat Protoc 2007, 2 (3): 541-546. 28. Wu Y, Tu W, Lam KT, Chow KH, Ho PL, Guan Y, Peiris JS, Lau YL. Lethal coinfection of influenza virus and Streptococcus pneumoniae lowers antibody response to influenza virus in lung and reduces numbers of germinal center B cells, T follicular helper cells, and plasma cells in mediastinal lymph Node. J Virol. 2015 Feb; 89 (4): 2013-23. 29.Xu Y, Hunt NH, Bao S. The correlation between proin flammatory cytokines, MAdCAM-1 and cellular infiltration in the inflamed colon from TNF-α gene knockout mice. Immunol Cell Biol 2007; 85: 633-639. 30. Yamashita T, Sasaki N, Kasahara K, Hirata K. Anti-inflammatory and immune-modulatory therapies for preventing atherosclerotic cardiovascular disease. J Cardiol. 2015 Jul; 66 (1): 1-8.

Prophylactic and therapeutic methods for inflammatory diseases including inflammatory factors such as intestinal diseases, infections, atherosclerosis and epithelial lesions have been developed, but these have been limited to the use of specific markers that induce or cause the disease. . In this regard, mucosal immunity was expected to suppress mucosal related diseases and inflammatory diseases. Therefore, the present application aims to propose a new method of anti-inflammatory therapy against various diseases using mucosal immunity of pneumococcal pneumoniae vaccine and other defense methods. . In addition, the mucosal vaccine using the immunotolerant properties of mucosal immunity was intended to prove that a new method for preventing or treating intestinal inflammation and infectious diseases.

In addition, we have developed a new method to protect against a wide range of inflammatory diseases, such as intestinal inflammation, respiratory infections and inflammation, as well as respiratory viral and bacterial infectious diseases by inoculating pneumococcal prophylactic vaccines on mucous membranes. Was intended.

However, the problem to be solved by the present application is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present application is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases, respiratory viral infection diseases, or bacterial infectious diseases other than pneumococci, including attenuated pneumococcal strains.

The above-mentioned means for solving the problems are merely exemplary, and should not be construed to limit the present invention. In addition to the exemplary embodiments described above, there may be additional embodiments and embodiments described in the drawings and detailed description of the invention.

According to the present invention, the pneumococcal vaccine can be inoculated into mucous membranes to prevent and / or treat inflammatory diseases by genes induced by immunization in the lung and spleen.

In particular, existing vaccines can only provide protection against certain antigenic components, but the use of pneumococcal mucosal vaccines in accordance with the present invention provides a wide range of diseases including the ability to protect against viral and bacterial infectious diseases as well as other organ inflammatory diseases. Prophylactic and / or therapeutic effects can be expected. In addition, the pneumococcal pneumoniae vaccine according to the present invention may provide a protective effect against various diseases without using an adjuvant unlike the conventional mucosal vaccine when administered to the mucosa.

1A and 1B are schematic diagrams of the results of pneumococcal vaccine function analysis by system biology analysis in the lungs (FIG. 1A) and spleen (FIG. 1B) according to one embodiment of the present disclosure. Mice were immunized three times at 2 week intervals with THpep27 mutant (Δpep27) and harvested lung and spleen after 2 weeks of final immunization, followed by high-speed mass sequencing by extracting total RNA, followed by gene expression for Ingenuity Pathway Analysis Analyzed. The genes of the immunized lung tissues in FIG. 1A suggest that they inhibit gastroenteritis and colon abnormalities, and the genes of the spleen in FIG. 1B show a protective effect against influenza virus infection.
2 is a schematic diagram of system biology analysis results in the lungs according to an embodiment of the present application. Mice were inoculated three times into the throat Δpep27 mutant at 2 week intervals, and 7 days after the last vaccination, mRNA was isolated from the lungs and subjected to rapid mass sequencing. IPA network analysis of lung sequence data is shown, green indicates gene expression is inhibited and red indicates gene induction.
3A to 3C are experimental results confirming that Treg cells are induced by inoculating Δpep27 in mice according to one embodiment of the present application. Figure 3a shows mice (n = 3) inoculated three times into the throat Δpep27 at intervals of two weeks, splenocytes were isolated 7 days after the last vaccination, Th1 (CD4, Tbet), Th2 (CD4, GATA3), Th17 Labeled with fluorescent cell markers for (CD4, RORγt) and Tregs (CD4, Foxp3). Markers were then detected by flow cytometry. 3B and 3C show the results of ELISA measuring cytokines of spleen cells (FIG. 3B) or serum (FIG. 3C) at 7 days after the last vaccination. Statistical significance was analyzed by ANOVA; *, P <0.05, **, P <0.01.
4A and 4B show experimental results of inducing IL-10 and IL-17 by inoculating Δpep27 in mice according to one embodiment of the present application. Mice (n = 3) were vaccinated three times in the throat with Δpep27 at 2 week intervals, and at day 7 after the last vaccination, mRNA was isolated from the lung and used for qPCR (FIG. 4A) or bronchoalveolar lavage fluid (BALF) It was used to measure the Cain level (FIG. 4B). Statistical significance was analyzed by ANOVA; *, P <0.05.
5 is an experimental result of activating the central memory T cells and memory Tfh cells in the spleen by inoculating Δpep27 in mice according to an embodiment of the present application. Mice (n = 3) were inoculated three times in the throat with Δpep27 at intervals of two weeks, and splenocytes were isolated 7 days after the last vaccination to isolate central memory T cells (CD4, CCR7, CD62L) and memory Tfh cells (CD4, CXCR5). , Fluorescent cell markers for CCR7). The fluorescent markers were then detected by flow cytometry. Statistical significance was analyzed by ANOVA; *, P <0.05.
6A and 6B show experimental results of inducing various immunoglobulin subtypes by inoculating Δpep27 in mice according to one embodiment of the present application. Mice (n = 6) were inoculated three times in the throat with Δpep27 at intervals of two weeks, and antibody titers were measured in serum (FIG. 6A) and bronchoalveolar lavage fluid (BALF) (FIG. 6B) 7 days after the last vaccination. Statistical significance was analyzed by ANOVA; * P <0.05, **, P <0.01.
7A to 7D are experimental results confirming that Δpep27 inoculation can protect against secondary pneumococcal infection after influenza infection according to one embodiment of the present application. Mice (9-10 / group) were inoculated three times with Δpep27 at weekly intervals, serum was collected from mice one week after the last immunization, and then three types of pneumococcal whole cells (FIG. 7A) or specific antigens ( IgG levels for Figure 7b) were measured by ELISA. Significant differences between the two groups were analyzed by unpaired t-test; *** p <0.001. After 10 days of influenza infection, pneumococcal D39 was infected via the intranasal route and survival was monitored for 14 days (FIG. 7C). Statistical significance was analyzed by Mentel-cox test; ** p <0.005. Twenty four hours after pneumococcal D39 infection, lung homogenates were incubated in serum to determine bacterial counts (FIG. 7D). Statistical significance was analyzed by One way ANOVA.
8A and 8B illustrate the protective effect of inhibiting influenza virus replication of the Δpep27 vaccine according to one embodiment of the present application. Mice (10 mice / group) were infected with the influenza virus, and then the body weights of the mice were observed for 11 days (FIG. 8A). Statistical significance was analyzed by one-way ANOVA; *** p <0.001. After harvesting the lung homogenate supernatant 5 days after influenza infection, the viral titers of each group were measured by TCID50 / ml (FIG. 8B). Significance was analyzed by one-way ANOVA; *** p <0.001.
9 is a test result of Δpep27 inoculation can prevent Gram negative bacterial infection according to an embodiment of the present application. Mice (3 mice / group) were inoculated with Δpep27 three times via the nasal route and mice were infected with K. pneumoniae 10 days after the last immunization. The bacteria count of each organ was determined by smear culture in blood agar 24 hours after pneumococcal infection; ** P <0.01.
10 is a test result of Δpep27 inoculation according to one embodiment of the present application can prevent Gram-positive bacterial infection. Mice (3 mice / group) were immunized three times with Δpep27 in the nasal cavity, and mice were infected with Staphylococcus aureus 10 days after the last immunization. The bacteria count of each organ was measured by smearing and incubating blood agar 24 hours after Staphylococcus aureus infection; * P <0.05.
11A and 11B show the results of experimenting whether Δpep27 inoculation inhibits weight loss in inflammatory bowel disease induced by dextran sulfate sodium (DSS) according to one embodiment of the present application. Weight loss was alleviated in colitis (inflammatory bowel disease) induced by inoculating the Δpep27 variant three times into the nasal cavity of mice (n = 5 / group) and then administering 5% DSS to drinking water via the oral route (FIG. 11A). A time course of basal weight loss percentage was observed for 9 days after treatment with DSS. Statistical significance was analyzed by Bonferroni's test after one way ANOVA analysis. Clinical disease activity index was assessed daily until treatment was stopped on day 9 (FIG. 11B). Progression of colitis was assessed by measuring weight loss, consistency of feces, rectal bleeding and / or blood volume in feces. Morbidity (piloerection, coma) in mice was also observed daily.
12A and 12B show the results of experimenting whether Δpep27 inoculation can prevent colon inflammation induced by dextran sulfate sodium (DSS) according to one embodiment of the present application. Weight loss was alleviated in colitis (inflammatory bowel disease) induced by Δpep27 variant inoculated three times into the nasal cavity of mice (n = 5 / group) followed by administration of 5% DSS to drinking water via the oral route (FIG. 12A). ). Colons were examined on day 9 of DSS treatment. Colon weight and length were measured on day 9 of DSS treatment (FIG. 12B).
Figure 13 is a test result of Δpep27 inoculation inhibits inflammatory cytokine gene expression in the colon according to an embodiment of the present application. Inflammatory bowel disease was induced in Balb c mice by adding 5% DSS to drinking water for 14 days. PBS + water was used as negative control. MRNA expression of IL-1β, IL-6, IL-17A and TNF-α was measured using real time quantitative PCR. The experimental values are expressed as mean ± SEM and P <0.05 was considered significant.

DETAILED DESCRIPTION Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present disclosure. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present application.

Throughout this specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding the other components unless specifically stated otherwise.

As used throughout this specification, the terms "about", "substantially" and the like are used at, or in the sense of, numerical values when a manufacturing and material tolerance inherent in the stated meanings is indicated, Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers. As used throughout this specification, the term "step to" or "step of" does not mean "step for."

Throughout this specification, the term "combination of these" included in the expression of the makushi form means one or more mixtures or combinations selected from the group consisting of constituents described in the expression of the makushi form, wherein the constituents It means to include one or more selected from the group consisting of.

Throughout this specification, description of "A and / or B" means "A, B, or A and B."

Throughout this specification, "pneumococcal" is a Gram-positive bacterium, also called pneumococcal or Streptococcus pneumoniae, and it is observed that the bacteria continue to be chained over time, resulting in the formation of streptococci. It is classified as a family and is known as a major cause of pneumonia.

Throughout this specification, "pep27" consists of 27 polypeptides and is secreted by vex transporters to induce growth inhibition and apoptosis, specifically membrane-bound histidine protein kinase (membrane- Expression is regulated by vncS, a bound histidine protein kinase, and vncR, a cytoplasmic effector, to induce cell death (Novak et al., 1999). The pep27 gene or a protein encoded therefrom may be named differently for each serotype of pneumococcal, and there may be a difference in the gene sequence of pep27 or a peptide sequence thereof, but as described above, the pep27 gene of the present invention is substantially pep27. If the gene performs the same function as is not limited to the serotype can be prepared by attenuating pneumococcal strain of the present invention by damaging it. Preferably it includes all of the pneumococcal genes that perform substantially the same function as the pep27 gene. More preferably, the pep27 gene of the present invention may be damaged by mutation of the gene encoding the amino acid of the pep27 peptide represented by SEQ ID NO: 1.

Throughout this specification, "attenuated" means that a toxic strain is modified in such a way as to be a less toxic strain or a weaker pathogen than before the modification, while such strain may still be able to divide in the host, causing clinical disease The ability to induce is significantly reduced. Preferably the attenuated strains of the present invention are strains whose toxicity or pathogenicity has been reduced to allow for administration for vaccines, and more preferably can cause clinical disease while still allowing the strain to replicate in the host. It means attenuating to the point of absence. Attenuated mutations can also be achieved by various methods for preparing attenuated mutations of the invention, for example point mutations, sequence exchange between associated viruses, or nucleotide deletions.

Throughout this specification, "mutation" refers to any action that changes the genetic function of a gene, and specifically, a mutation of a gene refers to a variation caused by a quantitative or qualitative change of a gene among various variations of an organism.

Throughout this specification, "prevention" means any action that inhibits or delays the onset of a disease by administration of a composition.

Throughout this specification, "treatment" means any action that improves or benefits the condition of a disease already caused by administration of the composition.

Throughout this specification, "vaccine" is an antigen used for immunity to prevent an infectious disease, which is made of an attenuated microorganism or virus, and is a hospital that is weakened or killed to artificially obtain immunogenicity for a specific infectious disease. It means the administration of microorganisms in a subject. When stimulated by the vaccine, the immune system in the individual is activated to produce antibodies, and in case of re-infection while maintaining its susceptibility, it is possible to overcome the disease by effectively producing antibodies in a short time. However, vaccines containing attenuated pneumococci according to the present invention, unlike the conventional vaccines, have the ability to prevent and treat a wide range of diseases including viral and bacterial infectious diseases as well as defense against certain antigenic components. Can have.

Throughout this specification, "ermB" refers to a gene that makes it resistant to macrolide, which has been used as an alternative to penicillin in the treatment of pneumococci, erythromycin, Clarithromycin, azithromycin, and the like.

Hereinafter, a pharmaceutical composition comprising the attenuated pneumococcal strain of the present application, and a use thereof for the prevention or treatment of an inflammatory disease, a respiratory viral infection, or a bacterial infectious disease will be specifically described with reference to embodiments, examples, and drawings. Explain. However, the present application is not limited to these embodiments, examples and drawings.

A first aspect of the present disclosure may provide a pharmaceutical composition for preventing or treating an inflammatory disease, a respiratory viral infection disease, or a bacterial infectious disease except pneumococci, including an attenuated pneumococcal strain. For example, the pharmaceutical composition may comprise a vaccine composition.

According to one embodiment of the present application, the attenuated pneumococcal strain may be a part or all of the deletion of the pep27 gene, for example, 1 to 53 of the nucleic acid sequence of the pep27 gene represented by SEQ ID NO: 1 It may be deleted, but may not be limited thereto.

According to one embodiment of the present application, the attenuated pneumococcal strain is an attenuated pneumococcal strain comprising a mutation substituted with an ermB cassette and deletions 1 to 53 of the pep27 gene nucleic acid sequence represented by SEQ ID NO: 1 It may be, but may not be limited thereto. For example, the attenuated pneumococcal strain may be an attenuated pneumococcal strain comprising the pep27 mutation disclosed in Korean Patent No. 10-1252911.

According to one embodiment of the present application, the inflammatory disease may be selected from asthma, bronchitis, rhinitis, inflammatory bowel disease, gastroenteritis, colitis, Crohn's disease, pancreatitis, atherosclerosis and arthritis, but may not be limited thereto. For example, preventing or treating an inflammatory disease may be an effect of inhibiting the expression of inflammation related genes, such as colitis genes. For example, the inflammatory disease may be related to intestinal and respiratory infections, but may not be limited thereto.

For example, the respiratory virus may be selected from metapneumovirus, coronavirus, enterovirus, respiratory syncytial virus, adenovirus, bocavirus, rhinovirus and influenza virus. The prevention or treatment of the respiratory viral infection disease may be an effect of inhibiting the replication of the virus. For example, the influenza virus may be influenza A, B or C, and may not be limited to the specific subtype.

In addition, the pharmaceutical compositions herein may also provide non-viral defense functions, in which case influenza as well as other viruses, such as, but not limited to, Respiratory syncytial virus and nasal virus (Rhinovirus) It can also provide protection against viruses, including).

According to one embodiment of the present application, the bacterial infectious disease may be selected from Gram-positive bacterial infectious diseases, Gram-negative bacterial infectious diseases and other infectious bacterial diseases, but may not be limited thereto. For example, the prevention or treatment of bacterial infectious diseases can be an effect of inhibiting the infection of Gram-positive and / or Gram-negative bacteria.

According to one embodiment of the present application, the Gram-positive bacteria may be selected from Staphylococcus, Streptococcus, Tetanus and Anthrax, and the Gram-negative bacteria may be selected from Salmonella, Heterogene, Pneumococcal, E. coli and Cholera. It may not be limited. For example, the Gram-positive bacteria may be Staphylococcus aureus.

According to one embodiment of the present application, the pharmaceutical composition may be immunized serotype-independently, but may not be limited thereto. The serotype-independent immunization pharmaceutical composition refers to a pharmaceutical composition that produces an antibody by producing an antibody regardless of the antigen, rather than producing an antibody specifically to the antigen.

According to one embodiment of the present application, the pharmaceutical composition may be non-invasive to the lung, spleen, blood or brain, but may not be limited thereto. The term "non-invasive" means that the pharmaceutical composition does not or does not penetrate quickly into other areas of the system other than the organs, tissues, and cells to which the pharmaceutical composition is directly administered, or is quickly removed, which means that the pharmaceutical composition is administered. It is opposite to the permeability that penetrates other areas of the whole body other than organs, tissues, and cells to damage the human body.

The term "administration" means introducing a composition of the invention to a subject in any suitable manner, and the route of administration of the composition of the invention may be administered via any general route as long as it can reach the desired tissue. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intraluminal administration, intradural, intramucosal administration may be, but is not limited thereto. Can be.

According to one embodiment of the present application, the pharmaceutical composition may be for administration intraperitoneally or mucosally, but may not be limited thereto. The location of the mucosa is not particularly limited and may be appropriately selected and administered by those skilled in the art among the body locations used for mucosal administration in the art.

According to one embodiment of the present application, the pharmaceutical composition may be for administration into the throat mucosa, but may not be limited thereto. Vaccination through the throat mucosa may use, for example, but not limited to aerosol or drip delivery system forms.

In the case of using the pharmaceutical composition according to the present invention, administration to the mucous membrane also shows effective immunogenicity in the individual, thereby eliminating the inconvenience of having previously been administered subcutaneously by injection, and especially in the injection. It may be advantageous to administer to infants suffering from, but may not be limited thereto. The individual to which the pharmaceutical composition according to the present invention can be administered is not limited, and may include, but is not limited to, a mammal such as a rat, a mouse, a livestock, and a human. The effective dosage range of the composition according to the present invention can be determined by sex, body surface area, type and severity of disease, age, sensitivity to drug, route and rate of administration, administration time, duration of treatment, target cell, expression level, etc. It may vary depending on various factors well known in the art, and can be easily determined by those skilled in the art.

According to one embodiment of the present application, the pharmaceutical composition may further include a pharmaceutically acceptable carrier or an adjuvant, but may not be limited thereto.

Carriers that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but may not be limited thereto.

As an adjuvant that may be used in the present invention, an adjuvant conventionally used in the art may be used without limitation, and the adjuvant may include cholera toxin binding unit, aluminum salts, lipid emulsion (MF-59), synthetic detergent (Tween). ), microspheres, liposomes, mucoadhesive polymers, and the like, but are not limited thereto. New formulations are being developed in this field, and these may also be used.

The pharmaceutical compositions according to the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, or sterile injectable solutions according to conventional methods. However, this may not be limited. In detail, when formulated, a diluent or excipient such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used may be prepared, but the present invention may not be limited thereto.

For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose in the compound. , Gelatin and the like can be mixed. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients, and liquid preparations include suspensions, solvents, emulsions, and syrups, and various excipients in addition to commonly used simple diluents such as water and liquid paraffin. For example, wetting agents, sweetening agents, fragrances, and preservatives may include, but may not be limited thereto.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspending agents, emulsions, lyophilized preparations and suppositories, and non-aqueous solvents and suspending agents include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and ethylol. Injectable esters such as late may be used but may not be limited thereto.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present application.

EXAMPLE

1. Materials and Methods

Attenuated  Pneumococcal Strains THpep27  Produce

THpep27 mutant, a pneumococcal strain used in the Examples herein, is a strain reported in the literature of Choi SY et al. (Inactivated pep27 mutant as an effective mucosal vaccine against a secondary lethal pneumococcal challenge in mice.Clin Exp Vaccine Res. 2013) It is the same strain except that it does not have an erythromycin resistance marker (ermAM) for selection in the pep27 mutant pneumococcal strain disclosed in Korean Patent No. 10-1252911.

Primer (5'-TGG CTT ACC GTT CGT) from a Cheshire cassette (Genbank accession No. FJ981645) with an erythromycin resistance marker (ermAM) that can be used as a transient marker for selection from Donald Morrison (University of Illinois of Chicago) Primers (5'-TCT CTA TCG GCC) to amplify upstream and downstream sequences using ATA G-3 '(SEQ ID NO: 2) and 5'-TCG ATA CCG TTC GTA TAA TGT-3' (SEQ ID NO: 3)) TCA AGC AG-3 '(SEQ ID NO: 4) and 5'-CTA TAC GAA CGG TAA GCC A GAT TTT CAC CAC TGC TTT CG-3' (SEQ ID NO: 5) and 5'-ACA TTA TAC GAA CGG TAT CGA AAG GCC PCR polymerase chain reaction (PCR) using D39 genomic DNA as a template with AGC AAG AGA CTA-3 '(SEQ ID NO: 6) and 5'-CTG CGA GGC TTG CAC TGT AG-3' (SEQ ID NO: 7) ). The linked product was then transformed with D39 to generate pep27 mutants.

Cheshire cassette cleavage was induced by addition of 1% L-fucose (Sigma, St. Louis, MO, USA). Fucos-treated cultures were plated in THY blood agar medium to obtain single colonies. The presence of the Cheshire cassette in each colony was confirmed by PCR using the following primers: 5'-TCT CTA TCG GCC TCA AGC AG-3 '(SEQ ID NO: 8) and 5'-CTG CGA GGC TTG CAC TGT AG-3' (SEQ ID NO: 9). Mutant (THpep27) sequences were identified by nucleotide sequencing (Cosmo, Seoul, Korea) as well as immunoblot analysis with Pep27 antibody (not shown).

To identify THpep27 mutants at the RNA level, RNA was isolated from early logarithmic bacteria using the hot phenol method described previously. After DNA was removed with DNase I (Takara, Tokyo, Japan), 1 microgram of bacterial RNA was reverse transcribed into cDNA using a random primer (Takara). Reverse transcription PCR was performed according to the manufacturer's instructions (Super Bio, American Building Restoration Products Inc., Franklin, WI, USA) using recommended primers.

Preparation of Other Bacterial Strains

The pneumococcal strains tested herein are shown in Table 1 below.

Pneumococcal ( S. pneumoniae ) serotype 2 (D39) wild-type strain, serotype 3 (A66.1), serotype 6B (BG7322) and THpep27 mutant strain (D39 Δpep27) were cultured and used in the laboratory (Kim et al, 2012). Pneumococci were incubated overnight on blood agar plates at 37 ° C. and then incubated at 37 ° C. for 3 hours in Todd-Hewitt broth with 0.5% yeast extract (THY; Difco Laboratories). Each bacterial culture was appropriately diluted and 10 μl of pneumococcal strain was throat-infected in CD1 mice.

Staphylococcus aureus ( S. aureus ATCC 25923) and pneumococcal bacillus ( K. pneumoniae , ATCC 9997) were purchased from the Korea Microbial Conservation Center (KCCM, Seoul) and extracted with brain heart infusion (BHI) Incubated overnight at 37 ° C. in medium, and then inoculated in BHI culture to 37 ° C. until OD ₅₅₀ = 0.5.

Influenza virus A / California / 04/2009 (H1N1) strains were cultured in eggs by the methods previously used (Shim et al, 2013).

In Vivo Infection Research

Four week old male CD1, BALB / c mice (Orient, Korea) were used for infection experiments. Animal use in this example was approved by Sungkyunkwan University Animal Ethics Committee according to the guidelines of the Korean Animal Protection Act.

In the vaccine efficacy evaluation, the days of survival measurement mice were inoculated with 1 × 10 ⁷ to 1 × 10 ⁸ Δpep27 strains three times in the throat ( in ) every two weeks. After 1-2 weeks of final immunization, mice throats were infected with 1 × 10 ⁷ to 1 × 10 ⁸ virulent strains D39 or 6B. Survival of the infected mice was monitored four times daily for the first five days, twice daily for five days thereafter, and daily thereafter for up to 14 days after infection.

To assess colonization inhibition, mice were inoculated with about 1 × 10 ⁷ to 1 × 10 ⁸ Δpep27 three times every two weeks, and 1 to 2 weeks after the final inoculation, the mice were given about 5 × 10 ⁶ to 1 × 10 ^Seven pneumococci were infected. Mice were sacrificed at the indicated times, aseptically removed, and then 1 ml of PBS (except blood) using a homogenizer (PRO Scientific Inc., Oxford, CT, USA, Model 200 Double insulated). Pneumococci were screened by homogenization at maximum speed on ice and serially diluted with sterile PBS and inoculated in blood agar medium containing 5-10 μg / ml gentamycin. Subsequently, the plate was incubated for about 18 hours at 37 ° C. with 95% air-5% CO ₂ to count colonies twice, and the average value was used.

By Δpep27 vaccine to investigate whether you can defend against Staphylococcus aureus and Klebsiella pneumoniae (K. pneumoniae) infection, throat three times Δpep27 inoculated into mice 10 days after inoculation and final Δpep27, a Staphylococcus aureus or Klebsiella pneumoniae, respectively. 1 x 10 ⁸ or 2 x 10 ⁶ were suspended in 50 μl PBS to infect the throat. Lung and nasal washes were then collected, homogenized and serially diluted 24 hours and 48 hours after infection, plated on BHI blood agar medium and incubated overnight at 37 ° C. to count bacteria.

Virus and Pneumococcal Infection Experiments

Mice (BALB / c females, 6-8 weeks old, Koatech, Korea) were inoculated three times at 1 week intervals by suspending about 1 × 10 ⁸ Δpep27 in 50 μl PBS and 10 days after the last inoculation, in 50 μl PBS H1N1 influenza virus was prepared so as to be 0.02 lethal dose (LD) and infected by a throat to monitor body weight daily. After 10-12 days after influenza infection, 1 × 10 ⁸ D39s were suspended in 50 μl PBS to infect the mouse throat and then survival was measured.

Spleen Cell Isolation

Mice were inoculated three times every two weeks with 1 × 10 ⁷ to 1 × 10 ⁸ of Δpep27 (THpep27 mutant strain). One week after the last inoculation, the spleens were taken out and treated with anti-CD3e (5 μl / ml; eBioscience) and anti-CD28 (3 μl / ml; eBioscience) antibodies to stimulate T lymphocytes (Bashour et al., 2014). ). Harvest after 24 hours incubation, the cytokine level of the culture medium was measured.

Cytokine measurement

The concentrations of bronchoalveolar lavage (BAL), interleukin (IL) -17, tumor necrosis factor (TNF) -α, interferon (IFN) -IL, IL-4 and IL-10 in bronchoalveolar lavage (BAL), serum and spleen cells Measurements were made as directed by the manufacturer using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, San Diego, Calif., USA).

IgG antibody titer and Ig subtype determination

Mice were inoculated 1 × 10 ⁷ to 1 × 10 ⁸ Δpep27 three times every two weeks. Seven days after the final inoculation, serum was collected by orbital blood collection and stored at -80 ° C until ELISA. Antibodies were titrated as previously described (Roche et al., 2007; Kim et al., 2012; Cohen et al. 2013) and Ig subtypes were measured using the mouse Ig isotyping ELISA kit (eBioscience, USA). .

IgG titers in serum in the co-infection study were determined in pneumococcal lysates (D39, A66.1, BG7322), or purified PspA protein in 96 well immune plates or PBS solutions coated with serotype 2 capsules (1 μg / ml) and measured by ELISA method (Kim et al., 2012; Cohen et al., 2013).

Virus and fungal counts in the lungs

Mice (BALB / c 4 mice / group) were throated with Δpep27, infected with H1N1 or H3N2 influenza virus as described above, and then lung samples were collected and analyzed as previously reported (Shim et al, 2013).

Five days after influenza virus infection, the mouse lungs were passed through a 70 μm strainer and centrifuged to store the supernatant at −80 ° C. until titer measurement. MDCK cells (2 × 10 ⁴ cells / well) were seeded in 96 well plates with MEM (BSA without 1% IgG, 1 × penicillin-streptomycin) medium for viral titer determinations, and then the plates at 37 ° C. Incubated for 4 hours. The cultured cells were then infected with the lung homogenate supernatant serially diluted 2-fold and incubated overnight. The supernatant was then discarded and measured using TCID ₅₀ / ml using anti-influenza A antibodies (Wu et al, 2015).

Real-time PCR

After celiac macrophages were isolated and cultured, total RNA was extracted using RNAiso plus (TAKARA, Japan). RT-PCR was performed using a one-step RT qPCR kit (Enzynomics, Korea). Gene specific primer sequences are as follows: IL-10 gene (Forward [F]: 5'- AGC CAC CTC ATG CTA GAG C (SEQ ID NO: 10), Reverse [R]: 5'- GCC TGG TCT GGC ATC ACT AC) (SEQ ID NO: 11)); Gene of IL-1β (F: 5'- CTG GTG TGT GAC GTT CCC AT (SEQ ID NO: 12); R: 5'- TGT CGT TGC TTG GTT CTC CT (SEQ ID NO: 13)); TNF-a gene (F: 5'-CAC AAG ATG CTG GGA CAG TGA (SEQ ID NO: 14); R: 5'- TCC TTG ATG GTG GTG CAT GA (SEQ ID NO: 15)). The GAPDH gene (primer F: 5'- TGC ATC CTG CAC CAC CAA (SEQ ID NO: 16); R: 5'- TCC ACG ATG CCA AAG TTG TC (SEQ ID NO: 17)) was used as a control.

PCR program was performed as follows: Holding (Holding; 95 ° C. 10 min); Every cycle for 40 cycles (95 ° C. 15 sec, 55 ° C. 30 sec, 72 ° C. 30 sec); Melt curve (95 ° C. 15 sec, 60 ° C. 1 min, 95 ° C. 15 sec).

High Throughput Sequencing

Do not anesthetize 1 x 10 ⁷ -1 x 10 ⁸ pneumococcal Δpep27 (THpep27: Choi et al., 2013) in mice (Balb / c 4 weeks old) to measure gene expression induced in lung and spleen after Δpep27 inoculation. And 3 times inoculated throat 2 weeks apart ( i. N. ). RNA was extracted using Trizol reagent (Invitrogen) in lungs and spleen and a sequencing library was constructed using 500 ng of total RNA. RNA libraries were constructed following standard protocols using the LEXOGEN Quant-Seq Library Prep Kit (Cat # 001.24) for sequencing. Gene expression was measured by high-throughput sequencing using Illumina NextSeq 500.

DNA processing steps

Illumina Casava1.8 software was used for the base call. Sequence reads were trimmed for adapter sequences, and were no longer processed for low complexity or low quality sequences using fastx_trimmer and then mapped to the mm10 whole genome using Bowtie2. Read count extraction and normalization was performed using edgeR. Experiments and system biology analysis using Ingenuity Pathway Analysis were performed in e-biogen (Seoul, Korea).

Gene expression analysis data was deposited in NCBI [GEO accession number GSE93718] (http://www.ncbi.nlm.nih.gov/geo/).

Determination of Defense from Intestinal Inflammatory Disease

Mice (C57BL / 6 males, 4 weeks old) were anesthetized by intraperitoneal injection of 100 μl of ketamine and inoculated 1 × 10 ⁷ to 1 × 10 ⁸ Δpep27 three times a week at intervals of one week. Mice were weighed and randomly divided into 4 groups using 2 animals per group. The mouse experimental groups used in each series of experiments were as follows: a control group not treated with dextran sulfate sodium (DSS), an experimental control group administered only 5% DSS, an experimental group administered only with Pep27, and Pep27 + 5% DSS. Administered group. Mice were inoculated with colitis by drinking DSS (5%, w / v) with an average molecular weight of 5000 (Sigma Chemical Co., St. Louis, Missouri, USA) for 14 consecutive days (Wirtz et al, 2007). DSS solution was prepared fresh daily. Control mice were allowed to drink only tap water.

All controls and experimental controls received the same amount of vehicle (saline) administered in the same manner as the DSS group during the study period.

Inhibition of DSS-induced Enteritis in Mice by ΔPep27 Vaccination>

Intestinal Disease (IBD) Sampling

After 14 days of 5% DSS administration, the experimental animals were sacrificed by CO ₂ asphyxiation and postmortem laparotomy. The entire colon from the cecum to the anus was removed, divided into proximal, middle and distal portions, and the selected tissues were separated, washed with phosphate buffered saline (PBS) and stored at -80 ° C until analysis.

Quantitative Histology of Colon Sections (Disease activity index: DAI)

The progression of colitis was assessed daily by measuring volume and weight loss, coherence of stool, rectal bleeding, and the presence of total blood in the stool. The morbidity (boredom, lethargy) of the mice was also monitored daily.

This parameter was scored according to the criteria presented below, which calculated the average daily disease activity index (DAI) for each animal as presented in a previously reported paper (Wirtz et al, 2007; Jawhara and Poulain, 2007). .

In addition, colorectal inflammation was assessed by visually measuring the length of the colon from the original unpulled rotatable junction to the anal margin. Pathological findings were recorded in detail in each group, and a system of visually scoring tissue from 0 to 5 was used to assess when and to what extent changes occurred in the most inflamed organs.

A previously validated pathological scoring system (Jawhara and Poulain, 2007; Xu et al, 2007) was used to assess the degree of colitis. All experiments were repeated at least twice, yielding the following in all groups:

1) weight loss, no change, 0; <5%, 1; 6-10%, 2; 11-20%, 3; > 20%, 4;

2) Stool consistency, normal or well formed pellets = 0; Dough-shaped bandages that do not stick to the anus = 1; Liquid stools 2 that remain attached to the anus; Slight bloodiness, 3; Complete liquid, bleeding or not excreted after 10 minutes; 4;

3) rectal bleeding, no blood cells, 0; Blood in the anus, 1; Blood on the fur, 2; Severe bleeding at work, 4 points.

4) general appearance, normal, 0; Mucus 1; Helpless and piloerect, 2; Helpless and bent. 3; Motionless, sick, 4.

Statistical analysis

All data are expressed as mean ± standard deviation of two independent experiments. Statistical comparisons were performed using Bonferroni's test using one-way ANOVA. P values less than 0.05 were considered statistically significant.

2. Results

2-1. Case # 1: With HTS  System biology analysis suggests protection from major lesions

2-1-1. Δpep27 inoculation protected mice from various lesions

System biology analysis in the lungs suggests that Δpep27 throat inoculation defends against abnormal changes in gastroenteritis and colon (FIG. 1A). In addition, system biology analysis in the spleen showed protection from influenza virus infection due to Δpep27 inoculation (FIG. 1B).

2-1-2. Δpep27 inoculation induced Treg cells

System biology analysis in the lung also shows that TGF-β is induced in the lung due to the inoculation of Δpep27 (FIG. 2). In Figure 2, green indicates that gene expression is suppressed and red means that the gene is induced. To confirm that Treg was induced by Δpep27 inoculation, splenocytes of inoculated mice were FACS analyzed. As a result, the Δpep27 inoculation increased the Th2, Th17 and Treg cell population but did not increase the Th1 cells (FIG. 3A), suggesting that these induced Treg cells may play a role in immune tolerance. Cytokine levels in splenocytes and serum were measured to support the induction of Tregs. Consistently, interferon (IFN) -γ, IL-4, IL-17 and IL-10 were significantly induced in the spleen (FIG. 3B). In addition, IFN-γ, IL-17 and IL-10 were significantly induced in serum, but IL-4 levels were not significantly changed (FIG. 3C). These results suggest that induction of Treg cells induces the production of the anti-inflammatory cytokine IL-10, thereby enhancing immune tolerance.

2-1-3. IL-10 Induction by Δpep27 Inoculation

IL-10 and IL-17 levels were measured in lung and bronchial alveolar lavage fluid (BAL) to support an immune tolerant response. Consistently, Δpep27 inoculation significantly increased both IL-10 and IL-17 levels and IL-17 levels of BAL in the lung (FIG. 4A), but did not significantly increase IL-10 levels in BAL (FIG. 4B).

2-1-4. Induction of memory response by Δpep27 inoculation

To confirm that throat inoculation can cause a memory response, splenocytes from the inoculated mice were harvested and the memory response measured. As a result, the Δpep27 inoculation increased the central memory T cells (CD4, CCR7, CD62L) and memory Tfh cells (CD4, CXCR5, CCR7) in the spleen (FIG. 5).

Antibody titers of various serum immunoglobulin subtypes were measured to reconfirm the memory response. As expected, Δpep27 vaccination induced various immunoglobulin subtypes such as IgG1, IgG2a, IgG2b and IgG3 (FIG. 6A). In addition, the secretory IgA levels of bronchial alveolar lavage (Bronchoalveolar Lavage, BAL) were also significantly increased by vaccination (FIG. 6B). Therefore, it was confirmed that the inoculation of the throat vaccine induces a memory response.

2-2. Case 2: Concurrent Infection Against Influenza Viruses

Streptococcus pneumoniae and influenza A virus (IAV) are the main causative agents of respiratory infections (Bosch et al, 2013, Shak et al, 2013). Pneumococcal or IAV itself causes respiratory disease, but secondary infections after influenza virus infection increase mortality (Mina and Klugman, 2013). However, the currently available pneumococcal polysaccharide conjugate vaccine PCV 13 did not effectively protect against secondary pneumococcal infection (Metzger et al, 2015).

2-2-1. Defending Secondary Pneumococcal Infection by Δpep27 Inoculation

Previous studies have shown that throat inoculation of mice with Δpep27 induces IgG and protects against heterologous pneumococcal infections (Kim et al, 2012). We have identified whether Δpep27 inoculation can increase antibody titers to specific antigens as well as whole bacterial cells. Experimental results showed that Δpep27 throat vaccination increased IgG titers against serotype 2 (D39) prefectures as well as serotypes 3 and 6B (FIG. 7A), resulting in significantly higher humoral immunity than both non-immunized and homologous pneumococcal controls. It was shown. When IgG titers for specific antigens, PspA proteins and serotype 2 polysaccharide capillaries were measured, Δpep27 inoculation increased PspA titers but did not increase titers for polysaccharide capillaries (FIG. 7B).

Mice were infected with H1N1 influenza virus at a 0.02 lethal dose (LD) to investigate whether Δpep27 sore throat protects against secondary pneumococcal infection. Ten days after influenza virus infection, toxic pneumococcal D39 strains were infected with mice and survival rates were measured. Unvaccinated mice (PBS / H1N1) after D39 infection survived pneumonia, while most of the mice vaccinated (THpep27 / H1N1) survived successfully (FIG. 7c), and these results showed that Δpep27 was vaccinated in mice. Suggests that not only can it be protected from influenza virus infection but also from secondary pneumococcal infection.

In addition, as a result of measuring the number of bacteria after pneumococcal infection after infection, a much smaller number of bacteria were detected in all vaccinated mouse lungs than in the non-inoculated control group (FIG. 7D). It has also been successfully defended against secondary pneumococcal infection after influenza infection.

2-2-2. Reduction of the number of viruses and bacteria in the lung due to Δpep27 inoculation

To investigate whether the influenza virus number was reduced by Δpep27 inoculation, it was measured whether the body weight was reduced after the influenza virus infection. Interestingly, all mice vaccinated with Δpep27 vaccine did not lose weight after influenza infection, but mice without vaccination significantly reduced body weight (FIG. 8A). Since Δpep27 vaccination inhibits weight loss due to influenza virus infection, the number of viruses in the lungs of influenza infected mice has been changed to TCID ₅₀ to further determine how influenza virus replication is affected in the lungs by Δpep27 vaccination. Measured. Surprisingly, vaccinated mice showed significantly lower virus counts than unvaccinated controls (FIG. 8B). This result indicates that influenza virus infection as well as pneumococcal infection are significantly reduced by Δpep27 throat vaccine inoculation.

2-3. Case 3: Other Bacterial Infections Against Δpep27 Inoculation

2-3-1. Gram-negative bacterial infection protection by Δpep27 inoculation

To determine whether Δpep27 vaccination could prevent the settlement of other bacterial infections, mice were vaccinated with Δpep27 vaccine and then infected with Gram-negative bacillus, Klebsiella pneumoniae , to determine the number of bacteria in the tissues. The vaccinated group was found to be able to prevent Gram-negative bacterial infection by Δpep27 vaccination since the number of pneumococcal in the lung and blood was significantly reduced compared to the non-vaccinated control group 24 hours after infection.

2-3-2. Gram-positive bacterial infection defense by Δpep27 inoculation

To reaffirm that the Δpep27 vaccination could prevent the settlement of other bacterial infections, mice were vaccinated with the Δpep27 vaccine and then infected with Gram-positive Staphylococcus aureus to determine the number of viable bacteria in the tissue. Interestingly, the Δpep27 vaccination group significantly reduced bacterial colony counts in the lung and nasal lavage of mice compared to the non-vaccinated controls (FIG. 10). These results demonstrate that Δpep27 vaccination can prevent other bacterial infections, such as Gram-positive bacteria.

2-4. Case 4: Intestinal Inflammatory Disease Defense

Oral immunotolerance has been studied in humans for rheumatoid arthritis (RA), allergic diseases, diabetes, arteriosclerosis, colitis, etc. (Faria and Weiner, 2005). Inoculation of Salmonella-derived secretion protein (SseB) induced intestinal and systemic IgA, Th1 and Th17 responses, and the oral lethal infection reduced the number of bacteria in the intestinal tissue and spleen (Pigny et al., 2016). However, no throat vaccine has been reported that protects against intestinal inflammatory diseases.

2-4-1. Δpep27  Intestinal Inflammatory Disease Defense by Vaccination

To determine if Δpep27 vaccination could inhibit intestinal inflammatory disease, mice were vaccinated and colitis was induced by DSS. As a result, 9 days after adding 5% DSS to drinking water, the disease index worsened and body weight was significantly decreased. Body weight was significantly reduced in mice treated with 5% DSS alone, but the group treated with Δpep27 and treated with 5% DSS lost significantly less weight than the DSS treated group (FIG. 11A).

When comparing the overall clinical disease activity index scores (p <0.05, Bonferroni test after one-way ANOVA, 6-9 days for DSS treatment), the 5% DSS-only group had a significantly greater stool consistency score than the Δpep27 + 5% DSS group. High (FIG. 11B). In other words, the DSS-induced colitis was worsened, and the DSS-treated group showed a higher clinical activity index, but the vaccine-treated group showed lower colitis than the normal group.

Shortening the colon length is used as an inflammatory marker in the DSS-induced colitis model. In fact, the length of the colon in the DSS-administered group was significantly shortened (FIG. 12A). Thus, the ratio of colon weight / colon length was significantly increased in the DSS group, whereas the Δpep27 vaccination group showed a near-normal ratio compared to the DSS group (FIG. 12B).

2-4-2. Inhibition of inflammatory cytokine levels by Δpep27 vaccination

To confirm that Δpep27 vaccination reduced intestinal inflammation, cytokine mRNA levels in the large intestine were measured. Experimental results showed that Δpep27 vaccination group significantly reduced basal IL-1β mRNA levels compared to non-immunized control group (FIG. 13). In addition, the mRNA levels of IL-17A, TNF-α and IL-6 were also inhibited by non-immunized controls by throat vaccination (FIG. 13), demonstrating that throat mucosal vaccination inhibits cytokine expression.

The above description of the present application is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present application. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the above description, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present application.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> PHARMACEUTICAL COMPOSITION CONTAINING ATTENUATED STREPTOCOCCUS          PNEUMOCOCCUS AND USES THEREOF <130> 1 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> pep27 of Streptococcus pneumoniae D39 <400> 1 atgagaaagg aatttcacaa cgttttatct agtgatcagt tacttacaga caaaaggcca 60 gcaagagact ataatagaaa atag 84 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 2 tggcttaccg ttcgtatag 19 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 3 tcgataccgt tcgtataatg t 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 3 <400> 4 tctctatcgg cctcaagcag 20 <210> 5 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer 4 <400> 5 ctatacgaac ggtaagccag attttcacca ctgctttcg 39 <210> 6 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer 5 <400> 6 acattatacg aacggtatcg aaaggccagc aagagacta 39 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 6 <400> 7 ctgcgaggct tgcactgtag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 7 <400> 8 tctctatcgg cctcaagcag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 8 <400> 9 ctgcgaggct tgcactgtag 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> IL223 primer F <400> 10 agccacctca tgctagagc 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-10 primer R <400> 11 gcctggtctg gcatcactac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1b primer F <400> 12 ctggtgtgtg acgttcccat 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1b primer R <400> 13 tgtcgttgct tggttctcct 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TNF-a primer F <400> 14 cacaagatgc tgggacagtg a 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-a primer R <400> 15 tccttgatgg tggtgcatga 20 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer F <400> 16 tgcatcctgc accaccaa 18 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer R <400> 17 tccacgatgc caaagttgtc 20

Claims (12)

For the prevention or treatment of inflammatory diseases, respiratory viral infections, or bacterial infectious diseases other than pneumococci, including attenuated pneumococcal strains deleted from 1 to 53 of the pep27 gene nucleic acid sequence represented by SEQ ID NO: 1 Pharmaceutical compositions.
delete delete The method of claim 1,
The inflammatory disease is selected from asthma, bronchitis, rhinitis, inflammatory bowel disease, gastroenteritis, colitis, Crohn's disease, pancreatitis, atherosclerosis and arthritis.
The method of claim 1,
The respiratory virus is selected from metapneumovirus, coronavirus, enterovirus, respiratory syncytial virus, adenovirus, bocavirus, rhinovirus and influenza virus.
The method of claim 1,
The bacterial infectious disease is selected from Gram-positive bacterial infectious diseases and Gram-negative bacterial infectious diseases.
The method of claim 6,
The Gram-positive bacteria are selected from Staphylococcus, Streptococcus, Tetanus and Anthrax, and The Gram-negative bacteria are selected from Salmonella, Heterogene, Pneumococcal, Escherichia coli and Cholera.
The method of claim 1,
A serotype-independent immunization.
The method of claim 1,
A pharmaceutical composition that is noninvasive to lungs, spleen, blood or brain.
The method of claim 1,
Pharmaceutical compositions for administration intraperitoneally or intramucosally.
The method of claim 10,
A pharmaceutical composition for administration into the throat mucosa.
The method of claim 1,
A pharmaceutical composition further comprising a pharmaceutically acceptable carrier or immune adjuvant.

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