RU2772131C2 - PHARMACEUTICAL COMPOSITION CONTAINING ATTENUATED Streptococcus pneumoniae STRAINS, AND ITS APPLICATION - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to the use of a pharmaceutical composition containing attenuated Streptococcus pneumoniae strain for the prevention or treatment of an inflammatory disease selected from asthma, pneumonia, sepsis, inflammatory bowel diseases, gastroenteritis, and colitis. At the same time, attenuated Streptococcus pneumoniae strain contains a mutant gene pep27, in which nucleotide residues are removed in positions 1-53 of a nucleotide sequence pep27 described in SEQ ID NO: 1.

EFFECT: specified pharmaceutical composition induces the immunization expression related to genes in the lung and spleen, thereby providing the prevention and/or treatment of inflammation-related diseases.

8 cl, 28 dwg, 1 tbl, 2 ex

Description

FIELD OF TECHNOLOGY

The invention relates to a pharmaceutical composition containing an attenuated strain of Streptococcus pneumoniae and its use for the prevention or treatment of inflammatory diseases, respiratory viral infections or bacterial infectious diseases.

In addition, the invention relates to a pharmaceutical composition containing a weakened strain of Streptococcus pneumoniae, and its use for the prevention or treatment of allergic diseases.

BACKGROUND OF THE INVENTION

According to published data, intranasal or oral administration of antigens induces regulatory T cells (Treg), which in turn induce mucosal tolerance in target organs (Faria and Weiner, 2005). It has also been reported (Faria and Weiner, 2005) that induction of mucosal tolerance suppresses various autoimmune diseases, including atherosclerosis in mice (Maron et al. , 2002). Accordingly, the principle of mucosal tolerance can be applied to animals and humans. Multiple mucosal stimulation by antigen challenge induces T cells to secrete anti-inflammatory cytokines such as IL-10 or TGF-β1 to protect tissues and induce mucosal tolerance (Faria and Weiner, 2005; Weiner, 2001). To maintain immune tolerance at the mucosal surface, a certain type of Treg cell is preferably induced, but the mechanism of their induction remains unclear.

Meanwhile, the currently used mucosal vaccines are too ineffective to elicit a sufficient immune response without adjuvants. Mucosal adjuvants are used for antigens which, when administered alone, induce low immune tolerance. An example of a mucosal adjuvant is the cholera toxin B subunit (CTB) (Faria and Weiner, 2005). However, the immune-enhancing effect of cholera toxin after intranasal inoculation implies recognition and mediation of bacteria (Kim et al., 2016) and therefore an important role for microorganisms in potentiation, suggesting that attenuated bacteria can be used as an adjuvant if necessary. In other words, the concept of using mucosal vaccines without adjuvants has not yet been proven.

In addition, it has not been reported anywhere in previous documents whether mucosal vaccines can affect various diseases other than those directly related to the antigens used in the vaccines. Current immunotherapy only protects against antigens used in vaccination. In particular, 23-valent polysaccharide vaccines or 13-valent conjugate vaccines are used to prevent pneumococcal infection. However, the 23-valent polysaccharide vaccine cannot induce the production of immunological memory cells, and the 13-valent conjugate vaccine can only protect against 13 out of 90 or more serotypes (Kalin, 1998).

Asthma is one of the most common chronic diseases in children and adults in the world (Anandan et al., 2010). Approximately 300 million people worldwide suffer from asthma, resulting in significant medical costs (WHO, 2007). According to forecasts, the number of asthma patients in the world will increase every year, and by 2025 the number of cases will increase by another 100 million people. According to statistics from the US Centers for Disease Control and Prevention, in 1980 the number of patients with asthma was 3.1% of the US population, and in 2010 it increased to 8.4%, while the upward trend continues (https://www. .cdc.gov/asthma).

In addition, 70% of patients with asthma also suffer from allergies [GINA guidelines, 2016]. Asthma was diagnosed in 1.66 million people in South Korea in 2015 (2015 Health Insurance Review and Evaluation Service statistics), making it the sixth most common disease among the top ten chronic diseases found in the South Korean population.

The occurrence of asthma and other allergic diseases is closely linked to environmental changes driven by Western lifestyles and urbanization (GINA Toolkit, 2016). Asthma is a heterogeneous disease with a variety of causes, usually caused by inflammation. Patients with asthma experience similar symptoms, but they are based on different mechanisms (GINA guidelines, 2016; National Asthma Education and Prevention Program, 2007). In addition, the types of airway inflammation differ from each other, depending on the types of asthma (GINA guidelines, 2016). Immunoglobulin E (IgE) mediated eosinophilic inflammation of the airways is a typical mechanism in the pathogenesis of asthma, and numerous pathology and asthma studies have focused on Th2-associated acquired immune responses (GINA guidelines, 2016). However, recent research has focused on innate immunity, microbiomes, microbes present in the body, and the like. (Bjorksten et al ., 2001; Kalliomaki et al ., 2001; Penders et al ., 2007; Hilty et al ., 2010; Green et al ., 2014; and Ozturk et al ., 2017).

According to the Asthma and Allergy Foundation of America (http://www.aafa.org/), currently available medications used to provide medical care for asthma can only reduce symptoms, but cannot completely cure asthma. Asthma is characterized by airway hyperresponsiveness (AHR) causing episodes of excessive bronchial constriction. Although regular administration of glucocorticosteroids by inhalation has significantly reduced mortality in recent years, asthma still causes approximately 250,000 deaths per year worldwide and is characterized by a high incidence (Suissa et al., 2000). Depending on the frequency of symptoms, asthma is divided into mild (1-2 times a month), moderate (1-2 times a week) and severe (asthma symptoms appear daily). In severe symptoms, daily administration of 3-4 drugs is required. Even mild symptoms need regular treatment. Because asthma is an inflammation of the airways that causes constriction of the bronchi, asthma patients who are short of breath should immediately spray a bronchodilator into the throat with an inhaler. In addition, some of the asthma patients and patients with severe asthma do not respond at all to corticosteroid hormones, so they may suffer from the disease throughout their lives or die without experiencing any clinical effects (Durham et al., 2011) . Newly marketed anti-IL-5 agents can alleviate asthmatic symptoms when administered once every four weeks. However, drugs or treatments that completely cure or prevent asthma have not yet been developed or described.

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL PROBLEM

Preventive and therapeutic methods have been developed for the treatment of inflammatory diseases due to inflammatory factors such as intestinal diseases, infections, atherosclerosis and epithelial lesions, but they are limited to the use of certain markers associated with inflammation or disease. In this regard, since mucosal immunity is believed to prevent mucosal and inflammatory diseases, the present invention provides a new anti-inflammatory and protective therapy against various diseases, which has the advantage of developing mucosal immunity under the action of pneumococcal whole cell vaccines. In addition, the present invention proved that the vaccine, which is injected into the mucosa to optimize the immune tolerance of mucosal immunity, can be used as a new therapeutic agent for the prevention or treatment of intestinal inflammation and infectious diseases.

In addition, the present invention provides a new method for protecting against a wide range of inflammatory diseases, including viral and bacterial infections of the respiratory tract, as well as disorders caused by inflammatory bowel disease and infection and inflammation of the respiratory tract, by inoculating a whole cell pneumococcal vaccine on the mucosa.

In addition, currently available asthma medications are divided into those that relieve symptoms by widening narrowed airways (bronchodilators) and those that control the disease by suppressing airway inflammation, thus preventing asthma attacks (anti-inflammatory drugs). However, medications cannot completely cure or prevent asthma. Thus, the present invention provides, as an agent for the prevention or treatment of allergic diseases including asthma, a pharmaceutical composition containing Streptococcus pneumoniae, and in particular a pharmaceutical composition containing a strain of Streptococcus pneumoniae that is attenuated enough to ensure safety even when administered intranasally. , intraperitoneal injection and intravenous injection, which overcomes the disadvantage that the use of Streptococcus pneumoniae in pharmaceutical compositions such as vaccines requires inactivation of Streptococcus pneumoniae, or separation and purification of individual components of Streptococcus pneumoniae due to the high toxicity of bacteria.

However, the objects to be achieved in the present invention are not limited to the foregoing, and other objects not mentioned may become apparent to those skilled in the art from the following description.

TECHNICAL SOLUTION

The present invention provides a pharmaceutical composition for the prevention or treatment of an inflammatory disease, a viral respiratory infection, or a bacterial infection other than Streptococcus pneumoniae, which contains an attenuated strain of Streptococcus pneumoniae.

In addition, the present invention provides the use of a strain of Streptococcus pneumoniae attenuated by a pep27 gene mutation for the prevention or treatment of various allergic diseases, including an allergic respiratory disease, and a pharmaceutical composition used therefor.

The solutions set forth above are illustrative only and should not be construed as limiting the present invention. In addition to the above illustrative embodiments, there may be other embodiments and examples, which are explained using the drawings and in the description.

BENEFICIAL EFFECTS

According to the present invention, mucosal inoculation of a pneumococcal whole cell vaccine induces the expression of immunization associated genes in the lungs and spleen, thereby providing for the prevention and/or treatment of inflammation-related diseases.

In particular, although conventional vaccines may only provide protection against specific antigenic ingredients, the use of the pneumococcal mucosal vaccine of the present invention is expected to provide a wide range of prophylactic and therapeutic effects, including protective mechanisms against inflammatory diseases of other organs, as well as viral and bacterial infections. diseases. In addition, when administered to the mucosa, the pneumococcal whole cell vaccine of the present invention can provide protective effects against various diseases even in the absence of any adjuvant, unlike conventional mucosal vaccines.

The present invention provides a method for the prevention or treatment of allergic diseases, including asthma, using a Streptococcus pneumoniae pep27 mutant. In particular, vaccination with the Streptococcus pneumoniae pep27 mutant may provide prevention or treatment of allergic respiratory diseases including asthma, allergic rhinitis, sinusitis and chronic obstructive pulmonary disease, as well as other allergic diseases such as urticaria, conjunctivitis, pollen allergy and atopy.

In addition, the mutant of Streptococcus pneumoniae pep27 according to the present invention has the advantage of ensuring safety even in intranasal administration, intraperitoneal injection and intravenous injection, since the mutant is sufficiently attenuated.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1 and 2 are schematic diagrams showing results of a Streptococcus pneumoniae vaccine performance analysis in the lungs (FIG. 1) and spleen (FIG. 2) obtained by systems biology analysis in accordance with an embodiment of the present invention. Mice are immunized with the THpep27 (Δpep27) mutant every two weeks for a total of three times. Two weeks after the last immunization, the lungs and spleens are isolated and total RNA is extracted from them. Gene expression is determined by high throughput sequencing and then analyzed using Ingenuity Pathway Analysis. In FIG. 1 shows that immunized lung tissue genes suppress gastroenteritis and colon anomaly, and FIG. 2 shows the protective effect of the spleen gene against influenza virus infection.

Figure 3 is a schematic diagram of the result of a systems biology analysis in the lung according to an embodiment of the present invention. Mice are intranasally inoculated with the Δpep27 mutant strain every two weeks for a total of three times. On the 7th day after the last immunization, mRNA was isolated from the lung and subjected to high throughput sequencing. Shown are the results of a network IPA analysis of lung sequence data indicating induction of IL-6, IL-23R, MUC2, IFN type 1, and TGF beta, but downregulation of CCR3.

Figures 4-6 show the results of an experiment to determine whether Δpep27 immunization can induce Treg cells in mice according to an embodiment of the present invention. Fig. 4: mice (n=3) are intranasally immunized with Δpep27 every two weeks for a total of three times and on the 7th day after the last immunization, spleen cells are isolated, labeled with fluorescent cell markers against Th1 (CD4, Tbet), Th2 (CD4, GATA3) , Th17 (CD4, RORγt) and Treg (CD4, Foxp3) and then detected by flow cytometry. Figures 5 and 6 show the levels of cytokines in splenocytes (figure 5) and serum (figure 6) obtained on the 7th day after the last immunization, measured by ELISA. Statistical significance is determined by ANOVA; *P<0.05, **P<0.01.

Figures 7 and 8 show the results of an experiment in which mice are immunized with Δpep27 to induce IL-10 and IL-17 according to an embodiment of the present invention. Mice (n=3) are immunized by intranasal inoculation with Δpep27 every two weeks for a total of three times. On the 7th day after the last immunization, mRNA was isolated from the lung and used to measure the expression levels of the genes IL-17 (Fig.7) and IL-10 (Fig.8) by qPCR. Statistical significance is determined by ANOVA; *P<0.05.

In FIG. 9 shows the results of an experiment in which mice are immunized with Δpep27 to activate central memory T cells and memory Tfh cells in the spleen according to an embodiment of the present invention. Mice (n=3) are immunized intranasally with Δpep27 every two weeks for a total of three times. On day 7 after the last immunization, splenocytes are isolated and labeled with fluorescent cell markers against central memory T cells (CD4, CCR7, CD62L) and memory Tfh cells (CD4, CXCR5, CCR7). The fluorescent markers are then detected by flow cytometry. Statistical significance is determined by ANOVA; *P<0.05.

In FIG. 10 and 11 show the results of an experiment in which mice are immunized with Δpep27 to induce the expression of immunoglobulins of different subtypes in accordance with an embodiment of the present invention. Mice (n=6) are immunized intranasally with Δpep27 every two weeks for a total of three times. On the 7th day after the last immunization in serum (Fig. 10) and bronchoalveolar lavage fluid (BAL) (Fig. 11) determine the antibody titers against three types of whole pneumococcal cells. Statistical significance is determined by ANOVA; * P<0.05, ** P<0.01.

In FIG. 12-15 show the results of an experiment to determine whether Δpep27 immunization can protect against secondary pneumococcal infection following influenza infection in accordance with an embodiment of the present invention. Mice (9-10 per group) are immunized intranasally with Δpep27 weekly for a total of three times. One week after the last immunization, mice are challenged intranasally with influenza virus. Blood samples are taken from the retroorbital sinus and then analyzed for cytokines (FIG. 12), or sera are collected from mice one week after the last immunization and IgG levels are analyzed against certain antigens by ELISA (FIG. 13). Significant differences between the two groups are determined using an unpaired t-test; *** p<0.001. Ten days after influenza infection, mice were infected with Streptococcus pneumoniae D39 by the intranasal route and then survival was recorded for 14 days (FIG. 14). Statistical significance is determined using the Mentel-Cox test; ** p<0.005. Twenty-four hours after challenge with Streptococcus pneumoniae D39, lung homogenates were incubated in serum to enumerate bacteria (FIG. 15). Statistical significance is determined using one-way ANOVA.

In FIG. 16 and 17 show the results of an experiment in which the Δpep27 vaccine is tested for inhibitory activity against influenza virus replication in accordance with an embodiment of the present invention. Mice (10 per group) are immunized intranasally with influenza virus and then their body weights are recorded for 11 days (FIG. 16). Statistical significance is determined using one-way ANOVA; *** p<0.001. Five days after influenza infection, lung homogenate supernatants were collected and virus titers in each group were measured by TCID ₅₀ /ml (FIG. 17). Significance is determined by one-way ANOVA; ***p<0.001.

In FIG. 18 shows the results of an experiment to determine whether Δpep27 immunization can prevent infection by Gram-negative bacteria according to an embodiment of the present invention. Mice (3/group) are immunized intranasally with Δpep27 three times. Ten days after the last immunization, mice are infected with K. pneumoniae. The number of bacteria in individual organs is determined 24 hours after infection with K. pneumoniae by spreading on blood agar plates; **P<0.01.

19 shows the results of an experiment to determine whether Δpep27 immunization can prevent infection by Gram-positive bacteria according to an embodiment of the present invention. Mice (3/group) are immunized intranasally with Δpep27 three times. Ten days after the last immunization, mice are infected with Staphylococcus aureus. The number of bacteria in individual organs is determined 24 hours after infection with S. aureus by spreading on blood agar plates; * P<0.05.

In FIG. Figures 20 and 21 show the results of an experiment to determine whether Δpep27 immunization can reduce weight loss from dextran sulfate sodium (DSS) induced inflammatory bowel disease, in accordance with an embodiment of the present invention. Mice (n=5 per group) are immunized intranasally with the Δpep27 mutant strain three times and then orally administered with 5% DSS in drinking water to induce colitis (inflammatory bowel disease). Immunized mice show a reduction in weight loss (FIG. 20). In this regard, the percentage loss of basal body weight is recorded within 9 days after DSS treatment. Statistical significance is determined by a one-way ANOVA followed by a Bonferroni test. The Clinical Disease Activity Index was assessed daily until completion of therapy on day 9 (FIG. 21). Colitis progression is assessed by measuring weight loss, stool consistency, rectal bleeding, and/or the amount of total blood in the stool. In addition, complications (piloerection and lethargy) are recorded daily in mice.

In FIG. 22 and 23 show the results of an experiment to determine whether Δpep27 immunization can prevent dextran sulfate sodium (DSS) colitis according to an embodiment of the present invention. Mice (n=5 per group) are immunized intranasally with Δpep27 three times and then administered orally with 5% DSS in drinking water to induce colitis (inflammatory bowel disease). Immunized mice show a reduction in weight loss (FIG. 20). The colon is examined on day 9 after DSS treatment. The length (FIG. 22) and weight (FIG. 23) of the colon was measured on day 9 after DSS treatment.

In FIG. 24 shows the results of an experiment to determine whether Δpep27 immunization can suppress the expression of inflammatory cytokine genes in the colon, in accordance with an embodiment of the present invention. Balb c mice are given 5% DSS in drinking water for 14 days to induce inflammatory bowel disease. PBS+water is used as a negative control. The mRNA expression levels of IL-1β, IL-6, IL-17A and TNF-α are measured by quantitative real-time PCR. Experimental data are expressed as mean±SEM. At P<0.05, the result is considered significant.

In FIG. 25 shows a timeline of testing the Δpep27 mutant strain for the ability to prevent allergic disease (A) and the results of testing the Δpep27 mutant strain for the ability to inhibit the secretion of various allergic cytokines (B) according to an embodiment of the present invention.

In FIG. 26 shows histochemical staining results showing the ability of the Δpep27 mutant strain to prevent allergic disease according to an embodiment of the present invention.

In FIG. 27 shows a timeline of testing a Δpep27 mutant strain for a therapeutic effect against an allergic disease (A) and test results showing the ability of the Δpep27 mutant strain to inhibit the secretion of various allergic cytokines (B) according to an embodiment of the present invention.

In FIG. 28 shows the results of histochemical staining showing the therapeutic effect of the Δpep27 mutant strain on an allergic disease according to an embodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Exemplary embodiments of the present invention will be described more fully with reference to the accompanying drawings. However, the invention may be embodied in various other forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that the present description is complete and complete and fully conveys the concept of the invention to those skilled in the art. In the drawings, elements not related to the description will be omitted for the sake of clarity.

Throughout the description, unless expressly stated otherwise, the word "comprise" and its variations such as "comprises" or "comprising" should be understood to mean the inclusion of the specified elements, but not the exclusion of any other elements.

Words denoting degrees such as "about", "substantially", etc., are used in the present description in the sense of "at or almost at, when tolerances are specified in the manufacture, design and use of materials corresponding to the specified circumstances", to prevent misuse by an unfair infringer of the advantages of the description of the invention, where exact or absolute numbers are indicated to facilitate understanding of the invention. The terms "stage something" as used throughout the description do not mean "stage for".

Throughout the description, the term "combination" included in the Markush description means a mixture or combination of one or more of the components, steps, operations and/or elements selected from the group consisting of the components, steps, operations and/or elements described in the Markush group, and thus means that the present invention includes one or more components, steps, operations and/or elements selected from the Markush group.

Throughout the description, the expression "A and/or B" means "A, B or A and B".

Throughout the description, "Streptococcus pneumoniae", also referred to as pneumococcus, is a Gram-positive bacterium of the genus Streptococcus and belongs to the Streptococcus family because bacteria appear to form chains as their cells divide over time along a single axis. This bacterium is known to be the main cause of pneumonia.

As used herein, "Pep27" refers to a 27 amino acid residue peptide secreted by the vex transporter system to cause growth inhibition and apoptosis. In more detail, pep27 expression induces programmed cell death in S. pneumoniae via signaling triggered through the membrane-bound histidine protein kinase vncS and the vncR response regulator, which is a cytoplasmic effector (Novak et al., 1999). The pep27 genes, or the proteins they encode, may be named differently, varying from one Streptococcus pneumoniae serotype to another, and there may also be a slight difference in the nucleotide or peptide sequence of pep27. However, provided that it performs essentially the function described above, any pep27 gene can be mutated and used regardless of serotypes to produce an attenuated strain of Streptococcus pneumoniae of the present invention. In particular, any Streptococcus pneumoniae gene that is functionally identical to the pep27 gene can be used in the present invention. More specifically, the pep27 gene of the present invention may be a mutated gene encoding the pep27 peptide sequence described in SEQ ID NO: 1.

As used herein, the term "attenuation" is intended to refer to the modification of a virulent strain to produce a less virulent strain or weaker pathogen. Such an attenuated strain is a strain that has a significantly lower virulence associated with clinical disease, but is still able to replicate in the host. In particular, the attenuated strain of the present invention has such low virulence or pathogenicity that it can be administered as a vaccine. More specifically, the pneumococcal strains of the present invention are attenuated to such an extent that they cannot cause clinical disease, but can replicate in the host. An attenuated mutant can be obtained by various methods such as point mutation, sequence exchange between related viruses, or nucleotide deletion.

In the present description, the term "mutation" is used to refer to all actions that cause a change in the genetic function of a gene. In more detail, the term "mutation" refers to a quantitative or qualitative change in a gene among different biological variations.

In the present description, the term "allergy" is used in relation to a number of disorders, diseases or abnormal conditions caused by hypersensitivity of the immune system to certain substances, that is, an overreaction of the immune system to foreign substances. Allergic diseases against which the composition of the present invention can be used are diseases resulting in particular from immediate type I hypersensitivity and delayed type IV hypersensitivity. Examples of type I hypersensitivity include bronchial asthma, rhinitis, atopic dermatitis, conjunctivitis, tympanitis, urticaria, and anaphylactic shock. Contact hypersensitivity, contact dermatitis, bacterial allergy, fungal allergy, viral allergy, drug allergy, thyroiditis, and allergic encephalitis are type IV delayed hypersensitivity. Type I immediate hypersensitivity is divided into two stages: in stage 1, allergen exposure disrupts the balance between the Th1 response, which is characterized by the production of IL12 and IFN-γ, which suppress the secretion of IgE and IgG1 and increase the secretion of IgG2a, and the Th2 response, which leads to the production of IL- 4, IL-5 and IL-13 in a Th2 bias so that IL-4 and IL-13 are produced in response to a Th2 dominant immune response, prompting B cells to produce allergen-specific IgE, which in turn binds to surface of mast cells and basophils, thereby preparing them for the development of allergies. Mast cells and basophils coated with IgE are sensitized to the allergen; Stage 2 allergy development is divided into early response and late response. In an early response, mast cells activated by repeated allergen exposure undergo degranulation, during which the cells release histamine, lipid metabolites, cytokines, and so on. from their granules, causing vasodilation, etc. In the late response, neutrophils, eosinophils, macrophages, Th2 cells, basophils, etc. penetrate into the corresponding tissues, causing inflammation, manifested as atopic dermatitis, rhinitis, asthma, and the like.

In the present description, the term "prophylaxis" refers to all actions that suppress or delay the onset of a disease by administering a composition in accordance with the present invention.

As used herein, the term "treatment" or "treat" refers to all actions taken to improve or beneficially alter a symptom associated with a disease by administering a composition in accordance with the present invention.

In the present description, the term "vaccine" refers to a biological preparation containing an antigenic substance that resembles a microorganism or virus that causes a disease, and is capable of providing active acquired immunity against such a disease. Vaccines are often made from weakened or killed bacteria or viruses. The introduction of vaccines is called vaccination and is carried out in order to provide artificially acquired immunogenicity against a specific infection. When stimulated by a vaccine, the individual's immune system is activated and begins to produce antibodies. Sensitization persists, and upon re-infection, antibodies can be efficiently generated within a short time, providing suppression of the disease. Meanwhile, due to the principle of immune tolerance, the vaccine containing attenuated Streptococcus pneumoniae according to the present invention can not only protect against a specific antigenic substance, but also exhibit a preventive and therapeutic effect on a wide range of diseases, including viral and bacterial infectious diseases, unlike conventional vaccines.

The term "ermB" as used herein refers to a gene that confers resistance to macrolides. Macrolides are a class of substitutes for penicillin in the treatment of diseases caused by Streptococcus pneumoniae and include erythromycin, clarithromycin and azithromycin.

The following is a detailed description of a pharmaceutical composition containing an attenuated strain of Streptococcus pneumoniae in accordance with the present invention, and its use for the prevention or treatment of inflammatory diseases, as well as respiratory viral or bacterial infectious diseases, with reference to illustrative embodiments and drawings. However, the present description should not be understood as being limited to the illustrative embodiments and drawings.

In the first aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases, respiratory viral infectious diseases, infectious diseases caused by bacteria other than Streptococcus pneumoniae, and the composition contains a weakened strain of Streptococcus pneumoniae. For example, a pharmaceutical composition may include a vaccine composition.

In an embodiment, the attenuated strain of Streptococcus pneumoniae may be a strain from which all or part of the pep27 gene has been removed. For example, nucleotide residues at positions 1 to 53 of the pep27 nucleotide sequence described in SEQ ID NO: 1 can be deleted without limitation.

According to an embodiment, an attenuated strain of Streptococcus pneumoniae may contain a mutant pep27 gene in which nucleotide residues at positions 1 to 53 of the pep27 nucleotide sequence described in SEQ ID NO: 1 are deleted and replaced with an ermB cassette, however, the present invention is not limited to such a strain. . For example, an attenuated strain of Streptococcus pneumoniae may be an attenuated strain of Streptococcus pneumoniae containing the pep27 mutant gene disclosed in Korean Patent No. 10-1252911.

According to an embodiment of the present invention, inflammatory diseases can be selected from, without limitation, asthma, bronchitis, rhinitis, inflammatory bowel disease, gastroenteritis, colitis, Crohn's disease, pancreatitis, atherosclerosis, and arthritis. For example, the prevention or treatment of inflammatory diseases may result from a reduction in the expression of a gene associated with inflammation, such as a gene associated with inflammation of the colon. Inflammatory diseases can be associated with, for example, intestinal and respiratory infectious diseases, but are not limited to.

Respiratory infectious diseases can be caused by a respiratory virus, which can be selected from, for example, metapneumovirus, coronavirus, enterovirus, respiratory syncytial virus, adenovirus, bocavirus, rhinovirus, and influenza virus. Prevention or treatment of infectious diseases caused by a respiratory virus may be the result of suppression of viral replication.

For example, the influenza virus may be influenza A, B, or C without being limited to specific subtypes.

In addition, the pharmaceutical composition of the present invention can provide a non-virus-specific protective function. In this context, the pharmaceutical composition may provide a protective function against influenza viruses and other viruses, including, without limitation, respiratory syncytial virus and rhinovirus.

According to an embodiment of the present invention, bacterial infectious diseases can be selected from, but not limited to, diseases caused by infection with Gram-positive bacteria, Gram-negative bacteria, and other infectious bacteria. For example, the prevention or treatment of bacterial infectious diseases may result from the suppression of infection by gram-positive bacterial and/or gram-negative bacteria.

According to an embodiment of the present invention, Gram-positive bacteria may be selected from Staphylococcus spp., Streptococcus spp., Clostridium tetani, and Bacillus anthracis, while Gram-negative bacteria may be selected from Salmonella spp. Shigella, Klepsiella pneumoniae, E. coli and Vibrio cholerae, but are not limited to. For example, Gram-positive bacteria may be Staphylococcus aureus.

In accordance with an embodiment of the present invention, the pharmaceutical composition can provide immunization in a serotype-independent manner, but is not limited to this. As used herein, a pharmaceutical composition that provides immunization in a serotype-independent manner is a pharmaceutical composition that induces the production of antibodies independent of antigens, but not the production of antibodies specific for particular antigens.

According to an embodiment of the present invention, the allergic disease may be an allergic respiratory disease selected from, but not limited to, asthma, allergic rhinitis, sinusitis, and chronic obstructive pulmonary disease.

According to an embodiment of the present invention, examples of allergic disease include, without limitation, food allergy, allergic tympanitis, anaphylactic shock, contact hypersensitivity, allergic contact dermatitis, bacterial allergy, fungal allergy, viral allergy, drug allergy, and allergic encephalitis, in addition to urticaria, conjunctivitis, allergy for pollen and atopy.

The pharmaceutical composition of the present invention can suppress the production of the Th1 cytokine and/or the Th2 cytokine, both of which are responsible for immune hypersensitivity. As used herein, the Th1 cytokine can be selected from interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-12 (IL-12), and the Th2 cytokine can be selected from interleukin-4 ( IL-4), interleukin-5 (IL-5) and interleukin-13 (IL-13), without any restrictions.

Levels of Th1 cytokine and/or Th2 cytokine can be measured in body fluids such as bronchoalveolar lavage fluid or serum without any limitation.

According to an embodiment of the present invention, the pharmaceutical composition may be non-invasive to the lung, spleen, blood or brain without any limitation. The term "non-invasive" as used herein means that the pharmaceutical composition does not penetrate into other systemic sites other than organs, tissues and cells into which the pharmaceutical composition is directly administered, or, despite penetration into other systemic sites, is rapidly eliminated from them, in contrast from the term "invasive", which refers to a composition capable of penetrating other systemic sites other than organs, tissues and cells into which the pharmaceutical composition is directly administered, thereby damaging the human body.

The term "administration" as used herein is intended to mean administering the composition of the present invention to an individual in a specific, appropriate manner. For the introduction of the composition of the present invention, you can use any method that ensures the delivery of the composition to the target tissue. For example, administration can be by, without limitation, oral, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, intranasal, intrapulmonary, intrarectal, intravesical, transdermal, and intramucosal routes.

According to an embodiment of the present invention, the pharmaceutical composition can be administered, without any limitation, by intraperitoneal or intramucosal route. The location of the mucous membrane into which the pharmaceutical composition is administered is not specifically limited. A person skilled in the art can correctly select a location for transmucosal administration among body sites.

In accordance with an embodiment of the present invention, the pharmaceutical composition can be configured, without any limitation, for administration through the mucosa of the nasopharynx. Vaccination through the mucous membrane of the nasopharynx can be carried out, for example, without limitation, using an aerosol or drops.

The pharmaceutical composition of the present invention, having effective immunogenicity in the individual even after mucosal administration, overcomes the disadvantage of conventional compositions that must be administered subcutaneously with a syringe, and has an advantage over conventional compositions in terms of administration to children who are afraid of injections with a syringe, but without any restrictions. There are no restrictions on the individual to whom the pharmaceutical composition of the present invention may be administered. An individual may include, without limitation, mammals such as humans, rats, mice, poultry, and so on. In addition, the vaccine composition of the present invention must be administered in a pharmaceutically effective amount. The pharmaceutically effective amount of the composition of the present invention varies depending on the sex, body surface area and age of the patient, type and severity of disease, drug sensitivity, route and frequency of administration, rate of elimination, time of administration, duration of treatment, type of target cells, expression level and other factors well known in the pharmaceutical art that can be readily determined by those skilled in the art.

According to an embodiment of the present invention, the pharmaceutical composition may further comprise, without any limitation, a pharmaceutically acceptable carrier or adjuvant.

Examples of a carrier suitable for use in the pharmaceutical composition of the present invention include, without limitation, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the present invention, you can use, without limitation, any adjuvant commonly used in the art. Examples of the adjuvant include, without limitation, cholera toxin binding subunit, aluminum salts, lipid emulsion (MF-59), synthetic detergent (Tween), microspheres, liposomes, and mucoadhesive polymers. It is also possible to use new forms of adjuvants if they have already been developed.

The pharmaceutical composition of the present invention can be prepared, without limitation, in the form of dosage forms, for example, oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., dosage forms for external use , suppositories or sterile injections, using traditional methods. In more detail, conventional diluents or adjuvants such as fillers, bulking agents, binders, humectants, disintegrants, surfactants, etc. can be used to prepare the composition without any limitation.

For example, solid agents for oral administration of the composition of the present invention may be in the form of tablets, pills, powders, granules, capsules, and the like. These solids are prepared using at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin. In addition, a lubricant such as magnesium stearate, talc, and the like can be added to the simple filler. Liquid agents for oral administration include suspensions, oral solutions, emulsions, syrups, and the like. In addition to the simple conventional diluent such as water or liquid paraffin, liquid agents for oral administration of the composition of the present invention may contain, without limitation, various liquid substances such as humectants, sweeteners, flavors, preservatives, and the like. .

Parenteral dosage forms of the composition of the present invention may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized forms and suppositories. As for non-aqueous solutions and suspensions, they can be prepared using propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyl oleate without any limitation.

METHOD FOR CARRYING OUT THE INVENTION

A better understanding of the present invention can be achieved with the help of the following examples, which are provided for illustration, however, they should not be construed as limiting the present disclosure.

[EXAMPLE 1] Analysis of the inhibitory potential of an attenuated strain of Streptococcus pneumoniae THpep27 against an inflammatory disease, a respiratory viral infectious disease, or an infectious disease caused by bacteria other than Streptococcus pneumoniae

1. Materials and methods

Obtaining an attenuated strain of Streptococcus pneumoniae THpep27

The mutant strain of Streptococcus pneumoniae THpep27 used in the examples of the present invention is the strain described by 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), which is similar to the pep27 mutant strain of Streptococcus pneumoniae disclosed in Korean Patent No. 10-1252911, except that for its selections do not use the erythromycin resistance marker (ermAM).

A Cheshire cassette (GenBank accession number FJ981645) carrying an erythromycin resistance marker (ermAM) that can be used as a temporary selection marker was amplified using primers (5'-TGG CTT ACC GTT CGT ATA G-3' (SEQ ID NO: 2) and 5'-TCG ATA CCG TTC GTA TAA TGT-3' (SEQ ID NO: 3)), which were provided by Dr. Donald Morrison (University of Illinois at Chicago) and ligated by polymerase chain reaction (PCR) to upstream and downstream sequences amplified using primers (5'-TCT CTA TCG GCC 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 AGC AAG AGA CTA-3' (SEQ ID NO: 6) and 5'-CTG CGA GGC TTG CAC TGT AG-3'(SEQ ID NO: 7) from D39 genomic DNA which serves as a template The ligation product is then used to transform D39 to obtain a pep27 mutant.

The Cheshire cassette is cut by adding 1% L-fucose (Sigma, St. Louis, MO, USA). The fucose-treated cultures were spread on THY blood agar plates to obtain single colonies. The presence of Cheshire cassettes 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). The mutant (THpep27) sequence was confirmed by nucleotide sequencing (Cosmo, Seoul, Korea) as well as by immunoblotting using an anti-Pep27 antibody (data not shown).

To confirm the THpep27 mutation at the RNA level, RNA is isolated from bacteria in the early exponential phase using the traditional hot phenol method. After DNA removal with DNase I (Takara, Tokyo, Japan), one microgram of bacterial RNA was reverse transcribed into cDNA using random primers (Takara). Reverse transcription PCR was performed using the recommended primer according to the manufacturer's instructions (Super Bio, American Building Restoration Products Inc., Franklin, WI, USA).

Obtaining other bacterial strains

Streptococcus pneumoniae strains used in the present description are shown in Table 1 below.

[Table 1]

Strain Characteristic Link D39 Encapsulated type, serotype 2 (Avery et al., 1944) A66.1 Serotype 3 (McDaniel et al., 1984) TIGR4 Serotype 4 (Aberge et al., 1995) BG7322 Serotype 6B (Briles et al., 1992) THpep27 D39 △pep27::
Cheshire ermB Em ^r (Choi et al., 2013)

S. pneumoniae strains serotype 2 (D39) wild type, serotype 3 (A66.1), serotype 6B (BG7322) and mutant strain THpep27 (D39 Δpep27) are cultured in a manner commonly used in the laboratory (Kim et al., 2012). Streptococcus pneumoniae is cultured overnight at 37° C. on blood agar plates and then at 37° C. for 3 hours in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY; Difco Laboratories). Each of the cultures of Streptococcus pneumoniae strains is appropriately diluted and administered intranasally (i.e.) in an amount of 10 μl to CD1 mice.

S. aureus (ATCC 25923) and K. pneumoniae (ATCC 9997), which were purchased from the Korean Microorganism Culture Center (KCCM, Seoul), are cultured overnight at 37°C in brain heart extract (BHI) broth containing defibrinated sheep blood, and then transferred to fresh BHI broth, in which the bacteria are cultured at 37°C to OD ₅₅₀ =0.5.

Influenza virus strain A/California/04/2009 (H1N1) is cultured in eggs as described previously (Shim et al., 2013).

In vivo infection study

Four week old male CD1, BALB/c mice (Orient, Korea) were used for infection experiments. The use of animals in experiments is approved by the Animal Ethics Committee of Sungkyunkwan University in accordance with the principles of the Korean Animal Welfare Law.

In a vaccine efficacy study, mice were vaccinated intranasally (i.n.) using 1×10 ⁷ -1×10 ⁸ cfu of the Δpep27 strain every one or two weeks for a total of three times and the survival time was measured. One to two weeks after the last immunization, mice are injected intranasally with 1×10 ⁷ -1×10 ⁸ of the virulent strain D39 or 6B. Survival of infected mice is recorded four times a day for the first five days, twice a day for the next five days, and once a day for up to 14 days after infection.

To assess the ability to inhibit colonization, mice were intranasally inoculated with 1×10 ⁷ -1×10 ⁸ CFU of the Δpep27 strain every two weeks for a total of three times. One to two weeks after the last vaccination, mice were challenged with 5×10 ⁶ -1×10 ⁷ cfu of Streptococcus pneumoniae. After killing the mice at a predetermined time, their larynx was removed under aseptic conditions and homogenized using a homogenizer (PRO Scientific Inc., Oxford, CT, USA, model 200 with double isolation) at maximum speed in 1 ml PBS (in the absence of blood) on ice and make serial dilutions in sterile PBS. The dilutions are distributed on blood agar plates containing 5-10 μg/ml gentamicin to screen for Streptococcus pneumoniae. The plates are then incubated at 37° C. for about 18 hours in an atmosphere containing 95% air and 5% CO ₂ and the resulting colonies are counted. This experiment is carried out twice and the average values of the measurement results are used.

To test whether the Δpep27 vaccine could protect against S. aureus and K. pneumoniae infection, mice were immunized intranasally three times with Δpep27. Ten days after the last Δpep27 immunization, mice are intranasally infected with a suspension of 1×10 ⁸ or 2×10 ⁶ cfu of S. aureus or K. pneumoniae in 50 μl of PBS. Then, 24 and 48 hours after infection, lung and nasal lavages are collected, homogenized and serially diluted to an appropriate degree. Serial dilutions are plated on BHI blood agar plates, cultured overnight at 37° C. and cells are counted.

Virus infection and Streptococcus pneumoniae experiment

Mice (male BALB/c, 6-8 weeks, Koatech, Korea) are immunized with a suspension containing approximately 1×10 ⁸ cfu of Δpep27 in 50 μl of PBS weekly for a total of three times. Ten days after the last immunization, mice are intranasally infected with a suspension of H1N1 influenza virus in 50 μl of PBS at a lethal dose (LD) of 0.02, followed by daily monitoring of body weight. 10-12 days after influenza challenge, mice are intranasally infected with a suspension of 1×10 ⁸ CFU D39 in 50 μl PBS and survival is measured.

Isolation of splenocytes

Mice are intranasally immunized with 1×10 ⁷ -1×10 ⁸ cfu of Δpep27 (THpep27 mutant strain) every two weeks for a total of three times. One week after the last immunization, the spleen is isolated and the resulting splenocytes are treated with anti-CD3e antibodies (5 µl/ml; eBioscience) and anti-CD28 antibodies (3 µl/ml; eBioscience) to stimulate T-lymphocytes (Bashour et al ., 2014). After incubation for 24 hours, the cells are harvested and the levels of cytokines are measured in the culture medium.

Measurement of cytokines

Levels of interleukin (IL)-17, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-4, and IL-10 in bronchoalveolar lavage (BAL), serum, and splenocytes are measured using an enzyme-linked immunosorbent assay kit ( ELISA) (BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions.

IgG antibody titer and Ig subtype determination

Mice are intranasally immunized with 1×10 ⁷ -1×10 ⁸ cfu of Δpep27 every two weeks for a total of three times. Seven days after the last immunization, serum samples are obtained from the retro-orbital sinus and stored at -80°C until ELISA. Antibodies are titrated as previously described (Roche et al., 2007; Kim et al., 2012; Cohen et al., 2013) and Ig subtypes are determined using the Mouse Ig Isotyping ELISA Kit (eBioscience, USA).

In coinfection studies, serum IgG titers are measured by ELISA using 96-well immunoplates coated with Streptococcus pneumoniae lysates (D39, A66.1 and BG7322) or serotype 2 capsules or purified PspA protein (1 µg/mL) in PBS (Kim et al., 2012; Cohen et al., 2013).

Counting viruses and bacteria in the lungs

After intranasal immunization with Δpep27 mice (BALB/c four/groups) are infected with H1N1 or H3N2 influenza virus as described above. Thereafter, lung samples are collected and analyzed as previously reported (Shim et al., 2013).

Five days after infection with the influenza virus, the lungs of mice are passed through a 70 μm sieve, followed by centrifugation. The supernatant thus obtained is stored at -80° C. until titration. To titrate the virus, a suspension of MDCK cells in MEM medium (1% BSA, no IgG, 1× penicillin-streptomycin) was seeded at a density of 2×10 ⁴ cells/well in 96-well plates, which were then incubated at 37°C for 4 hours. The cultured cells are then infected with 2-fold serial dilutions of the lung homogenate supernatant and incubated overnight. Then, after removing the supernatant, the virus titer is determined using an anti-influenza A antibody with TCID ₅₀ /ml (Wu et al., 2015).

real-time PCR

Total RNA is isolated from isolated and cultured peritoneal macrophages using RNAiso plus (TAKARA, Japan). RT-PCR was performed using a one-step RT-qPCR kit (Enzynomics, Korea). Gene-specific primers with the following sequences are used: 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)); IL-1β gene (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)); and the TNF-α 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 is carried out according to the following program: keeping; 95°C, 10 min; 40 cycles 95°C, 15 sec; 55°C, 30 sec, and 72°C, 30 sec; melting curve (95°C 15 sec; 60°C, 1 min; 95°C, 15 sec).

High throughput sequencing

To measure gene expression induced in the lungs and spleen after immunization with Δpep27, mice (Balb/c, 4 weeks old) were intranasally vaccinated with 1×10 ⁷ -1×10 ⁸ cfu of Streptococcus pneumoniae Δpep27 (THpep27: Choi et al., 2013) without anesthesia every two weeks for a total of three times. RNA was isolated from lungs and spleen using the Trizol reagent (Invitrogen) and sequencing libraries were constructed using 500 ng of total RNA. For use in subsequent sequencing, an RNA library was constructed using the LEXOGEN Quant-Seq Library Preparation Kit (Cat. No. 001.24) according to a standard protocol. Gene expression is measured by high throughput sequencing using an Illumina NextSeq 500.

DNA processing step

Recognition of nitrogenous bases is carried out using the Illumina Casava1.8 software. Sequence reads are arranged for adapter sequences, followed by filtering of low complexity, low quality sequence reads using fastx_trimmer. The resulting reads are mapped onto the entire mm10 genome using Bowtie2. Read extraction and data normalization is performed using edgeR. Systems biology experiments and analysis using Inwayuity Pathway Analysis are performed at e-Biogen (Seoul, Korea).

Gene expression analysis data are posted to the NCBI [GEO accession number GSE93718] (http://www.ncbi.nlm.nih.gov/geo/).

Measurement of protective potential against inflammatory bowel disease

After anesthesia with an intraperitoneal injection of 100 μl of ketamine, mice (male C57BL/6, 4 weeks old) were intranasally vaccinated with 1×10 ⁷ -1×10 ⁸ cfu of Δpep27 weekly for a total of three times. Mice were measured for body weight and randomly assigned to four experimental groups, two per group. The following experimental groups of mice were used in the series of experiments: control not treated with dextran sodium sulfate (DSS), experimental control treated with 5% DSS only, experimental group immunized with Pep27 only, and group treated with Pep27+5% DSS. Mice are given DSS (5%, w/v) having an average molecular weight of 5000 (Sigma Chemical Co., St. Louis, Missouri, USA) for 14 consecutive days to induce enterocolitis (Wirtz et al., 2007). In this regard, the DSS solution is replaced daily with fresh. Mice in the control group are allowed to drink only tap water.

All mice in the control group and the experimental control group were administered vehicle (saline) in amounts equivalent to those administered to the DSS receiving groups in the same manner as the DSS receiving group during the study period.

<ΔPep27 immunization assay for inhibition of DSS-induced enterocolitis in mice>

Bowel samples with inflammatory disease (IBD)

Fourteen days after administration of 5% DSS, experimental animals are euthanized by CO ₂ asphyxia followed by laparotomy. The entire colon from the caecum to the anus is removed and divided into proximal, middle and terminal sections. Selected tissues are separated, washed with phosphate-buffered saline (PBS) and stored at -80°C until analysis.

Quantification of colon fragments (disease activity index: DAI)

The progression of colitis is assessed daily by measuring the amount of drinking, weight loss, stool consistency, rectal bleeding, and the presence of total blood in the stool. Also evaluate clinical symptoms. Mice are also checked daily for morbidity (fatigue and lethargy).

These parameters are evaluated according to the criteria proposed below, which are used to calculate the daily average disease activity index (DAI) for each animal, as suggested in previous reports (Wirtz et al., 2007; Jawhara and Poulain, 2007).

In addition, inflammation of the colon is assessed with the naked eye by measuring the length of the colon, which remains undistended and extends from the sigmoid junction to the anal margin. Detailed pathogenic conclusions are given for each group. A system in which tissues are visually scored on a scale of 0 to 5 is used to determine the time for which the organ affected by the most severe inflammation undergoes a change, as well as the degree of change.

The pathology scoring system previously tested (Jawhara and Poulain, 2007; Xu et al., 2007) is being modified to assess colitis. All experiments are repeated at least two times and the results are calculated for all groups as follows:

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; pasty tourniquets not sticking to the anus (non-sticky, pasty, semi-formed), 1; sticky liquid which remains stuck to the anus, 2; sticky with blood, 3; very thin, bloody, or no ability to defecate after 10 minutes, 4;

3) Rectal bleeding: no blood, 0; visible blood in anus or rectum, 1; visible blood on coat, 2; significant bleeding from the rectum, 4;

4) General appearance: normal, 0; covered with mucus, 1; flaccid with piloerection, 2; flaccid and hunched over, 3; immobile and weak, 4.

Statistical analysis

All data are expressed as mean values of independent measurements with duplicates ± standard deviation. Statistical comparison is made by one-way ANOVA followed by Bonferroni's test. All P<0.05 values are considered significant.

2. Results

2-1. Case #1: Analysis of HTS and systems biology indicates the ability to protect against major lesions.

2-1-1. Immunization with Δpep27 protects mice from various lesions.

Systemic bioassay in the lung shows that intranasal immunization with Δpep27 protects against gastroenteritis and prevents colonic abnormalities (FIG. 1). In addition, systemic bioassay in the spleen shows that Δpep27 immunization protects against influenza virus infection (FIG. 2).

2-1-2. Immunization with Δpep27 induces Treg cells.

Systemic bioassay in the lungs also shows that Δpep27 immunization induces TGF-β expression in the lungs (FIG. 3). In FIG. 3 green means suppression of gene expression, and red means induction of gene expression. To confirm Treg induction by Δpep27 inoculation, splenocytes from inoculated mice were analyzed by FACS. As can be seen, Δpep27 immunization increases the population of Th2, Th17 and Treg cells, however, Th1 cells remain virtually unchanged (Figure 4), suggesting that such induced Treg cells play a role in immune tolerance. To confirm Treg induction, cytokine levels in splenocytes and serum are measured. In accordance with the above, there is a significant induction of interferon (IFN)-γ, IL-4, IL-17 and IL-10 in the spleen (figure 5). In addition, a significant induction of IFN-γ, IL-17 and IL-10 is also observed in serum, but without a significant change in the level of IL-4 (Fig. 6). These results imply that induction of Treg cells leads to the production of the anti-inflammatory cytokine IL-10, which enhances immune tolerance.

2-1-3. IL-10 induction by Δpep27 immunization

To confirm immune tolerant response, mRNA levels of IL-10 and IL-17 are measured in lung fluid and bronchoalveolar lavage fluid (BAL). Accordingly, Δpep27 immunization significantly increased both IL-10 and IL-17 mRNA levels in the lung (FIGS. 7 and 8).

2-1-4. Induction of anamnestic reaction as a result of Δpep27 immunization

To determine if intranasal vaccination causes a history reaction, splenocytes are collected from immunized mice and analyzed for history reaction. Δpep27 immunization was found to increase the population of central memory T cells (CD4, CCR7, CD62L) and Tfh memory cells (CD4, CXCR5, CCR7) in the spleen (FIG. 9).

To confirm the anamnestic reaction again, antibody titers of different subtypes of serum immunoglobulins are measured. As expected, immunization with the Δpep27 vaccine induces the expression of different immunoglobulin subtypes such as IgG1, IgG2a, IgG2b and IgG3 (FIG. 10). In addition, the level of IgG antibodies against three types of whole pneumococcal cells also significantly increased with vaccination (Fig. 11). Thus, it is confirmed that intranasal vaccination causes anamnestic reaction.

2-2. Case 2: Protection against co-infection with influenza viruses

Streptococcus pneumoniae and influenza A virus (IAV) are the main causes of respiratory infection (Bosch et al., 2013, Shak et al., 2013). Streptococcus pneumoniae or IAV itself causes respiratory illness, but mortality is increased by secondary infections following influenza virus infection (Mina and Klugman, 2013). However, the currently available pneumococcal polysaccharide conjugate vaccine, PCV 13, cannot effectively protect against secondary pneumococcal infection (Metzger et al., 2015).

2-2-1. Protection against secondary pneumococcal infection by Δpep27 immunization

It has previously been shown that intranasal inoculation of Δpep27 can induce IgG and protect mice from heterologous pneumococcal infection (Kim et al., 2012). In the present invention, a study is conducted to determine whether Δpep27 immunization can increase antibody titers against whole bacterial cells as well as specific antigens. The results demonstrate that intranasal inoculation of Δpep27 increases IgG titers against strains of capsular serotypes 3 and 6B, as well as whole cells of serotype 2 (D39). This increase is significantly higher than that observed in unimmunized controls, indicating that Δpep27 immunization induces humoral immunity against homologous and heterologous pneumococcal strains (Fig. 11). Regarding cytokine levels after influenza challenge, all INF-γ, TNF-α and IL-1β levels in immunized experimental groups are significantly lower than in non-immunized controls, suggesting that Δpep27 immunization induces immune tolerance 12). When IgG titers were measured against specific antigens such as PspA protein and serotype 2 capsular polysaccharide, Δpep27 immunization appeared to increase antibody titers against PspA protein, but not antibodies against serotype 2 capsular polysaccharide (FIG. 13).

To investigate the protective effect of intranasal immunization with Δpep27 against secondary pneumococcal infection, mice are challenged with a 0.02 lethal dose (LD50) of H1N1 influenza virus. Ten days after infection with influenza virus, mice are infected with virulent pneumococcal strain D39 and the survival rate is recorded. While unvaccinated mice (PBS/H1N1) die of pneumonia after D39 infection, most nasally immunized mice (THpep27/H1N1) successfully survive D39 infection (FIG. 14). This result suggests that intranasal immunization with Δpep27 may protect mice against secondary pneumococcal infection as well as influenza virus infection.

If mice are infected with Streptococcus pneumoniae after intranasal immunization, far fewer bacteria are found in the lungs of all vaccinated mice than in unvaccinated controls (Fig. 15), suggesting that intranasal immunization with Δpep27 also successfully protects mice from secondary pneumococcal infection after influenza infection. .

2-2-2. Reducing the number of viruses and bacteria in the lungs as a result of Δpep27 immunization

To establish whether Δpep27 immunization attenuates influenza virus viral load, body weight loss after infection with influenza virus is determined. Interestingly, none of the mice vaccinated with Δpep27 showed weight loss after influenza infection, while the unvaccinated mice showed significant weight loss (Fig. 16). Because Δpep27 vaccination protects against weight loss due to influenza virus infection, an additional study was conducted to test whether Δpep27 vaccination could influence influenza virus replication in the lungs by determining lung TCID ₅₀ in influenza-infected mice. Surprisingly, vaccinated mice showed significantly lower lung virus titers than unvaccinated control mice (FIG. 17). These results demonstrate that intranasal Δpep27 vaccination not only protects against pneumococcal infection, but also significantly attenuates influenza virus infection.

2-3. Case 3: Protection against other bacterial infections by Δpep27 immunization

2-3-1. Protection against Gram-negative bacteria infection by Δpep27 immunization

To investigate whether Δpep27 immunization can prevent colonization by other bacteria, mice are vaccinated with Δpep27 and then infected with the gram-negative bacterium Klebsiella pneumoniae, followed by tissue enumeration of bacteria. Twenty-four hours after challenge, significantly lower numbers of Klebsiella pneumoniae were found in the lungs and serum of the vaccinated groups than in the unvaccinated controls (FIG. 18). The results demonstrate that Δpep27 vaccination can prevent Gram-negative bacteria infection.

2-3-2. Protection against Gram-positive bacteria infection by Δpep27 immunization

To again investigate whether Δpep27 vaccination can prevent colonization of other bacteria, mice are vaccinated with Δpep27 and then infected with Gram-positive Staphylococcus aureus followed by viable tissue counts. Interestingly, lung and nasal lavage fluid from Δpep27 vaccinated animals showed significantly fewer colonies than unvaccinated controls (Fig. 19). This result demonstrates that Δpep27 vaccination can prevent infection by other bacteria such as Gram-positive bacteria.

2-4. Case 4: Protection against inflammatory bowel disease

The phenomenon of oral immune tolerance is studied in human diseases such as rheumatoid arthritis (RA), allergic diseases, diabetes, atherosclerosis, colitis (Faria and Weiner, 2005). Intranasal inoculation with an effector protein (SseB) derived from Salmonella has been described to induce intestinal and systemic IgA, Th1 and Th17 responses, after which bacterial loads in the intestinal tissues and in the spleen are reduced even with oral lethal infection (Pigny et al., 2016) . However, the administration of intranasal vaccines to protect against inflammatory bowel disease has not been previously reported.

2-4-1. Protection against inflammatory bowel disease by Δpep27 vaccination

To investigate whether Δpep27 vaccination can suppress inflammatory bowel disease, mice are immunized intranasally with the vaccine followed by DSS induction of colitis. As a result of the experiment, the disease activity index worsens from the ninth day after adding 5% DSS to drinking water, with a concomitant significant decrease in body weight in mice. While mice receiving only 5% DSS show significant weight loss, the experimental group immunized with Δpep27 and receiving 5% DSS showed significantly less weight loss than the group receiving only DSS (Figure 20).

Comparison of total clinical disease activity scores (p<0.05, one-way ANOVA followed by Bonferroni test, DSS treatment for 6-9 days) demonstrates that the group receiving only 5% DSS scores significantly higher in stool consistency, than the experimental group receiving Δpep27+5% DSS (figure 21). Thus, the results show that after the onset of DSS-induced colitis, the disease worsened in the DSS-only group with a high score of clinical disease activity, while the colitis in the vaccinated group was as mild as in the normal group.

Colonic shortening is used as a marker of inflammation in a model of DSS-induced colitis. In practice, the length of the colon in the groups receiving DSS is significantly reduced (Fig. 22). Accordingly, the ratio of colon mass to colon length increased significantly in the DSS group, but a nearly normal ratio was observed in the Δpep27 vaccinated group compared to the DSS group (Fig. 23).

2-4-2. Inhibition of inflammatory cytokines by Δpep27 vaccination

To confirm the restorative effect of Δpep27 vaccination on intestinal inflammation, cytokine mRNA levels in the colon are measured. The results of the experiment show that in the Δpep27 vaccinated group, there is a significant decrease in the level of pro-inflammatory IL-1β mRNA compared to the non-immunized control (Fig. 24). In addition, intranasal vaccination suppresses IL-17A, TNF-α, and IL-6 mRNA levels compared to non-immunized controls (FIG. 24), demonstrating that intranasal vaccination inhibits cytokine expression.

[ Example 2] Analysis of the inhibitory potential of an attenuated strain of Streptococcus pneumoniae THpep27 against an allergic disease

1 . Materials and methods

1.1. Attenuated strain of Streptococcus pneumoniae THpep27

The Streptococcus pneumoniae Δpep27 strain is the strain described by 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), which is similar to the pep27 mutant strain of Streptococcus pneumoniae disclosed in Korean Patent No. 10-1252911, except that for its selections do not use the erythromycin resistance marker (ermAM).

A Cheshire cassette (GenBank accession number FJ981645) carrying an erythromycin resistance marker (ermAM) that can be used as a temporary selection marker was amplified using primers (5'-TGG CTT ACC GTT CGT ATA G-3' (SEQ ID NO: 2) and 5'-TCG ATA CCG TTC GTA TAA TGT-3' (SEQ ID NO: 3)), which were provided by Dr. Donald Morrison (University of Illinois at Chicago) and ligated by polymerase chain reaction (PCR) to upstream and downstream sequences amplified using primers (5'-TCT CTA TCG GCC 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 AGC AAG AGA CTA-3' (SEQ ID NO: 6) and 5'-CTG CGA GGC TTG CAC TGT AG-3'(SEQ ID NO: 7) from D39 genomic DNA which serves as a template The ligation product is then used to transform D39 to obtain a pep27 mutant.

The Cheshire cassette is cut by adding 1% L-fucose (Sigma, St. Louis, MO, USA). The fucose-treated cultures were spread on THY blood agar plates to obtain single colonies. The presence of Cheshire cassettes 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). The mutant (THpep27) sequence was confirmed by nucleotide sequencing (Cosmo, Seoul, Korea) as well as by immunoblotting using an anti-Pep27 antibody (data not shown).

To confirm the THpep27 mutation at the RNA level, RNA is isolated from bacteria in the early exponential phase using the traditional hot phenol method. After DNA removal with DNase I (Takara, Tokyo, Japan), one microgram of bacterial RNA was reverse transcribed into cDNA using random primers (Takara). Reverse transcription PCR was performed using the recommended primers according to the manufacturer's instructions (Super Bio, American Building Restoration Products Inc., Franklin, WI, USA).

The thus obtained mutant strain of Streptococcus pneumoniae THpep27 is cultivated in THY broth (Todd-Hewitt broth containing 0.5% yeast extract; Difco Laboratories) at 37° C. until the OD ₅₅₀ reaches 0.3 (1×10 ⁸ cfu/ml). The harvested bacterial culture is washed with PBS and then diluted with filtered PBS to a final concentration of 1×10 ⁸ cfu/50 μl for use in immunization.

1.2. experimental animals

Female BALB/c mice (five weeks old, Orient, Korea) are received and then acclimatized for 7 days in an animal chamber before use. A 4:1 mixture of ketamine (ketamine injection, Yuhan Corporation, Korea) and xylazine (Rompun, Bayer Korea Ltd.) was diluted 2-fold with PBS. Mice are anesthetized by intraperitoneal injection of 100 μl of the diluted solution. Anesthesia is followed by infection and vaccination, and then experiments using animals in accordance with the principles of the Sungkyunkwan University Animal Ethics Committee.

1.3. Analysis of the preventive effect of the pep27 mutant on asthma

1) Mice are divided into three groups (n=7/group): normal group receiving sterile water (PBS); a group that is induced asthma using OVA (ovalbumin) and then receives sterile water; and a group in which asthma is induced after vaccination with the pep27 mutant (Δpep27).

2) On days 0, 7 and 14 after the start of the experiment, mice are anesthetized and intranasally (i.n.) vaccinated with Δpep27 in an amount of 1×10 ⁸ cfu/50 μl. On days 38 and 49 after the start of the experiment, mice are sensitized by intraperitoneal injection of 100 μl of sensitizing solution, which is prepared by mixing with 50 μg of OVA (chicken egg white albumin, Sigma Chemical Co., USA) and 2 mg of alum (aluminum hydroxide hydrate, Thermo Co., USA) in 100 µl of 0.9% saline (pH 4.0, Dyne Bio Inc., Korea) for 4 hours at 4°C. Each mouse is then challenged with 25 μl of a 0.4 mg/ml OVA solution in saline every day for six days from days 59 to 64 by dropwise addition of 12.5 μl of the solution to each of both intranasal areas (ultimately OVA is injected into total amount of 10 µg/mouse) to induce asthma. Twenty-four hours after the last OVA challenge, experimental animals are sacrificed, bronchoalveolar lavage fluid (BALF) is collected, and cytokine levels are measured. Lung tissues are cut, fixed and stained with hematoxylin-eosin (H&E) (FIGS. 25 and 26).

1.4. Analysis of the therapeutic effect in relation to asthma

1) Mice are divided into three groups (n=7/group): normal group receiving sterile water (PBS); a group in which asthma is induced using OVA (ovalbumin) (asthma induced group); and a group in which asthma is induced and then vaccinated with the pep27 mutant (Δpep27) (asthma treatment group).

2) Induced Asthma Group: Briefly, all groups except the normal group are sensitized on days 0 and 10 after the start of the experiment by intraperitoneal injection of 100 μl of sensitizing solution, which is prepared by mixing 50 μg of OVA (chicken egg white albumin, Sigma Chemical . Co., USA) and 2 mg of alum (aluminum hydroxide hydrate, Thermo Co., USA) in 100 µl of 0.9% saline (pH 4.0, Dyne Bio Inc., Korea) for 4 hours at 4° C. Ten days later, mice are challenged with 25 μl of a 0.4 mg/ml OVA solution in saline every day for six days from days 20 to 25 by injecting 12.5 μl of the solution into each of both intranasal areas (total OVA is 10 μg/ mouse). The normal group was given only saline (FIG. 27A).

3) Experimental Asthma Treatment Group: One week after the same OVA treatment as in point 2), mice were intranasally immunized with Δpep27 at 1×10 ⁸ cfu/50 μl once a week for a total of three times. Within three weeks of treatment, mice are treated with i.n. OVA at a dose of 10 µg/25 µl three days before the vaccine three times a week (9 times in total) to induce asthma. One week after the last vaccination, mice were treated for the last time with OVA and sacrificed 24 hours later (FIG. 27B).

1.5. Histochemical analysis

Lung and bronchial tissue is isolated and fixed with 10% (v/v) formaldehyde, followed by blocking with paraffin. The paraffin-blocked tissues were cut into 4 µm sections and then stained with H&E. H&E-stained tissues are photographed using an optical microscope. Histochemical analysis was commissioned by KNOTUS Co., Ltd. (Korea) (Fig. 28).

1.6. Cytokine analysis

Bronchoalveolar lavage fluid (BAL) is collected and cytokine levels are measured. Briefly, the trachea of the mouse is exposed and a catheter is inserted into the trachea. 1.0 ml of PBS for BAL is slowly instilled through the catheter into the bronchioles and approximately 0.9 ml is aspirated. This liquid is loaded as is, and then the lavage liquid is removed. The procedure is repeated twice more to obtain BALF with a total volume of 0.8 ml. The resulting BALF is centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant is then separated as BALF for cytokine measurement and stored at -70° C. in a freezer until use. Cytokine levels in BALF are measured using an ELISA kit (ELISA Ready-SET-Go!, eBioscience, San Diego).

1.7. Statistics

Experimental data are expressed as mean ± standard deviation. Statistical processing for measurements of cytokines is carried out using one-way ANOVA. Statistical comparison of each group is carried out according to the Bonferroni test (*P≤0.05; **P≤0.01; ***P≤0.001). All P<0.05 values are considered significant.

2. Results

2.1. Preventive efficacy of pep27 mutant (Δpep27) against asthma

2.1.1. Inhibition of asthma-induced cytokine secretion by Δpep27 vaccination

Measurement of alveolar cytokine levels using an ELISA kit showed that the respective levels of the allergy-inducing cytokines IL-4, IL-5 and IL-13 (Th2 cytokine; Cho et al., 2002) in the Δpep27 vaccinated group were significantly lower than in the asthma group, and about as low as in the normal group (Fig. 25).

2.1.2. Suppression of airway edema in asthma by Δpep27 vaccination

In allergic airway diseases, allergens cause inflammation, making the airways unusually thick (Fig. 26, asthma group). After immunohistochemical staining of inflammatory cells (hematoxylin-eosin, HE) and goblet cells (Iodic Acid-Schiff, PAS) at 400 magnification, it is observed that cells in the Δpep27 vaccinated group are characterized by significantly less inflammation than in the asthma group, with the level inflammation similar to the control (Fig. 26, vaccinated group).

2.2. Therapeutic efficacy of the pep27 (Δpep27) mutant in asthma

2.2.1. The therapeutic efficacy of Δpep27 vaccine is due to the suppression of asthma-related cytokines

Mice are immunized with the Δpep27 vaccine once a week for a total of three times, and one week after the last immunization, cytokine levels in BALF mice are quantified (FIG. 27A). The levels of Th2 cytokines (IL-4, IL-5, IL-13) are markedly elevated in the asthma group, but are about the same in the vaccinated group and in the normal group (Fig. 27B).

2.2.2. The therapeutic efficacy of the Δpep27 vaccine is due to the suppression of pulmonary inflammation

After completion of the experiment, the lungs of mice are stained with hematoxylin-eosin (HE). The Δpep27 vaccinated group (C) showed significant suppression of inflammation compared to the asthma induced group (B). In particular, the vaccinated group exhibited almost the same morphology as the normal control (A), as infiltration and mucosal secretion of inflammatory cells near the bronchi and blood vessels were suppressed (FIG. 28).

3. Discussion

et al., 2006).Articles reporting microbial prevention/treatment of asthma disclose that inhalation of inactivated Mycobacterium phlei suppresses bronchial hypersensitivity in children with moderate asthma (Ming et al., 2013) and inhibits IL-23R expression, regulating airway inflammation, mediated by IL-17-producing γΔT cells and thereby relieving asthma (Ming et al., 2017). In addition, intranasal immunization with an attenuated pertussis strain BPZE1 has been reported to reduce allergic airway inflammation and skin contact hypersensitivity in mice and, therefore, can be used to prevent and treat such diseases (Li et al., 2012). As described, immunization with diphtheria or attenuated tetanus toxin alone or in combination with whole cell pertussis vaccine suppresses allergen-induced Th2 immune responses in a mouse model and thus can be used to suppress airway inflammation or hypersensitivity (

et al., 2006).

In addition, Streptococcus pneumoniae infection induces regulatory T cells, thereby suppressing allergic airway disease (Preston et al., 2011). In addition, intranasal immunization with currently commercially available Streptococcus pneumoniae conjugate vaccines inhibited the progression of allergic airway disease, but no effect was obtained with intramuscular injection (Thorburn et al., 2010). In summary, injection of polysaccharides and attenuated Streptococcus pneumoniae toxin into the respiratory tract has been found to suppress allergic airway disease and induce regulatory T cells that inhibit the onset of allergic airway disease, thereby eliminating and suppressing immune responses to allergens (Thorburn et al. al. al., 2013). However, since all bacteria used in the prevention and treatment of asthma are highly toxic, with the exception of attenuated Bordetella pertussis, it is necessary to use inactivated bacteria or certain components of them. Furthermore, intranasal immunization has been reported to exhibit a higher inhibitory effect on airway allergic reactions than subcutaneous immunization (Takabayashi et al., 2003).

Bordetella pertussis infection induces Th1 responses (IFN-γ activation) in mouse models of allergic airway disease, which can exacerbate inflammation (Ennis et al., 2004; Cho et al., 2002; Kumar et al., 2004). Therefore, it is necessary to suppress allergic reactions without increasing the expression of IFN-γ. Measurement of the preventive effects on asthma after instillation of killed Streptococcus pneumoniae into the respiratory tract showed that the induction of the Th1 response occurs without significant suppression of IL-5 and IL-13 levels, so IFN-γ expression is significantly increased (Preston et al., 2007; Gibson et al., 2012; US patent No. 8226959). That is, despite the suppression of Th2 responses, the induction of Th1 responses cannot exclude the possibility of worsening inflammation. For example, Gibson et al. (2012; US patent No. 8226959) focus on certain components of Streptococcus pneumoniae, which have an inhibitory effect on allergic respiratory diseases.

However, by using the pep27 attenuated mutant (Δpep27) of the present invention, the expression of Th2 cytokines (IL-13 and IL-4) as well as Th1 cytokine (IFN-γ) can be significantly suppressed to normal levels similar to those in the normal group in terms of histological assessments. Therefore, the pharmaceutical composition containing the attenuated strain of Streptococcus pneumoniae according to the present invention is believed to be a safer agent for the prevention or treatment of allergic diseases than conventional vaccines or drugs.

The above description of the exemplary embodiments is provided for purposes of illustration, and those skilled in the art will appreciate that various changes and modifications may be made without changing the technical concept and essential features of the exemplary embodiments. Thus, it is clear that the above-described exemplary embodiments are illustrative in all aspects and do not limit the present invention. For example, each component described as having a single type may be implemented in a distributed manner. Likewise, components described as being distributable may be implemented in a combined manner.

It should be understood that the foregoing is an illustration of the present invention and should not be construed as a limitation to the disclosed specific embodiments, and that modifications to the disclosed embodiments, as well as other embodiments, are intended to be included within the scope of the appended claims. The invention is defined by the following claims, and the equivalent claims are to be included therein.

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SEQUENCE LIST

<110> RESEARCH & BUSINESS FOUNDATION SUNGKYUNKWAN UNIVERSITY

<120> PHARMACEUTICAL COMPOSITION CONTAINING ATTENUATED STRAINS

Streptococcus pneumonia AND ITS USE

<130> 19P0801

<150> KR 10-2017-0053512

<151> 2017-04-26

<150> KR 10-2018-0029765

<151> 2018-03-14

<150> PCT/KR 2018/004800

<151> 2018-04-25

<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>

<223> IL-10 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)

1. The use of a pharmaceutical composition containing an attenuated strain of Streptococcus pneumoniae , and further containing pharmaceutically acceptable excipients, for the prevention or treatment of an inflammatory disease selected from asthma, pneumonia, sepsis, inflammatory bowel disease, gastroenteritis and colitis, where the pharmaceutical composition induces the expression of immunization associated with genes in the lungs and spleen, thereby providing for the prevention and/or treatment of diseases associated with inflammation, and where the attenuated strain of Streptococcus pneumoniae contains a mutant pep27 gene in which the nucleotide residues at positions 1-53 of the pep27 nucleotide sequence described in SEQ ID NO: 1 are deleted. 2. Use according to claim 1, wherein the pharmaceutical composition provides immunization in a serotype-independent manner. 3. Use according to claim 1, wherein the attenuated strain of Streptococcus pneumoniae suppresses the production of a Th1 cytokine or a Th2 cytokine. 4. Application according to claim 3, where the Th1 cytokine is selected from interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-12 (IL-12), and the Th2 cytokine is selected from interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-13 (IL-13). 5. Use according to claim 1, wherein the pharmaceutical composition is non-invasive to the lungs, spleen, blood or brain. 6. Use according to claim 1, wherein the pharmaceutical composition is configured for administration by subcutaneous or intramucosal route. 7. Use according to claim 1, wherein the pharmaceutical composition is configured for administration by intranasal route. 8. Use according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.

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