WO2021080373A1 - Rv2299c-esat6-hspx-ripa fusion protein composition for boosting bcg vaccine - Google Patents

Abstract

The present invention relates to a BCG boosting composition and a BCG boosting method and, more specifically, to a BCG boosting composition comprising as an active ingredient a fusion protein of Rv2299c, ESAT6, HspX, and RipA, which are derived from M. tuberculosis and a BCG boosting method. The composition and method of the present invention boosts conventional BCG vaccines to augment immune responses in the human body.

Description

Composition for BCG vaccine booster of Rv2299c-ESAT6-HspX-RipA fusion protein

The present invention relates to a composition that has better performance than the existing BCG booster vaccine through the fusion of tuberculosis-derived proteins, and more specifically, to a fusion protein in which Rv2299c, ESAT6, HspX, and RipA proteins derived from M. tuberculosis are fused. It relates to a composition that can boost the BCG vaccine, an effective composition for defending against high-risk Mycobacterium tuberculosis, and a boosting method of a BCG vaccine using the same.

Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB) infection, is a major pathogen causing major public health problems with high morbidity and mortality worldwide. TB is a major disease in developing countries, with about 8 million new infections occurring every year and killing 2 million people, and is emerging as a problem in developed countries as well. It is also estimated that approximately 1.7 billion people, or 23% of the world's population, suffer from latent TB infection (LTBI) and are at risk for lifelong active tuberculosis.

Currently, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine is the only vaccine to prevent tuberculosis, but BCG does not provide sufficient lung protection against TB. Vaccine development is urgently needed (PMID: 11796598). For this reason, various types of adjuvants, antigens (Ag) targets and vaccine platforms have been developed to improve tuberculosis vaccines. Among them, in relation to BCG priming, a heterogeneous prime subunit vaccine boost therapy (adjuvanted subunit boost) was proposed as a promising vaccine strategy against Mtb infection (PMID: 23257069), and the heterogeneous prime boost strategy is a powerful vaccination method. Proved to be.

In a previous study, our group demonstrated that subunit vaccination of ESAT6 fused with Rv2299c augmented with MPL/DDA provides a high level of strong protection against the highly pathogenic strain Mtb HN878 Beijing clinical isolate (PMID: 28193909 ). ^{The improved protection with this vaccine was confirmed by a strong increase in durable and potent Th1 polarized multifunctional CD4 +} T cell immune responses in the lungs in comparison of BCG or ESAT6 alone and Rv2299c-ESAT6 vaccine in a standard mouse model (PMID: 28193909). .

Also recently, IL-17 and Th17 responses have been shown to be important for protective immunity against TB (20679438, 18209095, 20212094, 22003199, 23721367, 24831696, 25448107, 17351619). One of the Th17 responses is that CD4 ⁺ IL-17 ^{+ T cells promote recruitment faster than CD4 +} IFN-γ ⁺ T cells to the lungs of infected Mtb, which is particularly important in vaccine-mediated immunity (17351619, 21933877). However, the functional role of IL-17 in the protection against Mtb is not clear, and the role of the elevated IFN-γ on the antituberculosis action, especially in Mtb-infected macrophages, is unclear.

In the preceding patent, Korean Patent No. 1749165, the group of the present inventors treated immature dendritic cells (DC) with Rv2299c protein (HSP90 family) or a protein fused with Rv2299c and ESAT6 to induce maturation of immature dendritic cells (DC). Here, we identified the Rv2299c protein that effectively induces DC maturation, and investigated the anti-tuberculosis immunity mechanism through DC activation that elicits a strong Th1 type response. In addition, by testing the preventive vaccine effect of Rv2299c protein or RV2299c ESAT6 fusion protein on clinical isolation of Mtb HN878, it was confirmed that Rv2299c-mature DC induces Th1 cell response to anti-tuberculosis immune activity, and DC-activated protein-based As a novel concept of a vaccine, a fusion protein consisting of Rv2299c and ESAT6 was disclosed.

Such a fusion protein composed of Rv2299c and ESAT6 can be a promising vaccine target for boosting BCG, but a heterogeneous boost strategy capable of maintaining a stronger and longer boosting effect is required.

On the other hand, the HspX antigen of tuberculosis is an α-crystalline protein or one of the heat shock proteins of Mycobacterium tuberculosis called Hsp16.3, and can represent a good antigen target for T cell responses. HspX is in fact that this protein can be expressed primarily by tuberculosis during stationary growth or can account for up to 25% of total protein expression under low oxygen conditions. HspX protein can be an important antigenic target during latency, as M. tuberculosis bacteria trapped in granulomas can cause conditions similar to those mentioned above (Arshid Y., et al. 2018).

RipA (Rpf-interacting protein A) has been shown to stimulate the growth of Mycobacterium tuberculosis by interacting with Rpf protein, known as resuscitation-promoting factors, which have already been identified (17919286). The decisive role in cell division of actively growing Mtb is played by the endopeptidase, RipA (Chao et al., 2013; Ruggiero, Marasco et al., 2010). Since deletion of the gene encoding RipA leads to a decrease in growth and an abnormal phenotype composed of bacteria, it can be seen that RipA has a great effect on bacterial growth (Hett & Rubin, 2008).

However, there is no research on using HspX antigen and RipA antigen derived from Mycobacterium tuberculosis in a heterogeneous prime boost strategy by preparing a fusion protein in which a fusion protein backbone composed of Rv2299c and ESAT6 is added. In addition, in order to show the best vaccine efficacy, how to arrange the protein sequence is an important issue when constructing a multi-fusion vaccine.

Korean Patent No. 1270999 describes a method of differentiating immature dendritic cells into mature dendritic cells using the Rv2299c protein derived from Mycobacterium tuberculosis, but Rv2299c of the present invention in that a single protein is used to induce the differentiation of immature dendritic cells. -ESAT6-HspX-RipA fusion protein is different from the booster of BCG vaccine.

U.S. Patent No. 7670609 describes a vaccine against Mycobacteria tuberculosis (Mtb) in which one or more Mtb antigens and one or more Mtb resuscitation or activating antigens are overexpressed, but the Rv2299c-ESAT6-HspX-RipA fusion protein of the present invention is used as a BCG vaccine. There is a difference from using it as a booster for.

[Prior technical literature]

(Patent Document 1) Korean Patent Registration No. 1270999, Maturation method of dendritic cells using Rv2299c protein of Mycobacterium tuberculosis, 2013.05.29. Enrollment

(Patent Document 2) Korean Patent No. 1769165, a composition for promoting maturation of dendritic cells comprising a protein fused with Rv2299c or Rv2299c and ESAT6, registered on June 14, 2017.

(Patent Document 3) U.S. Patent No. 7670609, Recombinant BCG tuberculosis vaccine designed to elicit immune responses to Mycobacterium tuberculosis in all physiological stages of infection and disease, 2010 03.02. Enrollment

(Non-Patent Document 1) Brandt L. et al., Failure of the Mycobacterium bovis BCG vaccine: some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis, Infection and Immunity, 70(2), 672-678 , 2002.

(Non-Patent Document 2) Jonathan M. P. et al., Vaccination against tuberculosis: How can we better BCG? Microbial Pathogenesis xxx (2012) 1-15.

(Non-Patent Document 3) Han-Gyu C. et al., Rv2299c, a novel dendritic cell-activating antigen of Mycobacterium tuberculosis, fused-ESAT-6 subunit vaccine confers improved and durable protection against the hypervirulent strain HN878 in mice, Oncotarget. 8, 19947-19967, 2017.

(Non-Patent Document 4) Teresa M. et al., Mycobacterium bovis BCG-Specific Th17 Cells Confer Partial Protection against Mycobacterium tuberculosis Infection in the Absence of Gamma Interferon, Infection and Immunity, 78(10), 4187-4194, 2010.

(Non-Patent Document 5) Arshid Y. et al., HspX protein as a candidate vaccine against Mycobacterium tuberculosis: an overview, Frontiers in Biology, 13(4), 293-296, 2018.

(Non-Patent Document 6) Michelle B. R. et al., Transcriptional Profile of Mycobacterium tuberculosis Replicating in Type II Alveolar Epithelial Cells, Plos one, 10(4), e0123745, 1-22, 2015.

(Non-Patent Document 7) Chao M. C. et al., Protein Complexes and Proteolytic Activation of the Cell Wall Hydrolase RipA Regulate Septal Resolution in Mycobacteria, Plos one, 9(2), e1003197, 1-17, 2013.

(Non-Patent Document 8) Ruggiero A. et al., Structure and Functional Regulation of RipA, a Mycobacterial Enzyme Essential for Daughter Cell Separation, Structure, 8(9), 1184-1190, 2010.

(Non-Patent Document 9) Erik C. Hett, Eric J. Rubin, Bacterial Growth and Cell Division: a Mycobacterial Perspective, Microbiol. Mol. Biol. Rev. 72(1), 126-156, 2008.

(Non-Patent Document 10) Matthews K. et al., Predominance of interleukin-22 over interleukin-17 at the site of disease in human tuberculosis, Tuberculosis (Edinb), 91(6-3), 587-593, 2011.

(Non-Patent Document 11) Okamoto Y. et al., Essential role of IL-17A in the formation of a mycobacterial infection-induced granuloma in the lung, J Immunol. 184(8), 4414-22, 2010.

(Non-Patent Document 12) Khader S. A. et al., IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung, J Immunol. 187(10), 5402-72011, 2011.

(Non-Patent Document 13) Brandt L. et al., Failure of the Mycobacterium bovis BCG vaccine: some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis, Infect. Immun., 70(2), 672-8, 2002.

As described above, the development of a nontoxic immunomodulatory agent that enhances its own immune function and causes a strong immune response through the BCG-heterogeneous prime boost strategy and the analysis of multifunctional CD4+ T cells are becoming important tasks in cellular immunotherapy.

Accordingly, it is an object of the present invention to provide a BCG-heterogeneous prime boost composition. In addition, to determine the sequence of protein sequence of the multifusion protein for maximizing vaccine efficacy, and finally to provide a composition for a BCG vaccine booster comprising the Rv2299c-ESAT6-HspX-RipA protein as an active ingredient.

Another object of the present invention is to provide a BCG vaccine boosting method comprising the step of inoculating the BCG vaccine booster composition after the step of inoculating BCG.

In order to solve the above problems, the present invention is a BCG containing Rv2299c-ESAT6-HspX-RipA protein as an active ingredient based on preliminary experimental results for determining the location of the ESAT6 protein when adding a protein to the Rv2299c and ESAT6 fusion protein. It provides a composition for a vaccine booster.

In addition, the present invention provides a composition for a BCG vaccine booster, wherein Rv2299c, ESAT6, HspX and RipA of the Rv2299c-ESAT6-HspX-RipA fusion protein are proteins derived from M. tuberculosis, respectively.

The Rv2299c-ESAT6-HspX-RipA protein is preferably contained in an amount of 1 μg/ml to 20 μg/ml.

The present invention provides a composition for a BCG vaccine booster, characterized in that increasing ^{IL-17 +} IFN-γ ⁺ multifunctional T cells or IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ^{+ multifunctional T cells.}

In addition, the present invention provides a composition for a BCG vaccine booster, characterized in that it increases ^{CD4 +} CD44 ^{+ T cells.}

The present invention provides a composition for a BCG vaccine booster, characterized in that it increases ^{CD4 +} CD44 ^{+ T cells.}

The present invention also provides a composition for a BCG vaccine booster, characterized in that it reduces the bacterial load during infection with Mycobacterium tuberculosis.

The present invention provides a recombinant protein consisting of the amino acid sequence of SEQ ID NO: 1, and provides a composition for a BCG vaccine booster comprising the recombinant protein.

The present invention comprises the steps of: (a) inoculating BCG; (B) 12 weeks after the BCG inoculation of step (a), the step of inoculating the composition for the BCG vaccine booster; provides a BCG vaccine boosting method comprising a. The step (b) may be a BCG vaccine boosting method, which is a step of administering the composition for a BCG vaccine booster three times every three weeks. The BCG vaccine boosting method may be a BCG vaccine boosting method comprising increasing ^{IL-17 +} IFN-γ ⁺ multifunctional T cells or IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ^{+ multifunctional T cells.} have.

The BCG vaccine boosting method may be a BCG vaccine boosting method characterized by increasing ^{CD4 +} CD44 ^{+ T cells.}

In addition, the present invention provides a composition for enhancing immunity comprising the composition for the BCG vaccine booster.

As described above, according to the present invention, by utilizing the recombinant Rv2299c-ESAT6-HspX-RipA, which was not known at all, it is possible to effectively activate the body's immune response as a BCG-booster.

1 shows a timetable of BCG, booster inoculation and analysis of mice performed to see the BCG prime boosting effect on the highly pathogenic M2 strain of the Rv2299c-ESAT6-HspX-RipA fusion protein.

Figure 2 is a schematic diagram showing a cloning strategy for the production of Rv2299c-ESAT6-HspX-RipA fusion protein.

3 is a graph showing changes in cytokine after treatment with ESAT6 and RipA (1 or 5 μg/ml) of mouse splenocytes isolated after infection with Mycobacterium tuberculosis (H37Rv and K) to see the antigenicity of RipA.

FIG. 4 is a graph showing the results that when the Rv2882c antigen is added to the Rv2299c-ESAT6 fusion protein in the middle, the vaccine efficacy of the Rv2299c-ESAT6 fusion protein cannot be increased.

Figure 5 is a graph evaluated by cytokine staining of changes in T cell phenotype induced by pre-infection Rv2299c-ESAT6-HspX-RipA fusion protein vaccination, and IL-17, IFN-γ, TNF-α and IL-2 It is a graph showing the co-expression.

6 is a graph showing the degree of bacterial load in the lungs when non-vaccinated, BCG only inoculated and BCG inoculated, boosted with ESAT6, Rv2299c-ESAT6 and Rv2299c-ESAT6-HspX-RipA fusion proteins, and infected with M2 strain. (A) is a photograph of the upper lobe of the right lung of each group (G2-G6) immunized mice stained with H&E 10 weeks after Mtb M2 challenge (1X: scale bar = 2.0 mm). (B) is a graph showing the proportion of lung inflammation sites and the size of the lesions of mice immunized by each group (G2-G6). (C) is a graph showing the measurement of the lung CFU of each group 10 weeks after infection. Data from one of two independent experiments are described (7 mice per group at each specified time point).

Figure 7 is a graph evaluated by cytokine staining and IL-17, IFN-γ, TNF-α and IL-2 for the change in T cell phenotype induced by vaccination with Rv2299c-ESAT6-HspX-RipA fusion protein after infection. It is a graph showing the co-expression of.

8 shows the amino acid sequence of the Rv2299c-ESAT6-HspX-RipA fusion protein.

Until now, many studies have been conducted on proteins derived from M. tuberculosis in the immune response to immune cells, but studies on Rv2299c-ESAT6-HspX-RipA are not known.

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are illustrative to make the present invention easier to understand, and the contents of the present invention are not limited by the examples.

<Example 1> Materials and methods

1.1 Preparation of bacterial strains and Mycobacterium genus

Mtb M2 was obtained from the International Tuberculosis Research Institute (ITRC, Changwon, Gyeongsangnam-do, South Korea). Mycobacterium bovis BCG (pasteur strain 1173P2) was provided by Dr. Brooch of Pasteur Institute (Paris, France). All mycobacteria used in this study were prepared as previously described (Cha et al., 2015).

1.2 Animals, vaccinations and aerosol infections

Five to six week old female C57BL/6 mice without specific pathogens were purchased from Jackson Laboratories (BarHarbor, ME, USA). Mice are maintained at a constant temperature (24 ± 1℃ and humidity (50 ± 5%)) under barrier conditions at the BL-3 Biohazard Animal Facility of Yonsei University Medical Research Center. Animals are kept under standardized lighting control conditions (12 hours light). And a 12 hour dark cycle) with random access to water, were provided with a sterile commercial mouse diet.Mice were monitored daily and no mice showed clinical symptoms or disease during this experiment.

The BCG vaccine boosting method of the present invention includes (a) inoculating BCG, (b) inoculating a composition for a BCG vaccine booster 12 weeks after BCG inoculation in step (a). For vaccination, mice were first vaccinated with ^{BCG Pasteur 1173P2 through subcutaneous injection (2×10 5} CFU/mouse), and 12 weeks after post-BCG immunization vaccination, subunit vaccines were administered three times every three weeks. At this time, the subunit vaccine was applied by including ESAT6, Rv2299c-ESAT6, and Rv2299c-ESAT6-HspX-RipA proteins, respectively, as shown in Table 1.

G1 Naive G2 Infection G3 BCG G4 BCG + ESAT6 G5 BCG + Rv2299c-ESAT6 G6 BCG + Rv2299c-ESAT6-HspX-RipA

Four weeks after the final vaccination, spleen and lung cells were harvested and used to investigate immunogenicity (level of IFN-γ secretion and cycles of IFN-γ-producing T cells and Ag-specific T cells). To study the prophylactic effect of subunit vaccine alone, BCG immunization alone and BCG priming-subunit vaccine boosting groups were challenged to generate Mtb M2 strain and gas as described above (Cha et al., 2015b, Lee et al. , 2009). Briefly, mice were exposed to an inhalation chamber of an air infection device (Glas-Col, Terre Haute, IN, USA) at a predetermined M2 dose for 60 minutes to expose to approximately 150 CFU of viable Mtb. At 8 or 16 weeks post-challenge, spleen and lung cells were harvested from each group, and flow cytometry was used to evaluate the cycle of multifunctional T cells and T cell subtypes.

In order to evaluate the vaccine efficacy of the subunit protein as described above, the BCG vaccine vaccination, the fusion protein immunity cycle, the time of infection with the highly pathogenic M2 strain and the analysis timetable are shown in FIG. 1.

1.3 Antibodies and reagents

IFN-γ targeting PE-conjugated mAb, CD90.2 targeting BV605-conjugated mAb, CD4 targeting PerCP-Cy5.5-conjugated mAb, CD8 targeting BV785-conjugated mAb, CD44 targeting BV421-conjugated mAb, CD62L targeting Alexa700-conjugated mAb, FITC-conjugated mAb targeting IL-17, PE-Cy7-conjugated mAb targeting IL-2, and APC-conjugated mAb targeting TNF-a were purchased from eBioscience (San Diego, CA, USA). Phycoerythrin (PE)-conjugated rat anti-IgG1, rat anti-IgG2a and rat anti-IgG2b, APC-conjugated rat anti-IgG2a and rat anti-IgG1, FITC-conjugated rat anti-anti-IgG2b, and PE-Cy7 -Conjugated mouse anti-IgG1 and rat anti-IgG2b were obtained from eBioscience. These antibodies were used as isotype controls. ELISA kits for measuring TNF-α, IFN-γ, IL-2, IL-5, IL-10 and IL-17F were obtained from eBioscience.

1.4 Generation of recombinant Rv2299c-ESAT6-HspX-RipA

To generate the recombinant Rv2299c-ESAT6-HspX-RipA protein, genomic DNA from Mtb H37Rv (ATCC 27294) was used as a template, and the corresponding gene (frr) was amplified by PCR using the following primers.

Figure 2 is a schematic diagram showing a cloning strategy for the production of Rv2299c-ESAT6-HspX-RipA fusion protein. Using the Genomic DNA of Mycobacterium tuberculosis H37Rv as a template, HspX DNA and RipA DNA were overlapped through PCR to obtain a template, and HspX-RipA DNA with NotI restriction enzyme inserted at the 5'end and 3'end was prepared. It was inserted into the pET22b vector. Using the same method, using the Genomic DNA of Mycobacterium tuberculosis H37Rv as a template, Rv2299c and ESAT6 genes were overlapped through PCR to obtain a template using PCR method, and the 5'end NdeI and the 3'end of the HindIII restriction enzyme were inserted into the Rv2299c-ESAT6 DNA. Was additionally prepared and inserted into pET22b+(HspX-RipA) to complete pET22b+(Rv2299c-ESAT6)+(HspX-RipA).

Table 2 below shows the primers of each antigen used for the production of Rv2299c-ESAT6-HspX-RipA.

Primer Sequence Rv2299c-ESAT6-F CGTCTCGCGCGTACCTTGATGACAGAGCAGCAGTGG Rv2299c-ESAT6-R CCACTGCTGCTCTGTCATCAAGGTACGCGCGAGACG Rv2299c-NdeI-F CATATGAACGCCCATGTCGAGCAGTTG ESAT6-HindIII-R AAGCTTTGCGAACATCCCAGTGACGTT RipA-HindIII-F TCCGTCGACAAGCTTGATCCACAGACGGACACC RipA-R GGTGGTGGCCATGTACTCGATGTATCGGAC HspX-F TACATCGAGTACATGGCCACCACCCTTCCC HspX-NotI-R GCTCGAGTGCGGCCGCGTTGGTGGACCGGATCTG

The resulting PCR product was digested with NdeI and HindIII restriction enzymes and then inserted into pET-22b (+) vector (Novagen, Madison, WI, USA). Recombinant proteins were prepared as described above. In order to remove edotoxin contamination from the purified protein, the recombinant protein was incubated with polymyxin B-agarose (Sigma Chemical Co.) for 6 hours at 4°C. FIG. 8 shows Rv2299c-ESAT6-HspX-RipA of SEQ ID NO: 1 It shows the amino acid sequence of the fusion protein.

The amount of residual LPS in the Rv2299c-ESAT6-HspX-RipA formulation was evaluated using the LAL test kit (Lonza, Basel, Switzerland) according to the manufacturer's instructions. Purified Edotoxin-free Rv2299c-ESAT6-HspX-RipA was filter sterilized and frozen at 70°C. The purity of the Rv2299c-ESAT6-HspX-RipA protein was evaluated by CB staining and Western blotting analysis using anti-His antibody.

1.5 cytokine measurement

Sandwich ELISA was used according to a known method to determine the levels of TNF-α, IFN-γ, IL-2, IL-5, IL-10 and IL-17FA in the culture supernatant. These cytokine measurements were carried out as recommended by the manufacturer (eBioscience).

1.6 Analysis of intracellular cytokine and surface molecule expression by flow cytometry

For intracellular cytokine staining, single cell suspensions obtained from vaccinated animals (2×10 ⁶ cells) were ESAT6 (2 μg/ml), Rv2299c-ESAT6 (2 μg/ml) or Rv2299c-ESAT6-HspX-RipA ( 2 μg/ml) in the presence of GolgiStop at 37° C. for 12 hours (BD Biosciences). In addition, ESAT6, Rv2299c-ESAT6 or Rv2299c-ESAT6-HspX-RipA was used as a stimulator for intracellular cytokine staining after M2 challenge.

Cells were first blocked with an Fc block (anti-CD16/32) at 4° C. for 15 min, targeting CD90.2 for 30 min at 4° C. BV605-conjugated mAb, targeting CD4 PerCP-Cy5.5-conjugated mAb, targeting CD8 BV785-conjugated mAb, BV421-conjugated mAb targeting CD44, and Alexa700-conjugated mAb antibody targeting CD62L were stained. Cells were fixed and allowed to permeate with the Cytofix/Cytofirm kit (BD Biosciences) used according to the manufacturer's instructions. Intracellular IL-17, TNF-α, IL-2 and IFN-γ are FITC-conjugated anti-IL-17, APC-conjugated anti-TNF-α, PE-Cy7-conjugated anti-IL-2 and It was detected using a PE-conjugated anti-IFN-γ antibody. All antibodies were purchased from eBioscience (San Diego, CA) unless otherwise specified. Cells were analyzed using the commercially available software program FlowJo by FACSverse flow cytometry (Treestar, Inc., San Carlos, CA).

1.7 Bacterial Count and Histopathological Analysis

After the final vaccination and 10 weeks after the M2 challenge, 6 to 7 mice per group were euthanized with carbon dioxide, and the lungs and spleen were homogenized. Viable cell count was determined by plating on Middle Brook 7H11 agar (Difco Laboratories, Detroit, MI) through serial dilution of organ (half of the left lung and spleen) homogenate, which was 10% OADC (Difco Laboratories), amphoteric. It was supplemented with Sine B (Sigma-Aldrich, St. Louis, MO) and 2 μg/ml 2-thiophenecarboxylic acid hydrazide (Sigma-Aldrich). Colonies were counted after incubation at 37° C. for 4 weeks. For histopathological analysis, the upper lobe of the right lung was stained with hematoxylin and eosin and evaluated for severity of inflammation. As previously described (Cha et al., 2015a), the level of inflammation in the lungs was evaluated using the ImageJ (National Institutes of Health, Bethesda, ML) software program. In addition, the inflammatory response was evaluated based on the size of the lesion and the composition of immune cells. Data for CFU and lung inflammation were reported as median log10 CFU ± interquartile range (IQR).

1.8 Statistical Analysis

Each experiment was repeated at least 3 times and consistent results were obtained. The significance of the difference between the two groups was judged by the independent sample student t -test, and the difference between the three or more groups was Tukey's multiple comparison test using GraphPad Prism (version 4.03) statistical software (GraphPad Software, San Diego, CA, USA). It was evaluated by one-way ANOVA according to. Data in the graph are expressed as mean±SD. * p <0.05, ** p <0.01 and *** p <0.001 were considered statistically significant.

<Example 2> Antigenicity analysis of RipA using T cells of M. tuberculosis-infected mice

2.1 Experiment method

After isolation of splenocytes from mice infected with Mycobacterium tuberculosis (H37Rv and K), ESAT6 and RipA (1 or 5 ㎍/㎖) were treated, and then IFN-γ, IL-2, IL-10, IL-5, and TNF- α or IL-17AF cytokine was measured. Data are presented as the mean ± SD obtained from 3 experiments, and the independent sample t test was used to determine significance. *P <0.05, **p <0.01 and ***p <0.001 compared to each group (independent sample t test).

2.2 Analysis of experimental results

3 is a graph showing changes in cytokine after treatment with ESAT6 and RipA (1 or 5 μg/ml) of mouse splenocytes isolated after infection with Mycobacterium tuberculosis (H37Rv and K) in order to see the antigenicity of RipA . Since the antigenicity of RipA is currently unknown, cytokines were measured and antigenicity was analyzed. There was no difference between the groups in the secretion of IL-2 and TNF-α indicating T cell activity, but it was confirmed that IFN-γ, which is a Th1 response, was not secreted against RipA. However, it was confirmed that the secretion of IL-5 and IL-10, which is a Th2 reaction, was secreted by RipA, and the secretion of IL-17AF, which is a Th17 reaction, was increased. That is, it was confirmed that RipA expands the Th2/Th17 reaction, not the Th1 reaction.

< Example 3> Rv2299c - ESAT6 protein Between Rv2882c protein Add in the middle to evaluate vaccine efficacy

3.1 Experiment method

When constructing a multi-fusion protein, the order of each protein sequence is an important viewpoint in order to show optimal vaccine efficacy. First of all, in order to evaluate whether to increase the vaccine efficacy by producing Rv2299c-Rv2882c-ESAT6, which was inserted in the middle of the Rv2299c-ESAT6 fusion protein, the Rv2882c antigen, which has been known for vaccine efficacy, was injected 12 after inoculation of BCG as shown in Fig. 4A. From week to week, each fusion protein was immunized three times. Four weeks after the final immunization, lung tissue was analyzed by H&E staining 9 weeks after challenge with HN878 clinical tuberculosis strain, a highly pathogenic strain, and CFU was measured in the lungs and spleens of each group 9 weeks after infection (per group at each designated time point). 7 mice). One-way analysis of variance was followed by Tukey's multiple comparison test to evaluate significance. n.s.: not important, * p <0.05, ** p <0.01, *** p <0.001 and **** p <0.0001.

3.2 Analysis of experimental results

4 is a result of analyzing the vaccine effect of the fusion protein linked by adding Rv2282c to the middle of the Rv2299c-ESAT6 fusion protein. (B) is a photograph of H&E staining of the upper lobe of the right lung of each group (G1-G6) mouse 9 weeks after challenge with the highly pathogenic tuberculosis strain (HN878). (C) is a graph showing the measurement of the lung CFU of each group 9 weeks after infection.

4(B), it can be seen that the lesions are distinct in the lungs of mice infected with Mycobacterium tuberculosis (G2) compared to the lungs of mice that were not infected (G1), and ESAT6 (G4) compared to mice inoculated with only BCG (G3). ), Rv2299c-ESAT6(G5), Rv2299c-Rv2882C-ESAT6(G6) boosted lesions, but there was a clear difference between the groups boosted with Rv2299c-ESAT6(G5) and Rv2299c-Rv2882C-ESAT6(G6). There was no.

In addition, as shown in Figure 4 (C), as a result of measuring CFU in the lungs and spleens of each group 9 weeks after infection, the group boosted with Rv2299c-ESAT6 fusion protein (G5) was the group boosted with ESAT6 alone (G4 ), but there was no significant difference between the groups boosted with Rv2299c-ESAT6 and Rv2299c-Rv2882C-ESAT6.

As a result of the above, in the case of producing a multi-fusion protein by adding another antigen to the Rv2299c-ESAT6 fusion protein, it is better to add an antigen after the Rv2299c-ESAT6-Rv2882c rather than the RRv2299c-Rv2882c-ESAT6. Was expected to appear.

< Example 4> Mycobacterium tuberculosis Challenge I BCG + ESAT6 , Rv2299c - ESAT6 And Rv2299c -ESAT6-HspX-RipA- Immunized Induction of antigen-specific multifunctional T cells in the lungs of mice

4.1 Experimental method

Based on the above results, Rv2299c-ESAT6-HspX-RipA, in which HspX and RipA antigens were linked behind Rv2299c-ESAT6, was prepared to analyze whether it increases the immunological activity and vaccine efficacy of Rv2299c-ESAT6. For this, each group of mice was vaccinated and sacrificed as described in Materials and Methods. Four weeks after the final vaccination, each group of mice (n=5) was euthanized and the lung cells were stimulated with the indicated antigens for 12 hours at 37° C. in the presence of GolgiStop. ^{The number of antigen-specific CD4 +} T cells producing IL-17, IFN-γ and/or IL-2, TNF-α in cells isolated from the lungs of vaccinated mice was determined by multicolor flow cytometry. Lymphocytes were analyzed while gating. The pie chart shows the average number of cells co-expressing IL-17, IFN-γ and/or IL-2, TNF-α. Data are presented as mean±SD obtained from 5 mice in each group, and independent sample t tests were used to determine significance. *P <0.05, **p <0.01 and ***p <0.001 compared to the adjuvant alone group (independent sample t test).

4.2. Analysis of experimental results

Although consensus has not been reached on the protective correlation for tuberculosis vaccine development, recent studies have suggested a protective contribution of multifunctional T cells and a Th17-mediated immune response to Mtb infection in an animal model. Therefore, the present inventors found that antigen-specific IL-17, IFN-γ, TNF-α, and IL-2- multifunctional T cells were re-stimulated ex vivo with ESAT6, Rv2299c-ESAT6 and Rv2299c-ESAT6-HspX-RipA after final immunization. The generated frequency was evaluated. After stimulation with ESAT6, Rv2299c-ESAT6 and Rv2299c-ESAT6-HspX-RipA, lung CD4+ T cells were stained for intracellular cytokines, and then the phenotype of responding T cells was analyzed by flow cytometry.

Figure 5 is a graph evaluated by cytokine staining of changes in T cell phenotype induced by pre-infection Rv2299c-ESAT6-HspX-RipA fusion protein vaccination, and IL-17, IFN-γ, TNF-α and IL-2 It is a graph showing the co-expression. First, when stimulated with ESAT6, both groups boosted with BCG with ESAT6 (G4), Rv2299c-ESAT6 (G5) and Rv2299c-ESAT6-HspX-RipA (G6) antigen-specific IL-17 ⁺ IFN-γ ⁺ T cells Dilation occurred (gray circle outside the pie chart). But ESAT6 (G4) And Rv2299c-ESAT6 (G5) in the group boosting BCG with CD4 ⁺ CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ⁺ -multifunctional T cells had very little expansion, whereas Rv2299c-ESAT6-HspX -RipA (G6) It was confirmed that the group boosted with was significantly expanded. ^{In particular, CD4 +} CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ⁺ rarely observed in the BCG-boosted group (G5) with the Rv2299c-ESAT6 fusion protein composition known to be able to effectively mature DC. -Multifunctional T cells are Rv2299c-ESAT6-HspX-RipA (G6) The significant swelling in the group boosted with Rv2299c-ESAT6 can be said to be an unpredictable effect by boosting the composition containing Rv2299c-ESAT6.

Next, Rv2299c-ESAT6 boosting group when stimulated with Rv2299c-ESAT6 (G5) IL-17 ⁺ IFN-γ ⁺ T cell expansion occurs, but Rv2299c-ESAT6-HspX-RipA boosting group (G6) CD4 ⁺ CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ⁺ -expansion of multifunctional T cells also increased significantly in the Rv2299c-ESAT6-HspX-RipA boosting group (G6) Was confirmed.

Finally, when stimulated with Rv2299c-ESAT6-HspX-RipA, the group boosted with Rv2299c-ESAT6-HspX-RipA (G6) It was confirmed that antigen-specific IL-17 ⁺ IFN-γ ⁺ T cells expanded and CD4 ⁺ CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ⁺ multifunctional T cells expanded.

^{That is, after BCG inoculation, CD4 +} CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL in mice boosted with Rv2299c-ESAT6-HspX-RipA fusion protein compared to treatment with ESAT6 or Rv2299c-ESAT6 fusion protein. By confirming that the expansion of -2 ⁺ multifunctional T cells is superior, the Rv2299c-ESAT6-HspX-RipA fusion protein of the present invention is a boosting of the BCG vaccine, resulting in a remarkable vaccine boosting effect that could not be predicted by treatment with ESAT6 or Rv2299c-ESAT6 fusion protein alone. The presentation was confirmed by immunological analysis.

< Example 5> Rv2299c - ESAT6 - HspX - RipA Fusion protein Highly pathogenic M2 Significant BCG for strain Prime boosting effect

5.1 Experiment method

As in Example 1-7 above, mice were not inoculated, BCG only, and after BCG inoculation, boosting with ESAT6, Rv2299c-ESAT6 and Rv2299c-ESAT6-HspX-RipA fusion proteins, infection with M2 strain, bacterial load in the lungs The degree was measured. The upper lobe of the right lung of mice immunized with ESAT6, Rv2299c-ESAT6 and Rv2299c-ESAT6-HspX-RipA fusion proteins, respectively, was analyzed using H&E staining 10 weeks after Mtb M2 challenge (1X: scale bar = 2.0 mm), These were plotted with a box showing the proportion of the inflamed area and the size of the lesion. In addition, CFU of lung and spleen of each group was measured 10 weeks after infection. Data from one of two independent experiments are described (7 mice per group at each specified time point). One-way analysis of variance was followed by Tukey's multiple comparison test to evaluate significance. n.s.: not important, * p <0.05, ** p <0.01, *** p <0.001 and **** p <0.0001.

5. 2 Analysis of experimental results

6 is a graph showing the degree of bacterial load in the lungs when non-vaccinated, BCG only inoculated and BCG inoculated, boosted with ESAT6, Rv2299c-ESAT6 and Rv2299c-ESAT6-HspX-RipA fusion proteins, and infected with M2 strain. (A) is a photograph of the upper lobe of the right lung of each group (G2-G6) immunized mice stained with H&E 10 weeks after challenge with M2, a highly pathogenic tuberculosis strain (1X: scale bar = 2.0 mm). (B) is a graph showing the proportion of lung inflammation sites and the size of the lesions of mice immunized by each group (G2-G6). (C) is a graph showing the measurement of the lung and spleen CFU of each group 10 weeks after infection.

As shown in Figure 6 (A) and (B), the proportion of the inflammation site and the size of the lesion were the largest in the lungs of non-immunized mice (G2), and the group boosted with ESAT6 after BCG inoculation (G3) and BCG inoculation. In (G4), it was confirmed that the proportion of the inflamed area and the size of the lesion decreased. In addition, in the group boosted with Rv2299c-ESAT6 (G5) and Rv2299c-ESAT6-HspX-RipA fusion proteins (G6), after challenge with the M2 tuberculosis strain, the proportion of the inflammatory site and the size of the lesion decreased the most. There was no significant difference in the proportion of the inflammation site and the size of the lesion between the groups boosted with Rv2299c-ESAT6 (G5) and Rv2299c-ESAT6-HspX-RipA fusion protein (G6).

However, as shown in Figure 6 (C), as a result of measuring CFU in the lung and spleen of each group 10 weeks after infection, the Rv2299c-ESAT6-HspX-RipA fusion protein than the group (G5) boosted with the Rv2299c-ESAT6 fusion protein It was confirmed that the CFU was significantly reduced in the group boosted with (G6).

After BCG inoculation as a result of the above, when boosting with the Rv2299c-ESAT6-HspX-RipA fusion protein of the present invention, it was confirmed that the bacterial load in the lungs of infected mice was significantly reduced when infected with the highly pathogenic M2 strain. It was confirmed that the -ESAT6-HspX-RipA fusion protein acts as an effective booster for BCG inoculation.

< Example 6> Mtb M2 strain challenge After BCG + ESAT6 , Rv2299c - ESAT6 And Rv2299c-ESAT6-HspX-RipA- Immunized Induction of antigen-specific multifunctional T cells in the lungs of mice

6.1 Experimental method

Each group of mice was vaccinated and sacrificed as described in Materials and Methods. Four weeks after the final vaccination, the Mtb M2 strain was challenged, and after 10 weeks, each group of mice (n=7) was euthanized and the lung cells were stimulated with the indicated antigen at 37° C. for 12 hours in the presence of GolgiStop. Preventing IL-17 in the isolated from the lungs of the vaccinated mice cells, IFN-γ and / or IL-2, antigen-specific number of CD4 ⁺ T cells that produce the TNF-α is the flow cytometry of the multicolor CD4 ⁺ CD44 ⁺ Analyze while gating on lymphocytes. The pie chart shows the average number of cells co-expressing IL-17, IFN-γ and/or IL-2, TNF-α. Data are presented as mean±SD obtained from 5 mice in each group, and independent sample t tests were used to determine significance. *P <0.05, **p <0.01 and ***p <0.001 compared to the adjuvant alone group (independent sample t test).

6.2 Analysis of experimental results.

Based on the immunological contribution of Th1/Th17-biased immune status (Figure 5) and enhanced fungal control effect (Figure 6) before challenge after vaccination, the present inventors found IFN-γ ⁺ and IL-17 upon antigen restimulation. ^{+ It} was thought that the T cell response could be continuously induced or expanded even after infection. After 10 weeks of Mtb M2 challenge, lung cells (Figure 6) were stimulated in vitro with ESAT6, Rv2299c-ESAT6 and Rv2299c-ESAT6-HspX-RipA, and ^{the phenotype of antigen-specific CD4 +} T cells was evaluated using flow cytometry. .

Figure 7 is a graph evaluated by cytokine staining and IL-17, IFN-γ, TNF-α and IL-2 for the change in T cell phenotype induced by vaccination with Rv2299c-ESAT6-HspX-RipA fusion protein after infection. It is a graph showing the co-expression of. As in the pre-infection data (Fig. 5), all of the groups boosting BCG with ESAT6 (G4), Rv2299c-ESAT6 (G5) and Rv2299c-ESAT6-HspX-RipA (G6) when stimulated with ESAT6 were antigen-specific CD4 ⁺ CD44. ⁺ IL-17 ⁺ IFN-γ ⁺ T cell expansion occurred (gray circle outside the pie chart). But ESAT6 (G4) And a group boosted with Rv2299c-ESAT6 (G5) In the group where ^{the expansion of CD4 +} CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ⁺ multifunctional T cells was very small, Rv2299c-ESAT6-HspX-RipA boosted group (G6) It can be seen that it is remarkably expanded.

Next, when stimulated with Rv2299c-ESAT6, Rv2299c-ESAT6 (G5) Boosted Group CD4 ⁺ CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ expansion of the T cell only take place, CD4 ⁺ CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ T cells from the Rv2299c-ESAT6-HspX-RipA boosted group (G6) from Was significantly increased, CD4 ⁺ CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ⁺ expansion of multifunctional T cells also Rv2299c-ESAT6-HspX-RipA boosting group (G6) It can be seen that it has increased significantly.

Finally, even when stimulated with Rv2299c-ESAT6-HspX-RipA, the group boosted with Rv2299c-ESAT6-HspX-RipA (G6) It can be seen that the expansion of antigen-specific IL-17 ⁺ IFN-γ ⁺ and expansion of CD4 ⁺ CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ⁺ multifunctional T cells were observed.

These results show that the Rv2299c-ESAT6-HspX-RipA vaccination of the present invention is an antigen-specific multifunctional T cell CD4 ⁺ CD44 ⁺ IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ⁺ and IL-17 ⁺ IFN- It was confirmed that protection against Mtb strain infection can be improved by inducing a boost of γ ^{+ T cells.}

Upon stimulation with ESAT6, Rv2299c-ESAT6 and Rv2299c-ESAT6-HspX-RipA, mice immunized with Rv2299c-ESAT6-HspX-RipA were antigen-specific CD4 ⁺ CD44 ⁺ multifunctional T cells (IL-17+ IFN-γ + TNF- α + co-generation of IL-2). These results show that after BCG inoculation, the Rv2299c-ESAT6-HspX-RipA fusion protein was boosted to the BCG inoculum to induce a boost of antigen-specific multifunctional T cells, especially IL-17 ⁺ IFN-γ ⁺ T cells, to prevent infection with Mtb strains. It can be seen that the protection against it can be improved.

For a vaccine to induce protection against tuberculosis, it must rapidly recruit antigen-specific T cells into the lungs and activate infected phagocytes to control tuberculosis bacteria. In addition, cells that reach the site of infection must be able to survive in an environment full of phagocytic cells.

Immunological memory is characterized by increased levels of effector cells and the ability to respond faster and more strongly to a second encounter with a pathogen than during the primary reaction. In particular, a strong immune response is required for intracellular pathogens such as tuberculosis. Thus, the expansion of memory T cells, CD4 ⁺ CD44 ⁺ T cells, provides a pool of cells capable of rapidly responding to subsequent contact with Mycobacterium tuberculosis. In addition, CD4 ⁺ IFN-γ ⁺ T cells are classically thought to be essential for Mtb control, but the magnitude of the IFN-γ response does not provide an optimal correlation of protective efficacy against TB. ^{In addition, recent data revealed that CD4 +} T cells that produce a number of cytokines, including IFN-γ, TNF-α and IL-2, are associated with protection against Mtb infection. Therefore, ^{T cells that produce a number of cytokines in CD4 +} CD44 ⁺ T cells play an important role in the regulation of tuberculosis.

The functional role of IL-17 in the protection against Mtb in mice that induces the initial regulation of Mtb proliferation during Mycobacterium tuberculosis infection is not clear, and the synergy with IFN-γ in Mtb-infected macrophages is not clear. Our previous work has suggested a possible involvement of the Th17 response by examining the cytokine profile from dendritic cells activated by Rv2299c-ESAT6 co-cultured with naive CD4+ T cells in vitro, but the precise mechanism has been linked to its protective efficacy. A clear explanation was needed.

To address this question, we boosted with fusion protein after BCG vaccination and proceeded with infection with highly pathogenic tuberculosis strains to determine the protective efficacy of each therapy ^{and to see if there was a correlation with the phenotype of the protective CD4 + T cell subset.} . In the present study, we first evaluated the ability of the Rv2299c-ESAT6-HspX-RipA fusion protein vaccine to enhance and prolong the BCG-induced immune response in a rat tuberculosis infection model. It proved to be heavy-duty. In addition, we investigated why and how a Th17 response was necessary for the optimal vaccine efficacy of this subunit vaccine.

Our data show that BCG-primed Rv2299c-ESAT6-HspX-RipA booster vaccination simultaneously induced Th1/Th17 immune responses after final immunization upon ESAT6 restimulation in lung and spleen compared to BCG-primed ESAT6 and Rv2299c-ESAT6. Show. In addition, after BCG was amplified with Rv2299c-ESAT6-HspX-RipA, antigen-specific CD4 ⁺ CD44 ⁺ IL-17 ⁺ IFN-γ ^{+ co-producing three or two effector cytokines after in vitro stimulation with ESAT6} It was found that the number of T cells significantly increased. Thus, the ^{presence of immediate expandable IFN-γ/IL-17 to produce multifunctional CD4 +} T cells and their antigen-specific expansion induced by Rv2299c-E6-HspX-RipA boosting may contribute to enhanced protection against Mtb infection. I think it can. Overall, the present invention confirmed that the novel immunostimulatory antigen Rv2299c-ESAT6-HspX-RipA fusion protein can be effectively boosted after BCG inoculation.

Claims (12)

BCG vaccine booster composition comprising the Rv2299c-ESAT6-HspX-RipA fusion protein as an active ingredient. The method of claim 1, Rv2299c, ESAT6, HspX and RipA of the Rv2299c-ESAT6-HspX-RipA fusion protein is a BCG vaccine booster composition, characterized in that they are proteins derived from M. tuberculosis, respectively. The method of claim 1, The Rv2299c-ESAT6-HspX-RipA fusion protein is a BCG vaccine booster composition, characterized in that contained in a concentration of 1 ㎍ / ㎖ to 20 ㎍ / ㎖. The method of claim 1, The composition for a BCG vaccine booster is a composition for a BCG vaccine booster, characterized in that to increase ^{IL-17 +} IFN-γ ⁺ multifunctional T cells or IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ^{+ multifunctional T cells} The method of claim 1, The composition for BCG vaccine booster is a composition for BCG vaccine booster, characterized in that it increases ^{CD4 +} CD44 ^{+ T cells} The method of claim 1, The composition for BCG vaccine booster is a composition for BCG vaccine booster, characterized in that it reduces the bacterial load when infected with Mycobacterium tuberculosis Recombinant protein consisting of the amino acid sequence of SEQ ID NO: 1 Composition for a protein BCG vaccine booster comprising the recombinant protein of claim 7 (A) inoculating BCG; (B) BCG vaccine boosting method comprising the step of inoculating the BCG vaccine booster composition of any one of claims 1 to 6 and 8 after 12 weeks of BCG inoculation in step (a). The method of claim 9, The step (b) is a BCG vaccine boosting method in which a composition for a BCG vaccine booster is administered three times every three weeks. The method of claim 9, The BCG vaccine boosting method is a BCG vaccine boosting method comprising ^{increasing IL-17 +} IFN-γ ⁺ multifunctional T cells or IL-17 ⁺ IFN-γ ⁺ TNF-α ⁺ IL-2 ⁺ multifunctional T cells The method of claim 9, The BCG vaccine boosting method is a BCG vaccine boosting method characterized in that it increases ^{CD4 +} CD44 ^{+ T cells.}

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