JP4883816B2 - Method for detecting T cells (CD8 +) specific for Mycobacterium tuberculosis - Google Patents

Description

The present invention relates to a method for detecting T cells (CD8 ⁺ ) specific to Mycobacterium tuberculosis .

Several problems have been pointed out with BCG, which is known as a vaccine effective against Mycobacterium tuberculosis, and many attempts have been made to search for vaccines effective against M. tuberculosis consisting of peptides as component vaccines. (Patent Document 1 etc.).
As antigens of Mycobacterium tuberculosis, Ag85 family proteins such as Ag85A and Ag85B are major proteins common to Mycobacteria and are known to work as effective infection protective antigens.
The MPB / MPT51 molecule, which is a protein isolated from Mycobacterium tuberculosis and BCG, belongs to this family and its sequence has been elucidated (Non-patent Document 1). Non-patent document 2).
The inventors have already attempted to identify the T cell epitope of MPB / MPT51 using a DNA vaccine (Non-patent Document 3), but the epitope has not been identified, and more accurate identification is desired. It was.

Special table 2001-515359 Infection And Immunity, vol.65, No.9, p.3680-3685 (1997) Koide Y. et al., "Induction of protective cellular immunity against Mycobacterium tuberculosis using a DNA vaccine encoding MPB51 antigen carried by attenuated suicide Listeria monocytogenes and identification of T-cell epitopes of the antigen" Thirty-eight Research Conference on Tuberculosis and Leprosy, US-Japan Cooperative Medical Science Program, p32-38, 2003 The 76th Annual Meeting of the Bacteriological Society, Mina Suzuki et al. “Identification of T cell epitopes of MPB / MPT51, the major protein of Mycobacteria, using DNA vaccine” April 1-3, 2003

It is an object of the present invention to specify an epitope of Mycobacterium tuberculosis and provide an anti-Mycobacterium tuberculosis vaccine comprising a peptide corresponding to this epitope.
The present inventors have attempted to fix a T cell epitope in the MPB / MPT51 molecule using a DNA vaccine (Non-patent Document 3). However, the number of effective amino acids as an epitope is said to be 8 to 10, and the epitope considered to have been identified so far is much larger than this, and it has been required to identify the epitope more accurately.

The present inventors made an expression plasmid pCI-MPB51 of BCG-derived MPB51 molecule, immunized the mouse with this, and immunized mouse splenocytes with about 20 amino acids corresponding to M. tuberculosis or a part of BCG. The amount of IFN-γ was measured by co-culture with the synthetic peptide. Furthermore, flow cytometry was performed on CD8 ⁺ or CD4 ⁺ T cells using a synthetic peptide having about 10 amino acids in the peptide with a large amount of IFN-γ production. As a result, they succeeded in identifying a peptide fragment that specifically stimulates T cells (CD8 ⁺ ) to produce IFN-γ. As a result, this peptide with 10 amino acids that gives a remarkably high amount of IFN-γ increased the amount of IFN-γ by specifically stimulating T cells (CD8 ⁺ ) contained in spleen cells of immunized mice. Therefore, it is considered to correspond to the epitope of this T cell (CD8 ⁺ ).

As a result of the above-mentioned experiment, the present inventors have found that a peptide consisting of at least 8 consecutive amino acids from the C-terminal of the amino acid sequence (TLAGKGISVV) specifically stimulates T cells (CD8 ⁺ ), resulting in significantly higher IFN. The inventors have found that an amount of γ is given, and have completed the present invention.
That is, the present invention is the peptide (a) or (b).
(A) a peptide comprising at least 8 consecutive amino acids from the C-terminus of the amino acid sequence represented by SEQ ID NO: 1 (b) deletion of one or two amino acids except the C-terminus in the amino acid sequence of the peptide of (a), A peptide consisting of a substituted or added amino acid sequence and showing immunological activity against T cells (CD8 ⁺ )

It is a figure which shows the arrangement | sequence (sequence number 4) of MPT51 molecule | numerator. FIG. 3 is a diagram showing the results of IFN-γ ELISA of Example 1. The vertical axis represents the amount of IFN-γ. PPD (purified tuberculin) was used as a positive control at 5 μg / ml, and Medium (culture medium) was used as a negative control. FIG. 4 is a view showing the results of flow cytometry in Example 2. The vertical axis represents the number of intracellular IFN-γ positive cells (%). Control shows what does not add a peptide.

A peptide consisting of at least 8 consecutive amino acids from the C-terminal of the amino acid sequence represented by SEQ ID NO: 1 is a part of MPB / MPT51 molecule, which is a protein separated from M. tuberculosis and BCG, and is a T cell (CD8 ⁺ ) It is concluded that this is an epitope of this T cell (CD8 ⁺ ) because IFN-γ is specifically stimulated. Therefore, this peptide can be used as an anti-tuberculosis vaccine.
This peptide can be contained in a vaccine preparation together with an effective amount of an adjuvant and a pharmaceutically acceptable carrier as necessary.
This preparation can be administered to a patient by an appropriate route such as injection, subcutaneously, intradermally or intramuscularly. The dose of the vaccine varies depending on the presence or absence of an adjuvant to be administered together, but is generally administered in an amount of 1 μg to 100 mg per adult dose. When an adjuvant is administered together with the vaccine, generally an amount of 1 ng to 1 mg is administered per adult. As an adjuvant, dimethyl dioctadecylammonium bromide (DDA, Eastman Kodak), monophosphoryl lipid A (MPL, RIBI ImmunoChem Research Inc.), etc. can be used.

  Further, a mammal such as a mouse can be immunized with this peptide according to a conventional method, and lymphocytes and spleen cells of this mammal can be isolated and fused with a myeloma cell line according to a standard method to obtain a plurality of hybridomas. Further, cloning by limiting dilution is performed, and a hybridoma in which the hybridoma supernatant specifically reacts with the present peptide is selected, and an antibody (monoclonal antibody) produced by the selected hybridoma can be obtained. Since such an antibody reacts specifically with this peptide, it is useful for examining Mycobacterium tuberculosis or cleaning the vaccine when producing an anti-Mycobacterium tuberculosis vaccine. The method for detecting this antigen-antibody reaction is not particularly limited, but can be performed using immunoblotting, dot blotting, ELISA or the like.

Moreover, the oligonucleotide which consists of a base sequence which codes this peptide can be used as an anti- tubercle bacillus DNA vaccine by expressing this using the vector containing this.
As the vector, a vector that can autonomously replicate in a prokaryotic or eukaryotic host cell or can be integrated into a chromosome and contains a promoter at a position where transcription of the target DNA can be selected can be selected. Vectors include plasmids, phage-containing viruses, cosmids, and the like. The vector can further include a selection marker, a ribosome binding site, a replication origin, a terminator, an enhancer, a polylinker and the like as appropriate. Various expression vectors for prokaryotes such as bacteria and eukaryotes such as fungi, yeasts, insect cells, plant cells and animal cells can be used.
As the promoter, a promoter or the like previously incorporated into a commercially available expression vector can be appropriately selected and used. For example, trp promoter, lac promoter, PL promoter, PR promoter, etc. for prokaryotes, GAP promoter, ADH promoter, GPD promoter, etc. for yeast, cytomegalovirus promoter, SV40 early promoter, retrovirus promoter, mammary gland for animal cells Examples of cell-specific promoters include cauliflower mosaic virus 35S promoter for plant cells.
As the transformation method, calcium chloride method, calcium phosphate method, lithium acetate method, electroporation method, protoplast method, spheroplast method, lipofection method, Agrobacterium method and the like can be used.

The following examples illustrate the invention but are not intended to limit the invention.

Construction of Plasmid DNA Vaccine (pCI-MPT51) DNA encoding MPT51 was amplified by PCR from a plasmid pMB49 (obtained from Nagasaki University School of Medicine, Naoya Ohara) using primers (SEQ ID NOs: 2 and 3). pMB49 contains the MPB51 gene (Gene Bank / EMBL / CCBJ accession number: D26486) of M. bovis BCG Tokyo strain. The PCR fragment digested with Xba I was inserted into the expression plasmid pCI (Promega, Madison, Wis.) At the Xba I position located downstream of the cytomegal virus enhancer / promoter region.

Mouse immunity
HLA- A transgenic / mouse HMC class I-deficient (HHD) mice the plasmid DNA vaccine using a gene gun (Bio-Rad Laboratories) to (Institut Pasteur, France, obtained from Mr. FA Lemonnier) (pCI-MPT51 ). 0.5 mg of gold particles was coated with 1 μg of plasmid DNA vaccine. To immunize the mice, the abdominal skin was shaved and wiped with 70% ethanol. The mice were injected twice with 0.5 mg of gold particles at a time. Mice were bombarded 4 times with 2 μg of plasmid DNA in four portions at weekly intervals.

Peptide synthesis As shown in FIG. 1, peptides consisting of 20 amino acids were selected for MPB51 (SEQ ID NO: 4) so that 10 amino acids overlap, and 26 peptides were synthesized. However, the last peptide (255-266) consists of 12 amino acids.

Measurement of IFN-γ concentration by ELISA A suspension of spleen cells collected from an immunized mouse was cultured in RPMI / 10FCS in a 96-well plate in the presence of 5 μM of each peptide at 37 ° C. and 5% CO ₂ . . After 24 hours, the supernatant was collected and stored at −20 ° C. until IFN-γ concentration evaluation.
IFN-γ concentration was measured by ELIZA method. Coat 96-well ELISA plate (EIA / RIA plate A / 2) with capture antibody (anti-murine IFN-γ monoclonal antibody (mAb) R4-6A2; BD PharMingen) at 4 ° C overnight and contain 0.05% Tween-20 The plate was washed with PBS and blocked with Block Ace (Dainippon Seisakusho) at 37 ° C for 2 hours. After washing, the culture supernatant was added to the plate and incubated overnight at 4 ° C. After washing, anti-murine IFN-γ monoclonal antibody (mAb) (XMG1.2; BD PharMingen) labeled with 0.5 μg / ml biotin was added to the plate and incubated at room temperature for 1 hour. After washing, 0.1 μg / ml horseradish peroxidase (HRP) -conjugated streptavidine (Vector Laboratories, Inc.) was added. The plate was then incubated for 30 minutes at room temperature. After washing, bound HRP-bound streptavidin was measured using 3,3 ′, 5,5′-tetramethylbenzene dihydrochloride (TMB, Sigma-Aldrich Japan). After 5 minutes, the color reaction was terminated with 2M H ₂ SO ₄ and the absorbance at 450 nm was measured using an EZS-ABS microplate reader (IWAKI).
The results are shown in FIG. It was found that the 51st to 70th positions (P51) of MPB51 (FIG. 1, SEQ ID NO: 4) give the highest amount of IFN-γ.

Synthesis of Peptide Next, MPB51 (FIG. 1, SEQ ID NO: 4) (1) 51-70th, (2) 21-29th, (3) 53-61th (TLAGKGISV), and (4) 53-62 A peptide consisting of the second (TLAGKGISVV) was synthesized.

Evaluation by Flow Cytometry For the antigen-specific T cell subset, T cell phenotype and intracellular IFN-γ synthesis were measured by simultaneous flow cytometry evaluation method. In order to remove RBC (red blood cells), the immune spleen cells obtained in Example 1 were treated with ACK lysis buffer for 5 minutes at room temperature, washed twice with PRMI-1640 medium, and 1 × in PRMI / 10FCS. Resuspended at a concentration of 10 ⁷ cells / ml. The cells (200 μl) were incubated for 4 hours at 37 ° C. in the presence or absence of 5 μM of the synthetic peptide in a 1000-fold diluted Golgiplug stock solution (brefeldin A solution; BD PharMingen). The cells were then washed twice with FACS buffer (phosphate buffered saline containing 1% FCS and 0.1% NaN ₃ ) and fluorescein isothiocyanate (FITC) -conjugated anti-CD8 antibody (53-6.7, BD PharMingen) And CyChrome-conjugated anti-CD4 antibody (RM4-5, BD PharMingen) for 30 minutes on ice, washed twice, followed by intracellular cytokine staining (ICS) using the Cytofix / Cytoperm kit (BD PharMingen) . Intracellular cytokine staining for IFN-γ was performed using a phycoerythrin (PE) -conjugated anti-IFN-γ antibody (clone XMG1.2; BD PharMingen). Cells were washed twice and subsequently resuspended in FACS buffer. These were analyzed with an EPICS digital flow cytometer (EPICS XL; Beckman Coulter).

The results are shown in FIG. (4) It can be seen that the amount of IFN-γ produced by T cells (CD8) when the peptide consisting of positions 53 to 62 of MPB51 (SEQ ID NO: 4) is used is remarkably high. This is particularly remarkable when compared with the case of using (3) a peptide consisting of the 53rd to 61st MPB51.
That is, it is shown that T cells (CD8) produced IFN-γ in response to the 53rd to 62nd peptides of MPB51 (SEQ ID NO: 4).
This indicates that the sequence of (4) is MPT51, that is, a T cell epitope of Mycobacterium tuberculosis, and it can be said that its C-terminus is characterized as compared with (3). Furthermore, since it is considered that an effective epitope is usually 8 to 10 amino acids, it can be said that the epitope of MPT51 is a portion consisting of 8 to 10 amino acids including the C-terminus of the sequence of (4).

Claims (3)

Contacting a cell with a peptide comprising at least 8 consecutive amino acids from the C-terminus of the amino acid sequence represented by SEQ ID NO: 1 (TLAGKGISVV), and detecting the cell reacting with the peptide using flow cytometry A method for detecting T cells (CD8 ⁺ ) specific for MPB / MPT51 molecules in the cells. The cells are cultured in the presence of a peptide consisting of at least 8 consecutive amino acids from the C-terminus of the amino acid sequence represented by SEQ ID NO: 1 (TLAGKGISVV), T cells are isolated, and the T cells are separated from the anti-IFN-γ antibody and A method for detecting CD8 ⁺ T cells specific for MPB / MPT51 molecules in cells comprising detecting T cells that react with both antibodies using flow cytometry using anti-CD8 antibodies . The method according to claim 1 or 2, wherein the cell is a cell taken from a human.