METHOD FOR MEMORY B CELL-SPECIFIC DIFFERENTIATION INDUCTION, AND USES THEREOF

Abstract

The present invention relates to a method for memory B cell-specific differentiation induction and to uses thereof and, more specifically, to an anti-CD3 antibody or ligand in a biological sample obtained from and individual, a method for memory B cell-specific differentiation induction comprising a step of treating an anti-CD28 antibody or ligand, and a method for detection a memory B cell which is specific to a specific antigen by using same.

Claims

1. A method for specifically inducing differentiation of memory B cells comprising: treating an anti-CD3 antibody or ligand; and an anti-CD28 antibody or ligand in a biological sample obtained from a subject.

2. The method of claim 1, wherein the biological sample is selected from the group consisting of whole blood, plasma, serum, peripheral blood mononuclear cells (PBMCs), urine, feces, saliva, sputum, sweat, tears, tissues and combinations thereof.

3. The method of claim 1, further comprising: removing CD8.sup.+ T cells from the biological sample before treating the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand in the biological sample obtained from the subject.

4. The method of claim 1, wherein the differentiation of the memory B cells is induced to secrete IgG and IgM.

5. A method for detecting antigen-specific memory B cells comprising the steps of: (a) treating an anti-CD3 antibody or ligand; and an anti-CD28 antibody or ligand in a biological sample obtained from a subject; (b) treating an antigen in the biological sample; and (c) detecting an antibody specifically binding to the antigen.

6. The method of claim 5, further comprising: removing CD8.sup.+ T cells from the biological sample before treating the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand in the biological sample obtained from the subject in step (a).

7. The method of claim 5, wherein the antigen is at least one selected from the group consisting of a Mycobacterium tuberculosis antigen, an anthrax antigen, a Hepatitis A virus (HAV) antigen, a Hepatitis B virus (HBV) antigen, a Hepatitis C virus (HCV) antigen, a human immunodeficiency virus (HIV) antigen, an influenza virus antigen, a Herpes simplex virus (HSV) antigen, a Haemophilus influenzae type b (Hib) antigen, a Neisseria meningitidis antigen, a Corynebacterium diphtheria antigen, a Bordetella pertussis antigen, a Clostridium tetani antigen, a human papilloma virus (HPV) antigen, a Varicella virus antigen, an Enterococci antigen, a Staphylococcus aureus antigen, a Klebsiella pneumonia antigen, an Acinetobacter baumannii antigent, a Pseudomonas aeruginosa antigen, an Enterobacter antigen, a Helicobacter pylori antigen, a malaria antigen, a Dengue virus antigen, a MERS virus antigen, a Zika virus antigen, an Orientia tsutsugamushi antigen, a severe fever with thrombocytopenia syndrome Bunyavirus (SFTS Bunyavirus) antigen, a Japanese encephalitis virus antigen, a severe acute respiratory syndrome-corona virus (SARS-CoV) antigen, a severe acute respiratory syndrome-corona virus (SARS-CoV-2) antigen, an Ebola virus antigen, a hepatitis C virus antigen, a hepatitis B virus antigen, an acute respiratory syndrome virus antigen, a West Nile virus antigen, a vesicular stomatitis virus antigen, a Newcastle disease virus antigen, and a pneumococcal antigen.

8. The method of claim 5, wherein the detection of the antibody specifically binding to the antigen in step (c) is performed by at least one method selected from the group consisting of ELISPOT, ELISA, competitive ELISA, magnetic beads and immunochromatography.

9. The method of claim 5, further comprising: determining that the subject has a history of infection with bacteria or viruses expressing the antigen when the antibody specifically binding to the antigen is detected in step (c).

10. A composition for specifically inducing differentiation of memory B cells comprising an anti-CD3 antibody or ligand; and an anti-CD28 antibody or ligand.

Description

DESCRIPTION OF DRAWINGS

[0056] FIG. 1 illustrates a result of detecting IgM and IgG secreted using ELISPOT after treating isolated PBMC with anti-CD3 and anti-CD8 antibodies (immunoreceptor activity treated group: anti-CD3 antibody and anti-CD8 antibody treated group).

[0057] FIG. 2 illustrates a result of detecting secreted IgM and IgG using ELISPOT by removing CD4.sup.+ T cells or CD8.sup.+ T cells from isolated PBMC and then treating anti-CD3 and anti-CD8 antibodies.

[0058] FIGS. 3A and 3B illustrate results of detecting secreted IgM and IgG using ELISPOT by removing IgD or IgG from PBMC from which CD8.sup.+ T cells are removed and then treating anti-CD3 and anti-CD8 antibodies.

[0059] FIG. 4 illustrates a result of measuring the amount of antibodies through an ELISPOT experiment after isolating naive B cells from PBMC and treating polyclonal activators, TLR7/8 agonist (R848) and TLR9 agonist (CPG) at 1 ug/ml.

[0060] FIG. 5 illustrates a result of confirming whether to secrete IgM and IgG using ELISPOT by treating (i) anti-CD3 and anti-CD8 antibodies (immunoreceptor activity treated group), (ii) a TLR7/8 agonist (R848) or (iii) a TLR9 agonist (CPG) after isolating PBMC from the blood of monkeys which has been exposed or not to an MERS antigen (immunoreceptor activity treated group: anti-CD3 antibody and anti-CD8 antibody treated group, TLR agonist 1: TLR7/8 agonist (R848), TLR agonist 2: TLR9 agonist (CPG)).

[0061] FIGS. 6A and 6B are results of confirming whether to secrete IgM and IgG using ELISPOT by treating anti-CD3 and anti-CD8 antibodies (immunoreceptor activity treated group) after isolating PBMC from the blood of monkeys which has been exposed or not to a Dengue virus antigen (FIG. 6A) or a COVID-19 virus antigen (FIG. 6B) and removing CD8.sup.+ T cells (immunoreceptor activity treated group: anti-CD3 antibody and anti-CD8 antibody treated group).

MODES FOR THE INVENTION

[0062] Hereinafter, the present invention will be described in detail by the following Examples. However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.

Example 1: Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

[0063] The blood was isolated from a normal human and a non-human primate. The blood in an EDTA tube was mixed with 1×PBS, and then layers were slowly separated in 4 ml of Ficoll-Hypaque (Lymphoprep; Axis-Shield, 1114545). After centrifuging slowly at 4000 rpm for 20 minutes, when the layers were divided, white bands of PBMCs were collected using a pipette. The PBMCs were added with 1×PBS again and centrifuged at 1500 rpm for 5 minutes, and then a supernatant was removed. Thereafter, 1 ml of a medium was added to release the cells. The number of cells was counted using an Olympus R1 automatic cell counter.

Example 2: Induction of Differentiation of Memory B Cells by Treating Anti-CD3 Antibody or Ligand; and Anti-CD28 Antibody or Ligand

[0064] (1) Measurement of Antibody Secretion Amount According to Treatment of Anti-CD3 Antibody or Ligand; and Anti-CD28 Antibody or Ligand to PBMC

[0065] An anti-CD3 antibody or ligand; and an anti-CD28 antibody or ligand were treated to the PBMCs isolated in Example 1.

[0066] The PBMCs (1×10.sup.5 cells/well) isolated in Example 1 were added to a 96-well plated coated with 1 ug/ml of an anti-CD3 antibody (Clone OKT3, Biolegend Cat. No. 317302), treated with 1 ug/ml of an anti-CD28 antibody (Clone CD28.2, BD Cat. No. 555725), and then reacted in a 37° C., 5% CO.sub.2 incubator for 3 to 4 days.

[0067] Since the antibodies secreted from the memory B cells consisted of IgG and IgM types, the amounts of IgM and IgG secreted from the PBMC sample were then measured through an ELISPOT method.

[0068] Specifically, 10 ug/ml of anti-IgG and IgM antibodies were added into each plate by 50 ul, and then coated at 4° C. for 24 hours. The PBMCs stimulated with the anti-CD3 antibody and the anti-CD28 antibody were added to the coated plate, and then reacted in a 37° C., 5% CO.sub.2 incubator for 4 hours. Thereafter, an anti-IgG-FITC antibody and an IgM-Biotin antibody (Southern Biotech) reacted at room temperature for 2 hours, and FITC-HRP and Strep-AP (CTL) reacted at room temperature for 1 hour. Finally, a blue reducing agent was added and then reacted at room temperature for 15 minutes, and then IgM and IgG were measured.

[0069] The result thereof was illustrated in FIG. 1.

[0070] As illustrated in FIG. 1, it was confirmed that when the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand were treated to the PBMCs, the secretion amount of IgG and IgM-type antibodies significantly increased compared to PBMCs untreated with the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand. That is, the differentiation of memory B cells was induced by treatment of the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand, and as a result, it could be expected that the secretion of IgG and IgM from the differentiated B cells was increased.

[0071] (2) Induction of Differentiation of Memory B Cells in PBMCs from which CD4.sup.+ T Cells or CD8.sup.+ T Cells have been Removed

[0072] In order to optimize conditions in which the differentiation of the memory B cells was induced when the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand were treated in the experiment (1), CD4.sup.+ T cells or CD8.sup.+ T cells were removed from PBMCs and then the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand were treated by the same method as the experiment.

[0073] The CD4.sup.+ T cells or CD8.sup.+ T cells were removed from the PBMCs by the following method, respectively.

[0074] Specifically, PBMC-CD4 T cells (PBMCs from which CD4.sup.+ T cells were removed) and PBMC-CD8 T cells (PBMCs from which CD8.sup.+ T cells were removed) were isolated using an anti-CD4 microbead kit and an anti-CD8 microbead kit (Miltenyi Biotech, Auburn, Calif.). PBMCs (1×10.sup.7 cells) were added with 80 μl of a buffer (0.5% BSA and 2 mM EDTA in PBS), mixed well by adding 20 μl of CD4 microbeads or CD8 microbeads, mix well, and then reacted at 4° C. for 15 minutes. After the reaction was stopped and the PBMCs were centrifuged at 1500 rpm for 5 minutes, PBMC-CD4 T cells or PBMC-CD8 T cells not attached to a magnet were obtained using 10 ml of a buffer and an LS MACS column (Miltenyi Biotech, Auburn, Calif.), respectively.

[0075] The result thereof was illustrated in FIG. 2.

[0076] As illustrated in FIG. 2, it was confirmed that even if the PBMCs from which the CD4.sup.+ T cells have been removed were treated with the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand, the antibodies were not secreted at all. However, it was confirmed that as compared with PBMCs from which the CD8.sup.+ T cells were removed, the secretion amount of antibodies was significantly increased by treatment with the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand.

[0077] Therefore, the following experiment was performed using the PBMCs from which the CD8.sup.+ T cells were removed.

[0078] (3) Confirmation of Whether Memory B Cell-Specific Differentiation has been Induced

[0079] In the prior art, since substances treated to induce the differentiation of memory B cells were polycolonal activators, not only the memory B cells but also naive B cells were differentiated, so that it was impossible to specifically differentiate only B cells that actually have immune memory against a specific antigen.

[0080] Accordingly, the present inventors intended to confirm whether to specifically differentiate only memory B cells, not naive B cells by the method of treating the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand to the PBMCs.

[0081] Specifically, the remaining cells from which cells expressing IgD and IgG were removed from the cell surface corresponding to the PBMCs were isolated using BD FACS Aria II. Thereafter, the isolated cells (1×10.sup.5 cells/well) were added to a 96-well plated coated with 1 ug/ml of an anti-CD3 antibody (Clone OKT3, Biolegend Cat. No. 317302) at the previous day, treated with 1 ug/ml of an anti-CD28 antibody (Clone CD28.2, BD Cat. No. 555725), and then reacted in a 37° C., 5% CO.sub.2 incubator for 3 to 4 days, and thereafter, the antibody amounts were measured through the ELISPOT experiment.

[0082] The results thereof were illustrated in FIGS. 3A and 3B.

[0083] As can be seen in FIG. 3A, it was confirmed that in PBMCs from which cells expressing IgD on the surface have been removed, the secretion amount of IgM and IgG-type antibodies was still increased by treatment with the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand. In contrast, as can be seen in FIG. 3B, it was confirmed that in PBMCs from which cells expressing IgG on the surface were removed, even if the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand were treated, the secretion of IgM and IgG-type antibodies was not increased at all.

[0084] That is, it can be seen that naive B cells expressing IgD on the surface were not differentiated into cells secreting the antibodies even if the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand were treated, but the differentiation of only memory B cells expressing IgG on the surface has been induced by treatment of the anti-CD3 antibody or ligand; and the anti-CD28 antibody or ligand.

[0085] Polyclonal activators, substances used in previous studies, differentiate naive B cells that have not experienced the antigen to produce IgM antibodies on the surface that are not related to a memory of a specific antigen.

[0086] Therefore, the present inventors tried to confirm whether such a non-specific response existed by a method of treating PBMCs with TLR agonists, which were typically known as polyclonal activators.

[0087] Specifically, IgD.sup.+CD27.sup.− cells expressing naive B cells were isolated from the cell surface corresponding to the PBMCs using BD FACS Aria II. Thereafter, the isolated cells (1×10.sup.4 cells/well) were added in a 96-well plate, treated with 1 ug/ml of TLR7/8 agonist (R848) and TLR9 agonist (CPG), and then reacted in a 37° C., 5% CO.sub.2 incubator for 3 to 4 days, and thereafter, the antibody amount was measured through the ELISPOT experiment.

[0088] The result thereof was illustrated in FIG. 4.

[0089] As illustrated in FIG. 4, it was confirmed that the secretion amount of IgM was significantly increased in naive B cells treated with TLR7/8 agonist (R848) and TLR9 agonist (CPG). That is, it was found that the polyclonal activators induced differentiation of naive B cells that was not related to an antigen-specific memory.

Example 3: Detection of Memory B Cells Specific to Specific Antigen

[0090] After establishing a method for specifically differentiating only memory B cells in PBMCs through Examples 1 and 2, using the above method, it was evaluated to determine whether or not the memory B cells have been exposed to a specific antigen in a biological sample isolated from an actual subject.

[0091] Specifically, PBMCs of monkeys that had been tertiary-inoculated with MERS virus antigen protein and adjuvant in the thigh muscle were isolated. According to the method of the present invention, the isolated monkey PBMCs (5×10.sup.5 cells/well) were added to a 96-well plate coated with 1 ug/ml of an anti-CD3 antibody (Clone SP34, BD Cat. No. 557052) at the previous day, treated with 1 ug/ml of an anti-CD28 antibody (Clone CD28.2, BD Cat. No. 555725), and then reacted in a 37° C., 5% CO.sub.2 incubator for 3 to 4 days. Alternatively, the isolated monkey PBMCs were treated with a polyclonal activator, TLR7/8 agonist (R848, TLR agonist1) or TLR9 agonist (CPG, TLR agonist 2) instead of the anti-CD3 antibody and the anti-CD28 antibody and then reacted in a 37° C., 5% CO.sub.2 incubator for 3 to 4 days.

[0092] Thereafter, ELISPOT was performed by coating a MERS-specific protein.

[0093] The result thereof was illustrated in FIG. 5.

[0094] As can be seen in FIG. 5, it could be confirmed that among monkey PBMCs in which the differentiation of memory B cells was induced according to the method of the present invention (immunoreceptor activity treated group), IgG (red number) and IgM (blue number) that specifically recognized the MERS antigen were accurately detected in PBMCs of two monkeys that had been exposed to the MERS virus, but were not detected in PBMCs of two monkeys that had not been exposed to the MERS virus. On the other hand, it could be confirmed that in the monkey PBMCs in which the differentiation of memory B cells was induced according to the method of the present invention, the secretion of an IgG-type antibody that specifically recognized the MERS antigen was significantly higher detected than a group treated with a polyclonal activator, TLR7/8 agonist (R848, TLR agonist1) or TLR9 agonist (CPG, TLR agonist 2).

[0095] In addition, in the same method as the method, the experiment was performed using PBMCs of monkeys that have been tertiary-inoculated with the Dengue virus antigen protein and adjuvant in the thigh muscles or monkeys infected with severe acute respiratory syndrome-corona virus (SARS-CoV-2). As a result, it could be confirmed that in monkey PBMCs that have been exposed to each viral antigen, IgG (red number) and IgM (blue number) that specifically recognized a Dengue virus antigen (a) or a COVID-19 virus antigen (b) were accurately detected, and in monkey PBMCs that have not been exposed to each viral antigen, the antibodies were not detected.

INDUSTRIAL APPLICABILITY

[0096] According to the method of the present invention, since only memory B cells present in a biological sample may be specifically differentiated, it is possible to very accurately and quickly analyze whether a subject providing the biological sample has a history of infection with pathogenic substances containing a specific antigen.