WO2019221383A1 - Recombinant bcg employing pmyong2 vector system to express hiv-1 p24 and use thereof as hiv-1 vaccine - Google Patents
Recombinant bcg employing pmyong2 vector system to express hiv-1 p24 and use thereof as hiv-1 vaccine Download PDFInfo
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- WO2019221383A1 WO2019221383A1 PCT/KR2019/003597 KR2019003597W WO2019221383A1 WO 2019221383 A1 WO2019221383 A1 WO 2019221383A1 KR 2019003597 W KR2019003597 W KR 2019003597W WO 2019221383 A1 WO2019221383 A1 WO 2019221383A1
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Definitions
- the present invention relates to novel recombinant BCGs used in the prevention and / or treatment of human immunodeficiency virus type-1 (HIV-1) infections, vaccine compositions comprising them, methods for their preparation, and the like. . More specifically, the present invention provides a recombinant BCG using a novel shuttle vector for mycobacterium -E. Coli (Esherichia -coli ) (hereinafter “pMyong2”, Korean Patent No .: 1012916680000, US Patent No .: 8,841,432). It relates to a live vaccine platform.
- mycobacterium -E. Coli Esherichia -coli
- rBCG Mycobacterium bovis BCG
- HIV specific cellular immunity is primarily induced by HIV specific T cells with poly-functionality and proliferative capacity against immunodominant viral peptides, and because of this characteristic they are effective against HIV-1.
- CTLs virus-specific cytotoxic T lymphocytes
- BCG Mycobacterium bovis BCG
- EPI World Health Organization Expanded Program on Immunization
- BCG BCG Recombinant forms of BCG, which have been successfully used to express foreign antigens and induce immune responses, ie rBCG, are Borrelia burgdorferi , Streptococcus pneumoniae , Bordetella perturtu system (Bordetella pertussis), a rodent malaria (rodent malaria), resyu mania (leishmanial), measles virus, human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency including virus (SIV), vaccines against a variety of infectious agents It has been considered a candidate.
- the most practical and practical advantage of rBCG vectors is their high safety.
- rBCG exhibits good adjuvant properties, induces a long-lasting cellular immune response that persists for at least one to two years, is low in manufacturing cost, easy to administer, and usually only one or two immunizations. Need only.
- rBCG-based vaccines over other recombinant vaccine approaches suggest that rBCG can be a potent vaccine against HIV-1 infection, which can induce a safe, virus-specific immune response.
- the problem to be solved of the present invention is to provide a recombinant Mycobacterium bovis BCG (rBCG) expressing a p24 protein derived from HIV-1 and use thereof as an HIV-1 vaccine.
- rBCG Mycobacterium bovis BCG
- rBCG vectors as potential HIV-1 vaccines
- their practical application as HIV-1 vaccines has shown low immunogenicity due to lack of stability and low stability of heterologous expression of foreign genes in rBCG.
- Limited due to Therefore, to obtain sufficient immunogenicity and draw on the efficacy of protective vaccines, a high rBCG dose of approximately 10 to 100 times the dose required for actual BCG vaccination against tuberculosis in humans is required.
- In vivo administration of high doses of BCG may increase the risk of disseminated BCG of immuno-compromised vaccine recipients or act as a driving force for replication and spread of HIV-1 by excessive activation of T cells. Can be.
- a method of treating or preventing AIDS and / or tuberculosis by administering to a subject a vaccine composition comprising a therapeutically effective amount of the recombinant Mycobacterium bovis BCG as an active ingredient, a recombinant Mycobacterium bovis for preparing the vaccine Use of BCG to the use of recombinant Mycobacterium bovis BCG for treating or preventing AIDS and / or tuberculosis.
- the present inventors through the DSM 45126 T genomic analysis of Mycobacterium yongonense , a human pathogen member of the Mycobacterium avium complex ( MA ), Compared with the conventional pAL5000 vector system derived from Mycobacterium M. fortuitum, it is a linear plasmid capable of inducing increased heterologous gene expression in recombinant mycobacterial smegmatis (rSmeg) and rBCG. A new mycobacterial-E. coli shuttle vector system using a mycobacterial replicon of pMyong2 was introduced.
- rSmeg expressing HIV-1 p24 Gag using the pMyong2 vector system is enhanced immunity to HIV-1 p24 Gag in mice compared to rSmeg in the pAL5000 vector system or using the integrated plasmid pMV306 system. Response was induced, suggesting the viability of the pMyong2 vector system in rSmeg vaccine application.
- HIV-1 Gag-specific CD8 + T cell responses can be of great importance for immune regulation of HIV infection. Therefore, in the present invention, HIV-1 Gag p24 was selected as a target antigen for expression in the pMyong2 vector system. Therefore, in the present invention, pMyong2 vector system was adopted to improve the expression of foreign HIV-1 p24 Gag gene in rBCG (rBCG-pMyong2-p24). The efficacy of the pMyong2 vector system, ie its improved recombinant protein production, has been demonstrated in rBCG and rBCG-infected bone-marrow derived dendritic cells ("BMDCs"). In addition, to demonstrate vaccine efficacy, we explored its cellular and humoral immune responses to HIV Gag protein in vaccinated mice.
- BMDCs bone-marrow derived dendritic cells
- rBCG The best strategy for improving the potential of rBCG as an HIV vaccine is to use rBCG as a prime vaccine in a prime-booster vaccine protocol and to use a safe recombinant viral vector as a boost vaccine. As a result, this induces efficient virus-specific cellular immunity that persists long after immunization in animal models.
- the Th1 response induced by the rBCG vector may contribute to inducing Gag-specific CTL responses.
- a major barrier to the practical use of rBCG as an HIV-1 vaccine is that the low expression of foreign HIV-1 antigen in rBCG does not elicit sufficient virus-specific CTL responses to fight viral infection.
- strategies include the use of hemolysin-expressing BCG strains capable of eliciting larger CTL responses through the preferential cytoplasmic location of rBCG and codon optimization for HIV-1 gag p24 gene in the rBCG system.
- the pMyong2 shuttle vector system was applied to enhance the expression of HIV-1 gag p24 gene in rBCG.
- rBCG-pMyong2-p24 a tendency to be more attenuated in macrophage infection in rBCG-pMyong2-p24 that produced lower levels of CFU (colony-forming unit) than rBCG-pAL-p24 was observed (FIG. 2C), which is another vector. Presumably due to the higher copy number of pMyong2 in rBCG than those in the system. Immunization with lower doses or more attenuated rBCGs reduces the risks associated with high-dose skin administration, including adverse local skin reactions, possible association with Th2-type immune responses, or exacerbation of retroviral infections. Considering that it may be possible, rBCG-pMyong2-p24 may have additional advantages in the HIV vaccine protocol using rBCG.
- Multi-copy episomal vector-based Mycobacterial Escherichia coli shuttle vector systems are known to have drawbacks associated with the lack of stability of recombinant Mycobacteria compared to integrated plasmid systems.
- the pMyong2-p24 plasmid in rSmeg has been shown to gradually lose stability after five passages in an antibiotic-free medium.
- the present invention despite the use of the same pMyong2 vector system, allows pMyong2-p24 plasmid in rBCG (rBCG-pMyong2-p24) to maintain its stability even after 12 passages, whether or not antibiotics are added ( 3f).
- rBCG-pMyong2-p24 has the advantage over rSmeg-pMyong2-p24 in applications as an HIV-1 vaccine. .
- vaccine efficacy against HIV-1 in two different mycobacteria namely BCG ( rBCG-pMyong2-p24) and Smeg (rSmeg-pMyong2-p24) were compared using the same pMyong2 system.
- HIV-1 p24 antigen In the immune response to HIV-1 p24 antigen, although CTL response from immunized splenocytes, T cell proliferative capacity of infected BMDCs and most IFN- ⁇ ELISPOT levels were rBCG-pMyong2-p24 and rSmeg-pMyong2- Although nearly identical at p24, rBCG-pMyong2-p24 showed significantly improved IL-2 production in splenocytes and Th1-biased humoral immune responses compared to rSmeg-pMyong2-p24, which showed that rBCG-pMyong2-p24 This suggests that HIV-1 vaccine regimen may be superior to rSmeg-pMyong2-p24.
- the present invention compared the vaccine efficacy against HIV-1 between two different vaccine modules using rBCG-pMyong2-p24 and p24 proteins.
- the data herein indicate that rBCG-pMyong2-p24 had improved p24 specific IFN- ⁇ ELISPOT levels, CTL response and Th1-biased humoral immune response compared to p24 protein (FIGS. 8A-8C), which also showed The HIV-1 vaccine curing suggests that rBCG-pMyong2-p24 may be superior to the p24 protein.
- Gender differences are known to respond to various vaccines, including BCG, measles, mumps and rubella vaccines, and influenza vaccines. In general, in the adaptive immune response, women exhibit an improved humoral and cell mediated immune response compared to men. This is the reason why only female mice were selected for current vaccine studies in the present invention.
- rBCG-pMyong2-p24 of the pMyong2 vector system has higher levels of HIV-1 in rBCG than other BCG strains using pAL5000- (rBCG-pAL-p24) or pMV306-derived system (rBCG-pMV306-p24). It has been demonstrated to induce p24 Gag protein expression and to deliver more p24 antigen to phagocytes.
- the above-mentioned strains can also improve the T cell proliferative capacity of infected BMDCs in vaccinated mice and provide improved CTL response and Th1 bias compared to rBCG-pAL-p24 or rSmeg-pMyong2-p24. It has been shown that it can induce a humoral immune response.
- rBCG-pMyong2-p24 may be an efficient candidate as a prime vaccine in a heterologous prime-boost vaccine strategy for HIV-1 infection.
- the present invention provides recombinant Mycobacterium bovis BCG (rBCG) expressing a p24 protein derived from HIV-1.
- the p24 protein may be expressed by the pMyong2-p24 vector system disclosed in Figure 2a.
- the p24 protein may be encoded by a Gag gene derived from human immunodeficiency virus type 1 represented by the nucleotide sequence of SEQ ID NO: 2.
- the mycobacterium bovis BCG strain according to the present invention is Tokyo 172 strain.
- the present invention provides an HIV-1 vaccine composition comprising the recombinant mycobacterium bovis BCG as an active ingredient.
- the present invention also provides a method for treating or preventing AIDS and / or tuberculosis by administering to a subject a vaccine composition comprising a therapeutically effective amount of a recombinant mycobacterium bovis BCG according to the present invention.
- the present invention also provides the use of recombinant Mycobacterium bovis BCG for the production of vaccines for the prevention or treatment of AIDS and / or tuberculosis.
- the present invention also provides the use of recombinant Mycobacterium bovis BCG for the prevention or treatment of AIDS and / or tuberculosis.
- the present invention provides a vaccine composition for HIV infection or HIV and Mycobacterium tuberculosis co-infectious disease comprising the recombinant mycobacterium bovis BCG as an active ingredient.
- the recombinant mycobacterium bovis BCG in the vaccine composition according to the invention is alive.
- the vaccine according to the invention is further not artificially attenuated.
- the vaccine according to the invention may be one used in particular as a prime vaccine in a prime-boost vaccination protocol.
- the infection may be AIDS (AIDS, acquired immunodeficiency syndrome) or tuberculosis.
- AIDS AIDS, acquired immunodeficiency syndrome
- tuberculosis AIDS, acquired immunodeficiency syndrome
- RBCG-pMyong2-p24 according to the present invention has been shown to elicit enhanced HIV-1 p24 Gag expression in rBCG and infected antigen presenting cells (APC).
- rBCG-pMyong2-p24 compared with rBCG-pAL-p24 in the pAL5000 derived vector system, high levels of HIV-1 Gag-specific CD4 and CD8 T lymphocyte proliferation, gamma interferon ELISPOT cell induction, It has been shown to induce an enhanced p24 specific immune response, evidenced by antibody production and cytotoxic T cell (CTL) responses.
- CTL cytotoxic T cell
- rBCG-pMyong2-p24 according to the present invention has been shown to produce higher levels of p24-specific antibodies than rSmeg-pMyong2-p24 in the same pMyong2 vector system.
- rBCG-pMyong2-p24 recombinant BCG expressing p24, i.e. rBCG-pMyong2-p24, induces an enhanced immune response to HIV-1 infection using the pMyong2 vector system. Therefore, rBCG-pMyong2-p24 has the potential as a prime vaccine in a heterologous prime-boost vaccine strategy against HIV-1 infection.
- Figure 2a is a diagram showing a cleavage map of each p24 expression vector disclosed herein.
- mycobacterial-E. Coli shuttle vectors expressing the HIV p24 antigen express p24 under the control of the hsp65 promoter derived from Mycobacterium bovis BCG.
- Figure 2b is a diagram showing the growth curve of p24 rBCG strain in 7H9 medium to which ADC and 100 ⁇ g / ml kanamycin is added.
- kanamycin was excluded from the 7H9 medium. Growth curves were taken at each time point in culture aliquots and measured OD600.
- FIG. 2C shows a comparison of CFU levels of p24 rBCG strains in infection of mouse macrophage J774A.1 (left) and mouse bone marrow-derived dendritic cells (right).
- FIG. The data represent two independent experiments. (The results are expressed as mean ⁇ variance. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001 (Student's t-test).
- Figure 3a is a diagram confirming the expression of p24 in rBCG strain using ELISA.
- 3b is a diagram confirming p24 expression of rBCG strain using Western blotting analysis. Proteins were extracted from wild type BCG (lane 1) and rBCG strains (lane 2: rBCG-pMV306-p24, lane 3: rBCG-pAL-p24, lane 4: rBCG-pMyong2-p24). As a positive control, purified p24 protein was used (lane 5). M, molecular weight standard (Elpis Bio, Daejeon, Korea). Individual membranes are separated by white blanks and marker lanes by black vertical lines. In this figure the expression level of p24 is shown and the Western blotting images are cropped in full-length blots to improve clarity. Full-length blotting images are shown in FIG. 3C below.
- FIG. 3C is a diagram illustrating a full length original blotting image of the cropped image of FIG. 2C.
- FIG. Western blotting was performed using the antibodies shown in this figure. The cut part is shown by the solid line.
- FIG. 3D shows the stability of p24 expression in rBCG-pMyong2-p24 strains passaged in 7H10 agar plate containing kanamycin using Western blotting.
- Protein was extracted at each passage point in wild type BCG (lane 1) and rBCG-pMyong2-p24 strains (lane 2: first passage, lane 3: after fourth passage, lane 4: after sixth passage, lane 5: After passage 8, lane 6: after passage 10, lane 7: after passage 12).
- As a positive control purified p24 protein was used (lane 8).
- M molecular weight standard (Elpis Bio, Daejeon, Korea).
- P24 was detected using an Hsp65 antibody (Abcam) after cutting the membrane as an internal control in the upper size membrane. Individual membranes are separated by white blanks and marker lanes by black vertical lines.
- FIG. 3E illustrates a full length original blotting image of the cropped image of FIG. 2D. Western blotting was performed using the antibodies shown in this figure. The cut part is shown by the solid line.
- Figure 3f is confirmed by Western blotting the stability of p24 expression in rBCG-pMyong2-p24 strains passaged in 7H10 agar plate without kanamycin. Protein was extracted at each passage point in wild type BCG (lane 1) and rBCG-pMyong2-p24 strains (lane 2: first passage, lane 3: after fourth passage, lane 4: after fifth passage, lane 5: After the sixth passage, Lane 6: After the seventh passage, Lane 7: After the eighth passage, Lane 8: After the ninth passage, Lane 9: After the tenth passage, Lane 10: After the eleventh passage, Lane 11: The twelfth After passage). As a positive control, purified p24 protein was used (lane 12).
- M molecular weight standard (Elpis Bio, Daejeon, Korea).
- P24 was detected using an Hsp65 antibody (Abcam) after cutting the membrane as an internal control in the upper size membrane. Individual membranes are separated by white blanks and marker lanes by black vertical lines.
- FIG. 3G illustrates a full length original blotting image of the cropped image of FIG. 2F.
- Western blotting was performed using the antibodies shown in this figure. The cut part is shown by the solid line.
- 3H shows p24 post infection with wild type BCG and rBCG strains (rBCG-pMV306-p24, -pAL-p24, and -pMyong2-p24) in mouse macrophage J774A.1 (left) and mouse myeloid derived dendritic cells (right), respectively. It is a figure which measured the expression level of. The data represent two independent experiments. (The results are expressed as mean ⁇ variance. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001 (Student's t-test).
- FIG. 3i shows the expression level of p24 from BMDCs infected with different MOIs (1 and 10 MOI; multiplicity of infection) of rBCG-pAL-p24 and rBCG-pMyong2-p24 strains at Days 1 and 3 using ELISA to be. Results are expressed as mean ⁇ variance in duplicate wells. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001 (Student's t-test).
- FIG. 4A Schematic of T cell proliferation assay schedule.
- Two mice were injected with p24 protein (30 ⁇ g / mouse) and after 7 days the splenocytes of the mice were sorted into CD4 and CD8 T cells and labeled with CFSE.
- One day before co-culture DCs were infected with each strain (10 M.O.I.).
- gastric cells were analyzed for T cell proliferation;
- FIG. 5A-5C show in vivo immune responses induced by p24 rBCG strains, respectively:
- FIG. 5A Schematic of immunization performed for in vivo immunological analysis. At 4 week intervals, each group (5 mice / group) was immunized twice with wild type BCG, two types of rBCG strains and rSmeg strains, respectively. Four weeks after the last immunization, mice were sacrificed and spleen and blood samples collected for immunoassay;
- FIG. 5B Splenocytes of mice inoculated with the p24 rBCG strain were detected using IFN- ⁇ secretion level ELISPOT assay after in vitro stimulation.
- Figure 6 shows the humoral immune response induced by the p24 rBCG strain.
- p24 specific immunoglobulin subtypes IgG2a, IgG1 and total IgG
- p24 specific immunoglobulin subtypes IgG2a, IgG1 and total IgG
- OD values and IgG2a / IgG1 ratios for IgG2a and IgG1 subtypes were compared.
- Five mouse serum samples per group were analyzed. The data represent two independent experiments. The results are expressed as mean ⁇ variance values.
- mice immunized with rBCG strains show cytotoxic T lymphocyte responses in mice immunized with rBCG strains.
- the CTL response occurs in vitro by spleen cells (effector cells) stimulated by p24 and p24 epitope peptides (A9I) reacting with P815 cells (target cells).
- effector cells stimulated by p24 and p24 epitope peptides (A9I) reacting with P815 cells (target cells).
- a total of three mice per group were analyzed. The data represent two independent experiments. The results are expressed as mean ⁇ variance values. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001 (Student's t-test).
- FIG. 8a p24 protein (30 ⁇ g / mouse) and different CFUs ( Splenocytes from mice (3 mice / group) subcutaneously injected with rBCG-pMyong2-p24 strains (1 week apart, 2 injections) of 1 ⁇ 10 6 and 1 ⁇ 10 7 CFU) in vitro were stimulated with IFN- The result of ELISPOT analysis to compare the levels of ⁇ secretion. Representative images of ELISPOT membranes for each group are shown below the graph. (-), Negative control; (+), Positive control.
- the results are expressed as mean ⁇ variance in triplicate. ** P ⁇ 0.01; *** P ⁇ 0.001 (Student's t-test); (FIG. 8B) p24 specific immunoglobulin subtypes (IgG2a, IgG1 and total IgG) were detected by ELISA. Serum samples of three mice per group were analyzed. The results are expressed as mean ⁇ variance in triplicate. ** P ⁇ 0.01; *** P ⁇ 0.001 (Student's t-test); (FIG.
- the present invention is based on the discovery that recombinant Mycobacterium bovis BCG strains expressing HIV-1 p24 antigen can be used effectively in vaccines.
- the present invention relates to a recombinant mycobacterium bovis BCG strain expressing the p24 protein of HIV-1, wherein the p24 protein is expressed by the pMyong2-p24 vector disclosed in FIG. 2A.
- the p24 capsid (CA) protein expressed by the vector according to the invention is linked to the 3 'end of the MA (matrix protein) in an untreated Gag polyprotein.
- the p24 capsid (CA) protein contains two domains at the N and C termini that play important roles in HIV budding and capsid structure.
- P24 expressed by the vector according to the present invention is derived from HIV-1. Sequence variants thereof may be used as long as they are included in KM390026.1d or correspond to amino acids of SEQ ID NO: 1 or 3161 to 3856 nucleotides of sequence of SEQ ID NO: 4, and act as antigens for purposes according to the present application.
- P24 expressed by the strain according to the present application may act as an antigen for HIV infection when administered to a human body.
- p24 is expressed in particular by the pMyong2 vector disclosed in FIG. 2A.
- Recombinant BCG strain comprising pMyong2-p24 according to the present invention will induce higher levels of HIV-1 p24 protein expression and deliver more p24 antigen to macrophages compared to other rBCG strains using the pAL5000 or pMV306 induction system. It was found to be very good.
- the BCG recombinant strain comprising pMyong2-p24 according to the present invention improves the T cell proliferation ability of infected BMDCs (Bone Marrow-Derived Dendritic Cells) and improves T cell effector function and Th1-biased humoral immune response in inoculated mice. Has been shown to be able to induce.
- the rBCG-pMyong2-p24 strain according to the present invention is delayed in the initial growth, attenuated to present more antigens to phagocytes can maintain a stronger immune response.
- the present invention relates to a composition for immunogenicity, vaccine or immunotherapy for HIV infection comprising the strains disclosed herein.
- HIV is a type of retrovirus that destroys the human immune system. When HIV is infected, the host's immune system is degraded and progresses to AIDS, and the risk of opportunistic infections caused by bacteria, viruses, fungi, and parasites increases significantly. do.
- AIDS is an acquired immune deficiency syndrome that refers to a number of symptoms that result in a decrease in human immune function as a result of HIV infection.
- the vaccine composition according to the present invention delivers p24 specific immune responses, e.g., more p24 antigens to phagocytes, improves T cell proliferative capacity of BMDCs and improves T cell effector function and Th1-biased humoral immunity in inoculated mice
- the response can be elicited to prevent HIV infection or to alleviate, alleviate and / or treat symptoms caused by the infection.
- the vaccine is usually administered two or more times in the form of a prime-booster.
- the same vaccine is administered several times (homologus) or different kinds of vaccines containing the same antigen (heterologus).
- the vaccine according to the invention is used as a prime vaccine at homologous or heterologous prime-boost.
- the rBCG-pMyong2-p24 strain according to the present invention is particularly advantageous for use as a prime vaccine because the expression level of the p24 antigen is high and the antigen presenting ability is sufficient to induce sufficient p24-specific immune responses in naive individuals.
- the mycobacteria used in the compositions according to the invention are as described above, in particular live live bacteria can be used.
- the rBCG-pMyong2-p24 strain according to the present invention is delayed in the initial growth, it is naturally attenuated to present more antigens to phagocytes can maintain a stronger immune response.
- the term "vaccine” refers to a biological agent containing an antigenic substance that immunizes the living body, and refers to an immunogen that is immunized with the living body by injecting or injecting the living body for the prevention or treatment of AIDS and / or tuberculosis .
- the term "individual” means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, and the like. Mean mammal.
- an "immune response” refers to the activation of the host's immune system, eg, the mammalian immune system, in response to the introduction of an HIV-1 antigen, eg, a universal HIV-1 antigen, via a provided DNA plasmid vaccine. It is used herein to mean.
- the immune response can be in the form of a cellular or humoral response, or both.
- Vaccine compositions according to the invention may be formulated for systemic or topical administration and include pharmaceutically possible excipients, carriers and / or media such as phosphate buffered saline solutions, distilled water, water / oil emulsions, wetting agents, emulsifiers, pH adjusters and the like, but are not limited thereto.
- the vaccine composition according to the invention may comprise an adjuvant, in particular any substance or compound capable of promoting or increasing a T-cell mediated response, if necessary.
- the adjuvant may be used when the microorganisms killed by chemical or heat are included in the composition.
- Adjuvants are known in the art and include, for example, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, cross-linked polyacrylic acid polymers, dimethyldioctadecylammonium bromide (DDA), immunomodulators, lactoferrins, or IFN-gamma inducers, and the like. Can be.
- the crosslinked polyacrylic acid polymer comprises a Carbopol ⁇ c homopolymer or copolymer
- the lymphokine comprises IFNgamma, IL-1, IL-2 or IL-12
- the IFN-gamma inducer May include, but is not limited to, poly I: C.
- the synthesized sugar polymer, the aluminum hydroxide, aluminum phosphate, or aluminum potassium sulfate is included in about 0.05 to 0.1% by weight
- the cross-linked polyacrylic acid polymer may be included in 0.25% by weight but not limited thereto. It is not.
- compositions according to the invention may be administered topically or systemically, may be administered once or multiplely, may be administered by various routes, for example subcutaneously, intradermal, intramuscularly or intravenously, or by oral, nasal or inhalation routes. Can be. In one embodiment according to the invention it is administered once by intramuscular injection.
- Vaccine compositions according to the invention may be formulated in solid form, prepared in liquid solutions or suspensions suitable for the route of administration or dissolved or suspended in solution prior to injection.
- a "therapeutically effective amount” refers to a dosage required to elicit an antibody that is capable of significantly reducing the likelihood or seriousness of infection with human immunodeficiency virus type 1.
- the effective amount depends on the type and severity of the disease administered, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, the factors including the concurrent drug and other factors well known in the medical field. It can be easily determined by those skilled in the art in an amount that can be determined and the maximum effect can be obtained without any side effects in consideration of all the above factors.
- the dosage of the vaccine will depend on the route of administration as well as the age, weight and / or health of the patient.
- a suitable dosage can be, for example, 1 to 10 9 CFU. In one embodiment 10 6 CFU is used.
- the expression level of the antigen is excellent, and the antigen presenting ability is excellent, so that a lower dose or a lower dose compared to the existing strain vaccine can be used.
- prevention means any action that inhibits or delays the development of AIDS and / or tuberculosis by administration of a vaccine composition according to the invention.
- treatment refers to any action in which symptoms caused by AIDS and / or tuberculosis are improved or beneficially altered by administration of a vaccine composition according to the present invention.
- the invention relates to the vector disclosed in FIG. 2A for use in the expression of p24 in a strain according to the invention.
- the vector is most preferably represented by the nucleotide sequence of SEQ ID NO: 4.
- the invention also relates to a cell transformed with said vector.
- the cells in particular comprise Mycobacterium .
- mice Female BALB / c mice (approximately 25 g, 7 weeks old) were purchased from Orient-Bio (Seoul, Korea) and used for experiments at 8 weeks old. Mice were randomly divided into four groups of five mice per group.
- p24 protein was injected into the tail vein into two mice (BALB / c) (30 ⁇ g / mouse) and five mice (BALB / c) were bone marrow-derived dendritic cells (BMDCs) in each test. ) was used for the preparation.
- mice were wild-type, two recombinant BCG strains (rBCG-pAL-p24 and rBCG-pMyong2-p24) or rSmeg-pMyong2-p24 strains (1 in 100 ⁇ l PBS). ⁇ 10 6 CFU; 4 weeks apart) subcutaneously immunized at the tail base.
- PBS was injected subcutaneously.
- mice were euthanized by CO2 inhalation at each time point, and then the blood and spleen of the mice were removed to remove IFN- ⁇ ELISPOT, cytokine measurements, serum antibody detection (5 mice / group) and CTL analysis ( 3 groups / group).
- mice In addition, independent in vivo tests were performed to compare the differences in immune responses and different bacterial numbers induced by p24 protein treatment.
- BALB / c mice 3 mice / group) were replaced with p24 protein (30 ⁇ g / mouse) and 2 numbers of rBCG-pMyong2-p24 strains (1 ⁇ 10 6 and 1 ⁇ 10 7 CFU).
- p24 protein 30 ⁇ g / mouse
- rBCG-pMyong2-p24 strains (1 ⁇ 10 6 and 1 ⁇ 10 7 CFU.
- Three subcutaneous injections were made.
- PBS was injected subcutaneously.
- One week after the last immunization mice were euthanized by CO2 inhalation at each time point, and the blood and spleen of the mice were removed and used for immunological assays such as IFN- ⁇ ELISPOT, serum antibody detection and CTL analysis.
- rBCG-pMyong2-p24 BCG with pMyong2-p24 plasmid
- rBCG-pAL-p24 BCG with pAL-p24 plasmid
- pMV306-p24 To generate BCG with p24 plasmid (rBCG-pMV306-p24), three constructed plasmids, pMV306-p24, pAL-p24 and pMyong2-p24, were added to the competent BCG strain (Tokyo 172) in a Gene Pulser II electroporation device. Electroporation was performed using (Bio-Rad, Hercules, CA, USA).
- Transformants were selected on Middlebrook 7H10 medium (Difco Laboratories, Detroit, MI, USA) containing kanamycin (100 ⁇ g / ml) and OADC. Typically, transformant colonies were selected from plates and transferred to 7H9 medium (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% glycerol, 0.05% Tween-80, 10% ADC and kanamycin for 3-4 weeks. Incubated for Growth rate of the rBCG strain was measured by optical density (OD) at 600 nm.
- OD optical density
- Recombinant p24 protein was purified from Escherichia coli with slight modifications as described above.
- E. coli BL21 strain (RBC Bioscience, Taipei City, Taiwan) was transformed with pET23a-p24. Expression of the protein was induced by addition of 0.4 mM isopropyl ⁇ -D-thiogalactoside (IPTG, Duchefa Biochemie, Haarlem, The Netherlands). Bacteria cells were obtained and destroyed by sonication for 10 minutes on ice.
- the sonicated lysates were centrifuged at 1600 ⁇ g for 20 minutes at 4 ° C., and the pellets containing p24 protein were reproduced in binding buffer containing 4M urea (Urea, Sigma Aldrich, St. Louis, MO, USA). It was cloudy. Proteins were purified using Ni-NTA His binding resin (Merck, Darmstadt, Germany) and eluted with elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 250 mM imidazole) containing 4M urea. Purified protein was subsequently dialyzed against the elution buffer to remove imidazole, urea and residual salts.
- Bone marrow derived dendritic cells were prepared from bone marrow (BM) of 8- to 12-week-old BALB / c mice as described above. Briefly, BM cells were removed from the femur and tibia using serum-free Iscove's modified Eagle medium (IMDM; Gibco Invitrogen, UK).
- IMDM Iscove's modified Eagle medium
- the macrophage cell line J774.1 (ATCC TIB-67) of the mouse was supplemented with 10% (v / v) fetal bovine serum (FBS), 2 mM glutamine, and essential amino acids at 37 ° C. and 5% CO 2.
- FBS fetal bovine serum
- DMEM Dulbecco's modified Eagle's medium
- BMDCs were generated from mouse bone marrow as described above and 10% FBS (Gibco Invitrogen), recombinant mouse GM-CSF (1.5 ng / ml; PeproTech, Rocky Hill, NJ, USA), mouse IL at 37 ° C. and 5% CO 2.
- J774A.1 cells and BMDCs were infected with rBCG strains, ie rBCG-pMyong2-p24, -pAL-p24, and -pMV306-p24 and wild-type BCG strain (10 MOI) (3 pairs), followed by 4 hours After washing three times with PBS and incubated with fresh medium for 24 hours. After 24 hours, infected cells were lysed with 0.5% Triton X-100. Cell lysates were diluted with PBS and plated on Middlebrook 7H10 agar plates supplemented with OADC for calculation of colony forming units (CFUs). All infection groups were analyzed in duplicates in each experiment and a total of two independent experiments were performed.
- rBCG strains ie rBCG-pMyong2-p24, -pAL-p24, and -pMV306-p24 and wild-type BCG strain (10 MOI) (3 pairs
- infected cells were lysed with 0.5% Triton
- the expression level of p24 in each rBCG strain was measured using mouse anti-p24 monoclonal antibody (Abcam, Cambridge, USA; 1: 1,000 dilution). Mycobacterial Hsp65 (Abcam, 1: 1,000 dilution) was used as an internal control to confirm that protein concentrations were equivalent in all samples.
- p24 expression levels of the rBCG-pMyong2-p24 strain were also measured at various passage points (after 1, 4, 6, 8, 10 and 12 passages). Passage was performed from plate to plate (7H10 agar plate with or without kanamycin), and each experiment was performed after colonies from each passage were incubated in 7H9 broth medium for 3 weeks.
- a p24 ELISA kit was used (in the method suggested by the manufacturer) for the detection of p24 levels in the same amount of protein (in three sets of wells) (ABL, Rockville, USA). All groups were analyzed in two independent experiments.
- the J774.1 cells and BMDCs with 5 ⁇ 10 ⁇ 10 5 cells per well (24-well plate, 3 suits) was seeded, and cultured for 18 hours.
- Three different rBCG strains were infected with 10 multiplicity of infection (MOI) cells.
- MOI multiplicity of infection
- rBCG-pMyong2-p24 strains of different MOIs (1 and 10 MOIs) were infected with BMDCs to compare differences in p24 expression by different MOIs.
- J774.1 cells and BMDCs were incubated for 4 hours to allow phagocytosis of bacteria, and extracellular bacteria were removed by washing three times with PBS.
- Infected J774.1 cells and BMDCs were incubated for 24 hours and / or 72 hours.
- total protein in cell pellets was prepared by suspension in RIPA lysis buffer and p24 levels according to manufacturer's instructions using the p24 ELISA kit (ABL) (in three sets of wells). It was used for the measurement of. All infection groups were analyzed in duplicates in each experiment and a total of two independent experiments were performed.
- T cell proliferation assay For the T cell proliferation assay, the following experiment was performed. Two mice were injected intravenously with p24 protein (30 ⁇ g / mouse). After 7 days, splenocytes were washed with cryogenic FACS buffer [PBS containing 1% bovine serum albumin (BSA) and 1 mM EDTA] and 10% rat serum (Sigma Aldrich), 10% goat for 30 minutes on ice. Blocking with super block solution containing serum (Gibco Invitrogen), 10% mouse serum (Sigma Aldrich), and 2.4G2 monoclonal antibody (10 ⁇ g / ml; BD Biosciences, San Diego, CA, USA) blocking).
- BSA bovine serum albumin
- EDTA 10% rat serum
- BV421-conjugated anti-CD4 (Clone GK1.5, BD Biosciences) and PE-conjugated anti-CD8a (Clone 53-6.7, eBioscience, San Diego, CA, USA) for 30 minutes. Stain at C and wash three times with cryogenic FACS buffer. CD4 and CD8 T cell populations were sorted using the FACS AriaIII instrument (BD Biosciences). In addition, the day before co-culture, immature BMDCs were infected with wild type, two rBCG (rBCG-pMyong2-p24 and -pAL-p24) or rSmeg-pMyong2-p24 strains for 10 hours at 10 M.O.I.
- CFSE Invitrogen, Carlsbad, USA
- Sorted CD4 and CD8 T cells were stained with 5 ⁇ M CFSE at 37 ° C. for 4 minutes and on ice for 4 minutes. Thereafter, CFSE-labeled T cells and infected BMDCs were co-cultured for 4 days.
- mice IL-2 in supernatant (three sets of wells) co-cultured in the course of the T cell proliferation assay was measured according to the manufacturer's instructions (BioLegend, USA) using ELISA. All experiments were performed in duplicate.
- ELISPOT assays were performed using splenocytes from mice immunized with wild type and rBCG strains (5 mice / group). Briefly, a 96-well ELISPOT plate (PVDF membrane) is coated with mouse IFN- ⁇ (3 ⁇ g / ml, clone: AN-18) capture antibody (BD-Biosciences, San Diego, CA, USA) in PBS and overnight 4 Incubation was at ⁇ . Discard the capture antibody, wash the plate with PBS and PBS containing 0.05% Tween-20 (PBST) (three times each), and plate the plate with 200 ⁇ l RPMI 1640 medium containing 10% FBS for 3 hours at 37 ° C. It was.
- PBST 0.05% Tween-20
- splenocytes harvested from vaccinated mice were loaded with 5 x 10 5 cells per well.
- cells were stimulated in 3 sets with 5 ⁇ g / ml of purified p24 antigen or medium alone in a total volume of 200 ⁇ l. Plates were then incubated at 37 ° C. for 24 hours. Cells were stimulated with 5 ng / ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, USA) and 500 ng / ml ionomycin (Sigma-Aldrich) as positive controls.
- PMA phorbol 12-myristate 13-acetate
- each well was treated with biotin-labeled mouse IFN- ⁇ (3 ⁇ g / ml, clone: XMG1.2) detection antibody (BD-Biosciences) and plate Incubate at 4 ° C. overnight. The cells were washed again and horseradish peroxidase (HRP) -conjugated streptavidin was added to each well. HRP reactions were developed using 3-amino-9-ethylcarbazole (AEC) substrate reagent set (BD-Biosciences). The number of spot forming units (SFUs) per well was automatically counted using an ELISPOT reader (AID ELISPOT reader, Strasburg, Germany). All groups were analyzed in 3 sets and 2 independent experiments were performed.
- HRP horseradish peroxidase
- Splenocytes from immunized mice (5 mice / group) were adjusted to a concentration of 1 ⁇ 10 6 cells / well (96-well microplate, 200 ⁇ l volume, 3 sets) in RPMI 1640 medium containing 10% FBS and Purified p24 protein was added at a concentration of 5 ⁇ g / ml for in vitro stimulation.
- Cells were cultured and supernatants were obtained for IL-2 (BioLegend, San Diego, CA, USA), IL-6 (eBioscience) and IFN- ⁇ (BioLegend) cytokine measurements using an ELISA kit. All groups were analyzed in 3 sets and 2 independent experiments were performed.
- serum samples were collected using cardiac puncture methods after euthanasia through hyperrespiration of CO2 from immunized mice (5 mice / group).
- 96-well plates were coated overnight with purified p24 protein (5 ⁇ g / ml) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at 4 ° C. Plates were washed three times with PBST and PBS and blocked with 5% bovine serum albumin (BSA in PBST) for 1 hour at room temperature (RT). Serum samples were diluted with PBS at a ratio of 1:10 and 100 ⁇ l was added to each well (in three suits).
- CTL Cytotoxic T lymphocyte
- splenocytes from each immunized group of mice were labeled with major histocompatibility complex (MHC) class I-restricted p24 peptide A9I (AMQMLKETI) (10 ⁇ g / ml; Peptron, Daejeon, South Korea). Pulsed and incubated with IL-2 (30 U / ml; PeproTech, Rocky Hill, USA) in a 5% CO 2 incubator at 37 ° C. for 6 days.
- MHC major histocompatibility complex
- AMQMLKETI major histocompatibility complex
- Target cells ie P815 cells (H-2d) were prepared by incubation with A9I peptide (10 ⁇ g / ml) for 2 hours followed by co-culture of effectors and target cells.
- the cytotoxicity of the cells was assessed according to the manufacturer's protocol (CytoTox 96 non-radioactive cytotoxicity assay; Promega, Madison, USA) by lactate dehydrogenase (LDH) assay in U-shaped bottom 96-well plates.
- effector cells are matched to target cells (p24 pulsed P815 cells) in three different effector / target (E / T) ratios (10: 1, 20: 1 to 50: In the range of 1) for 6 hours; Then, the LDH value released from the cultured supernatant was detected using a spectrometer at 490 nm.
- the percentage of specific cell lysis was calculated using the following formula: [ ⁇ Experimental-Effector spontaneous-Target spontaneous] / ⁇ Target maximum-Target spontaneous ) ⁇ ] x 100 (%). All groups were analyzed in 3 sets and 2 independent experiments were performed.
- Example 1 rBCG-pMyong2-p24 Strain Induces Improved HIV-1 p24 Gag Expression in Bacteria and Infected Cells
- the rBCG-pMyong2-p24 strain may have less CFU than other strains (ie rBCG-pAL-p24, -pMyong2-p24 and wild-type BCG strains), presumably due to bacterial burden due to enhanced p24 expression. (FIG. 2C).
- BMDCs were infected with different M.O.I. (1 and 10 M.O.I.) of rBCG-pAL-p24 and rBCG-pMyong2-p24 for 1 and 3 days.
- M.O.I. 1 and 10 M.O.I.
- rBCG-pMyong2-p24 induced more p24 expression than the rBCG-pAL-p24 strain (FIG. 3I).
- rBCG-pMyong2-p24 increased the production of p24 in infected antigen presenting cells (APC) and bacteria, compared to the other two rBCG strains, rBCG-pAL-p24 and rBCG-pMV306-p24.
- Example 2 BMDCs Infected with rBCG-pMyong2-p24 Strain Induces Enhanced T Cell Proliferation in Mice Immunized with HIV-1 p24 Gag
- rBCG-pMyong2-p24 showing improved p24 protein production can improve T cell proliferation ability
- four different strains wild type BCG (as a control), two types of rBCG ( T cell proliferation in BMDCs infected with rBCG-pMyong2-p24 and rBCG-pAL-p24), and rSmeg-pMyong2-p24, respectively, was analyzed using the CFSE dilution method through mixed lymphocyte response (MLR).
- MLR mixed lymphocyte response
- FIG. 4A A schematic of the T cell proliferation assay is shown in FIG. 4A.
- BMDCs infected with two rBCG and one rSmeg strains induced significantly higher levels of CD4 and CD8 T cell proliferation than uninfected BMDCs.
- BMDCs infected with rBCG-pMyong2-p24 showed significantly higher levels of CD4 and CD8 T cell proliferation than those infected with the other two recombinant strains (rBCG-pAL-p24 and rSmeg-pMyong2-p24 strains) and wild-type BCG strains. Induced.
- rBCG-pMyong2-p24 and -pAL-p24 two types of rBCG strains (rBCG-pMyong2-p24 and -pAL-p24), rSmeg-pMyong2- Splenocytes were isolated from spleens of BALB / c mice (5 mice / group) subcutaneously immunized with p24 (FIG. 5A) and wild type BCG strain ( ⁇ 10 6 CFU) as a control and using IFN- ⁇ ELISPOT assay HIV-1 p24 Gag-specific T cell responses were analyzed.
- Splenocytes from mice subcutaneously immunized with three recombinant strains showed significantly higher SFUs than splenocytes from mice immunized with wild type BCG strain.
- splenocytes harvested from mice immunized with rBCG-pMyong2-p24 (987.78 ⁇ 195.11 SFUs / 10 6 splenocytes) showed two different strains: rBCG-pAL-p24 (479.56 ⁇ 213.90 SFUs / 10 6 splenocytes).
- the data of the present invention indicate that rBCG-pMyong2-p24 induced enhanced HIV-1 p24 Gag-specific production of IFN- ⁇ , a Th-1 signature cytokine, and by distorting the Th-1 type immune response Show feasibility to improve vaccine efficacy.
- splenocytes (5 mice / group) (FIG. 5A) obtained were stimulated in vitro with three sets of purified p24 protein (5 ⁇ g / ml). Induced cytokine production of IL-2, IFN- ⁇ and IL-6 in cell culture supernatants was detected.
- rBCG-pMyong2-p24 strain In two types of Th1 cytokines, IL-2 and IFN- ⁇ , and one pro-inflammatory cytokine, IL-6, the rBCG-pMyong2-p24 strain is always more than in wild type or two other recombinant strains, At all time points (Days 1 and 3), higher levels of cytokines were produced in splenocytes obtained from vaccinated mice (FIG. 5C and Table 1).
- rBCG-pMyong2-p24 showed significantly higher levels of total IgG than the other two recombinant strains (ie rBCG-pAL-p24 and rSmeg-pMyong2-p24) (FIG. 6).
- the higher IgG2a / IgG1 ratio indicates a more Th1-biased humoral immune response, which ratio is different from other strains in serum taken from mice immunized with rBCG-pMyong2-p24 (1.03 ⁇ 0.02).
- rBCG-pMyong2-p24 induces an enhanced HIV-1 p24 Gag-specific cytotoxic T lymphocyte (CTL) response in immunized mice
- CTL cytotoxic T lymphocyte
- two rBCGs rBCG-pMyong2-p24, -pAL-p24
- CTL activity in splenocytes from mice immunized with rSmeg-pMyong2-p24 or wild type BCG strain was assessed by LDH cytotoxicity assay.
- the immunization process is described in Figure 5a.
- P815 cells (H-2d) pulsed with A9I peptide for 2 hours were used as target cells and effector / target ratios were 10: 1, 20: 1, and 50: 1 as described above.
- Example 7 rBCG-pMyong2-p24 Strain Induces Improved HIV-1 p24 Gag-Specific Humoral and Cell Mediated Immune Responses in Immunized Mice Compared with p24 Protein Vaccination
- p24 specific IFN- ⁇ SFU was increased in a CFU dependent manner. However, splenocytes from mice injected with the p24 protein were unable to induce p24 specific IFN- ⁇ SFU (FIG. 8A). Similarly, p24 specific IgG2a antibodies also increased in a CFU dependent manner in serum samples from each immunized mouse. However, p24 specific IgG2a antibodies in the serum of mice injected with p24 protein showed lower levels compared to those of the rBCG-pMyong2-p24 injection group (FIG. 8B).
- HIV- 1 shows that it can have advantages as a vaccine curing method.
Abstract
The present invention relates to a recombinant BCG employing a pMyong2 vector system to express HIV-1 p24 and a use thereof as a HIV-1 vaccine. rBCG-pMyong2-p24, which is a pMyong2 vector system, was found to induce the upregulation of HIV-1 p24 gag expression in rBCG and infected antigen-presenting cells (APC) and to induce improved p24-specific immune responses in vaccinated mice, compared to rBCG-pAL-p24, in vector system-derived pAL5000, as verified by high levels of HIV-1 gag-specific CD4 and CD8 T lymphocyte proliferation, gamma interferon ELISPOT cell induction, antibody production, and cytotoxic T lymphocyte (CTL) responses. rBCG-pMyong2-p24 was identified to exhibit a higher p24-specific Ab production level than rSmeg-pMyong2-p24 in the same pMyong2 vector system. Therefore, the recombinant BCG employing rBCG-pMyong2-p24 to express HIV-1 p24 according to the present invention is identified to induce improved immune responses to HIV-1 infection in mouse model systems and thus can be expected to be used as a main vaccine in the heterogeneous prime-boost vaccination strategy against HIV-1 infection.
Description
본 발명은 인간면역결핍바이러스 타입-1(human immunodeficiency virus type-I, HIV-1) 감염에 대한 예방 및/또는 치료에 사용되는 신규한 재조합 BCG, 이를 포함하는 백신 조성물, 이의 제조 방법 등에 관한 것이다. 더욱 상세하게는, 본 발명은 신규한 마이코박테리아(
mycobacterium)-대장균(
Escherichia-coli)용 셔틀 벡터 (이하 “pMyong2”, 대한민국특허등록번호: 1012916680000, 미국특허등록번호: 8,841,432) 시스템을 사용한 재조합 BCG 생백신 플랫폼(live vaccine platform)에 관한 것이다.The present invention relates to novel recombinant BCGs used in the prevention and / or treatment of human immunodeficiency virus type-1 (HIV-1) infections, vaccine compositions comprising them, methods for their preparation, and the like. . More specifically, the present invention provides a recombinant BCG using a novel shuttle vector for mycobacterium -E. Coli (Esherichia -coli ) (hereinafter “pMyong2”, Korean Patent No .: 1012916680000, US Patent No .: 8,841,432). It relates to a live vaccine platform.
본 출원은 2018년 05월 14일에 출원된 대한민국 특허출원 제 10-2018-0055059호 및 2019년 03월 26일에 출원된 대한민국 특허출원 제 10-2019-0034183 호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다.This application claims the priority based on Korean Patent Application No. 10-2018-0055059, filed May 14, 2018 and Korean Patent Application No. 10-2019-0034183, filed March 26, 2019, All content disclosed in the specification and drawings of an application is incorporated in this application.
HIV-1 감염률은 전세계적으로 점차 감소하고 있는 추세지만, HIV-1에 대한 효율적인 예방 백신은 여전히 시급한 요구사항이다. 재조합 마이코박테리움 보비스(
Mycobacterium bovis) BCG(rBCG)는 HIV-1 백신 개발에서 유망한 균주이다. 이에 본 발명에서는 HIV-1 감염에 대한 백신 적용(vaccine application)에서 pMyong2 벡터를 이용한 HIV-1 p24 항원 Gag를 발현하는 rBCG(rBCG-pMyong2-p24)의 잠재력을 개시한다.HIV-1 infection rates are gradually decreasing worldwide, but an effective prophylactic vaccine against HIV-1 is still an urgent requirement. Recombinant Mycobacterium bovis BCG (rBCG) is a promising strain in the development of HIV-1 vaccines. Accordingly, the present invention discloses the potential of rBCG (rBCG-pMyong2-p24) expressing HIV-1 p24 antigen Gag using a pMyong2 vector in a vaccine application against HIV-1 infection.
HIV 감염 개체에서 HIV의 복제를 제어하는 고활성항바이러스 요법(highly activated antiretroviral therapy, HAART)의 기여에도 불구하고, 장기간의 치료 후 발생하는 약물-내성 바이러스 및 값비싼 약제의 비용 등은 여전히 해결되어야 할 여러 문제점들 중 하나로 남아 있다. 그러므로, 비록 새로운 HIV-1 감염 속도가 전세계적으로 점차 감소하고 있기는 하지만, 바이러스의 추가 확산을 억제하기 위하여 효율적인 예방 백신의 필요성은 여전히 요구되고 있다. HIV 특이적 세포성 면역은 주로 다기능성(poly-functionality), 그리고 면역우성(immunodominant) 바이러스 펩타이드에 대한 증식능력을 갖는 HIV 특이적 T 세포에 의해 유도되며, 이러한 특징으로 인해 HIV-1에 대한 효율적인 면역 반응이 발생할 수 있으므로 이러한 세포성 면역, 특히 바이러스-특이적 세포독성 T 림프구 (cytotoxic T lymphocytes, CTL)는 HIV-1에 대항하기 위한 숙주 면역 체계의 더 중요한 구성요소이어야 한다.Despite the contribution of highly activated antiretroviral therapy (HAART) to control the replication of HIV in HIV-infected individuals, the costs of drug-resistant viruses and expensive drugs that occur after prolonged treatment still need to be addressed. It remains one of several problems to do. Therefore, although the rate of new HIV-1 infection is gradually decreasing worldwide, there is still a need for an effective prophylactic vaccine to inhibit further spread of the virus. HIV specific cellular immunity is primarily induced by HIV specific T cells with poly-functionality and proliferative capacity against immunodominant viral peptides, and because of this characteristic they are effective against HIV-1. These cellular immunity, particularly virus-specific cytotoxic T lymphocytes (CTLs), should be a more important component of the host immune system against HIV-1 as immune responses may occur.
이러한 사실을 기반으로, 살아있는 바이러스 벡터 및 플라스미드 DNA 백신의 사용을 포함하는 강력한 HIV-1-특이적 CTL 및 Th1(Type 1 helper T cell)의 반응을 이끌어내 위한 여러 전략들이 개발 중에 있다. 그러나 이러한 각각의 접근법들에는 안전상의 쟁점을 포함한 여러 문제점들이 관련되어 있어 실제적, 실용적 사용이 제한되고 있다. 현재 전세계적으로 가장 광범위하게 투여되는 백신인 마이코박테리움 보비스 BCG(BCG)는 결핵(tuberculosis, TB)에 대항하기 위해 사용된 유일한 살아있는 약독화 백신으로서 80년 이상 사용되어 왔다. BCG는 아동의 파종성 질환(disseminated disease)을 예방할 수 있기 때문에, 1970년대 초반부터 아동 예방접종을 위한 세계보건기구 예방접종 확대계획(World Health Organization Expanded Program on Immunization(EPI))의 일부로서 사용되어 왔다.Based on this fact, several strategies are under development to elicit responses of potent HIV-1-specific CTLs and Type 1 helper T cells (Th1), including the use of live viral vectors and plasmid DNA vaccines. However, each of these approaches involves a number of problems, including safety issues, which limit practical and practical use. Mycobacterium bovis BCG (BCG), currently the most widely administered vaccine worldwide, has been used for more than 80 years as the only live attenuated vaccine used to fight tuberculosis (TB). Since BCG can prevent disseminated disease in children, it has been used as part of the World Health Organization Expanded Program on Immunization (EPI) for child vaccination since the early 1970s. come.
외래 항원을 발현하고 면역 반응을 유도하기 위해 성공적으로 사용되어온 BCG의 재조합 형태, 즉 rBCG는 보렐리아 부르그도르페리(
Borrelia burgdorferi), 스트렙토코쿠스 뉴모니아에(
Streptococcus pneumoniae), 보르데텔라 페르투시스(
Bordetella pertussis), 설치류 말라리아(
rodent malaria), 레슈마니아(
leishmanial), 홍역 바이러스, 인간면역결핍바이러스 1형(HIV-1) 및 유인원 면역결핍 바이러스(SIV)를 포함한, 다양한 감염성 병원체에 대한 백신 후보로 여겨져 왔다. rBCG 벡터의 가장 실제적·실용적 장점은 높은 안전성이다. 더불어 rBCG는 우수한 면역증강(adjuvant) 특성을 보이며, 적어도 1 내지 2년 동안 유지되는 긴 지속성 세포성 면역 반응을 유도하고, 제조비용이 낮으며, 투여가 용이하고, 대체로 단지 1회 또는 2회의 면역화만을 필요로 한다. 따라서 다른 재조합 백신 접근법들을 능가하는 rBCG-기반 백신의 상기 언급된 장점들은 rBCG가 안전한, 바이러스-특이적 면역 반응을 유도할 수 있는, HIV-1 감염에 대한 강력한 백신일 수 있음을 시사한다.Recombinant forms of BCG, which have been successfully used to express foreign antigens and induce immune responses, ie rBCG, are Borrelia burgdorferi , Streptococcus pneumoniae , Bordetella perturtu system (Bordetella pertussis), a rodent malaria (rodent malaria), resyu mania (leishmanial), measles virus, human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency including virus (SIV), vaccines against a variety of infectious agents It has been considered a candidate. The most practical and practical advantage of rBCG vectors is their high safety. In addition, rBCG exhibits good adjuvant properties, induces a long-lasting cellular immune response that persists for at least one to two years, is low in manufacturing cost, easy to administer, and usually only one or two immunizations. Need only. Thus the above-mentioned advantages of rBCG-based vaccines over other recombinant vaccine approaches suggest that rBCG can be a potent vaccine against HIV-1 infection, which can induce a safe, virus-specific immune response.
본 발명의 해결하고자 하는 과제는 HIV-1 유래의 p24 단백질을 발현하는 재조합 마이코박테리움 보비스(
Mycobacterium bovis) BCG (rBCG) 및 이를 HIV-1 백신으로 이용을 제공하고자 하는 것이다. The problem to be solved of the present invention is to provide a recombinant Mycobacterium bovis BCG (rBCG) expressing a p24 protein derived from HIV-1 and use thereof as an HIV-1 vaccine.
rBCG 벡터는 잠재적인 HIV-1 백신으로서의 가능성에도 불구하고, 이의 HIV-1 백신으로서의 실제적 적용은 rBCG 내에서 외래 유전자들의 이종성(heterologous) 발현의 낮은 안정성 및 양의 결핍으로 인한 낮은 면역원성(immunogenicity)으로 인해 제한된다. 그러므로 충분한 면역원성을 확보하고 방어적 백신의 효능을 끌어내기 위해서는, 인간에서 결핵에 대한 실제 BCG 예방접종에 필요한 용량의 대략 10 내지 100 배의 높은 rBCG 용량이 필요하다. 그러나 고용량의 BCG를 생체내로 투여하는 것은 면역-손상된(immuno-compromised) 백신 투여자의 파종성 BCG의 위험을 증가시키거나, T 세포의 과도한 활성화에 의한 HIV-1의 복제 및 확산을 위한 구동력으로서 작용할 수 있다.Despite the potential of rBCG vectors as potential HIV-1 vaccines, their practical application as HIV-1 vaccines has shown low immunogenicity due to lack of stability and low stability of heterologous expression of foreign genes in rBCG. Limited due to Therefore, to obtain sufficient immunogenicity and draw on the efficacy of protective vaccines, a high rBCG dose of approximately 10 to 100 times the dose required for actual BCG vaccination against tuberculosis in humans is required. In vivo administration of high doses of BCG, however, may increase the risk of disseminated BCG of immuno-compromised vaccine recipients or act as a driving force for replication and spread of HIV-1 by excessive activation of T cells. Can be.
이러한 이유로 안전성 보장을 위해 HIV-1에 대한 예방적 백신접종에서 rBCG의 저용량 면역화가 권장되어 왔고, 따라서 본 발명에서는 인간의 예방접종에 필요한, 더 낮은 용량에서도 효력을 보일 수 있는, HIV-1으로부터의 보호를 위한 rBCG 백신을 개발하고자 하였다. For this reason, low dose immunization of rBCG in prophylactic vaccination against HIV-1 has been recommended to ensure safety, and therefore the present invention is directed from HIV-1, which may be effective at lower doses required for human vaccination. An attempt was made to develop an rBCG vaccine for protection.
또한 상기 재조합 마이코박테리움 보비스 BCG의 치료학적 유효량을 유효성분으로 포함하는 백신 조성물을 개체에 투여하여 에이즈 및/또는 결핵을 치료 또는 예방하는 방법, 상기 백신을 제조하기 위한 재조합 마이코박테리움 보비스 BCG의 용도 내지 에이즈 및/또는 결핵을 치료 또는 예방하기 위한 재조합 마이코박테리움 보비스 BCG의 용도를 개발하고자 하였다.In addition, a method of treating or preventing AIDS and / or tuberculosis by administering to a subject a vaccine composition comprising a therapeutically effective amount of the recombinant Mycobacterium bovis BCG as an active ingredient, a recombinant Mycobacterium bovis for preparing the vaccine Use of BCG to the use of recombinant Mycobacterium bovis BCG for treating or preventing AIDS and / or tuberculosis.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned can be clearly understood by those skilled in the art from the following description. There will be.
상술한 과제를 해결하기 위하여, 본 발명자들은 마이코박테리움 아비움 복합체(M. avium complex, MAC)의 인간 병원체 구성원인 마이코박테리움 용고넨스(
Mycobacterium yongonense)의 DSM 45126
T 게놈 분석을 통해, 마이코박테리움 포르투이툼(M. fortuitum)으로부터 유래된 종래의 pAL5000 벡터 시스템을 사용한 것과 비교하여, 재조합 마이코박테리아 스메그마티스(rSmeg) 및 rBCG에서 증가된 이종성 유전자 발현을 유도할 수 있는 선형 플라스미드인 pMyong2의 마이코박테리아 레플리콘(replicon)을 사용하는 신규한 마이코박테리아-대장균 셔틀 벡터 시스템을 도입하였다.In order to solve the above problems, the present inventors through the DSM 45126 T genomic analysis of Mycobacterium yongonense , a human pathogen member of the Mycobacterium avium complex ( MA ), Compared with the conventional pAL5000 vector system derived from Mycobacterium M. fortuitum, it is a linear plasmid capable of inducing increased heterologous gene expression in recombinant mycobacterial smegmatis (rSmeg) and rBCG. A new mycobacterial-E. coli shuttle vector system using a mycobacterial replicon of pMyong2 was introduced.
나아가 본 명세서에서는 pMyong2 벡터 시스템을 사용하여 HIV-1 p24 Gag를 발현하는 rSmeg가, pAL5000 벡터 시스템에서 또는 통합형 플라스미드인 pMV306 시스템을 사용하는 rSmeg와 비교하여, 마우스에서 HIV-1 p24 Gag에 대한 향상된 면역 반응을 유도하였음을 보였고, 이것은 rSmeg 백신 적용에서 pMyong2 벡터 시스템의 실행가능성을 시사한다.Furthermore, in this specification, rSmeg expressing HIV-1 p24 Gag using the pMyong2 vector system is enhanced immunity to HIV-1 p24 Gag in mice compared to rSmeg in the pAL5000 vector system or using the integrated plasmid pMV306 system. Response was induced, suggesting the viability of the pMyong2 vector system in rSmeg vaccine application.
HIV-1 Gag-특이적 CD8+ T 세포 반응은 HIV 감염의 면역 조절에 대단히 중요할 수 있다. 따라서 본 발명에서는 pMyong2 벡터 시스템에서 발현하기 위한 표적 항원으로서 HIV-1 Gag p24를 선택하였다. 그러므로, 본 발명에서는 rBCG(rBCG-pMyong2-p24) 내에서 외래 HIV-1 p24 Gag 유전자의 발현을 향상시키기 위하여 pMyong2 벡터 시스템을 채택하였다. pMyong2 벡터 시스템의 효능, 즉 그것의 향상된 재조합 단백질 제조는 rBCG 및 rBCG로 감염된 일차 골수유래 수지상 세포(bone-marrow derived dendritic cells, 이하 "BMDCs")에서 증명되었다. 더불어, 백신 효능을 증명하기 위하여, 본 발명자들은 예방접종된 마우스에서 HIV Gag 단백질에 대한 그것의 세포성 및 체액성 면역 반응을 탐색하였다.HIV-1 Gag-specific CD8 + T cell responses can be of great importance for immune regulation of HIV infection. Therefore, in the present invention, HIV-1 Gag p24 was selected as a target antigen for expression in the pMyong2 vector system. Therefore, in the present invention, pMyong2 vector system was adopted to improve the expression of foreign HIV-1 p24 Gag gene in rBCG (rBCG-pMyong2-p24). The efficacy of the pMyong2 vector system, ie its improved recombinant protein production, has been demonstrated in rBCG and rBCG-infected bone-marrow derived dendritic cells ("BMDCs"). In addition, to demonstrate vaccine efficacy, we explored its cellular and humoral immune responses to HIV Gag protein in vaccinated mice.
HIV 백신으로서 rBCG의 잠재력을 개선하기 위한 최상의 전략은, 프라임-부스트(prime-booster) 백신 프로토콜에서 rBCG를 프라임 백신(prime vaccine)으로 사용하고 부스트 백신(booster vaccine)으로서 안전한 재조합 바이러스 벡터를 사용하는 것으로서, 이는 동물 모델에서 예방접종 후에 길게 지속되는 효율적인 바이러스-특이적 세포성 면역을 유도한다. 이러한 맥락에서, rBCG 벡터에 의해 유도된 Th1 반응은 Gag-특이적 CTL 반응을 유도하는 데 기여할 수 있다. 그러나 HIV-1 백신으로서 rBCG의 실제적 사용에 주된 장벽이 되는 것은 rBCG에서 외래 HIV-1 항원의 낮은 발현으로 인하여 바이러스 감염에 대항하기 위한 충분한 바이러스-특이적 CTL 반응을 유도하지 못한다는 것이다.The best strategy for improving the potential of rBCG as an HIV vaccine is to use rBCG as a prime vaccine in a prime-booster vaccine protocol and to use a safe recombinant viral vector as a boost vaccine. As a result, this induces efficient virus-specific cellular immunity that persists long after immunization in animal models. In this context, the Th1 response induced by the rBCG vector may contribute to inducing Gag-specific CTL responses. However, a major barrier to the practical use of rBCG as an HIV-1 vaccine is that the low expression of foreign HIV-1 antigen in rBCG does not elicit sufficient virus-specific CTL responses to fight viral infection.
이러한 한계를 극복하기 위하여, rBCG의 우선적 세포질 위치를 통해 더 큰 CTL 반응을 유도할 수 있는 용혈소(hemolysin)-발현 BCG 균주의 사용 및 rBCG 시스템에서 HIV-1 gag p24 유전자에 대한 코돈 최적화를 포함한 전략을 이용, 본 발명에서는 rBCG에서 HIV-1 gag p24 유전자의 발현을 향상시키기 위하여 pMyong2 셔틀벡터 시스템을 적용하였다. To overcome this limitation, strategies include the use of hemolysin-expressing BCG strains capable of eliciting larger CTL responses through the preferential cytoplasmic location of rBCG and codon optimization for HIV-1 gag p24 gene in the rBCG system. In the present invention, the pMyong2 shuttle vector system was applied to enhance the expression of HIV-1 gag p24 gene in rBCG.
본 발명에서는 비교를 위해 rBCG-pMyong2-p24가 감염된 대식세포 및 BMDCs에서 종래의 에피솜성(episomal) pAL5000 벡터(rBCG-pAL-p24) 및 통합형 pMV306 벡터(rBCG-pMV306-p24)(도 3a, 3b 및 3h)에서보다 더 많은 p24 단백질을 생성한 것을 확인하였고, BMDCs의 향상된 p24 특이적 T 세포 증식(도 4b 및 4c), T 세포 이펙터(effector) 기능(도 5b 및 5c), 특히 CTLs(도 7), 및 Th1-바이어스된 체액성 면역 반응(도 6)을 포함하여, rBCG-pMyong2-p24의 향상된 바이러스-특이적 백신 효능에 대한 기계론적 기초를 제공하였다.In the present invention, for comparison, conventional episomal pAL5000 vector (rBCG-pAL-p24) and integrated pMV306 vector (rBCG-pMV306-p24) in rBCG-pMyong2-p24-infected macrophages and BMDCs (FIG. 3A, 3B). And 3h), more p24 protein was produced than in BMDCs, with enhanced p24 specific T cell proliferation (FIGS. 4B and 4C), T cell effector function (FIGS. 5B and 5C), in particular CTLs (FIG. 7), and Th1-biased humoral immune response (FIG. 6), providing a mechanistic basis for the enhanced virus-specific vaccine efficacy of rBCG-pMyong2-p24.
본 발명에서는 rBCG-pAL-p24보다 더 낮은 수준의 CFU(colony-forming unit)를 생성한 rBCG-pMyong2-p24에서의 대식세포 감염에서 더 약독화되는 경향이 관찰되었는데(도 2c), 이는 다른 벡터 시스템에서의 그것들보다 rBCG에서 pMyong2의 더 높은 복사물 수로 인한 것으로 추측된다. 더 낮은 용량 또는 보다 약독화(attenuation)된 rBCG로의 면역화는, 불리한 국소 피부 반응, Th2-형 면역 반응과의 가능한 결부, 또는 레트로바이러스 감염의 악화를 포함한 고-투여량 피부 투여와 관련된 위험을 줄일 수 있을 것이라는 점을 고려하면, rBCG-pMyong2-p24는 rBCG를 사용하는 HIV 백신 프로토콜에서 추가적 장점을 가질 수 있다.In the present invention a tendency to be more attenuated in macrophage infection in rBCG-pMyong2-p24 that produced lower levels of CFU (colony-forming unit) than rBCG-pAL-p24 was observed (FIG. 2C), which is another vector. Presumably due to the higher copy number of pMyong2 in rBCG than those in the system. Immunization with lower doses or more attenuated rBCGs reduces the risks associated with high-dose skin administration, including adverse local skin reactions, possible association with Th2-type immune responses, or exacerbation of retroviral infections. Considering that it may be possible, rBCG-pMyong2-p24 may have additional advantages in the HIV vaccine protocol using rBCG.
다중-복사물 에피솜성 벡터-기반 마이코박테리아-대장균 셔틀 벡터 시스템은, 통합형 플라스미드 시스템과 비교하여, 재조합 마이코박테리아의 안정성 결핍과 관련된 결점을 가지는 것으로 알려져 있다. 실제로, rSmeg (rSmeg-pMyong2-p24)에서의 pMyong2-p24 플라스미드가 항생물질이 없는 매질에서 5회 계대 후에 안정성을 점차 잃어가는 것을 보였다. 그러나 놀랍게도, 본 발명은 동일한 pMyong2 벡터 시스템을 사용함에도 불구하고, rBCG (rBCG-pMyong2-p24)에서 pMyong2-p24 플라스미드가 항생물질의 첨가 여부와 관계없이 12회의 계대 후에도 그것의 안정성을 유지할 수 있다(도 3f). 이는 느리게 성장하는 마이코박테리아 용고넨스로부터 얻은 pMyong2 플라스미드가, Smeg와 같은 빠르게 성장하는 마이코박테리아의 경우 보다 BCG와 같은 느리게 성장하는 마이코박테리아에서 더 안정적일 수 있음을 시사한다. 항생물질이 없는 배지에서 플라스미드를 통합시키는 것의 안정성이 실제 사용하기 위한 재조합 생백신 제조에 중요한 것임을 고려할 때, rBCG-pMyong2-p24는 HIV-1 백신으로서의 적용에서 rSmeg-pMyong2-p24를 능가하는 장점을 가진다.Multi-copy episomal vector-based Mycobacterial Escherichia coli shuttle vector systems are known to have drawbacks associated with the lack of stability of recombinant Mycobacteria compared to integrated plasmid systems. Indeed, the pMyong2-p24 plasmid in rSmeg (rSmeg-pMyong2-p24) has been shown to gradually lose stability after five passages in an antibiotic-free medium. Surprisingly, however, the present invention, despite the use of the same pMyong2 vector system, allows pMyong2-p24 plasmid in rBCG (rBCG-pMyong2-p24) to maintain its stability even after 12 passages, whether or not antibiotics are added ( 3f). This suggests that the pMyong2 plasmid obtained from the slow-growing mycobacteria yonggonens may be more stable in slow-growing mycobacteria such as BCG than for the fast-growing mycobacteria such as Smeg. Given that the stability of integrating plasmids in antibiotic-free media is important for the production of recombinant live vaccines for practical use, rBCG-pMyong2-p24 has the advantage over rSmeg-pMyong2-p24 in applications as an HIV-1 vaccine. .
본 발명에서는 상이한 에피솜성 벡터 시스템에서의 rBCG 균주들, 즉 rBCG-pMyong2-p24 및 rBCG-pAL-p24을 사용하는 것에 더하여, 2개의 상이한 마이코박테리아에서의 HIV-1에 대한 백신 효능, 즉 BCG(rBCG-pMyong2-p24) 및Smeg(rSmeg-pMyong2-p24)를 동일한 pMyong2 시스템을 사용하여 비교하였다. HIV-1 p24 항원에 대한 면역 반응에서, 비록 면역화된 비장세포(splenocytes)로부터의 CTL 반응, 감염된 BMDCs의 T 세포 증식 능력 및 대부분의 IFN-γ ELISPOT 수준이 rBCG-pMyong2-p24 및 rSmeg-pMyong2-p24에서 거의 동일하긴 하였지만, rBCG-pMyong2-p24는 rSmeg-pMyong2-p24와 비교하여 비장세포 및 Th1-바이어스된 체액성 면역 반응에서 상당히 향상된 IL-2 생성을 나타냈고, 이것은 rBCG-pMyong2-p24가 rSmeg-pMyong2-p24보다 HIV-1 백신 양생법(regimen)에서 월등할 수 있음을 시사한다.In the present invention, in addition to using rBCG strains in different episomal vector systems, ie rBCG-pMyong2-p24 and rBCG-pAL-p24, vaccine efficacy against HIV-1 in two different mycobacteria, namely BCG ( rBCG-pMyong2-p24) and Smeg (rSmeg-pMyong2-p24) were compared using the same pMyong2 system. In the immune response to HIV-1 p24 antigen, although CTL response from immunized splenocytes, T cell proliferative capacity of infected BMDCs and most IFN-γ ELISPOT levels were rBCG-pMyong2-p24 and rSmeg-pMyong2- Although nearly identical at p24, rBCG-pMyong2-p24 showed significantly improved IL-2 production in splenocytes and Th1-biased humoral immune responses compared to rSmeg-pMyong2-p24, which showed that rBCG-pMyong2-p24 This suggests that HIV-1 vaccine regimen may be superior to rSmeg-pMyong2-p24.
더불어, 본 발명에서는 rBCG-pMyong2-p24 및 p24 단백질을 사용하여 2개의 상이한 백신 모듈 사이에서 HIV-1에 대한 백신 효능을 비교하였다. 본 명세서의 데이터는 rBCG-pMyong2-p24가 p24 단백질과 비교하여 향상된 p24 특이적 IFN-γ ELISPOT 수준, CTL 반응 및 Th1-바이어스된 체액성 면역 반응을 가졌음을 나타내며(도 8a~8c), 이는 또한 HIV-1 백신 양생법에서 rBCG-pMyong2-p24가 p24 단백질보다 월등할 수 있음을 시사한다. BCG, 홍역, 유행성이하선염 및 풍진 백신, 및 인플루엔자 백신을 포함하여, 다양한 백신에 대한 반응에는 성별의 차이가 있는 것으로 알려져 있다. 일반적으로, 적응성 면역 반응에서, 여성은 남성과 비교하여 향상된 체액성 및 세포 매개 면역 반응을 나타낸다. 이는 본 발명에서 현재 백신 연구에 오로지 암컷 마우스만을 선택한 이유이다.In addition, the present invention compared the vaccine efficacy against HIV-1 between two different vaccine modules using rBCG-pMyong2-p24 and p24 proteins. The data herein indicate that rBCG-pMyong2-p24 had improved p24 specific IFN-γ ELISPOT levels, CTL response and Th1-biased humoral immune response compared to p24 protein (FIGS. 8A-8C), which also showed The HIV-1 vaccine curing suggests that rBCG-pMyong2-p24 may be superior to the p24 protein. Gender differences are known to respond to various vaccines, including BCG, measles, mumps and rubella vaccines, and influenza vaccines. In general, in the adaptive immune response, women exhibit an improved humoral and cell mediated immune response compared to men. This is the reason why only female mice were selected for current vaccine studies in the present invention.
본 명세서에서는 pMyong2 벡터 시스템의 rBCG-pMyong2-p24가, pAL5000-(rBCG-pAL-p24) 또는 pMV306-유래 시스템 (rBCG-pMV306-p24)을 사용하는 다른 BCG 균주들보다 rBCG에서 고수준의 HIV-1 p24 Gag 단백질 발현을 유도하고 더 많은 p24 항원을 포식세포(phagocytes)로 전달하는 것을 증명하였다. In the present specification, rBCG-pMyong2-p24 of the pMyong2 vector system has higher levels of HIV-1 in rBCG than other BCG strains using pAL5000- (rBCG-pAL-p24) or pMV306-derived system (rBCG-pMV306-p24). It has been demonstrated to induce p24 Gag protein expression and to deliver more p24 antigen to phagocytes.
또한, 본 명세서에서는 상기 언급된 균주가 rBCG-pAL-p24 또는 rSmeg-pMyong2-p24와 비교하여, 예방 접종된 마우스에서 감염된 BMDCs의 T 세포 증식 능력을 향상시킬 수 있고 개선된 CTL 반응 및 Th1 바이어스된 체액성 면역 반응을 유도할 수 있음을 보였다.In addition, the above-mentioned strains can also improve the T cell proliferative capacity of infected BMDCs in vaccinated mice and provide improved CTL response and Th1 bias compared to rBCG-pAL-p24 or rSmeg-pMyong2-p24. It has been shown that it can induce a humoral immune response.
이러한 발견들은 rBCG-pMyong2-p24가 HIV-1 감염에 대한 이종성 프라임-부스트 백신 전략에서 프라임 백신으로서 효율적인 후보일 수 있음을 시사한다.These findings suggest that rBCG-pMyong2-p24 may be an efficient candidate as a prime vaccine in a heterologous prime-boost vaccine strategy for HIV-1 infection.
종합하면, 한 양태에서 본 발명은 HIV-1 유래의 p24 단백질을 발현하는 재조합 마이코박테리움 보비스 (
Mycobacterium bovis) BCG (rBCG)를 제공한다.Taken together, in one aspect the present invention provides recombinant Mycobacterium bovis BCG (rBCG) expressing a p24 protein derived from HIV-1.
일 구현예에서 상기 p24 단백질은 도 2a에 개시된 pMyong2-p24 벡터 시스템에 의해 발현되는 것일 수 있다. In one embodiment the p24 protein may be expressed by the pMyong2-p24 vector system disclosed in Figure 2a.
일 구현예에서 상기 p24 단백질은 서열번호 2의 염기 서열로 나타내는 인간면역결핍바이러스 타입 1 유래의 Gag 유전자가 코딩된 것일 수 있다. In one embodiment, the p24 protein may be encoded by a Gag gene derived from human immunodeficiency virus type 1 represented by the nucleotide sequence of SEQ ID NO: 2.
일 구현예에서 본 발명에 따른 마이코박테리움 보비스 BCG 균주는 Tokyo 172 균주이다.In one embodiment, the mycobacterium bovis BCG strain according to the present invention is Tokyo 172 strain.
또한, 본 발명에 따른 재조합 마이코박테리움 보비스 BCG를 유효성분으로 포함하는, HIV-1 백신 조성물을 제공한다. In addition, the present invention provides an HIV-1 vaccine composition comprising the recombinant mycobacterium bovis BCG as an active ingredient.
또한, 본 발명에 따른 재조합 마이코박테리움 보비스 BCG의 치료학적 유효량을 유효성분으로 포함하는 백신 조성물을 개체에 투여하여 에이즈 및/또는 결핵을 치료 또는 예방하는 방법을 제공한다.The present invention also provides a method for treating or preventing AIDS and / or tuberculosis by administering to a subject a vaccine composition comprising a therapeutically effective amount of a recombinant mycobacterium bovis BCG according to the present invention.
또한, 본 발명은 에이즈 및/또는 결핵의 예방 또는 치료용 백신의 제조를 위한, 재조합 마이코박테리움 보비스 BCG의 용도를 제공한다.The present invention also provides the use of recombinant Mycobacterium bovis BCG for the production of vaccines for the prevention or treatment of AIDS and / or tuberculosis.
또한, 본 발명은 에이즈 및/또는 결핵의 예방 또는 치료를 위한 재조합 마이코박테리움 보비스 BCG의 용도를 제공한다.The present invention also provides the use of recombinant Mycobacterium bovis BCG for the prevention or treatment of AIDS and / or tuberculosis.
또한, 본 발명에 따른 재조합 마이코박테리움 보비스 BCG를 유효성분으로 포함하는, HIV 감염증 또는 HIV 및 결핵균 동시 감염증에 대한 백신 조성물을 제공한다. In addition, the present invention provides a vaccine composition for HIV infection or HIV and Mycobacterium tuberculosis co-infectious disease comprising the recombinant mycobacterium bovis BCG as an active ingredient.
일 구현예에서 본 발명에 따른 백신 조성물에서 상기 재조합 마이코박테리움 보비스 BCG는 살아있는 것이다.In one embodiment the recombinant mycobacterium bovis BCG in the vaccine composition according to the invention is alive.
일 구현예에서 본 발명에 따른 백신은 추가로 인위적으로 약독화(attenuation)되지 않는 것이다. In one embodiment the vaccine according to the invention is further not artificially attenuated.
일 구현예에서 본 발명에 따른 백신은 특히 프라임-부스트 백신 접종 프로토콜에서 프라임 백신으로서 사용되는 것일 수 있다. In one embodiment the vaccine according to the invention may be one used in particular as a prime vaccine in a prime-boost vaccination protocol.
일 구현예에서 상기 감염증은 에이즈(AIDS, 후천성면역결핍증) 또는 결핵인 것일 수 있다. In one embodiment, the infection may be AIDS (AIDS, acquired immunodeficiency syndrome) or tuberculosis.
본 발명에 따른 rBCG-pMyong2-p24는 rBCG 및 감염된 항원 제시 세포(APC)에서 향상된 HIV-1 p24 Gag 발현을 이끌어 내는 것으로 나타났다.RBCG-pMyong2-p24 according to the present invention has been shown to elicit enhanced HIV-1 p24 Gag expression in rBCG and infected antigen presenting cells (APC).
또한, 본 발명에 따른 rBCG-pMyong2-p24는, pAL5000 유래 벡터 시스템에서의 rBCG-pAL-p24와 비교하여, 고수준의 HIV-1 Gag-특이적 CD4 및 CD8 T 림프구 증식, 감마 인터페론 ELISPOT 세포 유도, 항체 생성 및 세포독성 T 세포(CTL) 반응에 의해 증명되는, 향상된 p24 특이적 면역 반응을 유도하는 것으로 나타났다.In addition, rBCG-pMyong2-p24 according to the present invention, compared with rBCG-pAL-p24 in the pAL5000 derived vector system, high levels of HIV-1 Gag-specific CD4 and CD8 T lymphocyte proliferation, gamma interferon ELISPOT cell induction, It has been shown to induce an enhanced p24 specific immune response, evidenced by antibody production and cytotoxic T cell (CTL) responses.
나아가, 본 발명에 따른 rBCG-pMyong2-p24는 동일한 pMyong2 벡터 시스템에서 rSmeg-pMyong2-p24보다 높은 수준의 p24-특이적 항체를 생성함을 나타냈다.Furthermore, rBCG-pMyong2-p24 according to the present invention has been shown to produce higher levels of p24-specific antibodies than rSmeg-pMyong2-p24 in the same pMyong2 vector system.
결론적으로, 본 발명은 pMyong2 벡터 시스템을 사용하여 p24를 발현하는 재조합 BCG, 즉 rBCG-pMyong2-p24가 HIV-1 감염에 대한 향상된 면역 반응을 유도함을 나타낸다. 그러므로, rBCG-pMyong2-p24는 HIV-1 감염에 대한 이종성 프라임-부스트 백신 전략에서 프라임 백신으로서의 잠재력을 가진다.In conclusion, the present invention shows that recombinant BCG expressing p24, i.e. rBCG-pMyong2-p24, induces an enhanced immune response to HIV-1 infection using the pMyong2 vector system. Therefore, rBCG-pMyong2-p24 has the potential as a prime vaccine in a heterologous prime-boost vaccine strategy against HIV-1 infection.
도 1은 폴리아크릴아마이드 겔 전기 영동법(SDS-PAGE) 및 쿠마시 브릴리언트 블루 염색법(Coomassie brilliant blue staining)을 이용하여 p24 단백질의 순도를 확인한 도면이다. M, 분자량 표준(Elpis Bio, 대전, 대한민국). 레인 1, 정제된 p24 단백질 (10 μg).1 is a diagram confirming the purity of the p24 protein using polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining (Coomassie brilliant blue staining). M, molecular weight standard (Elpis Bio, Daejeon, Korea). Lane 1, purified p24 protein (10 μg).
도 2a는 본 명세서에서 개시하는 각각의 p24 발현 벡터의 개열지도를 나타난 도면이다. HIV p24 항원을 발현하는 마이코박테리아-대장균 셔틀 벡터로서 각각 pMV306-p24, pALp24 and pMyong2-p24 벡터가 마이코박테리움 보비스 BCG로부터 유래한
hsp65 프로모터의 조절 아래 p24를 발현한다.Figure 2a is a diagram showing a cleavage map of each p24 expression vector disclosed herein. As mycobacterial-E. Coli shuttle vectors expressing the HIV p24 antigen, the pMV306-p24, pALp24 and pMyong2-p24 vectors, respectively, express p24 under the control of the hsp65 promoter derived from Mycobacterium bovis BCG.
도 2b는 ADC와 100μg/ml의 카나마이신이 첨가 된 7H9 배지에서 p24 rBCG 균주의 성장 곡선을 나타낸 도면이다. 야생형 BCG 배양의 경우, 7H9 배지에서 카나마이신을 제외시켰다. 성장 곡선은 각 시점에서 배양 분취액을 취하고 OD600을 측정 하였다.Figure 2b is a diagram showing the growth curve of p24 rBCG strain in 7H9 medium to which ADC and 100μg / ml kanamycin is added. For wild type BCG culture, kanamycin was excluded from the 7H9 medium. Growth curves were taken at each time point in culture aliquots and measured OD600.
도 2c는 마우스 대식세포 J774A.1(왼쪽) 및 마우스 골수유래 수지상 세포(오른쪽)의 감염에서 p24 rBCG 균주의 CFU 수준을 비교하여 나타낸 도면이다. 데이터는 두 가지 독립적인 실험을 대표한다. (결과는 평균 ± 분산값으로 나타냈다. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test).FIG. 2C shows a comparison of CFU levels of p24 rBCG strains in infection of mouse macrophage J774A.1 (left) and mouse bone marrow-derived dendritic cells (right). FIG. The data represent two independent experiments. (The results are expressed as mean ± variance. * P <0.05; ** P <0.01; *** P <0.001 (Student's t-test).
도 3a는 ELISA를 사용하여 rBCG 균주에서의 p24 발현을 확인한 도면이다.Figure 3a is a diagram confirming the expression of p24 in rBCG strain using ELISA.
도 3b는 웨스턴 블롯팅 분석을 이용한 rBCG 균주의 p24 발현을 확인한 도면이다. 야생형 BCG (레인 1) 및 rBCG 균주 (레인 2: rBCG-pMV306-p24, 레인 3: rBCG-pAL-p24, 레인 4: rBCG-pMyong2-p24)로부터 단백질을 추출하였다. 양성 대조군으로는 정제된 p24 단백질을 사용하였다(레인 5). M, 분자량 표준(Elpis Bio, 대전, 대한민국). 개별 멤브레인은 흰색 공백으로 구분되며 마커 레인은 검정색 수직선으로 구분된다. 본 도면에는 p24의 발현 수준이 나타나 있으며 웨스턴 블롯팅 이미지는 선명도 향상을 위해 전체-길이 블롯에서 자른 것이다. 전체-길이 블롯팅 이미지는 하기 도 3c에 나타내었다.3b is a diagram confirming p24 expression of rBCG strain using Western blotting analysis. Proteins were extracted from wild type BCG (lane 1) and rBCG strains (lane 2: rBCG-pMV306-p24, lane 3: rBCG-pAL-p24, lane 4: rBCG-pMyong2-p24). As a positive control, purified p24 protein was used (lane 5). M, molecular weight standard (Elpis Bio, Daejeon, Korea). Individual membranes are separated by white blanks and marker lanes by black vertical lines. In this figure the expression level of p24 is shown and the Western blotting images are cropped in full-length blots to improve clarity. Full-length blotting images are shown in FIG. 3C below.
도 3c는 상기 도 2c의 자른 이미지의 전체 길이 원본 블롯팅 이미지를 나타낸 도면이다. 웨스턴 블롯팅은 본 도면에 표시한 항체를 이용하여 수행되었다. 자른 부분은 실선으로 표시하였다.FIG. 3C is a diagram illustrating a full length original blotting image of the cropped image of FIG. 2C. FIG. Western blotting was performed using the antibodies shown in this figure. The cut part is shown by the solid line.
도 3d는 카나마이신을 함유한 7H10 한천 플레이트(agar plate)에서 계대한 rBCG-pMyong2-p24 균주에서 p24 발현의 안정성을 웨스턴 블롯팅을 이용하여 확인한 도면이다. 단백질은 야생형 BCG (레인 1) 및 rBCG-pMyong2-p24 균주에서 각각의 계대 지점에서 추출하였다 (레인 2: 제 1계대, 레인 3: 제 4계대 후, 레인 4: 제 6계대 후, 레인 5: 제 8계대 후, 레인 6: 제 10계대 후, 레인 7: 제 12계대 후). 양성 대조군으로는 정제된 p24 단백질을 사용하였다 (레인 8). M, 분자량 표준(Elpis Bio, 대전, 대한민국). 상위 크기 멤브레인에서 내부 대조군으로서 멤브레인을 잘라낸 뒤 Hsp65 항체(Abcam)를 이용하여 p24를 검출하였다. 개별 멤브레인은 흰색 공백으로 구분되며 마커 레인은 검정색 수직선으로 구분된다.FIG. 3D shows the stability of p24 expression in rBCG-pMyong2-p24 strains passaged in 7H10 agar plate containing kanamycin using Western blotting. Protein was extracted at each passage point in wild type BCG (lane 1) and rBCG-pMyong2-p24 strains (lane 2: first passage, lane 3: after fourth passage, lane 4: after sixth passage, lane 5: After passage 8, lane 6: after passage 10, lane 7: after passage 12). As a positive control, purified p24 protein was used (lane 8). M, molecular weight standard (Elpis Bio, Daejeon, Korea). P24 was detected using an Hsp65 antibody (Abcam) after cutting the membrane as an internal control in the upper size membrane. Individual membranes are separated by white blanks and marker lanes by black vertical lines.
도 3e는 상기 도 2d의 자른 이미지의 전체 길이 원본 블롯팅 이미지를 나타낸 도면이다. 웨스턴 블롯팅은 본 도면에 표시한 항체를 이용하여 수행되었다. 자른 부분은 실선으로 표시하였다.FIG. 3E illustrates a full length original blotting image of the cropped image of FIG. 2D. Western blotting was performed using the antibodies shown in this figure. The cut part is shown by the solid line.
도 3f는 카나마이신이 없는 7H10 한천(agar) 플레이트에서 계대한 rBCG-pMyong2-p24 균주에서 p24 발현의 안정성을 웨스턴 블롯팅을 이용하여 확인한 도면이다. 단백질은 야생형 BCG (레인 1) 및 rBCG-pMyong2-p24 균주에서 각각의 계대 지점에서 추출하였다 (레인 2: 제 1계대, 레인 3: 제 4계대 후, 레인 4: 제 5계대 후, 레인 5: 제 6계대 후, 레인 6: 제 7계대 후, 레인 7: 제 8계대 후, 레인 8: 제 9계대 후, 레인 9: 제 10계대 후, 레인 10: 제 11계대 후, 레인 11: 제 12계대 후). 양성 대조군으로는 정제된 p24 단백질을 사용하였다 (레인 12). M, 분자량 표준(Elpis Bio, 대전, 대한민국). 상위 크기 멤브레인에서 내부 대조군으로서 멤브레인을 잘라낸 뒤 Hsp65 항체(Abcam)를 이용하여 p24를 검출하였다. 개별 멤브레인은 흰색 공백으로 구분되며 마커 레인은 검정색 수직선으로 구분된다.Figure 3f is confirmed by Western blotting the stability of p24 expression in rBCG-pMyong2-p24 strains passaged in 7H10 agar plate without kanamycin. Protein was extracted at each passage point in wild type BCG (lane 1) and rBCG-pMyong2-p24 strains (lane 2: first passage, lane 3: after fourth passage, lane 4: after fifth passage, lane 5: After the sixth passage, Lane 6: After the seventh passage, Lane 7: After the eighth passage, Lane 8: After the ninth passage, Lane 9: After the tenth passage, Lane 10: After the eleventh passage, Lane 11: The twelfth After passage). As a positive control, purified p24 protein was used (lane 12). M, molecular weight standard (Elpis Bio, Daejeon, Korea). P24 was detected using an Hsp65 antibody (Abcam) after cutting the membrane as an internal control in the upper size membrane. Individual membranes are separated by white blanks and marker lanes by black vertical lines.
도 3g는 상기 도 2f의 자른 이미지의 전체 길이 원본 블롯팅 이미지를 나타낸 도면이다. 웨스턴 블롯팅은 본 도면에 표시한 항체를 이용하여 수행되었다. 자른 부분은 실선으로 표시하였다.FIG. 3G illustrates a full length original blotting image of the cropped image of FIG. 2F. Western blotting was performed using the antibodies shown in this figure. The cut part is shown by the solid line.
도 3h는 마우스 대식세포 J774A.1(왼쪽) 및 마우스 골수유래 수지상 세포(오른쪽)에서 각각 야생형 BCG 및 rBCG 균주 (rBCG-pMV306-p24, -pAL-p24, 및 -pMyong2-p24) 감염 후, p24의 발현 수준을 측정한 도면이다. 데이터는 두 가지 독립적인 실험을 대표한다. (결과는 평균 ± 분산값으로 나타냈다. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test).3H shows p24 post infection with wild type BCG and rBCG strains (rBCG-pMV306-p24, -pAL-p24, and -pMyong2-p24) in mouse macrophage J774A.1 (left) and mouse myeloid derived dendritic cells (right), respectively. It is a figure which measured the expression level of. The data represent two independent experiments. (The results are expressed as mean ± variance. * P <0.05; ** P <0.01; *** P <0.001 (Student's t-test).
도 3i는 1일차 및 3일차에서 rBCG-pAL-p24 및 rBCG-pMyong2-p24 균주의 상이한 M.O.I.(1 및 10 M.O.I.; multiplicity of infection)에 감염된 BMDCs로부터 p24의 발현 수준을 ELISA를 이용하여 비교한 도면이다. 결과는 중복된 웰(duplicate wells)에서 평균 ± 분산값으로 나타냈다. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test).FIG. 3i shows the expression level of p24 from BMDCs infected with different MOIs (1 and 10 MOI; multiplicity of infection) of rBCG-pAL-p24 and rBCG-pMyong2-p24 strains at Days 1 and 3 using ELISA to be. Results are expressed as mean ± variance in duplicate wells. * P <0.05; ** P <0.01; *** P <0.001 (Student's t-test).
도 4a 내지 4d는 p24 rBCG 균주에 감염된 BMDCs에 의해 유도된 T 세포 증식 수준을 나타낸 도면들이다: (도 4a) T 세포 증식 분석 일정의 개략도. 마우스 2 마리에 p24 단백질(30㎍/마우스)을 주사하였고, 7일 후에 상기 마우스의 비장세포를 CD4 및 CD8 T 세포로 분류하고 CFSE로 표지하였다. 공동 배양하기 하루 전, DC를 각 균주(10 M.O.I.)에 감염시켰다. CFSE로 표지된 CD4 / CD8 T 세포 및 감염된 DC를 4일 동안 공동 배양한 후, 위 세포를 T 세포 증식에 대해 분석 하였다; (도 4b 및 4c) p24 rBCG 균주에 감염된 BMDCs의 CFSE-표지 CD4 및 CD8 T 세포 증식에 대한 유동세포계측분석(Flow cytometric analysis) 결과; (도 4d) MLR 분석법을 사용하여 CD4(좌측 패널) 및 CD8(우측 패널) 세포의 상등액에서 방출 된 IL-2의 ELISA 측정. 데이터는 세 가지의 독립적인 실험을 대표한다. 결과는 평균 ± 분산값으로 나타냈다. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test).4A-4D show T cell proliferation levels induced by BMDCs infected with the p24 rBCG strain: (FIG. 4A) Schematic of T cell proliferation assay schedule. Two mice were injected with p24 protein (30 μg / mouse) and after 7 days the splenocytes of the mice were sorted into CD4 and CD8 T cells and labeled with CFSE. One day before co-culture, DCs were infected with each strain (10 M.O.I.). After incubating CDSE / CD8 T cells labeled with CFSE and infected DCs for 4 days, gastric cells were analyzed for T cell proliferation; (FIGS. 4B and 4C) Flow cytometric analysis of CFSE-labeled CD4 and CD8 T cell proliferation of BMDCs infected with p24 rBCG strain; (FIG. 4D) ELISA measurement of IL-2 released from the supernatant of CD4 (left panel) and CD8 (right panel) cells using MLR assay. The data represent three independent experiments. The results are expressed as mean ± variance values. * P <0.05; ** P <0.01; *** P <0.001 (Student's t-test).
도 5a 내지 5c는 각각 p24 rBCG 균주에 의해 유도된 생체 내 면역 반응을 나타낸 도면이다: (도 5a) 생체 내 면역학적 분석을 위해 수행 된 면역화의 개략도. 4주 간격으로 각 군(5 마리/그룹)을 야생형 BCG, 두 종류의 rBCG 균주 및 rSmeg 균주로 각각 2회 면역시켰다. 최종 면역화 4주 후, 마우스를 희생시키고 면역 분석을 위해 비장 및 혈액 샘플을 수집하였다; (도 5b) p24 rBCG 균주를 접종한 마우스의 비장세포를 시험관 내 자극한 후 IFN-γ 분비 수준 ELISPOT 분석을 사용하여 검출하였다. 각 그룹의 ELISPOT 멤브레인 대표 이미지는 그래프 아래에 표시하였다. (-), 음성 대조군; (+), 양성 대조군; (도 5c) p24 rBCG 균주를 접종한 마우스의 비장세포를 p24로 시험관 내 자극한 후 IL-2, IFN-γ 및 IL-6 사이토카인(cytokine)의 수준을 ELISA 분석을 사용하여 검출하였다. 그룹 당 총 5마리의 마우스를 분석하였다. 데이터는 두 가지의 독립적인 실험을 대표한다. 결과는 평균 ± 분산값으로 나타냈다. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test).5A-5C show in vivo immune responses induced by p24 rBCG strains, respectively: (FIG. 5A) Schematic of immunization performed for in vivo immunological analysis. At 4 week intervals, each group (5 mice / group) was immunized twice with wild type BCG, two types of rBCG strains and rSmeg strains, respectively. Four weeks after the last immunization, mice were sacrificed and spleen and blood samples collected for immunoassay; (FIG. 5B) Splenocytes of mice inoculated with the p24 rBCG strain were detected using IFN-γ secretion level ELISPOT assay after in vitro stimulation. Representative images of ELISPOT membranes for each group are shown below the graph. (-), Negative control; (+), Positive control; (FIG. 5C) Spleen cells of mice inoculated with p24 rBCG strains were stimulated in vitro with p24 and levels of IL-2, IFN-γ and IL-6 cytokines were detected using ELISA analysis. A total of 5 mice per group were analyzed. The data represent two independent experiments. The results are expressed as mean ± variance values. * P <0.05; ** P <0.01; *** P <0.001 (Student's t-test).
도 6은 p24 rBCG 균주에 의해 유도된 체액성 면역 반응을 나타낸 도면이다. p24 특이 면역 글로불린 아형(IgG2a, IgG1 및 총 IgG)은 450nm에서 ELISA를 이용하여 측정하고, IgG2a 및 IgG1 아형에 대한 OD 값 및 IgG2a / IgG1의 비율을 비교 하였다. 그룹 당 5 마리의 마우스 혈청 샘플을 분석하였다. 데이터는 두 가지의 독립적인 실험을 대표한다. 결과는 평균 ± 분산값으로 나타냈다. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test).Figure 6 shows the humoral immune response induced by the p24 rBCG strain. p24 specific immunoglobulin subtypes (IgG2a, IgG1 and total IgG) were measured by ELISA at 450 nm, and the OD values and IgG2a / IgG1 ratios for IgG2a and IgG1 subtypes were compared. Five mouse serum samples per group were analyzed. The data represent two independent experiments. The results are expressed as mean ± variance values. * P <0.05; ** P <0.01; *** P <0.001 (Student's t-test).
도 7은 rBCG 균주로 면역화된 마우스에서의 세포 독성 T 림프구 반응을 나타낸 도면이다. CTL 반응은 시험관 내에서 p24에 의해 자극된 비장세포(이펙터 세포) 및 p24 에피토프 펩타이드 (A9I)가 P815 세포(표적 세포)를 반응시킴으로써 일어난다. 그룹 당 총 3마리의 마우스를 분석하였다. 데이터는 두 가지의 독립적인 실험을 대표한다. 결과는 평균 ± 분산값으로 나타냈다. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test).7 shows cytotoxic T lymphocyte responses in mice immunized with rBCG strains. The CTL response occurs in vitro by spleen cells (effector cells) stimulated by p24 and p24 epitope peptides (A9I) reacting with P815 cells (target cells). A total of three mice per group were analyzed. The data represent two independent experiments. The results are expressed as mean ± variance values. * P <0.05; ** P <0.01; *** P <0.001 (Student's t-test).
도 8a 내지 8c는 p24 단백질의 주사와 rBCG-pMyong2-p24 균주의 상이한 M.O.I.에 의한 p24 특이적 면역 반응의 비교 결과를 나타낸 도면들이다: (도 8a) p24 단백질 (30㎍/마우스) 및 상이한 CFUs (1 × 10
6 및 1 × 10
7 CFU)의 rBCG-pMyong2-p24 균주(1주 간격, 2회 주사)를 피하주사 한 마우스(3 마리/그룹)로부터 얻은 비장세포를 시험관 내 자극한 후 IFN-γ 분비 수준을 비교하기 위해 ELISPOT 분석한 결과이다. 각 그룹의 ELISPOT 멤브레인 대표 이미지는 그래프 아래에 표시하였다. (-), 음성 대조군; (+), 양성 대조군. 결과는 triplicate에서 평균 ± 분산값으로 나타냈다. **P<0.01; ***P<0.001 (Student's t-test); (도 8b) p24 특이적 면역 글로불린 아형 (IgG2a, IgG1 및 총 IgG)을 ELISA에 의해 검출한 결과이다. 그룹 당 3 마리 마우스의 혈청 샘플을 분석하였다. 결과는 triplicate에서 평균 ± 분산값으로 나타냈다. **P<0.01; ***P<0.001 (Student's t-test); (도 8c) p24 단백질 및 rBCG-pMyong2-p24 주입 마우스로부터 얻은 비장세포(A9I, p24 에피토프 펩타이드로 자극 됨; 이펙터 세포) 및 A9I 펩타이드 펄스된 P815 세포(표적 세포)의 반응으로 인한 세포 독성 T 림프구 반응을 나타내고 있는 도면이다. 그룹 당 3 마리 마우스의 혈청 샘플을 분석하였다. 결과는 triplicate에서 평균 ± 분산값으로 나타냈다. **P<0.01; ***P<0.001 (Student's t-test).8a to 8c show the comparison of the injection of p24 protein with the p24 specific immune response by different MOIs of the rBCG-pMyong2-p24 strain: (FIG. 8a) p24 protein (30 μg / mouse) and different CFUs ( Splenocytes from mice (3 mice / group) subcutaneously injected with rBCG-pMyong2-p24 strains (1 week apart, 2 injections) of 1 × 10 6 and 1 × 10 7 CFU) in vitro were stimulated with IFN- The result of ELISPOT analysis to compare the levels of γ secretion. Representative images of ELISPOT membranes for each group are shown below the graph. (-), Negative control; (+), Positive control. The results are expressed as mean ± variance in triplicate. ** P <0.01; *** P <0.001 (Student's t-test); (FIG. 8B) p24 specific immunoglobulin subtypes (IgG2a, IgG1 and total IgG) were detected by ELISA. Serum samples of three mice per group were analyzed. The results are expressed as mean ± variance in triplicate. ** P <0.01; *** P <0.001 (Student's t-test); (FIG. 8C) Cytotoxic T lymphocytes due to response of spleen cells (A9I, p24 epitope peptide stimulated; effector cells) and A9I peptide pulsed P815 cells (target cells) from p24 protein and rBCG-pMyong2-p24 injected mice It is a figure which shows reaction. Serum samples of three mice per group were analyzed. The results are expressed as mean ± variance in triplicate. ** P <0.01; *** P <0.001 (Student's t-test).
본 발명은 HIV-1 p24 항원을 발현하는 재조합 마이코박테리움 보비스(
Mycobacterium bovis) BCG 균주가 백신에 효과적으로 사용될 수 있다는 발견에 근거한 것이다.The present invention is based on the discovery that recombinant Mycobacterium bovis BCG strains expressing HIV-1 p24 antigen can be used effectively in vaccines.
이에 한 양태에서 본 발명은 HIV-1의 p24 단백질을 발현하는 재조합 마이코박테리움 보비스 BCG 균주에 관한 것으로, 상기 p24 단백질은, 도 2a에 개시된 pMyong2-p24 벡터에 의해 발현된다.In one embodiment, the present invention relates to a recombinant mycobacterium bovis BCG strain expressing the p24 protein of HIV-1, wherein the p24 protein is expressed by the pMyong2-p24 vector disclosed in FIG. 2A.
본 발명에 따른 벡터에 의해 발현되는 p24 캡시드(CA) 단백질은 처리되지 않은 Gag 폴리단백질에서 MA(매트릭스 단백질)의 3' 말단에 연결되어 있다. p24 캡시드 (CA) 단백질은 N 및 C 말단에 HIV 버딩 및 캡시드 구조에서 중요한 역할을 하는 두 개의 도메인을 포함하고 있다. 본 발명에 따른 벡터에 의해 발현되는 p24는 HIV-1 유래로 그 서열은 GenBank No. KM390026.1d에 포함되어 있거나, 서열번호 1의 아미노산 또는 서열번호 4의 서열의 3161 내지 3856 뉴클레오타이드에 해당되며, 본원에 따른 목적을 위해 항원으로 작용하는 한, 이의 서열변이체도 사용될 수 있다. 본원에 따른 균주가 발현하는 p24는 인체에 투여시 HIV 감염에 대한 항원으로 작용할 수 있다.The p24 capsid (CA) protein expressed by the vector according to the invention is linked to the 3 'end of the MA (matrix protein) in an untreated Gag polyprotein. The p24 capsid (CA) protein contains two domains at the N and C termini that play important roles in HIV budding and capsid structure. P24 expressed by the vector according to the present invention is derived from HIV-1. Sequence variants thereof may be used as long as they are included in KM390026.1d or correspond to amino acids of SEQ ID NO: 1 or 3161 to 3856 nucleotides of sequence of SEQ ID NO: 4, and act as antigens for purposes according to the present application. P24 expressed by the strain according to the present application may act as an antigen for HIV infection when administered to a human body.
본 발명에 따른 균주에서 p24는 특히 도 2a에 개시된 pMyong2 벡터에 의해 발현된다. 본 발명에 따른 pMyong2-p24를 포함하는 재조합 BCG 균주는 pAL5000 또는 pMV306 유도 시스템을 사용하는 다른 rBCG 균주와 비교하여 더 높은 수준의 HIV-1 p24 단백질 발현을 유도하고 더 많은 p24 항원을 대식세포로 전달할 수 있어, 매우 우수한 것으로 나타났다.In the strain according to the invention p24 is expressed in particular by the pMyong2 vector disclosed in FIG. 2A. Recombinant BCG strain comprising pMyong2-p24 according to the present invention will induce higher levels of HIV-1 p24 protein expression and deliver more p24 antigen to macrophages compared to other rBCG strains using the pAL5000 or pMV306 induction system. It was found to be very good.
나아가 본원에 따른 pMyong2-p24를 포함하는 BCG 재조합 균주가 감염된 BMDCs(Bone Marrow-Derived Dendritic Cells)의 T 세포 증식 능력을 향상시키고 접종 된 마우스에서 향상된 T 세포 이펙터 기능과 Th1-바이어스된 체액성 면역 반응을 유도 할 수 있는 것으로 나타났다. 또한 본원에 따른 rBCG-pMyong2-p24 균주는 초기 성장이 지연되어, 약독화됨으로써 더 많은 항원을 식세포에 제시하여 보다 강한 면역반응을 유지할 수 있다. 이러한 결과는 본 발명에 따른 rBCG-pMyong2-p24가 HIV-1 또는 HIV-1과 결핵과의 동시 감염에 효과적인 후보 백신이 될 수 있음을 나타내는 것이다.Furthermore, the BCG recombinant strain comprising pMyong2-p24 according to the present invention improves the T cell proliferation ability of infected BMDCs (Bone Marrow-Derived Dendritic Cells) and improves T cell effector function and Th1-biased humoral immune response in inoculated mice. Has been shown to be able to induce. In addition, the rBCG-pMyong2-p24 strain according to the present invention is delayed in the initial growth, attenuated to present more antigens to phagocytes can maintain a stronger immune response. These results indicate that rBCG-pMyong2-p24 according to the present invention can be an effective candidate vaccine for the simultaneous infection of HIV-1 or HIV-1 and tuberculosis.
이에 다른 양태에서 본 발명은 본 명세서에 개시된 균주를 포함하는 HIV 감염증에 대한 면역원성, 백신 또는 면역치료용 조성물에 관한 것이다. HIV는 인간의 면역체계를 파괴하는 레트로바이러스의 일종으로, HIV에 감염될 경우, 숙주의 면역시스템이 저하되어, 에이즈로 진행하며, 세균, 바이러스, 진균 및 기생충 등에 의한 기회감염의 위험이 현저히 증가된다. 에이즈는 후천성면역결핍증으로 HIV 감염의 결과로 인체 면역기능의 저하로 나타나는 여러 가지 증상을 일컫는 것이다.In another aspect, the present invention relates to a composition for immunogenicity, vaccine or immunotherapy for HIV infection comprising the strains disclosed herein. HIV is a type of retrovirus that destroys the human immune system. When HIV is infected, the host's immune system is degraded and progresses to AIDS, and the risk of opportunistic infections caused by bacteria, viruses, fungi, and parasites increases significantly. do. AIDS is an acquired immune deficiency syndrome that refers to a number of symptoms that result in a decrease in human immune function as a result of HIV infection.
본 발명에 따른 백신 조성물은 p24 특이적 면역반응 예를 들면 더 많은 p24 항원을 식세포로 전달하며, BMDCs의 T세포 증식 능력을 향상시키고 접종된 쥐에서 향상된 T 세포 이펙터 기능과 Th1-바이어스된 체액 면역 반응을 유도할 수 있어, HIV 감염을 예방하거나 또는 감염으로 인한 증상을 경감, 완화 및/또는 치료할 수 있다.The vaccine composition according to the present invention delivers p24 specific immune responses, e.g., more p24 antigens to phagocytes, improves T cell proliferative capacity of BMDCs and improves T cell effector function and Th1-biased humoral immunity in inoculated mice The response can be elicited to prevent HIV infection or to alleviate, alleviate and / or treat symptoms caused by the infection.
백신은 통상 프라임-부스트(prime-booster) 형태로 2회 이상 투여되는 것이 일반적이다. 이 경우 동일한 백신을 수회 투여하거나(homologus) 또는 동일한 항원을 포함하는 상이한 종류의 백신이 투여(이종, heterologus) 된다. 일 구현예에서 본원에 따른 백신은 상동 또는 이종 프라임-부스트에서 프라임 백신(prime vaccine)으로 사용된다. 본원에 따른 rBCG-pMyong2-p24 균주는 p24 항원의 발현량이 높고, 항원 제시능이 높아 naive한 개체에 충분한 p24-특이 면역 반응을 유도할 수 있기 때문에 특히 프라임 백신으로의 사용에 유리하다.The vaccine is usually administered two or more times in the form of a prime-booster. In this case, the same vaccine is administered several times (homologus) or different kinds of vaccines containing the same antigen (heterologus). In one embodiment the vaccine according to the invention is used as a prime vaccine at homologous or heterologous prime-boost. The rBCG-pMyong2-p24 strain according to the present invention is particularly advantageous for use as a prime vaccine because the expression level of the p24 antigen is high and the antigen presenting ability is sufficient to induce sufficient p24-specific immune responses in naive individuals.
본 발명에 따른 조성물에 사용되는 마이코박테리아는 앞서 설명한 바와 같으며, 특히, 살아있는 생균이 사용될 수 있다. 특히 본 발명에 따른 rBCG-pMyong2-p24 균주는 초기 성장이 지연되어, 자연적으로 약독화됨으로써 더 많은 항원을 식세포에 제시하여 보다 강한 면역반응을 유지할 수 있다.The mycobacteria used in the compositions according to the invention are as described above, in particular live live bacteria can be used. In particular, the rBCG-pMyong2-p24 strain according to the present invention is delayed in the initial growth, it is naturally attenuated to present more antigens to phagocytes can maintain a stronger immune response.
본 발명에서 용어 "백신"은 생체에 면역을 주는 항원성 물질을 함유한 생물학적인 제제로서, 에이즈 및/또는 결핵의 예방 또는 치료를 위하여 생물체에 주입 또는 주사하여 생체에 면역이 생기게 하는 면역원을 말한다.As used herein, the term "vaccine" refers to a biological agent containing an antigenic substance that immunizes the living body, and refers to an immunogen that is immunized with the living body by injecting or injecting the living body for the prevention or treatment of AIDS and / or tuberculosis .
본 발명에서 "개체"란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간 또는 비-인간인 영장류, 생쥐(mouse), 쥐(rat), 개, 고양이, 말 및 소 등의 포유류를 의미한다.As used herein, the term "individual" means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, and the like. Mean mammal.
본 발명에서 "면역 반응"은 제공된 DNA 플라스미드 백신을 통해 HIV-1 항원, 예를 들면, 보편적인 HIV-1 항원의 도입에 대한 응답으로, 숙주의 면역계, 예를 들면, 포유동물의 면역계의 활성화를 의미하기 위해 본원에서 이용된다. 면역 반응은 세포성 또는 체액성 반응, 또는 둘 모두의 형태일 수 있다.In the present invention an "immune response" refers to the activation of the host's immune system, eg, the mammalian immune system, in response to the introduction of an HIV-1 antigen, eg, a universal HIV-1 antigen, via a provided DNA plasmid vaccine. It is used herein to mean. The immune response can be in the form of a cellular or humoral response, or both.
본 발명에 따른 백신 조성물은 전신 또는 국소 투여용으로 제형화 될 수 있으며, 약학적으로 가능한 부형제, 담체 및/또는 매체 예를 들면 인산완충 식염수 용액, 증류수, 수/유 에멀젼, 습윤제, 에멀젼화제, pH 조절제 등을 포함할 수 있으나 이로 제한되는 것은 아니다.Vaccine compositions according to the invention may be formulated for systemic or topical administration and include pharmaceutically possible excipients, carriers and / or media such as phosphate buffered saline solutions, distilled water, water / oil emulsions, wetting agents, emulsifiers, pH adjusters and the like, but are not limited thereto.
본 발명에 따른 백신 조성물은 필요한 경우 면역보조제(adjuvant), 특히 T-세포 매개 반응을 촉진 또는 증가시킬 수 있는 임의의 물질 또는 화합물을 포함할 수 있다. 일 구현예에서 상기 면역보조제는 화학적 또는 열에 의해 사멸된 균이 조성물에 포함되는 경우에 사용될 수 있다. 면역보조제는 당업계에 공지되어있으며, 예를 들면 알루미늄 하이드록사이드, 알루미늄 포스페이트, 알루미늄 포타슘 설페이트, 가교 폴리아크릴산 중합체, DDA (dimethyldioctadecylammonium bromide), 면역 조절물질, 락토페린, 또는 IFN-gamma 유도제 등이 사용될 수 있다. 일 구현예에서, 상기 가교 폴리아크릴산 중합체는 Carbopol￠c 단일중합체 또는 공중합체를 포함하고, 상기 림포카인은 IFNgamma, IL-1, IL-2 또는 IL-12를 포함하며, 상기 IFN-gamma 유도제는 poly I:C를 포함할 수 있으나 이로 제한하는 것은 아니다. 일 구현예에서, 합성된 당 폴리머인 상기 알루미늄 하이드록사이드, 알루미늄 포스페이트, 또는 알루미늄 포타슘 설페이트는 약 0.05 내지 0.1 중량%로 포함되고, 상기 가교 폴리아크릴산 중합체는 0.25중량%로 포함될 수 있으나 이로 제한하는 것은 아니다.The vaccine composition according to the invention may comprise an adjuvant, in particular any substance or compound capable of promoting or increasing a T-cell mediated response, if necessary. In one embodiment, the adjuvant may be used when the microorganisms killed by chemical or heat are included in the composition. Adjuvants are known in the art and include, for example, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, cross-linked polyacrylic acid polymers, dimethyldioctadecylammonium bromide (DDA), immunomodulators, lactoferrins, or IFN-gamma inducers, and the like. Can be. In one embodiment, the crosslinked polyacrylic acid polymer comprises a Carbopol ￠ c homopolymer or copolymer, and the lymphokine comprises IFNgamma, IL-1, IL-2 or IL-12, wherein the IFN-gamma inducer May include, but is not limited to, poly I: C. In one embodiment, the synthesized sugar polymer, the aluminum hydroxide, aluminum phosphate, or aluminum potassium sulfate is included in about 0.05 to 0.1% by weight, the cross-linked polyacrylic acid polymer may be included in 0.25% by weight but not limited thereto. It is not.
본 발명에 따른 조성물은 국소 또는 전신 투여될 수 있으며, 일회 또는 다회 투여될 수 있으며, 다양한 경로, 예를 들면 피하, 피내, 근육내 또는 정맥으로 투여되거나, 또는 경구, 비강 또는 흡입 경로로 투여될 수 있다. 본 발명에 따른 일 구현예에서는 근육내 주사로 일회 투여된다.The compositions according to the invention may be administered topically or systemically, may be administered once or multiplely, may be administered by various routes, for example subcutaneously, intradermal, intramuscularly or intravenously, or by oral, nasal or inhalation routes. Can be. In one embodiment according to the invention it is administered once by intramuscular injection.
본 발명에 따른 백신 조성물은 투여 경로에 적합한 액상 용액 또는 현탁제로 제조되거나 또는 주사하기 전에 용액에 용해 또는 현탁되는 고형의 형태로 제형화될 수 있다.Vaccine compositions according to the invention may be formulated in solid form, prepared in liquid solutions or suspensions suitable for the route of administration or dissolved or suspended in solution prior to injection.
본 발명에서 "치료학적 유효량"이란 인간면역결핍바이러스 타입 1에 감염될 확률 또는 감염의 심각성을 상당히 감소시킬 수 있을 정도의 항체를 유발하는데 필요한 투여량을 말한다. 상기 유효량은 투여되는 질환 종류 및 중증도, 환자의 연령 및 성별, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정되며 상기 요소를 모두 고려하여 부작용 없이 최대 효과를 얻을 수 있는 양으로, 당업자에 의해 용이하게 결정될 수 있다.As used herein, a "therapeutically effective amount" refers to a dosage required to elicit an antibody that is capable of significantly reducing the likelihood or seriousness of infection with human immunodeficiency virus type 1. The effective amount depends on the type and severity of the disease administered, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, the factors including the concurrent drug and other factors well known in the medical field. It can be easily determined by those skilled in the art in an amount that can be determined and the maximum effect can be obtained without any side effects in consideration of all the above factors.
백신의 투여량은 투여경로는 물론 환자의 연령, 체중 및/또는 건강상태에 따라 달라질 것이다. 예를 들면 적절한 투여량은 예를 들면 1 내지 10
9 CFU 일 수 있다. 일 구현예에서는 10
6 CFU가 사용된다. 본 발명에 따른 균주의 경우, 항원의 발현량이 우수하고, 항원 제시능 등이 우수하여, 이보다 더 적은 투여량 또는 기존 균주 백신과 비교하여 더 적은 투여량을 사용할 수 있음은 물론이다.The dosage of the vaccine will depend on the route of administration as well as the age, weight and / or health of the patient. For example, a suitable dosage can be, for example, 1 to 10 9 CFU. In one embodiment 10 6 CFU is used. In the case of the strain according to the present invention, the expression level of the antigen is excellent, and the antigen presenting ability is excellent, so that a lower dose or a lower dose compared to the existing strain vaccine can be used.
본 발명에서 사용되는 용어, "예방"이란 본 발명에 따른 백신 조성물의 투여에 의해 에이즈 및/또는 결핵을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" means any action that inhibits or delays the development of AIDS and / or tuberculosis by administration of a vaccine composition according to the invention.
본 발명에서 사용되는 용어, "치료"란 본 발명에 따른 백신 조성물의 투여에 의해 에이즈 및/또는 결핵에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "treatment" refers to any action in which symptoms caused by AIDS and / or tuberculosis are improved or beneficially altered by administration of a vaccine composition according to the present invention.
또 다른 양태에서 본 발명은 본 발명에 따른 균주에서 p24의 발현에 사용되는 도 2a에 개시된 벡터에 관한 것이다.In another aspect the invention relates to the vector disclosed in FIG. 2A for use in the expression of p24 in a strain according to the invention.
일 구현예에서 상기 벡터는 가장 바람직하게는 서열번호 4의 염기서열로 나타낸다.In one embodiment the vector is most preferably represented by the nucleotide sequence of SEQ ID NO: 4.
또 다른 양태에서 본 발명은 또한 상기 벡터로 형질 전환된 세포에 관한 것이다. 상기 세포는 특히 마이코박테리움(
Mycobacterium)을 포함한다.In another embodiment the invention also relates to a cell transformed with said vector. The cells in particular comprise Mycobacterium .
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예]EXAMPLE
[실험 방법][Experimental method]
1. 마우스 및 면역화 과정1. Mouse and Immunization Process
암컷 BALB/c 마우스(약 25 g, 생후 7주령)를 Orient-Bio사(한국, 서울)로부터 구매하였고 생후 8주령이 되었을 때 실험에 사용하였다. 마우스들은 그룹 당 5마리의 마우스로 구성되는 4개 그룹으로 무작위로 나누었다.Female BALB / c mice (approximately 25 g, 7 weeks old) were purchased from Orient-Bio (Seoul, Korea) and used for experiments at 8 weeks old. Mice were randomly divided into four groups of five mice per group.
T 세포 증식 분석을 위해, p24 단백질을 꼬리 정맥을 통해 2마리의 마우스(BALB/c)에 주사하고(30μg/마우스) 5마리의 마우스(BALB/c)를 각 테스트에서 골수유래 수지상 세포(BMDCs) 제조에 사용하였다.For T cell proliferation analysis, p24 protein was injected into the tail vein into two mice (BALB / c) (30 μg / mouse) and five mice (BALB / c) were bone marrow-derived dendritic cells (BMDCs) in each test. ) Was used for the preparation.
예방 접종(vaccination) 테스트를 위해, BALB/c 마우스를 야생형, 2 종류의 재조합 BCG 균주(rBCG-pAL-p24 및 rBCG-pMyong2-p24) 또는 rSmeg-pMyong2-p24 균주로 2회(100μl PBS 중의 1 Х 10
6 CFU; 4주 간격으로) 꼬리 기부에서 피하로 면역화하였다. 음성 대조군 그룹의 경우, PBS를 피하로 주사하였다. 최종 면역화 후 4주 후에, 마우스를 각 시점에서 CO2 흡입에 의해 안락사 시킨 후, 마우스들의 혈액 및 비장을 제거하여 IFN-γ ELISPOT, 사이토카인 측정, 혈청 항체 검출(5마리/그룹) 및 CTL 분석(3마리/그룹)과 같은 면역학적 검정에 사용하였다.For the vaccination test, BALB / c mice were wild-type, two recombinant BCG strains (rBCG-pAL-p24 and rBCG-pMyong2-p24) or rSmeg-pMyong2-p24 strains (1 in 100 μl PBS). Х 10 6 CFU; 4 weeks apart) subcutaneously immunized at the tail base. For the negative control group, PBS was injected subcutaneously. Four weeks after the last immunization, mice were euthanized by CO2 inhalation at each time point, and then the blood and spleen of the mice were removed to remove IFN-γ ELISPOT, cytokine measurements, serum antibody detection (5 mice / group) and CTL analysis ( 3 groups / group).
또한, p24 단백질 처리에 의해 유도된 면역 반응들의 차이 및 상이한 박테리아 수를 비교하기 위하여 독립적인 생체 내 테스트를 수행하였다. 이 경우, 1주 간격으로, BALB/c 마우스(3마리/그룹)를 p24 단백질(30μg/마우스) 및 상이한 수의 rBCG-pMyong2-p24 균주(1 Х 10
6 및 1 Х 10
7 CFU)으로 2회 피하 주사하였다. 음성 대조군 그룹의 경우, PBS를 피하로 주사하였다. 최종 면역화 후 1주 뒤 마우스를 각 시점에서 CO2 흡입에 의해 안락사 시키고, 마우스들의 혈액 및 비장을 제거하여 IFN-γ ELISPOT, 혈청 항체 검출 및 CTL 분석과 같은 면역학적 검정에 사용하였다.In addition, independent in vivo tests were performed to compare the differences in immune responses and different bacterial numbers induced by p24 protein treatment. In this case, at weekly intervals, BALB / c mice (3 mice / group) were replaced with p24 protein (30 μg / mouse) and 2 numbers of rBCG-pMyong2-p24 strains (1 Х 10 6 and 1 Х 10 7 CFU). Three subcutaneous injections were made. For the negative control group, PBS was injected subcutaneously. One week after the last immunization mice were euthanized by CO2 inhalation at each time point, and the blood and spleen of the mice were removed and used for immunological assays such as IFN-γ ELISPOT, serum antibody detection and CTL analysis.
2. HIV-1 p24 Gag를 발현하는 rBCG 균주의 생성2. Generation of rBCG Strains Expressing HIV-1 p24 Gag
HIV-1 p24 Gag를 발현하는 rBCG 균주의 3개의 상이한 유형, 즉 pMyong2-p24 플라스미드를 갖는 BCG(rBCG-pMyong2-p24), pAL-p24 플라스미드를 갖는 BCG(rBCG-pAL-p24), 및 pMV306-p24 플라스미드를 갖는 BCG(rBCG-pMV306-p24)를 생성하기 위하여, 3개의 구성된 플라스미드, 즉 pMV306-p24, pAL-p24 및 pMyong2-p24를 컴피턴트 BCG 균주 (Tokyo 172)에 Gene Pulser II 전기천공 장치(Bio-Rad, Hercules, CA, USA)를 사용하여 전기천공 하였다. 형질전환체는 카나마이신(100μg/ml) 및 OADC가 함유된 Middlebrook 7H10 배지(Difco Laboratories, Detroit, MI, USA) 상에서 선택하였다. 전형적으로, 형질전환체 콜로니를 플레이트로부터 선택하고, 0.5% 글리세롤, 0.05% Tween-80, 10% ADC 및 카나마이신이 보충된 7H9 배지(Difco Laboratories, Detroit, MI, USA)로 옮겨서 3주 내지 4주 동안 배양하였다. rBCG 균주의 성장률을 600 nm에서의 광학 밀도(OD)에 의해 측정하였다.Three different types of rBCG strains expressing HIV-1 p24 Gag: BCG with pMyong2-p24 plasmid (rBCG-pMyong2-p24), BCG with pAL-p24 plasmid (rBCG-pAL-p24), and pMV306- To generate BCG with p24 plasmid (rBCG-pMV306-p24), three constructed plasmids, pMV306-p24, pAL-p24 and pMyong2-p24, were added to the competent BCG strain (Tokyo 172) in a Gene Pulser II electroporation device. Electroporation was performed using (Bio-Rad, Hercules, CA, USA). Transformants were selected on Middlebrook 7H10 medium (Difco Laboratories, Detroit, MI, USA) containing kanamycin (100 μg / ml) and OADC. Typically, transformant colonies were selected from plates and transferred to 7H9 medium (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% glycerol, 0.05% Tween-80, 10% ADC and kanamycin for 3-4 weeks. Incubated for Growth rate of the rBCG strain was measured by optical density (OD) at 600 nm.
3. 대장균(E. coli)으로부터의 p24 단백질의 생산3. Production of p24 Protein from E. coli
재조합 p24 단백질은 앞서 기술된 것에서 약간 변형시켜서 대장균으로부터 정제하였다. 융합 단백질의 발현 및 정제를 위해, 대장균 BL21 균주(RBC Bioscience, Taipei City, 대만)을 pET23a-p24로 형질전환시켰다. 단백질의 발현은 0.4 mM의 아이소프로필 β-D-싸이오갈락토시드 (IPTG, DuchefaBiochemie, Haarlem, 네덜란드)를 첨가함으로써 유도하였다. 박테리아 세포를 수득하고 얼음에서 10분 동안 초음파처리에 의해 파괴하였다. 초음파처리된 용해물을 1600 Х g에서 20분 동안 4℃에서 원심분리하고, p24 단백질을 함유한 펠릿을 4M 유레아(Urea, Sigma Aldrich, St. Louis, MO, USA)를 함유한 결합 완충액에 재현탁하였다. 단백질을 Ni-NTA His 결합 수지(Merck, Darmstadt, 독일)를 사용하여 정제하고, 4M 유레아를 함유한 용출 완충액(300 mM NaCl, 50 mM 인산 나트륨 완충액, 250 mM 이미다졸)으로 용출하였다. 정제된 단백질을 용출 완충액에 대해 연속적으로 투석하여 이미다졸, 유레아 및 잔류 염을 제거하였다. p24 단백질의 순도를 도데실 황산 나트륨-폴리아크릴아미드 겔 전기영동(SDS-PAGE; 12% 겔)에 의해 평가하였다. 겔은 쿠마시 브릴리언트 블루 염색 방법을 사용하여 시각화하였다(도 1).Recombinant p24 protein was purified from Escherichia coli with slight modifications as described above. For expression and purification of the fusion protein, E. coli BL21 strain (RBC Bioscience, Taipei City, Taiwan) was transformed with pET23a-p24. Expression of the protein was induced by addition of 0.4 mM isopropyl β-D-thiogalactoside (IPTG, Duchefa Biochemie, Haarlem, The Netherlands). Bacteria cells were obtained and destroyed by sonication for 10 minutes on ice. The sonicated lysates were centrifuged at 1600 Х g for 20 minutes at 4 ° C., and the pellets containing p24 protein were reproduced in binding buffer containing 4M urea (Urea, Sigma Aldrich, St. Louis, MO, USA). It was cloudy. Proteins were purified using Ni-NTA His binding resin (Merck, Darmstadt, Germany) and eluted with elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 250 mM imidazole) containing 4M urea. Purified protein was subsequently dialyzed against the elution buffer to remove imidazole, urea and residual salts. Purity of the p24 protein was assessed by dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE; 12% gel). Gels were visualized using Coomassie Brilliant Blue staining method (FIG. 1).
4. 마우스에서 골수 유래 수지상 세포의 생산4. Production of Bone Marrow-derived Dendritic Cells in Mice
골수유래 수지상 세포(Bone marrow derived dendritic cells, BMDCs)를 앞서 기술한 것과 같이 8주 내지 12주령 BALB/c 마우스의 골수(BM)로부터 제조하였다. 간단히 설명하면, BM 세포를 serum-free Iscove's modified Eagle medium (IMDM; Gibco Invitrogen, UK)를 사용하여 대퇴골 및 경골에서 제거하였다. 단일 세포 현탁액을 24-well 플레이트에 10% FBS(Gibco Invitrogen), 재조합 마우스 GM-CSF(1.5ng/ml; PeproTech, Rocky Hill, NJ, USA) 및 마우스 IL-4(1.5ng/ml; PeproTech, USA), 페니실린(100units/ml; Gibco Invitrogen), 스트렙토마이신(100 μg/ml; Gibco Invitrogen), 겐타마이신(50μg/ml; Gibco Invitrogen), L-글루타민(2 mM; Gibco Invitrogen), 및 β-머캡토에탄올(50nM; GibcoInvitrogen)이 보충된 최종 부피 2 ml의 complete IMDM에 well 당 1 Х 10
6 세포의 농도로 시딩(seeding)하였다. 배지의 절반을 6일 동안 격일로 동등한 부피의 complete IMDM으로 교체하였다. BMDCs를 이용한 각각의 실험을 준비하기 위하여 5마리의 마우스를 사용하였고, BMDCs를 분화시키기 위하여 5개의 24-well 플레이트를 사용하였다.Bone marrow derived dendritic cells (BMDCs) were prepared from bone marrow (BM) of 8- to 12-week-old BALB / c mice as described above. Briefly, BM cells were removed from the femur and tibia using serum-free Iscove's modified Eagle medium (IMDM; Gibco Invitrogen, UK). Single cell suspensions were placed in 24-well plates in 10% FBS (Gibco Invitrogen), recombinant mouse GM-CSF (1.5 ng / ml; PeproTech, Rocky Hill, NJ, USA) and mouse IL-4 (1.5 ng / ml; PeproTech, USA), penicillin (100 units / ml; Gibco Invitrogen), streptomycin (100 μg / ml; Gibco Invitrogen), gentamicin (50 μg / ml; Gibco Invitrogen), L-glutamine (2 mM; Gibco Invitrogen), and β- The final volume of 2 ml complete IMDM supplemented with mercaptoethanol (50 nM; GibcoInvitrogen) was seeded at a concentration of 1 Х 10 6 cells per well. Half of the medium was replaced with an equivalent volume of complete IMDM every other day for six days. Five mice were used to prepare each experiment with BMDCs and five 24-well plates were used to differentiate BMDCs.
5. rBCG 균주로 감염된 J774A.1 및 BMDCs에서의 CFU 검정5. CFU assay in J774A.1 and BMDCs infected with rBCG strain
마우스의 대식세포 세포주 J774.1(American Type Culture Collection, ATCC TIB-67)을 37℃ 및 5% CO2에서 10%(v/v) 소 태아 혈청(FBS), 2 mM 글루타민, 및 필수 아미노산이 보충된 Dulbecco's modified Eagle's medium(DMEM; Thermo Scientific, Rockford, IL, USA)에서 유지시켰다. 앞서 기술된 바와 같이 마우스 골수로부터 BMDCs를 생성시켰고 37℃ 및 5% CO2에서 10% FBS(Gibco Invitrogen), 재조합 마우스 GM-CSF(1.5ng/ml; PeproTech, Rocky Hill, NJ, USA), 마우스 IL-4(1.5ng/ml; PeproTech, USA), 페니실린(100units/ml; Gibco Invitrogen), 스트렙토마이신(100μg/ml; Gibco Invitrogen), 겐타마이신(50μg/ml; Gibco Invitrogen), L-글루타민(2mM; Gibco Invitrogen), 및 β-머캡토에탄올(50nM; Gibco Invitrogen)이 보충된 Iscove's modified Eagle medium(IMDM; Gibco Invitrogen, UK)에서 유지시켰다.The macrophage cell line J774.1 (ATCC TIB-67) of the mouse was supplemented with 10% (v / v) fetal bovine serum (FBS), 2 mM glutamine, and essential amino acids at 37 ° C. and 5% CO 2. Were maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Scientific, Rockford, IL, USA). BMDCs were generated from mouse bone marrow as described above and 10% FBS (Gibco Invitrogen), recombinant mouse GM-CSF (1.5 ng / ml; PeproTech, Rocky Hill, NJ, USA), mouse IL at 37 ° C. and 5% CO 2. -4 (1.5 ng / ml; PeproTech, USA), penicillin (100 units / ml; Gibco Invitrogen), streptomycin (100 μg / ml; Gibco Invitrogen), gentamicin (50 μg / ml; Gibco Invitrogen), L-glutamine (2 mM) Gibco Invitrogen), and Iscove's modified Eagle medium (IMDM; Gibco Invitrogen, UK) supplemented with β-mercaptoethanol (50 nM; Gibco Invitrogen).
J774A.1 세포 및 BMDCs를 rBCG 균주들, 즉 rBCG-pMyong2-p24, -pAL-p24, 및 -pMV306-p24 및 야생형 BCG 균주(10 M.O.I.)(3개 한 벌로)으로 4시간 동안 감염시키고, 이어서 PBS로 3회 세척 후 새로운 배지와 함께 24시간 동안 배양하였다. 24시간 후, 감염된 세포들을 0.5% Triton X-100으로 용해시켰다. 세포 용해물을 PBS로 희석하고 콜로니 형성 단위(CFUs)의 계산을 위해 OADC가 보충된 Middlebrook 7H10 한천(agar) 플레이트 위에 플레이팅하였다. 모든 감염 그룹을 각 실험에서 3개 한 벌로 분석하였고, 총 2개의 독립 실험을 수행하였다.J774A.1 cells and BMDCs were infected with rBCG strains, ie rBCG-pMyong2-p24, -pAL-p24, and -pMV306-p24 and wild-type BCG strain (10 MOI) (3 pairs), followed by 4 hours After washing three times with PBS and incubated with fresh medium for 24 hours. After 24 hours, infected cells were lysed with 0.5% Triton X-100. Cell lysates were diluted with PBS and plated on Middlebrook 7H10 agar plates supplemented with OADC for calculation of colony forming units (CFUs). All infection groups were analyzed in duplicates in each experiment and a total of two independent experiments were performed.
6. rBCG 균주에서 p24 Gag 발현 수준의 측정6. Measurement of p24 Gag Expression Level in rBCG Strains
rBCG 균주들에서 p24 Gag 발현 수준을 측정하기 위하여, 웨스턴 블롯팅 및 ELISA 분석을 수행하였다. 간단히 말하면, 배양된 rBCG 균주들의 펠릿(pellet)을 라이소자임(100μg/ml), DNase(5U/ml), 및 단백질 가수분해효소 억제제가 보충된 B-PER 완충액(Thermo scientific, Rockford, IL, USA)에 현탁시켰다. 그런 후, 현탁액을 얼음에서 5분 동안 초음파처리하고(펄스: 0.3초, 중단: 0.7초) 13,000 rpm에서 4℃에서 15분 동안 원심분리하였다. 수성 상(aqueous phase)의 동일한 양의 단백질을 웨스턴 블롯팅 분석에 사용하였다. 각 rBCG 균주에서 p24의 발현 수준은 마우스 항-p24 단클론성 항체(Abcam, Cambridge, USA; 1:1,000 희석)를 사용하여 측정하였다. 단백질 농도가 모든 샘플에서 동등했는지를 확인하기 위하여 마이코박테리아 Hsp65(Abcam, 1:1,000 희석)를 내부 대조군으로서 사용하였다. p24의 안정적인 발현을 평가하기 위하여, 다양한 계대 지점(1, 4, 6, 8, 10 및 12회 계대 후)에서 rBCG-pMyong2-p24 균주의 p24 발현 수준 또한 측정하였다. 계대 과정은 플레이트에서 플레이트로(카나마이신이 있거나 없는 7H10 한천 플레이트) 수행하였고, 각 계대로부터 얻은 콜로니들을 7H9 broth medium에서 3주 동안 배양한 후에 각각의 실험을 수행하였다. 추가적으로, p24 ELISA 키트를 사용 (제조사에 의해 제안된 방법으로)하여 동일한 양의 단백질에서 p24 수준의 검출을 위해 사용하였다(3개 한 벌의 well에서)(ABL, Rockville, USA). 모든 그룹을 2개의 독립적인 실험으로 분석하였다.In order to measure p24 Gag expression levels in rBCG strains, Western blotting and ELISA analysis were performed. In brief, pellets of cultured rBCG strains were loaded with B-PER buffer (Thermo scientific, Rockford, IL, USA) supplemented with lysozyme (100 μg / ml), DNase (5 U / ml), and protease inhibitors. Suspended in. The suspension was then sonicated on ice for 5 minutes (pulse: 0.3 seconds, interruption: 0.7 seconds) and centrifuged for 15 minutes at 4 ° C. at 13,000 rpm. The same amount of protein in the aqueous phase was used for western blotting analysis. The expression level of p24 in each rBCG strain was measured using mouse anti-p24 monoclonal antibody (Abcam, Cambridge, USA; 1: 1,000 dilution). Mycobacterial Hsp65 (Abcam, 1: 1,000 dilution) was used as an internal control to confirm that protein concentrations were equivalent in all samples. To assess stable expression of p24, p24 expression levels of the rBCG-pMyong2-p24 strain were also measured at various passage points (after 1, 4, 6, 8, 10 and 12 passages). Passage was performed from plate to plate (7H10 agar plate with or without kanamycin), and each experiment was performed after colonies from each passage were incubated in 7H9 broth medium for 3 weeks. In addition, a p24 ELISA kit was used (in the method suggested by the manufacturer) for the detection of p24 levels in the same amount of protein (in three sets of wells) (ABL, Rockville, USA). All groups were analyzed in two independent experiments.
7. rBCG 균주로 감염된 BMDCs 및 J774.1 세포에서 p24 Gag 발현 수준의 측정7. Measurement of p24 Gag Expression Levels in BMDCs and J774.1 Cells Infected with rBCG Strains
rBCG 감염을 위해, J774.1 세포 및 BMDCs를 well 당 5~10 Х 10
5 개의 세포로(24-well 플레이트, 3개 한 벌) 시딩하고 18시간 동안 배양하였다. 3개의 상이한 rBCG 균주를 세포에 10의 감염 다중성(multiplicity of infection, M.O.I.)으로 감염시켰다. 또한, 상이한 M.O.I.(1 및 10 M.O.I.)의 rBCG-pMyong2-p24 균주를 BMDCs에 감염시켜서 상이한 M.O.I.에 의한 p24발현의 차이를 비교하였다. J774.1 세포 및 BMDCs를 4시간 동안 인큐베이션하여 박테리아의 식세포 작용을 허용하였고, 세포외 박테리아를 PBS로 3회 세척하여 제거하였다. 감염된 J774.1 세포 및 BMDCs는 24시간 및/또는 72시간 동안 인큐베이션하였다. For the rBCG infection, the J774.1 cells and BMDCs with 5 ~ 10 Х 10 5 cells per well (24-well plate, 3 suits) was seeded, and cultured for 18 hours. Three different rBCG strains were infected with 10 multiplicity of infection (MOI) cells. In addition, rBCG-pMyong2-p24 strains of different MOIs (1 and 10 MOIs) were infected with BMDCs to compare differences in p24 expression by different MOIs. J774.1 cells and BMDCs were incubated for 4 hours to allow phagocytosis of bacteria, and extracellular bacteria were removed by washing three times with PBS. Infected J774.1 cells and BMDCs were incubated for 24 hours and / or 72 hours.
세포에서의 p24 발현을 분석하기 위하여, 세포 펠릿 중의 총 단백질을 RIPA 용해 완충액에서의 현탁에 의해 제조하고 p24 ELISA 키트(ABL)(3개 한 벌의 well에서)를 사용하여 제조사 설명서에 따라 p24 수준의 측정을 위해 사용하였다. 모든 감염 그룹을 각 실험에서 3개 한 벌로 분석하였고, 총 2개의 독립적인 실험을 수행하였다.To analyze p24 expression in cells, total protein in cell pellets was prepared by suspension in RIPA lysis buffer and p24 levels according to manufacturer's instructions using the p24 ELISA kit (ABL) (in three sets of wells). It was used for the measurement of. All infection groups were analyzed in duplicates in each experiment and a total of two independent experiments were performed.
8. T 세포 증식 분석8. T Cell Proliferation Assay
T 세포 증식 분석을 위해 다음과 같은 실험을 수행하였다. 마우스 2 마리에 p24 단백질(30μg/마우스)을 정맥내 주사하였다. 7일 후, 비장세포(splenocytes)를 극저온 FACS 완충액 [1% 소 혈청 알부민(BSA) 및 1mM EDTA를 함유한 PBS]으로 세척하고 얼음에서 30분 동안 10% 래트 혈청(Sigma Aldrich), 10% 염소 혈청(Gibco Invitrogen), 10% 마우스 혈청(Sigma Aldrich), 및 2.4G2 단클론성 항체(10 μg/ml; BD Biosciences, San Diego, CA, USA)를 함유한 수퍼 블럭(super block) 용액으로 블로킹(blocking)하였다. 세포를 계속해서 BV421-콘쥬게이트된 항-CD4(Clone GK1.5, BD Biosciences) 및 PE-콘쥬게이트된 항-CD8a(Clone 53-6.7, eBioscience, San Diego, CA, USA)으로 30분 동안 4℃에서 염색하고 극저온 FACS 완충액으로 3회 세척하였다. FACS AriaIII 기기(BD Biosciences)를 사용하여 CD4 및 CD8 T 세포 집단을 분류하였다. 또한 공동 배양하기 하루 전 날, 미성숙 BMDCs를 야생형, 2개의 rBCG(rBCG-pMyong2-p24 및 -pAL-p24) 또는 rSmeg-pMyong2-p24 균주로 10의 M.O.I.에서 24시간 동안 감염시켰다. 증식 분석은 형광 세포질 추적 염료인 CFSE(Invitrogen, Carlsbad, USA)를 사용하여 수행하였다. 분류된 CD4 및 CD8 T 세포를 5μM CFSE로 4분 동안 37℃에서 및 4분 동안 얼음에서 염색하였다. 그런 후, CFSE가 표지된 T 세포 및 감염된 BMDCs를 4일 동안 공동 배양하였다.For the T cell proliferation assay, the following experiment was performed. Two mice were injected intravenously with p24 protein (30 μg / mouse). After 7 days, splenocytes were washed with cryogenic FACS buffer [PBS containing 1% bovine serum albumin (BSA) and 1 mM EDTA] and 10% rat serum (Sigma Aldrich), 10% goat for 30 minutes on ice. Blocking with super block solution containing serum (Gibco Invitrogen), 10% mouse serum (Sigma Aldrich), and 2.4G2 monoclonal antibody (10 μg / ml; BD Biosciences, San Diego, CA, USA) blocking). Cells were continued for 30 minutes with BV421-conjugated anti-CD4 (Clone GK1.5, BD Biosciences) and PE-conjugated anti-CD8a (Clone 53-6.7, eBioscience, San Diego, CA, USA) for 30 minutes. Stain at C and wash three times with cryogenic FACS buffer. CD4 and CD8 T cell populations were sorted using the FACS AriaIII instrument (BD Biosciences). In addition, the day before co-culture, immature BMDCs were infected with wild type, two rBCG (rBCG-pMyong2-p24 and -pAL-p24) or rSmeg-pMyong2-p24 strains for 10 hours at 10 M.O.I. Proliferation assays were performed using CFSE (Invitrogen, Carlsbad, USA), a fluorescent cytoplasmic tracer dye. Sorted CD4 and CD8 T cells were stained with 5 μM CFSE at 37 ° C. for 4 minutes and on ice for 4 minutes. Thereafter, CFSE-labeled T cells and infected BMDCs were co-cultured for 4 days.
T 세포와 감염된 BMDCs을 공동 배양한지 4일 뒤에, 공동 배양된 세포(3개 한 벌의 well)를 얼음에서 30분 동안 수퍼 블락(super block) 용액으로 블로킹하고 CD4 BV421-콘쥬게이트된 항-CD4(Clone GK1.5, BD Biosciences) 및 PE-콘쥬게이트된 항-CD8a(Clone 53-6.7, eBioscience)로 30분 동안 4℃에서 염색하였다. 세포 사이클 프로파일은 FACS LSRFortessa(BD Biosciences)를 사용하여 측정하였고, 측정값은 Flowjo 소프트웨어를 사용하여 분석하였다(도 4a). 모든 실험은 3개 한 벌로 수행하였다.Four days after co-culture of T cells and infected BMDCs, co-cultured cells (three sets of wells) were blocked with super block solution for 30 minutes on ice and CD4 BV421-conjugated anti-CD4 (Clone GK1.5, BD Biosciences) and PE-conjugated anti-CD8a (Clone 53-6.7, eBioscience) for 30 minutes at 4 ° C. Cell cycle profiles were measured using FACS LSRFortessa (BD Biosciences), and measurements were analyzed using Flowjo software (FIG. 4A). All experiments were performed in three sets.
9. IL-2 ELISA9.IL-2 ELISA
상기 T 세포 증식 분석 과정에서 공동 배양된 상층액(3개 한 벌의 well)의 마우스 IL-2의 양을 ELISA를 사용하여 제조사의 설명서(BioLegend, USA)를 따라 측정하였다. 모든 실험을 이중으로 수행하였다.The amount of mouse IL-2 in supernatant (three sets of wells) co-cultured in the course of the T cell proliferation assay was measured according to the manufacturer's instructions (BioLegend, USA) using ELISA. All experiments were performed in duplicate.
10. Enzyme-Linked ImmunoSpot (ELISPOT) 분석10.Enzyme-Linked ImmunoSpot (ELISPOT) Analysis
야생형 및 rBCG 균주로 면역화된 마우스(5 마리/그룹)로부터 얻은 비장세포를 사용하여 다음과 같이 ELISPOT 검정을 수행하였다. 간단히 설명하면, 96- well ELISPOT 플레이트(PVDF 막)를 PBS 중의 마우스 IFN-γ(3μg/ml, 클론: AN-18) 포획 항체(BD-Biosciences, San Diego, CA, USA)로 코팅하고 밤새 4℃에서 인큐베이션 하였다. 포획 항체를 버리고, 플레이트를 0.05% Tween-20 (PBST)을 함유한 PBS 및 PBS(각각 3회)로 세척하고, 플레이트를 10% FBS를 포함한 200μl의 RPMI 1640 배지로 3시간 동안 37℃에서 블로킹하였다. 블로킹 후, 예방접종된 마우스로부터 채취한 비장세포를 각 well 당 5 x 10
5개씩 로딩하였다. 각각의 치료 그룹에 대해, 세포를 3개 한 벌로 총 200μl의 부피 중 5μg/ml의 정제된 p24 항원 또는 배지 단독으로 자극하였다. 그 후 플레이트를 37℃에서 24시간 동안 인큐베이션하였다. 세포를 양성 대조군으로서 5ng/ml의 포르볼 12-미리스테이트 13-아세테이트(PMA)(Sigma-Aldrich, St. Louis, USA) 및 500ng/ml의 이오노마이신(Sigma-Aldrich)으로 자극하였다. PBST 및 PBS(각각 3회)로 세척한 후, 각 well을 비오틴(biotin)이 표지된 마우스 IFN-γ(3μg/ml, 클론: XMG1.2) 검출 항체(BD-Biosciences)로 처리하고 플레이트를 밤새 4℃에서 인큐베이션하였다. 세포를 다시 세척하고 양고추냉이 과산화효소(HRP)-콘쥬게이트된 스트렙트아비딘(streptavidin)을 각 well에 첨가하였다. HRP 반응은 3-아미노-9-에틸카르바졸(AEC) 기질 시약 세트(BD-Biosciences)를 사용하여 전개하였다. well 당 스팟 형성 단위(SFUs)의 수를 ELISPOT 판독기(AID ELISPOT 판독기, Strasburg, 독일)를 사용하여 자동으로 계수하였다. 모든 그룹을 3개 한 벌로 분석하였고 2개의 독립적인 실험을 수행하였다.ELISPOT assays were performed using splenocytes from mice immunized with wild type and rBCG strains (5 mice / group). Briefly, a 96-well ELISPOT plate (PVDF membrane) is coated with mouse IFN-γ (3 μg / ml, clone: AN-18) capture antibody (BD-Biosciences, San Diego, CA, USA) in PBS and overnight 4 Incubation was at 캜. Discard the capture antibody, wash the plate with PBS and PBS containing 0.05% Tween-20 (PBST) (three times each), and plate the plate with 200 μl RPMI 1640 medium containing 10% FBS for 3 hours at 37 ° C. It was. After blocking, splenocytes harvested from vaccinated mice were loaded with 5 x 10 5 cells per well. For each treatment group, cells were stimulated in 3 sets with 5 μg / ml of purified p24 antigen or medium alone in a total volume of 200 μl. Plates were then incubated at 37 ° C. for 24 hours. Cells were stimulated with 5 ng / ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, USA) and 500 ng / ml ionomycin (Sigma-Aldrich) as positive controls. After washing with PBST and PBS (3 times each), each well was treated with biotin-labeled mouse IFN-γ (3 μg / ml, clone: XMG1.2) detection antibody (BD-Biosciences) and plate Incubate at 4 ° C. overnight. The cells were washed again and horseradish peroxidase (HRP) -conjugated streptavidin was added to each well. HRP reactions were developed using 3-amino-9-ethylcarbazole (AEC) substrate reagent set (BD-Biosciences). The number of spot forming units (SFUs) per well was automatically counted using an ELISPOT reader (AID ELISPOT reader, Strasburg, Germany). All groups were analyzed in 3 sets and 2 independent experiments were performed.
11. rBCG 균주로 면역화된 마우스에서 사이토카인 생성의 측정11. Measurement of cytokine production in mice immunized with rBCG strains
면역화된 마우스(5마리/그룹)로부터 얻은 비장세포를 10% FBS를 포함한 RPMI 1640 배지에서 1 Х 10
6 세포/웰(96-well 마이크로 플레이트, 200μl 부피, 3개 한 벌로)의 농도로 조정하고, 정제된 p24 단백질을 시험관 내 자극을 위해 5μg/ml의 농도로 첨가하였다. 세포를 배양하고, 상층액을 ELISA 키트를 사용하여 IL-2(BioLegend, San Diego, CA, USA), IL-6(eBioscience) 및 IFN-γ(BioLegend) 사이토카인 측정을 위해 수득하였다. 모든 그룹을 3개 한 벌로 분석하였고 2개의 독립적인 실험을 수행하였다.Splenocytes from immunized mice (5 mice / group) were adjusted to a concentration of 1 Х 10 6 cells / well (96-well microplate, 200 μl volume, 3 sets) in RPMI 1640 medium containing 10% FBS and Purified p24 protein was added at a concentration of 5 μg / ml for in vitro stimulation. Cells were cultured and supernatants were obtained for IL-2 (BioLegend, San Diego, CA, USA), IL-6 (eBioscience) and IFN-γ (BioLegend) cytokine measurements using an ELISA kit. All groups were analyzed in 3 sets and 2 independent experiments were performed.
12. 혈청 항체 검출12. Serum Antibody Detection
혈청 항체 비율을 검출하기 위하여, 면역화된 마우스(5마리/그룹)로부터 CO2의 과호흡을 통한 안락사 후에 심장 천공 방법을 사용하여 혈청 샘플을 수집하였다. 96-well 플레이트를 밤새 4℃에서 0.05 M 탄산염-중탄산염 완충액(pH 9.6) 중의 정제된 p24 단백질(5μg/ml)로 코팅하였다. 플레이트를 PBST 및 PBS로 3회 세척하고 상온(RT)에서 1시간 동안 5% 소 혈청 알부민(PBST 중의 BSA)으로 블로킹하였다. 혈청 샘플을 PBS로 1:10의 비율로 희석하고, 100μl를 각 well에 첨가하였다(3개 한 벌로). 플레이트를 2시간 동안 상온에서 인큐베이션하고, PBST 및 PBS로 3회 세척하고, 1시간 동안 비오티닐화(biotinylated)된 래트 항-마우스 IgG2a, IgG1(BD Biosciences, 1:1,000 희석) 및 총 IgG(eBioscience, 1:1,000 희석) 항체와 함께 인큐베이션하였다. 그런 후, 플레이트를 다시 세척하고, HRP 콘쥬게이트된 스트렙트아비딘(eBioscience)과 함께 30분 동안 상온에서 인큐베이션하였다. 최종 세척 단계 후에, 모든 well을 BD OptEIA 기질(BD Biosciences)과 10분 동안 반응시킨 후 반응을 1N H2SO4를 사용하여 반응을 중단시켰다. 분광계를 사용하여 450 nm의 파장에서 광학 밀도(OD)를 측정하였다.To detect serum antibody ratios, serum samples were collected using cardiac puncture methods after euthanasia through hyperrespiration of CO2 from immunized mice (5 mice / group). 96-well plates were coated overnight with purified p24 protein (5 μg / ml) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) at 4 ° C. Plates were washed three times with PBST and PBS and blocked with 5% bovine serum albumin (BSA in PBST) for 1 hour at room temperature (RT). Serum samples were diluted with PBS at a ratio of 1:10 and 100 μl was added to each well (in three suits). Plates were incubated at room temperature for 2 hours, washed three times with PBST and PBS, biotinylated rat anti-mouse IgG2a, IgG1 (BD Biosciences, 1: 1,000 dilution) and total IgG (eBioscience) for 1 hour. , 1: 1,000 dilution). The plates were then washed again and incubated with HRP conjugated streptavidin (eBioscience) for 30 minutes at room temperature. After the final wash step, all wells were reacted with BD OptEIA substrate (BD Biosciences) for 10 minutes and then the reaction was stopped using 1N H 2 SO 4. Optical density (OD) was measured at a wavelength of 450 nm using a spectrometer.
13. 세포독성 T 림프구(Cytotoxic T lymphocyte, CTL) 분석13. Cytotoxic T lymphocyte (CTL) analysis
유도된 CTL 반응은 앞서 기술된 것에서 약간의 변형을 가하여 측정하였다. 간단히 설명하면, 이펙터 세포의 경우, 각각의 면역화된 그룹의 마우스로부터 얻은 비장세포를 주요 조직적합성 복합체(MHC) class I-제한 p24 펩타이드 A9I (AMQMLKETI)(10μg/ml; Peptron, 대전, 대한민국)를 사용하여 펄스하고 6일 동안 IL-2(30U/ml; PeproTech, Rocky Hill, USA)와 함께 37℃에서 5% CO2 인큐베이터에서 인큐베이션하였다. 표적 세포, 즉 P815 세포(H-2d)를 A9I 펩타이드(10μg/ml)와 함께 2시간 동안 인큐베이션한 후 이펙터 및 표적 세포의 공동 배양에 의해 제조하였다. 세포의 세포독성을 U자형 바닥 96-well 플레이트에서 락트산 탈수소효소(lactate dehydrogenase, LDH) 분석법에 의해 제조사의 프로토콜(CytoTox 96 비-방사성 세포독성 분석; Promega, Madison, USA)을 따라 평가하였다. 간단히 말하면, 이펙터 세포(항원에 의해 자극된 비장세포)를 표적 세포(p24 펄스된 P815 세포)에 3개 한 벌로 상이한 이펙터/표적(E/T) 비율(10:1, 20:1 내지 50:1의 범위)로 6시간 동안 첨가하였다; 그런 후, 배양된 상층액으로부터 방출된 LDH값을 490nm에서 분광계를 사용하여 검출하였다. 특이적 세포 용해의 백분율을 다음 식을 사용하여 계산하였다: [{실험(Experimental) - 자생적 이펙터(Effector spontaneous) - 자생적 표적(Target spontaneous)}/{최대 표적(Target maximum) - 자생적 표적(Target spontaneous)}] x 100(%). 모든 그룹을 3개 한 벌로 분석하였고 2개의 독립적인 실험을 수행하였다.The induced CTL response was measured with some modification in what was described above. Briefly, for effector cells, splenocytes from each immunized group of mice were labeled with major histocompatibility complex (MHC) class I-restricted p24 peptide A9I (AMQMLKETI) (10 μg / ml; Peptron, Daejeon, South Korea). Pulsed and incubated with IL-2 (30 U / ml; PeproTech, Rocky Hill, USA) in a 5% CO 2 incubator at 37 ° C. for 6 days. Target cells, ie P815 cells (H-2d), were prepared by incubation with A9I peptide (10 μg / ml) for 2 hours followed by co-culture of effectors and target cells. The cytotoxicity of the cells was assessed according to the manufacturer's protocol (CytoTox 96 non-radioactive cytotoxicity assay; Promega, Madison, USA) by lactate dehydrogenase (LDH) assay in U-shaped bottom 96-well plates. In short, effector cells (antigen-stimulated splenocytes) are matched to target cells (p24 pulsed P815 cells) in three different effector / target (E / T) ratios (10: 1, 20: 1 to 50: In the range of 1) for 6 hours; Then, the LDH value released from the cultured supernatant was detected using a spectrometer at 490 nm. The percentage of specific cell lysis was calculated using the following formula: [{Experimental-Effector spontaneous-Target spontaneous] / {Target maximum-Target spontaneous )}] x 100 (%). All groups were analyzed in 3 sets and 2 independent experiments were performed.
14. 통계적 분석14. Statistical Analysis
제공된 모든 데이터는 평균 ± 표준 편차로서 표시하였다. 스튜던트 t-테스트는 변량을 비교하기 위해 마이크로소프트 엑셀 소프트웨어를 이용하여 수행하였고, 가능성 값(probability values)이 0.05보다 적을 때 차이를 통계학적으로 유의미한 것으로 간주하였다.All data provided are expressed as mean ± standard deviation. Student's t-test was performed using Microsoft Excel software to compare the variances and considered the difference as statistically significant when the probability values were less than 0.05.
[실시예]EXAMPLE
실시예 1. rBCG-pMyong2-p24 균주는 박테리아 및 감염 세포에서 향상된 HIV-1 p24 Gag 발현을 유도한다Example 1 rBCG-pMyong2-p24 Strain Induces Improved HIV-1 p24 Gag Expression in Bacteria and Infected Cells
본 발명에서는 HIV-1 p24 Gag 예방접종을 위한 rBCG의 제조에서 pMyong2 벡터 시스템의 유용성을 조사하기 위하여, 상이한 마이코박테리움-대장균 셔틀 벡터, 즉 pMyong2-TOPO, pAL-TOPO, 및 pMV306을 각각 사용하여 p24를 발현하는 3가지 유형의 rBCG 균주, 즉 rBCG-pMyong2-p24, rBCG-pAL-p24 및 rBCG-pMV306-p24를 생성하였다(도 2a). 7H9 broth(100μg/ml의 카나마이신 포함)에서 30일 동안 3개의 rBCG 균주의 성장률을 비교하였고, 그 결과 rBCG 및 야생형 BCG 균주가 거의 동일한 성장률을 나타냈다(도 2b). 추가적으로, 대식세포 및 DCs에서 이러한 rBCG 균주들의 생존을 조사하기 위하여, 감염된 세포를 0.05% Triton X-100(in PBS)으로 용해시키고 7H10 한천 플레이트 상에 플레이팅하였다. 두 세포에서 모두, 아마도 향상된 p24 발현에 의한 박테리아 부담(bacterial burden)으로 인해 rBCG-pMyong2-p24 균주는 다른 균주들(즉 rBCG-pAL-p24, -pMyong2-p24 및 야생형 BCG 균주)보다 더 적은 CFU를 나타냈다(도 2c).In the present invention, to investigate the usefulness of the pMyong2 vector system in the preparation of rBCG for HIV-1 p24 Gag vaccination, different mycobacterium-E. Coli shuttle vectors, ie pMyong2-TOPO, pAL-TOPO, and pMV306, respectively, are used. Three types of rBCG strains expressing p24 were generated, namely rBCG-pMyong2-p24, rBCG-pAL-p24 and rBCG-pMV306-p24 (FIG. 2A). The growth rates of the three rBCG strains for 30 days in 7H9 broth (including 100 μg / ml kanamycin) were compared, with the result that the rBCG and wild-type BCG strains showed almost the same growth rate (FIG. 2B). In addition, to examine the survival of these rBCG strains in macrophages and DCs, infected cells were lysed with 0.05% Triton X-100 (in PBS) and plated on 7H10 agar plates. In both cells, the rBCG-pMyong2-p24 strain may have less CFU than other strains (ie rBCG-pAL-p24, -pMyong2-p24 and wild-type BCG strains), presumably due to bacterial burden due to enhanced p24 expression. (FIG. 2C).
3개의 rBCG 균주의 박테리아에서 p24의 발현 수준을 비교하기 위하여, 배양된 박테리아의 용해 후에 p24에 대한 ELISA(도 3a) 및 웨스턴 블롯팅(도 3b) 분석을 수행하였다. 모든 rBCG 균주는 p24 단백질을 발현할 수 있었다. rSmeg-pMyong2-p24 균주와 유사하게, rBCG-pMyong2-p24 균주는 다른 벡터 시스템에서의 균주보다 대략 2 또는 3배 더 높은 수준의 p24를 발현하였다. rBCG-pAL-p24는 rBCG-pMV306-p24보다 약간 더 높은 수준의 p24를 생성하였다(도 3a 및 3b).To compare the expression level of p24 in bacteria of three rBCG strains, ELISA (FIG. 3A) and Western blotting (FIG. 3B) analyzes were performed on p24 after lysis of cultured bacteria. All rBCG strains were able to express the p24 protein. Similar to the rSmeg-pMyong2-p24 strain, the rBCG-pMyong2-p24 strain expressed approximately 2 or 3 times higher levels of p24 than strains in other vector systems. rBCG-pAL-p24 produced slightly higher levels of p24 than rBCG-pMV306-p24 (FIGS. 3A and 3B).
p24의 안정적인 발현을 평가하기 위하여, 카나마이신이 있거나 없는 7H10 한천 플레이트 상에서 다양한 계대 지점에서의 rBCG-pMyong2-p24에서 p24의 발현 수준을 또한 웨스턴 블롯팅 분석에 의해 측정하였다. rBCG-pMyong2-p24 균주는 카나마이신이 있거나 없는 7H10 한천 플레이트 상에서 12회의 계대 후에도 안정적인 p24 발현을 보였다(도 3d 및 3f). 추가적으로, 3개의 rBCG 균주로 감염된 마우스 대식세포(J774A.1) 및 BMDCs에서 p24의 발현 수준을 ELISA를 이용하여 조사하였다. 관찰된 경향은 용해된 rBCG 균주로 관찰된 것과 유사하였다(도 3h). To assess stable expression of p24, expression levels of p24 at rBCG-pMyong2-p24 at various passage points on 7H10 agar plates with or without kanamycin were also determined by Western blotting analysis. The rBCG-pMyong2-p24 strain showed stable p24 expression even after 12 passages on 7H10 agar plates with or without kanamycin (FIGS. 3D and 3F). Additionally, expression levels of p24 in mouse macrophages (J774A.1) and BMDCs infected with three rBCG strains were examined using ELISA. The observed trend was similar to that observed with lysed rBCG strains (FIG. 3H).
또한, 상이한 M.O.I.에 따르는 p24 발현 수준을 비교하기 위하여, BMDCs를 rBCG-pAL-p24 및 rBCG-pMyong2-p24의 상이한 M.O.I.(1 및 10 M.O.I.)로 1일 및 3일 동안 감염시켰다. 그 결과는 두 가지 균주의 증가된 M.O.I.가 p24 발현 증가에 영향을 미친 것으로 나타났다. 그러나, 상기에서 제시된 것과 같이, rBCG-pMyong2-p24는 rBCG-pAL-p24 균주보다 더 많은 p24 발현을 유도하였다(도 3i).In addition, to compare p24 expression levels according to different M.O.I., BMDCs were infected with different M.O.I. (1 and 10 M.O.I.) of rBCG-pAL-p24 and rBCG-pMyong2-p24 for 1 and 3 days. The results showed that increased M.O.I. of the two strains affected the increase in p24 expression. However, as suggested above, rBCG-pMyong2-p24 induced more p24 expression than the rBCG-pAL-p24 strain (FIG. 3I).
전체적으로, 다른 두 개의 rBCG 균주, 즉 rBCG-pAL-p24 및 rBCG-pMV306-p24와 비교하여, rBCG-pMyong2-p24는 감염된 항원 제시 세포(APC) 및 박테리아에서 p24의 생성을 증가시켰다.Overall, rBCG-pMyong2-p24 increased the production of p24 in infected antigen presenting cells (APC) and bacteria, compared to the other two rBCG strains, rBCG-pAL-p24 and rBCG-pMV306-p24.
실시예 2. rBCG-pMyong2-p24 균주로 감염된 BMDCs는 HIV-1 p24 Gag로 면역화된 마우스에서 향상된 T 세포 증식을 유도한다Example 2. BMDCs Infected with rBCG-pMyong2-p24 Strain Induces Enhanced T Cell Proliferation in Mice Immunized with HIV-1 p24 Gag
본 발명에서는 향상된 p24 단백질 생성을 보이는 rBCG-pMyong2-p24가 T 세포 증식 능력을 개선시킬 수 있는지의 여부를 측정하기 위하여, 4개의 상이한 균주, 즉 야생형 BCG(대조군으로서), 2가지 유형의 rBCG(rBCG-pMyong2-p24 및 rBCG-pAL-p24), 및 rSmeg-pMyong2-p24으로 각각 감염된 BMDCs에서의 T 세포 증식을 혼합된 림프구 반응(mixed lymphocyte response, MLR)을 통한 CFSE 희석 방법을 사용하여 분석을 수행하였다. 동일한 pMyong2 벡터 시스템, 즉 rBCG-pMyong2-p24 및 rSmeg-pMyong2-p24를 사용하여 2개의 상이한 종 사이의 HIV-1 p24 Gag 특이적 면역 반응을 유도하는 능력을 비교하기 위하여 rSmeg-pMyong2-p24 균주를 또한 포함시켰다. T 세포 증식 분석의 개략적인 내용은 도 4a에 도시하였다.In the present invention, to determine whether rBCG-pMyong2-p24 showing improved p24 protein production can improve T cell proliferation ability, four different strains, wild type BCG (as a control), two types of rBCG ( T cell proliferation in BMDCs infected with rBCG-pMyong2-p24 and rBCG-pAL-p24), and rSmeg-pMyong2-p24, respectively, was analyzed using the CFSE dilution method through mixed lymphocyte response (MLR). Was performed. To compare the ability to induce HIV-1 p24 Gag specific immune responses between two different species using the same pMyong2 vector system, ie rBCG-pMyong2-p24 and rSmeg-pMyong2-p24, the rSmeg-pMyong2-p24 strain was used. Also included. A schematic of the T cell proliferation assay is shown in FIG. 4A.
결과적으로 2개의 rBCG 및 1개의 rSmeg 균주로 감염된 모든 BMDCs는 감염되지 않은 BMDCs보다 상당히 더 높은 수준의 CD4 및 CD8 T 세포 증식을 유도하였다. 특히, rBCG-pMyong2-p24로 감염된 BMDCs는 다른 2개의 재조합 균주(rBCG-pAL-p24 및 rSmeg-pMyong2-p24 균주) 및 야생형 BCG 균주로 감염된 것들보다 상당히 더 높은 수준의 CD4 및 CD8 T 세포 증식을 유도하였다. 그러나, rBCG-pAL-p24로 감염된 BMDCs와 rSmeg-pMyong2-p24로 감염된 것들 사이에서는 CD4 및 CD8 T 세포 둘 다의 증식에서 유의한 차이는 관찰되지 않았다(도 4b 및 4c). 자극된 CD4 및 CD8 T 세포에서 IL-2 수준의 비교는 또한 T 세포 증식 분석에서 관찰된 것들과 유사한 경향성을 나타냈다(도 4d).As a result, all BMDCs infected with two rBCG and one rSmeg strains induced significantly higher levels of CD4 and CD8 T cell proliferation than uninfected BMDCs. In particular, BMDCs infected with rBCG-pMyong2-p24 showed significantly higher levels of CD4 and CD8 T cell proliferation than those infected with the other two recombinant strains (rBCG-pAL-p24 and rSmeg-pMyong2-p24 strains) and wild-type BCG strains. Induced. However, no significant difference was observed in the proliferation of both CD4 and CD8 T cells between BMDCs infected with rBCG-pAL-p24 and those infected with rSmeg-pMyong2-p24 (FIGS. 4B and 4C). Comparison of IL-2 levels in stimulated CD4 and CD8 T cells also showed similar trends as those observed in T cell proliferation assays (FIG. 4D).
실시예 3. rBCG-pMyong2-p24 균주는 피하 면역화에 의해 생성된 마우스 비장에서 향상된 HIV-1 p24 Gag 특이적 IFN-γ 스팟 형성 세포(spot forming cells, SFC)를 유도한다Example 3 rBCG-pMyong2-p24 Strain Induces Improved HIV-1 p24 Gag Specific IFN-γ Spot Forming Cells (SFC) in Mouse Spleens Generated by Subcutaneous Immunization
rBCG-pMyong2-p24의 예방접종 후에 T 세포 반응이 개선되었는지 여부를 측정하기 위하여, 3개의 상이한 균주, 즉 2개 유형의 rBCG 균주(rBCG-pMyong2-p24 및 -pAL-p24), rSmeg-pMyong2-p24(도 5a) 및 대조군으로서 야생형 BCG 균주(~10
6 CFU)으로 피하(subcutaneously) 면역화된 BALB/c 마우스(5마리/그룹)의 비장으로부터 비장세포를 분리하고 IFN-γ ELISPOT 분석법을 사용하여 HIV-1 p24 Gag-특이적 T 세포 반응에 대해 분석하였다. 3개의 재조합 균주로 피하 면역화된 마우스로부터 얻은 비장세포는 야생형 BCG 균주로 면역화된 마우스로부터 얻은 비장세포보다 상당히 더 높은 SFUs를 나타냈다. 특히, rBCG-pMyong2-p24 (987.78 ± 195.11 SFUs/10
6 비장세포)로 면역화된 마우스로부터 채취한 비장세포는 다른 2개의 균주, 즉 rBCG-pAL-p24(479.56 ± 213.90 SFUs/10
6 비장세포) 및 rSmeg-pMyong2-p24 (647.00 ± 151.01 SFUs/10
6 비장세포)로 면역화된 마우스로부터 채취한 비장세포보다 상당히 더 높은 SFUs를 유발하였다(도 5b). 그러나, 예방 접종된 마우스로부터의 p24 특이적 IFN-γ SFUs에서 rBCG-pAL-24와 rSmeg-pMyong2-p24 균주 사이에 유의한 차이는 관찰되지 않았다(도 5b).In order to determine whether the T cell response improved after vBC v-pMyong2-p24 vaccination, three different strains, two types of rBCG strains (rBCG-pMyong2-p24 and -pAL-p24), rSmeg-pMyong2- Splenocytes were isolated from spleens of BALB / c mice (5 mice / group) subcutaneously immunized with p24 (FIG. 5A) and wild type BCG strain (˜10 6 CFU) as a control and using IFN-γ ELISPOT assay HIV-1 p24 Gag-specific T cell responses were analyzed. Splenocytes from mice subcutaneously immunized with three recombinant strains showed significantly higher SFUs than splenocytes from mice immunized with wild type BCG strain. In particular, splenocytes harvested from mice immunized with rBCG-pMyong2-p24 (987.78 ± 195.11 SFUs / 10 6 splenocytes) showed two different strains: rBCG-pAL-p24 (479.56 ± 213.90 SFUs / 10 6 splenocytes). And significantly higher SFUs than splenocytes harvested from mice immunized with rSmeg-pMyong2-p24 (647.00 ± 151.01 SFUs / 10 6 splenocytes) (FIG. 5B). However, no significant difference was observed between rBCG-pAL-24 and rSmeg-pMyong2-p24 strains in p24 specific IFN-γ SFUs from vaccinated mice (FIG. 5B).
종합하면, 본 발명의 데이터는 rBCG-pMyong2-p24가, Th-1 시그니처 사이토카인인, IFN-γ의 향상된 HIV-1 p24 Gag-특이적 생성을 유도하였고, Th-1형 면역 반응을 왜곡시킴으로써 백신 효능을 향상시키기 위한 실행가능성을 보여준다.Taken together, the data of the present invention indicate that rBCG-pMyong2-p24 induced enhanced HIV-1 p24 Gag-specific production of IFN-γ, a Th-1 signature cytokine, and by distorting the Th-1 type immune response Show feasibility to improve vaccine efficacy.
실시예 4. rBCG-pMyong2-p24 균주는 예방접종된 마우스에서 얻은 비장세포에서 Th1 또는 전-염증성(pro-inflammatory) 사이토카인의 향상된 생성을 유도한다Example 4 rBCG-pMyong2-p24 Strain Induces Enhanced Production of Th1 or Pro-inflammatory Cytokines in Splenocytes from Immunized Mice
rBCG 균주 및 rSmeg-pMyong2-p24로 두 번 면역화한 후 4주 뒤에 얻어진 비장세포(5마리/그룹)(도 5a)를 정제된 p24 단백질(5μg/ml)로 3개 한 벌로 시험관 내에서 자극시키고, 세포 배양 상층액 중의 IL-2, IFN-γ 및 IL-6의 유도된 사이토카인 생성을 검출하였다. 두 종류의 Th1형 사이토카인, 즉 IL-2 및 IFN-γ, 그리고 하나의 전-염증성 사이토카인, 즉 IL-6에서, rBCG-pMyong2-p24 균주는 야생형 또는 다른 2개의 재조합 균주에서보다 항상, 모든 시점(1일차 및 3일차)에서, 예방 접종된 마우스로부터 얻은 비장세포에서 보다 더 높은 수준의 사이토카인을 생성하였다(도 5c 및 표 1).Four weeks after immunization twice with rBCG strain and rSmeg-pMyong2-p24, splenocytes (5 mice / group) (FIG. 5A) obtained were stimulated in vitro with three sets of purified p24 protein (5 μg / ml). Induced cytokine production of IL-2, IFN-γ and IL-6 in cell culture supernatants was detected. In two types of Th1 cytokines, IL-2 and IFN-γ, and one pro-inflammatory cytokine, IL-6, the rBCG-pMyong2-p24 strain is always more than in wild type or two other recombinant strains, At all time points (Days 1 and 3), higher levels of cytokines were produced in splenocytes obtained from vaccinated mice (FIG. 5C and Table 1).
실시예 5. rBCG-pMyong2-p24 균주는 면역화된 마우스에서 HIV-1 p24 Gag-특이적 Th1-바이어스된 체액성 반응을 유도한다Example 5 rBCG-pMyong2-p24 Strain Induces HIV-1 p24 Gag-Specific Th1-Biased Humoral Response in Immunized Mice
본 발명에서는 rBCG-pMyong2-p24가 면역화된 마우스에서 Th1-바이어스된 체액성 반응을 유도하는지의 여부를 측정하기 위하여, 각각 Th1 및 Th2 반응의 공지된 마커인 HIV-1 p24 Gag-특이적 IgG2a 및 IgG1의 수준을 분석하였다. 도 6에서 나타난 바와 같이, 2개의 rBCG 및 rSmeg-pMyong2-p24 균주는 야생형보다 상당히 더 높은 수준의 IgG2a 동형(isotype)을 유도하였다. IgG1 동형과 관련하여, 3개의 재조합 균주가 유사한 수준의 IgG1을 유도하였다; 그러나, 그 결과는 통계학적 유의미성에는 도달하지 못하였다. 총 IgG의 경우에, rBCG-pMyong2-p24는 다른 두 개의 재조합 균주(즉 rBCG-pAL-p24 및 rSmeg-pMyong2-p24)보다 상당히 더 높은 수준의 총 IgG를 나타냈다(도 6). In the present invention, in order to determine whether rBCG-pMyong2-p24 induces a Th1-biased humoral response in immunized mice, HIV-1 p24 Gag-specific IgG2a and the known markers of Th1 and Th2 responses, respectively, The level of IgG1 was analyzed. As shown in Figure 6, the two rBCG and rSmeg-pMyong2-p24 strains induced significantly higher levels of IgG2a isotype than the wild type. With respect to IgG1 isotypes, three recombinant strains induced similar levels of IgG1; However, the results did not reach statistical significance. For total IgG, rBCG-pMyong2-p24 showed significantly higher levels of total IgG than the other two recombinant strains (ie rBCG-pAL-p24 and rSmeg-pMyong2-p24) (FIG. 6).
종합적으로, IgG2a/IgG1 비율은 더 높을수록 더욱 Th1-바이어스된 체액성 면역 반응을 나타내며, 상기 비율은 rBCG-pMyong2-p24(1.03 ± 0.02)로 면역화된 마우스에서 채취한 혈청에서, 다른 균주들(야생형 BCG = 0.91 ± 0.71; rBCG-pAL-p24 = 0.88 ± 0.21; rSmeg-pMyong2-p24 = 1.01 ± 0.17)로 면역화된 마우스에서 채취한 혈청에서의 비율보다 더 높았고, 이러한 결과는 rBCG-pMyong2-p24 균주가 면역화된 마우스에서 향상된 HIV-1 p24 Gag-특이적 Th1-바이어스된 체액성 반응을 유도할 수 있음을 보여준다.Overall, the higher IgG2a / IgG1 ratio indicates a more Th1-biased humoral immune response, which ratio is different from other strains in serum taken from mice immunized with rBCG-pMyong2-p24 (1.03 ± 0.02). Wild type BCG = 0.91 ± 0.71; rBCG-pAL-p24 = 0.88 ± 0.21; rSmeg-pMyong2-p24 = 1.01 ± 0.17), which was higher than the ratio in serum taken from mice immunized with rBCG-pMyong2-p24 It is shown that the strain can induce an enhanced HIV-1 p24 Gag-specific Th1-biased humoral response in immunized mice.
실시예 6. rBCG-pMyong2-p24 균주는 면역화된 마우스에서 향상된 HIV-1 p24 Gag-특이적 세포독성 T 림프구 반응을 유도한다Example 6 rBCG-pMyong2-p24 Strain Induces Enhanced HIV-1 p24 Gag-Specific Cytotoxic T Lymphocyte Responses in Immunized Mice
본 발명에서는 rBCG-pMyong2-p24가 면역화된 마우스에서 향상된 HIV-1 p24 Gag-특이적 세포독성 T 림프구(CTL) 반응을 유도하는지의 여부를 측정하기 위하여, 2개의 rBCG(rBCG-pMyong2-p24, -pAL-p24), rSmeg-pMyong2-p24 또는 야생형 BCG 균주로 면역화된 마우스로부터의 비장세포에서 CTL 활성을 LDH 세포독성 분석을 통해 평가하였다. 면역화 과정은 도 5a에서 기술하였다. A9I 펩타이드로 2시간 동안 펄스된 P815 세포(H-2d)가 표적 세포로서 사용되었고, 이펙터/표적 비율은 앞서 기술된 것과 같이 10:1, 20:1, 및 50:1이었다. 도 7에서 나타난 바와 같이, 50:1의 E:T 비율에서, rBCG-pMyong2-p24로 면역화된 마우스의 CTL이 다른 균주들로 면역화된 것들보다 상당히 더 높은 수준의 HIV-1 p24 Gag 특이적 표적 세포 용해(lysis)를 유도할 수 있음을 보여준다(도 7). 그러나, rBCG-pMyong2-p24와 rSmeg-pMyong2-p24 균주 사이에서 유의미한 차이는 관찰되지 않았다(도 7).In the present invention, to determine whether rBCG-pMyong2-p24 induces an enhanced HIV-1 p24 Gag-specific cytotoxic T lymphocyte (CTL) response in immunized mice, two rBCGs (rBCG-pMyong2-p24, -pAL-p24), CTL activity in splenocytes from mice immunized with rSmeg-pMyong2-p24 or wild type BCG strain was assessed by LDH cytotoxicity assay. The immunization process is described in Figure 5a. P815 cells (H-2d) pulsed with A9I peptide for 2 hours were used as target cells and effector / target ratios were 10: 1, 20: 1, and 50: 1 as described above. As shown in Figure 7, at an E: T ratio of 50: 1, CTLs of mice immunized with rBCG-pMyong2-p24 had significantly higher levels of HIV-1 p24 Gag specific target than those immunized with other strains. It can be shown that it can induce cell lysis (FIG. 7). However, no significant difference was observed between rBCG-pMyong2-p24 and rSmeg-pMyong2-p24 strains (FIG. 7).
실시예 7. rBCG-pMyong2-p24 균주는 p24 단백질 예방접종과 비교하여, 면역화된 마우스에서 향상된 HIV-1 p24 Gag-특이적 체액성 및 세포 매개된 면역 반응을 유도한다Example 7 rBCG-pMyong2-p24 Strain Induces Improved HIV-1 p24 Gag-Specific Humoral and Cell Mediated Immune Responses in Immunized Mice Compared with p24 Protein Vaccination
본 발명에서는 p24 단백질과 rBCG-pMyong2-p24 균주들의 상이한 CFU 사이의 p24 특이적 면역 반응을 비교하기 위하여, 다음과 같은 그룹으로 독립적인 생체내 실험을 수행하였다. i) PBS 대조군, ii) p24 단백질(30μg/마우스) 주사, iii) rBCG-pMyong2-p24(1 Х 10
6 CFU) 주사, 및 iv) rBCG-pMyong2-p24(1 Х 10
7 CFU) 주사(1주 간격, 2회 피하 주사) 그룹. 면역화 과정은 실험 방법 단락에서 기술하였다. 마지막 면역화 후에, p24-특이적 IFN-γ ELISPOT, IgG 아형 분석 및 CTL 분석을 수행하였다. IFN-γ ELISPOT 분석의 경우, p24 특이적 IFN-γ SFU가 CFU 의존적 방식으로 증가하였다. 그러나, p24 단백질이 주사된 마우스로부터 얻은 비장세포는 p24 특이적 IFN-γ SFU를 유도할 수 없었다(도 8a). 이와 유사하게, 각각의 면역화된 마우스로부터 얻은 혈청 샘플에서 p24 특이적 IgG2a 항체 또한 CFU 의존적 방식으로 증가하였다. 그러나, p24 단백질이 주사된 마우스의 혈청에서 p24 특이적 IgG2a 항체는 rBCG-pMyong2-p24 주사 그룹의 그것들과 비교하여 더 낮은 수준을 나타냈다(도 8b). In the present invention, in order to compare p24 specific immune responses between different CFUs of p24 protein and rBCG-pMyong2-p24 strains, independent in vivo experiments were performed in the following groups. i) PBS control, ii) p24 protein (30 μg / mouse) injection, iii) rBCG-pMyong2-p24 (1 Х 10 6 CFU) injection, and iv) rBCG-pMyong2-p24 (1 Х 10 7 CFU) injection (1 Weekly interval, 2 subcutaneous injections) group. Immunization procedures are described in the Experimental Methods section. After the last immunization, p24-specific IFN-γ ELISPOT, IgG subtype analysis and CTL analysis were performed. For IFN-γ ELISPOT assays, p24 specific IFN-γ SFU was increased in a CFU dependent manner. However, splenocytes from mice injected with the p24 protein were unable to induce p24 specific IFN-γ SFU (FIG. 8A). Similarly, p24 specific IgG2a antibodies also increased in a CFU dependent manner in serum samples from each immunized mouse. However, p24 specific IgG2a antibodies in the serum of mice injected with p24 protein showed lower levels compared to those of the rBCG-pMyong2-p24 injection group (FIG. 8B).
또한 p24 단백질과 rBCG-pMyong2-p24 균주들의 상이한 CFU 사이의 p24 특이적 CTL 반응을 비교하였다. 본 명세서의 데이터는 rBCG-pMyong2-p24의 p24 특이적 CTL 반응이 CFU 의존적 방식으로 증가하였고 항상 p24 단백질의 그것보다 상당히 더 높았음을 나타냈다(도 8c).We also compared the p24 specific CTL response between p24 protein and different CFUs of the rBCG-pMyong2-p24 strains. The data herein indicated that the p24 specific CTL response of rBCG-pMyong2-p24 was increased in a CFU dependent manner and was always significantly higher than that of the p24 protein (FIG. 8C).
종합적으로, 본 명세서의 데이터는 rBCG-pMyong2-p24가 CFU 의존적 방식으로 p24-특이적 Th1-바이어스된 세포성 및 체액성 면역 반응을 유도할 수 있고, p24 단백질 예방접종 모듈과 비교하여, HIV-1 백신 양생법으로서의 장점을 가질 수 있음을 보여준다.Overall, the data herein indicate that rBCG-pMyong2-p24 can induce p24-specific Th1-biased cellular and humoral immune responses in a CFU dependent manner, and compared to the p24 protein vaccination module, HIV- 1 shows that it can have advantages as a vaccine curing method.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
Claims (12)
- 서열번호 1의 아미노산 서열로 나타내는 인간면역결핍바이러스 타입 1 유래의 p24 단백질을 발현하는 재조합 마이코박테리움 보비스(Mycobacterium bovis) BCG로서, 상기 p24 단백질은 도 2a에 개시된 pMyong2-p24 벡터 시스템에 의해 발현되는 것을 특징으로 하는, 재조합 마이코박테리움 보비스 BCG.As a recombinant Mycobacterium bovis BCG expressing p24 protein from human immunodeficiency virus type 1 represented by the amino acid sequence of SEQ ID NO: 1, said p24 protein is expressed by the pMyong2-p24 vector system disclosed in FIG. 2A Recombinant Mycobacterium bovis BCG.
- 제 1 항에 있어서, The method of claim 1,상기 p24 단백질은 서열번호 2의 염기 서열로 나타내는 인간면역결핍바이러스 타입 1 유래의 Gag 유전자가 코딩된 것인, 재조합 마이코박테리움 보비스 BCG.The p24 protein is a recombinant Mycobacterium bovis BCG encoded by the Gag gene derived from human immunodeficiency virus type 1 represented by the nucleotide sequence of SEQ ID NO: 2.
- 제 1 항에 있어서, The method of claim 1,상기 마이코박테리움 보비스 BCG는, Tokyo 172 균주인 것인, 재조합 마이코박테리움 보비스 BCG.The mycobacterium bovis BCG is a recombinant strain of Mycobacterium bovis BCG, which is a Tokyo 172 strain.
- 제 1 항 내지 제 3 항 중 어느 한 항에 따른 재조합 마이코박테리움 보비스 BCG를 유효성분으로 포함하는, HIV-1 백신 조성물.The HIV-1 vaccine composition comprising the recombinant mycobacterium bovis BCG according to any one of claims 1 to 3 as an active ingredient.
- 제 1 항 내지 제 3 항 중 어느 한 항에 따른 재조합 마이코박테리움 보비스 BCG를 유효성분으로 포함하는, HIV 감염증 또는 HIV 및 결핵균 동시 감염증에 대한 백신 조성물. A vaccine composition for HIV infection or HIV and Mycobacterium tuberculosis infection comprising the recombinant mycobacterium bovis BCG according to any one of claims 1 to 3 as an active ingredient.
- 제 5 항에 있어서, The method of claim 5,상기 재조합 마이코박테리움 보비스 BCG는 살아있는 것인, 백신 조성물. The recombinant mycobacterium bovis BCG is alive, vaccine composition.
- 제 5 항에 있어서, The method of claim 5,상기 백신은 추가로 약독화(attenuation) 되지 않은 것인, 백신 조성물.The vaccine composition is further attenuated.
- 제 5 항에 있어서, The method of claim 5,상기 백신은 프라임-부스트 백신 접종법에서 프라임 백신으로 사용되는 것인, 백신 조성물.The vaccine is used as a prime vaccine in prime-boost vaccination, vaccine composition.
- 제 5 항에 있어서, The method of claim 5,상기 감염증은 에이즈 또는 결핵인, 백신 조성물.The infectious disease is AIDS or tuberculosis.
- 제 1 항 내지 제 3 항 중 어느 한 항에 따른 재조합 마이코박테리움 보비스 BCG의 치료학적 유효량을 유효성분으로 포함하는 백신 조성물을 개체에 투여하여 에이즈 및/또는 결핵을 치료 또는 예방하는 방법.A method of treating or preventing AIDS and tuberculosis by administering to a subject a vaccine composition comprising a therapeutically effective amount of the recombinant mycobacterium bovis BCG according to any one of claims 1 to 3.
- 에이즈 및/또는 결핵의 예방 또는 치료용 백신의 제조를 위한, 제 1 항 내지 제 3 항 중 어느 한 항에 따른 재조합 마이코박테리움 보비스 BCG의 용도.Use of the recombinant mycobacterium bovis BCG according to any one of claims 1 to 3 for the manufacture of a vaccine for the prevention or treatment of AIDS and / or tuberculosis.
- 에이즈 및/또는 결핵의 예방 또는 치료를 위한, 제 1 항 내지 제 3 항 중 어느 한 항에 따른 재조합 마이코박테리움 보비스 BCG의 용도.Use of the recombinant mycobacterium bovis BCG according to any one of claims 1 to 3 for the prevention or treatment of AIDS and / or tuberculosis.
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| US17/055,394 US11512317B2 (en) | 2018-05-14 | 2019-03-27 | Recombinant BCG expressing HIV-1 p24 using pMyong2 vector system and use thereof |
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| KR1020190034183A KR102079761B1 (en) | 2018-05-14 | 2019-03-26 | A recombinant BCG expressing HIV-1 p24 using a pMyong2 vector system and use as a HIV-1 vaccine thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006076343A1 (en) * | 2005-01-12 | 2006-07-20 | Albert Einstein College Of Medicine Of Yeshiva University | Mycobacteria expressing hiv-1 and malaria antigens |
| KR101291668B1 (en) * | 2011-04-21 | 2013-08-01 | 서울대학교산학협력단 | Shuttle Vectors for Mycobacteria-Escherichia coli and Uses Thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2006076343A1 (en) * | 2005-01-12 | 2006-07-20 | Albert Einstein College Of Medicine Of Yeshiva University | Mycobacteria expressing hiv-1 and malaria antigens |
| KR101291668B1 (en) * | 2011-04-21 | 2013-08-01 | 서울대학교산학협력단 | Shuttle Vectors for Mycobacteria-Escherichia coli and Uses Thereof |
Non-Patent Citations (4)
| Title |
|---|
| DATABASE GenBank 25 August 2017 (2017-08-25), "gag protein, partial [Human immunodeficiency virus 1", Database accession no. ASV46276.1 * |
| KIM, B. J. ET AL.: "Development of a Live Recombinant BCG Expressing Human Immunodeficiency Virus Type 1 (HIV-1) Gag Using a pMyong2 Vector System: Potential Use As a Novel HIV-1 Vaccine", FRONTIERS IN IMMUNOLOGY, vol. 9, 27 March 2018 (2018-03-27), pages 1 - 13 * |
| KIM, B. J. ET AL.: "Recombinant Mycobacterium smegmatis with a pMyong2 vector expressing Human Immunodeficiency Virus Type I Gag can induce enhanced virus-specific immune responses", SCIENTIFIC REPORTS, vol. 7, 16 March 2017 (2017-03-16), pages 1 - 11 * |
| LEE, H. ET AL.: "The development of a novel Mycobacterium-Escherichia coli shuttle vector system using pMyong2, a linear plasmid from Mycobacterium yongonense DSM 45126T", PLOS ONE, vol. 10, no. 3, 30 March 2015 (2015-03-30), pages 1 - 21, XP055653645 * |
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