KR20180080999A - Recombinant Mycobacterium smegmatis with vector expressing Human Immunodeficiency Virus Type 1 p24 and vaccine comprising the same - Google Patents

Abstract

According to the present invention, disclosed are a recombinant Mycobacterium smegmatis strain expressing p24 protein of human immunodeficiency virus type I, and a vaccine composition for HIV infection including the same. The vaccine according to the present invention expresses an antigen at a high level to effectively present the same to phagocytes, improves T cell proliferative capacity of dendritic cells, and can induce a humoral immune response which is Th1-biased with an improved T cell effector function from an inoculated mice, thereby being effectively used for a co-infection of HIV-1 and tuberculosis.

Description

{Recombinant Mycobacterium smegmatis with vector expressing human pneumococcal vaccinia virus and pneumococcal vaccine containing the p24 protein of HIV 1,

This is a technical field related to a strain vaccine.

It focuses on the development of vaccine cell immunity induction strategies, especially CTL1, for the development of vaccines against diseases such as AIDS and tuberculosis. Early laboratory experiments in laboratory animals and humans indicate that recombinant vectors contained in live bacteria can cause CD4 and CD8 T-lymphocyte responses against a variety of pathogenic microorganisms. Of particular note, the most effective strategy for inducing cellular immune responses is the heterozygous prime / boost regimen. Recently, rSmeg (recombinant M. smegmatis ) strains containing live recombinant vectors have been shown to induce a cellular immune response to HIV-1 infection by enhancing the active secondary immune response after boosting, particularly by expanding the compartmental CTL of the large pool, And could act as a priming vector for vaccination.

This is due to the ability of Smeg to induce differentiation into antigen-specific memory CD8 T cells, but it has its limitations. In other words, it is a problem to induce a small number of antigen-specific CD8 T cells as compared to those induced by other vectors. This limitation is partly due to the fact that only a small amount of antigen is expressed in vivo by rSmeg, Lt; RTI ID = 0.0 > MHC < / RTI > class I presentation due to limited access of the antigen to the cytoplasm.

Thus, in order to improve the efficacy of rSmeg as a priming vector in prime / boost vaccination, it is necessary to develop a suitable mycobacterial vector system and strains containing it that are particularly effective in inducing the transferred gene to the MHC class I processing pathway .

U.S. Patent No. 7,612,173 (published October 18, 2007) U.S. Publication No. 2008/0254061 (published October 16, 2008)

The present application is directed to providing a suitable mycobacterial vector system and a strain and vaccine comprising it that are particularly effective in deriving the transferred gene to the MHC class I processing pathway in order to improve the efficacy of rSmeg as a priming vector in prime / boost vaccination do.

In one embodiment, the present invention provides a recombinant Mycobacterium smegmatis that expresses the p24 protein from human immunodeficiency virus type I.

In the strain according to the present invention, the p24 protein is expressed by the pMyong2-p24 plasmid shown in Fig.

In one embodiment, the Mycobacterium smegmatis strain according to the present invention is deposited on November 24, 2017 with accession number KCTC (Korean Collection for Type Cultures) 13403BP.

The present invention also provides vaccine compositions for HIV infection or HIV and Mycobacterium tuberculosis co-infections comprising a strain according to the invention and a pharmaceutically acceptable excipient.

In a vaccine according to the present invention, the strain is live.

In one embodiment, the vaccine according to the present invention is a strain that is not further artificially attenuated.

The vaccine according to the present invention can be used as a priming vaccine, especially in the prime-boost vaccination method.

The vaccine according to the present invention may be used for AIDS or tuberculosis.

The present application also provides the pMyong2-p24 plasmid disclosed in Figure 1 for use in the expression of p24 antigen in a strain according to the present invention.

The present invention also provides a cell comprising a plasmid according to the present invention.

The rSmeg-pMyong2-p24 strain according to the present invention can induce higher levels of HIV-1 p24 Gag protein expression and deliver more p24 antigen into the phagocyte as compared to other rSmeg strains using the pAL5000 or pMV306 induction system. Furthermore, these strains have been shown to enhance the T cell proliferative capacity of infected BMDC and induce enhanced T cell effector function and Th1-biased humoral immune response in inoculated mice.

In addition, the strain rSmeg-pMyong2-p24 according to the present invention has a delayed initial growth, attenuates the effect, and can present more antigen to phagocytes to maintain a stronger immune response. These results indicate that rSmeg-pMyong2-p24 according to the present invention can be a candidate vaccine effective for co-infection of HIV-1 or HIV-1 with tuberculosis.

Figure 1 is one embodiment according to the present p24 expressed in vector map, a p24 expression produced mycobacteria - Escherichia coli shuttle vector map shows the diagrammatically, pMV306-p24, p24 and pAL-pMyong2-p24 vectors of M. bovis BCG hsp65 Under the control of the promoter, p24 was expressed, and the insert was inserted in the XbaI and EcoRV sites of each vector in the 5 '->3' direction.
FIG. 2 shows the results of observing the expression level of p24 by mouse macrophages infected with recombinant M. smegmatis (rSmeg) and rSmeg strain according to one embodiment of the present invention: (a) p24 expression in rSmeg was confirmed by ELISA; (b) Expression of p24 in rSmeg was confirmed by Western blot, and wild-type M. smegmatis (lane 1) and rSmeg strains (lane 2, rSmeg-pMV306-p24, lane 3, rSmeg-pAL-p24; lane 4, rSmeg-pMyong2-p24). Purified p24 protein was used as a positive control (lane 5). M is a molecular weight standard; a (Elpis Bio, Taejeon, Korea DokDo -MARK TM); (left) and BMDCs (right) infected with the rSmeg strain (rSmeg-pMV306-p24, rSmeg-pAL-p24, rSmeg-pMyong2-p24).
FIG. 3 shows the results of BMDCs-induced T cell proliferation in p24 recombinant M. smegmatis strains: (a) Schedule diagram for T cell proliferation assay; (b and c) is the result of flow cytometry analysis of cleavage of CFSE-labeled CD4, CD8 T cells induced by BMDCs infected with the p24 recombinant M. smegmatis strain; (d) IL-2 ELISA in the supernatants of CD4 (left) and CD8 (right) T cells used in the mixed lymphocyte reaction (MLR) assay. The data represent representative results of three independent experiments. All statistical analyzes were calculated by comparing the values of rSmeg-pMyong2-p24. Results were expressed as mean ± variance. * P < 0.05; ** P < 0.01; *** P <0.001.
Figure 4 shows the in vivo induced immune response results by p24 rSmeg strains: (a) a schematic view of the immunization schedule for in vivo immunological analysis; (b) In vitro stimulation of mouse splenocytes immunized with p24 rSmeg strains, and the secretion potency of IFN-γ was analyzed by ELISPOT, and representative images of each group of ELISPOT were shown at the bottom of the graph; (c) ELISA for IL-2, IFN-γ, TNF-α, and IL-10 cytokines after stimulation with splenocytes in mice immunized with p24 recombinant rSmeg strains in vitro -24 for p24 and for IFN-y and IL-10 for 72 hours; (d) The OD values for the IgG2a and IgG1 subtypes were compared with the IgG2a / IgG1 ratio as a result of p24-specific immunoglobulin subtype (IgG2a, IgG1) ELISA confirmed at 450 nm. All statistical analyzes were calculated by comparing the values of rSmeg-pMyong2-p24. Results were expressed as mean ± variance. * P < 0.05; ** P < 0.01; *** P <0.001.
Figure 5 shows the results of cytotoxic T lymphocyte response of mice immunized with rSmeg strains: (a) CTL response results induced by reacting Ag85B-transformed P815 cells with Ag85B-stimulated splenocytes in vitro; (b) P815 cells transfected with p24 and splenocytes stimulated with p24 in vitro. Two independent experiments showed typical results. All statistical analyzes were performed in comparison with the values of rSmeg-pMyong2-p24. Results were expressed as mean ± variance. * P < 0.05; ** P &lt; 0.01; *** P <0.001.
Figure 6 shows the growth curves of the p24 recombinant strains (rSmeg-pMV306-p24, rSmeg-pAL-p24, rSmeg-pMyong2-p24) and the wild-type M. smegmatis strain of the present invention. When the strain rSmeg-pMyong2-p24 was cultured for 5 days in 7H9 broth (with 100 μg / ml of kanamycin) together with other comparative strains (rSmeg-pMV306-p24, rSmeg-pAL-p24 and wild type M. smegmatis ) Indicates that the growth is slower than the other strains at the initial stage (6 to 48 hours). This difference is an indirect result that the vector according to the present invention is intended to maintain a high copy number and that the rSmeg-pMyong2-p24 strain according to the present invention is a more attenuated strain than the other recombinant strains in macrophage and mouse infection. It can be effectively used advantageously.
Fig. 7 shows the results of infection of wild type M. smegmatis and p24 recombinant strains (rSmeg-pMV306-p24, rSmeg-pAL-p24, rSmeg-pMyong2- p24) on macrophages (J774A.1) and BMDC Were fused and cultured in 7H10 solid medium (with 100 μg / ml of kanamycin). As a result of confirming intracellular CFU by infecting macrophage and BMDC with another recombinant strain including strain rSmeg-pMyong2-p24 according to the present invention, the rSmeg-pMyong2-p24 strain according to the present invention showed relatively low CFU as compared with other recombinant strains Respectively. These results indicate that the strains into which the vectors according to the present invention are introduced are attenuated compared with other recombinant strains, indicating that they can be advantageously used effectively as a vaccine.

Herein, recombinant Mycobacterium smegmatis expressing HIV-1 p24 Gag antigen It is based on the discovery that strains can be used effectively for vaccines.

In one embodiment, the present invention relates to a recombinant Mycobacterium smegmatis strain expressing the p24 protein of human immunodeficiency virus type I, wherein the p24 protein is expressed by the pMyong2-p24 plasmid shown in Fig. 1 Lt; / RTI &gt;

The p24 capsid (CA) protein expressed by the vector according to the present invention is linked to the 3 'end of MA (matrix protein) in untreated Gag polyprotein. The p24 capsid (CA) protein contains two domains that play important roles in HIV budding and capsid structures at the N and C ends. P24 expressed by the vector according to the invention is derived from human immunodeficiency virus type I and its sequence is GenBank No. 1. Sequence variants thereof, which are included in KM390026.1d or correspond to 3161 to 3850 nucleotides of the sequence of SEQ ID NO: 1, and which function as an antigen for the purposes herein, may also be used. The p24 expressed by the strain according to the present invention can act as an antigen against HIV infection upon administration to the human body.

In a strain according to the present invention, p24 is expressed in particular by the pMyong2 vector disclosed in Fig. Smeg strains containing pMyong2-p24 according to the present invention can induce higher levels of HIV-1 p24 protein expression and transfer more p24 antigen into phagocytes as compared to other rSmeg strains using the pAL5000 or pMV306 induction system, It is very good. In one embodiment 24 is particularly represented by the sequence of SEQ ID NO: 1, wherein the pMyong2 vector disclosed in FIG.

The strain according to the present invention was deposited on November 24, 2017 with the deposit number KCTC 13403BP at the Microbiological Resource Center of the Korea Research Institute of Bioscience and Biotechnology.

Further, the present invention provides a method for improving the T cell proliferation ability of a BMDC (Bone Marrow-Derived Dendritic Cell) infected with a Smeg recombinant strain containing pMyong2-p24 and inducing an enhanced T cell effector function and a Th1 biased humoral immune response in the inoculated mouse . In addition, rSmeg-pMyong2-p24 strain according to the present invention is delayed in early growth and attenuated, so that more antigen can be presented to phagocytes to maintain a stronger immune response. These results indicate that rSmeg-pMyong2-p24 according to the present invention can be a candidate vaccine effective for co-infection of HIV-1 or HIV-1 with tuberculosis.

In another aspect, this disclosure relates to an immunogenicity, vaccine or immunotherapeutic composition for an HIV infection comprising a strain as disclosed herein.

HIV is a type of retrovirus that destroys the human immune system. When infected with HIV, the host's immune system is lowered, progressing to AIDS, and significantly increasing the risk of opportunistic infection by bacteria, viruses, fungi and parasites do.

AIDS is a condition called AIDS that refers to a number of symptoms that result from a decrease in human immune function as a result of HIV infection.

The vaccine composition according to the present invention can be used to deliver a p24-specific immune response, for example, more p24 antigen to the phagocytic cells, to enhance the T cell proliferative capacity of BMDC and to provide enhanced T cell effector function and Th1- biased humoral immune response in inoculated mice And may prevent, prevent, or alleviate symptoms of HIV infection or symptoms due to infection.

Vaccines are generally administered in the prime-boost form two or more times. In this case, the same vaccine may be administered several times (homologus) or a different kind of vaccine containing the same antigen may be administered (heterologous).

In one embodiment, the vaccine according to the present invention is used as a priming vaccine in homologous or heterozygous prime-boost. The rSmeg-pMyong2-p24 strain according to the present invention is advantageous for use as a priming vaccine because it can induce a p24-specific immune response sufficient for a naive individual because of high p24 antigen expression and high antigen presentation capability.

The mycobacteria used in the composition according to the present invention are as described above, and in particular live bacteria can be used. In particular, the strain rSmeg-pMyong2-p24 according to the present invention is delayed in initial growth and naturally attenuated, so that more antigen can be presented to phagocytes to maintain a stronger immune response.

The vaccine composition according to the present invention may be formulated for systemic or topical administration and may be formulated with pharmaceutically acceptable excipients, carriers and / or vehicles such as phosphate buffered saline solution, distilled water, water / oil emulsion, wetting agent, emulsifying agent, pH And the like, but are not limited thereto.

The vaccine composition according to the present invention may also include any substance or compound which, if necessary, can facilitate or increase the extent of the T-cell mediated response. In one embodiment, the zvant may be used in the case where a chemical or thermally killed microorganism is included in the composition. Azubent is well known in the art and includes, for example, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, crosslinked polyacrylic acid polymers, dimethyldioctadecyl-ammonium bromide, immunomodulators, lactoferrin, or IFN- Can be used. In one embodiment, the crosslinked polyacrylic acid polymer comprises a Carbopol? C homopolymer or copolymer, wherein the lymphokine comprises IFN-gamma, IL-1, IL-2 or IL-12, gamma inducers may include, but are not limited to, poly I: C. In one embodiment, the synthesized sugar polymer comprises about 0.05 to 0.1 wt% of the aluminum hydroxide, aluminum phosphate, or aluminum potassium sulfate, and the crosslinked polyacrylic acid polymer may include, but is limited to, 0.25 wt% It is not.

The compositions according to the present invention may be administered topically or systemically and may be single or multiple doses and may be administered by various routes such as subcutaneous, intradermal, intramuscular or intravenous, or by oral, nasal or inhalation routes have. In one embodiment according to the present application, it is administered once by intramuscular injection.

The vaccine composition according to the present invention may be formulated as a liquid solution or suspension suitable for the route of administration or as a solid form dissolved or suspended in a solution prior to injection.

The dosage of the vaccine will depend on the route of administration as well as the age, weight and / or health condition of the patient. For example, a suitable dosage can be, for example, 1 to 10 ⁹ cfu. In one implementation, 10 ⁶ cfu is used. In the case of the strains according to the present invention, it is of course possible to use a smaller dose or a smaller dose as compared with a conventional strain vaccine, because the strain exhibits excellent antigen expression and antigen presentation ability.

In another embodiment, the present disclosure relates to the vector disclosed in Figure 1 used for the expression of p24 in a strain according to the present invention.

In one embodiment, the vector is represented by the sequence of SEQ ID NO: 1.

In another embodiment, the disclosure also relates to a cell transformed with said vector. Such cells include, in particular, mycobacteria.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Experimental Method

Mouse and Immune Process

Female BALB / c mice (~ 25 g, 7 weeks old) were purchased from Orient Bio (Korea) and experiments were conducted using 8-week old mice. Each strain (wild type M. smegmatis , recombinant M. smegmatis ; rSmeg-pMV306-p24, rSmeg-pAL-p24, rSmeg-pMyong2-p24) was subcutaneously inoculated . Two weeks after the last immunization, each mouse was euthanized via CO ₂ and respiration, and the spleen was detached and used for immunohistochemical analysis.

Human immunodeficiency virus  (HIV-1) expressing the Gag p24 antigen Myco bacteria - Construction of Escherichia coli shuttle vector

In order to prepare a mycobacteria-E. coli shuttle vector capable of expressing the P24 antigen, the promoter portion of the heat shock protein 65 kDa (heat shock protein 65, hsp65 ) was amplified from the DNA of M. bovis BCG and the DNA base corresponding to the p24 antigen The sequences were amplified in pNL4-3-deltaE-EGFP vector (NIH AIDS Reagent Program, Germantown, MD, USA) and then ligated by overlapping PCR. The primers used were as follows: M. bovis BCG hsp65 promoter of the primers to amplify the region sequences: Forward: 5'-TT GGTACC GGTGACCACAACGACGCGC -3 '( restriction enzyme corresponding to the Kpn I, the underlined part); And Reverse: 5'-CTGCACTATAGGCATTGCGAAGTGATTCCT-3 '. Primer sequence to be amplified by P24 gene: Forward: 5'-AGGAATCACTTCGCAATGCCTATAGTGCAG-3 '; And Reverse: 5'-AA TCTAGA CTACAAAACTCTTGCCTTATGGCCAGG-3 '( Xba I, restriction enzyme corresponding to the underlined region). For the overlapping PCR to link the amplified two genes, the forward of the hsp65 promoter and the reverse primer of the p24 gene were used.

Then, the overlapping sequences (phsp-p24) Kpn I and Xba I (NEB, Ipswich, MA, USA) cut with restriction enzyme T4 ligase (TaKaRa, Kyoto, Japan) with mycobacteria were cloned into Escherichia coli shuttle vectors pMV306 . Using the prepared pMV306-p24 as a template, phsp-p24 was amplified by PCR and then cloned again into pAL-TOPO and pMyong2-TOPO vectors. The primer sequence amplifying the Phsp-p24 nucleotide sequence is as follows: forward: 5'-TT GATATC GGTGACCACAACGACGCGC-3 '( EcoR V, restriction enzyme corresponding to the underlined region) and reverse primer of p24. The amplified PCR product was then cloned into pAL- and pMyong2-TOPO vectors after cutting with EcoR V and Xba I.

Recombination from E. coli Ag85B and  Production of p24 protein

E. coli strain BL21 (RBC Bioscience, Taipei City, Taiwan) was transformed with pET23a-Ag85B or p24 for the expression and purification of recombinant proteins. Protein expression was induced by adding 0.4 mM isopropyl [beta] -D-thiogalactoside (IPTG, Duchefa Biochemie, Haarlem, Netherlands). The transformed bacteria were collected and sonicated in ice for 10 minutes to destroy them. The supernatant was centrifuged (1,600 x g, 20 min, 4 ° C) and the pellet was resuspended in binding buffer (Sigma Aldrich, St. Louis, MO, USA) containing 4 M urea. Proteins were purified using Ni-NTA His binding resin (Merck, Darmstadt, Germany) and dissolved in elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 250 mM imidazole) containing 4 M urea. The purified protein was continuously dialyzed against the elution buffer to remove imidazole, urea, and residual base.

Recombination expressing HIV-1 p24 Gag M. smegmatis  ( rSmeg ) Production of strain

The pMV306-p24, pAL-p24, and pMyong2-p24 plasmids prepared above were designated as M. smegmatis mc ² 155 were transformed with electroporation (using the Gene Pulser II electroporation apparatus Bio-Rad, Hercules, Calif., USA). Transformed recombinant strains (rSmeg-pMV306-p24, rSmeg-pAL-p24, rSmeg-pMyong2-p24) were cultured in Middlebrook 7H10 solid medium (Difco Laboratories, Detroit, MI, USA) supplemented with OADC and kanamycin USA). The selected colonies were transferred to 7H9 liquid medium (Difco Laboratories) supplemented with 0.5% glycerol, 0.05% Tween-80, 10% ADC and kanamycin and cultured for 3 days. To compare the growth rate of wild-type M. smegmatis with recombinant strains, growth rates were compared by measuring absorbance over time using strains set at 0.2 OD at 600 nm.

rSmeg  Comparison of Expression Patterns of p24 Gag in Recombinant Strain

In order to compare the amount of p24 Gag expression between the recombinant strains prepared above, Western blot and ELISA were performed as follows. First, the cultured recombinant strains were suspended in B-PER buffer (Thermo Scientific, Rockford, Ill., USA) supplemented with lysozyme (100 μg / ml), DNase sonication (pulse: 0.3 sec, stop: 0.7 sec). After that, the pulverized liquid was centrifuged (13,000 rpm, 15 min, 4 ° C) and the supernatant was taken to quantify the protein (Quick Start ^TM Bradford Protein Assay; Bio-Rad, USA). The protein samples isolated from each recombinant strain were mixed with the sample buffer and heated at 100 ° C for 5 minutes. The heated sample was loaded onto a 12.5% SDS-PAGE gel and transferred to a nitrocellulose (NC) membrane. Membrane was placed in 5% skim milk (in TBST) and blocked for 1 hour at room temperature. Subsequently, mouse anti-p24 monoclonal antibody (Abcam, Cambridge, USA; 1: 1,000 dilution) was added and incubated overnight at 4 ° C. Then, HRP-conjugated goat anti-mouse secondary antibody (Abcam, 1: 2,000 dilution) was added and incubated at room temperature for 1 hour. The membrane was washed with TBST (0.05% Tween-20) at all stages. The immune response signal is transferred to the membrane via WEST-one ^TM Western blot detection system (iNtRON, Kyungkido, Republic of Korea) was treated with LAS-3000 (Fujifilm, Tokyo, Japan). Using mycobacterial Hsp65 (Abcam, 1: 1,000 dilution) as an internal control, confirm that all samples were dispensed in equal amounts.

In order to confirm the stable expression of p24 by the rSmeg-pMyong2-p24 strain, the expression of p24 was confirmed by separating proteins at various time points (1, 4, 6, 8, 10 and 12 passages). The passages were carried out in 7H10 solid medium with or without kanamycin. Colonies were selected for each passage and cultured in 7H9 liquid medium for 3 days. P24 ELISA (ABL, Rockville, USA) was also performed using the same amount of protein.

Differentiation of dendritic cells derived from mouse bone marrow cells

BALB / c mouse bone marrow cells from 8 to 12 weeks of age were isolated from the femur and tibial bone using serum-free Iscove's modified Eagle medium (IMDM; Gibco Invitrogen, UK) (Gibco Invitrogen), recombinant mouse GM-CSF (1.5 ng / ml; PeproTech, USA) supplemented with 1 × 10 ⁶ cells (100 μg / ml; Gibco Invitrogen), gentamicin (50 μg / ml) and mouse IL-4 (1.5 ng / ml; PeproTech, USA) (Gibco Invitrogen), L-glutamine (2 mM; Gibco Invitrogen), and? -mercaptoethanol (50 nM; Gibco Invitrogen) were dispensed into 24-well plates and differentiated for 6 days.

Differentiated BMDC (bone marrow derived dendritic cells, bone marrow derived dendritic cells) were either infected with wild-type M. smegmatis or each p24 recombinant strain or treated with lipopolysaccharide (LPS, 1 μg / ml; Sigma Aldrich) Dendritic cells were matured.

Analysis of p24 expression pattern in infected dendritic cells and J774.1 cells

Each recombinant strain corresponding to 10 MOIs was infected with J774A.1 and BMDC (5-10 x 10 ⁵ cells / well; 24-well plate) cultured for 18 hours. Two hours after infection, the cells were washed 3 times with PBS to remove extracellular bacteria. After infected J774A.1 and BMDC were cultured for 24 hours, the cells were lysed with RIPA lysis buffer, and the expression of p24 in the cells was analyzed by p24 ELISA.

T cell proliferation assay

To perform the T cell proliferation assay, CD4 and CD8 T cells immunized with p24 protein and DCs infected with wild type and recombinant strains were used. First, splenocytes were isolated from mice pretreated with p24 protein in the tail vein for 7 days. The splenocytes were washed with ice-cold FACS buffer (PBS containing 1% bovine serum albumin (BSA) and 1 mM EDTA) Respectively. Thereafter, a super block solution (Sigma Aldrich), 10% goat sera (Gibco Invitrogen), 10% mouse sera (Sigma Aldrich), and 2.4G2 monoclonal antibody (10 μg / ml; BD Biosciences, San Diego , CA, USA), and the blocking was carried out on ice for 30 minutes. After staining for 30 minutes with ice-cold PBS421-conjugated anti-CD4 (Clone GK1.5, BD Biosciences) and PE-conjugated anti-CD8a (Clone 53-6.7, eBioscience, San Diego, CA, USA) and washed three times with -cold FACS buffer. The CD4 and CD8 T cell populations were then isolated via FACS AriaIII instrument (BD Biosciences).

In addition, wild type and three types of rSmeg strains (rSmeg pMV306-p24, pAL-p24 and pMyong2-p24) were infected with immature DCs at 10 M.O.I. for 24 hours. Isolated CD4 and CD8 T cells were stained with 5 μM CFSE for 4 minutes at 37 ° C and for 4 minutes on ice. CD4 and CD8 T cells stained with infected DCs and CFSE were co-cultured, blocked with super block solution for 30 min on ice, and incubated for 30 min with BV421-conjugated anti-CD4 antibody and PE-conjugated anti-CD8a antibody and stained with ice. Cell division was measured using FACS LSRFortessa (BD Biosciences) and analyzed using Flowjo software. All experiments were performed independently three times.

IL-2 ELISA assay

The supernatant of co-cultured cells for T cell proliferation was taken and the expression level of IL-2 was measured by ELISA according to the manufacturer's method (BioLegend, San Diego, CA, USA).

Enzyme-Linked Immuno  Spot ( ELISPOT ) assay

ELISPOT assays were performed using splenocytes from mice immunized with wild type and recombinant strains. (3 μg / ml, clone AN-18) capture antibody (BD Biosciences) was coated on 96-well PVDF membrane ELISPOT plates and incubated overnight at 4 ° C. Capture antibody was discarded and 200 μl of RPMI 1640 medium containing 10% FBS was added to each well and blocked at 37 ° C for 3 hours. After blocking, the spleen cells of each experimental group were added to each well at a level of 5 × 10 ⁵ cells. Each experimental group was treated with 5 μg / ml of p24 antigen or treated with no stimulation (no stimulation). Phorbol 12-myristate 13-acetate (PMA) (5 μg / ml; Sigma-Aldrich) and ionomycin (500 ng / ml; Sigma-Aldrich) were used as positive controls. The plate is incubated at 37 ° C for one day. After washing three times with PBST and PBS, biotin-labeled mouse IFN-γ (3 μg / ml, clone XMG1.2) detection antibody (BD Biosciences) was added to each well and incubated overnight at 4 ° C. After each well was washed, horseradish peroxidase (HRP) -conjugated streptavidin was treated and developed with 3-amino-9-ethylcarbazole (AEC) substrate reagent (BD Biosciences). The number of spot forming units (SFUs) per well was calculated using an ELISPOT reader (AID ELISPOT Reader, Strassberg, Germany).

rSmeg  Cytokine analysis in mice immunized with recombinant strains

Splenocytes from mice immunized with each strain were seeded at a density of 1 × 10 ⁶ cells in a 96-well plate and stimulated with 5 μg / ml p24 antigen in each well. The cells were then cultured and cultured for 72 hours with IL-2 (BioLegend), TNF-α (eBioscience) (24 hours culture), IL-10 (R & D Systems, Minneapolis, MN, USA), and IFN- ) Were subjected to ELISA.

Serum antibody detection

To determine the antibody ratio in the serum, mice immunized with each strain were euthanized by CO ₂ and respiration and serum samples were prepared by heart puncture. P24 protein (5 μg / ml) was mixed with 0.05 M carbonate-bicarbonate buffer (pH 9.6) in a 96 well plate and coated on each well (4 ° C overnight). Each well was washed three times with PBST and PBS, blocked with 5% bovine serum albumin (BSA, in PBST) and incubated at room temperature for one hour. Serum samples were diluted in PBS at a ratio of 1:10, treated in each well, and incubated at room temperature for 2 hours. The cells were then washed three times each with PBST and PBS and treated with IgG2a and IgG1 antibody (BD Biosciences, 1: 1,000 dilution) for 1 hour at room temperature. After washing each well, HRP conjugated streptavidin was stained for 30 minutes at room temperature, developed with BD OptEIA substrate (BD Biosciences), and terminated with 1 NH ₂ SO 4. The optical density (OD) was measured at 450 nm with a spectrophotometer.

Cytotoxic  T lymphocyte ( CTL ) analysis

Plasmid (pcDNA3.3-p24 or Ag85B) containing p24 or Ag85B-ESAT-6 was transferred to P815 cells (H-2 ^d ) into lipofectamine 2000 (Invitrogen, Carlsbad, USA). The nucleotide sequence of each expression vector p24 (amplified from pNL4-3 deltaE-EGFP-vector) or a composite of Ag85B-ESAT6 (amplified from genomic DNA of M. tuberculosis ATCC 27294) pcDNA ^TM And cloned into a 3.3-TOPO® TA cloning vector (Invitrogen). Transfected cells were used as target cells (5 x 10 ⁴ cells) for CTL assay. Untransfected cells were used as negative controls. To identify antigen expression (p24 and Ag85B-ESAT6) in transfected cells, western blot is performed on the lysates of each cell. To confirm the expression of Ag85B-ESAT6, rabbit anti-Mycobacterium tuberculosis Ag85B antibody (Abcam, 1: 1,000 dilution) and HRP-conjugated goat anti-rabbit secondary antibody (Abcam, 1: 2,000 dilution) were used. Β-actin (Santa Cruz Biotechnology, Texas, USA; 1: 1,000 dilution) antibody was used as an internal control to confirm the same amount of protein in each lane.

Spleen cells (5 x 10 ⁶ cells / well) of each mouse were treated with p24 and Ag85B antigen (5 μg / ml) for 6 days and used as effector cells. To further confirm the HIV-1 gag specific CTL response, MHC class I-restricted p24 peptide, A9I (AMQMLKETI) (10 μg / ml; Peptron, Daejeon, South Korea) and IL-2 (30 U / ml; Spleen cells treated with PeproTech, Rocky Hill, USA) were also used as effector cells. In this case, P815 cells and A9I peptide (10 μg / ml) were incubated for 2 hours and used as target cells.

To assess cytotoxicity, a lactate dehydrogenase (LDH) assay (CytoTox 96 Non-Radioactive Cytotoxicity Assay; Promega, Madison, USA) was performed. Effector cells and target cells were co-cultured for 6 hours at different effector / target (E / T) ratios of 10: 1, 20: 1 and 50: 1, Were measured with a spectrophotometer. The ratio of antigen-specific cytotoxicity was calculated by the formula: [(Experimental Effector spontaneous Target spontaneous) / (Target maximum target spontaneous)] x 100 (%).

Growth curve measurement

The recombinant strains (rSmeg-pMV306-p24, rSmeg-pAL-p24, rSmeg-pMyong2-p24) prepared above were supplemented with 7H9 liquid medium (supplemented with 0.5% glycerol, 0.05% Tween-80, 10% ADC and 100 μg / ml kanamycin), and the experiment was conducted to measure the growth rate by adjusting the optical density at 600 nm to 0.2. In the case of wild type M. smegmatis , it was cultured in 7H9 liquid medium without kanamycin. After that, the culture was taken at each time zone and the absorbance at OD ₆₀₀ was measured to prepare a growth graph.

Experiment of cell line infection

Bone marrow-derived dendritic cells (BMDCs) isolated from mouse macrophage cell line J774A.1 and mouse were cultured in Dulbecco's modified Eagle's medium (DMEM, supplemented with 10% FBS, 2 mM glutamine, penicillin, streptomycin, essential amino cells were cultured in Iscove's modified Eagle medium (IMDM supplemented with 10% FBS, 1.5 ng / ml GM-CSF, 1.5 ng / ml IL-4, penicillin, streptomycin, gentamicin, L-glutamine and β-mercaptoethanol) . On the day before the strain infection, each cell was cultured in a 24-well plate at 5 × 10 ⁵ cells / well. Each recombinant strain (rSmeg-pMV306-p24, rSmeg-pAL-p24, rSmeg-pMyong2-p24) and wild type M. smegmatis strains were prepared at 10 MOI and infected with the cells. Two hours after the infection, the cells were washed three times with PBS to remove the non-extracellular matrix, and then cultured for 24 hours in a fresh medium. After 24 hours, the cell culture medium was removed, and the cells were washed three times with PBS. Then, the cells were detached with 0.05% Triton X-100 and lysed. The homogenized cell lysate was diluted to the appropriate dilution and counted colony forming colonies (CFUs) grown in 7H10 solid medium (containing 10% OADC and 100 μg / ml kanamycin).

Example  1. According to the present invention rSmeg - pMyong2 Bacteria and infected mouse macrophages by the -p24 strain HIV in the cell line -1 p24 Gag expression increased

To determine the optimal recombinant strains for the production of the HIV-1 p24 Gag vaccine and to analyze its usefulness, three types of p24 expression vectors, rSmeg-pMyong2-p24, rSmeg-pAL-p24 and rSmeg-pMV306- p24. &lt; / RTI &gt; The vector used here is shown in Fig. When the growth rates of the three strains were compared in the 7H9 medium (including 100 μg / ml of kanamycin) for 5 days, the strain rSmeg-pMyong2-p24 according to the present invention was delayed in growth from the initial stage of growth, Respectively. However, after 48 hours, all three strains showed almost the same growth rate.

Expression levels of p24 in three kinds of rSmeg strains were analyzed by ELISA and Western blotting. The results are shown in Figures 2a and 2b. P24 was expressed in all rSmeg strains, but the expression level of rSmeg-pMyong2-p24 strain was about 5 to 10 times higher than that of the other two strains.

To analyze the stability of p24 expression, p24 expression levels were then cultured in various passages of rSmeg-pMyong2-p24 strain on kanamycin-containing or 7H10 agar plates. In the medium containing kanamycin, the rSmegpMyong2-p24 strain also expressed a stable p24 antigen even after the 12th passage. However, in the case of growing on kanamycin-free medium, the stability of expression of p24 sharply decreased after 6 passages. This is because the high copy number pMyong2-TOPO vector that can induce the overexpression of exogenous p24 is disappeared.

We also analyzed ELISA in infected J774A.1 cells and mouse BMDC to determine whether the difference in the expression level of p24 in the three rSmeg strains can be replicated in infected macrophages and infected BMDCs. The result is shown in Figure 2c. Similar trends were observed in infected J774A.1 cells and mouse BMDC as those observed in lysed bacteria, but the difference was more pronounced. Taken together, these results show that rSmeg-pMyong2-p24 according to the present invention increases p24 production in bacteria as well as infected macrophages, as compared to rSmeg-pAL-p24 and rSmeg-pMV306-p24. These results suggest that rSmeg-pAL-p24 according to the present invention is more likely to induce an enhanced p24 antigen-specific immune response compared to the other two strains by presenting more p24 antigen to the antigen presenting cells upon inoculation to an animal or a human And can be effectively used as a vaccine.

Example  2. rSmeg - pMyong2 infected with the -p24 strain BMDC  HIV-1 on p24 Gag due to Induce enhanced T cell proliferation from immunized mouse T cells

To test whether increased p24 protein production by rSmeg-pMyong2-p24 enhanced T cell proliferative capacity, we improved the T cell proliferative capacity and we performed an infected T cell proliferation assay. T cell proliferation of infected BMDCs with three different strains of Smeg-pMyong2-p24, rSmeg-pAL-p24 and rSmeg-pMV306-p24 and CFSE dye dilution in mixed lymphocyte reaction (MLR) analysis as wild- Respectively. A schematic diagram for T cell proliferation assay is shown in Figure 3A. All BMDCs infected with four Smeg strains (three rSmeg and wild type) induced significantly higher levels of CD4 T cell proliferation compared to BMDCs not infected with M. smegmatis strain. Of particular note, BMDC infected with rSmeg-pMyong2-p24 was significantly higher in CD4 and CD8 T cell proliferation compared to the other two rSmeg and wild-type strains. The rSmeg strains rSmeg-pAL-p24 and rSmeg-pMV306-p24 were not different. In CD4 T cell proliferation, BMDCs infected with rSmeg-pAL-p24 or rSmeg-pMV306-p24 showed similar wild-type proliferation levels (Fig. 3b and c). The results of the comparison of the amount of IL-2 from the stimulated CD4 and CD8 T cells were also similar to those of the T cell proliferation assay. As a result, BMDCs infected with rSmeg-pMyong2-p24 were found to induce significantly higher levels of IL-2 secretion from both CD4 and CD8 T cells than BMDC and wild type infected with the other two rSmeg strains 3d). Taken together, these results indicate that rSmeg-pMyong2-p24 according to the present invention can enhance T cell proliferation by enhancing antigen presentation to antigen-presenting cells such as DC, thereby enhancing the efficacy of the vaccine.

Example  3. rSmeg - pMyong2 Avoidance of -p24 strain Avoid immunization  Improved HIV-1 p24 Gag specificity in mouse spleen produced by immunization IFN -γ Spot  Forming cell ( SFC ) Judo.

Three types of rSmeg strains, rSmeg-pMyong2-p24, rSmeg-pAL-p24, rSmeg-pMV306-p24 (~10 ⁶ CFU), and rSmeg-pMyong2-p24 were tested to test whether rSmeg- Splenocytes were isolated from the spleen of subcutaneously immunized BALB / c mice in each of the wild-type strains, and HIV-1 p24 Gag-specific T cell responses were assayed using IFN-y ELISPOT assay (Fig. 4A). Splenocytes from mice immunized with two types of rSmeg strains, rSmeg-pMyong2-p24 and rSmeg-pAL-p24 showed significantly higher SFU than the control strain rSmeg-pMV306-p24. Notably, spleen cells of mice immunized with rSmeg-pMyong2-p24 (146.33 ± 66.91 SFU / 5 × 10 ⁵ pancreatic cells) were rSmeg-pAL-p24 (63.63 ± 15.72 SFU / 5 × 10 ⁵ spleen cells) and rSmeg- showed significantly higher SFU compared to splenocytes immunized with pMV306-p24 (63.63 ± 15.72 SFU / 5 × 10 ⁵ spleen cells) (FIG. 4b). Interestingly, significant differences in SFC levels were observed between two rSmeg strains, rSmeg-pAL-p24 and rSmeg-pMV306-p24, which showed little difference in T cell proliferation or p24 expression. These results indicate that an enhanced effect in rSmeg-pMyong2-p24 vaccinated animals can induce T cell function.

Example  4. rSmeg - pMyong2 by -p24 strain Th1  Related to the immune response Saito  Generate cine.

2, IFN-?, TNF-? and IL-10 (Fig. 4A) were stimulated in vitro with purified p24 protein Cytokines were measured in cell culture supernatants. In the case of rSmeg-pMyong2-p24 immunized splenocytes, the concentration of all Th1-immunoreactive cytokines was significantly higher compared to the control and other strains as follows: IL-2, rSmeg-pMV306-p24 versus rSmeg- pAL-p24 vs. rSmeg-pMyong2-p24: 1.37 + 0.37 vs. 2.27 ± 0.54 vs. 5.95 + 0.66 pg / ml; IFN-γ: 11.78 ± 2.16 vs. 22.67 ± 5.44 vs. 42.95 ± 3.21 pg / ml and TNF-α: 16.00 ± 2.26 vs.21.08 ± 3.77 vs. 28.92 + - 3.41 pg / ml. The rSmeg-pMyong2-p24 strain also showed increased levels of IL-10 release but was similar in all strains used: rSmeg-pMV306-p24 versus rSmeg-pAL-p24 versus rSmeg-pMyong2-p24: 43.88 ± 3.87 Versus 52.12 +/- 3.85 vs. 55.72 +/- 3.12 pg / ml) (Figure 4c).

Example  5. rSmeg - pMyong2 HIV-1 p24 Gag specific in mice immunized with the -p24 strain Th1  Inducing bias body fluid response

In immunized rats. In order to test whether rSmeg-pMyong2-p24 induces a Th1-biased humoral response, the concentrations of HIV-1 p24 Gag specific IgG2a and IgG1, known as markers for Th1 and Th2 responses, were analyzed, respectively. Sera of wild-type strains were assayed as negative control BALB / c mice immunized subcutaneously with different strains of three kinds of rSmeg-pMyong2-p24, rSmeg-pAL-p24 and rSmeg-pMV306- As a result, rSmeg-pMyong2-p24 and rSmeg-pAL-p24 all showed significantly higher levels of IgG2a isotype than the wild-type, except for rSmeg-pMV306-p24. Regarding the IgG1 isotype, rSmeg-pMyong2-p24 induced lower levels of IgG1 than rSmeg-pAL-p24; However, this was not statistically significant (P = 0.146). High IgG2a / IgG1 ratios indicate a higher Thl-Bias body fluid immune response and IgG2a / IgG1 ratios are higher than other types of Smeg (wild type = 1.05; rSmeg-pAL-p24 = 1.03; rSmeg-pMV306-p24 = 0.97) (Fig. 4d), which was higher than that immunized with rSmeg-pMyong2-p24 strain (1.21) (Fig. 4d) compared to that immunized with rSmeg-pMyong2- Specific &lt; / RTI &gt; Th1-biased body fluids response and, as a result, can be effectively used as a vaccine.

Example  6. rSmeg - pMyong2 -p24 Strain-immunized  Improved HIV-1 p24 Gag-specific cytotoxic T lymphocyte response in mice

In order to test whether the mice immunized with rSmeg-pMyong2-p24 induce an improved HIV-1 p24 Gag-specific cytotoxic T lymphocyte (CTL) response, four different types of Smeg strains, 3 rSmeg: rSmeg-pMyong2-p24 , rSmeg-pAL-p24 and rSmeg-pMV306-p24 and splenocytes immunized with the wild-type strain were assayed using the LDH cytotoxicity assay. The immunized procedure is described in FIG. 4A. P815 cells (H-2 ^d ) transfected with a plasmid carrying the p24 or Ag85B-ESAT-6 fusion gene (pcDNA3.3-p24 or pcDNA3.3-Ag85B-ESAT-6) were used as target cells and the effector (E) / Target cells (T) are 10: 1, 20: 1 and 50: 1, respectively. Expression of each transfected P815 cells was confirmed by Western blot analysis. As shown in Figure 5, at an E: T ratio of 50: 1, the CTL of mice immunized with rSmeg-pMyong2-p24 showed significantly higher levels of HIV-1 p24 Gag-specific target cell fusion (Fig. 5B). These results suggest that rSmeg-pMyong2-p24 is the most abundant of p24 expressing antigen-presenting cells (APCs) among the four types of Smeg strains. However, there was no significant difference in Ag85B-specific CTL death among the 4 strains (Fig. 5A). The reason is that almost the same level of Ag85B can be presented to APC regardless of vector type or Smeg strain. In fact, M. Ag85B orthologue (fbpB) (GenBank Accession No. NC_008596) with amino acid sequence homology exceeding 70% with tuberculosis Ag85B was also found in M. smegmatis strain 700084 / mc ² 155 (NC_008596, NC_008596) genome. Thus, a similar level of Ag85B-specific CTL response found between mice vaccinated with the Smeg strain (one wild type and three recombinant strains) is believed to be due to the presence of the M. smegmatis Ag85B orthologue, which can be expressed in similar amounts between strains . However, since the results of FIG. 5A were shown to induce a cytotoxic T lymphocyte response to Ag85B, a tuberculous antigen, this indicates that the strain according to the present invention can act as a tuberculosis vaccine. In addition, p24 peptide A9I was also used for cytotoxicity analysis to identify and compare p24-specific CTL responses by rSmeg strains. This peptide is known as the major histocompatibility complex (MHC) class I restricted p24 epitope of BALB / c mice (H-2 ^d ). The results showed that strains rSmeg-pMyong2-p24 were able to induce stronger CTL responses (more than 50% of cytotoxicity) compared to P815 target cells pulsed with A9I peptide compared to other rSmeg strains. These results indicate that the rSmeg-pMyong2-p24 strain according to the present invention can induce an improved HIV-1 p24 Gag-specific CTL response in immunized mice and thus can be used as an effective vaccine.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

Here we present three different vector systems (two episomal vectors under the mycobacterial hsp65 promoter, with 2-6 replications per cell, in order to search for an appropriate Mycobacterium vector system that promotes the vaccine efficacy of rSmeg, The pAL5000 derived vector and the replication number were 37 times more than the pAL5000 vector. The pMyong2 derived vector and one integrating vector pMV306) were compared. The results showed that the best expression was achieved in rSmeg-pMyong2-p24 with the pMyong2 vector (Fig. 2a and b). In addition, rSmeg-pMyong2-p24 stably expressed the p24 antigen even after passage 12. In addition, a more significant difference in p24 expression was seen in infected phagocytes (Fig. 2c), indicating a bias associated with enhanced p24-specific T cell proliferation of BMDC (Fig. 3b and c), T cell effector function (Fig. 4b and c) , Especially CTL (Figure 5), and rSmeg-pMyong2-p24 (Figure 4d).

Comparison of the growth rate of 3 rSmeg strains in 7H9 medium showed that the rSmeg-pMyong2-p24 strains had a growth delay over the interval of 0 to 48 hours compared to other rSmeg strains. This difference may be due to the high copy number of the vector according to the invention acting on the pressure. This finding suggests that rSmeg-pMyong2-p24 may be more attenuated in macrophage or in vivo mouse infections than other rSmeg strains. In fact, we found that rSmeg-pMyong2-p24 formed 2-3 times less colonies (CFU) than rSmeg-pAL-p24 or rSmeg-pMV306-p24 after infection with macrophages. This finding suggests that attenuated Smeg maintains a stronger immune response by presenting more antigen to phagocytes than wild type . Junqueira-Kipnis, AP et al. Prime-Boost with Mycobacterium smegmatis Recombinant Vaccine Improves Protection in Mice Infected with Mycobacterium tuberculosis . PLoS One 8, doi: 10.1371 / journal.pone.0078639 (2013)), these results show the additional advantage that the rSmeg-pMyong2-p24 strain according to the present invention can more effectively function as a vaccine.

In summary, here we demonstrate that rSmeg-pMyong2-p24 according to the present invention induces higher levels of HIV-1 p24 Gag protein expression as compared to other rSmeg strains that use the pAL5000 or pMV306 induction system and deliver more p24 antigen to phagocytes Proved to be possible. Furthermore, it has been shown that these strains can enhance T cell proliferative capacity of infected BMDC and induce enhanced T cell effector function and Th1-biased humoral immune response in inoculated mice. These results indicate that rSmeg-pMyong2-p24 according to the present invention can be a candidate vaccine effective for co-infection of HIV-1 or HIV-1 with tuberculosis.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

<110> Seoul National R & DB Foundation <120> Recombinant Mycobacterium smegmatis with vector expressing Human          Immunodeficiency Virus Type I p24 and vaccine comprising the same <130> DP201712003P <150> KR 10-2017-0001715 <151> 2017-01-05 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 7445 <212> DNA <213> Artificial Sequence <220> <223> pMyong2-p24 <220> <221> CDS &Lt; 222 > (3161) .. (3856) <223> HIV type 1, p24 <220> <221> misc_feature &Lt; 222 > (2780). (2785) <223> EcoRV restriction site for cloning <220> <221> misc_feature &Lt; 222 > (3860) .. (3865) <223> XbaI restriction site for clonning <400> 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg 240 gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgc ccttaggcgg 300 gcaacacgac atctcaatag ttggcacgga gctcctaaac gacgacagcc ccgctaatgc 360 ggggccgcaa cgtcgatttg gctctacttc gtctctggag tttcgtctct ggagtttcag 420 gtccgagcgg ctaactcggt cctaggcgtc tcccgtgccg cgcccccgaa gggacagtct 480 tgctggactg gtctcacggt agcgcatacc cgaatcagat tcgtggtgac gcgcaaaacc 540 gtgatctcac atttacagat tcccgtgcat ccgtgtgcac ggattcacgc ccatctgggc 600 attcacgtgg ccgttttggt gcctccgtct gggctctcgt cgctcccggc ggcgcgtcgt 660 ggtgacaacc gcgttcgacg cggcagagat tgccgccgcg gtcccggaat ctggctcacc 720 gtgcagcggt acagcgtcag ctcgactgcg cagaatccgc gccaaggaag cccgcagggc 780 gtcgatcgaa cgctgtgggg cggtgccggc cgcggtgccg atgtggtcgt cgcgggagtt 840 gtggactgcc gatctgcggg tgcttttgtc gggtccggag ttcagcacgc gccgggtgat 900 ttcggcggcg acggtgctcg ctgtcgcggt ggcgatggcc gagttcgccg accatgccac 960 gggccgcaat gtggcggtga caaacgaggt tctggccgag cgcgcgaggt gttctaagcg 1020 ttcggtgacc gcggcgcgcg gggtgttgaa agcgttgggt gtcgcggtgg aagcggtacg 1080 tgggcatggc tctgcgacca cacacacggt cggtaatcga ccgagcattt ggcacctggt 1140 aagccgacgc cagcccacca tcgacaaccc gcccacggcc ccgcagaacg gccgcggcga 1200 gcctgccgat acggtgcccg atcgcggcca gagcgcgccc gtggctgtgg agacttgcga 1260 cctaccacca tcccgtaggg ataggtgggt aactcctgtt gagaattact caccaagcac 1320 gcgcgagcgc gcgagcgcgg aaaattcttc cccaaaacaa acacaaccgg cgcggtcgcg 1380 gcggcgctac cgcgccacgc cgcgccccct ggacgttcag cgactcgccg ccggcctggt 1440 cacgccggca gtcggccacg gcccagacaa cgacgggcgg cgaaccgcgc tgatcgccgg 1500 cctggagcag ggccatatcg gggccatctg cgacgcgatc acgacggccg gcatcgacgc 1560 gccgcctgg actccgaaga cgctcacggc ggcactgaac gccgacgcgc gggtgaccgg 1620 ctggtcgtgg ccggatcgca tcgaacgtcc tggcgcgttc ctggcgtcgc ggctgcgccg 1680 cctgcccgca cggcccgaca ccagtggccc ggttgacaac ggcctggatc aggcccgtag 1740 gacacccgtt gagccgtcag cggcccgtgt agcgccggta cagacggccg ctggccgcgc 1800 gtacgcccgt gcgttgttcg ccgagcagcg acggcaccgg gtgaccgccg ccaatgccca 1860 gtcagccgcg gtgccggtgc gccaaagtgc gccagaaacc gcggtgtgcg caacgtgcgg 1920 atgctcggac gcaccacggc ggcggttcct gccaacgcgg cgggctcaca tttgcgatgc 1980 ctgtttccaa ggatgtggtg gtgggcaggc gcgtactggt cgcgtcggaa cggtcggcag 2040 cagttccgcg gtgccacagt gccagtagtc gggcagggct tgcacgggga tgcggaccca 2100 tccgccggcc gcggccggcg atgggtccag tgtcgcgtta gggccgtcgt ccagccgcgc 2160 cagctcgcgg tcggatgcga tcagcttggc accgtagatg tcgtcgtggc ggcggtccca 2220 cgtgttggtg atgcggtcgc ggtaccagcc tgtccagtct tcgccggtgc cgccggcggt 2280 gagcccggcc cggtaccacg cttcgaggcg atcacggccc cgtgcgtggt tgatggtctg 2340 ggccgcagcc cagtccgcgt cacggagggc tctgtcggct tcgtccatga acccgcgccg 2400 ggtcatgtcg atgatggctg gcgacggggc gtcgtagggg cctggcatgg gcggaggcag 2460 tcggaactgt tgcggtttgc ttgcgttcag ttcctcgtcc tgctggtggg tgcggtcgtg 2520 cagggcaagg ctgtcgcggt accaggcgtc gagcgcggtg cgccagtccc cgccagtggc 2580 gtagggctcc cacggttgcg gtggcagggt ggtgaactcg tgcaggacca ggccgtcgat 2640 gtcgtcgcgc agttcttcac cggcgatggc ggcgatgcgg gcgcagtagc cgggcagatg 2700 gctacgccag tcggcgggca gttcgccgct gcgcgcccac cgcagcacat cggcccacaa 2760 cccaagggcg aattctgcag atatcggtga ccacaacgac gcgcccgctt tgatcgggga 2820 cgtctgcggc cgaccattta cgggtcttgt tgtcgttggc ggtcatgggc cgaacatact 2880 cacccggatc ggagggccga ggacaaggtc gaacgagggg catgacccgg tgcggggctt 2940 cttgcactcg gcataggcga gtgctaagaa taacgttggc actcgcgacc ggtgagtcgt 3000 aggtcgggac ggtgaggcca ggcccgtcgt cgcagcgagt ggcagcgagg acaacttgag 3060 ccgtccgtcg cgggcactgc gcccggccag cgtaagtagc ggggttgccg tcacccggtg 3120 acccccggtt tcatccccga tccggaggaa tcacttcgca atg cct ata gtg cag 3175                                             Met Pro Ile Val Gln                                               1 5 aac ctc cag ggg caa atg gta cat cag gcc ata tca cct aga act tta 3223 Asn Leu Gln Gly Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu                  10 15 20 aat gca tgg gta aaa gta gta gaa gag aag gct ttc agc cca gaa gta 3271 Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val              25 30 35 ata ccc atg ttt tca gca tta tca gaa gga gcc acc cca caa gat tta 3319 Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu          40 45 50 aat acc atg cta aac aca gtg ggg gga cat caa gca gcc atg caa atg 3367 Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met      55 60 65 tta aaa gag acc atc aat gag gaa gct gca gaa tgg gat aga ttg cat 3415 Leu Lys Glu Thr Ile Asn Glu Glu Ala Glu Trp Asp Arg Leu His  70 75 80 85 cca gtg cat gca ggg cct att gca cca ggc cag atg aga gaa cca agg 3463 Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg                  90 95 100 gga agt gac ata gca gga act act agt acc ctt cag gaa caa ata gga 3511 Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly             105 110 115 tgg atg aca cat aat cca cct atc cca gta gga gaa atc tat aaa aga 3559 Trp Met Thr His Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg         120 125 130 tgg ata ctg gga tta aat aaa ata gta aga atg tat agc cct acc 3607 Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr     135 140 145 agc att ctg gac ata aga caa gga cca aag gaa ccc ttt aga gac tat 3655 Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr 150 155 160 165 gta gac cga ttc tat aaa act cta aga gcc gag caa gct tca caa gag 3703 Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Ser Gln Glu                 170 175 180 gta aaa aat tgg atg aca gaa acc ttg ttg gtc caa aat gcg aac cca 3751 Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro             185 190 195 gat tgt aag act att tta aaa gca ttg gga cca gga gcg aca cta gaa 3799 Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Gly Ala Thr Leu Glu         200 205 210 gaa atg atg aca gca tgt cag gga gtg ggg gga cct ggc cat aag gca 3847 Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala     215 220 225 aga gtt ttg tagt ctagagggcc caattcgccc tatagtgagt cgtattacaa 3900 Arg Val Leu 230 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 3960 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 4020 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca 4080 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 4140 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 4200 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 4260 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 4320 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 4380 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 4440 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaatt 4500 cagggcgcaa gggctgctaa aggaagcgga acacgtagaa agccagtccg cagaaacggt 4560 gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa 4620 agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat 4680 ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct 4740 gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat 4800 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag 4860 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg 4920 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca 4980 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc 5040 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg 5100 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatcccac cttgctcctg 5160 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta 5220 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag 5280 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac 5340 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg 5400 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg 5460 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg 5520 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg 5580 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaatt gaaaaaggaa 5640 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 5700 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 5760 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 5820 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 5880 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 5940 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 6000 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 6060 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 6120 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 6180 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 6240 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 6300 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 6360 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 6420 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 6480 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 6540 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 6600 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 6660 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 6720 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 6780 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 6840 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 6900 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 6960 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 7020 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 7080 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 7140 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 7200 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 7260 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 7320 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 7380 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 7440 ggaag 7445

Claims (9)

The recombinant Mycobacterium smegmatis expressing the p24 protein derived from human immunodeficiency virus type 1, wherein the p24 protein is expressed by the pMyong2-p24 plasmid shown in Fig. 1, Mycobacterium smegmatis strain.
The method according to claim 1,
Wherein the Mycobacterium smegmatis strain is deposited with KCTC 13403BP.
A vaccine composition for HIV infection or HIV and Mycobacterium infections co-infected with a strain according to claims 1 or 2 and a pharmaceutically acceptable excipient, wherein said strain is live, HIV infection or simultaneous infection with HIV and Mycobacterium tuberculosis &Lt; / RTI &gt;
4. The vaccine composition according to claim 3, wherein the vaccine is not further attenuated, for HIV infection or simultaneous infection of HIV and Mycobacterium tuberculosis.
4. The vaccine composition of claim 3, wherein the vaccine is used as a priming vaccine in a prime-boost vaccination.
4. The vaccine composition of claim 3, wherein the infectious disease is AIDS or tuberculosis, HIV infection or simultaneous infection of HIV and Mycobacterium tuberculosis.
1. A pMyong2-p24 plasmid as shown in Fig. 1 contained in the strain of claim 1 or 2.
8. The pMyong2-p24 plasmid according to claim 7, wherein said plasmid is represented by the sequence of SEQ ID NO: 1.
9. A cell comprising the plasmid according to claim 7 or 8.

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