Vaccine taking bacillus calmette guerin vaccine infected macrophage debris as antigen as well as construction method and application of vaccine

Abstract

The invention relates to the field of vaccines, in particular to a vaccine with BCG (bacillus calmette guerin) infected macrophage debris as an antigen and a construction method and application of the vaccine, the BCG infected macrophage debris is used as the vaccine antigen, and the method comprises the following steps: S1, BCG is used for infecting macrophages; s2, cracking the infected cells by means of repeated freeze thawing and ultraviolet inactivation to obtain an antigen composition containing cell debris; and S3, co-incubating the dendritic cells differentiated in vitro and the antigen composition to form the cell vaccine. The method disclosed by the invention has the beneficial technical effects that the safety of immunization of the bacillus calmette-guerin vaccine related vaccine through a vein way is enhanced, and compared with a viable bacteria immunization way, the method disclosed by the invention can improve the safety; and the venous immunity can stimulate antigen-specific immune response. The novel cell vaccine not only can maintain the immunocompetence of the bacillus calmette guerin vaccine, but also can stimulate the anti-tuberculosis infection immune protection effect.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccines, and in particular to a vaccine using BCG-infected macrophage fragments as antigens, and a construction method and use thereof. Background Art

[0002] Tuberculosis is one of the main killers affecting human health. According to the latest statistical report of the World Health Organization, there will be approximately 10.6 million new cases of tuberculosis and 1.3 million deaths worldwide in 2022. Vaccines are one of the most effective ways to fight infectious diseases in human history. At present, the only available tuberculosis vaccine is Bacille Calmette-Guérin (BCG). Most countries, especially those with a high incidence of tuberculosis, implement BCG immunization; in countries with a low incidence of tuberculosis, such as the United States, the Netherlands and Italy, only high-risk groups are vaccinated. In children and adolescents, BCG can provide a high level of protective efficacy; however, as the vaccine recipients age, the immune memory induced by BCG gradually decreases, resulting in poor prevention of adult tuberculosis. Therefore, in order to better control the tuberculosis epidemic, it is necessary to develop more effective new tuberculosis vaccines for booster immunization, or to further improve the existing BCG.

[0003] One of the difficulties in the development of tuberculosis vaccines is that tuberculosis bacteria have up to 4,000 proteins. During early infection, intracellular proliferation, immune escape, latent persistence, and recurrent infection, the expression profiles of various antigens continue to change. Therefore, although subunit vaccines are safer, the number of tuberculosis antigens they contain is limited, and they cannot cover antigens that are highly expressed in different infection phases. The protective efficacy of the specific immune response they induce is often limited. BCG has most of the antigens of Mycobacterium tuberculosis, and the induced immune response is more comprehensive. In addition, as the vaccine with the largest number of vaccinations to date, the safety of BCG has also been repeatedly verified. Therefore, modifying BCG is a feasible strategy for tuberculosis vaccine design.

[0004] In the initial development stage, BCG was administered orally. In 1928, it was discovered that the subcutaneous route of BCG administration was safer than the oral route, and it has been used ever since. However, although subcutaneous administration is safer, this route cannot produce sufficiently lasting immune memory in the lungs, which is considered to be one of the possible reasons for the poor immune protection of the vaccine. This is because the subcutaneous inoculation site is far away from the lungs, and its antigens have very little contact with the antigen-presenting cells in the lungs, and cannot induce sufficiently effective immune memory in the lungs, the main site for tuberculosis to establish infection. As early as the 1970s, researchers proposed that the immune protection of BCG could be enhanced by changing the immune route. However, due to safety concerns, the route of BCG administration is still subcutaneous administration. In recent years, researchers have confirmed the excellent protective effect of intravenous BCG in non-human primates: 6 of the 10 macaques who received intravenous BCG were fully able to resist the infection attack of tuberculosis, and no lesions were detected in the lungs. Studies have confirmed that the enhancement of respiratory innate immune responses and adaptive CD4 T cell immunity is associated with the ability of intravenous BCG to induce protection. However, for safety reasons, it is unrealistic to directly transfuse live BCG bacteria into human blood, which limits the practical application value of intravenous immunization. Although the injection of killed bacteria will significantly improve safety, there are reports that inactivated BCG does not provide sufficient protection.

[0005] Therefore, it is necessary to modify the vaccine on this basis, without using live BCG bacteria for intravenous administration to ensure safety, and through vaccine preparation strategies, without significantly reducing the immunogenicity of BCG and the immune protection efficacy against tuberculosis infection. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a vaccine using BCG-infected macrophage fragments as antigens and a construction method and use thereof, so as to solve the problem that live BCG bacteria are low in safety when directly injected intravenously into human blood, while injection of dead bacteria leads to insufficient immune protection, so as to achieve the use of non-infectious BCG components to induce sufficiently effective immune protection against tuberculosis infection.

[0007] In order to achieve the above object, a vaccine construction method is provided, wherein BCG-infected macrophage fragments are used as vaccine antigens, and the method comprises the following steps:

[0008] S1. Infect macrophages with BCG;

[0009] S2. Lysing infected cells by repeated freezing and thawing and UV inactivation to obtain an antigen composition containing cell fragments;

[0010] S3. Co-culture the in vitro differentiated dendritic cells with the antigen composition to form a cell vaccine.

[0011] Preferably, the BCG vaccine is Mycobacterium bovis.

[0012] Preferably, the antigen composition comprises Mycobacterium tuberculosis antigen fragments cleaved by proteases and captured by cells.

[0013] Preferably, the dendritic cells are professional antigen presenting cells.

[0014] Preferably, the vaccine is in the form of dendritic cells loaded with the cell fragments.

[0015] Preferably, the vaccine is for intravenous administration, intramuscular injection, subcutaneous injection or inhalation administration.

[0016] The present invention also provides a vaccine, which also includes other technical features. The vaccine is used for intravenous inoculation, intramuscular injection, subcutaneous injection or inhalation inoculation.

[0017] The present invention also provides a use of a vaccine. The vaccine cell fragments and the dendritic cell vaccine loaded with the fragments can be used alone or in combination with other tuberculosis vaccines.

[0018] Compared with the prior art, the present invention has the following beneficial technical effects:

[0019] The present invention significantly enhances the safety of BCG-related vaccines for immunization via intravenous route, while maintaining the BCG immune activity without a significant decrease. First, compared with live bacteria immunization, this treatment method can improve safety; second, through the dendritic cell vaccine that recognizes cell fragments, intravenous immunization can stimulate a more comprehensive antigen-specific immune response. The experimental results show that this new cell vaccine can not only maintain the immune activity of BCG, but also stimulate effective immune protection against tuberculosis infection. Therefore, the present invention provides a new cell vaccine with potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Flow chart of the preparation of dendritic cell vaccine loaded with BCG-infected cell fragments in Example 1 of the present invention.

[0021] Figure 2The following is a schematic diagram of the experimental process and results of evaluating the ability of macrophage fragments infected with BCG to stimulate macrophage maturation by BIMCD in Example 2 of the present invention. A is a schematic diagram of the experimental process for evaluating the ability of macrophage fragments infected with BCG to stimulate macrophage maturation by BIMCD; B is a comparison of the secretion of interleukin-1b, interleukin-6, interleukin-10, interleukin-12, and tumor necrosis factor-a by dendritic cells stimulated by each group; C is a comparison of the percentage of positive cells expressed on the surface of dendritic cells stimulated by each group; D is a comparison of the expression of leukocyte differentiation antigen 80, leukocyte differentiation antigen 83, and leukocyte differentiation antigen 86 on the surface of dendritic cells stimulated by each group, And the comparison chart of the average fluorescence intensity of major histocompatibility complex I and major histocompatibility complex II; E is the flow cytometry schematic diagram of each group of stimulated dendritic cells expressing leukocyte differentiation antigen 80, leukocyte differentiation antigen 83 and leukocyte differentiation antigen 86, as well as major histocompatibility complex I and major histocompatibility complex II; F is the fluorescence intensity scatter plot of each group of fluorescent small molecule compounds; G is the peak graph of the fluorescence intensity of DC positive cell markers in each group; H is the data summary analysis chart of each group of fluorescent small molecule compounds.

[0022] Figure 3 The following is a schematic diagram of the experimental process and results of the specific immune response induced in the spleen of mice by the dendritic cell vaccine BIMHDC loaded with macrophage fragments infected with BCG in this Example 3. Among them, A is a schematic diagram of the experimental process of the specific immune response induced in the spleen of mice by the dendritic cell vaccine BIMHDC loaded with macrophage fragments infected with BCG; B is a dot diagram showing the results of the specific immune response induced in the spleen of mice by the dendritic cell vaccine BIMHDC loaded with macrophage fragments infected with BCG; C is a positive dot diagram of the secretion of interferon-γ per million cells by spleen cells under the stimulation of polypeptide after specific stimulation by mouse ELISpot assay; D is a positive dot diagram of the secretion of interferon-γ per million cells by spleen cells under the stimulation of tuberculosis after specific stimulation by mouse ELISpot assay. Figure 3 is a graph showing the positive spots of interferon-γ secreted by spleen cells per million cells; E is a schematic diagram showing the results of flow cytometry detection of interferon-γ, interleukin-2 and tumor necrosis factor-α secreted by spleen cells after specific stimulation; F is a graph showing the percentage of CD4 cells specifically secreted by spleen cells in each group after specific stimulation by flow cytometry; G is a graph showing the percentage of CD8 cells specifically secreted by spleen cells in each group after specific stimulation by flow cytometry.

[0023] Figure 4The following are schematic diagrams of the experimental process and results of the specific immune response induced in the lungs of mice by the dendritic cell vaccine BIMHDC loaded with macrophage fragments infected with BCG in this Example 4; wherein, A is a schematic diagram of the experimental process of the specific immune response induced in the lungs of mice by the dendritic cell vaccine BIMHDC loaded with macrophage fragments infected with BCG; B is a dot diagram showing the results of the specific immune response induced in the lungs of mice by the dendritic cell vaccine BIMHDC loaded with macrophage fragments infected with BCG; C is a positive spot of the mouse ELISpot assay to evaluate the secretion of interferon-γ per million cells by lung cells under specific post-stimulation polypeptide stimulation. Figure; D is a positive spot diagram of interferon-γ secreted per million cells by lung cells stimulated by tuberculosis after specific stimulation evaluated by mouse enzyme-linked immunosorbent assay; E is a schematic diagram of the results of interferon-γ, interleukin-2 and tumor necrosis factor-α secreted by lung cells after specific stimulation detected by flow cytometry; F is a diagram showing the percentage of interferon-γ, interleukin-2 and tumor necrosis factor-α specifically secreted by lung cells in each group after specific stimulation detected by flow cytometry, accounting for the percentage of CD4 cells; G is a diagram showing the percentage of interferon-γ, interleukin-2 and tumor necrosis factor-α specifically secreted by lung cells in each group after specific stimulation detected by flow cytometry, accounting for the percentage of CD8 cells.

[0024] Figure 5 Schematic diagram of the experimental process and results for evaluating the protective efficacy of BIMHDC, a dendritic cell vaccine loaded with macrophage fragments infected with BCG, against tuberculosis infection. A is a schematic diagram of the experimental process for evaluating the protective efficacy of BIMHDC, a dendritic cell vaccine loaded with macrophage fragments infected with BCG, against tuberculosis infection; B is a comparison of the load of tuberculosis in the spleen of each group; C is a comparison of the load of tuberculosis in the lungs of each group; D is a comparison of the acid-fast staining sections of the lungs of each group DETAILED DESCRIPTION

[0025] In order to make the purpose and principle of the present invention more clear, it is further described below in conjunction with the accompanying drawings and specific embodiments.

[0026] Example 1: Preparation of dendritic cell vaccine loaded with BCG-infected macrophage fragments

[0027] See also Figure 1 This embodiment provides a method for preparing BCG-infected macrophage fragments as antigens, and the specific steps include:

[0028] S1. Use mouse bone marrow differentiated macrophages, i.e. bone marrow-derived macrophages: collect bone marrow cells from the femur and tibia of BALB / c mice, resuspend them in DMEM / F12 medium (Corning, 10-092-CVa) at a concentration of 2×105 cells / ml, and the medium is supplemented with 10% fetal bovine serum, 1% penicillin & streptomycin and 100 active units / ml of recombinant mouse macrophage colony stimulating factor (rmM-CSF; Peprotech, 250-05), the culture temperature is 37°C, and the culture conditions are 5% CO2.

[0029] S2: After 4 days of culture, the medium was replaced by half with DMEM / F12 medium containing the above components. After 7 days, the cells were washed three times with phosphate buffered saline and digested with Cellstripper cell digestion and dissociation solution (Corning, 25-056-CI) at 37°C for 5 minutes.

[0030] S3: Collect the digested cells by blowing with a pipette, wash three times with cell culture medium, and count the cells. Infect the above macrophages with BCG: Inoculate the macrophages at a concentration of 2×106 cells / well into a flat-bottomed six-well cell culture plate, and then infect with BCG at a multiplicity of infection = 1, and incubate at 37°C for 24 hours. Use macrophages not infected with BCG as negative control.

[0031] S4: The cells were washed three times with phosphate buffer and resuspended, and repeated freezing and thawing (quick freezing in liquid nitrogen and thawing at room temperature) for at least five times to lyse the cells and release the BCG fragments cut by cell digestion, and then ultraviolet sterilization was performed for 1 hour to kill the BCG bacteria. The sterilized cell fragments (named BIMCD, the same below) were harvested and stored in a -80°C refrigerator for further use.

[0032] S5: One week before mouse immunization, prepare mouse bone marrow differentiated dendritic cells: collect bone marrow cells from the femur and tibia of BALB / c mice, resuspend them at a concentration of 2×105 cells / ml in RPMI 1640 medium (Hycolne, SH30022.01B), and culture the medium with 10% fetal bovine serum, 1% penicillin & streptomycin, and 20 ng / ml recombinant mouse granulocyte-macrophage colony stimulating factor (rmGM-CSF; Peprotech, 315-03) at 37°C and 5% CO2.

[0033] S6: After 4 days of culture, the RPMI 1640 medium containing the above components was replaced by half. After 7 days, the cells were blown with phosphate buffered saline to collect non-adherent cells, which were washed three times and resuspended in cell culture medium for cell counting.

[0034] S7: The dendritic cells (1×10 6 cells / mouse) were co-incubated with BIMCD at a multiplicity of infection of 1 for 30 minutes. The cell vaccine was named BIMHDC, and then mice were immunized by intravenous immunization.

[0035] Example 2: Ability of BCG-infected macrophage fragments to induce dendritic cell maturation in vitro

[0036] See also Figure 2 The steps to evaluate the ability of BIMCD to stimulate dendritic cells include:

[0037] S1: BIMCD was used to incubate dendritic cells for 24 hours, and then the cells and culture supernatant were collected ( Figure 2 A).

[0038] S2: Enzyme-linked immunosorbent assay was used to detect inflammatory factors in the supernatant, and flow cytometry was used to detect the expression of leukocyte differentiation antigen and major histocompatibility complex.

[0039] S3: Collect the stimulated cells and incubate them with the fluorescent small molecule compound Dextran to detect the phagocytic ability of the fluorescent small molecule compound (the maturity of dendritic cells after stimulation is inversely proportional to their phagocytic ability).

[0040] In this part of the experiment, culture medium and blank cell fragments not infected with BCG were used as negative controls, and lipopolysaccharide stimulation was used as a positive control to compare the stimulating effects of inactivated BCG, BCG, and BIMCD.

[0041] Finally, after the dendritic cells stimulated by different components were co-incubated with the fluorescent small molecule Dextran, it was found that the phagocytic ability of the dendritic cells stimulated by BIMCD was significantly reduced ( Figure 2 FH). The above results show that BIMCD can significantly induce the maturation of dendritic cells. Example 3: Specific immune response induced by dendritic cell vaccine loaded with BCG-infected macrophage fragments in mouse spleen

[0042] See also Figure 3 The immunogenicity of the cell vaccine was evaluated using a mouse immune model.

[0043] S1: BALB / c mice were immunized intravenously, the number of dendritic cells was 1×106 cells / mouse, and the multiplicity of infection of BIMCD and dendritic cells was 1. Phosphate buffer, blank dendritic cells, and dendritic cells loaded with macrophage fragments not infected with BCG were used as negative controls, and BCG (1×106 colony forming units / mouse) was used as a positive control.

[0044] S2: Eight weeks after the mice were immunized, the spleens of the mice were taken and digested into single cell suspensions, and specific stimulation with Ag85AB peptide and tuberculin was performed to evaluate the antigen-specific immune response ( Figure 3 A).

[0045] Mouse ELISpot assay was used to evaluate the ability of spleen cells to secrete interferon-γ after specific stimulation. Figure 3 B shows the spot diagram. Figure 3 CD shows the summary data analysis. Then, flow cytometry was used to detect specific stimulation (see schematic diagram for details). Figure 3 E), CD4 cells ( Figure 3 F) and CD8 cells ( Figure 3 G) The ratio of specific secretion of interferon-γ, interleukin-2 and tumor necrosis factor-α.

[0046] Example 4: Dendritic cell vaccine loaded with BCG-infected macrophage fragments induces specific immune response in mouse lungs

[0047] See also Figure 4 , mice were immunized using the above immunization scheme, and the mouse lungs were taken and digested into single cell suspensions eight weeks after immunization ( Figure 4 A) Mouse ELISpot assay to evaluate the ability of lung cells to secrete interferon-γ after specific stimulation. Figure 4 B shows the spot diagram. Figure 3 C-3D shows the summary data analysis. Then, flow cytometry was used to detect specific stimulation (see schematic diagram for details). Figure 4 E), CD4 cells ( Figure 4 F) and CD8 cells ( Figure 4 G) The ratio of cells specifically secreting interferon-γ, interleukin-2, and tumor necrosis factor-α.

[0048] Example 5: Protective efficacy against tuberculosis infection induced by dendritic cell vaccine loaded with BCG-infected macrophage fragments

[0049] See also Figure 5 The mice were immunized using the above immunization scheme. Eight weeks after immunization, the mice were challenged with tuberculosis aerosols and sacrificed five weeks later. The colony counts in the lungs and spleens, as well as the pathological analysis of the lungs were performed ( Figure 5A). The results showed that compared with negative control groups such as phosphate buffered saline, BCG and BIMHDC vaccinations significantly reduced lung ( Figure 5 B) and spleen ( Figure 5 C), and the inflammatory level of BCG and BIMHDC vaccines was reduced in the acid-fast stained lung sections ( Figure 5 D) This result indicates that BIMHDC can induce immune protection against tuberculosis infection similar to that of BCG.

[0050] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent substitutions or changes according to the technical solutions and novel concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for constructing a vaccine, characterized in that: Using BCG-infected macrophage fragments as vaccine antigens, the method comprises the following steps: S1. Infect macrophages with BCG; S2. Lysing infected cells by repeated freezing and thawing and UV inactivation to obtain an antigen composition containing cell fragments; S3. Co-culture the in vitro differentiated dendritic cells with the antigen composition to form a cell vaccine.

2. A vaccine construction method as claimed in claim 1, characterized in that The BCG vaccine is Mycobacterium bovis.

3. A vaccine construction method as claimed in claim 1, characterized in that The antigen composition comprises tuberculosis antigen fragments cleaved by protease and captured by cells.

4. A vaccine construction method as claimed in claim 1, characterized in that The dendritic cells are professional antigen presenting cells.

5. A vaccine constructed by the method according to any one of claims 1 to 4, characterized in that The vaccine is in the form of dendritic cells loaded with the cell fragments.

6. A vaccine as claimed in claim 5, characterized in that The vaccine is used for intravenous inoculation, intramuscular injection, subcutaneous injection or inhalation inoculation.

7. A use of the vaccine as claimed in claim 5, characterized in that The vaccine cell fragments and the dendritic cell vaccine loaded with the fragments can be used alone or in combination with other tuberculosis vaccines.

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