Application of subcutaneous combined inoculation of bacillus calmette guerin vaccine and complete freund's adjuvant in tuberculosis prevention

Abstract

The invention relates to the field of vaccines, in particular to application of a bacillus calmette-guerin vaccine and a complete freund's adjuvant in preparation of tuberculosis prevention vaccines. The invention provides a novel inoculation strategy of subcutaneous combined inoculation of the bacillus calmette guerin vaccine and the complete freund's adjuvant, the safety consistent with that of the existing tuberculosis prevention vaccine in the clinical stage is achieved, and the immune protection effect higher than that of the existing tuberculosis prevention vaccine in the clinical stage and the preclinical research stage is achieved. The compound is expected to be used as a novel tuberculosis vaccine for preventing tuberculosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of vaccines, in particular, the application of subcutaneous co-administration of Bacillus Calmette-Guerin (BCG) and Complete Freund's Adjuvant in the prevention of tuberculosis. BACKGROUND

[0002] Tuberculosis (TB) is the leading cause of death from infectious disease globally. The only licensed TB vaccine is Bacillus Calmette-Guerin (BCG; live attenuated Mycobacterium bovis), which is administered by intradermal injection at birth. Although the vaccine is protective against disseminated tuberculosis in infants, its efficacy against pulmonary tuberculosis in adolescents and adults varies significantly. Currently, 16 candidate vaccines are being evaluated in clinical trials, and several more are in preclinical development. M72:AS01E is one of the most advanced subunit vaccines in the pipeline, which was recently evaluated in a phase III clinical trial (NCT01755598) and showed a 54% protection against latent TB infection in HIV-negative individuals when administered as an intramuscular emulsion. In an earlier phase II trial (NCT02075203), BCG revaccination (re-vaccination) failed to provide protection against primary Mycobacterium tuberculosis (Mtb) infection but prevented persistent Mtb infection. The efficacy of TB vaccines currently in clinical development is below 60%, a threshold insufficient to meet the global goal of TB eradication, highlighting the urgent need for new vaccine platforms that can induce potent and long-lasting immunity against Mtb.

[0003] Adjuvants not only stimulate immune responses effectively, but also protect antigens from degradation and facilitate their delivery to target tissues. Current TB vaccine adjuvants have evolved from traditional aluminum adjuvants (which are difficult to induce potent Th1 / CD8+ T cell immunity against intracellular M. tuberculosis) to liposomal and emulsion systems in clinical development. Among them, AS01 (containing MPL and QS-21) showed the best efficacy in humans: a phase III clinical trial (NCT01755598) in patients with latent TB infection showed that it achieved a protection rate of 54% in a 3-year follow-up period. CAF01 (DDA / TDB) can safely induce Th1 / Th17 responses, but its effect on controlling pulmonary infection after booster immunization is limited; while GLA-SE (TLR4 agonist + squalene) and IC31 (KLK / ODN1a) enhance the strength and persistence of multifunctional CD4+ T cell responses in early trials. New platforms such as dextran / CpG nanoparticles (GamTBVac, phase IIa) and Advax microparticles (preclinical stage) also show good immunogenicity, however, only AS01 has been fully validated in clinical applications. It is worth noting that Complete Freund's Adjuvant (CFA) is mainly composed of inactivated H37Ra strain and mineral oil. However, the protective effect of the combination of BCG and CFA subcutaneous vaccination strategy on tuberculosis has not been reported. SUMMARY

[0004] The purpose of the present application is to provide a new tuberculosis prevention strategy of subcutaneous immunization of BCG combined with CFA and its application. In the early stage of the present application, it was found through Mtb H37Rv lung load detection and lung histopathology analysis that subcutaneous vaccination can produce stronger protection effect against tuberculosis. It is worth noting that all 10 mice receiving BCG+CFA subcutaneous immunization showed a high degree of protection, and 8 mice were not detected with Mtb in the body.

[0005] In a first aspect of the present application, the use of BCG combined with CFA in the preparation of a tuberculosis prevention vaccine is provided.

[0006] Further, in the tuberculosis prevention vaccine, BCG and CFA are subcutaneously inoculated.

[0007] In a second aspect of the present application, a tuberculosis prevention vaccine is provided, and the active ingredients thereof include BCG and CFA.

[0008] Further, the BCG (2 mg / ml, i.e. 2×10 7CFU / ml) and complete Freund's adjuvant (MedChemExpress, Cat# HY-153808) at a volume ratio of 1:1. After mixing, each 100 μL contained 10 6 CFU BCG (50 μL volume) and 50 μL complete Freund's adjuvant.

[0009] Further, the tuberculosis prevention vaccine is a combination of the BCG vaccine and complete Freund's adjuvant for subcutaneous vaccination. The vaccination procedure is only on day 0, and only one time.

[0010] In a third aspect, the present application provides a method for preparing the tuberculosis prevention vaccine as described above, comprising the following steps:

[0011] A) The BCG strain culture is centrifuged at 12,000xg for 3 minutes at 4°C, washed once with ice-cold phosphate buffer PBS, and then resuspended in PBS. The McFarland method is used to quantify the concentration to 2 mg / ml, i.e. 2x10 7 CFU / ml;

[0012] B) Equal volumes of 2 mg / ml BCG bacteria solution and complete Freund's adjuvant are mixed in an ultrasonic tube, and the final concentration of BCG is 1 mg / ml; ultrasonic emulsification is performed twice;

[0013] C) Stirring emulsification is performed for 10 minutes using an electric emulsifier (BDYQ1001; Biodragon), and the emulsion is used in a state of not spreading in water for 5 minutes.

[0014] The present application has the following advantages:

[0015] The present application provides a new vaccination strategy of subcutaneous combination of BCG vaccine and complete Freund's adjuvant, which achieves the same safety as the existing clinical stage tuberculosis prevention vaccine, and achieves a higher immune protection effect than the existing clinical stage and preclinical research stage tuberculosis prevention vaccine. It is expected to be used as a new tuberculosis vaccine for the prevention of tuberculosis. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 . Lung bacterial load detection results of mice in different immunization groups after infection;

[0017] Figure 2 . Lung HE staining results of mice in different immunization groups after infection;

[0018] Figure 3 . Lung bacterial load detection results of mice in different immunization groups after infection;

[0019] Figure 4 . Lung HE staining results of mice in different immunization groups after infection;

[0020] Figure 5 Safety test results of different immunization groups. DETAILED DESCRIPTION

[0021] The specific embodiments provided by the present application are described in detail below with reference to the examples.

[0022] Example 1:

[0023] Analysis of the immune protection effect of subcutaneous immunization of BCG+CFA on tuberculosis using C57BL / 6 mice

[0024] The control mice (containing the following three groups: 1. PBS group: the subcutaneous immunization component is sterile PBS, the amount is 100 ul; 2. BCG group: the subcutaneous immunization component is 1 mg / ml BCG bacterial solution resuspended in PBS, the amount is 100 ul; 3. CFA group: the subcutaneous immunization component is a suspension of equal volume of complete Freund's adjuvant and PBS emulsified sufficiently, the amount is 100 ul) and 10-12 week old female C57BL / 6 mice subcutaneously immunized with BCG+CFA (100 ul in total, volume ratio 1:1) for one month, 6-10 mice per group, were placed in ABSL-3 for 1 week, then infected with H37Rv strain by aerosol route, about 100 CFU / mouse. Four weeks after infection, the mice were sacrificed. The effect of subcutaneous immunization of BCG+CFA on tuberculosis prevention was analyzed by detecting the following indicators:

[0025] 1. Lung bacterial load: under sterile conditions, collect the lung, add PBS homogenate, 10-fold dilution, then inoculate on 7H10 agar medium, 37°C culture for 4 weeks, observe the growth of Mtb, calculate the CFU number of viable bacteria, and count the lung bacterial load. The results showed that subcutaneous vaccination of BCG combined with CFA can produce stronger protection effect against tuberculosis. It is worth noting that all 10 mice subcutaneously immunized with BCG+CFA showed high protection, among which 8 mice did not detect Mtb in the body Figure 1 ).

[0026] 2. Lung histopathology detection: under sterile conditions, collect the lung, take part of the lung tissue and fix it with 4% paraformaldehyde, dehydrate, wax-embed, section, H&E and acid-fast staining, observe the lung histopathology changes under microscope. The results showed that the lung pathological changes of mice subcutaneously vaccinated with BCG combined with CFA were significantly reduced compared with the control group, and the immune cell infiltration and lung tissue damage were also significantly reduced, which was better than the BCG group Figure 2 ).

[0027] From C57BL / 6 mice, subcutaneous immunization BCG+CFA, after immunization infection Mtb H37Rv, after one month of infection, through the mouse lung CFU detection, and lung pathological damage analysis, found that subcutaneous immunization BCG+CFA significantly reduced the infection of mice lung bacterial load, reduce pathological changes, better than the clinical use of vaccine BCG.

[0028] The above results show that subcutaneous vaccination of BCG combined with complete Freund's adjuvant (CFA) can produce stronger protection against tuberculosis, and the effect is better than the first-line tuberculosis vaccine BCG.

[0029] Example 2:

[0030] Using C57BL / 6 mice, subcutaneous immunization BCG+CFA for three months, analysis of BCG and CFA combined subcutaneous immunization of tuberculosis immune protection effect:

[0031] The control mice (including the following three groups: 1. PBS group: the subcutaneous immunization component is sterile PBS, the amount is 100ul; 2. BCG group: the subcutaneous immunization component is 1mg / ml BCG bacteria liquid resuspended in PBS, the amount is 100ul; 3. CFA group: the subcutaneous immunization component is an equal volume of complete Freund's adjuvant and PBS suspension, the amount is 100ul) and 10-12 week old female C57BL / 6 mice subcutaneously immunized with BCG+CFA (100ul, volume ratio 1:1) for three months, each group of 7, placed in ABSL-3 for 1 week, infected with H37Rv strain by aerosol route, about 100 CFU / mouse. Four weeks after infection, the mice were sacrificed. By detecting the following indexes, the effect of subcutaneous immunization of BCG+CFA on tuberculosis prevention was analyzed:

[0032] 1. Lung bacterial load: under sterile conditions, collect the lung, add PBS homogenate, 10-fold dilution, then inoculate on 7H10 agar medium, 37℃ culture for 4 weeks, observe the growth of Mtb, calculate the CFU number of viable bacteria, and count the lung bacterial load. The results show that subcutaneous vaccination of BCG combined with complete Freund's adjuvant (CFA) can produce stronger protection against tuberculosis. It is worth noting that all 7 mice subcutaneously immunized with BCG+CFA showed a high degree of protection, and 6 mice had no Mtb detected in the body. Figure 3 ).

[0033] 3. Lung histopathology detection: under sterile conditions, collect the lung, take part of the lung tissue and fix it with 4% paraformaldehyde, dehydrate, wax immersion, embedding, section, H&E and acid-fast staining, observe the lung histopathology under a microscope. The results show that the lung pathological changes of mice subcutaneously vaccinated with BCG combined with complete Freund's adjuvant (CFA) are significantly reduced compared with the control group, and the immune cell infiltration and lung tissue damage are also significantly reduced, which is better than the BCG groupFigure 4 ).

[0034] From C57BL / 6 mice, subcutaneous immunization BCG+CFA, after immunization infection Mtb H37Rv, after three months of infection, by mouse lung CFU detection, and lung pathological damage analysis, found that subcutaneous immunization BCG+CFA significantly reduced the bacterial load of the infected mouse lung, reduced pathological changes, better than the effect of clinical use of vaccine BCG.

[0035] The above results show that subcutaneous vaccination of BCG combined with complete Freund's adjuvant (CFA) can produce stronger protection against tuberculosis, and the effect is better than the first-line tuberculosis vaccine BCG.

[0036] Example 3:

[0037] Using SCID mice, subcutaneous immunization BCG+CFA, analysis of the safety of BCG and CFA combined subcutaneous immunization:

[0038] Control mice (including the following three groups: 1. PBS group: subcutaneous immunization component is sterile PBS, 100ul; 2. BCG group: subcutaneous immunization component is 1mg / ml BCG bacterial solution resuspended in PBS, 100ul; 3. CFA group: subcutaneous immunization component is an equal volume of complete Freund's adjuvant and PBS suspension, 100ul) and 6-8 week old female SCID mice subcutaneously immunized with BCG+CFA (100ul, volume ratio 1:1), 6 in each group, by continuously detecting the body temperature, body weight, whole blood cell count, C-reactive protein, survival curve, kidney function and liver function indicators, to analyze the safety of subcutaneous immunization BCG+CFA; multiple index results show that the safety of subcutaneous immunization BCG+CFA has no significant difference with the clinical use of vaccine BCG (P>0.05). Figure 5 ).

[0039] SCID mice, subcutaneous immunization BCG+CFA, after immunization, by detecting the body temperature, body weight, whole blood cell count, C-reactive protein, survival curve, kidney function and liver function indicators, found that the safety of subcutaneous immunization BCG+CFA has no significant difference with the clinical use of vaccine BCG.

[0040] The new vaccination strategy of subcutaneous vaccination of BCG combined with CFA has achieved the same safety as the existing clinical stage of tuberculosis prevention vaccine. It is expected to be used as a new tuberculosis vaccine for tuberculosis prevention.

[0041] The preferred embodiments of the present application have been disclosed with the above particularity, but the application is not limited to the embodiments disclosed, and variations and modifications can be made by those skilled in the art without deviating from the spirit of the application, and such variations and modifications are intended to be included within the scope of the application as defined in the following claims.

Claims

1. Use of BCG combined with complete Freund's adjuvant in the preparation of a tuberculosis preventive vaccine.

2. Use of BCG in combination with Complete Freund's Adjuvant for the preparation of a vaccine for the prevention of tuberculosis according to claim 1, characterized in that, The tuberculosis preventive vaccine is subcutaneously inoculated with BCG combined with complete Freund's adjuvant.

3. A tuberculosis prophylactic vaccine, characterized by comprising a protein having an amino acid sequence represented by SEQ ID NO: 1 or a fragment thereof. The active ingredients include BCG and complete Freund's adjuvant, and the volume ratio of the BCG and the complete Freund's adjuvant is 1:

1.

4. The tuberculosis prophylactic vaccine according to claim 3, characterized in that, The tuberculosis preventive vaccine is subcutaneously inoculated with BCG combined with complete Freund's adjuvant.

5. The tuberculosis prophylactic vaccine according to claim 4, characterized in that, The inoculation procedure is to inoculate only on day 0 and only once.

6. A method of preparing a tuberculosis prophylactic vaccine as claimed in claim 3, characterized in that, The method comprises the following steps: A) BCG strain culture was centrifuged at 12,000 x g for 3 minutes at 4°C, washed once with ice-cold phosphate-buffered saline (PBS), and resuspended in PBS. The McFarland method was used to quantify the suspension at 2 mg / ml, i.e., 2 x 10 7 CFU / ml; B) Mix equal volumes of 2 mg / ml BCG bacterial liquid and complete Freund's adjuvant in an ultrasonic tube, so that the final concentration of the BCG is 1 mg / ml; and perform ultrasonic emulsification twice; C) Stir and emulsify for 10 min, and drop into water; and use in a state of not diffusing for 5 min.

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