A recombinant bcg vaccine for treating tuberculosis and use thereof

Abstract

The application belongs to the technical field of medicines, and particularly discloses a recombinant BCG vaccine for treating tuberculosis and application thereof. Mb2258 The recombinant BCG vaccine is obtained by knocking out genes of a traditional BCG vaccine strain, and can not only prevent the occurrence and development of tuberculosis, but also relieve the progress of tuberculosis as a therapeutic immunoregulatory drug. The effect of the recombinant BCG vaccine on preventing and treating tuberculosis is superior to that of a wild BCG vaccine strain, and therefore the recombinant BCG vaccine is a new type of tuberculosis immunotherapy drug with good potential.

Description

Technical Field

[0001] This invention belongs to the field of medicine and health, specifically relating to a recombinant BCG strain BCG△Mb2258 and the application of this recombinant strain in the prevention and treatment of tuberculosis. Background Technology

[0002] Tuberculosis (TB) is a major chronic and fatal infectious disease caused by Mycobacterium tuberculosis (Mtb). The World Health Organization's "Global Tuberculosis Report 2022" shows that approximately 10.6 million new TB cases were diagnosed globally in 2021, an increase of 4.5% compared to 2020; and approximately 1.6 million TB deaths occurred in 2021, exceeding the death tolls of 2020 and 2019, returning to 2017 levels. Tuberculosis control has stagnated or even reversed, and the situation is far from optimistic.

[0003] Currently, the only vaccine approved for clinical use in the prevention of tuberculosis is the BCG vaccine (Bovisbacille Calmette-Guérin, BCG). It has a protective effect of about 50-80% against tuberculous meningitis in newborns and preschool children, but its protective effect against pulmonary tuberculosis varies greatly among individuals, and it has no obvious protective effect against pulmonary tuberculosis in adults (Trunz BB, Fine P&Dye C (2006), Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. Lancet 367:1173–1180; Helen A Fletcher (2016), Sleeping Beauty and the Story of the Bacille Calmette-Guérin Vaccine. mBio30;7(4):e01370-16). Therefore, the development of a new tuberculosis vaccine is urgently needed. On the other hand, these vaccines generally only provide preventative protection and do not have a therapeutic effect. In addition to active tuberculosis patients, a large proportion of the population are latent tuberculosis carriers, meaning they are infected with Mtb but do not show obvious symptoms. This group is substantial and urgently needs therapeutic drugs to slow the progression of the disease. For example, the injectable Mycobacterium bovis (trade name: Microcard) developed by Zhifei Biological Products is used not only as an adjunct treatment for tuberculosis but also to prevent the progression of latent tuberculosis infection to tuberculosis, thus having broader clinical application value.

[0004] The Mtb genome encodes more than ten eukaryotic-like protein kinases and phosphatases, which inhibit host immune responses and promote Mtb survival by directly targeting host proteins. Among them, PtpA, encoded by the Mb2258 gene in BCG, is a low-molecular-weight tyrosine phosphatase. Studies have shown that PtpA is a key Mtb effector protein that directly targets the host, directly inhibiting host cell phagosome acidification and suppressing the activation of innate immune signaling pathways (Wang J et al., (2015), Mycobacterium tuberculosis suppresses innate immunity by coopting the hostubiquitin system. Nature Immunology 16(3):237-45; Bach H et al., (2008), Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B. Cell Host Microbe 15; 3(5):316-22). Based on this, this invention conducted research and found unexpected results. Summary of the Invention

[0005] This invention constructs a recombinant BCG strain with the Mb2258 gene knocked out to relieve the inhibitory effect of the PtpA effector protein in traditional BCG on the host's innate immunity, thereby more effectively enhancing the body's immune level. Using an Mtb-mouse infection model, the recombinant BCG strain BCG△Mb2258 was found to prevent tuberculosis, consistent with the expected functional outcome of this gene. However, the study unexpectedly discovered that it can also serve as a therapeutic immunomodulatory drug to alleviate tuberculosis progression, with a therapeutic effect superior to wild-type BCG strains. This is entirely different from the usual practice of recombinant BCG strains only serving as vaccines to prevent tuberculosis, thus completing this invention.

[0006] This invention constructs a recombinant BCG strain by knocking out the Mb2258 gene on the basis of wild-type BCG strain. Mtb-mouse infection experiments show that this recombinant BCG strain is more effective than the traditional wild-type BCG strain in both preventing and treating tuberculosis.

[0007] A first aspect of the present invention is to provide a BCG recombinant strain (BCG△Mb2258) for therapeutic use, wherein the Mb2258 gene in the BCG recombinant strain is completely knocked out. The amino acid sequence of the Mb2258 gene is SEQ ID No.: 1.

[0008] Furthermore, the Mb2258 gene homologous arm sequence used in the knockout process is SEQ ID No.: 2–5.

[0009] A second aspect of the present invention is to provide a method for constructing the above-mentioned recombinant BCG bacteria for therapeutic purposes, wherein the recombinant BCG bacteria are obtained by scarless knockout of the Mb2258 gene in a wild-type BCG strain using phage homologous recombination technology.

[0010] A third aspect of the present invention provides a recombinant BCG vaccine for treating tuberculosis, which contains a recombinant BCG strain according to the stated therapeutic use as an active ingredient.

[0011] Specifically, the medicament comprises a culture of the recombinant BCG strain or a component thereof for the therapeutic purpose. Further, the medicament also includes a pharmaceutically acceptable adjuvant.

[0012] In particular, the fourth aspect of the present invention provides the use of the recombinant BCG strain described for therapeutic purposes in the preparation of a medicament for treating tuberculosis.

[0013] Specifically, the drug comprises the recombinant BCG strain and its culture or components for the therapeutic purpose.

[0014] More specifically, the drug also includes pharmaceutically acceptable adjuvants, in the form of a drug with suitable dosage forms and routes of administration, etc.

[0015] In the course of studying the inhibitory effect of removing the PtpA effector protein (i.e., knocking out the Mb2258 gene) from traditional BCG on the host's innate immunity, this invention unexpectedly discovered that it has a good application in the treatment of tuberculosis, and experiments have confirmed that it has application value in the development of therapeutic immunomodulatory drugs. Attached Figure Description

[0016] Figure 1 PCR identification of the knockout results of the Mb2258 gene in the recombinant BCG strain BCG△Mb2258;

[0017] Figure 2 Flowchart for evaluating the protective efficacy of recombinant BCG strain BCG△Mb2258 as a prophylactic vaccine against TB in mice;

[0018] Figure 3 Comparison of Mtb bacterial load in lung tissues of mice vaccinated with different BCG strains as a preventive treatment for TB.

[0019] Figure 4 Pathological staining results of lung tissue from mice vaccinated against TB with different BCG strains;

[0020] Figure 5Flowchart for evaluating the efficacy of recombinant BCG strain BCG△Mb2258 as a therapeutic immunomodulatory drug for TB in mice;

[0021] Figure 6 Comparison of Mtb bacterial load in lung tissue of mice inoculated with different BCG strains as therapeutic immunomodulatory drugs for TB.

[0022] Figure 7 Pathological staining results of lung tissue from mice inoculated with different BCG strains as therapeutic immunomodulatory drugs for TB. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings to provide a better understanding of the invention. However, the following embodiments do not limit the invention.

[0024] Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared according to conventional methods, unless otherwise specified.

[0025] The sequence information used in the embodiments is as follows:

[0026] SEQ ID No.: 1Mb2258 gene encoding amino acid sequence

[0027] MSDPLHVTFVCTGNICRSPMAEKMFAQQLRHRGLGDAVRVTSAGTGNWHVGSCADERAA

[0028] GVLRAHGYPTDHRAAQVGTEHLAADLLVALDRNHARLLRQLGVEAARVRMLRSFDPRSG

[0029] THALDVEDPYYGDHSDFEEVFAVIESALPGLHDWVDERLARNGPSSEQ ID No.: 2Mb2258 Upstream primer sequence of the left homologous arm of the gene

[0030] TTTTTTTCCATAAATTGGTACACGCCATGGTCAATGCC

[0031] SEQ ID No.: Downstream primer sequence of the left homologous arm of the 3Mb2258 gene

[0032] TTTTTTTTCATTTCTTGGTCAGACACCTAGCGCCTC

[0033] SEQ ID No.: Upstream primer sequence of the right homologous arm of the 4Mb2258 gene

[0034] TTTTTTTTCCATAGATTGGATGCCCCGCCTAGCGTTCCTG

[0035] SEQ ID No.: Downstream primer sequence of the right homologous arm of the 5Mb2258 gene

[0036] TTTTTTTTCCATCTTTTGGGTCGAGGCGGCGTTTGCTGG

[0037] SEQ ID No.: Sequencing primer sequence for the left homologous arm of the 6Mb2258 gene

[0038] AACAGCTTGTTCGACGGGAT

[0039] SEQ ID No.: Sequencing primer sequence for the right homologous arm of the 7Mb2258 gene

[0040] ACGTTTTGGCATTCTTGCCC

[0041] Example 1: Construction and identification of recombinant BCG strain BCG△Mb2258

[0042] This study constructed a recombinant BCG strain (BCG△Mb2258) with the Mb2258 gene deletion based on wild-type BCG (Japanese strain) using homologous recombination technology. The main procedures are as follows:

[0043] 1.1 Construction of p0004S-△Mb2258 plasmid

[0044] Using BCG whole-genome DNA as a template, two pairs of primers targeting the left and right arms of the Mb2258 gene were designed: Mb2258-LS TTTTTTTTCCATAAATTGGTACACGCCATGGTCAATGCC (SEQ ID No.: 2), Mb2258-LA TTTTTTTTCCATTTCTTGGTCAGACACCTAGCGCCTC (SEQ ID No.: 3), and Mb2258-RS TTTTTTTTCCATAGATTGGATGCCCCGCCTAGCGTTCCTG (SEQ ID No.: 4), Mb2258-RA TTTTTTTTCCATCTTTTGGGTCGAGGCGGCGTTTGCTGG (SEQ ID No.: 5). Homologous arm sequences of the left and right sides of the Mb2258 gene were amplified by polymerase chain reaction (PCR). Reaction conditions: 95℃ for 3 min; 95℃ for 15 s; 58℃ for 15 s; 72℃ for 1 min, 30 cycles; 72℃ for 5 min. The left and right homologous arms of the Mb2258 gene and the p0004S plasmid were digested with Van91I (cat#FD0714, Thermo Scientific). The digested fragments were ligated, transformed into DH5α competent cells, and plated on LB agar plates containing 150 μg / mL hygromycin (cat#10843555001, Roche). Single colonies were picked, plasmids were extracted, and positive clones were confirmed by Van91I digestion.

[0045] 1.2 Construction of the phAE159-△Mb2258 shuttle plasmid

[0046] The p0004S-ΔMb2258 and phAE159 plasmids were digested with PacI (cat#FD2204, Thermo Scientific) restriction enzyme, and the digested fragments were ligated. HB101 competent cells were transformed using a phage in vitro packaging kit (cat#MP5120, Epicentre) and plated on LB agar plates containing 150 μg / mL hygromycin. Single colonies were picked, plasmids were extracted, and positive clones were confirmed by PacI digestion.

[0047] 1.3 Phage amplification

[0048] 5 μg of phAE159-△Mb2258 plasmid was electroporated into Mycobacterium smegmatis mc 2 155 competent cells were added to 700 μL of 7H9 culture medium and recovered at 37°C for 4 h. The recovered bacterial culture was then mixed with 0.7% agar and spread on 7H10 plates. The cells were incubated at 30°C in the dark for 2–3 days.

[0049] Pick 4–5 plaques into an EP tube, add 200 μL of MP buffer, and incubate overnight at 4°C. Take 50 μL of the supernatant and add it to 300 μL of freshly cultured Mycobacterium smegma. 2 Add 155g of bacterial culture to 0.7% agar and mix well. Spread the mixture onto 7H10 plates. Once the plaques have expanded and connected, add an appropriate amount of MP buffer and incubate overnight at 4°C. Aspirate the supernatant and filter it through a 0.22μm filter. Store the phages at 4°C.

[0050] The bacteriophages were serially diluted, and 100 μL of each dilution was added to freshly cultured Mycobacterium smegmatis mcg. 2 Mix 155% bacterial suspension with 0.7% agar, spread it on 7H10 plates, and incubate at 30°C in the dark for 2–3 days. Calculate the plaque titer.

[0051] 1.4 Construction of the BCG△Mb2258 recombinant BCG strain

[0052] To prepare competent strains of wild-type BCG (Japanese strain), the competent BCG and phage were mixed at a ratio of 1:10 and incubated in the dark for 3–4 h. Then, 10 mL of 7H9 medium (containing OADC enrichment broth) was added, and the mixture was incubated at 37°C for 16–20 h. The BCG was collected by centrifugation and spread on 7H10 plates containing 75 μg / mL hygromycin. The plates were incubated at 37°C for 4–6 weeks, and positive clones were selected.

[0053] Identification of 1.5BCG△Mb2258 recombinant BCG strain

[0054] Single colonies from the above plates were picked and incubated statically at 37°C for 3 weeks in 7H9 medium. The bacterial culture was collected, and genomic DNA was extracted from the strains. Primers were designed based on the left and right homologous arm sequences of the Mb2258 gene: Mb2258-upAACAGCTTGTTCGACGGGAT (SEQ ID No.: 6) and Mb2258-down ACGTTTTGGCATTCTTGCCC (SEQ ID No.: 7) for PCR amplification and identification. PCR conditions: 95°C for 3 min; 95°C for 15 s; 58°C for 15 s; 72°C for 3 min, 30 cycles; 72°C for 5 min. Strains correctly identified by PCR were sent for bacterial genome sequencing to confirm that the Mb2588 gene fragment in the recombinant BCG vaccine was completely knocked out.

[0055] The results are as follows Figure 1As shown, PCR identification confirmed that the Mb2258 gene (1012 bp) could be detected in wild-type BCG, while the Mb2258 gene could not be detected in the recombinant BCG strain BCG△Mb2258, but was replaced by the homologous recombination hygromycin resistance gene (4120 bp). At the same time, gene sequencing results confirmed that the Mb2258 gene was completely knocked out in the recombinant BCG vaccine.

[0056] Example 2: Detection of the protective efficacy of recombinant BCG strain BCG△Mb2258 as a prophylactic vaccine against TB at the mouse level.

[0057] This embodiment uses the recombinant BCG strain BCG△Mb2258 constructed in Example 1 to verify its preventive effect as a prophylactic vaccine against tuberculosis in mice. The specific experimental steps and results are as follows:

[0058] refer to Figure 2 The experimental flowchart illustrates the use of both intradermal and mucosal inoculation methods. Each group of C57BL / 6 mice was intradermally inoculated with 2 × 10⁶ cells / mL. 6 1×10 via individual or mucosal inoculation 7 Mice were infected with wild-type BCG or recombinant BCG△Mb2258 at a bacterial count of 100 cells / mL, with the uninoculated group serving as a control. Four weeks after inoculation, mice in each group were infected with Mycobacterium tuberculosis H37Rv strain via nebulization in a biosafety level 3 laboratory, with an infection quantity of 100 cells / mL.

[0059] Four weeks after infection, mice were euthanized by cervical dislocation, and lung tissue was isolated for colony counting. The Mtb load in the lung tissue of each group of mice was calculated. Simultaneously, the lung tissue of each group of mice was fixed in 4% paraformaldehyde and embedded in paraffin. Further hematoxylin-eosin staining and acid-fast staining were used to observe the differences in lung tissue pathology and Mtb load among the groups.

[0060] As shown in the figure, compared with unvaccinated control mice, intradermal and mucosal inoculation with BCG or recombinant BCG△Mb2258 were effective in protecting against Mtb infection. Mucosal inoculation was more effective than intradermal inoculation. Furthermore, regardless of whether mucosal or intradermal inoculation was performed, the protective effect of the recombinant BCG△Mb2258 strain was superior to that of the wild-type BCG strain. Under intradermal inoculation conditions, the bacterial load in the lung tissue of mice inoculated with the recombinant BCG△Mb2258 strain was 1–2 orders of magnitude lower than that of unvaccinated mice, and 0.5–1.5 orders of magnitude lower than that of mice inoculated with the wild-type BCG strain. Under mucosal inoculation conditions, the bacterial load in the lung tissue of mice inoculated with the recombinant BCG△Mb2258 strain was 1.5–2 orders of magnitude lower than that of unvaccinated mice, and 0.5–1.5 orders of magnitude lower than that of mice inoculated with the wild-type BCG strain (e.g., ...). Figure 3Furthermore, regardless of whether the inoculation was intradermal or mucosal, the lung tissue of mice inoculated with recombinant BCG ΔMb2258 showed less inflammatory cell infiltration and more intact alveolar tissue structure compared to the lung tissue of mice not vaccinated with BCG and mice vaccinated with wild-type BCG (e.g., Figure 4 ).

[0061] In conclusion, compared with traditional BCG strains, the recombinant BCG△Mb2258 strain can more effectively enhance the body's immunity against Mtb infection, thus exhibiting superior efficacy in preventing tuberculosis compared to traditional BCG. Furthermore, mucosal BCG vaccination provides better protection than intradermal BCG vaccination.

[0062] Example 3: Detection of the efficacy of recombinant BCG strain BCG△Mb2258 as a therapeutic immunomodulatory drug for TB in mice.

[0063] This embodiment uses the recombinant BCG strain BCG△Mb2258 constructed in Example 1 to verify its therapeutic effect as a therapeutic immunomodulatory drug on mice infected with tuberculosis at the mouse level. The specific experimental steps and results are as follows:

[0064] refer to Figure 5 The experimental flowchart illustrates how C57BL / 6 mice were infected with Mycobacterium tuberculosis H37Rv strain via nebulization in a biosafety level 3 laboratory. The Mtb bacterial load was 100 cells / mL. Three days after infection, each group of mice was inoculated intradermally with 1 × 10⁻⁶ Mtb cells / mL. 6 Wild-type BCG or recombinant BCG△Mb2258 of three different strains (Shanghai D2 strain, Japanese strain, and Pasteur strain) were used as negative controls, while the group vaccinated with prophylactic microcardiogram (Zhifei Biological) served as a positive control. Subsequent intradermal vaccinations were administered every two weeks for a total of three doses.

[0065] Two weeks after the third immunization, mice were sacrificed by cervical dislocation, and lung tissue was isolated for colony counting. The Mtb load in the lung tissue of each group of mice was calculated. At the same time, the lung tissue of each group of mice was fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin-eosin staining and acid-fast staining were used to observe the differences in lung tissue pathology and Mtb load among the groups of mice.

[0066] like Figure 6As shown, compared with unvaccinated mice, there was no significant difference in the bacterial load in the lung tissue of mice intradermally inoculated with three different strains of wild-type BCG, suggesting that wild-type BCG strains have no significant effect on the treatment of tuberculosis. Simultaneously, comparisons revealed that the bacterial load in the lung tissue of mice inoculated with three different strains of recombinant BCG△Mb2258 was significantly lower than that of the unvaccinated group and the mice inoculated with wild-type BCG, with an average decrease of approximately 50%–90%, suggesting that intradermal inoculation with different strains of recombinant BCG△Mb2258 has a significant therapeutic effect on tuberculosis (e.g., Figure 6 Furthermore, the bacterial load in the lung tissue of mice inoculated with recombinant BCG△Mb2258 (Japanese strain) and recombinant BCG△Mb2258 (Pasteur strain) was comparable to that in mice inoculated with prophylactic microcard, while the bacterial load in the lung tissue of mice inoculated with recombinant BCG△Mb2258 (Shanghai D2 strain) was lower than that in mice inoculated with prophylactic microcard, with an average decrease of approximately 50%.

[0067] Pathological analysis results were consistent with the above findings: lung tissue from mice inoculated with recombinant BCG ΔMb2258 strain showed less inflammatory cell infiltration and more intact alveolar tissue structure compared to lung tissue from mice not vaccinated with BCG or vaccinated with wild-type BCG (e.g., Figure 7 In all treatment groups (including negative and positive controls, different BCG vaccine treatments, and recombinant BCG vaccine treatments), mice inoculated with recombinant BCG△Mb2258 (Shanghai D2 strain) showed the least inflammatory cell infiltration and the best alveolar tissue structure. In conclusion, the recombinant BCG△Mb2258 strain can significantly enhance the host's resistance to Mtb infection, especially by effectively reducing Mtb survival levels in the lungs and alleviating histopathological damage, thus demonstrating good therapeutic effects for tuberculosis. These research data suggest that recombinant BCG△Mb2258, as an immunotherapy drug for tuberculosis, will exert its anti-infective effect by enhancing the host's immune response. Combined with existing anti-tuberculosis drugs, it will have therapeutic effects on active tuberculosis (infection by susceptible and drug-resistant bacteria) and latent tuberculosis infection.

[0068] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, any changes and modifications made without departing from the concept of the present invention should be covered within the patent protection scope of the present invention.

Claims

1. The use of a recombinant BCG strain for therapeutic purposes in the preparation of a drug for treating tuberculosis, characterized in that, The recombinant BCG strain used for therapeutic purposes is a wild-type BCG strain with the Mb2258 gene completely knocked out, and the amino acid sequence encoded by the Mb2258 gene is shown in SEQ ID No:

1.

2. The application according to claim 1, characterized in that, The Mb2258 gene in the recombinant BCG strain was completely knocked out using phage-mediated homologous recombination technology.

3. The application according to claim 2, characterized in that, It was obtained by knocking out the Mb2258 gene in wild-type BCG strains using phage-mediated homologous recombination technology.

4. The application according to any one of claims 1 to 3, characterized in that, The drug comprises a culture of the recombinant BCG strain used for therapeutic purposes.

5. The application according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable adjuvants.

Images