A mycobacterium tuberculosis fusion antigen and subunit vaccine thereof

Abstract

The present application provides a kind of fusion antigen comprising Mycobacterium tuberculosis latent infection and acute infection antigen, comprising Rv2029c, Rv2028c, Rv3126c and Ag85A, and on this basis, tuberculosis subunit vaccine is constructed, can be used as the vaccine for preventing acute tuberculosis infection and treating latent tuberculosis infection, can also be used to prepare new vaccine for preventing or treating other mycobacterium infection related diseases.We optimize the dosage and inoculation site of the new vaccine, and determine its immunogenicity as a BCG booster vaccine.In acute infection and latent infection mouse models, the fusion antigen can provide effective protection against tuberculosis infection.Therefore, the fusion antigen and subunit vaccine involved in the present application are expected to become a new type of preventive tuberculosis vaccine and latent infection exposure vaccine.

Description

Technical Field

[0001] This invention belongs to the field of vaccines and relates to Mycobacterium tuberculosis fusion antigen and its subunit vaccine, specifically to a fusion antigen containing latent infection and acute infection antigens, and its application in tuberculosis subunit vaccines. Background Technology

[0002] Tuberculosis is one of the leading killers affecting human health. According to the latest statistics from the World Health Organization, there were approximately 10.6 million new cases of tuberculosis and 1.6 million deaths worldwide in 2021. my country ranks third in the world in terms of tuberculosis burden, with approximately 780,000 new cases each year (1). It is estimated that one-quarter of the world's population are carriers of Mycobacterium tuberculosis, in a state of latent infection (2).

[0003] Studies have shown that after initial exposure to Mycobacterium tuberculosis, 30% of people will develop primary infection, of which 85% to 90% will develop asymptomatic latent tuberculosis infection (LTBI) (3). This population maintains a persistent Mycobacterium tuberculosis-specific immune response, which to some extent limits the replication and dissemination of Mycobacterium tuberculosis (4). Nevertheless, due to factors such as aging, decreased immunity, and human immunodeficiency virus (HIV) infection, 5%-10% of those with latent tuberculosis infection will develop active tuberculosis in their lifetime (5,6). A 2018 clinical cohort study showed that the probability of developing tuberculosis annually in those with latent tuberculosis infection is 8.8 times higher than in those without infection (7). Therefore, the large population of those with latent tuberculosis infection (approximately 2 billion people) is a major source of active tuberculosis, and vaccines / treatments targeting those with latent infection can help control the tuberculosis epidemic more effectively.

[0004] Currently, the only vaccine for preventing tuberculosis is Bacille Calmette-Guérin (BCG). Infant BCG vaccination is effective in preventing invasive childhood tuberculosis: meningeal tuberculosis and miliary tuberculosis (8). However, with age, the immune memory induced by BCG gradually fades, resulting in insufficient protection against adult tuberculosis (9). Adult revaccination of BCG poses a significant safety risk. Therefore, new tuberculosis vaccines are needed to boost BCG immunization. Currently, most new tuberculosis subunit vaccines that have entered clinical trials are based on antigens expressed by Mycobacterium tuberculosis during the replication phase (10). These vaccines are beneficial in preventing new tuberculosis infections, but they cannot eliminate established latent infections or prevent their recurrence (11,12). The addition of latent period-associated antigens helps overcome this deficiency (13,14). Currently, research institutions have constructed multiple polyphasic protein vaccines containing latent period-associated antigens, which have shown immunoprotective effects in animal models, and some have entered clinical trials (13). However, after latent infection is established, Mycobacterium tuberculosis mainly resides in lung tissue (15,16), and systemic vaccination with protein vaccines cannot induce sufficiently effective immune memory in the lung mucosa. Therefore, the successful development of a truly effective vaccine targeting latent tuberculosis infection remains a long and arduous task.

[0005] Therefore, developing novel tuberculosis subunit vaccines with a fresher approach is of great significance for enhancing their effectiveness in clinical application. On the one hand, it is necessary to screen for novel antigens with higher levels of protective efficacy (Mycobacterium tuberculosis has about 4,000 proteins), especially latent infection antigens or combinations of antigens, in order to induce a more effective antigen-specific immune response. On the other hand, it is necessary to select appropriate vaccine formulations based on this, in order to induce effective immune surveillance in lung tissue, the site where Mycobacterium tuberculosis resides for a long time. Summary of the Invention

[0006] The technical problem to be solved by this invention is:

[0007] On the one hand, latent infection antigens with high levels of anti-tuberculosis infection protection efficacy were screened and validated using mouse infection models; on the other hand, a recombinant subunit vaccine containing the fusion antigen of the acute and latent infection phases of Mycobacterium tuberculosis was constructed, and the vaccine's immunoprotective efficacy was validated in acute infection mouse models and latent infection mouse models, respectively.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] The present invention provides a fusion antigen comprising latent infection and acute infection antigens of Mycobacterium tuberculosis, wherein the fusion antigen comprises Rv2029c, Rv2028c, Rv3126c and Ag85A.

[0010] The fusion antigens provided by this invention comprise antigen combinations in which the full-length or partial sequences of Rv2029c, Rv2028c, Rv3126c and Ag85A proteins are fused in various sequences and linkages, including but not limited to A986 (sequence No. 1, containing Ag85A, Rv2029c, Rv2028c, and Rv3126c in sequence), 986A (sequence No. 2, containing Rv2029c, Rv2028c, Rv3126c, and Ag85A in sequence), and 896A (sequence No. 3, containing Rv2028c, Rv2029c, Rv3126c, and Ag85A in sequence), etc.

[0011] The present invention also provides a tuberculosis subunit vaccine comprising the above-mentioned fusion antigen, including recombinant viral vector vaccines, protein vaccines, peptide vaccines and nucleic acid vaccines.

[0012] The present invention also provides the use of the tuberculosis subunit vaccine in the preparation of vaccines for the prevention of acute tuberculosis and post-exposure tuberculosis vaccines for latent infection and / or in the preparation of novel vaccines for the prevention or treatment of other mycobacterial infection-related diseases.

[0013] The aforementioned uses can be achieved by using fusion antigen and subunit vaccines alone, or in combination with other tuberculosis vaccines.

[0014] The subunit vaccines are administered via intramuscular injection, subcutaneous immunization, and inhalation.

[0015] First, this invention screened nine latent tuberculosis infection-associated antigens as potential candidate antigens. By constructing a recombinant DNA vaccine, three antigens (Rv2029c, Rv2028c, and Rv3126c) were selected based on their immunotherapeutic effect against acute tuberculosis infection in mice and their high reactivity in latently infected individuals. Second, we constructed a fusion antigen expressing the three latent-phase antigens and one acute-phase antigen, Ag85A, designated "986A". Based on this, a recombinant subunit vaccine expressing this fusion antigen was constructed, named SeV986A. The gene and protein sequences of the antigen combination "986A" are provided in the attached sequence listing. Subsequently, in mouse models, the dosage, titer, and injection site of SeV986A were optimized, and its immunogenicity in BCG-immunized and unimmunized mice was determined. Finally, using mouse models of acute and latent tuberculosis infection, we verified the immunoprotective efficacy of SeV986A against tuberculosis infection.

[0016] The beneficial effects of this invention: Safe and effective vaccination is usually the most important means of controlling infectious diseases. Currently, the only vaccine for preventing tuberculosis is the BCG vaccine, but this vaccine is only effective in children and provides insufficient protection for adults. There is an urgent need to develop new tuberculosis vaccines to supplement the BCG vaccine. Furthermore, the large population with latent tuberculosis infection (approximately 2 billion people) is the main source of active tuberculosis; however, there is currently a lack of tuberculosis vaccines targeting this latent infection population. Therefore, implementing booster immunization with a novel tuberculosis vaccine targeting latent infection in the eligible BCG-immunized population is expected not only to achieve protective effects by enhancing the gradually weakening immune response after BCG vaccination, but also to help prevent the recurrence of latent tuberculosis infection. Therefore, this research has significant strategic importance and practical social benefits in global tuberculosis control efforts. Attached Figure Description

[0017] Figure 1 This shows detailed information on the encoding genes of the nine latent infection-related candidate antigens screened in Example 1.

[0018] Figure 2 In Examples 2 and 3, the latent infection antigens of tuberculosis were screened for protective efficacy using a mouse DNA vaccine treatment model of tuberculosis infection and a clinical cohort, respectively.

[0019] Figure 3 Example 4 illustrates the method of constructing the 986A chimeric gene, the protein expression of the recombinant subunit vaccine SeV986A, and the three-dimensional protein structure of 986A.

[0020] Figure 4 Example 4 illustrates a preliminary test to evaluate the protective efficacy of the 986A chimeric gene vaccine against acute tuberculosis infection.

[0021] Figure 5 Example 5 illustrates the optimization of the inoculation dose, titer, and route of SeV986A.

[0022] Figure 6 Example 6 illustrates the evaluation of SeV986A's ability to induce antigen-specific cellular immune responses in the lungs.

[0023] Figure 7 Example 6 shows a schematic diagram of flow cytometry gating detection.

[0024] Figure 8 Example 6 shows the detection of SeV986A-induced IL-17 response in the lungs and immune response in the spleen cells.

[0025] Figure 9 Example 7 demonstrates the efficacy of SeV986A vaccine-induced immune protection using a mouse acute infection model.

[0026] Figure 10 Example 8 demonstrates the efficacy of SeV986A vaccine-induced immune protection using a mouse latent infection model. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0028] Example 1: Screening for candidate antigens for latent tuberculosis infection based on literature reports or predictions.

[0029] 1. Candidate antigens were screened based on the high reactivity reported in the literature among people with latent tuberculosis infection.

[0030] 2. Based on the structural prediction of antigens, nine latent tuberculosis infection-related antigens were screened as candidate antigens.

[0031] 3. For detailed information on candidate antigens, please see [link / reference]. Figure 1 .

[0032] Example 2: Validating the protective efficacy of nine candidate antigens using a mouse acute infection model.

[0033] 1. Based on the sequences of nine candidate latent infection antigens and acute infection antigen Ag85A, from Mycobacterium tuberculosis strain H37Rv...

[0034] The nucleic acid sequence was amplified and inserted into the DNA vaccine vector pVAX1 to prepare 10 recombinant DNA vaccines.

[0035] 2. Using an acute infection model in mice, BALB / c mice were immunized with three doses of recombinant DNA vaccine five weeks after infection with Mycobacterium tuberculosis, with each dose two weeks apart. Four weeks after the last immunization, colony smears were performed to identify the different strains of the vaccine.

[0036] The ability of the vaccine to resist tuberculosis infection was compared with that of rifampin, a first-line anti-tuberculosis chemical drug, as a positive control. Figure 2 A).

[0037] 3. The study results showed that five antigens could reduce the bacterial load in mouse lungs by more than 0.5 log, namely Rv3126c, Rv1813, Rv2031, Rv2028c, and Rv2029c. Figure 2 B).

[0038] Example 3: Verifying the human reactivity of three candidate latent infection antigens

[0039] 1. The clinical cohort used included 98 patients with active tuberculosis (ATB), 20 people with latent tuberculosis infection (LTBI), 30 healthy volunteers (HC), and 32 non-tuberculosis patients with other lung diseases (Non-TB), with whole blood collected intravenously.

[0040] 2. Latent infection antigens Rv2029c, Rv2028c, and Rv3126c, and the non-latent tuberculosis infection antigen ESAT-6 / CFP10 were used as negative controls. Whole blood samples from the above cohorts were stimulated with these antigens. The supernatant was collected by centrifugation after 48 hours and subjected to IFN-γ ELISA detection.

[0041] 3. The study results showed that after in vitro stimulation with Rv2029c, Rv2028c, and Rv3126c proteins, IFN-γ secretion in latently infected individuals was significantly higher than that in patients with active tuberculosis, healthy volunteers, and non-pulmonary tuberculosis patients with other lung diseases. Figure 2 CE). In contrast, latently infected individuals and tuberculosis patients showed comparable antigen-specific responses to the tuberculosis antigen ESAT-6 / CFP10, and significantly higher responses than healthy volunteers and non-pulmonary tuberculosis patients with other lung diseases. Figure 2 F). Therefore, the above data proves that latently infected individuals exhibit high reactivity to these three antigens.

[0042] Example 4: Construction and Validation of an Immunogen Combination Containing 3 Latent Infection Antigens and 1 Acute-Phase Dominant Antigen 1. The three latent infection antigens Rv2029c, Rv2028c, and Rv3126c, along with the acute-phase dominant antigen Ag85A, were ligated using HL5linker and named the chimeric 986A gene (sequence No. 1). The chimeric sequence is as follows: Figure 3 As shown in Figure A, the gene and protein sequences of chimeric 986A are shown in Sequence No. 1 of the sequence listing. The chimeric gene was constructed by linking the DNA sequences encoding Ag85A, Rv2029c, Rv2028c, and Rv3126c using the HL5 linker, and then constructing the chimeric gene using a standard cloning procedure.

[0043] 2. The three-dimensional structure of the 986A protein was predicted using the FastAF2 server by uploading the amino acid sequence encoded by the chimeric 986A gene. The accuracy of the predicted protein structure was measured by average accuracy, where structures with an average accuracy >70 were considered well-folded regions. The predicted structure was displayed using the PyMol program. Figure 3 C).

[0044] 3. Using pVAX1 as a DNA vaccine vector, a recombinant DNA vaccine pVAX1-986A expressing the chimeric 986A gene was constructed. The immunoprotective efficacy of this antigen combination was confirmed using an acute infection mouse model. Figure 4 AB).

[0045] 4. The DNA sequence of chimeric antigen 986A was inserted into the SeV vector to construct the SeV986A vaccine. The construction method is as follows:

[0046] The 986A chimeric gene was mixed with plasmids pGEM-L, pGEM-N, and pGEM-P, which cloned the SeVL, NP, and P / C genes downstream of the T7 promoter. The plasmid mixture was transfected into LLC-MK2 cells infected with the vaccine virus vTF7-3 containing the phage T7 RNA polymerase gene. Cell extracts from transfected LLC-MK2 cells were used to detect the expression of the chimeric antigen. Western blotting experiments using mouse anti-986A serum confirmed the expression of the chimeric 986A protein in lysates of LLC-MK2 cells infected with SeV986A. Figure 3 B).

[0047] Example 5: Optimization of SeV986A inoculation dosage, titer, and route.

[0048] 1. The immunization schedule for SeV986A was optimized. First, the number of vaccinations was optimized by administering 1, 2, and 3 doses, with each dose spaced 2 weeks apart, via intramuscular immunization of BALB / c mice at a dose of 1×10⁻⁶. 7 CIU (Cell Infectious Units) used PBS (Phosphate Buffered Saline) and the recombinant Sendai virus vector vaccine SeVS1 expressing irrelevant antigens as negative controls, and BCG as a positive control. Figure 5 A).

[0049] 2. For example Figure 5 As shown in Figure B, antigen-specific IFN-γ responses were detectable in all immunization groups. The response was highest with two doses of the vaccine, indicating that two doses were sufficient to produce an antigen-specific immune response.

[0050] 3. Try reducing the immunization dose to 2×10 6 CIU ( Figure 5 C), the results were found to be unsatisfactory. Figure 5 D), hint 1×10 7 CIU is superior.

[0051] 4. Finally, optimize the vaccination route and compare intramuscular injection and nasal drop administration. Figure 5 C), it was found that the two had similar immunogenicity ( Figure 5D). Considering the convenience of the vaccination method, subsequent studies chose the intramuscular injection route.

[0052] Example 6: SeV986A Immunogenicity Detection

[0053] 1. Considering that most people in the world have received BCG vaccination in infancy, we tested the efficacy of SeV986A as a booster vaccine for BCG, using PBS and the recombinant Sendai virus vector vaccine SeVS1 expressing irrelevant antigens as negative controls. Figure 6 A). Two weeks after the final dose of vaccine, the mice were euthanized, and the antigen-specific cellular immune response of lung cells was detected using enzyme-linked immunosorbent assay (ELISPOT) and flow cytometry.

[0054] 2. Lung tissue was aseptically collected and digested into a single-cell suspension. In the ELISPOT assay, stimulation with Ag85A, Rv2029c, Rv2028c, and Rv3126c proteins was used, with incubation in culture medium serving as a negative control. Results showed that mice immunized with SeV986A exhibited a certain degree of specificity in response to these single antigens, and SeV986A enhancement increased the BCG-induced Ag85A response by approximately 10-fold. Figure 6 B). Boosting immunization with SeV986A also enhances the strength of immunity against Rv2029c, Rv2028c, and Rv3126c. Figure 6 B).

[0055] 3. In flow cytometry analysis, the induction levels of antigen-specific IFN-γ, IL-2, and TNF-α positive T cells were compared among different vaccine groups. Figure 6 C), See the diagram of the gating mechanism for flow cytometry detection. Figure 7 Consistent with ELISPOT results, SeV986A, as a booster vaccine for BCG, significantly enhanced the response to Ag85A in vitro stimulation, and significantly increased the secretion of IFN-γ and TNF-α cytokines in CD4+ and CD8+ T cells. Figure 6 CD). In addition, SeV986A, as a booster vaccine for BCG, can significantly enhance the immune response of CD4+ T cells to antigen 986 stimulation, enhance the production of IFN-γ and IL-2 cytokines, and also increase the IFN-γ secretion level in CD8+ T cells (CD). Figure 6 EF). Furthermore, enhanced immunization with SeV986A also increased the secretion level of Ag85A-specific IL-17 to some extent, and in CD4+ T cells, a significantly higher IL-17 response was observed after stimulation with antigen 986. Figure 8 AB). ELISPOT( Figure 8 C) and flow cytometry ( Figure 8 DE also measured antigen-specific T cell immune responses in spleen cells, confirming that both BCG and SeV986A enhancement groups induced higher levels of Ag85A-specific CD4+ T cells. Figure 8 D), while the SeV986A enhancement group induced a stronger 986-specific CD4+ T cell response (D). Figure 8 E). These data collectively indicate that booster immunization with SeV986 significantly enhances BCG-induced antigen-specific CD4+ and CD8+ T cell responses.

[0056] Example 7: Verification of the protective efficacy of SeV986A using a mouse acute infection model

[0057] 1. To confirm the immunoprotective efficacy of this novel vaccine against acute tuberculosis infection, mice immunized with BCG were used to simulate adults who received BCG in infancy. These mice were then given two intramuscular immunizations of SeV986A, two weeks apart. Two weeks after the last immunization, the mice were challenged with tuberculosis aerosols and sacrificed five weeks later. PBS, SeVS1 expressing irrelevant antigens, BCG immunization alone, and SeV986A immunization alone served as controls. Figure 9 A).

[0058] 2. The study results showed that, compared with SeVS1, both BCG and SeV986A vaccination alone significantly reduced lung ( Figure 9 B) and spleen ( Figure 9 The amount of tuberculosis bacteria in C). Additionally, compared to BCG or SeV986A vaccination alone, the amount of bacteria in the lungs was further reduced in the BCG primiparous-SeV986A booster group. Figure 9 B). Furthermore, decreased inflammation levels were observed in acid-fast stained lung sections of mice in the BCG, SeV986A, and BCG primiparous-SeV986A booster groups. Figure 9 (D) This result indicates that immunization with the recombinant vaccine SeV986A alone can induce immune protection against tuberculosis infection and can enhance the immune protection of BCG as a booster vaccine.

[0059] Example 8: Verification of the protective efficacy of SeV986A using a mouse latent infection model

[0060] 1. To confirm the immunoprotective efficacy of this novel vaccine against latent tuberculosis infection, mice immunized with BCG were used to simulate adults who received BCG in infancy. Four weeks after BCG vaccination, mice were challenged with aerosols of Mycobacterium tuberculosis. After a four-week period to establish infection, the mice were given eight weeks of chemotherapy with the anti-tuberculosis chemotherapeutic drugs isoniazid and pyrazinamide in their drinking water to eliminate chemotherapy-sensitive bacteria and simulate the latent infection state in humans. Immediately after chemotherapy, mice were immunized with two doses of SeV986A to induce an antigen-specific immune response and inhibit the reactivation of lingering Mycobacterium tuberculosis. Four weeks after the last immunization, mice were treated with dexamethasone to suppress the host immune response, simulating the reactivation of Mycobacterium tuberculosis due to age-related immunodeficiency in latently infected individuals. Eight weeks later, lungs and spleens were homogenized and plated to assess residual Mycobacterium tuberculosis, and acid-fast staining of the lungs was performed to assess lung pathology (Figure 1).

[0061] 10A).

[0062] 2. Compared with PBS and the first-generation vaccine SeV85AB, SeV986A significantly reduced lung ( Figure 10 B) and spleen ( Figure 10 CFU in C). The expression profiles of four T cell exhaustion markers, CD38, LAG3, PD1, and TIM3, were assessed by flow cytometry. It was found that in tuberculosis-specific CD4+ T cells, the levels of these four T cell exhaustion markers were significantly reduced in the SeV986A immunized group. Figure 10 DG) indicates that T cell exhaustion in chronic latent infection is reduced after SeV986A inoculation. Furthermore, in acid-fast stained sections, the SeV986A group showed significantly less inflammatory infiltration and the lowest inflammation score (DG). Figure 10 (HI). Therefore, SeV986A significantly improved the ability of mice to resist latent tuberculosis infection.

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Claims

1. A fusion antigen comprising latent and acute infection antigens of Mycobacterium tuberculosis, said fusion antigen as shown in sequence No.

1.

2. A tuberculosis subunit vaccine comprising the fusion antigen of claim 1, including a protein vaccine or a peptide vaccine.

3. Use of the fusion antigen of claim 1 or the tuberculosis subunit vaccine of claim 2 in the preparation of vaccines for the prevention of acute tuberculosis and post-exposure tuberculosis vaccines for latent infection.

4. As described in claim 3, the fusion antigen and subunit vaccine can be used alone or in combination with other tuberculosis vaccines.

5. As described in claim 3, the subunit vaccine is administered via intramuscular injection, subcutaneous immunization, or inhalation.

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