Screening method for mRNA vaccine candidate antigen epitopes and application thereof

Abstract

This invention belongs to the field of bioimmunotechnology, specifically relating to a method and application for screening candidate antigen epitopes for mRNA vaccines that can effectively activate the body's immunity against tuberculosis infection. The screening method comprises the following steps: screening for population-specific tuberculosis protective antigens based on reverse prediction from a TCR immune repertoire; construction of mRNA vaccines using candidate antigens and identification of the optimal antigen; and evaluation of the in vivo immunogenicity, immunogenicity, and immunoprotective effects of the mRNA vaccine. This technical solution combines population-specific common HLA allele profiles with existing tuberculosis-specific TCR databases to target and predict broad-spectrum protective antigens recognized by TCRs shared under the major HLA backgrounds in the population.

Description

Technical Field

[0001] This invention belongs to the field of bioimmunotechnology, specifically relating to a method and application for screening candidate antigen epitopes for mRNA vaccines that can effectively activate the body's immunity against tuberculosis infection. Background Technology

[0002] Bacillus calmette-guerin (BCG) has been widely used worldwide for tuberculosis prevention since its discovery and is currently the only licensed tuberculosis vaccine. Although BCG is highly effective in preventing severe tuberculosis in children (such as tuberculous meningitis) (protection rate up to 80%), its protective efficacy declines sharply after puberty, offering little protection to adults, who account for over 90% of global tuberculosis cases. Adults are the primary source of tuberculosis transmission. To overcome the limitations of BCG, the global scientific community has long been committed to developing novel tuberculosis vaccines. Currently, 17 candidate vaccines are in different stages of clinical trials, covering various technical approaches including viral vector vaccines, whole cell / extract vaccines, recombinant subunit vaccines, recombinant BCG, and mRNA vaccines.13 Among them, the subunit vaccine M72AS01E, developed by GlaxoSmithKline, consists of two tuberculosis antigens, MTB39A and MTB32A, and is supplemented with the adjuvant AS01E. Phase II clinical trial results showed that it significantly reduced the incidence of pulmonary tuberculosis in HIV-negative adults with latent tuberculosis infection. Within three years of vaccination, its overall protective efficacy was 49.7%, making it the new vaccine with the closest protective efficacy to BCG. However, although the development of existing vaccines has made some progress, no effective vaccine that can completely replace BCG has yet been found. Furthermore, current vaccine development faces several core bottlenecks, such as the inability of traditional vaccines to activate a durable cellular immune response, the over-reliance of subunit vaccines on adjuvants, and the numerous safety concerns associated with recombinant BCG. Therefore, the discovery of new vaccines is urgently needed.

[0003] The development of tuberculosis vaccines faces numerous bottlenecks, with the core issue being the insufficient discovery of dominant neoantigens. Currently, antigen discovery heavily relies on a limited library of known antigens (such as Ag85 / ESAT-6 / CFP-10). However, the immune responses induced by existing antigens are insufficient in both breadth and persistence. Therefore, the discovery of dominant neoantigens is urgently needed. In recent years, the development of high-throughput sequencing technology, especially T-cell receptor (TCR) sequencing, has provided revolutionary tools for solving these problems. This technology can deeply analyze the in vivo TCR library after Mtb infection, directly reflecting the actual immune response profile of the host. Based on this, a new strategy of "reverse antigenic epitope prediction based on TCR sequencing" has emerged. This method uses the specific TCRs actually produced in individuals infected with Mycobacterium tuberculosis as "molecular probes" to predict their corresponding antigens. However, the extreme polymorphism of the HLA system limits the universality of antigens. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for screening candidate antigen epitopes for mRNA vaccines that can effectively activate the body's immunity against tuberculosis infection, so as to provide empirical evidence and solutions to overcome the bottlenecks of insufficient universality and protective efficacy of existing tuberculosis vaccines.

[0005] To achieve the above objectives, this application employs the following technical solution:

[0006] A method for screening candidate antigenic epitopes for mRNA vaccines, comprising the following steps:

[0007] S1. Screening of protective tuberculosis antigens in the population based on reverse prediction of TCR immune repertoire;

[0008] S2. Construction of mRNA vaccines based on candidate antigens and identification of the optimal antigen;

[0009] S3. Evaluation of the in vivo immunogenicity, immunogenicity, and immunoprotective effect of the mRNA vaccine.

[0010] Furthermore, in step S1, the screening of population tuberculosis protective antigens based on reverse prediction from the TCR immune repertoire is as follows:

[0011] S11. Establish a localized TCR immune genome database;

[0012] S12. Identify shared specific clonal clusters and simultaneously predict potential HLA-restricted alleles for each TCR cluster;

[0013] S13. Screening recipient populations for common HLA-II class allele-restricted TCR clusters;

[0014] S14. By integrating TCR clustering, HLA restriction, and Mycobacterium tuberculosis whole genome peptide libraries, the precise antigenic epitopes targeted by TCR clusters are predicted in reverse, and a candidate antigenic epitope library is constructed.

[0015] Furthermore, in step S11, the tuberculosis TCR sequence dataset published in the study by Musvosvi et al. is obtained, and a localized TCR immune repertoire database is established.

[0016] Furthermore, in step S12, the GLIPH2 algorithm is used to perform similarity clustering on the TCR CDR3 region to identify shared specific clonal groups.

[0017] Furthermore, in step S13, based on the high-frequency HLA allele profile of the bone marrow donor program population, TCR clusters with common HLA-II class allele restriction in the recipient population are screened.

[0018] Furthermore, in step S14, the NetMHCIIpan tool is used to reverse predict the precise antigenic epitopes targeted by the TCR cluster.

[0019] Furthermore, the predicted protective antigenic epitopes will be used to construct novel thermostable mRNA vaccines based on the antigens screened above, thus preparing novel mRNA freeze-dried vaccines.

[0020] Furthermore, in step S3, the immunogenicity and protective efficacy of the antigen are rapidly evaluated through a mycobacterial growth inhibition test.

[0021] The application of an mRNA vaccine candidate antigen epitope, wherein the antigen selected by any of the above methods activates the host's immune response to resist Mycobacterium tuberculosis infection.

[0022] The beneficial effects of this invention are:

[0023] This technical solution combines population-specific common HLA allele profiles with existing tuberculosis-specific TCR databases to target and predict broad-spectrum protective antigens recognized by TCRs shared in the population's major HLA background. Attached Figure Description

[0024] Figure 1 This invention enables the successful expression of the Mycobacterium tuberculosis-specific protein Rv2351c in the lungs of mice immunized with an mRNA vaccine.

[0025] Figure 2 The bacterial load in lung tissue of mice immunized with mRNA vaccine 14 days after infection with Mycobacterium tuberculosis. Detailed Implementation

[0026] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, and should not be construed as limiting the technical solution of the present invention.

[0027] This technical approach combines population-specific common HLA allele profiles with existing tuberculosis-specific TCR databases to target and predict broad-spectrum protective antigens recognized by TCRs shared within the predominant HLA background of the population. LNP-mRNA vaccine technology has been established as an ideal platform for delivering these newly discovered advantageous antigens.

[0028] This application provides a method for screening candidate antigenic epitopes for mRNA vaccines, comprising the following steps:

[0029] S1. Screening of protective antigens for tuberculosis in the population based on reverse prediction of TCR immune repertoire.

[0030] The steps for screening for protective tuberculosis antigens in the population based on reverse prediction from the TCR immune repertoire are as follows:

[0031] S11. Establish a localized TCR immune genome database;

[0032] S12. Identify shared specific clonal clusters and simultaneously predict potential HLA-restricted alleles for each TCR cluster;

[0033] S13. Screening recipient populations for common HLA-II class allele-restricted TCR clusters;

[0034] S14. By integrating TCR clustering, HLA restriction, and Mycobacterium tuberculosis whole genome peptide libraries, the precise antigenic epitopes targeted by TCR clusters are predicted in reverse, and a candidate antigenic epitope library is constructed.

[0035] In step S11, the tuberculosis TCR sequence dataset published in the study by Musvosvi et al. (Nature Medicine 2023) is obtained, and a localized TCR immune repertoire database is established.

[0036] In step S12, the GLIPH2 algorithm is used to perform similarity clustering on the TCR CDR3 region to identify shared specific clonal groups.

[0037] In step S13, based on the high-frequency HLA allele profile (CWD) of the bone marrow donor program (CMDP), TCR clusters with common HLA class II allele restriction in the recipient population are screened.

[0038] In step S14, the NetMHCIIpan tool is used to reverse predict the precise antigenic epitopes targeted by the TCR cluster.

[0039] S2. Construction of mRNA vaccines based on candidate antigens and identification of the optimal antigen.

[0040] The predicted protective antigenic epitopes were used to construct novel thermostable mRNA vaccines based on the antigens screened above, and novel mRNA freeze-dried vaccines were prepared.

[0041] S3. Evaluation of the in vivo immunogenicity, immunogenicity, and immunoprotective effect of the mRNA vaccine.

[0042] The immunogenicity and protective efficacy of the antigen can be rapidly evaluated using the mycobacterial growth inhibition assay.

[0043] This application also relates to the application of an mRNA vaccine candidate antigen epitope, wherein the antigen selected by any of the above methods activates the host immune response to resist Mycobacterium tuberculosis infection.

[0044] like Figure 1 and Figure 2 As shown, an animal immunization model was established in C57BL / 6 mice using low-dose nebulized Mtb H37Rv infection. The immunoprotective efficacy of the vaccine was determined by flow cytometry, tissue HE staining, mycobacterial growth inhibition assay, and organ colony forming units (CFU) counting. The specific steps are as follows:

[0045] (1) Further screening of antigens based on in vivo experiments: After immunization, mice were sacrificed, and the inhibition rate of Mtb H37Rv by spleen mononuclear cells was measured by the Mycobacterium growth inhibition assay (MGIA). Antigens corresponding to mRNA vaccines with high inhibition rates were screened, and the above antigens were prepared into a multi-target antigen combination to prepare mRNA vaccines for subsequent vaccine evaluation. Finally, it was found that the Rv2351c protein had the highest inhibition rate.

[0046] (2) Evaluation of the immunoprotective effect of the novel mRNA vaccine: 14 days after the last immunization, a low-dose MtbH37Rv infection model was established using a nebulized infection system. Mice were sacrificed for H&E staining of lung tissue and detection of serum inflammatory factors. Mice in each group that were immunized and nebulized were sacrificed on the 14th day after infection. Splenic mononuclear cell suspensions were prepared and analyzed by flow cytometry. At the same time, CFU in the lungs of mice were selected for post-infection tissue detection to assess bacterial load.

[0047] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method of screening for mRNA vaccine candidate epitopes, characterized in that, The following steps are used: S1. Screening of protective tuberculosis antigens in the population based on reverse prediction of TCR immune repertoire; S2. Construction of mRNA vaccines based on candidate antigens and identification of the optimal antigen; S3. Evaluation of the in vivo immunogenicity, immunogenicity, and immunoprotective effect of the mRNA vaccine.

2. The method for screening candidate antigenic epitopes for mRNA vaccines according to claim 1, characterized in that, In step S1, the screening steps for population tuberculosis protective antigens based on reverse prediction from the TCR immune repertoire are as follows: S11. Establish a localized TCR immune genome database; S12. Identify shared specific clonal clusters and simultaneously predict potential HLA-restricted alleles for each TCR cluster; S13. Screening recipient populations for common HLA-II class allele-restricted TCR clusters; S14. By integrating TCR clustering, HLA restriction, and Mycobacterium tuberculosis whole genome peptide libraries, the precise antigenic epitopes targeted by TCR clusters are predicted in reverse, and a candidate antigenic epitope library is constructed.

3. The method for screening candidate antigenic epitopes for mRNA vaccines according to claim 2, characterized in that, In step S11, the tuberculosis TCR sequence dataset published in the study by Musvosvi et al. is obtained, and a localized TCR immune repertoire database is established.

4. The method for screening candidate antigenic epitopes for mRNA vaccines according to claim 2, characterized in that, In step S12, the GLIPH2 algorithm is used to perform similarity clustering on the TCR CDR3 region to identify shared specific clonal groups.

5. The method for screening candidate antigenic epitopes for mRNA vaccines according to claim 2, characterized in that, In step S13, based on the high-frequency HLA allele profile of the bone marrow donor program population, TCR clusters with common HLA-II class allele restriction in the recipient population are screened.

6. The method for screening candidate antigenic epitopes for mRNA vaccines according to claim 2, characterized in that, In step S14, the NetMHCIIpan tool is used to reverse predict the precise antigenic epitopes targeted by the TCR cluster.

7. The method for screening candidate antigenic epitopes for mRNA vaccines according to claim 1, characterized in that, The predicted protective antigenic epitopes were used to construct novel thermostable mRNA vaccines based on the antigens screened above, and novel mRNA freeze-dried vaccines were prepared.

8. The method for screening candidate antigenic epitopes for mRNA vaccines according to claim 1, characterized in that, In step S3, the immunogenicity and protective efficacy of the antigen are rapidly evaluated by mycobacterial growth inhibition assay.

9. The application of a candidate antigen epitope for an mRNA vaccine, characterized in that, The antigen selected by any one of claims 1 to 8 above activates the host's immune response to resist Mycobacterium tuberculosis infection.

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