Abstract
Background:Vaccination is one of the effective measures to prevent latent tuberculosis infection (LTBI) from developing into active tuberculosis (TB). Applying bioinformatics methods to pre-evaluate the biological characteristics and immunogenicity of vaccines can improve the efficiency of vaccine development. Objectives: To evaluate the immunogenicity of tuberculosis vaccine W541 and explore the application of bioinformatics technology in tuberculosis vaccine research.Methods: This study concatenated the immunodominant sequences of Ag85A, Ag85B, Rv3407 , and Rv1733c to construct the W541 DNA vaccine. Then, bioinformatics methods were used to analyze the physicochemical properties, antigenicity, allergenicity, toxicity, and population coverage of the vaccine, identify its epitopes, and perform molecular docking with MHC alleles and Toll-like receptor 4 (TLR4) of the host. Finally, the immunogenicity of the vaccine was evaluated through animal experiments.Results:the W541 vaccine protein is a soluble cytoplasmic protein with a half-life of 1.1 hours in vivo and an instability index of 45.37. It has good antigenicity and wide population coverage without allergenicity and toxicity. It contains 138 HTL epitopes, 73 CTL epitopes, 8 linear and 14 discontinuous epitopes of B cells, and a strong affinity for TLR4. Immune simulations showed it could effectively stimulate innate and adaptive immune responses. Animal experiments have confirmed that the W541 DNA vaccine could effectively activate the Th1- and Th17-type immune responses, producing high levels of IFN-γ and IL-17A, but could not significantly increase antibody levels. Conclusion: the W541 DNA vaccine can induce strong cellular immune responses. However, further optimization of the vaccine design is needed to make the expressed protein more stable in vivo. Bioinformatics analysis could reveal vaccines’ physicochemical and immunological information, which is critical for guiding vaccine design and development.
Keywords: Tuberculosis; DNA vaccine; Bioinformatic analysis; Simulated immunization; Immunogenicity
1. Introduction
Latent tuberculosis infection (LTBI) is characterized by the presence of specific immune responses to Mycobacterium tuberculosis(M.tb ) previously infected without clinical evidence of active tuberculosis (TB)(1). Currently, about 23% of the world’s population is in an LTBI state, in which 5-15% of those with LTBI may develop into active TB in their lifetime; LTBI has become an essential source of active TB(2). According to the 2015 WHO Guidelines for the Management of Latent Tuberculosis Infection, individuals with LTBI can take anti-TB drugs to avoid developing active TB(2). Considering that LTBI has no clinical symptoms, it appears that vaccination-based preventive treatment is more acceptable than chemotherapy. However, the Bacillus Calmette-Guérin (BCG) vaccine, widely used for tuberculosis prevention, has a poor preventive effect on LTBI(3). M72/AS01E, which was in phase IIb clinical trial and developed by GlaxoSmithKline Plc., has only a 54.0% protective efficacy against LTBI developing into active pulmonary TB(4). The phase III clinical trial of the M.vaccae vaccine produced by Anhui ZhiFeiLongKeMa Biopharmaceutical Co., Ltd showed a protective efficacy of 54.7% against LTBI(5). These data suggest that developing an effective LTBI preventive and therapeutic vaccine has broad prospects.
According to research reports, the Ag85 complex is the main secretory protein ofM.tb , consisting of three proteins: Ag85A, Ag85B, and Ag85C. It accounts for 30% of the total secreted protein In the M.tb H37Rv strain and can be isolated from early cultures. It has mycobacterial acid transferase activity, allowing trehalose to transfer and deposit on the cell wall ofM.tb , playing an essential role in the final stage of M.tbcell wall synthesis(6, 7). Ag85A and Ag85B contain multiple human T-cell epitopes, and CD4+ T cells from TB patients could respond to the whole Ag85A or Ag85B polypeptides to produce interferon-gamma (IFN-γ)(8). Our previous animal experimental studies(9-13)and the clinical trials reported(8, 13-25) have shown that Ag85A and Ag85B had high immunogenicity, could induce Th1-type responses and cytotoxic T lymphocytes, reduce bacterial loads in lung and other tissues, alleviate lung lesions, and had better protective or therapeutic effects on TB or mouse model with latent tuberculosis infection (LTBI). At present, many new TB vaccines internationally chose Ag85A and/or Ag85B protein as vaccine antigens(15, 16), among which multiple vaccines have entered clinical trials, such as AERAS-402 (including Ag85A, Ag85B, and TB10.4)(17), MVA85A \ Ad5Ag85A \ ChAdOx1 85A (all including Ag85A)(8, 13-20), TB/FLU-04L (including Ag85A and ESAT6)(21), GamTBvac (including Ag85A, ESAT6, and CFP10)(22), H1/IC31 (including Ag85B and ESAT6)(23), H4: IC31 (including Ag85B and TB10.4) [], H56: IC31 (including Ag85B, ESAT6, and Rv2660c )(24), AEC/BC02 (including Ag85B, ESAT6, and CFP10)(25).
Rv3407 is a protein consisting of 99 amino acids, specifically expressed during the M.tb transition from dormancy to reactivation. It may be a kind of antitoxin, only slightly expressed inM.tb virulent strains and not expressed in BCG strains(26-28). Schuck D et al. have revealed that the Rv3407 protein could induce abundant IFN-γ and robust Th1-type cell-mediated immune responses in individuals with LTBI and was notably deficient in active TB patients, indicating that the Rv3407protein may confer significant protection against dormant M.tbinfection in susceptible populations(29). Reece et al. engineered therv3407 gene into the BCG vaccine and immunized mice with the recombinant BCG vaccine, and found that this modified BCG vaccine stimulated high levels of IFN-γ production in mice and markedly enhanced protection against TB(30). Our research group also found through animal experiments that the mice immunized with the rv3407 DNA vaccine could produce higher levels of antigen-specific IFN-γ in the culture supernatant of splenic lymphocytes, had more Th1 cells and an increased Th1/Th2 cells ratio in the whole blood, could reduce the bacterial load in the lungs of mouse models with acute infection or LTBI, and alleviate the degree of lung lesions(31, 32).
Rv1733cis a major dormancy antigen highly expressed by latentM.tband can be well recognized by T cells from individuals with LTBI(33). Zhang W et al. immunized mice with a DNA vaccine encoding Rv1733cand exhibited higher splenocyte stimulation index and IFN-γ, IL-2, and IL-4 levels than those injected with saline(34). Our research group used animal experiments to compare the preventive and therapeutic effects of MTB ag85ab and 7 types of LTBI DNA vaccines on a mouse LTBI model, and it showed that the ag85ab, rv2659c , andrv1733c DNA vaccines reduced the bacterial load and degree of lung lesions in the mouse LTBI model(32). Additionally, Coppola M et al. immunized mice with synthetic Rv1733c long peptides (28 amino acid sequences located at positions 57-84, IPFAAAAGTAVQDSRSHVYAHQAQTRHP) and exhibited significantly increased expression of IFN-γ, TNF-α, and specific antibody, and reduced the pulmonaryM.tb load. The findings suggest that Rv1733c has the potential for the prevention or treatment of TB(33).
Based on this, we chose the full-length amino acid sequence of Ag85A and 308 amino acids of Ag85B as the vaccine backbone, added 51 amino acids ofRv3407 protein (including 15 amino acids at positions 16-30 and 36 amino acids at positions 61-96) and 28 amino acids of Rv1733cprotein (located at positions 57-84), and then concatenated them to construct a new TB DNA vaccine, named W541 based on the number of recombinant plasmids been constructed by our research group over the years, aiming to elicit synergistic protective immunity on TB and LTBI.
The development of bioinformatics and the application of big data analytics have provided convenient conditions for the design and development of vaccines, allowing researchers to have the opportunity to understand vaccine-related information in advance, thereby gaining a deeper understanding of vaccine characteristics and making corresponding optimizations to improve the efficiency of vaccine development (35-38). In this study, we used bioinformatics techniques to analyze various physicochemical properties and immunological characteristics of the W541 DNA vaccine. Then, we verified the immunogenicity of the W541 vaccine through animal experiments, exploring the feasibility of employing bioinformatics analysis methods as a means of preliminary assessment during tuberculosis vaccine development to aid vaccine research.
Material and method
The flow chart of the study design was shown in figure 1.