4. Discussion
In recent years, the rapid development of bioinformatics methods and the application of Internet databases have provided practical approaches for selecting protein-dominant epitopes and the pre-evaluation of vaccines (64-66), accelerating the vaccine research and development process. In this study, we employed bioinformatic methods to analyze the physicochemical properties, structure, safety, and immunological functions of a multi-stage DNA vaccine W541 constructed by tandem immunodominant sequences of ag85A, ag85B, Rv1733c , andRv3407 antigens. Furthermore, we evaluated its immunogenicity through a murine model to verify the results of bioinformatics analysis and lay a foundation for further optimization of the vaccine.
The bioinformatic analysis revealed that the W541 vaccine protein was a soluble protein with a molecular weight of 74 kDa, which exhibited excellent antigenicity and broad population coverage without allergenicity or toxicity. These fundamental data strongly support the suitability of W541 as a vaccine candidate based on the essential criteria.
There is a close relationship between the secondary structure of a protein and B-cell epitopes. The structures of irregular coiling and β-turns, being more loosely arranged, are prone to distortion and spiralization, leading to their exposure to the protein surface. These regions typically harbor a greater abundance of B-cell epitopes. The predictive analysis revealed that within the W541 vaccine protein, the sequence length of irregular coiling and β-turns constitutes 53.98% of the total sequence length, most located on the surface of protein molecules. Furthermore, the protein harbors 8 linear B-cell epitopes and 14 discontinuous epitopes. These structures and epitopes provide the foundation for eliciting effective humoral immune responses in the host. The immune stimulation in silico revealed that the W541 vaccine protein could induce a heightened antibody response. However, in animal experiments, most mice in the W541 DNA vaccine group did not exhibit a significant increase in antibody levels. This phenomenon may be attributed to the subcellular localization (in the cytoplasm and lysosomes) and poor stability of the W541 protein, which consequently affects the vaccine’s efficacy in eliciting a robust humoral immune response. The W541 vaccine protein expressed in the cytoplasm and lysosomes lost the opportunity to bind to BCR. In addition, it had a short half-life and poor stability, indicating that the vaccine protein is prone to degradation, leading to loss of conformational epitopes, which is detrimental to B cell activation and antibody production(65). Emerging evidence suggests that B cells and humoral immunity can regulate various immune responses of the intracellular pathogen M. tuberculosis (67). In addition, unstable proteins may undergo degradation before being captured by macrophages and DC cells, compromising their antigen presentation effectiveness. Ultimately, this could also impact the activation of TH cells (66). Therefore, optimizing the vaccine by increasing signal peptides to alter the subcellular localization of vaccine proteins or optimizing sequences to enhance their stability may help improve the immunogenicity of the vaccine (68-70). Studies have shown that lysine residues exposed on protein surfaces can bind to ubiquitin and promote protein degradation by proteases (71, 72). Additionally, the glycine, lysine, arginine, and cysteine residues at the N-terminus and middle portion of proteins have marked effects on protein stability (73). In the future, we will focus on fine-tuning the structure of the W541 vaccine to enhance the immunogenicity of the vaccine protein.
TLRs play a crucial role in activating the anti-TB immune responses, and TLR agonists are considered a promising class of vaccine adjuvants. TLR4 is an essential receptor involved in MTB recognition within cells, expressed in both immune and non-immune cells, and its structure includes the extracellular domain, transmembrane domain, and intracellular domain(74). TLR4 forms homodimers to recognize pathogen-associated molecular patterns such as lipopolysaccharides (LPS), lipoteichoic acid (LTA), dsRNA, etc. After TLR4 activation, it can initiate innate immune responses and regulate the migration, maturation, and function of antigen-presenting cells while facilitating adaptive immune responses(75, 76).M.tbdormancy-related proteins Rv2659c and Rv1738 can mediate the production of inflammatory cytokines through the TLR4 pathways(77). Furthermore, the activated TLR4 can limit the survival of M.tb by inducing cellular autophagy(78). These studies demonstrate the beneficial inclusion of TLR4 agonists in the design of TB vaccines. The relatively stable binding between TLR and ligand is fundamental for TLR activation. Molecular docking simulations in this study have demonstrated that the expressed W541 protein in cells can establish stable interactions with TLR4, thereby possessing the potential to activate TLRs. Simulation immune results also proved that the W541 vaccine can effectively trigger the body’s innate immune responses.
The prominent immune characteristics of TB patients include impaired Th1 cell-mediated immune function or imbalanced Th1/Th2 cell immune responses, which represent the primary risk factors of TB(79, 80). Hence, effectively activating the host’s Th1-type cell-mediated immune response is essential for TB vaccines to exert their protective effects against M.tb (81). IFN-γ plays a crucial role in the defense against M.tb infection by promoting the proliferation and differentiation of Th0 cells into Th1 cells and activating macrophages(82). The bioinformatics analysis of this study showed that the W541 vaccine protein was an antigen with a majority of T-cell epitopes, containing a total of 138 HTL epitopes, in which 101 HTL epitopes could induce the production of IFN-γ. In addition, it also included a smaller number of HTL epitopes that could induce the production of IL-4, IL-6, and IL-10. The simulated immunization with the W541 vaccine demonstrated that the W541 DNA vaccine could effectively activate TH cells and elicit a robust release of IFN-γ and small amounts of TGF-β, IL-10, and IL-12 brief secretion. In contrast, the secretion of other cytokines (including IL-4 and IL-6) was not observed. Animal experiments have confirmed that mice in the vaccine group exhibited significantly higher levels of IFN-γ secretion in spleen cells compared to the control group, and there was no significant increase in the secretion levels of Th2-type cytokines (IL-4, IL-6, and IL-10). These results collectively validate the consistency between most of the immunoinformatics analysis results and animal experimentation results of the vaccine, demonstrating a favorable structure-function relationship.IL2 is also a representative multifunctional cytokine mainly secreted by CD4+ T cells in the Th1-type immunity, which can activate T cells and promote cytokine production, activate macrophages, enhance the killing activity of NK cells, and promote the production of immunoglobulins by B cells, playing an essential role in the body’s anti-TB immunity. In this study, immunoinformatics prediction showed a significant increase in IL2, but animal experiments exhibited that the W541 immunization could not effectively induce the production of IL2. The possible reasons for this are: (1) W541 immunization expressed high levels of antigens in mice, mainly inducing effector memory T cells (TEM) and producing predominant high IFN- γ/ low IL-2 reaction. After weakened vaccine expression, it may mainly induce central memory T cells (TCM), possibly with a predominant low IFN- γ/ high IL2 reaction. (2) It may also be because mouse spleen cells were stimulated by the specific antigen W540 for a shorter time (24 hours), which generally requires culture for 72 hours to induce high levels of IL2 production(83). In addition, the experiment results showed that the level of IL-17A in the vaccine group was significantly higher compared to the control group. IL-17A was produced by activated T cells and mediated the production of inflammatory molecules, chemokines, antimicrobial peptides, and remodeling proteins (84), playing an essential role in the immune response to M.tb (85). The W541 vaccine protein contains 138 CTL epitopes, which can effectively activate cytotoxic T cells, confirmed in immune simulations. Cytotoxic T cells are crucial in clearingM.tbinfection by promoting target cell apoptosis or clearing infected target cells and persisting bacteria through the perforin-granzyme pathway(86-89). Numerous HTL and CTL epitopes within the W541 vaccine protein have been experimentally validated. For instance, KLIANNTRV has been identified as an HLA-A2-specific CD8+ immunodominant antigen peptide (90), PBMCs from TB patients exhibit a strongly proliferative response to a peptide (DQSGLSVVMPVGGQSSFY) derived from Ag85(91).
5. Conclusions
In summary, although the results of bioinformatics analysis may not encompass all biological effects elicited by vaccines in vivo, the results of the existing analysis showed a strong positive correlation with animal experiments. The research findings indicate that the W541 DNA vaccine composed of antigen Ag85A, Ag85B, Rv3407 , andRv1733c sequences contains a large number of HTL and CTL epitopes, which can activate TH cells and TC cells, mainly inducing Th1 and Th17 immune responses of the body. However, the protein expressed by the vaccine in vivo was not stable and could not effectively induce humoral immune responses. Therefore, we will optimize the vaccine design to address the issues with W541 and further evaluate the immunogenicity and protective efficacy of the vaccine in a mouse LTBI model.
Funding: This study was supported by a grant from the National Key R&D Program of China (2022YFA1303500-003).
Institutional Review Board Statement: The animal study was reviewed and approved by The Animal Ethical Committee of the Eighth Medical Center of the Chinese PLA General Hospital (Approved Number: 309202108250973).
Data Availability Statement:The original contributions presented in the study are included in the article and Supplementary Material. Further inquiries can be directed to the corresponding author.
Conflict of interest: The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.