Simulation immune results also proved that this W541 vaccine can effectively trigger the body’s innate immune responses. The prominent immune characteristics of TB patients include impaired Th1 cellmediated immune function or imbalanced Th1/Th2 cell immune responses, which represent the primary risk factors of TB [87,88]. 14 discontinuous B cell epitopes, and has a strong affinity for TLR4. Immune simulations have shown that it can effectively stimulate innate and adaptive immune responses. Animal experiments confirmed that this W541 DNA vaccine could effectively activate Th1 and Th17type immune responses, generating high levels of IFN and IL17A, 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 KPT276 stable in vivo. Bioinformatics analysis could reveal the physicochemical and immunological information of vaccines, which is critical for guiding vaccine design and development. Keywords:bioinformatic analysis, DNA vaccine, immunogenicity, simulated immunization, tuberculosis In this study, we construct the novel multistage DNA vaccine, W541, against Mycobacterium tuberculosis. Then, bioinformatics methods were applied to analyze various physicochemical properties and immunological characteristics of the vaccine. Finally, the immunogenicity of the vaccine was evaluated in animal experiments. The results suggested that the W541 DNA vaccine can induce strong cellular immune responses, and further optimization Mouse monoclonal to KT3 Tag.KT3 tag peptide KPPTPPPEPET conjugated to KLH. KT3 Tag antibody can recognize C terminal, internal, and N terminal KT3 tagged proteins KPT276 of this vaccine is possible. Bioinformatics analysis could reveal vaccines physicochemical and immunological information, which is critical for guiding vaccine design and development. == 1. Introduction == Latent tuberculosis infection (LTBI) is characterized by the presence of specific immune responses toMycobacterium tuberculosis(M. tuberculosis) in previously infected individuals without clinical evidence of active tuberculosis (TB) [1]. Currently, approximately 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 a major source of active TB [2]. According to the 2015 WHO Guidelines for the Management of Latent Tuberculosis Infection, people with LTBI can take antiTB drugs to prevent them from developing active TB [2]. Considering that LTBI has no clinical symptoms, vaccinationbased prophylaxis seems more acceptable than chemotherapy. However, the Bacillus CalmetteGurin (BCG) vaccine, widely used for tuberculosis KPT276 prevention, has a poor preventive effect on LTBI [3]. M72/AS01E, which was developed by GlaxoSmithKline Plc. and in phase IIb clinical trial, has a protective efficacy of only 54.0% against LTBI developing into active pulmonary TB [4]. The phase III clinical trial of theM. vaccaevaccine produced by Anhui ZhiFeiLongKeMa Biopharmaceutical Co., Ltd. showed a protective efficacy of 54.7% against LTBI [5]. These data suggest that developing an effective preventive and therapeutic vaccine against LTBI has broad prospects. Nucleic acid vaccines represent the third revolution in the history of vaccine development. Compared with other types of vaccines, DNA vaccines offer the following advantages [1]: They can induce a comprehensive immune response, activating both humoral and cellular immunity, particularly inducing the production KPT276 of cytotoxic T lymphocyte (CTL) responses that recognizeM. tbinfected cells and clear pathogens within them. This constitutes an effective pathway for eliminatingM. tblurking in macrophages, compensating for the weak CTL responses elicited by BCG vaccine, recombinant protein vaccines, and inactivated vaccines [2]. Vaccination with DNA vaccines is relatively safe, posing no risk of disease caused by bacterial virulence or residual virulent virus particles. It is safer for immunocompromised individuals than BCG [3]. Easy preparation and low production costs [4]. Plasmid DNA vaccines exhibit better stability and are easy to store and transport. Reports on the use of DNA vaccines to prevent or treat human diseases, such as AIDS [6], hepatitis B [7], hepatitis C [8], COVID19 [9], and tumor (including breast cancer, prostate cancer, and lymphoplasmacytic lymphoma) [10,11,12], among others, are gradually increasing. DNA vaccines KPT276 have emerged as a hot topic in vaccine research, but so far, there is no research.