Introduction
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) invasion, which is one of the main causes of death from infectious diseases and the leading cause of death from drug resistance (1). According to the Global Tuberculosis Report 2022 published by World Health Organization (WHO), there were about 10.6 million new cases, 1.6 million dead cases of TB, and 450,000 multidrug-resistant/rifampicin-resistant TB (MDR/RR-TB) cases worldwide in 2021(1). It can be seen that TB remains a major infectious disease threatening human health. The diagnosis and treatment of MDR/RR-TB is a major clinical problem. The global pandemic COVID-19 has also brought great challenges to the prevention and control of TB(2).
TB is not only a bacterial infectious disease but also an immune disease(3). The occurrence and development of TB are closely related to immunodeficiency(4, 5), imbalance of Th1/Th2 immune response(6), and hypoimmunity(7-9). Chemotherapy only with antituberculosis drugs needs 6-9 months or even longer to kill the vast majority of MTB in the lesion(10, 11). However, there may still be a small amount of persisting MTB in vivo, especially in macrophages, which is difficult to remove and becomes a ”time bomb” for TB recurrence(12). Antituberculosis immunoadjuvant therapy with immunomodulators has great potential in preventing latent MTB reactivation and treating active TB patients(13). It can correct low or abnormal immune function, inhibit the adverse immune response and inflammatory injury, and improve the immune function and curative effect. In recent years, immunoadjuvant therapy for TB has made great progress. Some immunomodulators have entered clinical trials or been marketed, mainly including immunoactive substances, immunotherapeutic vaccines, chemical agents(14), traditional Chinese medicine, and cell therapy (13).
TB immunotherapeutic vaccine is to regulate or selectively induce the potential of the immune system of MTB-infected people, to achieve the purpose of suppressing immune damage, recovering immune balance, improving immunity, and inhibiting or killing MTB in vivo(15). It is mainly used to prevent individuals with latent tuberculosis infection (LTBI) from turning into active TB or help active TB patients recover faster. Using a vaccine for the prophylactic treatment of high-risk populations with MTB infection is simple, convenient, economical, and has few side effects(16). At present, there are the following types of TB therapeutic vaccines: (1) Inactivated vaccines: Of the TB inactivated vaccines prepared from non-tuberculous Mycobacteria ,Vaccae (Prepared from inactivated Mycobacterium vaccae ) (17) and Utilins (18) (prepared from inactivated Mycobacterium phlei ) have obtained new drug certificates in China. DAR-901(18) (SRL172, prepared from inactivated M. kyogaense ) and MIP(19) (prepared from inactivated M.indicus pranii ) have entered clinical trials. (2) Subunit vaccine: Of the subunit vaccines prepared from some cell components of the MTB complex, BCG polysaccharide and nucleic acid injection (trade name Siqikang) has obtained a new drug certificate in China (18); RUTI (prepared from MTB H37Rv cultured under low oxygen, low pH and low nutrient conditions by crushing, detoxification and then embedding in liposomes) (18), and 4 recombinant protein vaccines (M72/AS01E, H56/IC31, ID93/GLA-SE, and AEC/BC02) have entered Phase I or II clinical trials(20-22); (3) DNA vaccine: Of the DNA vaccines constructed from the genes encoding MTB antigen and eukaryotic expression vectors, only Korean GX-70 (composed of 4 MTB antigen plasmids and Flt3 ligand) has entered phase I clinical trial (ClinicalTrials.gov Identifier: NCT03159975), but this study has been withdrawn. It is proved that DNA vaccine could provide remarkable protective efficacy and strong therapeutic effect on mouse MDR-TB models(23-25).
MTB Ag85A and Ag85B are secreted proteins and antigens recognized by host innate immune cells at an early stage, with good immunogenicity. However, the adaptive immune response in the mouse lungs arrests the proliferation of MTB and results in a 10 to 20-fold reduction in the mRNA expression of the secreted Ag85 complex(26, 27). The down-regulation of gene expression significantly reduces the frequency of Ag85A/Ag85B-specific CD4+ effector T cells activated during the MTB infection. Therefore, the ag85 antigens have become popular candidate targets for developing new TB vaccines(22). Our previous studies have demonstrated that the ag85a/b chimeric DNA vaccine could induce significant Th1 and CTL cellular immune responses, relieve lung tissue lesions, reduce the bacterial load in organs, and have a significant treatment effect on MTB-infected mice (28). To solve the problem of relatively low immunogenicity of DNA vaccines and the need for very high doses in large animal and human clinical trials(29, 30), our team used electroporation (EP) technology to deliver different doses of MTB ag85a/b chimeric DNA vaccine and compared their immunotherapeutic effect with traditional intramuscular injection (IM). The results showed that EP immunization can improve the immunogenicity of low-dose DNA vaccines and reduce the amount of plasmid DNA used. The therapeutic effect of the 50μg DNA EP group on the mouse TB model had no significant difference with the 100μg DNA IM group. They all could significantly reduce the bacterial load of the lung and spleen, and lung lesion area, resulting in a good immunotherapeutic effect (31).
At present, the pathogenesis of MTB and the interaction between MTB and host have not been fully elucidated, which is a challenge to the research and development of an effective vaccine for TB. After the DNA vaccine is expressed in vivo, the correlation and mechanism of its inducing protective immunity have also not been completely determined. First, we need to understand the interaction between the DNA vaccine and the host, the key anti-TB targets of the proteins expressed by the DNA vaccine, and the body’s multiple anti-TB systems regulated by the DNA vaccine. Second, it is necessary to deeply understand the protective immune response of DNA vaccine in TB treatment, determine whether it can repair the pathological damage caused by MTB infection, help to inhibit and eliminate MTB, and find out what indexes is helpful to evaluate the effectiveness of the new TB vaccine. Third, we need to understand the possibility of DNA vaccine inducing pathological immune responses to determine the risk of possible adverse reactions to the vaccine. In recent years, the development of the frontier disciplines of systems biology has provided a powerful tool for the study of the pharmacological mechanism of vaccines(32-34). Therefore, this study used gene chip technology to obtain the gene expression profiles of experimental animals, and used bioinformatics methods to identify the differential expression levels of genes from mouse peripheral blood mononuclear cells (PBMCs) before and after MTB infection and before and after ag85a/b DNA vaccine treatment. This is the first attempt to analyze the pathogenic targets of MTB and the therapeutic targets ofag85a/b DNA vaccine at the level of gene transcription, and then to elaborate the molecular mechanism of DNA vaccine in regulating disease network and playing the role of anti-TB by combining pathway analysis and functional analysis, etc. At the same time, we analyzed whether exists differences in the effective dose, action target, and action mechanism of the two DNA immunization methods by comparing the differentially expressed (DE) genes before and after immunotherapy with different doses of ag85a/b DNA IM and EP. In addition, the immune characteristics of the ag85a/b DNA vaccine were verified through animal experiments, and the protective immune response of the vaccine was analyzed by comparing the therapeutic effects. Finally, the expression levels of 3 MTB pathogenic target genes found in this study were verified in TB patients by real-time reverse transcription-quantitive polymerase chain reaction (RT-qPCR) to determine the reliability of the gene expression profiling results. In addition, we downloaded gene expression datasets from the GEO database to compare with our expression profile results. The same MTB pathogenic target genes and therapeutic target genes were screened to verify our expression profile results.