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.