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.