Abstract
Background:Vaccination is one of the effective measures to prevent latent
tuberculosis infection (LTBI) from developing into active tuberculosis
(TB). Applying bioinformatics methods to pre-evaluate the biological
characteristics and immunogenicity of vaccines can improve the
efficiency of vaccine development. Objectives: To evaluate the
immunogenicity of tuberculosis vaccine W541 and explore the application
of bioinformatics technology in tuberculosis vaccine research.Methods: This study concatenated the immunodominant sequences
of Ag85A, Ag85B, Rv3407 , and Rv1733c to construct the W541
DNA vaccine. Then, bioinformatics methods were used to analyze the
physicochemical properties, antigenicity, allergenicity, toxicity, and
population coverage of the vaccine, identify its epitopes, and perform
molecular docking with MHC alleles and Toll-like receptor 4 (TLR4) of
the host. Finally, the immunogenicity of the vaccine was evaluated
through animal experiments.Results:the W541 vaccine protein is a soluble cytoplasmic protein with a
half-life of 1.1 hours in vivo and an instability index of 45.37. It has
good antigenicity and wide population coverage without allergenicity and
toxicity. It contains 138 HTL epitopes, 73 CTL epitopes, 8 linear and 14
discontinuous epitopes of B cells, and a strong affinity for TLR4.
Immune simulations showed it could effectively stimulate innate and
adaptive immune responses. Animal experiments have confirmed that the
W541 DNA vaccine could effectively activate the Th1- and Th17-type
immune responses, producing high levels of IFN-γ and IL-17A, 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 stable in vivo. Bioinformatics analysis could
reveal vaccines’ physicochemical and immunological information, which is
critical for guiding vaccine design and development.
Keywords: Tuberculosis; DNA vaccine; Bioinformatic analysis;
Simulated immunization; Immunogenicity
1. Introduction
Latent
tuberculosis infection (LTBI) is characterized by the presence of
specific immune responses to Mycobacterium tuberculosis(M.tb ) previously infected without clinical evidence of active
tuberculosis (TB)(1). Currently,
about 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 an essential source of active TB(2).
According
to the 2015 WHO Guidelines for the Management of Latent Tuberculosis
Infection, individuals with LTBI can take anti-TB drugs to avoid
developing active
TB(2).
Considering that LTBI has no clinical symptoms, it appears that
vaccination-based preventive treatment is more acceptable than
chemotherapy. However, the Bacillus Calmette-Guérin (BCG) vaccine,
widely used for tuberculosis prevention, has a poor preventive effect on
LTBI(3). M72/AS01E, which was in phase IIb clinical trial and developed
by GlaxoSmithKline Plc., has only a 54.0% protective efficacy against
LTBI developing into active pulmonary TB(4). The phase III clinical
trial of the M.vaccae vaccine produced by Anhui ZhiFeiLongKeMa
Biopharmaceutical Co., Ltd showed a protective efficacy of 54.7%
against LTBI(5). These data suggest that developing an effective LTBI
preventive and therapeutic vaccine has broad prospects.
According to research reports, the
Ag85 complex is the main secretory protein ofM.tb , consisting of three
proteins: Ag85A, Ag85B, and Ag85C. It accounts for 30% of the total
secreted protein In the M.tb H37Rv strain and can be
isolated from early cultures. It has mycobacterial acid transferase
activity, allowing trehalose to transfer and deposit on the cell wall ofM.tb , playing an essential role in the final stage of M.tbcell wall synthesis(6, 7).
Ag85A
and Ag85B contain multiple human T-cell epitopes, and
CD4+ T cells from TB patients could respond to the
whole Ag85A or Ag85B polypeptides to produce interferon-gamma
(IFN-γ)(8).
Our previous animal experimental studies(9-13)and the clinical trials
reported(8, 13-25) have shown that Ag85A and Ag85B had high
immunogenicity, could induce Th1-type responses and cytotoxic T
lymphocytes, reduce bacterial loads in lung and other tissues, alleviate
lung lesions, and had better protective or therapeutic effects on TB or
mouse model with latent tuberculosis infection (LTBI). At present, many
new TB vaccines internationally chose Ag85A and/or Ag85B protein as
vaccine antigens(15, 16), among which multiple vaccines have entered
clinical trials, such as AERAS-402 (including Ag85A, Ag85B, and
TB10.4)(17), MVA85A \ Ad5Ag85A \ ChAdOx1
85A (all including Ag85A)(8, 13-20), TB/FLU-04L (including Ag85A and
ESAT6)(21), GamTBvac (including Ag85A, ESAT6, and CFP10)(22), H1/IC31
(including Ag85B and ESAT6)(23), H4: IC31 (including Ag85B and TB10.4)
[], H56: IC31 (including Ag85B, ESAT6, and Rv2660c )(24),
AEC/BC02 (including Ag85B, ESAT6, and CFP10)(25).
Rv3407 is a protein consisting of 99 amino acids, specifically
expressed during the M.tb transition from dormancy to
reactivation. It may be a kind of antitoxin, only slightly expressed inM.tb virulent
strains
and not expressed in BCG strains(26-28).
Schuck
D et al. have revealed that the Rv3407 protein could induce
abundant IFN-γ and robust Th1-type
cell-mediated immune responses in individuals with LTBI and was notably
deficient in active TB patients, indicating that the Rv3407protein may confer significant protection against dormant M.tbinfection in susceptible populations(29). Reece et al. engineered therv3407 gene into the
BCG
vaccine and immunized mice with the recombinant BCG vaccine, and found
that this modified BCG vaccine stimulated high levels of IFN-γ
production in mice and markedly enhanced protection against TB(30). Our
research group also found through animal experiments that the mice
immunized with the rv3407 DNA vaccine could produce higher levels
of
antigen-specific
IFN-γ in the culture supernatant of splenic lymphocytes, had more Th1
cells and an increased Th1/Th2 cells ratio in the whole blood, could
reduce the bacterial load in the lungs of mouse models with acute
infection or LTBI, and alleviate the degree of lung lesions(31, 32).
Rv1733cis a major dormancy antigen highly expressed by latentM.tband can be well recognized by T cells from individuals with LTBI(33).
Zhang W et al. immunized mice with a DNA vaccine encoding Rv1733cand exhibited higher splenocyte stimulation index and IFN-γ, IL-2, and
IL-4 levels than those injected with saline(34).
Our
research group used animal experiments to compare the preventive and
therapeutic effects of MTB ag85ab and 7 types of LTBI DNA vaccines on a
mouse LTBI model, and it showed that the ag85ab, rv2659c , andrv1733c DNA vaccines reduced the bacterial load and degree of
lung lesions in the mouse LTBI model(32). Additionally, Coppola M et al.
immunized mice with synthetic Rv1733c long peptides (28 amino
acid sequences
located
at positions 57-84, IPFAAAAGTAVQDSRSHVYAHQAQTRHP) and exhibited
significantly increased
expression
of IFN-γ, TNF-α, and specific antibody, and reduced the pulmonaryM.tb load. The findings suggest that Rv1733c has the
potential for the prevention or treatment of TB(33).
Based
on this, we chose the full-length amino acid sequence of Ag85A and 308
amino
acids of Ag85B as the vaccine backbone, added 51 amino acids ofRv3407 protein (including 15 amino acids at positions 16-30 and
36 amino acids at positions 61-96) and 28 amino acids of Rv1733cprotein (located at positions 57-84), and then concatenated them to
construct a new TB DNA vaccine, named W541 based on the number of
recombinant plasmids been constructed by our research group over the
years, aiming to elicit synergistic protective immunity on TB and LTBI.
The
development of bioinformatics and the application of big data analytics
have provided convenient conditions for the design and development of
vaccines, allowing researchers to have the opportunity to understand
vaccine-related information in advance, thereby gaining a deeper
understanding of vaccine characteristics and making corresponding
optimizations to improve the efficiency of vaccine development (35-38).
In this study, we used bioinformatics techniques to analyze various
physicochemical properties and immunological characteristics of the W541
DNA vaccine. Then, we verified the immunogenicity of the W541 vaccine
through animal experiments, exploring the feasibility of employing
bioinformatics analysis methods as a means of preliminary assessment
during tuberculosis vaccine development to aid vaccine research.
Material
and method
The flow chart of the study
design was shown in figure 1.