Long-Term Immunogenicity and In Vitro Prophylactic Protective Efficacy of M. tuberculosis Fusion Protein DR2 Combined with Liposomal Adjuvant DIMQ as a Boosting Vaccine for BCG.

The resuscitation of dormant Mycobacterium tuberculosis is an important cause of adult tuberculosis (TB) transmission. According to the interaction mechanism between M. tuberculosis and the host, the latency antigen Rv0572c and region of difference 9 (RD9) antigen Rv3621c were selected in this study to prepare the fusion protein DR2. Stimulating clinically diagnosed active tuberculosis infections (i.e., TB patients), latent tuberculosis infections, and healthy controls confirmed that T lymphocytes could recognize DR2 protein in the peripheral blood of TB-infected individuals more than subcomponent protein. The DR2 protein was then emulsified in the liposome adjuvant dimethyl dioctadecyl ammonium bromide, and imiquimod (DIMQ) was administered to C57BL/6 mice immunized with Bacillus Calmette-Guérin (BCG) vaccine to evaluate their immunogenicity. Studies have shown that DR2/DIMQ, a booster vaccine for BCG primary immunization, can elicit robust CD4+ Th1 cell immune response and predominant IFN-γ+ CD4+ effector memory T cells (TEM) subsets. Furthermore, the serum antibody level and the expression of related cytokines increased significantly with the extension of immunization time, with IL2+, CD4+, or CD8+ central memory T cells (TCM) subsets predominant in the long term. This immunization strategy showed matched prophylactic protective efficacy by performing in vitro challenge experiment. This result provides robust evidence that the novel subunit vaccine prepared by fusion protein DR2 combined with liposomal adjuvant DIMQ is a promising TB vaccine candidate for further preclinical trials as a booster vaccine for BCG.

Construction and expression of Mycobacterium tuberculosis fusion protein AR2 and its immunogenicity in combination with various adjuvants to form vaccine.

Tuberculosis (TB) is recognized as a highly infectious disease worldwide, and Bacille Calmette-Guerin (BCG) remains the only TB vaccine licensed for clinical use. As there is little evidence that BCG is effective in adults, there is an urgent need for a safe and effective vaccine to control TB in adults. In this study, we tested the immunomodulatory efficiency of the fusion protein AR2. whole blood IFN-γ release assay (WBIA) was used to detect antigen specificity. The immunogenicity of the vaccine was tested in C57BL/6 mice, and confirmed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and qRT-PCR. The fusion protein AR2 was successfully constructed and expressed. The level of IFN-γ in the peripheral blood of subjects stimulated by AR2 was significantly higher than in those induced by all subcomponent proteins. AR2-specific IgG and the Th1 cytokines IFN-γ, TNF-α, and iNOS were significantly increased in the group treated with the fusion protein and compound adjuvant (AR2+DMC). Likewise, the number of IFN-γ+ CD4+, IFN-γ+CD8+, and IL-4+ CD8+ T lymphocytes increased significantly. The combination of the fusion protein and the compound adjuvant (AR2+DMC) may be a suitable candidate for an enhanced TB vaccine. This study provides theoretical and experimental support for future research to enhance the effectiveness of TB vaccines and provides an experimental basis for evaluating the influence of different adjuvants on vaccine efficacy.

Use of DosR and Rpf antigens from Mycobacterium tuberculosis to screen for latent and relapse tuberculosis infection in a tuberculosis endemic community of Huainan City.

The dormancy survival regulator (DosR) antigens upgraded during latency and resuscitation-promoting factors (Rpfs) expressed over the reactivation from dormant Mycobacterium tuberculosis (M. tuberculosis) could be used to diagnose tuberculosis (TB) at different stages. We performed a retrospective cohort study based on four groups, including healthy controls (HCs), active tuberculosis infections (ATBs), latent tuberculosis infections (LTBIs), and relapse tuberculosis infections (RTBs) enrolled between November 2020 and June 2021. Compared to the fusion protein E6-C10, combined with early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate of 10 kDa (CFP-10), the DosR- or Rpf-encoded antigens could not elicit significant IFN-γ concentration for the diagnosis of ATB. Of note, the DosR antigens produce significantly more antigen-specific IFN-γ in LTBIs than Rpfs, and the levels of antigen-specific IFN-γ elicited in RTBs stimulated by Rpfs were higher than the DosR antigens. Among the DosR antigens, Rv2003c was the most immunogenic in diagnosing LTBIs, followed by Rv2007c and Rv2005c. As far as Rpfs are concerned, Rv0867c was the best antigen to identify RTBs, followed by Rv2389c and Rv1009. Both Rv2450c and Rv1884c showed relatively limited IFN-γ concentration in RTBs. Besides, the selected DosR antigens and Rpfs showed ideal specificity and inadequate sensitivity, which could have been enhanced by the fusion antigens prepared by the DosR antigens or Rpfs, respectively. The results of this study can provide more accurate detection methods for LTBIs and RTBs and could be used for screening the dormant M. tuberculosis throughout reactivation.