Search

A member of the pe / ppe gene family, lipX ( pe11 ), is capable of directing persistent Mycobacterium tuberculosis and avoiding host immune responses. Some studies have indicated that LipX (PE11) can detect humoral antibodies in tuberculosis patients. Hence, information on immune mediators' responses to this protein is essential to understand its protective efficacy against M. tuberculosis infections. This study aimed to examine the response of immune mediators to pCDNA3.1- lipX expression in vivo. In the experiment, pCDNA3.1- lipX was injected into BALB/c strain male mice aged between 6 and 8 weeks, and they were compared to groups injected with pCDNA3.1 and without injection. The injection was carried out three times intramuscularly every two weeks. Blood was taken retro-orbitally and used for humoral response analysis by Western blotting against LipX-His protein. Simultaneously, the splenocytes were cultured and induced with LipX-His protein for cellular immunity analyses. Our study showed that the recombinant DNA of pCDNA3.1- lipX induced a humoral and cellular immune response, especially in IL-4, IL-12, and IFN-γ, which are the primary cellular responses to M. tuberculosis infections. However, additional studies, such as a challenge study, are needed to strengthen the argument that this plasmid construction is feasible as a tuberculosis seed vaccine candidate.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb). Bacille Calmette-Guérin (BCG) is the only licensed vaccine against TB, and it is effective in children but not in adults. The Vaccine Research Team, Department of Microbiology FKUI has developed a DNA-based TB vaccine candidate pcDNA3.1-rpfB. This candidate induces immune responses in mice, but its potency is unknown. The gold standard for potency testing of TB vaccine is the challenge method. The BSL3 animal laboratory for the challenge method is currently unavailable at FKUI. Therefore, mycobacterial growth inhibition assay (MGIA) was used as a preliminary test before the in vivo challenge test was conducted. The principle of MGIA is to reculture Mtb in a Mycobacteria Growth Indicator Tube (MGITTM) from co-cultured Mtb with mammalian cells that have been previously treated with pcDNA3.1-rpfB, pcDNA3.1 (negative control), and BCG (positive control). MGITTM shows the time to positivity, which is the time that has lapsed until a positive growth of Mtb is detected. In addition, measurements of interferon (IFN)? levels by enzyme-linked immunosorbent assay were carried out. This study concluded that pcDNA3.1-rpfB can inhibit the growth of Mtb in vitro and showed no statistical difference from BCG. The IFN? levels from co-culturing did not correlate with the level of inhibition of the growth of Mtb in vitro. [ABSTRACT FROM AUTHOR]

Resuscitation-promoting factor D (RpfD) is a protein involved in the resuscitation of dormant bacteria. A new tuberculosis vaccine carrying the rpfD gene has been successfully constructed, pcDNA3.1-rpfD. It was demonstrated that this vaccine exhibits cellular and humoral immune responses. Therefore, within this study, the efficacy of this new vaccine candidate was evaluated using mycobacterial growth inhibition assay (MGIA). MGIA is a functional assay that measures the complex host immune response, peripheral blood mononuclear cell (PBMC) and splenocyte from BALB/c mice against mycobacteria. With BACTECTM MGITTM 960 automated system, the effect of vaccination on bacterial growth was reported as a time to positivity (TTP) in hours. The mean of TTP from the vaccinated group (both pcDNA3.1-rpfD and BCG) was higher than the negative control group. These results suggest that pcDNA3.1-rpfD may be effective in controlling tuberculosis growth and may provide a clue for the development of the tuberculosis vaccine. In addition, despite previous evidence that IFNγ was essential for tuberculosis immunity, IFNγ (interferon gamma) production was found not to be correlated with mycobacterial inhibition. Therefore, these findings offer an alternative method to evaluate vaccine candidates than the assessment using IFNγ only. [ABSTRACT FROM AUTHOR]