ABSTRACT
Bacillus Calmette-Guérin (BCG) is the only widely used prophylactic tuberculosis (TB) vaccine that can prevent severe TB in infants. However, it provides poor protection in adults, and therefore, there is ongoing research into new TB vaccines and immunization strategies with more durable immune effects. The recombinant BCG and BCG prime-protein booster are two important vaccine strategies that have recently been developed based on BCG and could improve immune responses. In this study, three immune strategies based on four protective antigens, namely, ESAT-6, CFP-10, nPPE18, and nPstS1, were applied to construct recombinant rBCG-EPCP009, EPCP009 subunit protein, and BCG prime-EPCP009 booster vaccine candidates. The short- and long-term immune effects after vaccination in Balb/c mice were evaluated based on humoral immunity, cellular immunity, and the ability of spleen cells to inhibit in vitro mycobacterial growth. At 8 and 12 weeks after the initial immunization, splenocytes from mice inoculated with the BCG prime-EPCP009 protein booster secreted higher levels of PPD- and EPCP009-specific IFN-γ, IL-2, TNF-α, IL-17, GM-CSF, and IL-12 and had a higher IFN-γ+CD4+ TEM:IL-2+CD8+ TCM cell ratio than splenocytes from mice inoculated with the rBCG-EPCP009 and EPCP009 proteins. In addition, the EPCPE009-specific IgG2a/IgG1 ratio was slightly higher in the BCG prime-EPCP009 protein booster group than in the other two groups. The in vitro mycobacterial inhibition assay showed that the splenocytes of mice from the BCG prime-EPCP009 protein booster group exhibited stronger inhibition of Mycobacterium tuberculosis (M. tuberculosis) growth than the splenocytes of mice from the other two groups. These results indicate that the BCG prime-EPCP009 protein booster exhibited superior immunogenicity and M. tuberculosis growth inhibition to the parental BCG, rBCG-EPCP009, and EPCP009 proteins under in vitro conditions. Thus, the BCG prime-EPCP009 protein booster may be important for the development of a more effective adult TB vaccine.
ABSTRACT
Background: Ethionamide (ETH), a structural analogue of isoniazid (INH), is used for treating multidrug-resistant tuberculosis (MDR-TB). Due to the common target InhA, INH and ETH showed cross-resistance in M. tuberculosis. This study aimed to explore the INH and ETH resistant profiles and genetic mutations conferring independent INH- or ETH-resistance and INH-ETH cross-resistance in M. tuberculosis circulating in south of Xinjiang, China. Methods: From Sep 2017 to Dec 2018, 312 isolates were included using drug susceptibility testing (DST), spoligotyping, and whole genome sequencing (WGS) to analyze the resistance characteristics for INH and/or ETH. Results: Among the 312 isolates, 185 (58.3%) and 127 (40.7%) belonged to the Beijing family and non-Beijing family, respectively; 90 (28.9%) were INH-resistant (INHR) with mutation rates of 74.4% in katG, 13.3% in inhA and its promoter, 11.1% in ahpC and its upstream region, 2.2% in ndh, 0.0% in mshA, whilst 34 (10.9%) were ETH-resistant (ETHR) with mutation rates of 38.2% in ethA, 26.2% in inhA and its promoter, and 5.9% in ndh, 0.0% in ethR or mshA; and 25 (8.0%) were INH-ETH co-resistant (INHRETHR) with mutation rates of 40.0% in inhA and its promoter, and 8% in ndh. katG mutants tended to display high-level resistant to INH; and more inhA and its promoter mutants showed low-level of INH and ETH resistance. The optimal gene combinations by WGS for the prediction of INHR, ETHR, and INHRETHR were, respectively, katG+inhA and its promoter (sensitivity: 81.11%, specificity: 90.54%), ethA+inhA and its promoter+ndh (sensitivity: 61.76%, specificity: 76.62%), and inhA and its promoter+ndh (sensitivity: 48.00%, specificity: 97.65%). Conclusion: This study revealed the high diversity of genetic mutations conferring INH and/or ETH resistance among M. tuberculosis isolates, which would facilitate the study on INHR and/or ETHR mechanisms and provide clues for choosing ETH for MDR treatment and molecular DST methods in south of Xinjiang, China.
