Objective: To examine the association between the cross-resistance to ethionamide (Eto) and isoniazid (INH) and mutations of drug resistant genes in Mycobacterium tuberculosis (MTB), so as to provide the evidence for clinical diagnosis and treatment for multidrug-resistant (MDR) tuberculosis. Methods: Totally 126 MTB clinical isolates were selected, including 88 MDR-MTB clinical isolates and 38 INH- and rifampicin (RFP)-sensitive isolates. The resistance to INH and Eto was tested in MTB clinical isolates using the drug susceptibility test, and the mutations in the spacer region of INH and Eto resistance-related katG, inhA, ethA, mshA, ndh, spacer region of oxyR-ahpC and inhA promoter were detected using PCR assay. The phenotypic resistance served as a gold standard, and the sensitivity, specificity and accuracy of gene mutation tests were calculated for detection of MTB clinical isolates cross-resistant to INH and Eto. Results: Of the 126 MTB clinical isolates, there were 37 isolates cross-resistant to INH and Eto (29.37%), 51 isolates with resistance to INH and susceptibility to Eto (40.48%), 4 isolates with susceptibility to INH and resistance to Eto (3.17%) and 34 isolates with susceptibility to INH and Eto (26.98%). Among the 41 Eto-resistant MTB clinical isolates, there were 37 isolates with resistance to INH (90.24%). There were 64 MTB clinical isolates detected with katG mutations (50.79%), 4 isolates with mutation in the spacer region of oxyR-ahpC (3.17%), 2 isolates with inhA mutations (1.59%), and these isolates were all resistant to INH. There were 11 MTB clinical isolates detected with mutation in the inhA promoter (8.73%) and one isolate with ndh mutation, and all these isolates were cross-resistant to INH and Eto. There were 23 MTB clinical isolates detected with ethA mutations (18.25%) and 40 isolates with mshA mutations (31.75%), in which Eto-susceptible and -resistant isolates were detected. The diagnostic sensitivity, specificity and accuracy of inhA promoter tests for detection of cross-resistance to INH and Eto were 29.73% (95%CI: 16.44%-47.17%), 100.00% (95%CI: 87.36%-100.00%) and 63.38% (95%CI: 51.76%-73.63%) in MTB clinical isolates. Conclusions The prevalence of INH resistance is high in Eto-resistant MTB clinical isolates. Mutation in the inhA promoter region correlates with the cross-resistance to INH and Eto in MTB clinical isolates, and detection of mutation in the inhA promoter may be feasible to detect the cross-resistance to INH and Eto in MTB clinical isolates.

Objective To evaluate the antigenicity of two proteins of Mycobacteium tuberculosis (M. tuberculosis), Dnak(Rv0350) and MPT83(Rv2873), in order to provide a scientific basis for immuno-logical diagnosis of tuberculosis and research on vaccines. Methods The two antigen proteins, Dnak (Rv0350) and MPT83(Rv2873), were cloned, expressed and purified using the methods of genetic recom-bination and protein purification technology. Blood samples were collected from subjects including tuberculo-sis patients ( TB) , non-tuberculosis patients with other pulmonary diseases ( non-TB) and healthy volunteers (HV). To analyze the immunological properties of the recombinant Dnak (Rv0350) and MPT83 (Rv2873) proteins, they were used as antigens to detect humoral and cellular immunity in the subjects with enzyme linked immunosorbent assay ( ELISA ) and effector T cell enzyme-linked immunospot assay ( ELISPOT ) . Results The recombinant and purified Dnak (Rv0350) and MPT83 (Rv2873) proteins of M. tuberculosis were successfully obtained and used as antigens in the detection of humoral and cellular immunity in the sub-jects. Specific antibodies ( IgG) in the serum samples of 135 TB, 56 non-TB and 94 HV were tested with ELISA. The results showed that the sensitivity, specificity and accuracy of Dnak ( Rv0350 ) protein were 77. 80％ (105/135), 56. 70％ (85/150) and 66. 67％ (190/285). Similarly, the sensitivity, specificity and accuracy of MPT83 (Rv2873) protein were 76. 30％ (103/135), 43. 30％ (65/150) and 58. 95％(168/285). Cellular immunity was tested with the levels of IFN-γproduced by effector T lymphocytes after stimulating peripheral blood monouclear cells ( PBMC) collected form subjects of 59 TB, 65 non-TB and 64 HV with Dnak (Rv0350) and MPT83 (Rv2873) protein antigens. The results showed that the sensitivity, specificity and accuracy of Dnak (Rv0350) and MPT83 (Rv2873) proteins were 66. 10％ (39/59), 62. 79％ (81/129) and 63. 83％ (120/188), and 47. 46％ (28/59), 79. 84％ (103/129) and 69. 68％(131/188), respectively. Conclusions M. tuberculosis Dnak (Rv0350) and MPT83 (Rv2873) proteins have good antigenicity and could stimulate T cells to produce stronger immune responses. The two proteins used in combination might have promising potential in the research of immunodiagnosis of tuberculosis and the development of new anti-tuberculosis vaccines.

Objective:To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis (TB) vaccine and effective prevention and control of TB.Methods:Three full-length Mycobacterium ( M.) tuberculosis protein antigens (EsxH, Rv2628, and HspX) and two epitope-predicted and optimized epitope-dominant protein antigens (nPPE18 and nPstS1) were selected, from which five protein antigens were used to construct a protein antigen composition EPRHP014, including a fusion expression multi-component protein antigen (EPRHP014f) and a multi-component mixed protein antigen (EPRHP014m) formed with the five single protein using clone, purification, and purification respectively. Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant, and the BCG vaccine was used as a control. ELISA detected the titer of serum-specific antibodies, the secretion of various cytokines was detected by ELISpot and Luminex, and immune protection was observed by the M.tuberculosis growth inhibition test in vitro. The results were statistically analyzed by t-test or rank sum test, and P<0.05 was considered a statistically significant difference. Results:Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a, and the antibody titers were similar to those of mice immunized with BCG, with no statistical significance ( P>0.05). The number of spot-forming cells (SFC) secreting IFN-γ and IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group ( P<0.05), but there was no significant difference in the number of SFC for IFN-γ and IL-4 induced between EPRHP014m group and BCG group ( P>0.05). The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group ( P<0.05), but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group ( P>0.05). There was no significant difference in the secretions of IL-6, IL-10, IL-12, and GM-CSF between the EPRHP014f and BCG groups ( P>0.05). EPRHP014m group, EPRHP014f group, and BCG group had obvious antibacterial effects in vitro, and the difference was insignificant ( P>0.05). Conclusion:Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization, and have a strong ability to inhibit the growth of M. tuberculosis in vitro, indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.

Objective:The cellular immunity of 5 Mycobacterium tuberculosis recombinant proteins and their compositions was evaluated.Method:A total of 88 fresh venous blood from peripheral heparin anticoagulant population, 42 of which were from tuberculosis patients treated by The Tuberculosis Prevention and Treatment Center of Changping District, Beijing, and 46 of healthy volunteers were provided by the Infection Diseases of Chinese Center for Disease Control and Prevention. Healthy volunteers without a history of tuberculosis exposure and any clinical signs and symptoms. Using the Mycobacterium tuberculosis standard strain H37Rv DNA as a template, complete genes of the selected 5 recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c by PCR amplified; 5 recombinant proteins were cloned, expressed and purified as stimulants by genetic recombination and protein purification techniques, and the effector T cell enzyme-linked immunospot assay (ELISPOT) was used to detect cellular immunity in the population.Results:The recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c were successfully cloned, expressed and purified; And the sensitivities were 50.00%, 71.43%, 69.04%, 73.81% and 76.19%, and the specificities were 86.96%, 76.09%, 71.74%, 39.13% and 36.96%. In addition, the positive predictive value, negative predictive value, area under the curve and Youden index were 52.46% to 77.78%, 62.96% to 74.47%, 0.511 to 0.754 and 0.129 to 0.475, respectively. Except for Rv1411c and Rv3418c, the number of spot-forming cell (SFC) detected by Rv3874, Rv3875 and Rv2031c in tuberculosis patients was higher than healthy volunteers, and the differences were statistically significant ( P<0.001). Among the 26 compositions composed of 5 recombinant proteins, the sensitivity was 80.95% to 95.24%, and the specificity was 68.89% to 24.44%. As the number of recombinant proteins in the composition increases, the sensitivity gradually increased, but the specificity decreased. Conclusion:The recombinant proteins of Mycobacterium tuberculosis Rv3874, Rv3875 and Rv2031c have strong ability to stimulate T cells to produce immune response, and have certain antigenicity. The efficacy of Rv1411c and Rv3418c alone as diagnostic antigens is not ideal, and the composition composed of multi-component antigens has certain application value. This article provides experimental evidence for the immune diagnosis of tuberculosis and the preparation of new anti-tuberculosis vaccines.

Objective:To analyze the resistance mutational profiles of multi-drug resistant Mycobacterium tuberculosis in China and the correlation between major mutation types and genotypes based on the whole-genome sequencing data. Methods:Search and download of the genome-wide sequencing data of M. tuberculosis published in China by August 2019 on NCBI database were conducted. Mutation frequency of drug resistance-related gene loci based on whole-genome sequencing was used to predict the molecular susceptibility of strains, and the correlation between mutation types and genotypes was analyzed. Results:According to the results of molecular resistance and susceptibility profiles, 1 024 MDR strains were identified from 2 019 M. tuberculosis strains. The major mutation types of resistance-related genes to common drugs were katG S315T (73.2%, isoniazid), rpoB S450L (63.1%, rifampicin), rpsL K43R (70.0%, streptomycin), embB M306V (37.4%, ethambutol), pncA_promoter T (-11)C (7.9%, pyrazinamide), gyrA A90V (32.3%, fluoroquinolones), rrs A1401G (67.7%, second-line injection drugs), fabG1_promoter C (-15) T (87.0%, Ethionamide), folC I43T (30.4%, P-aminosalicylic acid). Among them, the frequencies of katG S315T, embB M306V, rpsL K43R, gyrA A90V in lineage 2 were significantly higher than those in lineage 4, and folC I43T was only found in lineage 2. The proportion of katG S315T was significantly higher in the ancient Beijing genotype compared to the modern genotype, in contrast, the proportion of rpsL K43R was significantly higher in modern Beijing genotype, the differences were significant (all P<0.05). Conclusions:The results showed the main mutation types of resistance-related genes of MDR strains to many commonly used anti-tuberculosis drugs in China based on whole-genome sequencing, providing a basis for the development of sensitive and specific rapid molecular detection methods. At the same time, it was also found that the major mutation types of MDR-related genes were related to the genotype of the strains.

Objective:The cellular immunity of 5 Mycobacterium tuberculosis recombinant proteins and their compositions was evaluated.Method:A total of 88 fresh venous blood from peripheral heparin anticoagulant population, 42 of which were from tuberculosis patients treated by The Tuberculosis Prevention and Treatment Center of Changping District, Beijing, and 46 of healthy volunteers were provided by the Infection Diseases of Chinese Center for Disease Control and Prevention. Healthy volunteers without a history of tuberculosis exposure and any clinical signs and symptoms. Using the Mycobacterium tuberculosis standard strain H37Rv DNA as a template, complete genes of the selected 5 recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c by PCR amplified; 5 recombinant proteins were cloned, expressed and purified as stimulants by genetic recombination and protein purification techniques, and the effector T cell enzyme-linked immunospot assay (ELISPOT) was used to detect cellular immunity in the population.Results:The recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c were successfully cloned, expressed and purified; And the sensitivities were 50.00%, 71.43%, 69.04%, 73.81% and 76.19%, and the specificities were 86.96%, 76.09%, 71.74%, 39.13% and 36.96%. In addition, the positive predictive value, negative predictive value, area under the curve and Youden index were 52.46% to 77.78%, 62.96% to 74.47%, 0.511 to 0.754 and 0.129 to 0.475, respectively. Except for Rv1411c and Rv3418c, the number of spot-forming cell (SFC) detected by Rv3874, Rv3875 and Rv2031c in tuberculosis patients was higher than healthy volunteers, and the differences were statistically significant ( P<0.001). Among the 26 compositions composed of 5 recombinant proteins, the sensitivity was 80.95% to 95.24%, and the specificity was 68.89% to 24.44%. As the number of recombinant proteins in the composition increases, the sensitivity gradually increased, but the specificity decreased. Conclusion:The recombinant proteins of Mycobacterium tuberculosis Rv3874, Rv3875 and Rv2031c have strong ability to stimulate T cells to produce immune response, and have certain antigenicity. The efficacy of Rv1411c and Rv3418c alone as diagnostic antigens is not ideal, and the composition composed of multi-component antigens has certain application value. This article provides experimental evidence for the immune diagnosis of tuberculosis and the preparation of new anti-tuberculosis vaccines.

Objective To investigate the cross-reactive immune responses to Mycobacterium vac-cae (M. vaccae), Mycobacterium tuberculosis (M. tuberculosis, H37Rv) and Mycobacterium bovis Bacillus Calmette-Guerin ( BCG) for providing reference for the development of new vaccines with M. vaccae. Meth-ods M. vaccae (ATCC95051), M. tuberculosis (H37Rv) and BCG (China strain) were cultured on L-J solid media and harvested. Total bacterial protein antigens prepared by ultrasonic disruption were used to im-munize BALB/c mice. IgG antibodies in serum samples were detected with enzyme-linked immunosorbent assay ( ELISA) to evaluate humoral immune responses. Cellular immunity was assessed by detecting various cytokines with cytokine release assay ( CRA) . Results The mice that were respectively immunized with the three mycobacterial antigens could produce high titers of antibodies ( IgG) and high levels of IFN-γand IL-2, but low levels of IL-4 and IL-10. Results of the cross reactivity tests showed that ATCC95051, H37Rv and BCG were able to cross-react with the immunized mice, and all of them induced high levels of IFN-γ, IL-2 and IgG antibodies. Conclusions The three Mycobacteria mainly elicited Th1 immune responses. There were cross-reactive immune responses to M. vaccae, M. tuberculosis and BCG, which might provide ref-erence for using M. vaccae in the development of new anti-tuberculous vaccines.

Objective:To evaluate the humoral and cellular immunoreactivity of recombinant Mycobacterium tuberculosis ( M. tuberculosis) PstS1 and HspX protein antigens in order to provide reference for immunodiagnosis of tuberculosis and screening of candidates for vaccine antigens. Methods:Purified recombinant M. tuberculosis PstS1 and HspX proteins were obtained using molecular cloning expression and Ni 2+ affinity chromatography. Blood samples and epidemiological data of healthy individuals and patients with M. tuberculosis infection were collected. Specific IgG antibodies and IFN-γ-producing antigen-specific T cells were respectively detected by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT) with the recombinant proteins used as antigens. The humoral and cellular immunoreactivity of the recombinant PstS1 and HspX proteins were assessed with statistical analysis of data. Results:Both the recombinant PstS1 and HspX proteins could induce the secretion of IFN-γ by more specific effector T cells in patient with M. tuberculosis infection, and the differences between the infection and healthy control groups were statistically significant ( P<0.05). The specificity and sensitivity of the recombinant PstS1 and HspX as the diagnostic antigens of ELISPOT were 92.11% (35/38) and 65.96% (31/47), and 68.42% (26/38) and 91.49% (43/47), respectively. The two proteins also possessed some humoral immunoreactivity, but statistically significant difference was only observed in the HspX-specific antibody level between the two groups ( P<0.05). Conclusions:Both the recombinant PstS1 and HspX proteins had good cellular immunoreactivity and were the immunodominant antigens of cellular immunity. They performed well in cellular immunodiagnosis and were good potential candidate antigens for anti-tuberculosis vaccines.

Objective To investigate the single nucleotide polymorphism (SNP) of toxinantitoxin-chaperone (TAC) system of Mycobacterium (M.) tuberculosis with different genotypes and its biological significance.Methods A total of 183 clinical M.tuberculosis isolates were collected for spoligotyping.The sequences of higA,higB and Rv1957 were obtained by using PCR and DNA sequencing.The sequences were compared for possible mutations.Functional consequences of nonsynonymous SNPs were predicted by using I-Mutant 2.0 servers.Results Among the 183 M.tuberculosis isolates,138(75.41％) belonged to the Beijing family,while 45(24.59％) belonged to the non-Beijing family.A total of 149(81.42％) isolates showed polymorphisms in the TAC system.We discovered 6 nonsynonymous SNPs and 2 synonymous SNPs.All the synonymous mutations occurred in higA gene,while nonsynonymous SNPs were found in the higA,higB and Rv1957 genes either.All the synonymous mutations and 4 nonsynonymous SNPs were restricted to the Beijing family strains and only 2 nonsynonymous SNPs were observed in the non-Beijing family strains.Of the 6 nonsynonymous SNPs studied,4 were predicted to have ability to affect the stability of respective protein.Conclusion The SNPs in the coding sequences of TAC system in clinical isolates can be relatively high and the Beijing family strains are with higher polymorphism,which might benefit to adapt to different host environment.

Objective:To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates (a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m) and to provide a new antigen combination for the development of tuberculosis vaccines.Methods:Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed. Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant (50 μg/mouse) for three times with an interval of 10 d. The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples. Serum antibody titers and cytokine levels were measured by ELISA, Luminex technique and enzyme-linked immunospot assay (ELISPOT). Mycobacterial growth inhibition assay (MGIA) was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro. One-way analysis of variance and t-test were used for statistical analysis. Results:Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level. The levels of cytokines related to Th1 (IL-2, TNF-α, IFN-γ), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) as well as other proinflammatory cytokines (GM-CSF, IL-12) were higher in the EPDPA015f group than in the adjuvant group ( P<0.05). The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10 6. The results of MGIA showed that the numbers of Mtb (lgCFU) in the PBS, adjuvant, EPDPA015f and EPDPA015m groups were 3.46±0.11, 3.51±0.06, 2.98±0.09 and 3.19±0.08, respectively. The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups ( P<0.001, P<0.001, P<0.01). Conclusions:The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses, and exhibited superior in vitro inhibitory activity against the growth of Mtb. EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine