• Tuberculosis and Respiratory Diseases
• /
• v.57 no.2
• /
• pp.125-131
• /
• 2004

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate BCG vaccine is urgently needed. We constructed three recombinant Mycobacterium bovis BCG (rBCG) strains over-expressing antigen (Ag) 85A, Ag85B, or both of M. tuberculosis using their own promoter and secretory sequence, or hsp60 promoter. SDS-PAGE analysis of rBCG proteins showed overexpression of Ag85A and Ag85B proteins in higher level than of those in their parental strain of BCG. In addition, rBCG(rBCG/B.FA) over-expressing Ag85A and Ag85B induced strong IFN-${\gamma}$ production in splenocytes. However, there was no significant difference in protective efficacy between rBCG and their parental BCG strain. In this study, therefore, rBCG over-expressing Ag85A, Ag85B, or both failed to show enhanced protection against M. tuberculosis infection in a mouse model.

• Tuberculosis and Respiratory Diseases
• /
• v.60 no.2
• /
• pp.187-193
• /
• 2006

Background : Normal cell proliferation and viability is strongly depends on the availability of metabolic energy and the maintenance of the appropriate adenylate-nucleotide pools. Hypothetically, changes in adenylate kinase (AK) expression could therefore be associated with adaptation to altered growth characteristics or inversely altered growth characteristics of proliferating cells could drive the changes in the metabolic profile. This study investigated whether the expression of either AK1 or a Mycobacterium tuberculosis adenylate kinase mutant which has the same catalytic activity of AK1 could affect the growth rate of slow-growing BCG. Method : Recombinant BCGs, which were cloned the human muscle-type adenylate kinase synthetic gene (AK1) and adenylate kinase mutation gene (AKmtDM) of Mycobacterium tuberculosis into the Mycobacterium/E.coli expression vectors, were constructed. Recombinant BCGs and wild-type BCG were cultured in 7H9 media and the optical density at 600nm was measured at intervals of 2-3 days. Result : There wasn't the growth rate change induced by AK1 or AKmtDM expression in recombinant BCGs. Conclusion : The expression of AK1 or Mycobacterium tuberculosis adenylate kinase mutant in BCG does not affect the growth rate of BCG.

• Parasites, Hosts and Diseases
• /
• v.52 no.3
• /
• pp.251-256
• /
• 2014

A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased $CD4^+$ and $CD8^+$ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.

• Pediatric Infection and Vaccine
• /
• v.4 no.1
• /
• pp.106-115
• /
• 1997

Objective : To evaluate the immunogenicity and safety afforded by the HG-II$^{(R)}$ recombinant hepatitis B vaccine given to healthy neonates and children and to find the influence of preceding BCG vaccination on immunogenicity. Methods : Three doses of recombinant hepatitis B vaccine with a dose of $10{\mu}g$ were given at birth, 1 and 6 months of age. This study was conducted in three hospitals (Gyeongsang National University Hospital(Group A), Kangnam General Hospital(Group B) and Younsei University Hospital(Group C)) from April, 1995 to June, 1996. Group A and Group B received 2nd dose of hepatitis B vaccine at 1 week after and before BCG vaccine, respectively. Antibidy levels, at 1 month after the 3rd dose of hepatitis B vaccine were determined by a radioimmunoassay. Results : 1) One hundred four infants and ten children were enrolled : 55 infants and 43 infants received 2nd dose of hepatitis B vaccine at 1 week after( After BCG Group) and before BCG vaccine(Before BCG Group), respectively. 2) The seropositive rate was 99.1%, and geometric mean anti-HBs titer was 131.2mIU/ml. 3) The geometric mean titers were 105.5mIU/ml and 162.8mIU/ml in After BCG and Before BCG Group, respectively(p<0.025). 4) Among 359 episodes of vaccination, the occurrence of systemic and local side reaction were reported in 7.8% and 1.4%, respectively. Conclusion : Recombinant hepatitis B vaccine(HG-II$^{(R)}$))was highly immunogenic and safe. The significantly lower geometric mean antibody titer in the BCG preceding group was observed. Well-designed controlled study with the large number of sample size will be required to show the influence of preceding BCG vaccination.

• The Journal of the Korean Society for Microbiology
• /
• v.34 no.6
• /
• pp.555-559
• /
• 1999

The 38 kDa protein of Mycobacterium tuberculosis, which was known previously as antigen 5, has been extensively used in the serodiagnosis of tuberculosis. In an attempt to develop and evaluate a serodiagnostic test using the antigen, we expressed the 38 kDa protein in BCG and its seroreactivity was compared to that expressed in Escherichia coli. The coding region of the 38 kDa protein was amplified by PCR, and the gene was cloned into a Mycobacterium-E. coli shuttle expression vector pYMC-his and pQE30 expression vector and expressed in BCG and E. coli, respectively. Both recombinant 38 kDa proteins showed strong seroreactivity against pooled serum from tuberculosis patients. There was no significant difference in seroreactivity between the two recombinant antigens in sera from the far advanced tuberculosis patients. However, of 25 tuberculosis patients graded as "minimal" by chest X-ray, 5 (20.0%) were seropositive by r38 kDa expressed in E. coli, while 8 (32.0%) by that expressed in BCG. Likewise, higher seroreactivity by r38 kDa expressed in BCG was found in sera from the moderately advanced tuberculosis. This study thus indicates that the recombinant 38 kDa expressed in BCG is more effective than that expressed in E. coli in detecting antibodies to the native 38 kDa protein of M. tuberculosis in sera from minimally affected tuberculosis patients.

• BMB Reports
• /
• v.34 no.4
• /
• pp.342-346
• /
• 2001

The Mycobacterium bovis BCG panB gene, encoding ketopantoate hydroxymethyltransferase (KPHMT), was cloned from a ${\lambda}gt11$ genomic library and sequenced. The DNA sequence encodes a protein that contains 281 amino acid residues (M, 29,337) with a high similarity to the KPHMTs. Subcloning of a 846 by open reading frame (ORF), but not a 735 by ORF, into the vector pUC19 led to complementation of the panB mutant of Escherichia coli. The BCG pang gene was overexpressed in E. coli and the KPHMT purified to homogeneity The recombinant protein was further confirmed by an enzymatic assay.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.6
• /
• pp.1022-1029
• /
• 2018

Tuberculosis (TB) remains a serious health issue around the word. Adenovirus (Ad)-based vaccine and modified vaccinia virus Ankara (MVA)-based vaccine have emerged as two of the most promising immunization candidates over the past few years. However, the performance of the homologous and heterologous prime-boost immunization regimens of these two viral vector-based vaccines remains unclear. In the present study, we constructed recombinant Ad and MVA expressing an Ag85B-TB10.4 fusion protein (AdH4 and MVAH4) and evaluated the impact of their different immunization regimens on the humoral and cellular immune responses. We found that the viral vector-based vaccines could generate significantly higher levels of antigen-specific antibodies, $IFN-{\gamma}$-producing splenocytes, $CD69^+CD8^+$ T cells, and $IFN-{\gamma}$ secretion when compared with bacillus Calmette-$Gu{\acute{e}}rin$ (BCG) in a mouse model. AdH4-containing immunization regimens (AdH4-AdH4, AdH4-MVAH4, and MVAH4-AdH4) induced significantly stronger antibody responses, much more $IFN-{\gamma}$-producing splenocytes and $CD69^+CD8^+$ T cells, and higher levels of $IFN-{\gamma}$ secretion when compared with the MVAH4-MVAH4 immunization regimen. The number of $IFN-{\gamma}$-producing splenocytes sensitive to $CD8^+$ T-cell restricted peptides of Ag85B (9-1p and 9-2p) and Th1-related cytokines ($IFN-{\gamma}$ and $TNF-{\alpha}$) in the AdH4-MVAH4 heterologous prime-boost regimen immunization group was significantly higher than that in the other viral vector-based vaccine- and BCG-immunized groups, respectively. These results indicate that an immunization regimen involving AdH4 may have a higher capacity to induce humoral and cellular immune responses against TB in mice than that by regimens containing BCG or MVAH4 alone, and the AdH4-MVAH4 prime-boost regimen may generate an ideal protective effect.

• Tuberculosis and Respiratory Diseases
• /
• v.58 no.2
• /
• pp.142-152
• /
• 2005

Background : Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 70 (Hsp70) of Mycobacterium tuberculosis. Method : M. tuberculosis genes encoding AK, NdK, and Hsp70 proteins were amplified by PCR and cloned into E. coli expression vector, pQE30. Recombinant AK, NdK, and Hsp70 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and $IFN-{\gamma}$ after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. Result : Vaccination with recombinant proteins AK, NdK, and Hsp70 delivered in DDA elicited significant level of antibody and $IFN-{\gamma}$ responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis BCG. Conclusion : Recombinant proteins AK, NdK, and Hsp70 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant.

• Tuberculosis and Respiratory Diseases
• /
• v.52 no.5
• /
• pp.497-505
• /
• 2002

Background : INF-${\gamma}$ plays an important role in the host response to a mycobacterial infection. A complete IFN-${\gamma}$ receptor 1 deficiency is a life threatening condition because it renders patients highly susceptible to a mycobacterial infection. Several mutations in the IFN-${\gamma}$ receptor and STAT1 gene have been identified in the rare mycobacterial infections. These mutations have partial function of the IFN-${\gamma}$ receptor and similar pathologic features to clinical tuberculosis. Materials and Methods : The function of the IFN-${\gamma}$ receptor was evaluated in the patients with clinical tuberculosis. In addition, the DNA coding sequence of the IFNgR1 and STAT1 gene was also analyzed in disseminated tuberculosis patients who might have a defective IFN-${\gamma}$ receptor. Results : The cell surface expression levels of HLA-DR and CD64 in the PMBC after being stimulation with IFN-${\gamma}$ (100IU/ml, 1000IU/ml) were increased in both controls and patients. However, the rate of increase in both groups was similar. The production of TNF-${\alpha}$ in the response to stimulation with LPS was higher in the both groups ($850.7{\pm}687.8$ vs. $836.7{\pm}564.3$ pg/ml). Pretreatment with IFN-${\gamma}$ prior to LPS stimulation resulted in further increase in TNF-${\alpha}$ production between both groups ($2203.5{\pm}242.5$ vs. $2227.5{\pm}560.4$ pg/ml). However, the rate of the increase in TNF-${\alpha}$ production in the both groups was similar. The known mutations in the IFNgR1 and STAT1 coding sequences were not found in the genomic DNA of patients with disseminated tuberculosis. Conclusion : The functional and genetic defects of the IFN-${\gamma}$ receptor were not identified in clinical tuberculosis. This suggests the defective IFN-${\gamma}$ receptor that predispoe patients to a BCG or NTM infection can not alone account for the cases of clinical tuberculosis.