Chinese Journal of Antituberculosis

The present status and future developments in new tuberculosis vaccine research

WU Xue-qiong

Chinese Journal of Antituberculosis. 2012, 34(3): 133-137.

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Immunotoxicological evaluation about new tuberculosis vaccines

DU Wei-xin,DONG Na,CHEN Bao-wen, XU Miao, YANG Lei, LU Jin-Biao, SHEN Xiao-Bing, WANG Guo-Zhi

Chinese Journal of Antituberculosis. 2012, 34(3): 138-144.

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Objective To develop a method for the immunotoxicological evaluation of vaccines in a guinea pig model, and to preliminarily evaluate the immunotoxicological effects of a new tuberculosis vaccine (smegmatis vaccine). Methods (1) Thirty guinea pigs were prepared the allergy models, and 15 as negative controls. The total IgG1 antibody in serum and the content of allergic media in the bronchoalveolar lavage fluid and the culture supernatants from the peripheral blood basophils and mast cells in peritoneal lavage fluid in vitro simulated were detected by ELISA. (2) The guinea pigs were divided into 3 groups as follow: allergy model, smegmatis vaccine and negative control. Their immunotoxicological effects were preliminarily evaluated using the methods developed above. Results The optimal experimental conditions for the detection of total IgG1 antibody in sera from guinea pigs included that 6 μg/ml goat anti-guinea pig IgG1 antibody was coated, the serum sample was diluted 1:10⁵, and horseradish peroxidase labeled goat anti-guinea pig IgG1 antibody was diluted 1:20 000. In the culture supernatant of peripheral blood basophils simulated in vitro in the allergy model group and negative control group, the histamine contents were （99.37±15.34）nmol/L and （29.94±5.86）nmol/L, and leukotriene levels were （13.09±3.87）pg/ml and （2.22±0.53）pg/ml, respectively. In the supernatant of bronchoalveolar lavage fluid, the histamine contents were (73.42±18.60) nmol/L and (31.00±12.09) nmol/L, and leukotriene levels were (123.20±16.57) pg/ml and (39.05±16.94) pg/ml, respectively. In the culture supernatant of peritoneal lavage fluid simulated in vitro, the histamine contents were (39.59±12.94) nmol/L and (14.35±5.85) nmol/L, and leukotriene levels were (6.79±2.58) pg/ml and (3.09±1.18) pg/ml, respectively. Both of the contents of histamine and leukotriene in three kinds of samples mentioned above were significantly different between the allergy model group and control group (histamine: t=12.60, t=5.41, t=5.20, P<0.05; leukotriene: t=8.46, t=9.15, t =3.85, P＜0.05). The abnormal immunotoxicological effects were not observed in the safety evaluation of smegmatis vaccine by the methods above. Conclusion The method for immunotoxicological evaluation of vaccines was preliminarily established. The smegmatis vaccine proved preliminarily to be safe.

Immunogenicity of the recombinant M. smegmatis expressing Ag85B-ESAT-6 fusion protein of M. tuberculosis in mice

WANG Ping, WANG Li-mei, ZHANG Wei, BAI Yin-lan, KANG Jian, HAO Yan-fei, LUO Tai-lai, XU Zhi-kai

Chinese Journal of Antituberculosis. 2012, 34(3): 145-149.

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Objective To evaluate the immunogenicity of the recombinant M. smegmatis expressing Ag85B-ESAT-6 fusion protein of M. tuberculosis (Ag85B-ESAT-6-rM.s) in mice. Methods Six-week-old BALB/c mice were immunized by subcutaneous injection on the back with 1×10⁶ colony-forming unit (CFU) of Ag85B-ESAT-6-rM.s per mouse. The mice immunized by M. smegmatis (M.s), BCG, Ag85B-ESAT-6 fusion protein and saline used as control. The percentages of CD4⁺ and CD8⁺ T cells in the peripheral blood mononuclear cells (PBMC) of mice were detected once a week for six weeks after immunization. The proliferation of splenic lymphocytes of mice was assayed by MTS［3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt］, and the numbers of splenic lymphocytes secreting interferon γ (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4) were detected by enzyme-linked immunospot assay (ELISPOT) at 6 weeks after immunization. Results The Ag85B-ESAT-6-rM.s could significantly stimulate the generation of CD4⁺ and CD8⁺ T cells in the PBMC from immunized mice, and its percentages of CD4⁺ and CD8⁺ T cells ［(59.6±1.5)%］were significantly higher than that in BCG group ［(48.8±2.3)%］ (t=12.252, P<0.05) and M.s group ［(45.7±1.6)%］(t=20.166, P<0.05). Ag85B-ESAT-6-rM.s could stimulate the proliferation of splenic lymphocytes in immunized mice, and its stimulation index (3.23±0.31) was equivalent to that in BCG group (2.95±0.36). However, the level of IFN-γ in mice immunized by Ag85B-ESAT-6-rM.s ［(167.5±36.6) spot forming cells (SFC)/10⁶］was significantly higher than that in BCG group ［(98.5±26.9)SFC/10⁶ ］(t=4.804, P<0.05), and the level of IFN-γ was also higher than levels of IL-2 and IL-4 (t=6.174 and 6.449, P<0.05) in mice immunized by Ag85B-ESAT-6-rM.s. Conclusion The expression of Ag85B-ESAT-6 fusion protein in M. smegmatis could improve the immunogenicity, and Ag85B-ESAT-6-rM.s could induce immune response preventing M. tuberculosis infection in mice. Ag85B-ESAT-6-rM.s as a TB candidate vaccine is worthy of further study.

Development of BCG reference system of evaluating the protective efficacy of vaccine in China

CHEN Bao-wen，SHEN Xiao-bing，DU Wei-xin，SU Cheng，YANG Lei，LU Jin-biao，WANG Guo-zhi

Chinese Journal of Antituberculosis. 2012, 34(3): 150-153.

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Objective To develop BCG reference system to evaluate protective efficacy of new tuberculosis vaccine in China. Methods The guinea pigs were immunized by subcutaneous injection with 1/10 human dose (0.005 mg/0.2 ml) of BCG strains (D ₂ PB 302), and the saline was used as the control. A total of 4 experiments were performed respectively. Each guinea pig was infected by subcutaneous injection with 0.5 ml of Mtb suspension (200 to 2000 CFU/ml) at 5 weeks (testⅠand test Ⅱ), 28 weeks (test Ⅲ), and 60 weeks (test Ⅳ) after immunization, respectively. The guinea pigs were sacrificed 6 weeks after the infection. The pathological changes of livers, spleens and lungs of the guinea pigs were observed. The spleens were taken, homogenized and then cultured. Mtb colonies in the spleens were enumerated and the results were expressed as logarithm values. Results The lesion indexes of livers, spleens and lungs in test Ⅰ, Ⅱ, Ⅲ, Ⅳ were (10.0±3.8), (6.7±2.1), (10.0±4.5), and (14.4±4.8), respectively, significantly lower than those in the control groups［(46.9±4.8), t=－0.63, P<0.01; (36.3±6.0), t=－4.63, P<0.01; (57.5±6.2), t=－6.24, P<0.01; (56.9±5.0), t=－6.16, P<0.01, respectively］. Mtb colonies in the spleens in test Ⅰ, Ⅱ, Ⅲ, Ⅳ were lg (0.49±0.49), lg (0.38±0.38), lg (2.16±0.93), and lg (1.46±0.69), respectively, significantly lower than those in the control groups［lg (5.41±0.18), t=－9.43, P<0.01; lg (5.11±0.19), t=－11.22, P<0.01; lg (5.44±0.27), t=－3.39, P<0.05; lg (5.43±0.20), t=－5.55, P<0.01, respectively］. Conclusion BCG reference system used to evaluate protective efficacy of new tuberculosis vaccine is reliable and stable. The BCG used in our country exhibits excellent protective efficacy.

Construction and immunogenicity of Mycobacterium tuberculosis subunit vaccine ESAT6-RpfE

LI Zhi, DA Ze-jiao, ZHANG Guo-ping, LI Rui-ying, LIU Wan-bo, SU Juan, ZHANG Ying, ZHU Bing-dong

Chinese Journal of Antituberculosis. 2012, 34(3): 154-160.

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Objective To construct and express Mycobacterium tuberculosis esat6-rpfE fused gene, and to purify the fusion protein and study its immunogenicity. Methods The esat6 and rpfE genes were amplified from the genomic DNA of Mycobacterium tuberculosis reference strain H37Rv and clinical stain respectively by PCR, and inserted into the plasmid pET30a. The fusion protein ESAT6-RpfE was expressed in E. coli BL21(DE3) and purified after two successive chromatographic purification steps. In the first experiment, the mice C57BL/6 were divided randomly into five groups named ESAT6, RpfE, ESAT6-RpfE, PBS and BCG, the adjuvant used was dimethyl-dioctyldecyl ammonium bromide (DDA). In the second experiment, the mice were divided randomly into three groups called ESAT6-RpfE, PBS and BCG, the adjuvants used were DPG ［DDA+poly(I:C)+Gelatin］. The mice were immunized subcutaneously at weeks 0, 3 and 6. The level of IFN-γ secreted by spleen lymphocytes stimulated by antigen ESAT6 or RpfE was detected at the 8th week after the last immunization. The antibodies IgG2b and IgG1 in the sera were dectected by ELISA. Results The esat6 and rpfE gene sequences cloned were consistent with GenBank report. The recombinant fusion protein ESAT6-RpfE was expressed in inclusion bodies. The counts of spleen lymphocytes secrecing RpfE-specific IFN-γ in the subunit vaccine group［ (427.13±100.46)］ were significantly higher than those in PBS group (26.13±22.34, q=7.924，P＜0.001), and the counts of spleen lymphocytes secrecing ESAT6-specific IFN-γ in the subunit vaccine group［ (506.50±140.40) ］were significantly higher than those in PBS group (19.25±14.75，q=11.36，P＜0.001) and BCG group (125.5±46.41，q=8.882, P＜0.001). The mice immunized by ESAT6-RpfE protein can induced antigenspcific antibody. Conclusion The recombinant fusion protein ESAT6-RpfE was successfully expressed and obtained. It can induce antigen-specific immune responses in C57BL/6 mice. ESAT6 and RpfE fusion protein can be used as a candidate of the new TB subunit vaccine to further study.

Evaluation on effectiveness and safety of BCG CpG DNA combination adjuvant system (BC-C02)

ZHAO Ai-hua，LI Feng-xiang，KOU Li-jie，LI Chang-gui，QIAO Lai-yan, WANG Peng，CHEN Bao-wen，XU Miao，DU Wei-xin，SHEN Xiao-bing，SU Cheng，LU Jin-biao，YANG Lei，WANG Guo-zhi

Chinese Journal of Antituberculosis. 2012, 34(3): 161-167.

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Objective To evaluate the effectiveness and safety of an aluminum salt and BCG CpG DNA combination adjuvant system (BC-C02). Methods Cell surface marker and intracellular cytokines were measured by FACS and ELISPOT to evaluated the immunostimulatory effect of BCG CpG DNA adjuvant. The effects of CpG DNA adjuvant on H1N1 vaccine were evaluated by humoral immunity and protective efficacy of H1N1 vaccine. The mice were divided into 7 groups: PBS control, low dose H1N1, low dose H1N1+CpG DNA, middle dose H1N1, middle dose H1N1+CpG DNA, high dose H1N1, and high dose H1N1+CpG DNA, 20 mice per group. The effect of BC-C02 adjuvant on a new TB vaccine was evaluated by the therapeutic effect of vaccine in guinea pigs infected with M. tuberculosis, in which low dose，middle dose and high dose of vaccine (Ag85B+ BC-C02) were set, and the saline and adjuvant were used as contol, 10 guinea pigs per group. The IgE level of BC-C02 in mice and general allergic reaction of BC-C02 in guinea pigs were measured to evaluate its immunotoxicologic effect and safety. Results BC-C02 could stimulate B cells proliferation (CD19⁺% of CpG DNA was 41% and that of saline was 27%, t=－4.40, P<0.05), up-regulate the expression of TLR9 in B cells (CD19⁺TLR-9⁺% of CpG DNA was 2.8% and that of saline was 1.1%, t=－5.92, P<0.05), promote the expressions of MHC Ⅱ, TLR-9 and IL-12 in macrophages (F4/80⁺I-A/I-E⁺% of CpG DNA was 82% and that of saline was 31%, F4/80⁺I-A/I-E⁺TLR-9⁺IL-12⁺% of CpG DNA was 2.1% and that of saline is 0.1%, t_MHC-Ⅱ=－4.32, t_{TLR-9 and IL-12}=4.80, P<0.05). In the experiment of CpG DNA as H1N1 vaccine adjuvant, the titers of HI antibody were detected at 5 d, 7 d, 14 d and 28 d after the immunization, the antibody titers in PBS conrol group were all less than 10; the antibody titers in low dose H1N1 group < that in middle dose H1N1 group < that in high dose H1N1 group; the antibody titers in all groups of antigen with CpG DNA were higher than that in the corresponding group without CpG DNA. The protective efficacy of H1N1 vaccine showed that the survival rates of PBS, low dose H1N1, low dose H1N1+CpG DNA, high dose H1N1, high dose H1N1+CpG DNA groups were 30%，50%，90%，100%，100%, respectively, CpG DNA could promote 40% of survival rate compared with the groups without adjuvant. After the guinea pigs infected with M. tuberculosis were treated with Ag85B+ BC-C02, there were TB lesions in all of livers, lungs and spleens of saline group; there were no TB lesions in 30% of animals, and less than 30 of mean TB lesion scores (liver, lung and spleen) in 60%～70% of animals in vaccine groups. BC-C02 adjuvant could not induce the produce of antibody IgE in mice and general allergic reactions in guinea pigs. Conclusion BC-C02 combination adjuvant system can induce cellular and humoral immune responses, improve the protective effectiveness of vaccines, and be used as a candidate adjuvant of new vaccine.

Immune efficacy of tuberculosis DNA vaccine produced by magnifying technology

WU Xiao-li，BI Lan，ZHAO Ya-jie，LUO Dan，QI Jian-xin，WANG Jiong，LI Zhong-ming

Chinese Journal of Antituberculosis. 2012, 34(3): 168-171.

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Objective To study the immunogenicity of Mycobacterial tuberculosis Ag85A DNA vaccine with pilot scale production in mice. Methods Twenty BALB/c mice were randomly divided into 2 groups, and then immunized intramuscularly for 3 times with Mtb Ag85A DNA or PBS respectively. The mice were killed and the whole blood samples were collected at 3 weeks after the last immunization. The specific antibody and cytokines (IL-4) in sera were detected. The Mtb Ag85A specific interferon-γ (IFN-γ) level and the percentages of CD4⁺ or CD8⁺ T cell in mouse spleen cells were also detected. Results The data obtained by statistical analyses demonstrated that: (1) The absorbance values of Ag85A-specific antibody in the sera of each group mice before and after immunization were all less than 0.2. (2) The IL-4 level in sera from Ag85A DNA group［(146.2±34.3) pg/ml］was significantly lower than that from PBS group ［(177.7±28.1) pg/ml］(t=2.244，P=0.038). The IFN-γ level in sera from Ag85A DNA group ［(129.6±159.0) pg/ml］was higher than that from PBS group ［(76.5±21.5) pg/ml］, but there was no significant difference (t=－1.047，P=0.309). (3) The data from the ELISPOT assay for IFN-γ showed that the number of spot-forming spleen cells in Ag85A DNA group(103.60±112.14) was significantly higher than that in PBS group(5.78±5.83) (t=36.538，P=0.018). (4) The percentage (21.57%) of CD4⁺ T cells in Ag85A DNA group was higher than that (12.17%) in PBS group (t=3.043，P=0.038); however, there was no significant difference on the percentage of CD8⁺ T cells between the two groups (13.70% vs. 10.57%, t=0.847，P=0.445). Conclusion The Th1 cellular immunity was mainly stimulated in the mice immunized by Mtb Ag85A DNA vaccine. But Th2 cellular immune response was not significantly induced in these mice.

Analysis of acceptability survey on the trinity tuberculosis prevention new service model in health administration departments and tuberculosis prevention institutes of Guangxi

WU Teng-yan, HUANG Shu-hai，LIU Fei-ying

Chinese Journal of Antituberculosis. 2012, 34(3): 172-175.

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Objective To understand the acceptability on the trinity tuberculosis prevention new service model in the health administration departments and the tuberculosis prevention institutes of Guangxi, in order to analyze the feasibility and further improve the management practices of cooperation between medical agencies and TB institutions. Methods Convenience sampling method and questionnaire survey were used. One of the key people in the health administration department and the tuberculosis prevention institute of 14 cities and 27 counties was selected respectively for this survey. Results Most of the interviewees accepted the trinity tuberculosis prevention model, the acceptability proportion among the leaders of health administration departments, leaders in charge of TB control institutions, and directors of TB institutions were 73.7%（28/38）, 75.0% （30/40）and 66.7%（26/39）respectively. The main supporting reason is that this model could help each department better play its own advantages, and effectively use the medical resources, and facilitate patient’s diagnosis and treatment. The main reason not in favor of trinity is the insufficient public health resources in the general hospitals now. Conclusion The implementation of the trinity tuberculosis prevention model is feasible in Guangxi, however this model needs to expand in a stepwise way under the basis of improving the existing rules and regulations.

Study on the effect evaluation of the Fifth Round of Global Fund Program on Mtb and HIV co-infection control

YIN Ji-guo, HE Wei-hua, LIAN Zu-yin, HE Fu-chun, YUAN Xiao-yan, YU Xiao-feng, ZHANG Yong, CHU Guang-sheng

Chinese Journal of Antituberculosis. 2012, 34(3): 176-180.

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Objective To evaluate the effect of the Fifth Round of Global Fund Program on Mtb/HIV co-infection control in Suizhou city. Methods Quarterly and annual reports, special survey data were collected between the fourth quarter of 2006 and the fourth quarter of 2010 from Suizhou CDC. Database was established for statistical analysis. Results During the program implementation period, 4077 TB patients were registered(among which, 21 cases were detected from TB screening among HIV/AIDS patients), the remaining 4056 cases received HIV antibody test, the examination rate reached 100.00%（4056/4056）, and HIV positive detection rate was 0.15%(6/4056). Regarding TB screening among HIV/AIDS patients, among 1521 cases of HIV/AIDS patients being followed-up, 92.64% (1409/1521) received TB screening. The corresponding TB detection rate was 1.49% (21/1409), which was 9.93 times of HIV detection rate among TB patients. A total of 27 Mtb/HIV patients were registered, all of them were administered anti-TB medication, the cure and treatment completion rate were 81.48% (22/27), treatment failure rate was 3.70% (1/27) and the death rate was 11.11% (3/27). The detection rate of active tuberculosis among HIV/AIDS patients decreased significantly from 5.17%(12/232) to 0.67%(2/300) in past 5 years（χ²=13.267，P=0.000）.Conclusion The work of Mtb/HIV co-infection control should be focused on detection of TB cases among people living with HIV/AIDS. TB detection among HIV/AIDS patients, HIV tests among TB patients and anti-TB medication for Mtb/HIV patients are important in control of Mtb/HIV epidemic, and should carry out on a long-term basis.

Immunotherapeutic effect of Ag85A DNA vaccine on tuberculosis

LIANG Yan, WU Xue-qiong, ZHANG Jun-xian, BAI Xue-juan, YANG You-rong, LI Ning, YU Qi, LI Zhong-ming, WANG Lan, SHI Ying-chang

Chinese Journal of Antituberculosis. 2012, 34(3): 181-183.

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Tuberculosis is a respiratory infectious disease caused by Mycobacterium tuberculosis, and the situation of tuberculosis (TB) in the world is still very severe. While the effect of currently available vaccine Bacillus Calmette-Guérin (BCG) against TB, is not ideal. A new tuberculosis vaccine has become one of the hotspots in the research of tuberculosis at home and abroad. This article will introduce the immunogenicity and therapeutic effects of Mtb Ag85A DNA vaccine in the mouse tuberculosis model from our research team since 1998. The results showed that Mtb Ag85A DNA vaccine could induce humoral immune and Th1 cell immune responses in mice, reduce M. tuberculosis, especially multi-drug-resistant M. tuberculosis in the organs of mice. The vaccine combined with conventional chemotherapy can improve the immunity, and effectively kill or inhibit M. tuberculosis, so as to improve the effect of chemotherapy.

Advancement Of BCG immumotherapy in appling foundation and clinical research

Chinese Journal of Antituberculosis. 2012, 34(3): 184-187.

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Research progress in BCG prime-boost vaccination strategy against tuberculosis

Chinese Journal of Antituberculosis. 2012, 34(3): 188-191.

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