Objective By perform the immunological characteristics and preclinical animal safety study of recombinant Mycobacterium tuberculosis fusion protein ESAT-6-CFP-10 (Which is the Chinese generic name of the drug determined by the National Pharmacopoeia Committee, EC stands for recombinant fusion protein 6 kDa early secretory antigenic target (ESAT-6) and 10 kDa culture filtrate protein (CFP-10))(EC), to discuss its clinical application prospect. Methods (1) Immunological characteristics study: 6 female guinea pigs were sensitized with Mycobacterium tuberculosis live bacteria solution (H37Ra), 5 weeks later, intradermally injected 0.2ml EC (2.5μg/ml); and 4 female guinea pigs were sensitized with live BCG bacteria solution, 5 weeks later, intradermally injected 0.2ml EC (20μg/ml). Observed the mean diameter of the induration or redness reaction ((longitudinal diameter + transverse diameter)/2, ≥5 mm is considered positive, <5 mm is considered negative). (2) Acute toxicity test: 80 ICR mice (Institute of Cancer Research (USA)) were divided into single intramuscular injection group and intradermal injection group, each group with 40, half male and half female. And then, each group was divided into 4 subgroups, each subgroup with 10, half male and half female. High dose subgroup was injected with EC of 53.61μg/0.1ml, while low does subgroup was 0.2μg/0.1ml, solvent control subgroup was injected with diluent (0.1ml) of EC, and the blank control subgroup would receive no injection. These mice were observed for abnormality in appearance, motor function, body weight, various organs and skin of injection site. (3) Guinea pig systemic active allergy test: 24 guinea pigs were divided into 4 subgroups, each subgroup with 6, half male and half female. Subgroups were injected intraperitoneally every other day with EC of 5μg/kg (high dose), 0.5μg/kg (low dose), bovine Serum Albumin, and 0.9%NaCl (2 ml) respectively, after 3 successive times, the sensitization finished. On the 12th day after the last sensitization, the sensitized guinea pigs were irritated by rapid intravenous injection with 2 times doses or more sensitizer. During sensitization, each animal’s symptom was observed daily and the body mass of each animal was measured on the first and last sensitization and irritation days. (4) Intradermal irritation experiment: 6 New Zealand rabbit was injected intradermally with EC of 10μg(0.2 ml)/spot, 5 spots on each side, observed the intradermal irritation reaction on the injection site. Results (1) Immunological characteristics study: In 4 BCG live bacteria solution sensitized guinea pigs, the skin tests with EC showed 0 positivity, and in 6 live MTB (H37Ra) sensitized guinea pigs, skin tests showed 6 with positivity. (2) Acute toxicity test: No significant acute toxic reactions or acute target organ poisoning were observed in all mice. The mean body masses of EC intradermally injected mice group at the time of: before injection, 1 d,3 d,5 d,7 d,8 d,10 d,12 d,14 d post injection, respectively in high dose subgroup and low dose subgroup were (20.6±1.3) g-(24.9±2.1) g,(20.5±1.6) g-(26.0±3.1) g;and compare to blank control group ((21.0±1.1) g-(25.3±2.3) g) in the corresponding time, the t values were 0.571-0.392;0.695-0.615;while P values were 0.575-0.700;0.496-0.546, respectively; the differences were not statistically significant. The mean body masses of EC intramuscularly injected mice group at the time of: before injection, 1 d,3 d,5 d,7 d,8 d,10 d,12 d,14 d post injection, respectively in high dose subgroup and low dose subgroup were (21.0±1.5) g-(26.2±1.9) g,(20.5±2.1) g-(25.8±3.8) g;and compare to blank control group ((21.2±1.7) g-(25.8±3.1) g) in the corresponding time, the t values were 0.360-0.318;0.900-0.006;while P values were 0.723-0.754;0.380-0.995;the differences were not statistically significant. (3) Guinea pig systemic active allergy test: no allergic reactions in guinea pigs. The average body mass of guinea pigs in the high,the low dose group of EC and 0.9%NaCl injected negative control group at the time of the first sensitization, the last sensitization, irritation and post irritation were (327.5±24.3) g,(347.2±32.7) g,(402.2±34.9) g;(331.3±26.7) g,(346.2±32.0) g,(411.3±38.9) g;(329.5±27.4) g,(348.3±27.0) g,(399.4±25.4) g, respectively. Compare to 0.9%NaCl injected negative control group, the t values were 0.328,0.181,0.284;0.474,0.366,0.875;while P values were 0.757,0.864,0.788;0.656,0.730,0.422, respectively, the differences were not statistically significant. (4) Intradermal irritation experiments in rabbits: for rabbits with single intradermal injection with 10μg(0.2 ml)/spot EC, no significant irritation reaction were observed. Conclusion EC is able to differentiate tuberculosis infection from BCG immunization. With good preclinical safety, it is expected to be applied in the in vivo diagnosis for Mycobacterium tuberculosis infection.
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