Recombinant tuberculosis vaccine AEC/BC02 induces antigen-specific cellular responses in mice and protects guinea pigs in a model of latent infection.

PURPOSE: To preliminarily evaluate the immunogenicity and efficacy of the recombinant tuberculosis vaccine AEC/BC02 in which Ag85b and fusion protein ESAT6-CFP10 were combined with bacillus Calmette-Guérin CpG and an aluminum salt-based adjuvant system. METHODS: Groups of BALB/c mice were immunized intramuscularly three times at 10-day intervals with AEC/BC02 or the adjuvant alone and the vaccine-induced cell-mediated immune responses were evaluated. The efficacy of AEC/BC02 was evaluated in two guinea pig models, one a model of prevention and the other a model of latent infection. RESULTS: The AEC/BC02 vaccine induced strong cellular immune responses characterized by a high frequency of antigen-specific interferon-γ-secreting T cells in mice at different time points after the last vaccination. In the preventive model of guinea pig, AEC/BC02 did not protect against Mycobacterium tuberculosis as a pre-exposure vaccine. However, in a latent infection model of guinea pig, it effectively controlled the reactivation of M. tuberculosis and lowered the bacterial load in the lung and spleen. CONCLUSION: These results indicate AEC/BC02 can protect against reactivation of latent infection and may function as a therapeutic vaccine.

Efficacy and safety of recombinant Mycobacterium tuberculosis ESAT-6 protein for diagnosis of pulmonary tuberculosis: a phase II trial.

BACKGROUND: This study aimed to determine the efficacy and safety of recombinant Mycobacterium tuberculosis ESAT-6 protein for diagnosis of pulmonary tuberculosis (TB). MATERIAL AND METHODS: A phase II trial was performed in 158 patients with pulmonary TB (145 initially-treated and 13 re-treated) and 133 healthy subjects. Skin testing was carried out by injecting purified protein derivative (PPD) (on left forearm) or recombinant ESAT-6 protein at a dosage of 2, 5, or 10 µg/mL (on the right forearm) in each subject. Reaction activity and adverse events were monitored at 24, 48, and 72 h following the injection. Receiver operating characteristic curves were plotted to determine the areas under the curves (AUCs) and the cut-off induration diameters for the optimal diagnostic performance. RESULTS: The reaction activity was significantly increased upon recombinant ESAT-6 injection in pulmonary TB patients compared with healthy subjects. In pulmonary TB patients, the reaction was dose-dependent, and at 48 h, 10 µg/mL recombinant ESAT-6 produced a reaction similar to that produced by PPD. The AUCs for a 10 µg/mL dosage were 0.9823, 0.9552, and 0.9266 for 24 h, 48 h, and 72 h, respectively, and the induration diameters of 4.5-5.5 mm were the optimal trade-off values between true positive rates and false positive rates. No serious adverse events occurred in any subjects. CONCLUSIONS: Recombinant ESAT-6 protein is efficacious and safe for diagnosing pulmonary TB. Based on the reaction, performance, safety, and practicability, we recommend that 10 µg/mL at 48 h with an induration cut-off value of 5.0 mm be used.

Preclinical study and phase I clinical safety evaluation of recombinant Mycobacterium tuberculosis ESAT6 protein.

BACKGROUND: To investigate the ability of rESAT6 to identify different mycobacteria-sensitized guinea pigs and its safety in preclinical and phase I clinical study. MATERIAL AND METHODS: Guinea pigs were sensitized with different Mycobacteria. After sensitization, all animals were intradermally injected with rESAT6 and either PPD or PPD-B. At 24 h after the injection, the erythema of the injection sites were measured using a double-blind method. For the preclinical safety study, different doses of rESAT6 and BSA were given 3 times intramuscularly to guinea pigs. On day 14 after the final immunization, the guinea pigs were intravenously injected with the same reagents in the hind legs and the allergic reactions were observed. A single-center, randomized, open phase I clinical trial was employed. The skin test was conducted in 32 healthy volunteers aged 19-65 years with 0.1 µg, 0.5 µg, and 1 µg rESAT6. Physical examination and laboratory tests were performed before and after the skin test and adverse reactions were monitored. The volunteers' local and systemic adverse reactions and adverse events were recorded for 7 days. RESULTS: Positive PPD or PPD-B skin tests were observed in all Mycobacteria-sensitized guinea pigs; the diameters of erythema were all >10 mm. The rESAT6 protein induced a positive skin test result in the guinea pigs sensitized with MTB, M. bovis, M. africanum and M. kansasii; the diameters of erythema were 14.7±2.0, 9.3±3.8, 18.7±2.4, and 14.8±4.2 mm, respectively. A negative skin test result was detected in BCG-vaccinated and other NTM-sensitized guinea pigs. The rESAT6 caused no allergic symptoms, but many allergic reactions, such as cough, dyspnea, and even death, were observed in the guinea pigs who were administered BSA. During the phase I clinical trial, no adverse reactions were found in the 0.1 µg rESAT6 group, but in the 0.5 µg rESAT6 group 2 volunteers reported pain and 1 reported itching, and in the 1 µg rESAT6 group there was 1 case of pain, 1 case of itching, and 1 case of blister. No other local or systemic adverse reactions or events were reported. CONCLUSIONS: The rESAT6 can differentiate effectively among MTB infection, BCG vaccination, and NTM infection and is safe in healthy volunteers.

[The effect of various antigen compounds of tuberculosis composite antigen vaccine on the immune function in mice].

OBJECTIVE: To study the immune function of mice immunized by different combinations of antigen 85b (Ag85b), fusion protein culture filtered protein 10 (CFP-10), early secreted antigenic target 6 kDa protein (ESAT-6) and heat shock protein X (Hsp X) with combined adjuvants of Bacille Calmette-Guerin (BCG) CpG and aluminum. METHODS: According to antigen combinations, 48 BALB/c mice were divided into 8 groups: (1) group A: Ag85b + CFP-10/ESAT-6 + HspX + adjuvant; (2) group B: CFP-10/ESAT-6 + HspX + adjuvant; (3) group C: Ag85b + HspX + adjuvant; (4) group D: Ag85b + CFP-10/ESAT-6 + adjuvant; (5) group E: Ag85b + adjuvant; (6) group F: CFP-10/ESAT-6 + adjuvant; (7) group G: HspX + adjuvants; (8) control group: saline (6 mice per group). The mice were subcutaneously immunized 3 times. One week after the third subcutaneous immunization, spleens were collected for enzyme-linked immunospot (ELISPOT) assay to detect IFN-γ and IL-4 secretion, and for the lymphocyte proliferation assay to observe antigen-specific lymphocyte proliferation. Serum samples were separated for enzyme-linked immunosorbent assay (ELISA) to detect the titers of antigen-specific IgG, IgG(1) and IgG(2a) antibodies. RESULTS: The amount of IFN-γ spots in Group E [median(quartile), 122.8 (78.4 - 184.4)] was significantly more than that in group C [14.3 (6.5 - 14.6)] and the control group [0.5 (0.5 - 1.3)] (u = 0.0, P < 0.01). The amount of IL-4 spots in Group D stimulated with Ag85b and CFP-10/ESAT-6 [173.5 (78.8 - 233.4), 132.8 (50.3 - 159.4)] were significantly more than those in the control group [0.5 (0.5 - 1.3), 5.3 (2.9 - 6.5)] (u = 0.0, P < 0.01). The level of stimulation index of lymphocyte proliferation in Group A, C, D, E (2.42 ± 0.50, 2.18 ± 0.37, 2.86 ± 0.51, 2.70 ± 0.15) was significantly higher than that of the control group (1.11 ± 0.13) (F = 20.96, P < 0.01). The level of antigen-specific IgG, IgG(1), IgG(2a) antibody titers induced by Hsp X [lg(antibody dilution degree), 3.90 - 5.21] was significantly higher than those induced by Ag85b (3.30 - 4.51) and CFP-10/ESAT-6 (3.10 - 4.05) (F = 63.8 - 70.4, P < 0.01). CONCLUSIONS: With the use of adjuvants, different antigen combinations showed different influences on the immune function in mice. A combination of 3 antigens did not elicit the best immune effect, suggesting that the interaction among antigens may affect their immunity.

The development and preliminary evaluation of a new Mycobacterium tuberculosis vaccine comprising Ag85b, HspX and CFP-10:ESAT-6 fusion protein with CpG DNA and aluminum hydroxide adjuvants.

Ag85b and HspX of Mycobacterium tuberculosis (Mtb) (H37Rv) were expressed and purified in this study. These two proteins were combined with another fusion protein CFP-10:ESAT-6 (C/E) (Ag), then mixed with the adjuvants CpG DNA and aluminum hydroxide and used to vaccinate mice and guinea pigs challenged with Mtb (H37Rv). The number of spleen lymphocytes secreting Ag85b, HspX and C/E-specific interferon-gamma were significantly higher in the Ag+Al+CpG group than in the Ag and CpG groups. The combination of Ag, Al and CpG induced the highest concentrations of anti-Ag85b, anti-HspX and anti-C/E immunoglobulin G in mouse serum. Mouse peritoneal macrophages from the Ag+Al+CpG group secreted significantly higher levels of interleukin-12 compared with macrophages from the other groups. The total mean liver, lung and spleen lesion scores and bacterial loads in the spleen in guinea pigs vaccinated with Ag+Al+CpG were lower than those of the other groups, but no significant difference was found. These results show that the mixture of Ag85b, HspX and C/E with a combination of CpG and aluminum adjuvants can induce both humoral and cellular immune responses in mice, whereas it plays only a small role in the control of disease progression in guinea pigs challenged with Mtb.

[Preparation of two antigens--Ag85b and HspX of Mycobacterium tuberculosis H37Rv and the effects of their co-administration with adjuvants in mice].

OBJECTIVE: To synthesize two antigens-Ag85b and HspX of Mycobacterium tuberculosis H37Rv with molecular biological methods and to observe their biologic activity after co-administration of adjuvants (aluminum and/or CpG) in mice. METHODS: Recombinant expression plasmids pET30a-Ag85b and pET30a-HspX were constructed. The objective DNA fragments was characterized with restriction enzyme. Then the recombinant plasmids were transformed into E. coli BL-21, and two proteins were expressed by induction of isopropyl beta-D-1-thiogalactopyranoside. After purification with anion exchange column Source30, QHP, and hydrophobic chromatography column, two proteins were identified by amino acid sequencing. After the successful preparation of these two antigens, they were co-administered in mice with adjuvants of aluminum and/or CpG (Ag85b, Ag85b + Al, Ag85b + CpG, Ag85b + Al + CpG; HspX, HspX + Al, HspX + CpG, HspX + Al + CpG); one group received normal saline and served as the control. Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma (IFN-gamma); in addition, lymphocytes proliferation test was performed to observe lymphocytes proliferation after in vitro stimulated with two antigens. RESULTS: The purity of two proteins reached 95% after purification. The N-terminal amino acid sequence (15 aa) of the purified proteins was same as the target sequence. For Ag85b, the secreted specific IFN-gamma from isolated splenic lymphocytes after having been stimulated in vitro with Ag85b (80 microg/ml) remarkably increased in Ag85b + CpG group, Ag85b + Al group, and Ag85b + CpG + Al group; the changes were significantly different between these three groups and control group (P < 0.05). For HspX, the changes were significantly different between HspX + Al + CpG group and normal sodium group, although remarked increase of IFN-gamma was also observed in HspX group, HspX + Al group, and HspX + CpG group. CONCLUSIONS: Ag85b and HspX were successfully expressed and purified. A cell-mediated immunity may be induced when the antigens are co-administered with adjuvants of aluminum and/or CpG in mice, indicating that the recombinant proteins are bioactive.