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Anti-drug antibody (ADA) positivity is correlated with disease relapse risk when treated with monoclonal antibody (mAb) therapeutics. ADA evaluation can assist with interpreting pharmacokinetic, pharmacological, and toxicology results. Here, we established an ADA assay based on two steps of acid dissociation combined with a bridging immunoassay to provide a comprehensive validation strategy. The three-tiered sample analysis process included screening, confirmation, and titration assays using therapeutic HLX26 (targeting lymphocyte activation gene-3 [LAG-3]) as an example. The cut points were determined by testing 50 individual normal human serum samples, including screening cut point (SCP) (SNR: 1.08), confirmatory cut point (CCP) (% inhibition: 12.65), and titration cut point (TCP) (sample-to-noise ratio [SNR]: 1.17). The assay sensitivity, low positive control (LPC), and high positive control (HPC) titer acceptable range were also set up as 33.0 ng/mL, 41.0 ng/mL, and 320-1280, respectively. After full validation, both the intra-assay and inter-assay precision testing passed with coefficient of variations (CVs) < 20%. The assay enabled excellent drug tolerance up to 768.0 µg/mL at the HPC level and 291.0 µg/mL at the LPC level, while the tolerance of target interference was up to 74.0 ng/mL of soluble LAG3. Moreover, no false-positive results were observed in the presence of 5% hemolyzed serum samples and 150 mg/dL of triglyceride in the serum samples, no hook effect was observed, and the stability performed normally under room temperature for 24 h, 2-8 °C for 7 d, and six freeze/thaw cycles. In summary, this ADA assay is feasible and could be used for evaluating the immunogenicity of HLX26 in clinical trials.
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BACKGROUND: Diagnostics to identify tuberculosis infection are limited. We aimed to assess the diagnostic accuracy and safety of ESAT6-CFP10 (EC) skin test for tuberculosis infection in Chinese adults. METHODS: We conducted 2 randomized, parallel-group clinical trials in healthy participants and tuberculosis patients. All participants were tested with the T-SPOT.TB test, then received an EC skin test and tuberculin skin test (TST). The diameter of skin indurations and/or redness at injection sites were measured at different time periods. A bacillus Calmette Guerin (BCG) model was established to assess the diagnosis of tuberculosis infection using an EC skin test. RESULTS: In total, 777 healthy participants and 96 tuberculosis patients were allocated to receive EC skin test at 1.0 µg/0.1 mL or 0.5 µg/0.1 mL. The area under the curve was 0.95 (95% confidence interval [CI], .91-.97) for the EC skin test at 1.0 µg/0.1 mL at 24-72 hours. Compared with the T-SPOT.TB test, the EC skin test demonstrated similar sensitivity (87.5, 95% CI, 77.8-97.2 vs 86.5, 95% CI, 79.5-93.4) and specificity (98.9, 95% CI, 96.0-99.9 vs 96.1, 95% CI, 93.5-97.8). Among BCG vaccinated participants, the EC skin test had high consistency with the T-SPOT.TB test (96.3, 95% CI, 92.0-100.0). No serious adverse events related to the EC skin test were observed. CONCLUSIONS: The EC skin test demonstrated both high specificity and sensitivity at a dose of 1.0 µg/0.1 mL, comparable to the T-SPOT.TB test. The diagnostic accuracy of the EC skin test was not impacted by BCG vaccination. CLINICAL TRIALS REGISTRATION: NCT02389322 and NCT02336542.
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Tuberculosis Latente , Mycobacterium tuberculosis , Tuberculosis , Adulto , China , Humanos , Sensibilidad y Especificidad , Prueba de Tuberculina , Tuberculosis/diagnósticoRESUMEN
BACKGROUND: Cell lytic enzyme is a kind of highly evolved protein, which can destroy the cell structure and kill the bacteria. Compared with antibiotics, cell lytic enzyme will not cause serious problem of drug resistance of pathogenic bacteria. Thus, the study of cell wall lytic enzymes aims at finding an efficient way for curing bacteria infectious. Compared with using antibiotics, the problem of drug resistance becomes more serious. Therefore, it is a good choice for curing bacterial infections by using cell lytic enzymes. Cell lytic enzyme includes endolysin and autolysin and the difference between them is the purpose of the break of cell wall. The identification of the type of cell lytic enzymes is meaningful for the study of cell wall enzymes. OBJECTIVE: In this article, our motivation is to predict the type of cell lytic enzyme. Cell lytic enzyme is helpful for killing bacteria, so it is meaningful for study the type of cell lytic enzyme. However, it is time consuming to detect the type of cell lytic enzyme by experimental methods. Thus, an efficient computational method for the type of cell lytic enzyme prediction is proposed in our work. METHODS: We propose a computational method for the prediction of endolysin and autolysin. First, a data set containing 27 endolysins and 41 autolysins is built. Then the protein is represented by tripeptides composition. The features are selected with larger confidence degree. At last, the classifier is trained by the labeled vectors based on support vector machine. The learned classifier is used to predict the type of cell lytic enzyme. RESULTS: Following the proposed method, the experimental results show that the overall accuracy can attain 97.06%, when 44 features are selected. Compared with Ding's method, our method improves the overall accuracy by nearly 4.5% ((97.06-92.9)/92.9%). The performance of our proposed method is stable, when the selected feature number is from 40 to 70. The overall accuracy of tripeptides optimal feature set is 94.12%, and the overall accuracy of Chou's amphiphilic PseAAC method is 76.2%. The experimental results also demonstrate that the overall accuracy is improved by nearly 18% when using the tripeptides optimal feature set. CONCLUSION: The paper proposed an efficient method for identifying endolysin and autolysin. In this paper, support vector machine is used to predict the type of cell lytic enzyme. The experimental results show that the overall accuracy of the proposed method is 94.12%, which is better than some existing methods. In conclusion, the selected 44 features can improve the overall accuracy for identification of the type of cell lytic enzyme. Support vector machine performs better than other classifiers when using the selected feature set on the benchmark data set.
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Biología Computacional , Endopeptidasas/aislamiento & purificación , N-Acetil Muramoil-L-Alanina Amidasa/aislamiento & purificación , Proteínas/aislamiento & purificación , Algoritmos , Secuencia de Aminoácidos/genética , Aminoácidos/genética , Antibacterianos/química , Bacterias/efectos de los fármacos , Bacterias/patogenicidad , Endopeptidasas/química , Endopeptidasas/genética , Humanos , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/genética , Proteínas/química , Proteínas/genética , Máquina de Vectores de SoporteRESUMEN
The high global burden of tuberculosis (TB) underscores the urgent need for an effective TB vaccine since the only licensed Bacillus Calmette-Guérin (BCG) vaccine is ineffective in preventing adult pulmonary TB and affords no protection against latent TB infection (LTBI). Herein we investigated the potential of Mycobacterium tuberculosis (Mtb) antigen proteins AEC comprised of Ag85b and ESAT6-CFP10 proteins in conjunction with aluminum (Al) and polyriboinosinic-polyribocytidylic acid (poly-IC) as a novel subunit vaccine against TB. The immunogenicity and protection induced by the adjuvanted vaccine were evaluated in two animal models. Mice vaccinated with AEC/Al/poly-IC exhibited significant antigen-specific humoral immune responses and cell-mediated immunity as determined by immunoassay and multicolor flow cytometric assay, and the protective effect of the vaccine was demonstrated in a guinea pig model of latent Mtb infection. Compared to the control group, the mean pathological scores and bacterial loads in lungs and spleens of AEC/Al/poly-IC-immunized guinea pigs were significantly reduced. These data indicate that the AEC/Al/poly-IC is highly immunogenic in mice and can effectively protect guinea pigs against latent Mtb infection; it may represent a promising candidate vaccine for the control of latent TB.
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Adyuvantes Inmunológicos/administración & dosificación , Aluminio/inmunología , Tuberculosis Latente/inmunología , Mycobacterium tuberculosis/inmunología , Fragmentos de Péptidos/inmunología , Poli I-C/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Antígenos Bacterianos/inmunología , Vacuna BCG/inmunología , Proteínas Bacterianas/inmunología , Femenino , Cobayas , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Inmunización/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Bazo/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Vacunación/métodos , Vacunas de Subunidad/inmunologíaRESUMEN
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.
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Antígenos Bacterianos/inmunología , Tuberculosis Latente/inmunología , Mycobacterium tuberculosis/inmunología , Células TH1/inmunología , Vacunas contra la Tuberculosis/inmunología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Vacuna BCG/inmunología , Carga Bacteriana/inmunología , Modelos Animales de Enfermedad , Cobayas , Interferón gamma/inmunología , Interferón gamma/metabolismo , Tuberculosis Latente/microbiología , Tuberculosis Latente/prevención & control , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Bazo/microbiología , VacunaciónRESUMEN
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.
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Antígenos Bacterianos , Proteínas Bacterianas , Proteínas Recombinantes , Tuberculosis Pulmonar/diagnóstico , Adulto , Análisis de Varianza , Antígenos Bacterianos/efectos adversos , Antígenos Bacterianos/genética , Área Bajo la Curva , Proteínas Bacterianas/efectos adversos , Proteínas Bacterianas/genética , China , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/genética , Pruebas CutáneasRESUMEN
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.
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Antígenos Bacterianos/efectos adversos , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/efectos adversos , Proteínas Bacterianas/inmunología , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/inmunología , Adulto , Anciano , Animales , Antígenos Bacterianos/administración & dosificación , Proteínas Bacterianas/administración & dosificación , Relación Dosis-Respuesta Inmunológica , Evaluación Preclínica de Medicamentos , Femenino , Cobayas , Humanos , Inmunización , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/administración & dosificación , Tuberculina/inmunología , Prueba de Tuberculina , Adulto JovenRESUMEN
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.
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Antígenos Bacterianos/inmunología , Vacunas contra la Tuberculosis/inmunología , Vacunas Sintéticas/inmunología , Adyuvantes Inmunológicos , Animales , Vacuna BCG/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Mycobacterium bovis/inmunología , Tuberculosis/prevención & controlRESUMEN
OBJECTIVE: To establish the guinea pig model of latent Mycobacterium tuberculosis (MTB) H37Rv infection, and to study the multiplication dynamics of MTB in vivo, and the relationship between latent MTB infection and PPD skin test. METHODS: Sixty-two guinea pigs were randomly divided into the model group (n = 42) and the control group (n = 20), and the model group was subdivided into a 4 weeks group (n = 12), an 8 weeks group (n = 21) and a 12 weeks group (n = 9), challenged by 500 CFU H37Rv with restored toxicity. After 2 weeks challenge, the model groups were treated with isoniazid (INH, 10 mg/kg) + pyrazinamidum aldinamide (PZA, 40 mg/kg) for 4 weeks, 8 weeks and 12 weeks respectively. The natural recurrence of tuberculosis was observed in the model 4 weeks group, and the natural and induced recurrence by dexamethasone was observed in the model 8 weeks group and 12 weeks group. PPD skin test, the pathologic changes, and MTB quantity of organs were observed. RESULTS: In the control group, the average MTB quantity of spleen was 3.3 lg CFU after 2 weeks challenge, and the average MTB quantity of spleen and lung in guinea pigs were 4.5 lg CFU and 1.8 lg CFU respectively after 6 weeks challenge, and they reached 5.3 lg CFU and 5.4 lg CFU at 18 weeks respectively. The latent MTB infection of the model 4 weeks group recurred naturally 12 weeks after stopping treatment. The latent MTB infection of the model 8 weeks group recurred naturally and by dexamethasone treatment. The latent MTB infection of the model 12 weeks group did not recur naturally, but dexamethasone induced recurrence. The positive PPD response correlated with recurrence. CONCLUSIONS: A latent MTB infection model was established successfully by H37Rv challenge and treatment with INH and PZA. The latent MTB infection may recur naturally or by induction. The PPD response was related to tuberculosis recurrence.
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Modelos Animales de Enfermedad , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/patología , Animales , Cobayas , Masculino , Tuberculosis/microbiologíaRESUMEN
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.
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Aciltransferasas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Hidróxido de Aluminio/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Recuento de Colonia Microbiana , Femenino , Cobayas , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Hígado/patología , Pulmón/patología , Linfocitos/inmunología , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Bazo/inmunología , Bazo/microbiología , Bazo/patología , Vacunas de Subunidad/inmunologíaRESUMEN
It is well known that a deletion within chromosome 22q11.2 has been identified in most cases of congenital heart disease (CHD) with DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS). Whether the 22q11.2 deletion is associated with isolated CHD is controversial. Our data is consistent with previous publications which show that the 22q11.2 deletion is associated with isolated CHD even though it is rare.
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Deleción Cromosómica , Síndrome de DiGeorge/genética , Cardiopatías Congénitas/genética , Síndrome de DiGeorge/diagnóstico , Cardiopatías Congénitas/diagnóstico , HumanosRESUMEN
OBJECTIVE: To create and evaluate the PCR restriction fragment length polymorphism (PCR-RFLP) based on hsp65 gene as a method for rapid identification of Mycobacteria to the species level. METHODS: hsp65 gene was amplified from the DNA of mycobacterial reference strains and the PCR products were subjected to digestion by two restriction endonucleases Hae III and Bstp I, then loaded onto a 4% MetaPhor agorose. The size of the restricted fragments of each species (strains) was determined according to the position of the fragments on the gel, by which the differential DNA fingerprint was confirmed. RESULTS: A total of 40 Mycobacterium species (strains) was analyzed, in which six reference strains of Mycobacterium tuberculosis Complex had two different electrophoresis patterns, and thirty-four reference species of non-tuberculosis Mycobacteria had unique pattern. CONCLUSION: PCR-RFLP Based upon hsp65 gene can be used for identification of Mycobacterium species, and the method is more rapid and simple and easy-to-use for mycobacterial species identification.
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Proteínas Bacterianas/genética , Chaperonina 60/genética , Mycobacterium/clasificación , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Dermatoglifia del ADN , Enzimas de Restricción del ADN , ADN Bacteriano , Mycobacterium/genética , Mycobacterium/aislamiento & purificación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificaciónRESUMEN
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.
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Aciltransferasas/aislamiento & purificación , Adyuvantes Inmunológicos/uso terapéutico , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Mycobacterium tuberculosis/metabolismo , Aciltransferasas/administración & dosificación , Aciltransferasas/uso terapéutico , Animales , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/uso terapéutico , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/uso terapéutico , Escherichia coli , Inmunidad Celular , Interferón gamma , Ratones , Mycobacterium tuberculosis/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/uso terapéuticoRESUMEN
OBJECTIVE: To study the effect of Mycobacterium smegmatis vaccine on the level of nitric oxide (NO) produced by peritoneal macrophages in immunized mice. METHODS: Balb/c mice were randomized into low-dose, middle-dose, and high-dose groups (injected with different doses of Mycobacterium smegmatis vaccine) and a control group (injected with normal saline). Then the peritoneal macrophages were cultured with lipopolysaccharide in vitro. The supernatants were collected and the concentrations of NO were analyzed through the reaction with Griess reagents. RESULTS: The levels of NO produced by the peritoneal macrophages in the control group, low-dose group, middle-dose group, and high-dose group were (3.50 +/- 3.11), (16.63 +/- 6.47), (13.97 +/- 6.20), and (7.55 +/- 2.26) ng/ml, respectively. The levels of NO in all dosing groups were significantly different from that in control group (P < 0.01). CONCLUSION: Mycobacterium smegmatis vaccine can promote the peritoneal macrophages to produce NO in mice.
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Vacunas Bacterianas/uso terapéutico , Macrófagos Peritoneales/metabolismo , Mycobacterium smegmatis , Óxido Nítrico/metabolismo , Animales , Lipopolisacáridos , Ratones , Ratones Endogámicos BALB CRESUMEN
OBJECTIVE: To analyze the genotypes of intraspecies of common mycobacteria. METHODS: The genotypes of 94 strains of mycobacteria from DSMZ, as compared with 12 reference strains, were studied by 16S-23S rRNA internal transcription space (ITS) sequence analysis. RESULTS: The sequencing of 16S-23S rRNA ITS of the 106 strains of common mycobacteria were completed. All the mycobacteria could be discriminated to several genotypes except 4 M. intracellulare, 4 M. avium, 6 M. marinum and 2 M. malmoense which were identical to their reference strains. CONCLUSIONS: 16S-23S rRNA ITS sequence analysis is a reliable method to discriminate mycobacteria in interspecies even in intraspecies.
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Mycobacterium/clasificación , Mycobacterium/genética , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , ADN Espaciador Ribosómico/genética , Genes de ARNr , Genotipo , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
A method for the identification of Mycobacterium species using reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The fingerprints library was constructed on the basis of RP-HPLC chromatograms of the mycolic acids derivatives from 49 Mycobacterium species cultures. Two inoculation loops of Mycobacterium, for fast growth with one week incubation or for slow growth with three weeks incubation, were saponified for 1 h and stocked at 4 degrees C. The mycolic acids from each culture of Mycobacterium species were acidified, extracted, derivatized, and analyzed by the RP-HPLC method. On the basis of the HPLC patterns of relative retention time and relative peak height, the identifications of Mycobacterium species were performed. This established method has a good precision of retention times with the relative standard deviations (RSDs) ranging from 0.13% to 1.07%. The mycolic acids fingerprints library of HPLC patterns was set up, including 49 species that were recorded in "Bergey's Manual of Systematic Bacteriology". Three patterns were observed from the chromatographic behaviors of mycolic acids derivatives, including single peak-cluster, double peak-clusters, triple and multiple peak-clusters. Forty-one species were successfully identified according to the different relative retention times of the peaks and the relative peak heights. The established method can identify Mycobacterium species with rapidity and high reliability.
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Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Mycobacterium/química , Mycobacterium/clasificación , Ácidos Micólicos/análisis , Ácidos Micólicos/química , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: To investigate the presence of rifampicin-dependent Mycobacterium tuberculosis strains by use of a guinea pig model of tuberculosis of rifampicin-dependent Mycobacterium tuberculosis. METHODS: Guinea pigs were randomly divided into groups of infection by rifampicin-dependent Mycobacterium tuberculosis strains (1130 strain, 1219 strain, b858 strain), rifampicin-resistant Mycobacterium tuberculosis strain (1290 strain) and ATCC 35810 strain and each group was further divided into an experimental group and a control group. The guinea pigs were challenged with 1130 strain, 1219 strain, b858 strain, 1290 strain and ATCC 35810 strain to establish the tuberculosis model. The experimental groups were treated with rifampicin. The parameters including macroscopic visceral pathological change index, visceral weight index (spleen, lungs and liver), the colony-forming units (CFU) quantity of visceral Mycobacterium tuberculosis culture (spleen, lungs) and tissue pathology of guinea pigs were observed. RESULTS: At the 7th week after challenged with 1130 strain, 1219 strain, 1290 strain and b858 strain, all animals were sacrificed. The macroscopic visceral pathological change indices of the experimental group were 68.7 +/- 13.8, 60.0 +/- 13.5, 70.0 +/- 5.8 and 23.8 +/- 18.9, whereas all those parameters of the control group were 76.2 +/- 18.9, 40.0 +/- 16.8, 63.8 +/- 10.3 and 22.5 +/- 15.5 respectively, and there was no significance between the experimental group and the control group (t = 0.64, 1.85, 0.35 and 0.10, all P > 0.05). The spleen weight indices of experimental group were 0.229 +/- 0.048, 0.256 +/- 0.067, 0.324 +/- 0.054 and 0.199 +/- 0.029, whereas all those parameters of control groups were 0.278 +/- 0.025, 0.216 +/- 0.076, 0.368 +/- 0.033 and 0.213 +/- 0.038 respectively, and there was no significance between the experimental group and the control group (t = 1.75, 0.79, 1.41 and 0.57, all P > 0.05). The CFU quantity of spleen Mycobacterium tuberculosis culture of the experimental group were 4.98 +/- 0.30, 4.68 +/- 1.26, 5.07 +/- 0.47 and 3.85 +/- 0.45, whereas all those parameters of control groups were 4.90 +/- 1.03, 4.79 +/- 0.45, 5.08 +/- 0.55 and 4.23 +/- 0.95 respectively, and there was no significance between the experimental group and the control group (t = 0.11, 0.15, 0.03 and 0.73, all P > 0.05); Moreover, the tissue pathology of both groups was similar. CONCLUSIONS: The tuberculosis model of rifampicin-dependent Mycobacterium tuberculosis strains was similar to the model of rifampicin-resistant Mycobacterium tuberculosis in guinea pigs. Rifampicin-dependency was not evident in this guinea pig tuberculosis model.