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

Abstract

BackgroundTo investigate the ability of rESAT6 to identify different mycobacteria-sensitized guinea pigs and its safety in preclinical and phase I clinical study.Material/MethodsGuinea 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.ResultsPositive 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.ConclusionsThe rESAT6 can differentiate effectively among MTB infection, BCG vaccination, and NTM infection and is safe in healthy volunteers.

Safety Evaluation of Recombinant Fusion Protein RP22 as a Skin Test Reagent for Tuberculosis Diagnosis: A Phase I Clinical Trial

“…In animal studies, intradermal tests with ESAT6 and a combination of ESAT6 with CFP10 protein had shown safety and sensitivity to inducing specific skin test responses [17,18]. Several large and ongoing follow-up studies in humans have also shown specificity and sensitivity, proof of safety, feasibility, and dosage tolerability of a recombinant dimeric version of ESAT6 (rdESAT6) [19,20].…”

Teste intradérmico com proteínas recombinantes de Mycobacterium bovis como antígenos em Cavia porcellus

“…Para esse procedimento, além das técnicas de lise mecânica como a desrupção ultrassôni-ca e método físico como o choque térmico (congelamento/ descongelamento), é necessário lançar mão do processo de lise química utilizando agentes caotrópicos como a uréia e o hidrocloreto de guanidina que rompem as interações não covalentes, aumentam a solubilidade das substâncias apolares e desfazem as interações hidrofóbicas desnaturando as proteínas. No entanto, se levarmos em consideração que a imunotoxicidade é a questão mais importante à cerca dos reagentes utilizados para o diagnóstico intradérmico que depende de uma resposta imunológica (Du et al 2013), esses agentes desnaturantes precisam ser totalmente removidos das amostras proteicas por apresentarem toxicidade celular. Contudo, o processo de diálise de 72 h empregado nesse estudo parece não ter sido suficiente para completa remoção desses reagentes das amostras proteicas.…”

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

“…They showed that in treated TB patients, the responses to recombinant dimmer ESAT-6 were optimal at 0.1 μg (in 0.1 mL) [ 23 ]. Our phase I study showed that intradermal injection of 0.1 mL of 1 (15 cases), 5 (18 cases), and 10 μg/mL (10 cases) recombinant ESAT-6 protein is safe for human volunteers, and only a few local adverse events such as pain, itching, and blister occurred following ESAT-6 injection [ 17 ]. In the present study, administration of 0.1 mL of all doses, ranging from 2 to 10 μg/mL of ESAT-6, was well-tolerated by healthy volunteers and patients with pulmonary TB.…”

“…However, its promise for the use in the skin test has never been evaluated in humans. In a phase I study, we performed a preliminary assessment of the safety of a recombinant M. tuberculosis ESAT-6 protein in Chinese volunteers [ 17 ]. Then, we performed a phase II trial to examine the diagnostic performance and safety of the recombinant M. tuberculosis ESAT-6 protein used as a skin test for the diagnosis of pulmonary TB.…”