RESUMO
Mycobacterium bovis BCG has been used for a century as the only licensed vaccine against tuberculosis. Owing to its strong adjuvant properties, BCG has also been employed as an oncological immunotherapeutic as well as a live vaccine vector against other pathogens. However, BCG vaccination has limited efficacy in protecting against adult forms of tuberculosis (TB), raises concerns about its safety in immunocompromised populations, compromises the diagnosis of TB through the tuberculin test and lacks predictability for successful antigen expression and immune responses to heterologous antigens. Together, these factors propelled the construction and evaluation of auxotrophic BCG strains. Auxotrophs of BCG have been developed from mutations in the genes required for their growth using different approaches and have shown the potential to provide a model to study M. tuberculosis, a more stable, safe, and effective alternative to BCG and a vector for the development of recombinant live vaccines, especially against HIV infection. In this review, we provide an overview of the strategies for developing and using the auxotrophic BCG strains in different scenarios.
RESUMO
BACKGROUND: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug-resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. RESULTS: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. CONCLUSIONS: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii.
Assuntos
Marcadores Genéticos/genética , Pichia/genética , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.
Assuntos
Animais , Feminino , Camundongos , Plasmídeos/genética , Plasmídeos/imunologia , Vacina BCG/genética , Vacina BCG/imunologia , Vetores Genéticos/imunologia , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Escherichia coli/genética , Vetores Genéticos , Camundongos Endogâmicos BALB CRESUMO
Objetivo: Identificar la presencia del fenotipo auxotrofo en cepas de P. aeruginosa aisladas de pacientes con fibrosis quística o con VIH/SIDA. Métodos: Se realizó un estudio observacional descriptivo longitudinal utilizando la metodolog¡a de Barth y Pitt. En la determinación de la auxotrofía se realizó la siembra de la dilución 1:100 de cada cepa en un medio nutricionalmente completo agar Pseudomonas P (King A) y en un medio incompleto Agar Medio Mínimo y para la determinación del requerimiento específico en las auxotrofas se utilizaron 22 soluciones madre de aminoácidos en su forma L. Resultados: Se identificaron 20 cepas auxotrofas, once procedentes de pacientes VIH/SIDA y nueve de pacientes con fibrosis quística. Todos los aislamientos nutricionalmente dependientes proced¡an de muestras de esputo. El aminoácido simple de mayor requerimiento fue la metionina (7 de 20 aislamientos auxotrofos) seguido de arginina, lisina y glutamina. Conclusiones: Estos resultados sugieren que la P. aeruginosa auxotrofa coloniza a los pacientes VIH/SIDA durante el curso de la infección pulmonar al igual que en los pacientes con fibrosis quística. Confirmamos la auxotrofía como un fenotipo exclusivo de infecciones respiratorias.
Objective: To identify the presence of auxotrophic phenotype in strains of P. aeruginosa isolated from patients with cystic fibrosis or HIV/AIDS. Methods: We conducted a longitudinal descriptive study using the methodology of Barth and Pitt. For determination of auxotrophy, a seeding of a 1:100 dilution of each strain was made on nutritionally complete medium P Pseudomonas agar (King A) and in the incomplete medium minimal medium agar. For determining the specific requirement of auxotrophs, 22 amino acids solutions in their L-form were used. Results: We identified 20 auxotrophic strains, eleven from HIV/AIDS patients and nine frompatients with cystic fibrosis. All isolates that were nutritionally dependent came from sputum samples. The amino acid with the highest requirement was methionine (7 of 20 auxotrophs isolates) followed by arginine, lysine and glutamine. Conclusions: These results suggest that auxotrophic P. aeruginosa colonizes HIV/AIDS patients during the course of pulmonary infection, as in patients with cystic fibrosis. Auxotrophy was confirmed as a unique phenotype of respiratory infections.
Assuntos
Humanos , HIV , Escarro , Fenótipo , Fibrose Cística , Pseudomonas aeruginosa/isolamento & purificação , Síndrome da Imunodeficiência Adquirida , Epidemiologia Descritiva , Estudos Longitudinais , Estudos Observacionais como AssuntoRESUMO
Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.