Tesis doctoral de Ainhoa Arbués Arribas
Tuberculosis is an old disease whose history is closely related to that of mankind; and its causative agent, mycobacterium tuberculosis, may have killed more persons than any other microbial pathogen. Although tuberculosis is treatable by drug therapy and there is a vaccine currently in use, bcg, the emergence of drug resistant strains and the variable efficacy of bcg against pulmonary manifestations of the disease make tuberculosis still today a global public health problem. given the variable protective efficacy generated by bcg, a worldwide coordinated effort is focused in the development of more efficacious vaccines. The starting point of the present study was the gene phop, which codifies for a transcription factor that regulates ~2% of m. Tuberculosis genome and is involved in virulence. The strain so2, a phop mutant constructed in m. Tuberculosis mt103 clinical isolate, is highly attenuated and confers good protection against tuberculosis in relevant animal models, hence pointing to the phop mutant as a promising vaccine candidate. Although the attenuated phenotype of so2 strain has been well studied, additional requirements have been proposed for live attenuated m. Tuberculosis candidates. For this reason, the present work has been focused on further characterization of so2 mutant strain and the construction of a new generation of phop-based vaccines that could be evaluated in human clinical trials. with the aim to characterize the so2 vaccine candidate in more depth, a complementation study in immunocompetent mice was performed. We found that phop complementation is unable to restore virulence of so2, pointing out to the presence of some additional genetic lesion contributing (together with phop mutation) to the attenuation of the strain in this mouse model. Biochemical analyses of the lipid composition of the mycobacterial envelope of so2 showed that this strain is devoid in the synthesis of the virulence-associated lipids phthiocerol dimycocerosates (pdim). Additional studies in mice demonstrated that together phop mutation and pdim deficiency contribute to the sound attenuation and protective properties characteristic of the so2 strain. rationally attenuated m. Tuberculosis mutants are promising new live vaccine candidates against tuberculosis; however, additional safety and stability criteria are required for entry in phase i trials. Two non-reverting independent mutations and elimination of antibiotic resistance markers were recommended in the geneva consensus document for live tuberculosis vaccines established in 2005. On the basis of the results obtained with so2, we chose to delete the fadd26 gene, which is essential for pdim synthesis, as a second stable mutation to fulfill these criteria. An unmarked stable m. Tuberculosis double deletion mutant in phop and fadd26 was constructed and named mtbvac. Western-blot analysis and transcriptional profile of the strain, together with the absence of replication in bone marrow-derived macrophages, demonstrated the phop-deficient phenotype of mtbvac. On the other hand, the lipid profile and inability to fix the dye neutral red confirmed the absence of several mycobacterial envelope complex lipids. These findings demonstrate that mtbvac is the first live attenuated m. Tuberculosis vaccine candidate to fulfill the geneva consensus requirements. preclinical characterization of so2 strain demonstrated proof of principle for adequate safety and protective efficacy of pdim-deficient phop-based vaccine candidates. In order to confirm that no unexpected collateral side effects have arisen during the construction process of mtbvac, these phenotypes were tested in relevant animal models.The results with the intermediate construct mtbvac::hyg showed that is more attenuated than bcg pasteur in immunodeficient scid mice and presents a protective efficacy profile identical to so2 in guinea pigs. Preliminary safety data with the final unmarked strain mtbvac demonstrate that this vaccine candidate presents comparable safety to bcg in a subcutaneous scid model using 50 times the human dose (in an endpoint-defined experiment). Based on these preclinical studies we deduce that data generated from proof-of-principle studies with the prototype so2 strain provides strong scientific support for the safety, attenuation and protective efficacy of mtbvac. Therefore, mtbvac is in position to move from research to development and be evaluated for the first time in humans. although the attenuated phenotype of mtbvac should primarily be attributed to the inactivation of phop gene and pdim deficiency, it can not be excluded that additional polymorphisms (like those in the wild-type mt103 strain) might have contributed to the attenuation features and vaccine properties of mtbvac. The comparative analysis of mtbvac genome using m. Tuberculosis h37rv as reference, revealed the presence of several genes absent from h37rv as well as multiple single nucleotide polymorphisms (snps) between the two strains. Interestingly, we identified two mtbvac-specific snps in papa2 and ppsb that could be responsible for the sulfolipid and pdim deficiency of mt103 and so2 strains, respectively.
Datos académicos de la tesis doctoral «Construction and characterization of a new generation of phop-based vaccines against tuberculosis«
- Título de la tesis: Construction and characterization of a new generation of phop-based vaccines against tuberculosis
- Autor: Ainhoa Arbués Arribas
- Universidad: Zaragoza
- Fecha de lectura de la tesis: 08/10/2010
Dirección y tribunal
- Director de la tesis
- Carlos Martín Montañés
- Tribunal
- Presidente del tribunal: rafael Gómez-lus lafita
- christophe Guilhot (vocal)
- Jesús anguel Gonzalo asensio (vocal)
- brigitte Gicquel (vocal)