The current worldwide epidemic of Mycobacterium tuberculosis infection is a huge global health problem. Widespread BCG vaccination remains a useful tool in combating this epidemic; however, its variable efficacy requires urgent development of novel vaccines against Mycobacterium tuberculosis. Such a candidate vaccine is a serotype 5 adenovirus expressing antigen 85A from M. tuberculosis (Ad85A). In animal models Ad85A confers significant protection when administered intra-nasally. The work in this thesis demonstrates that intra-nasal immunisation with Ad85A results in inhibition of M. tuberculosis growth in the lung early after infection, in contrast to the late inhibition induced by parenterally administered vaccines. Early inhibition correlates with the presence in the lung of a highly activated population of antigen-specific CD8 T cells, maintained for at least 6 months post-immunisation by persistent antigen. For intra-nasal Ad85A to be effective, the vaccine must be delivered into the lower respiratory tract, as immunisation targeting only the nasal-associated lymphoid tissue (NALT) does not result in protection. Following a change of animal facility, the lung immune response to intra-dermal immunisation with Ad85A increased and this route of immunisation now induced protection, though growth of M. tuberculosis was inhibited only late after infection. However, this response and protection can be altered by exposure to environmental mycobacteria. Further experiments showed that simultaneous respiratory and parenteral immunisations (SIM) act additively, where local lung immunity inhibits the growth of M. tuberculosis early after infection and systemic immunity protects later. SIM regimes generate greatly improved protection over either immunisation alone and do not depend on priming and boosting.

Made available in DSpace on 2014-07-29T15:13:53Z (GMT). No. of bitstreams: 1 TeseEdiane_Batista.pdf: 714564 bytes, checksum: 03efd3ac163525d9d401336bdd75356e (MD5) Previous issue date: 2008-08-05 / Several aspects of the bovine tuberculosis were analyzed in this study. The immunogenicity of recombinant MPT-51 (rMPT-51), Ag-85 and M. bovis-BCG were characterized in an immunosorbent assay, where 208 serum samples from positive intradermal tuberculin test (ITT) animals and 54 serum samples from ITT negative animals where analyzed. M. bovis-BCG and Ag-85 were strongly recognized by antibodies from naturally infected cattle. Additionally, the clinical status of the animals were correlated with the ITT positivity, with the specific production of IL-4 by TCD4 and TCD8 positive lymphocytes, and with nitric oxide (NO) production by macrophages from naturally tuberculosis infected bovine peripheral blood. ITT positive animals showed TCD4+IL4+ cells specific to M. bovis-BCG extract. High background levels of TCD8+IL-4+ lymphocytes were observed in ITT positive animals independent of the stimuli. When cell cultures where stimulated with M. bovis-BCG protein extract, there was no observed difference in NO production between the groups. Naturally tuberculosis infected bovine presented TCD4+IL4+ cells specific for M. bovis- BCG and a preserved NO production. Finnally, the immunogenicity of rMPT-51 use as a proteic sub-unit vaccine was evaluated in BALB/c mice, with two different adjuvants, incomplete Freund and CpG DNA. For this, mice were immunized and challenged with M. tuberculosis. Immunization with rMPT-51 antigen and either adjuvant induced, in the lungs, a migration increase of TCD5+IFN + cells specific for rMPT-51, when compared to controls (P<0.05). rMPT-51 plus CpG DNA presented a better performance among the different vaccination schemes tested, in part due to the ability of stiulate TCD5+IFN + cells and hampering the bacterial load, thus preserving the functional integrity of challenged mice lungs / Nesta tese foram avaliados vários aspectos da tuberculose bovina e do Mycobacterium bovis. Primeiro caracterizou-se a imunogenicidade do MPT-51, Ag 85 e BCG em ensaio imunoenzimático, onde foram utilizadas 208 amostras de soro de animais TTI positivo e 54 amostras de bovinos negativos. O BCG-M. bovis e o Ag 85 foram fortemente reconhecidos pelos anticorpos dos bovinos naturalmente infectados. Segundo correlaciounou-se o estado clínico do animal à positividade ao teste intradérmico, com a produção específica de IL-4 por linfócitos TCD4+ e TCD8+ e a produção de óxido nítrico por macrófagos do sangue periférico de bovinos naturalmente infectados com tuberculose. Foi observado que os bovinos TTI positivos apresentaram células CD4+IL-4+ específicas para extrato protéico de M. bovis-BCG. Observaram-se altos níveis basais de linfócitos TCD8+IL-4+ independente de estímulos nos animais reatores. Quando as culturas foram estimuladas com extrato protéico de BCG, não houve diferença quanto à produção de NO. Os bovinos tuberculosos, naturalmente infectados, apresentaram células TCD4+IL4+ específicas para M. bovis-BCG, preservando a produção de NO pelos macrófagos. Finalmente, a imunogenicidade do MPT-51 como vacina de sub-unidade protéica foi avaliada em camundongos BALB/c, testando o MPT-51 com dois adjuvantes, o incompleto de Freund e o CpG DNA. Os camundongos foram imunizados e posteriormente desafiados com M. tuberculosis. No pulmão, a imunização com antígeno rMPT-51 a 20&#956;g/ml + adjuvantes de Freund Incompleto e CpG DNA induziu aumento da migração de células TCD5+IFN- + específicas para o MPT-51 para o local da infecção, quando comparados com o controle (p<0,05). O MPT-51+CpG DNA, apresentou melhor desempenho dentre os esquemas de vacinação adotados, devido a capacidade de estimular a produção de células TCD5+IFN- + e a diminuição da carga bacteriana, preservando assim a integridade funcional do pulmão dos camundongos, quando desafiados