Brucellosis is the most common zoonotic disease worldwide. The intracellular localization of Brucella hinders the action of drugs that poorly cross cell membrane barriers. Additionally, when the immune response fails to clear the infection, chronic brucellosis ensues that becomes more challenging to treat with antibiotics. Therefore, two approaches, intracellular drug delivery and immunostimulation, have been explored in this dissertation, with an aim to develop a better therapeutic against Brucella infection in mice. First, to overcome the cell membrane barriers, drug loaded nanoparticles were tested to treat B. melitensis infection in mice. Gentamicin loaded block-ionomer complexes (BICs) and magnetite block-ionomer complexes (MBICs) were tested in vitro and along with clusters of MBICs (MBIClusters) were tested in vivo as tools to deliver gentamicin intracellularly. While these complexes showed very high efficacy compared to free gentamicin against Brucella in macrophage cell culture, they failed to show similar efficacies in mice. Histopathological examination of kidneys from mice treated with MBICs or MBIClusters showed deposition of brown pigment-laden macrophages in peri-renal adipose tissue and the pigment was confirmed as MBICs or MBIClusters based on special staining for iron. Additionally, it was found that doxycycline-gentamicin (DG) treatment results in better clearance of Brucella from infected mice compared to doxycycline alone. Secondly, two vaccine candidates, irradiated B. neotomae (IBN) and outer membrane vesicles (OMVs), were tested as immunostimulants to treat chronic B. melitensis infection in mice in combination with antibiotics. The non-ionic block co-polymer Pluronic P85, when mixed with OMVs as an adjuvant showed significantly higher protection against B. melitensis challenge in vaccinated mice compared to those vaccinated with OMVs alone. When tested as immunostimulants, there was no additive effect of vaccines and antibiotics on Brucella clearance from mice. However, IBN enhanced the production of IFN-γ while OMVs were associated with enhanced antibody production. This enhancement in the immune system resulted in the control of Brucella growth after the end of treatment. When given without antibiotics, vaccine alone failed to clear any Brucella from infected mice. The use of these vaccine candidates in combination with antibiotics shows a potential to prevent relapses in cases of brucellosis. / Ph. D.

Made available in DSpace on 2014-08-20T13:32:49Z (GMT). No. of bitstreams: 1 tese_katia_leston_bacelo.pdf: 1389207 bytes, checksum: b5ad0bea30c73a3ecb9d5b196b2a8b64 (MD5) Previous issue date: 2013-12-10 / A major challenge for the development of vaccines based on purified or recombinant protein subunits, and synthetic peptides resides in the fact that these are poorly immunogenic and mobilize insufficient immunoprotective response. Adjuvants are often used in association with these subunits in order to amplify and direct the immune response induced. Nowadays, there is a wide range of compounds that demonstrate adjuvant activity, however, few are approved for human use, and these often fail to induce appropriate immune response against a particular pathogen. Thus, there is a need to develop new adjuvants that are safe, effective and represent an alternative to currently available. In the present study, we used as a model antigen the non- identical portion of the Leptospira LigA protein (Leptospiral immunoglobulin-like protein A), an outer membrane protein of great interest as a mediator pathogenic mechanisms, used in serological diagnosis and as experimental vaccines. This antigen was formulated with various adjuvants, and the formulations tested for their immunoprotective potential in hamsters, challenged with a virulent strain of L. interrogans serovar Copenhageni. For this, the LigAni protein was produced in recombinant form (rLigAni) using Escherichia coli as the expression system and associated to xanthan polysaccharide, in its variants xanthan pruni strains 106 (X1) and 101 (X2), commercial xanthan and also to oligodinucleotídeo CpG (CpG ODN), carbon nanotubes (CNTs) and aluminum hydroxide (Alhydrogel). Formulations containing rLigAni associated with xanthan polysaccharide and CNTs induced significant IgG antibody titers, comparable to that induced when the protein was associated with Alhydrogel. Protection against lethal challenge was observed in 100%, 100%, 67% and 50% of the hamsters immunized with rLigAni-X1, rLigAni- CpG-X1, rLigAni-Alhydrogel and rLigAni-X2, respectively (Fisher test P < 0.05). The preparations containing rLigAni associated with CNTs, although induced an antibody response, failed to confer immunoprotection. Additionally, xanthan and CNTs adjuvants were not toxic to Chinese hamster ovary (CHO) cells, in vitro. The results of this study indicate xanthan as a new adjuvant for subunit vaccines against leptospirosis, presenting the ability to potentiate the immune response against the antigen, besides biocompatibility and the possibility of reduction of number of doses required for protection. / O maior desafio para o desenvolvimento de vacinas baseadas em subunidades proteicas, recombinantes ou purificadas e peptídeos sintéticos, reside no fato destas serem pouco imunogênicas e mobilizarem uma resposta imunoprotetora insuficiente. Adjuvantes são utilizados associados a estas subunidades com o intuito de amplificar e direcionar a resposta imune induzida. Na atualidade, existe uma grande gama de compostos que demostram ação adjuvante, contudo, poucos são aprovados para uso humano, e estes muitas vezes falham em induzir resposta imune adequada contra determinado agente patogênico. Assim, existe a necessidade do desenvolvimento de novos adjuvantes que sejam seguros, efetivos e representem uma alternativa aos atualmente disponíveis. No presente estudo, utilizamos como antígeno modelo a porção não-idêntica da proteína LigA (Leptospiral immunoglobulin-like protein A) de Leptospira spp., uma proteína de membrana externa de grande interesse como mediadora de mecanismos de patogenicidade, utilizada em sorodiagnóstico e em vacinas experimentais. Esta proteína foi associada a diferentes adjuvantes, e as formulações testadas quanto ao seu potencial imunoprotetor em hamsters desafiados com cepa virulenta de Leptospira interrogans sorovar Copenhageni. Para isso, a proteína LigAni foi produzida em sua forma recombinante (rLigAni) utilizando Escherichia coli como sistema de expressão e associada ao polissacarídeo xantana, em suas variantes xantana pruni cepas 106 (X1) e 101 (X2), xantana comercial, e também ao oligodinucleotídeo CpG (CpG ODN), nanotubos de carbono (CNTs) e hidróxido de alumínio (Alhydrogel). Formulações contendo rLigAni associada ao polissacarídeo xantana e aos CNTs induziram títulos de anticorpos IgG significativos e comparáveis aos induzidos quando a proteína foi associada ao Alhydrogel. Proteção contra o desafio letal foi observada em 100%, 100%, 67% e 50% dos hamsters imunizados com rLigAni-X1, rLigAni-CpG-X1, rLigAni-Alhydrogel e rLigAni-X2, respectivamente (Fisher test P < 0,05). As preparações contendo rLigAni associada aos CNTs, embora tenham induzido resposta de anticorpos, falharam em conferir imunoproteção. Adicionalmente, os adjuvantes xantana e CNTs não se mostraram tóxicos em células de ovário de hamster Chinês (CHO), in vitro. Os resultados desse estudo apontam a xantana como um novo adjuvante para vacinas de subunidade contra leptospirose, apresentando a propriedade de potencializar a resposta imune contra o antígeno, além de biocompatibilidade e a possibilidade de redução no número de doses requeridas para proteção.