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Development of recombinant vaccines against Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is an important human pathogen, causing pneumonia, otitis media and meningitis, affecting mainly children, the elderly and immunodeficient individuals. The currently available pneumococcal vaccine consists of a mixture of polisaccharides (PS) from 23 different strains. Because of the induction of a strictly serotype-specific protection, production of this vaccine requires an enormous structure. Furthermore, PS vaccines induce a thymus-independent immune response, which is not effective in children. The use of protein antigens in alternative presentation systems could broaden vaccine coverage as well as induce a switch from a thymus-independent response to a thymus-dependent one. Several pneumococcal proteins, such as pneumolysin and PspA have been investigated. The surface antigen, PsaA, has been shown to be immunogenic and protective in experimental animals and seems to be fairly conserved in the 23 vaccine strains. We propose to investigate the potential of PsaA in 4 different vacci ne approaches: 1) conjugated PS-recombinante PsaA; 2) recombinant BCG expressing PsaA-3) recombinant lactobacilus expressing PsaA; and 4) a PsaA-DNA vaccine. Development of a conjugated PS-PsaA vaccine could result in an efficient and broad-range thymus-dependent antigen. Efficient because for capsulated bacteria, PS are the best antigens. Broad specificity and thymus dependency would be ensured by covalent linkage to PsaA. The development of the conjugated PsaA-PS vaccine will involve the following steps: i) expression of PsaA in E. coli and its purification; ii) fermentation of the prevalent S. pneumoniae strain and its PS purification; and iii) PsaA-PS conjugation and purification. Recombinant BCG vaccines have been considered strong potential candidates for live antigen-presenting systems, specially for developing countries. The recombinant BCG-PsaA vaccine will require high level expression of the antigen through a strong mycobacterial promotor, pBlaF* (a mutated M.fortuitum beta-lactamase promotor) in a replicative vector. Mycobacterial expression systems, to be used in humans, will also be investigated through 2 strategies: i) constitution of a leuD- BCG auxotroph through allelic exchange, using a truncated leuD gene in a suicide vector containing a SacB counterselectable marker to select for double crossover events. The leuD-BCG auxotroph can then be transfected with a vector containing the heterologous expression system and the leuD gene; ii) constitution of a beta-lactamase knock-out BCG using the SacB counterselectable marker to investigate its potential as a locus for integration of an antigen expression system by allelic exchange. GFP (green fluorescent protein) would be used as an expression marker. Development of the recombinant lactobacilus expressing PsaA will first require the establishment of a suitable expression vector. The PsaA gene will then be cloned in this vector and the recombinant lactobacilus analyzed for expression of the heterologous protein. Induction of an immune response and immune protection against S. pneumoniae following oral immunization with the recombinant Lactobacilus-PsaA will be analyzed. A strategy which has been seen with enthusiasm in the development of new vaccines is the constitution of DNA vaccines, which induces production of the antigen inside the cells. The PsaA-DNA vaccine will be constructed in the mammalian expression vector pcDNA3 and different administration routes will be tested. A second antigen, the conserved part of PspA, can also be included and double expression will be tested.The immunological variability of PsaA in Brazilian strains will be investigated by Western Blot and the genes of the most prevalent strains will be sequenced. The induced humoral immune response against PsaA will be characterized in mice for all the produced vaccines. Cross reactivity and protection against heterologous strains will also be determined. Possible cellular immune responses will be evaluated by cytokine profile analysis of sera and respective splenocite culture supernatants of immunized mice. This project intends to explore several possibilities of using recombinant PsaA as an antigen in different presentation systems which may lead to the development of an effective broad-range pneumococcal vaccine, while consolidating our competence in recombinant and conjugated vaccine development and testing. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
CHRIST, ANA P.; RODRIGUEZ, DUNIA; BORTOLATTO, JULIANA; BORDUCCHI, ERICA; KELLER, ALEXANDRE; MUCIDA, DANIEL; SILVA, JOAO S.; LEITE, LUCIANA C. C.; RUSSO, MOMTCHILO. Enhancement of Th1 Lung Immunity Induced by Recombinant Mycobacterium bovis Bacillus Calmette-Guerin Attenuates Airway Allergic Disease. American Journal of Respiratory Cell and Molecular Biology, v. 43, n. 2, p. 243-252, AUG 2010. Web of Science Citations: 12.

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