The overall goal of this grant is to achieve a detailed immunochemical characterization of Pseudomonas aeruginosa high molecular weight polysaccharide (PS) antigen which could lead to a vaccine against infection based upon these antigens. The goals include structural characterizations of PS and comparison with corresponding LPS O-specific side chains, testing of the safety and immunogenicity in animals and humans, protection experiments in animal models, characterization of the humoral and cellular immune response to these antigens and detailed characterization of T-cells and cell circuits which regulate the immune response to these PS. P.aeruginosa infections remain among the most difficult to treat. Immunocompromised states which predispose patients to P.aeruginosa infection may mean that the immune system needs additional effectors to resist infection. One of these effectors is antibody specific for the lipopolysaccharide (LPS) O side chain, which bears the serotype determinant. High molecular weight polysaccharides are large versions of the O side chain. They are immunogenic in animals and humans and engender both humoral and cellular immunity which augment host resistance to infection. The variation in O side chain structure is intimately related to the specificity of the host's immune response. Among P.aeruginosa strains there are major variations which translate into 7-10 serotypes causing most of the infection. Among some of these serotypes there are subtypes. One goal of this proposal is to compare serotype and subtype specific immunity using a series of structurally characterized high molecular weight polysaccharide antigens. A second goal is to prepare a human hexavalent vaccine for safety and immunogenicity evaluations. This multivalent high molecular weight polysaccharide vaccine represents a first generation application of the technology developed to produce these antigens.
A third aim i s to prepare and characterize polysaccharide-protein conjugates using as a carrier a toxoid of P.aeruginosa exotoxin A which contains a deletion of the active- site residue (glutamic acid 553) in the enzymatic portion of the toxin. We will analyze in detail the cells and cell circuits involved in response to the conjugates because of potential problems in the long-term use of protein-polysaccharide conjugates due to suppression of the immune response to the haptenic portion of the conjugate. Finally we will expand our investigations into the properties of two small molecular weight neutral polysaccharides that appear to be antigenically common to all strains of P.aeruginosa. We will analyze their occurrence, prepare and characterize monoclonal and polyclonal antibodies to them and evaluate the ability of these antibodies to provide protection in animal and mediate in vitro killing of bacteria. These antibodies will be compared with antibodies specific for the O side chain which are known to be protective and opsonic. These comparisons could shed light on the differences between protective and non-protective antibodies directed towards surface polysaccharide antigens of bacteria.
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