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Contenido archivado el 2024-04-30

Peptide and DNA-based strategies for vaccination against major human infections

Objetivo



Immunization with bacterial plasmids containing the genes encoding antigens (DNA vaccines) have been shown to generate effective humoral and cellular immune responses. For the design of effective DNA vaccines there are two important considerations; the choice of antigen(s) and the mode of administration to obtain the desired immune response. The DNA vaccine construct can contain more than one gene, each coding for an entire protein. In contrast to immunization with fragments or short peptides derived from recombinant proteins, DNA vaccines allow expression of proteins in their native conformation thus inducing better antibody responses. Furthermore, the problems of MHC restriction at the T cell level are avoided since it is likely that T cell epitopes for the majority of MHC molecules will be generated. However there are several disadvantages to using genes coding for full length proteins. First, bacterial or protozoal DNA with different codon usage may not be easily expressed in mammalian cells. This may be more pronounced with longer molecules.
Secondly, there can be certain parts of the molecule that may contain harmful epitopes or immunodominant epitopes may reside in polymorphic regions. Thirdly, mammalian glycosylation may alter the conformation of the expressed molecule such that antibody recognition is affected. We propose to address some of these problems of DNA vaccination by constructing synthetic genes encoding regions of protective antigens lacking T cell epitopes that are harmful or polymorphic. These will be produced in a polyepitope string together with B cell regions and mimotopes of conformational B cell epitopes. The peptides containing the T cell epitopes will be selected to contain the peptide motifs able to bind a wide range of MHC Class I and Class II molecules. We will study proteins from four pathogens, HIV, Measles Virus, Borrelia burgdorferi and the malaria parasite, Plasmodium chabaudi. T and B cell epitopes of defined proteins of these pathogens will be identified by analysing peptides digested by proteosomes or presented on different MHC molecules In the case of conformational B cell epitopes, mimotopes will be identified using phage display libraries. Polypeptides strings will then be constructed using these epitopes and flanking sequences will be modified such that the correct epitopes are generated for CD8+ and CD4+T cell responses.
Synthetic genes based on the polyepitopes and containing additional targeting sequences to ensure optimal presentation in the MHC Class I (CD8+ T cell responses) and MHC Class II (CD4+ T cell responses) pathways will be constructed. These genes will be incorporated into DNA vaccines and the conditions for optimal immunization in animal models will be investigated. The antigens chosen allow us to acquire fundamental information on the diversity of epitopes generated and the impact of genetic variability on the epitope specific immunity and immune escape. With these studies we hope to establish conditions that permit the selective induction of effective protective immune responses and avoid undesired immune responses.

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MRC National Institute for Medical Research
Aportación de la UE
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Dirección
The Ridgeway Mill Hil
NW7 1AA London
Reino Unido

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