Exploring the potential of novel multivalent DNA-based vaccines
Fissolo, Nicolas Miguel
LicenseStandard (Fassung vom 03.05.2003)
In this dissertation, we exploited the DNA vaccination approach to test in the mouse some aspects relevant for the design of optimal CTL-stimulating, multiepitope vaccines. We have used three different ways to prime multispecific CD8+ T cell responses: 1) We have cloned a polytope DNA vaccine that encodes 10 epitopes binding MHC class I molecules encoded by the K, D or L locus (of H-2d, H-2b and H-2k haplotype mice). Vaccination of different mouse strains showed that Ld-restricted CD8+ T cell responses down regulated copriming of CD8+ T cell responses to other epitopes, irrespective of their restriction or epitope specificity. When the pt10 domain was fused to a 77 residue hsp 73-binding domain derived from the SV40 T-Ag (to generate a T77-pt10 fusion protein) immunogenicity of the polytope vaccine was greatly enhanced. Priming of multispecific CD8+ T cell responses was efficiently elicited even under conditions under which the ‘suppressive’, Ld-dependent immunodominance operated. Thus, presentation of an hsp7 bound "polytope" facilitates copriming of CD8+ T cells. 2) We have cloned the sequence encoding the polymerase (Pol) protein of hepatitis B virus (HBV) into different expression vectors. This sequence contains a primary reading frame encoding Pol (the expression of which is under heterologous HCMV, SV40 or metallothionin promoter control) and an alternative reading frame encoding all three (large, middle and small) hepatitis B surface antigen (HBsAg) variants. Pol-encoding vectors coexpressed Pol and readily detectable amounts of HBsAg. Endogenous HBV promoter sequences drive HBsAg expression from these constructs. DNA immunization of mice efficiently coprimes CD8+ T cell responses to Pol and HBsAg epitopes. Immunodominant Ld-restricted CD8+ T cell responses to HBsAg down modulated priming of CD8+ T cell responses to other HBsAg epitopes but not to the Kd-Pol140-148 epitope. Thus, two frame expression constructs offer an additional chance to incorporate an extended repertoire of antigenic information into synthetic genes delivered as DNA vaccines (or by recombinant viruses). 3) We constructed a vector that encodes the small hepatitis B surface antigen (HBsAg, S) in the primary ORF, and the C-terminal (residue 344-832) polymerase (Pol) fragment in the alternative (out-of-frame) reading frame. This DNA vaccine efficiently primes multispecific, HLA-A2-restricted CD8+ T cell responses to epitopes of HBsAg and Pol. Generation of immunogenic epitopes from products translated in an alternative ORF was independent of the level of expression of this product, but dependent on the type of heterologous promoter sequences that controls transcription (with e.g. CMV-derived promoters being more efficient than SV40-derived promoters).
Subject HeadingsHepatitis-B-Surface-Antigen [GND]
Gene expression [MeSH]
Vaccines. Testing [MeSH]