Prime-boost vaccination strategy

#The present invention relates to a method for inducing an immune response to an antigen in a subject.

#Measles is a highly contagious viral disease that has persisted for more than 1000 years since it was first described (Babbott and Gordon, 1954). Severe infection may lead to pneumonia, encephalitis (brain inflammation) and death. Although measles can be effectively prevented by a live-attenuated vaccine (LAV) it still causes approximately 800,000 deaths every year, predominantly among children in developing countries (Cutts and Steinglass, 1998).

#The inability to control measles using the LAV is largely due to neutralization of the vaccine by maternal antibodies. In order to avoid neutralization by maternal antibodies the LAV is generally administered between 12 and 18 months, However maternal antibodies may decline more rapidly in infants of developing countries (Gans et al., 1998). As a consequence, there is a window between 6 and 18 months of age during which infants may lack both passive and active immunity.

#FIG. 1: Plant transformation vector constructs for expression of MV-H protein in tobacco. The T-DNA region inserted into the plant genome contains the nopaline synthase expression cassette (KanR),which confers kanamycin resistance on transformed cells, and the MV-H protein expression cassette. The MV-H protein expression cassette comprises a cauliflower mosaic virus 35S promoter (35S-Pro) fused to a tobacco etch virus 5′-untranslated region (TEV) and cauliflower mosaic virus terminator sequence (35S-Ter). The pBinH/KDEL and pBinSP/H/KDEL constructs contain an SEKDEL peptide sequence (KDEL) fused to the C-terminal end of the H protein for retention in the endoplasmic reticulum. The pBinSP/H/KDEL construct also contains a plant signal peptide (SP) fused to the N-terminal end of the H protein.

#FIG. 2: Transgene expression and production of recombinant MV-H protein in transgenic tobacco. (A) Northern blot comparing the level of MV-H gene expression of the six highest expressing T0 transgenic tobacco lines obtained for each MV-H construct. Each lane contained 10 μg of total RNA and was probed with a 32P-labeled MV-H cDNA probe. (B) EUSA analysis of MV-H protein expression in each of the T0 transgenic tobacco lines shown in (A) detected with a rabbit anti-measles polyclonal antibody. Four independent control transgenic lines transformed with a pBin construct lacking the MV-H gene, were included in analyses.

#FIG. 3: Detection of MV-H protein in pBinH/KDEL T1 transgenic lines. Selected kanamycin resistant progeny from the three highest T0 expressing lines (8B, 12C and 39H) were analysed for MV-H protein expression using ELISA. The analysis was performed using either a rabbit anti-measles polyclonal antibody or MV-positive human serum. Control extract is from a transgenic tobacco line transformed with a pBin construct lacking the MV-H gene.

#Experimental Protocol

#Construction of Transgenic Tobacco Plants Producing H Protein

#Three constructs were generated for the expression of MV-H protein in tobacco plants (FIG. 1) (a) pBinH—H protein alone, (b) pBinDEL—addition of a C-terminal endoplasmic reticulum (ER)-retention sequence and (c) pBinSP/H/KDEL—addition of both an N-terminal plant signal peptide and a C-terminal ER-retention sequence.