Materials and methods relating to improved vaccination strategies

#The present invention relates to materials and methods for improving vaccination strategies. Particularly, but not exclusively, the invention relates to “prime-boost” vaccination protocols in which the immune system is induced by a priming composition and boosted by administration of a boosting composition. The invention further relates to novel tetrameric soluble class I MHC/peptide complexes as a tool for directly monitoring vaccination regimens and determining novel epitopes.

#Recent advances in our ability to monitor frequency of antigen CTL responses in ex-vivo assays are rapidly improving our capacity to compare different vaccination protocols. In particular, the use of tetrameric soluble class I MHC/peptide complexes (tetramers) provides an opportunity to greatly accelerate development of new vaccines by allowing rapid and accurate analysis of human CTL responses 1-3.

#It has become clear that heterologous prime-boost vaccination protocols, based on repeated injections of non-cross reactive vectors encoding the same antigenic protein, result in strong CTL responses, probably due to focusing of the immune response towards epitopes contained within the recombinant target proteins 4. Recent results have demonstrated that combination of priming with plasmid DNA and boosting with recombinant defective vaccinia virus MVA generate high levels of specific immunity 5-10.

#Table 1 Poly-CTL epitope construct (SEQ ID NO:2). The poly-CTL epitope gene (mel3) (SEQ ID NO:1) was constructed as a single string of epitopes. Mel3 cassette was cloned into four distinct vectors: naked DNA, vaccinia virus WR, MVA and SFV.

#FIG. 1 efficient processing and presentation of mel3 CTL. HLA A2 and HLA A1 positive B cells were infected with mel3. MVA and used as targets of CTL clones specific to each epitope contained within the mel.3 poly-CTL construct. Specificity of each CTL clone and percentage of specific lysis are shown above each panel. Target cells were pulsed with relevant peptide (+ peptide), unpulsed (− peptide), infected either with mel3 MVA (MVA.mel3) or an irrelavant vaccinia (irr vac). Mouse NP 366-374 specific CTL were used as effectors cells against mouse MC57 fibroblasts infected with mel3.MVA.

#Each bar corresponds to a different effector to target ratio: black bars 10:1, open bars 3:1 dotted bars 1:1.

#Materials and Methods

#Plasmid DNA Construct

#The DNA vector pSG2, used throughout the study, was derived from pRc/CMV (Invitrogen, Paisley, UK) by removing the BamH1 fragment that contains the SV40 origin of replication and neomycin resistance marker and replacing the CMV promoter with a longer version of the same promoter containing intron A. The resulting plasmid contains the CMV promoter with intron A for expression in eukaryotic cells, followed by a multiple cloning site and the bovine growth hormone poly-A sequence. The plasmid is incapable of replication in mammalian cells. The gene encoding the mel3 sequence (Table 1) was introduced into the multiple cloning site using standard methods. Plasmid DNA for injection was purified using anion-exchange chromatography (Qiagen, Hilden, Germany) and diluted in phosphate buffered saline (PBS) at 1 mg DNA/ml.