DNA fusion vaccines encoding the idiotypic (Id) single chain variable fragments of B-cell malignancies fused to the potato virus X coat protein (PVXCP) were shown to enhance anti Id antibody and T-cell responses which resulted in protection in lymphoma and myeloma models. I further explored the use of PVXCP to enhance induction of anti -tumour immunity. The aims of this study were to generate two types of Id tumour vaccines against the BCL1 lymphoma, a multimeric Id-PVXCP fusion protein and a PVX particle displaying Id antigen, and to compare their performance with the DNA fusion vaccine. The multimeric fusion protein vaccine was produced in plants using the HyperTrans expression system. Three constructs which directed expression to the cytoplasm, for retention in the endoplasmic reticulum (ER) and for secretion were tested. The construct that directed the fusion protein to retain in the ER provided high protein expression and was used for further studies. The fusion protein was purified by antibody affinity chromatography and size exclusion. ELISA confirmed the integrity of the expressed protein and protein multimerisation was shown by transmission electron microscopy (TEM). This vaccine induced both anti-Id and anti-PVXCP antibody responses in mice to the levels comparable to the DNA vaccine and it provided protection against the lymphoma challenge. The plant viral particle (PVP) Id vaccine was produced by linking BCL]IgG to PVX via biotin-streptavidin. The PVP vaccine induced both anti-Id and anti-PVXCP antibodies to significantly higher levels than the DNA vaccine. PVP provided protection to the levels comparable to the DNA vaccine. The PVP vaccine also induced cellular responses as high numbers of PVXCP-specific IFN-y and IL-2 secreting cells were detected. In vivo PVX bound to CD 11 c + dendritic cells (DCs) and induced activation of all CD 11 c + DC subsets as determined by up-regulation of activation markers. PVX-activated DCs also changed their cytokine, chemokine and chemokine receptor expression profiles toward activated DC phenotypes. These data suggest that PVX enhanced immunity to the linked tumour antigen with the involvement of T cells and DCs. The feasibility of genetically linking BCL] scFv to PVX was also determined. BCLI scFv sequence was fused to the N-terminus of PVXCP sequence and inserted into a PVX-based vector to enable expression of the chimeric viral particle (CVP) in plants. CVP was assembled in plants as judged by the ability to infect host plants both locally and systemically. The CVP RNA and proteins (both fusion protein and PVXCP) were detected in all infected leaves. BCLI scFv on the CVP surface was detected by TEM with goldlabelled anti -BCLI antibody. However, low amount of CVP was obtained and therefore this vaccine production strategy requires further optimisation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595522 |
| Date | January 2010 |
| Creators | Jobsri, Jantipa |
| Contributors | Stevenson, Freda ; Savelyeva, Natalia |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/362530/ |
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