Orf virus (ORFV) has the potential to be developed as a vaccine vector. Its ability to stimulate non-specific as well as specific immune responses in permissive and non-permissive hosts stands it in good stead to be utilised as such a tool. The fusion of immunogenic peptides to vaccinia virus (VACV) structural proteins have been shown to improve their immunogenicity due to presentation of the foreign antigens in a particulate form that can stimulate both B and T cells. The aims of this study were to fuse foreign antigens to ORFV structural proteins to demonstrate proof-of-concept that such surface display could also render the foreign antigens more immunogenic.
Little is known about ORFV structure and morphogenesis. When this study commenced, the ORFV genome had recently been sequenced and this revealed a large number of homologues in common with VACV. It was thus assumed that both viruses may share structural similarities and that ORFV also assumes the different morphological forms such as the mature virion (MV) and extracellular virion (EV) that are present in VACV. The MV and EV forms are both infectious, with the EV containing an additional membrane acquired from the trans-Golgi network during viral morphogenesis. Furthermore, specific viral proteins are associated with both the MV and EV membranes.
Six ORFV structural proteins ORFV 089, 10 kDa, F1, that are homologues of structural membrane proteins A13, A27 and H3 of VACV MVs, together with ORFV 109, ORFV 110 and B2, that are homologues of structural membrane proteins A33, A34 and F13 of VACV EVs were selected as possible candidates for manipulation. At present, there is some information available only for 10 kDa, F1 and B2. The 10 kDa is required for virus assembly, F1 for mediating cell attachment while B2 has been shown to induce significant antibody responses in sheep. Indeed proteomic analyses predicted similarities in the topologies of all of these proteins with their VACV counterparts.
Using this information, preliminary studies were conducted to generate recombinant ORFVs (rORFVs) which had FLAG fused to the terminus of the protein that was exposed on the surface of the virus particle. Three rORFVs 10 kDa, F1L and 110 were successfully generated. Immunogold labelling of FLAG proteins on virus particles isolated from lysed cells showed that FLAG-10 kDa and FLAG-F1 were displayed on the surface of MV particles whereas FLAG-ORFV 110 could not be detected. Western blot analyses of solubilised recombinant ORFV 110-FLAG particles revealed that FLAG-ORFV 110 was abundant and undergoes post-translational modification indicative of endoplasmic reticulum trafficking whereas FLAG-10 kDa and FLAG-F1 did not appear to be subjected to post-translational modifications. Fluorescent microscopy confirmed the prediction that ORFV 110-FLAG localised to the Golgi in virus-infected cells and immunogold labelling of EVs showed that ORFV110-FLAG became exposed on the surface of EV-like particles as a result of egress from the cell, suggesting that the membranes had been acquired from the Golgi. These modifications also appeared to have minimal effect on the infectivity of these rORFVs.
The study was extended by replacing the small FLAG peptide with an immunogenic protein (EG95), derived from the oncosphere of the zoonotic parasite Echinococcus granulosus. This protein is known to confer protection in immunised animals. Three rORFVs were generated in which a truncated version of the protein, EG95[Delta]TM, was fused to 10 kDa in the absence (rORFV 699) or presence (rORFV 700) of a linker, and also to F1 (rORFV 701). Western blot analyses of these solubilised particles demonstrated that the fusion proteins appeared to be post-translationally modified while immunogold labelling using anti-EG95 monoclonal antibodies successfully demonstrated the surface labelling on these rORFVs.
In order to test the immunogenicity of these rORFVs, prime-boost experiments in sheep were conducted using rORFVs 699, 700 and 701 and a glutathione-S-transferase (GST-EG95) based vaccine. The results showed the production of EG95-specific antibodies. In particular, antibody production by group rORFV 701 compared favourably with a control group that was primed and boosted by GST-EG95 vaccine. This was despite the slightly slower growth rates of rORFVs 700 and 701 and the decreased infectivity of all three rORFVs discovered in in vitro experiments.
In conclusion, these studies indicated the feasibility of this strategy to manipulate ORFV structural proteins for use as an agent for vaccine delivery.
| Identifer | oai:union.ndltd.org:ADTP/234761 |
| Date | January 2009 |
| Creators | Tan, Joanne Li-Ching, n/a |
| Publisher | University of Otago. Department of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Joanne Li-Ching Tan |
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