Prevention of disease with vaccine technologies is one of the most significant medical achievements to date. However, many vaccines are proven to be ineffective for various infections and cancers. These situations require facilitated targeting of the antigen into immune cells. Adjuvants are a non-antigen component included in a vaccine formulation to increase the effectiveness of that particular antigen (Pashine, Valiante et al. 2005) and can consist of either a delivery vehicle or an immunopotentiator. The present study explored both of these adjuvant types; using virus-like particles (VLPs) as potential delivery systems, as well as the C5a agonist as an immuno-potentiator.
This research also explored the hypothesis that conjugation of the C5a agonist to Streptococcus will form antibodies (Ab) in vivo against Streptococcus and that they are protective. In order to investigate the pathway this vaccine employed to create antibodies in order for the vaccine to be traced in live animals in real-time using magnetic resonance imaging (MRI) techniques. Additionally, this project examined if combination of Streptococcus with C5a molecular adjuvants resulted in enhancement of various immune processing steps, in particular: cell binding; cell uptake; antigen (Ag) processing; and/or the in vivo Ab production level; as well as investigation of vaccine biodistribution and bioavailability, in real-time, using MRI imaging with a view to determine its suitability for the development of a mucosal meningococcal vaccine.
This study aims to load the VLPs with a tracer via the assembly/disassembly method and subsequently traced in cell cultures. Vaccines can be made more effective through optimising the antigen delivery system to enhance the immune response to the antigens. An optimum delivery system is more economical, safe and efficient. One of the most promising delivery systems for vaccine use is the VLPs. VLPs are comprised of recombinant structural viral coat proteins which are self-assembled into protein spheres closely resembling the parent virus particles (May, Gleiter et al. 2002). VLPs are often found to behave in a similar manner to native viruses (Hagensee, Benyunes et al. 1994; Rodgers, Chang et al. 1994; Pawlita, Muller et al. 1996; Krauzewicz, Stokrova et al. 2000; Rodgers and Cook 2005; Bucarey, Noriega et al. 2009; Chen, Guo et al. 2011). In this study, a novel potential vaccine carrier was investigated. VLPs derived from the major capsid protein VP1 of the Murine Polyomavirus (MPyV) were used.
VLP production in a baculovirus expression system; purification; and assembly/disassembly of the VLPs were extensively optimized in Chapter 3. MPyV VLPs were expressed in baculovirus expression systems where they self- assembled in vivo and were purified from the culture. This was an essential part of developing the necessary techniques for subsequent experiments and to establish suitable laboratory protocols for this section and for the overall thesis research project. Furthermore, packaging the VLPs with a fluorescent marker to visualise and trace them in cell culture was also optimised. This study demonstrated that VLPs can be successfully expressed in Baculovirus systems, purified and then packaged with BSAFITC molecules. Traceable VLPs were made by packaging FITC or chemical coupling with FITC. The assembly/disassembly process was optimised to ensure maximum numbers of VLPs were formed for subsequent packaging purposes. Results suggested that approximately 14% of the VP1 protein can be retrieved from the total VP1 expressed in insect cells, VLP size varies after the assembly/disassembly process and the presence of Ca+ was not critical for VLP reassembly under in vitro conditions. From the results presented in this chapter it can be concluded that the development of future vaccines with VLPs will require an even greater understanding of the control mechanisms governing viral assembly and disassembly.
Hence in the next chapter experiments in which the VLPs were packaged with florescent marker and applied to cell cultures will be described. The MuPy VLPs were successfully packaged with a fluorescent marker and loaded onto CHO cells. Results showed that the packaged VLPs internalised more readily and in a more organised pattern than the tagged VLPs. This suggested that packaging the VLPs using the assembly/disassembly method did not disrupt the receptor recognition and binding of the VLPS. Results showed that to some extent the tagged VLPs appeared to hinder the uptake into the cells. Hence, the creation of a traceable VLP which could be traced within the cells, organs or whole animals would offer great potential for a better technology in investigating VLP modifications. This can be a powerful tool which will overcome most of the current problems that hinder gene therapy or vaccines from reaching clinical trials.
The C5a agonist agonist (YSFKPMPLaR) was utilised in order to improve the immunogenicity of the Group A Streptococcus (GAS) -derived J8 peptide. C5a agonists have been developed from the C-terminal portion of the C5a protein of the complement system (Short, Paczkowski et al. 1999). Certain components of the complement system can be used as molecular adjuvants for inducing acquired immune responses via more specific pathways or activation (Sanderson 2008). One such component is the extremely potent pro-inflammatory peptide C5a which plays a vital in vivo role in greatly reducing inflammatory injury. Several studies have demonstrated that C5a agonist activity is capable of inducing antigen responses (Sanderson 2008). In the current study the C5a agonist was labelled with a tracer for visualisation purposes to determine the yield of internalisation and pathways of entry into immune cells. In addition the scramble-C5a agonist and FITC-NH2 were the controls for this set of experiments.
The C5a agonist was also conjugated with potential vaccine candidates with a view to developing a new vaccine strategy for both J8 and MUC1.. The C5a agonist readily internalises into the human polymorphonuclear leukocytes specifically through the C5a receptor. Also, when the C5a agonist is conjugated to other peptides such as J8 it displayed a good affinity for the C5a receptors hence were used later in the in vivo studies as discussed in the next chapter. On the other hand, the conjugated MUC1 peptides to the C5a agonist had a very poor binding affinity. This was attributed to poor sequence synthesis rather then any real effects of MUC1 on the C5a agonist activities. Results from this series of experiments show that in addition to the finding that the CFA proved a much more effective adjuvant than the C5a agonist in raising immune responses in mice against the J8 peptide, the incorporation of a poly A probably plays a significant role in their immunogenicity.