Evaluation of chimeric virus-like particles, capsomeres and bacterial minicells as vaccine delivery platforms

Abstract

Malaria is a major public health problem, with an estimated 207 million cases and 627,000 deaths each year as well as associated socio-economic costs. Considerable inroads have been made in the reduction of malaria-related deaths and illness through the widespread use of preventative measures, but an effective vaccine is likely to hold our greatest chance of effectively eliminating the risk of Plasmodium infection. However, the most advanced anti-malarial vaccine candidate, RTS,S, has an efficacy rate of approximately 30%, and, alternative novel vaccine delivery platforms are likely to be required.During my doctoral studies, I have evaluated chimeric virus-like particles (VLPs), chimeric capsomeres and bacterial minicells targeted to dendritic cells as novel vaccine delivery platforms, in a rodent model of malaria.Chimeric VLPs are formed when recombinant viral structural proteins with foreign antigenic epitopes assemble into a highly repetitive array which resembles the native form of the virus. These VLPs are self-adjuvanting and highly immunogenic with regard to inducing antibody responses. Capsomeres are pentamers of viral capsid proteins which have not formed into VLPs. These are also immunogenic but require co-administration with adjuvants to induce comparable antibody titres to VLPs. Chimeric VLPs and capsomeres with surface exposed Plasmodium yoelii circumsporozoite (PyCSP) CD8+ and CD4+ T cell and B cell epitopes were constructed using a recently established murine polyomavirus VP1 structural protein scaffold. These constructs were designed so that the platform could be evaluated for their ability to induce cell-mediated immunity as well as antibody responses sufficient to protect against a complex pathogen. Individual and pooled VLPs or pooled capsomere constructs with (pooled VLPs and capsomeres) or without (VLPs only) adjuvant were evaluated in mice for immunogenicity (T cell and antibody responses) and protective capacity in both homologous and heterologous (DNA prime/VLP boost) immunisation regimens. I established that homologous regimens of VLPs and capsomeres induced moderate CD8+ T cell immune responses, and these responses were significantly enhanced by the prime/boost regimen. Both platforms were inefficient at generating a robust CD4+ T cell response, however, capsomere induced responses were superior to those induced by VLPs. Furthermore, I found that both VLPs and to a lesser extent capsomeres, induced robust antibody responses, and that induced antibodies recognised conformational epitopes on the surface of the sporozoite. Neither VLPs nor capsomeres were able to protect mice from sporozoite challenge, with data suggesting that the magnitude of the response was insufficient.Bacterial minicells are small anucleate cells which bud off from bacteria during binary fission as a result of premature septum formation. They have shown potential as a drug delivery platform but have not been well studied as a potential vaccine platform. Minicells produced by transformed E.coli DS410 bacteria containing recombinant protein and homologous mammalian expression plasmid DNA were purified using a novel purification method. Antigen-loaded minicells were then targeted to dendritic cells (DCs) via endocytic receptors DEC205 or Clec9a using bi-specific antibodies to enhance uptake and antigen presentation by dendritic cells and subsequent enhancement of immunogenicity. This platform was evaluated in an ovalbumin model as well as a murine malaria model to assess immunogenicity and protection capacity. In OTI and OTII ovalbumin models, ovalbumin packaged minicells induced ova-specific antibodies but failed to induce proliferation of adoptively transferred cells or kill ovalbumin CD8+ T cell epitope pulsed lymphocytes in an in vivo CTL assay. The amount of target protein in the ovalbumin packaged minicells was only 0.1 µg/dose, and subsequent studies indicated that this antigen load was insufficient for inducing the sought immune responses. In the malaria model, minicells containing P. yoelii apical membrane antigen-1 (PyAMA-1) protein and plasmid DNA for mammalian expression of PyAMA-1, with a protein antigen dose of approximately 1 µg, was evaluated. Blood cytokine and white blood cell activation marker assays, and antibody assays did not indicate any Plasmodium-specific responses, and mice were not protected from sporozoite challenge. In each minicell experiment, targeting DCs did not alter the immune responses, however, this was likely dose-related rather than a platform-specific issue.The potential and limitations of chimeric VLPs, capsomeres and bacterial minicell vaccine delivery platforms were identified which will be useful for future research. Importantly, it was established that chimeric VLPs and capsomeres induce moderate levels of cellular immune responses which complements their capabilities to induce robust antibody responses.