Browsing by Author "Pillay, Sirika"

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    Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles.
    (Elsevier, 2008) Halsey, Richard J; Tanzer, Fiona L; Meyers, Ann; Pillay, Sirika; Lynch, Alisson; Shephard, Enid; Williamson, Anna-Lise; Rybicki, Edward P
    HIV-1 Pr55 Gag virus-like particles (VLPs) are strong immunogens with potential as candidate HIV vaccines. VLP immunogenicity can be broadened by making chimaeric Gag molecules: however, VLPs incorporating polypeptides longer than 200 aa fused in frame with Gag have not yet been reported. We constructed a range of gag-derived genes encoding in-frame C-terminal fusions of myristoylation-competent native Pr55Gag and p6-truncated Gag (Pr50Gag) to test the effects of polypeptide length and sequence on VLP formation and morphology, in an insect cell expression system. Fused sequences included a modified reverse transcriptase-Tat-Nef fusion polypeptide (RTTN, 778 aa), and truncated versions of RTTN ranging from 113 aa to 450 aa. Baculovirus-expressed chimaeric proteins were examined by western blot and electron microscopy. All chimaeras formed VLPs which could be purified by sucrose gradient centrifugation. VLP diameter increased with protein MW, from ∼100 nm for Pr55Gag to ∼250 nm for GagRTTN. The presence or absence of the Gag p6 region did not obviously affect VLP formation or appearance. GagRT chimaeric particles were successfully used in mice to boost T-cell responses to Gag and RT that were elicited by a DNA vaccine encoding a GagRTTN polypeptide, indicating the potential of such chimaeras to be used as candidate HIV vaccines.
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    HIV-1 sub-type C chimaeric VLPs boost cellular immune responses in mice
    (BioMed Central Ltd, 2010) Pillay, Sirika; Shephard, Enid; Meyers, Ann; Williamson, Anna-Lise; Rybicki, Edward P
    Several approaches have been explored to eradicate HIV; however, a multigene vaccine appears to be the best option, given their proven potential to elicit broad, effective responses in animal models. The Pr55Gag protein is an excellent vaccine candidate in its own right, given that it can assemble into large, enveloped, virus-like particles (VLPs) which are highly immunogenic, and can moreover be used as a scaffold for the presentation of other large non-structural HIV antigens. In this study, we evaluated the potential of two novel chimaeric HIV-1 Pr55Gag-based VLP constructs - C-terminal fusions with reverse transcriptase and a Tat::Nef fusion protein, designated GagRT and GagTN respectively - to enhance a cellular response in mice when used as boost components in two types of heterologous prime-boost vaccine strategies. A vaccine regimen consisting of a DNA prime and chimaeric HIV-1 VLP boosts in mice induced strong, broad cellular immune responses at an optimum dose of 100 ng VLPs. The enhanced cellular responses induced by the DNA prime-VLP boost were two- to three-fold greater than two DNA vaccinations. Moreover, a mixture of GagRT and GagTN VLPs also boosted antigen-specific CD8+ and CD4+ T-cell responses, while VLP vaccinations only induced predominantly robust Gag CD4+ T-cell responses. The results demonstrate the promising potential of these chimaeric VLPs as vaccine candidates against HIV-1.
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    Optimization of chimaeric HIV-1 virus-like particle (VLP) production and immunogenicity testing of VLPs in mice
    (2008) Pillay, Sirika; Rybicki, Ed; Meyers, Ann
    The devastating effect the HIV pandemic has had on the human population in the last twenty five years has highlighted the great need to develop a prophylactic HIV vaccine. The manufacture of a vaccine has proven difficult though, with a number of successful designs in animal models having little success in humans. In view of this, there has been a need for novel vaccine approaches that are able to elicit effective cellular and humoral immune responses, both of which are believed to be important in the eradication of the virus. One such approach is the use of HIV-1 Gag VLPs as vaccine candidates. In this study, the production of two chimaeric (Gag VLP vaccine candidates (GagRT and GagTN) was optimized in insect cells, and their ability to enhance a murine immune response in a DNA prime-VLP boost vaccine strategy was evaluated.