Browsing by Subject "HIV vaccine"
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- ItemRestrictedChimaeric 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 PHIV-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.
- ItemOpen AccessInvestigation of particulate HIV-1 Env vaccine candidates using Zera® and SpyTag/SpyCatcher technologies(2022) Ximba, Phindile Thobeka; Rybicki, Ed; Williamson, Anna-Lise; Meyers, AnnThe HIV-1 envelope glycoprotein (Env) is the primary focus of prophylactic HIV vaccine development. However, the unusually low density of Env spikes on the virion (≈14 spikes/virion) is unfavourable for eliciting high titre, long-lasting antibody responses. It is possible that increasing the Env spike density of particulate vaccine candidates generated by protein body formation or via the display of Env on nanoparticles could improve the induction of long-lasting neutralising antibodies (NAbs). For this thesis, two different nanoparticle approaches were therefore investigated. The HIV-1 Env sequence used for both approaches was derived from the superinfecting subtype C CAP256 virus. This was truncated to remove the transmembrane domain, and engineered to contain a flexible linker (FL) in place of the furin cleavage site and an I559P mutation to generate soluble, stable and cleavageindependent gp140 proteins. The first approach investigated the impact of genetically fusing a 27 kDa proline-cysteine-rich domain of the ɣ-zein maize seed storage protein - Zera® - to either the N- or C-terminus of CAP256 gp140. Fusion of Zera® to a protein of interest can promote the self-assembly of large protein bodies (PBs) containing the protein of interest, thereby improving yields of the recombinant protein and enabling easy isolation using gradient ultracentrifugation. The purification of Zera-induced Env PBs from infiltrated Nicotiana benthamiana plants was not optimal. Consequently, the generation of Zera®-induced gp140 protein bodies was evaluated in a mammalian expression system. Stable HEK293 cell lines expressing Zera®-gp140 or gp140-Zera® were generated. A mixture of small PB-like structures was observed in cells expressing gp140-Zera®. However, no PB-like structures were seen in cells expressing Zera®-gp140. The immunogenicity of Zera®-gp140 and gp140-Zera® was evaluated by in rabbits. Binding and Tier 1A neutralising serum titres were higher for gp140-Zera® than for Zera®-gp140. Neither gp140-Zera® nor Zera®-gp140-specific sera neutralised a Tier 1B pseudovirus or the autologous Tier 2 CAP256SU pseudovirus, suggesting that Zera® might have compromised the structure of the Zera®-tagged gp140 proteins. The second approach investigated the two-component SpyCatcher/SpyTag technology. The stable HEK293 cell line expressing CAP256 gp140-SpyTag (gp140-ST) was generated, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced in E. coli and purified by ultracentrifugation. The gp140-ST trimers and the SCAP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). SDS-PAGE, dynamic light scattering and negative stain electron microscopy indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs. The second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens and then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. These results demonstrate that careful selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of HIV-1 envelope trimers is an important consideration and that this could improve the effect of nanoparticle-displayed gp140.
- ItemOpen AccessStability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media(BioMed Central Ltd, 2012) Lynch, Alisson; Meyers, Ann; Williamson, Anna-Lise; Rybicki, EdwardBACKGROUND:HIV-1 Pr55gag virus-like particles (VLPs) expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability.FINDINGS:We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose) for their ability to stabilise HIV-1 Pr55gag VLPs during prolonged storage. Transmission electron microscopy (TEM) was done on VLPs stored at two different concentrations of the media at three different temperatures (4degreesC, -20degreesC and 70degreesC) over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at 70degreesC retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. CONCLUSIONS: Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at 70degreesC for 12 months is most effective in retaining VLP stability.