Browsing by Author "Jaffer, Mohamed A"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
- ItemRestrictedA deletion and point mutation study of the human papillomavirus type 16 major capsid gene(2006) Varsani, Arvind; Williamson, Anna-Lise; Jaffer, Mohamed A; Rybicki, Edward PRecombinant human papillomavirus (HPV) virus-like particles (VLPs) made from the major capsid protein L1 are promising vaccine candidates for use as vaccines against genital and other HPV infections, and particularly against HPV-16. However, HPV-16 genotype variants have different binding affinities for neutralising mouse Mabs raised against HPV-16 L1 VLPs. This paper analyses, using a panel of well-characterised Mabs, the effects on the antigenicity of various C- and N-terminal deletants of HPV-16 L1 made in insect cells via recombinant baculovirus, of an A→T mutation at residue 266 (A266T), and of a C→G mutation at conserved position 428 (C428G). The effects of these changes on assembly of the variant L1s were studied by electron microscopy. Binding of Mab H16:E70 to A266T was reduced by almost half in comparison to wild type L1. Retention of the C-terminal region 428–483 was critical for the binding of conformation-specific Mabs (H16:V5, H16:E70, H16:U4 and H16:9A) whereas deletion of the nuclear localisation signal (NLS) or the C428G mutation or an N-terminal deletion (residues 2–9) did not affect the antigenicity. The N-terminal deletion resulted in a mixed population of 30 and 55 nm VLPs, which differs from the same construct expressed in Escherichia coli, whereas pentamer aggregates resulted from deletion of the 428–465 region or the C428G mutation. The results have implications both for considering use of single-genotype HPV vaccines, and for design of novel second-generation vaccines.
- ItemRestrictedAn investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins(Springer Verlag (Germany), 2008) Windram, Oliver P; Weber, Brandon; Jaffer, Mohamed A; Rybicki, Edward P; Shepherd, Dionne N; Varsani, ArvindDevelopment of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (*55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6–8 kb.
- ItemRestrictedA modeled structure of an aptamer-gp120 complex provides insight into the mechanism of HIV-1 neutralization(American Chemical Society, 2010) Joubert, Marisa K; Kinsley, Nichole; Capovilla, Alexio; Sewell, B Trevor; Jaffer, Mohamed A; Khati, MakobetsaThe HIV-1 envelope glycoprotein, gp120, is a key target for a class of drugs called entry inhibitors. Here we used molecular modeling to construct a three-dimensional model of an anti-gp120 RNA aptamer, B40t77, alone and in complex with gp120. An initial model of B40t77 was built from the predicted secondary structure and then subjected to a combination of energy minimization and molecular dynamics. To model the B40t77-gp120 complex, we docked the B40t77 predicted structure onto the CD4-induced epitope of the gp120 crystal structure. A series of gp120 point mutations in the predicted B40t77-gp120 interface were measured for their binding affinity for B40t77 by surface plasmon resonance. According to the model, of the 10 gp120 amino acids that showed a reduction in the level of binding when mutated to alanine, all of them are modeled as making direct contact with B40t77 as part of a hydrogen bonding network. Comparison by electron microscopy of the B40t77-gp120 complex with gp120 alone revealed that only the longest dimension of the complex significantly increased in length, in a manner consistent with the predicted model. Binding assays revealed that B40t77 can weaken the binding of gp120 to the monoclonal antibodies B6, B12, and 2G12, none of which have binding sites that overlap with B40t77, as well as strengthen the binding to the antibody 19b. Thus, B40t77 may induce distant conformational changes in gp120 that disrupt its association with host cells and may suggest a mechanism for aptamer neutralization of HIV-1.
- ItemRestrictedSubcellular organization of N2-fixing nodules of cowpea (Vigna unguiculata) supplied with silicon.(Springer, 2001) Nelwamondo, Azwianewi; Jaffer, Mohamed A; Dakora, Felix DProvision of silicon (0, 0.048, 0.096, 0.24, 0.48, and 0.96 g/1) in the form of silicic acid (H4SiO4) to nodulated cowpea plants(Vignia unguiculata [L.] Walp.) grown in liquid culture resulted in considerable changes in the internal organization of nodule structure. Compared to the control plants which received no added silicate, bacteroid numbers increased significantly (P ≤ 0.05) at silicate concentrations of both 0.096 and 0.48 g/1. The number of symbiosomes also increased by 3.2-fold at the silicate concentration of 0.96 g/1 compared to the control. In contrast, the size of bacteroids and symbiosomes decreased significantly (P ≤ 0.05) inside nodules of silicate-treated plants. The peribacteroid space was also decreased considerably (P ≤ 0.05) with the application of 0.096 and 0.96 g of silicate per liter to plants. However, the size of intercellular spaces adjacent to infected and uninfected interstitial cells within the nodule medulla increased significantly (P ≤ 0.05) at 0.096 g of silicate per liter followed by a sharply marked (P ≤ 0.05) decrease with each subsequent increase in silicate application. The result was a large decrease (P≤0.05) in the area of bacteria-infected tissue occupied by intercellular space at the highest silicate concentration, which was caused by a significant (P ≤ 0.05) increase in cell wall thickness. Our findings show that the positive effects of silicon on N2 fixation might actually be due to an increased number of bacteroids and symbiosomes.