1. Expression of HIV-1 antigens in plants as potential subunit vaccines Ann Meyers, Ereck Chakauya, Enid Shephard, Fiona L Tanzer, James Maclean, Allison Lynch, Anna-Lise Williamson, and Edward P Rybicki BMC Biotechnology 8: 53 23 June 2008 Presented by Eddie Moat
14. Summary of the recombinant constructs used for Agrobacterium -mediated transient and transgenic expression. All genes were Nicotiana spp. codon-optimised M = myristylated; NM = non-myristylated Objective Comp . Transient expression is a viable alternative to transgenic plants Usefulness of Codon Opt. The influence of Subcellular localization & Myristylation of recombinant proteins Efficacy of best vaccine candidate in BALb/c mice Vector Subcellular Target Insert Clone pTRAc cytosol p24 pTRA-Cp24 p17/p24 pTRA-Cp17/p24-M pTRA-Cp17/p24-NM gag pTRA-Cgag pTRAkc-ERH ER p24 pTRA-ERp24 p17/p24 pTRA-ERp17/p24-M pTRA-ERp17/p24-NM gag pTRA-ERgag pTRAkc-rbcS1-cTP chloroplast p24 pTRA-CPTp24 p17/p24 pTRA-CPTp17/p24-M pTRA-CPTp17/p24-NM gag pTRA-CPTgag
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16. Comparison of yields of recombinant HIV-1 p24, p17/24 and Gag proteins obtained in N. benthamiana by (A) A. tumefaciens- mediated transient expression or (B) transgenic expression. Levels of protein are expressed as μg p24 per kg of fresh leaf weight. All genes were Nicotiana spp. codon-optimised. C, ER and CPT in construct names indicate whether the protein remained in the cytosol, or was targeted to the endoplasmic reticulum or the chloroplast, respectively. D: regenerants died during transplantation -: no transgenic plants generated A B Recombinant protein Pr55Gag GagC 7 – 44 D GagER 8 – 13 0.13 – 7 GagCPT 1 – 29 0.03 – 48 p24 p24C 0.01 0.04 p24ER 3691 – 16148 0.01 – 1.19 p24CPT 937 – 4014 636 – 2994 p17/p24 p17/p24C 220 - p17/p24ER 220 <1 p17/p24CPT 4800 5–230
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18. The effect of intracellular localisation on accumulation of myristylated p17/24. Transient expression of HIV-1 p17/24 protein in Agrobacterium -infiltrated N. benthamiana was measured by HIV-1 p24 ELISA 4 days after infiltration.
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20. Which Subcellular location best yields HIV p17/p24? Western blot analysis of HIV p17/24 from infiltrated N. benthamiana leaves and fractionation of purified protein .
26. Gag-specific serum total IgG and IgG subtypes . Gag-specific serum total IgG and IgG subtypes . Mouse groups were inoculated with the indicated antigens as specified in the methods and serum isolated on day 40 for antibody measurements by ELISA quantifying total IgG and IgG subtypes IgG1, IgG2a, IgG2b and IgG3 against Gag. Pooled serum samples from 5 mice per group were tested at 1:1000 dilution. (A) Bars represent the average absorbance ± SD for triplicate values, and represent the level of total IgG and IgG subtypes IgG1, IgG2a, IgG2b and IgG3 against Gag above pre-bleed absorbance values which were not more than 0.003. Ratio of absorbance values to prebleed values is shown on the right. (B) Ratio of IgG2a vs IgG1 for the indicated groups of mice.
