eddie senior seminar! 1
TRANSCRIPT
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 Biotechnology8: 53
23 June 2008
Presented by Eddie Moat
HIV
HIV Mechanism
Epidemic• HIV-1 has infected more than 40 million people worldwide• Sub-Saharan Africa has the majority of new infections with 32.1%• HIV-1 Subtype C• Necessitates the development of cheap, effective vaccines
Possible Vaccines
• Universal Vaccine?• DNA Vaccines?• Sub Saharan African
Isolates• SAAVI
Plant Advantage
• Expression host (Nicotiana Tabacum, Nicotiana spp., alfafa rice, and etc.)
• Equivalent Protein Folding• Cost• Highly Scalable• Oral delivery
»Hypothesis• Expression of HIV-1 (subtype C) antigens in plants will
render a viable plant-produced HIV-1 vaccine.
» Objective• Prove Transient expression is a viable alternative to
transgenic plants• Show that HIV-1 Pr55Gag is a promising vaccine
candidate• The influence of Subcellular localization of recombinant
protein• Efficacy of “best vaccine candidate” in BALb/c mice
Expression Systems• Agrobacterium tumefaciens-mediated transient expression*• Generation of stable Transgenic plants
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
Transient..viable alternative?• Vs. Transgenic plants• Short production• Codon Optomization• High Protein yields
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
Experiment
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
Show that HIV-1 Pr55Gag is a promising vaccine candidate
TMV Expression (viral vector)
•Usefulness of:●Codon Optimization
Methods2.N. Bathamiana infected w/ pBSGgags RNA3.Immunotrapped onto copper grids4.Coated w/ HIV p17 antiserum5.Stained w/ 2% uranyl acetate
Yields of recombinant proteins obtained in Nicotiana spp. using the TMV expression system.
Recombinant protein (non-targeted)
TMV Expression (μg p24/kg)
Full length Pr55Gagwtgag <0.01ngag 0.46 – 2.0
p24wtp24 172 – 13900np24 230 – 17350hp24 167 – 1000
Levels of protein are expressed as μg p24 per kg of fresh leaf weight.wt = wildtype codon usage; n = Nicotiana spp. codon-optimised; h = human codon-optimised
The HIV-1 Gag-derived protein
• Pr55Gag• P17 (MA)• P24(Ca)• VLPs
• p17/ p24 produce CTL epitopes
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
Summary of the recombinant constructs used for
Agrobacterium-mediated transient and transgenic
expression.
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-CPTgagAll genes were Nicotiana spp. codon-optimisedM = myristylated; NM = non-myristylated
Experiment
• Tumefaciens-mediated transient expression & Transgenic expression– Recombinant Proteins: Pr55Gag, p24, p17/p24– Subcellular localization (C, ER, CPT)
Methods• Agroinfiltrated into N. benthamiana• p24 antigen ELISA
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
Transient vs. transgenic expression in specific locations
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.
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
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
• Effects of Myristylated p17/24– Compared G2A-mutated p17/24 (NM) and p17/24 (M)
expression
Method– Attachment at N-term glycine– Mutated by PCR (G (GGT) to A(GCT))– Amplified by pTHGagC
ExperimentMyristylation
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
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.
What’s the best candidate vaccine?
• Pr55Gag, p24 or p17/24?• Pr55Gag expression negligible• p24ER yielded highest levels of p24/kg FW
BUTp17/p24CPT• <epitopes• May form VLPs• More effective cell response
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
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.
Experiment• pTRAk-CPT p17/p24-M vaccination of BALB/c miceMethods
Cellular and humoral response of mice to pTRAk-CPT p17/p24
Mice Groups Day 0 Day 28
1 Group pTHGagC -
2 Group pTHGagC pTHGagC
3 Group pTHGagC 646 ng p17/p24
4 Group pTHGagC 64 ng p17/p24
5 Group pTHGagC 100 ng Leaf protein
6 Group pTHGagC + 64 ng p17/p24
7 Group pTHGagC + 646 ng p17/p24
8 Group pTHGagC + 100 ng Leaf protein
All mice were sacrificed on 40th Day
Method cont.
• Spleens harvested 40th day (5/group)• IFN-γ ELISPOT assay• Peptides GagCD8 and GagCD4 were
used as stimuli in assay• CTL analyzer
Cellular and humoral response of mice to pTRAk-CPT p17/p24
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
FN-γ ELISPOT analysis of Gag T cell responses after vaccination of BALB/c mice
Results
• Gag CD8+ Tcell increased 2.3 fold (64 ng of p17/ p24)
• 646 nd p17/ p24 same increase• GagCD4+T cell increase 4.7 fold (64 ng of p17/p
p24)• 646 ng p17/ p24 same increase• p17/p24 no response• pThGagC + pTHGagC = pTHGagC + p17/ p24
Gag specific serum total IgG and IgG subtypes
Methods• Measurement of Antibody• Elisa and Western Blot• Abs. (450 nm) value for total IgG, IgG1, IgG2a, IgG2b,
and IgG3• W.B. Determined presence of antibodies (Goat anti-
mouse IgG)
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
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.
HIV-1 Antibodies determined