eddie senior seminar! 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 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