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Expression of HPV-11 L1 protein in transgenic Arabidopsis thaliana and Nicotiana tabacum.

Kohl TO, Hitzeroth II, Christensen ND, Rybicki EP - BMC Biotechnol. (2007)

Bottom Line: Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro.Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity.This has important and potentially negative implications for the production of HPV-11 vaccines in plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, PO Observatory 7925, Cape Town, South Africa. thomas.kohl@jefferson.edu

ABSTRACT

Background: We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine.

Results: Transformation of plants was only achieved with the HPV-11 L1 gene with the C-terminal nuclear localization signal (NLS-) encoding region removed, and not with the full-length gene. The HPV-11 L1 NLS- gene was stably integrated and inherited through several generations of transgenic plants. Plant-derived HPV-11 L1 protein was capable of assembling into virus-like particles (VLPs), although resulting particles displayed a pleomorphic phenotype. Neutralising monoclonal antibodies binding both surface-linear and conformation-specific epitopes bound the A. thaliana-derived particles and - to a lesser degree - the N. tabacum-derived particles, suggesting that plant-derived and insect cell-derived VLPs displayed similar antigenic properties. Yields of up to 12 microg/g of HPV-11 L1 NLS- protein were harvested from transgenic A. thaliana plants, and 2 microg/g from N. tabacum plants - a significant increase over previous efforts. Immunization of New Zealand white rabbits with approximately 50 microg of plant-derived HPV-11 L1 NLS- protein induced an antibody response that predominantly recognized insect cell-produced HPV-11 L1 NLS- and not NLS+ VLPs. Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro.

Conclusion: We expressed the wild-type HPV-11 L1 NLS- gene in two different plant species and increased yields of HPV-11 L1 protein by between 500 and 1000-fold compared to previous reports. Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity. This has important and potentially negative implications for the production of HPV-11 vaccines in plants.

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Immunogenicity of plant extracts. Analysis of serum from New Zealand white rabbits #11 and #12 immunized with A. thaliana-derived HPV-11 L1 NLS- VLPs and rabbits #13 to #18 immunized with N. tabacum-derived HPV-11 L1 NLS- VLPs. Rabbits were immunized on days 1, 28 and 56 and serum from days 1, 15, 28, 42, 56 and 70 post injection was tested by ELISA in wells coated with 0.4 μg insect cell-derived HPV-11 L1 NLS- VLPs. Data from rabbit #12 is incomplete as it had to be euthanized due to growth of an abscess on the neck. Error bars represent the standard deviation calculated from triplicate analysis of samples.
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Figure 5: Immunogenicity of plant extracts. Analysis of serum from New Zealand white rabbits #11 and #12 immunized with A. thaliana-derived HPV-11 L1 NLS- VLPs and rabbits #13 to #18 immunized with N. tabacum-derived HPV-11 L1 NLS- VLPs. Rabbits were immunized on days 1, 28 and 56 and serum from days 1, 15, 28, 42, 56 and 70 post injection was tested by ELISA in wells coated with 0.4 μg insect cell-derived HPV-11 L1 NLS- VLPs. Data from rabbit #12 is incomplete as it had to be euthanized due to growth of an abscess on the neck. Error bars represent the standard deviation calculated from triplicate analysis of samples.

Mentions: Eight New Zealand white rabbits were immunized with concentrated transgenic A. thaliana and N. tabacum protein extracts. Serum collected on days 1, 15, 28, 42, 56 and 70 was analysed for anti-HPV-11 L1 reactive antibodies by ELISA and results are shown in Figure 5. Rabbits #11 and #12 were immunised with T3 generation transgenic A. thaliana-derived L1 protein extract calculated to contain between 21 μg and 43 μg.


Expression of HPV-11 L1 protein in transgenic Arabidopsis thaliana and Nicotiana tabacum.

Kohl TO, Hitzeroth II, Christensen ND, Rybicki EP - BMC Biotechnol. (2007)

Immunogenicity of plant extracts. Analysis of serum from New Zealand white rabbits #11 and #12 immunized with A. thaliana-derived HPV-11 L1 NLS- VLPs and rabbits #13 to #18 immunized with N. tabacum-derived HPV-11 L1 NLS- VLPs. Rabbits were immunized on days 1, 28 and 56 and serum from days 1, 15, 28, 42, 56 and 70 post injection was tested by ELISA in wells coated with 0.4 μg insect cell-derived HPV-11 L1 NLS- VLPs. Data from rabbit #12 is incomplete as it had to be euthanized due to growth of an abscess on the neck. Error bars represent the standard deviation calculated from triplicate analysis of samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2235857&req=5

Figure 5: Immunogenicity of plant extracts. Analysis of serum from New Zealand white rabbits #11 and #12 immunized with A. thaliana-derived HPV-11 L1 NLS- VLPs and rabbits #13 to #18 immunized with N. tabacum-derived HPV-11 L1 NLS- VLPs. Rabbits were immunized on days 1, 28 and 56 and serum from days 1, 15, 28, 42, 56 and 70 post injection was tested by ELISA in wells coated with 0.4 μg insect cell-derived HPV-11 L1 NLS- VLPs. Data from rabbit #12 is incomplete as it had to be euthanized due to growth of an abscess on the neck. Error bars represent the standard deviation calculated from triplicate analysis of samples.
Mentions: Eight New Zealand white rabbits were immunized with concentrated transgenic A. thaliana and N. tabacum protein extracts. Serum collected on days 1, 15, 28, 42, 56 and 70 was analysed for anti-HPV-11 L1 reactive antibodies by ELISA and results are shown in Figure 5. Rabbits #11 and #12 were immunised with T3 generation transgenic A. thaliana-derived L1 protein extract calculated to contain between 21 μg and 43 μg.

Bottom Line: Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro.Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity.This has important and potentially negative implications for the production of HPV-11 vaccines in plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, PO Observatory 7925, Cape Town, South Africa. thomas.kohl@jefferson.edu

ABSTRACT

Background: We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine.

Results: Transformation of plants was only achieved with the HPV-11 L1 gene with the C-terminal nuclear localization signal (NLS-) encoding region removed, and not with the full-length gene. The HPV-11 L1 NLS- gene was stably integrated and inherited through several generations of transgenic plants. Plant-derived HPV-11 L1 protein was capable of assembling into virus-like particles (VLPs), although resulting particles displayed a pleomorphic phenotype. Neutralising monoclonal antibodies binding both surface-linear and conformation-specific epitopes bound the A. thaliana-derived particles and - to a lesser degree - the N. tabacum-derived particles, suggesting that plant-derived and insect cell-derived VLPs displayed similar antigenic properties. Yields of up to 12 microg/g of HPV-11 L1 NLS- protein were harvested from transgenic A. thaliana plants, and 2 microg/g from N. tabacum plants - a significant increase over previous efforts. Immunization of New Zealand white rabbits with approximately 50 microg of plant-derived HPV-11 L1 NLS- protein induced an antibody response that predominantly recognized insect cell-produced HPV-11 L1 NLS- and not NLS+ VLPs. Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro.

Conclusion: We expressed the wild-type HPV-11 L1 NLS- gene in two different plant species and increased yields of HPV-11 L1 protein by between 500 and 1000-fold compared to previous reports. Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity. This has important and potentially negative implications for the production of HPV-11 vaccines in plants.

Show MeSH
Related in: MedlinePlus