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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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Detection of HPV-11 L1 protein in plant extracts using ELISA. (A) VLP characterisation by ELISA, utilising conformation-specific and linear MAbs, of HPV-11 L1 NLS- transgenic T3 generation A. thaliana protein extract from pooled lines and (B) N. tabacum protein extract from individual lines and generations. (for example: X1.2 represents line X1, T2 generation). Non-transgenic A. thaliana or N. tabacum protein extract was used as negative control; the positive control consisted of non-transgenic protein extract spiked with 0.4 μg/well insect cell-derived HPV-11 L1 NLS- VLPs. Error bars represent the standard deviation calculated from triplicate analysis of samples.
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Figure 2: Detection of HPV-11 L1 protein in plant extracts using ELISA. (A) VLP characterisation by ELISA, utilising conformation-specific and linear MAbs, of HPV-11 L1 NLS- transgenic T3 generation A. thaliana protein extract from pooled lines and (B) N. tabacum protein extract from individual lines and generations. (for example: X1.2 represents line X1, T2 generation). Non-transgenic A. thaliana or N. tabacum protein extract was used as negative control; the positive control consisted of non-transgenic protein extract spiked with 0.4 μg/well insect cell-derived HPV-11 L1 NLS- VLPs. Error bars represent the standard deviation calculated from triplicate analysis of samples.

Mentions: The final product resulting from homogenization, filtration and centrifugation of transgenic leaf material was analysed by standard techniques and showed that HPV-11 L1 NLS- protein was indeed present in the concentrated plant protein extracts (Figures 2 and 3).


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

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

Detection of HPV-11 L1 protein in plant extracts using ELISA. (A) VLP characterisation by ELISA, utilising conformation-specific and linear MAbs, of HPV-11 L1 NLS- transgenic T3 generation A. thaliana protein extract from pooled lines and (B) N. tabacum protein extract from individual lines and generations. (for example: X1.2 represents line X1, T2 generation). Non-transgenic A. thaliana or N. tabacum protein extract was used as negative control; the positive control consisted of non-transgenic protein extract spiked with 0.4 μg/well insect cell-derived HPV-11 L1 NLS- VLPs. 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 2: Detection of HPV-11 L1 protein in plant extracts using ELISA. (A) VLP characterisation by ELISA, utilising conformation-specific and linear MAbs, of HPV-11 L1 NLS- transgenic T3 generation A. thaliana protein extract from pooled lines and (B) N. tabacum protein extract from individual lines and generations. (for example: X1.2 represents line X1, T2 generation). Non-transgenic A. thaliana or N. tabacum protein extract was used as negative control; the positive control consisted of non-transgenic protein extract spiked with 0.4 μg/well insect cell-derived HPV-11 L1 NLS- VLPs. Error bars represent the standard deviation calculated from triplicate analysis of samples.
Mentions: The final product resulting from homogenization, filtration and centrifugation of transgenic leaf material was analysed by standard techniques and showed that HPV-11 L1 NLS- protein was indeed present in the concentrated plant protein extracts (Figures 2 and 3).

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