Production of human papillomavirus type 16 virus-like particles in transgenic plants

Abstract

Cervical cancer is linked to infection with human papillomaviruses (HPV) and is the third most common cancer among women worldwide. There is a strong demand for the development of an HPV preventive vaccine. Transgenic plants expressing the HPV major capsid protein L1 could be a system to produce virus-like particles for prophylactic vaccination or could even be used as edible vaccines to induce an L1-specific prophylactic immune response. Here, we describe the generation of transgenic tobacco and potato plants carrying the HPV type 16 major structural gene L1 under the control of the cauliflower mosaic virus 35S promoter. All attempts to express either the original, unmodified L1 gene or an L1 gene with a codon usage optimized for expression in plants failed. Surprisingly, small amounts of the protein were detected using an L1 gene optimized for expression in human cells. However, Northern blot analysis revealed that most of the L1 transcripts were degraded. Introduction of the translational enhancer Omega derived from the tobacco mosaic virus strongly increased transcript stability and resulted in accumulation of L1 protein to approximately 0.5 to 0.2% of total soluble protein in transgenic tobacco and potato plants, respectively. The plant-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles. Furthermore, we did not find any indications of protein modification of the L1 protein produced in plants. Plant-derived L1 was as immunogenic as L1 expressed in baculovirus-infected insect cells. Feeding of tubers from transgenic potatoes to mice induced an anti-L1 antibody response in 3 out of 24 mice, although this response was only transient in two of the mice. Our data, however, indicate that an anti-L1 response was primed in about half of the 24 animals.

FIG. 1.

Expression analysis of L1h-expressing tobacco plants. (a) Total RNA was isolated from leaves of tissue-cultured plantlets of 12 different transgenic lines (lanes 1 to 12) and wild-type (wt) controls (lanes 13 and 14). Fifteen micrograms of total RNA was loaded per lane and hybridized with L1h and Rbcs cDNA, respectively. (b) Immunodetection of L1 protein accumulation. Protein extracts were prepared from leaves of tissue-cultured plants. Eleven transgenic lines (lanes 2 to 11) and wild-type plants (lane 1 and 14) were analyzed by Western blotting using a rabbit-derived polyclonal anti-L1 antiserum (dilution 1:5,000) and a goat-derived anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:20,000). Equal amounts of protein were loaded to each lane. L1h protein migrated at approximately 55 kDa. (c) Expression-construct L1h used to generate the lines shown in (a) and (b). L1h is driven by the CaMV 35S promoter. Transcription is terminated at the octopine synthase polyadenylation signal (OCS).

FIG. 2.

Expression analysis of OD-L1h-transgenic tobacco plants. (a) Total RNA was isolated from greenhouse-grown source leaves of 10 different transgenic lines (lines 7, 35, 41, 44, 46, 50, 65, 71, 84, and 86) and wild-type (wt) plants (lanes 11 and 12). Thirty micrograms of RNA was loaded per lane and hybridized with L1-h and Rbcs cDNA, respectively. (b) Immunoblot analysis of L1 protein accumulation in OD-L1h expressing tobacco plants. Protein extracts were prepared from mature source leaves of 10 different transgenic lines (7, 35, 41, 44, 46, 50, 65, 71, 84, and 86) and wild-type plants (lane 7 and 13). Equal amounts of leaf protein (25 μg) were loaded onto each lane. Western blotting was performed using a rabbit-raised polyclonal anti-L1 antiserum (dilution 1:5,000) and a goat-raised anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:20,000). As a control and in order to assess percentage of L1 accumulation, 80 ng of L1 protein purified from insect cells was loaded (lane 1). L1h protein migrated at approximately 55 kDa. Protein migrating at ca. 65 kDa is assumed to be a cross-reacting plant protein. (c) Expression-construct OD-L1h used to generate the lines shown in panels a and b.

FIG. 3.

Accumulation of L1 protein in potato tubers of OD-L1h-expressing potato plants. Protein extracts were prepared from freshly harvested potato tubers of eight selected transgenic lines (lanes 2 to 8, corresponding to lines 7, 14, 27, 28, 30, 33, 34, and 37) and a wild-type (wt) control (lane 10). Thirty micrograms of soluble tuber protein were loaded onto each lane. As a control, 40 ng of L1 protein purified from insect cells were loaded (lane 1). Western blotting was performed as described in the legend to Fig. 2.

FIG. 4.

Purification of VLPs from transgenic potato plants. (a) Detection of L1 antigen by antigen-capture ELISA in fractions of CsCl gradient. The arrow indicates the fraction in which VLPs were detected. (b) Electron microscopy of potato plant-derived VLPs. Fractions of CsCl gradients with a density of approximately 1.32 g/ml were analyzed by negative staining. Bar = 200 nm. (c) Sucrose sedimentation analysis of L1 derived from transgenic tobacco leaves. Soluble proteins from tobacco leaf (upper right) and potato tuber (lower left and right) extracts were fractionated by sucrose density gradient centrifugation (fraction 1 corresponds to tube bottom). As control, purified VLPs from insect cells were loaded onto the gradients (upper left). As a 60 S sedimentation marker, empty VLPs of adeno-associated virus were used (45). As negative controls, extracts from tubers of nontransgenic plants were separated (upper left, open squares). Closed circles indicate the refractive index of the respective fraction.

FIG. 5.

VLPs derived from transgenic plants are immunogenic. A total of six mice were immunized twice at a 4-week intervals with 40 ng of VLPs purified from plants (top) or from baculovirus-infected insect cells (bottom), respectively. Sera from the immunized mice were then tested in an ELISA against insect cell-derived VLPs.

FIG. 6.

Oral vaccination of mice with transgenic potato tubers. Five groups of mice were fed four times within 46 days with 5 g of L1-transgenic (groups 1 to 3, mice 1 to 8, 9 to 16, 17 to 24, respectively) potato tubers. As control (groups 4 and 5, mice 25 to 27 and 28 to 30), six mice were fed with nontransgenic tubers. Groups 2 and 5 received tubers spiked with CpG plasmid DNA; mice of group 2 received the tubers with cholera toxin B-peptide as adjuvants. Serum samples were collected before the first and after each of the four feedings and tested in an ELISA for the presence of L1-specific IgG and IgM antibodies. Bars represent standard deviation from duplicate wells. (Note that serum of mouse 3 of group 1 was no longer available after the third feeding).

FIG. 7.

Priming of anti-L1 antibody responses in mice after oral uptake of transgenic plant material. To detect priming of a humoral immune response by ingestion of L1-transgenic potato tubers, all mice received a boost with a subimmunogenic dose of purified VLPs injected s.c. Sera were collected 21 days after the boost and tested in an ELISA for the presence of anti-L1-specific antibodies (IgG and IgM). A cutoff line was arbitrarily set according to the reactivity in the two control groups, groups 4 and 5 (mice 25 to 30). Sera of 12 of the remaining 21 mice from groups 1 to 3 that were fed with L1-transgenic tubers showed a reaction in the ELISA above this cutoff. Bars represent standard deviation from duplicate wells. (Note that sera from mice 1, 2, and 12 were not available for the assay.)