An effective orally delivered HPV prophylactic vaccine could facilitate efforts to control cervical HPV disease, particularly in low-resource settings, where the disease is most prevalent. The results of our study demonstrate that HPV11 L1 capsid protein can be expressed in an edible plant (i.e., potato tuber) to form empty capsids that are appropriately antigenic, as judged by the ability to bind antibodies that react specifically with, and efficiently neutralize, native HPV11 virions (28
). Importantly, our results demonstrate that ingestion of transgenic L1 tubers activates anti-VLP immune responses that can be boosted by subsequent administration of purified VLPs. From this, we conclude that HPV transgenic L1 plants offer a feasible and potentially useful alternative strategy for immunization against anogenital HPV disease.
The requirement for coadministered adjuvant for induction of observed responses to potato-derived antigen suggests that the effective dose of HPV11 L1 VLPs in these experiments was relatively low. Previously, the induction of anti-VLP responses in mice following oral administration of as little as 1 μg of purified insect cell-derived VLPs was reported (13
). In the present study, responses induced by ingestion of transgenic L1 potato were dependent on the coadministration of adjuvant. Consistent with this observation, our quantitative analysis of L1 expression in transgenic tuber indicated that the concentration of L1 VLPs was ~20 ng per g of fresh tuber. Thus, the effective oral dose level of VLPs in these experiments was ~100 ng per 5-g feeding, or roughly 1/10 the amount of immunogen previously determined to represent the minimum oral dose level of purified insect cell-derived VLPs without adjuvant (13
). Such a low level of expression in plants precludes the direct use of the material from the present study in human subjects; nevertheless, the present results constitute an important proof of principle for this immunization strategy.
The ability of an immunogen to establish a memory response is a key element in the design of an efficacious vaccine. Responses to oral boosting observed in mice that ingested transgenic L1 potato with adjuvant indicated the generation of antigen-specific memory cells. The absence of response to oral boosting in mice fed only nontransgenic potato supports this conclusion. The ability of plant-expressed L1 to establish VLP-specific immune memory, even though VLPs were expressed at a relatively low level in potato, encourages further study of this vaccine material.
Previous studies have shown that transgene expression in plants often leads to low expression levels, presumably due to RNA instability or to the use of codons that are unfavorable in plants (24
). It has also been shown that resynthesis of complete genes and their adaptation to the plant host can mediate much higher levels of heterologous protein expression (19
). Our analysis of the HPV11 L1 coding sequence revealed several internal polyadenylation signals, intron splice recognition sequences, and mRNA-destabilizing motifs that could yield truncated mRNA or decrease mRNA stability in plant cells. The synthetic L1 sequence spares those signals and additionally provides a pattern of codon usage that is highly preferred for expression in dicotyledonous plants (2
). With the optimized version of L1, we observed expression of L1 protein only in plants transformed with a truncated form that lacked the C-terminal arm domain, which contains a well-characterized NLS sequence (23
). Our observations that (i) full-length HPV11 L1 protein directed fused GFP specifically to the nuclei of plant cells and (ii) full-length L1 protein was not expressed in transgenic potato plants are consistent with the conclusion that expression of full-length L1 protein is not well tolerated in plants. Relatively low expression and poor transformation efficiency with the truncated L1 gene suggest that even with the C-terminal NLS removed, L1 protein could be toxic to plant cells. Use of developmentally regulated or chemically inducible promoters to control expression is likely to solve this problem. Preliminary results for HPV11 L1 expression in tomato show promise for a fruit-specific promoter strategy (data not shown).
The study of plant-based expression and oral delivery of vaccine antigens has expanded greatly in recent years, with a large number of papers showing faithful expression of antigenic proteins (22
). Three human clinical trials with orally delivered vaccines produced in plants have been published, all showing stimulation of immune responses against the recombinant antigens expressed in edible plant tissues. The first (40
) used potatoes expressing E. coli
labile toxin B subunit (LT-B), a strong mucosal immunogen that binds gangliosides displayed on epithelial cell surfaces. Ingestion of raw potato containing up to 750 μg of LT-B on days 0, 7, and 21 resulted in toxin-neutralizing serum IgG antibodies in 10 of 11 subjects as late as day 59 and LT-B-specific IgA in fecal samples of some volunteers. A human study with potato-expressed capsid protein of another enteric pathogen, Norwalk virus, also showed promising results (41
), with 95% of subjects showing increases in antibody-secreting cells of the IgA subtype. However, the serum IgG and fecal IgA levels produced by orally delivered antigen in that study were less impressive than in the LT-B study, suggesting that higher doses were needed. The only published human study with a nonenteric vaccine used hepatitis B virus surface antigen expressed in lettuce and stimulated serum IgG at protective levels in two of three volunteers with two doses containing only 1 μg of antigen (17
Recent evidence of the remarkable protective efficacy of a parenterally administered HPV VLP vaccine (18
) bodes well for the possibility of controlling cervical HPV disease through vaccination. Alternative immunization strategies are needed, however, to address the difficulties associated with the distribution of parenteral vaccines in developing regions. As with other promising noninvasive methods of VLP administration (3
), the low cost and ease of delivering an edible HPV vaccine could facilitate vaccine distribution in economically disadvantaged regions, which carry a large burden of anogenital HPV disease.