Plant-Produced Cottontail Rabbit Papillomavirus L1 Protein Protects against Tumor Challenge: a Proof-of-Concept Study

ABSTRACT The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants.

Dynamics of the VIGS-Mediated Chimeric Silencing of the Nicotiana benthamiana ChlH Gene and of the Tobacco Mosaic Virus Vector

The ChlH gene, encoding for the H subunit of the magnesium chelatase enzyme, was silenced in Nicotiana bentahamiana plants by virus-induced gene silencing (VIGS), using tobacco mosaic virus (TMV) expression vector. Strong silencing of the ChlH target gene was initiated only in the apical tissues, in which the endogenous transcription level of the target gene and the level of TMV vector RNA were both very high. The virus vector was also targeted by VIGS, and its suppression was correlated with the silencing of the ChlH mRNA. In the apical tissues, the suppression of both the virus vector and the ChlH mRNA led to a reduction of the silencing pressure and, consequently, to partial recovery of the new growth from the silencing. As the virus vector and the target mRNA levels increased, silencing was reestablished. The feedback regulation system, caused by the transient increase and reduction in levels of the virus vector and ChlH mRNA, led to a fluctuation of the silenced and recovered phenotypes in the plant apex. This TMV-vector mediated silencing system differed from previously analyzed VIGS systems; although the TMV vector was initially targeted by the silencing system, it was not permanently suppressed, indicating that, in this system, TMV was able to effectively escape post-transcriptional gene silencing.