Abstract
Human papillomaviruses (HPV) cause cervical cancer and have recently also been implicated in mouth, laryngeal and anogenital cancers. There are three commercially available prophylactic vaccines that show good efficacy; however, efforts to develop second-generation vaccines that are more affordable, stable and elicit a wider spectrum of cross-neutralising immunity are still ongoing. Testing antisera elicited by current and candidate HPV vaccines for neutralizing antibodies is done using a HPV pseudovirion (PsV)-based neutralisation assay (PBNA). PsVs are produced by transfection of mammalian cell cultures with plasmids expressing L1 and L2 capsid proteins, and a reporter gene plasmid, a highly expensive process. We investigated making HPV-16 PsVs in plants, in order to develop a cheaper alternative. The secreted embryonic alkaline phosphatase (SEAP) reporter gene and promoter were cloned into a geminivirus-derived plant expression vector, in order to produce circular dsDNA replicons. This was co-introduced into Nicotiana benthamiana plants with vectors expressing L1 and L2 via agroinfiltration, and presumptive PsVs were purified. The PsVs contained DNA, and could be successfully used for PBNA with anti-HPV antibodies. This is the first demonstration of the production of mammalian pseudovirions in plants, and the first demonstration of the potential of plants to make DNA vaccines.
Highlights
The fact that infectious Human papillomaviruses (HPV) virions are produced in vivo only in terminally differentiated keratinocytes[8] has severely hindered studies of virus replication and vaccine development, due to a lack of an efficient and reliable way to culture the virus[9]
In order to develop constructs to be used as the reporter genes for the PsVs, the self-replicating plant expression vector pRIC3.0 was modified by inserting a mammalian expression cassette that includes the secreted embryonic alkaline phosphatase (SEAP) gene
Autonomous replication of the constructs in plants would result in replicons of 4.8 kb and 6.6 kb for mSEAP and iSEAP, respectively (Fig. 1), a size range within the size limit (< 8 kb) to be packaged by the co-expressed HPV capsid proteins
Summary
The fact that infectious HPV virions are produced in vivo only in terminally differentiated keratinocytes[8] has severely hindered studies of virus replication and vaccine development, due to a lack of an efficient and reliable way to culture the virus[9]. The currently-accepted method of PsV production utilises the human cell line HEK293TT for high-titre production of HPV PsVs encapsidating a secreted alkaline phosphatase-expressing (SEAP) reporter plasmid with a SV40 origin of replication to allow multiplication in these cells[10,11] While this is a robust and effective method of PsV production, the protocol is both highly expensive and time-consuming. Several groups have reported the successful production of papillomavirus L1 capsid proteins in plants Both transgenic and transient expression of L1 has been done by us and by others, and spontaneous VLP assembly for HPV types 8, 11 and 16 has been shown, with varying degrees of efficiency[18,19,20,21,22,23]. Several investigations have shown that use of geminivirus-derived vectors, and especially of Bean yellow dwarf mastrevirus (BeYDV)-derived vectors, is a successful strategy for high-level protein production for products as diverse as candidate vaccine proteins or whole monoclonal antibodies[25,27,28]
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