Production of Human papillomavirus pseudovirions in plants and their use in pseudovirion-based neutralisation assays in mammalian cells

www.nature.com/scientificreports OPEN received: 21 August 2015 accepted: 04 January 2016 Published: 08 February 2016 Production of Human papillomavirus pseudovirions in plants and their use in pseudovirion-based neutralisation assays in mammalian cells Renate L Lamprecht1, Paul Kennedy1, Suzanne M Huddy1, Susanne Bethke2, Megan Hendrikse1, Inga I Hitzeroth1 & Edward P Rybicki1,3 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. Human papillomaviruses (HPV) are the most common agents of viral infections of the human reproductive tract that are transmitted through sexual contact. Infection and persistence of the oncogenic high-risk HPV-type infections, such as HPV types 16 and 18, are linked to cervical cancer and other anogenital and oropharyngeal cancers in humans. The non-oncogenic or low-risk types of HPV cause common skin and genital warts and other lesions. More than a hundred HPV types have been identified of which twelve have been linked to cervical cancer1–3. HPV is a virus with a double-stranded circular DNA genome of ~8 kb, and small non-enveloped isometric particles with a diameter of 55–60 nm. The capsid of the virus is composed of the main capsid protein L1 and the minor capsid protein L2. Although L2 is not required for capsid formation, it is thought to play a number of essential roles in viral DNA encapsidation, and in the viral infectious entry pathway to effectively deliver the viral DNA into the host cell4,5. Two prophylactic vaccines – Gardasil (Merck) and Cervarix (GSK) - were approved by the U.S. Food and Drug Administration (FDA) in 2006 and 2009 respectively, in order to combat the development of HPV-associated cancers. These prophylactic vaccines exploit the fact that the HPV L1 self-assembles into virus-like particles (VLPs) that are both morphologically correct and highly immunogenic6,7. Merck’s second-generation vaccine Gardasil-9, approved in November 2014, is comprised of VLPs from nine different HPV types, and has the potential of preventing up to 90% of cervical, vulvar, vaginal and anal cancers. 1 Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, 7701, South Africa. 2Pharmaceutical Product Development, Fraunhofer IME, Aachen, 52074, Germany. 3Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, 7701, South Africa. Correspondence and requests for materials should be addressed to I.I.H. (email: ) Scientific Reports | 6:20431 | DOI: 10.1038/srep20431 1 www.nature.com/scientificreports/ The fact that infectious HPV virions are produced in vivo only in terminally differentiated keratinocytes8 has severely hindered studies of virus replication and vaccine development, due to a lack of an efficient and reliable way to culture the virus9. Testing of neutralisation of infectivity in vivo or in vitro has also been hindered: however, several methods have recently been developed to produce structurally authentic HPV pseudovirions (PsVs). It was shown that HPV VLPs produced by co-expression of L1 and L2 could package non-papillomaviral DNA in vitro, with similar efficiency to packaging of the papillomaviral genomic DNA. PsVs could be produced by co-transfection of L1 and L2-encoding expression vector plasmids and a plasmid encoding a reporter gene into cultured mammalian cells, and were able to transfer plasmid DNA into epithelial cells in the same manner as HPV virions would transfer the viral genomic DNA. PsVs have since then become indispensable for their use in the pseudovirion-based neutralisation assay (PBNA), now considered the gold standard test of immunogenicity for candidate HPV vaccines. 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 specifically in these cells10,11. While this is a robust and effective method of PsV production, the protocol is both highly expensive and time-consuming. Transient expression of recombinant proteins in plants, mainly via infiltration of plants with recombinant Agrobacterium tumefaciens (agroinfiltration), has become a viable alternative to other more established production systems12,13. Transient expression is preferred to the establishment of transgenic plant lines as (1) it is much less time-consuming, (2) transient expression generally results in higher protein yields, (3) scale up and good manufacturing practices are adaptable, and (4) waste generated is more easily contained13–15. The development of industrial-scale vacuum infiltration equipment has shown transient expression to be a highly effective tool for large-scale production of even complex VLPs such as candidate influenza or orbivirus vaccines16,17. 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 efficiency18–23. In all instances the plant-produced VLPs were morphologically similar to VLPs produced in other systems, and elicited similar immunological responses. While expression of HPV L2 proteins is far less well studied, and plant-made L1 +  L2 VLPs have not been reported in the literature, our group has successfully expressed HPV-16 L2 in N. benthamiana via agroinfiltration24. The use of replicating DNA virus-derived vectors for transient expression in plants has recently bee (...truncated)