Comparative analysis of recombinant Human Papillomavirus 8 L1 production in plants by a variety of expression systems and purification methods

Abstract

Human papillomavirus 8 (HPV-8), one of the high-risk cutaneous papillomaviruses (cHPVs), is associated with epidermodysplasia verruciformis and nonmelanoma skin cancer in immuno-compromised individuals. Currently, no vaccines against cHPVs have been reported; however, recent studies on cross-neutralizing properties of their capsid proteins (CP) have fostered an interest in vaccine production against these viruses. We examined the potential of producing HPV-8 major CP L1 in Nicotiana benthamiana by agroinfiltration of different transient expression vectors: (i) the binary vector pBIN19 with or without silencing suppressor constructs, (ii) the nonreplicating Cowpea mosaic virus-derived expression vector pEAQ-HT and (iii) a replicating Tobacco mosaic virus (TMV)-based vector alone or with signal peptides. Although HPV-8 L1 was successfully expressed using pEAQ-HT and TMV, a 15-fold increase was obtained with pEAQ-HT. In contrast, no L1 protein could be immune detected using pBIN19 irrespective of whether silencing suppressors were coexpressed, although such constructs were required for identifying L1-specific transcripts. A fourfold yield increase in L1 expression was obtained when 22 C-terminal amino acids were deleted (L1ΔC22), possibly eliminating a nuclear localization signal. Electron microscopy showed that plant-made HPV-8 L1 proteins assembled in appropriate virus-like particles (VLPs) of T = 1 or T = 7 symmetry. Ultrathin sections of L1ΔC22-expressing cells revealed their accumulation in the cytoplasm in the form of VLPs or paracrystalline arrays. These results show for the first time the production and localization of HPV-8 L1 protein in planta and its assembly into VLPs representing promising candidate for potential vaccine production.

In planta production of a candidate vaccine against bovine papillomavirus type 1

“…Particles with a diameter of *30 nm were observed in VLP preparations, which is consistent (Matic et al 2012) with representing T = 1 particles of BPV (Fig. 3).…”

Assembly and Purification of Polyomavirus-Like Particles from Plants

“…DVLP was generated by the removal of a polybasic motif from the N-terminus of VP1. The motif is known to be involved in nuclear localisation [26], and the removal of the nuclear localisation signal from the related HPV L1 protein can improve expression of that protein in plants [23]. The present study shows that this is also the case for MPyV VP1, even though nuclear localisation was not fully excluded by DVP1.…”

“…It is expected that both intracapsomeric and intercapsomeric disulphide bridges are more efficiently formed during in vivo assembly than in vitro and this results in VLPs from eukaryotic cells possessing greater stability than in vitro-assembled VLPs [43]. Despite limited optimisation performed during this study and the analysis of just two gradient fractions, the recovery of purified VLPs approaches the highest reported levels of HPV L1 protein in crude extracts of plant tissue [21,23]. Purification could certainly be optimised with improved gradient and ultracentrifugation conditions, which could also benefit from a pre-enrichment of VLPs by, for example, PEG precipitation [14].…”

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“…To increase the level of targeted proteins in the host plant, scientists have been employing other physiological, biochemical and molecular approaches (Matic et al, 2012). At the gene-transfer-system level, some interesting assays have focused on using bacterial plasmids containing expression cassettes, while others exploit viral vector systems to express heterologous genes and their proteins .…”