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

摘要

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 (L1????C22), 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 L1????C22?￠????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.