Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco
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Human Papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death for women. The major capsid L1 protein self-assembles in Virus Like Particles (VLPs), which are highly immunogenic and suitable for vaccine production. In this study, a plastid transformation approach was assessed in order to produce a plant-based HPV-16 L1 vaccine. Transplastomic plants were obtained after transformation with vectors carrying a chimeric gene encoding the L1 protein either as the native viral (L1v gene) or a synthetic sequence optimized for expression in plant plastids (L1pt gene) under control of plastid expression signals. The L1 mRNA was detected in plastids and the L1 antigen accumulated up to 1.5% total leaf proteins only when vectors included the 5′-UTR and a short N-terminal coding segment (Downstream Box) of a plastid gene. The half-life of the engineered L1 protein, determined by pulse-chase experiments, is at least 8 h. Formation of immunogenic VLPs in chloroplasts was confirmed by capture ELISA assay using antibodies recognizing conformational epitopes and by electron microscopy.
KeywordsPlastid transformation HPV16 L1 Plant vaccines Tobacco Nicotiana tabacum
This research was partially supported by grants from the Italian Ministry of Health F.S.N. 2003 and Italian Ministry of Research DD 1105/2002. PL was the recipient of a Ph.D. studentship of the University of Naples “Federico II”; part of the studentship was spent at Rutgers University. Anita Morgese and Alfonso Piccolo are gratefully acknowledged for technical help. We thank Dr. M. Müller (Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, 69120 Heidelberg, Germany) for providing anti-L1 monoclonal Mabs, rabbit anti-L1 polyclonal serum and baculovirus-derived VLPs. PL thanks in particular Dr. Z. Svab for stimulating discussions and guidance during his stay at Rutgers University.
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