Transient expression of chimeric Human papillomavirus 16 L1 protein carrying the M2e influenza epitope in Nicotiana benthamiana

Human papillomavirus 16 (HPV-16) L1 has the capacity to self-assemble into pentamers or virus-like particles (VLPs) that are highly immunogenic. In this report, we investigate the potential for using HPV-16 L1 as a carrier of two heterologous epitopes of influenza A virus: (i) M2e, ectodomain of the M2 protein, that is highly conserved among all influenza A isolates, or (ii) M2e8, shorter version of M2e containing the N terminal highly conserved SLLTEVET epitope, that is common for both M1 and M2 influenza proteins. Structural models available for HPV-16 L1 showing that helix 4 (h4), and the coil region between h4 and ?-sheet J were projected outwards in each of the five L1 monomers, led us to choose these two positions for the insertion of the influenza epitopes. A synthetic HPV-16 L1 gene optimized with human codon usage and fused to a calreticulin-endoplasmic reticulum targeting signal at the C terminus was used as a master gene to create four chimeric sequences: ChiM2e_h4, ChiM2e_c (with the M2e epitope introduced into the h4 and coil, respectively), ChiM2e8_h4, and ChiM2e8_c (with the M2e8 epitope introduced into the h4 and coil, respectively). All chimeric constructs transiently expressed in plants using a Cowpea mosaic virus-derived expression vector pEAQ-HT (Sainsbury et al., 2009) were recognized by a panel of linear and conformation-specific anti HPV-16 L1 MAbs. Preliminary results indicated that ChiM2e_h4 was the construct with the highest expression level in plants. This chimeric protein also strongly reacted with the anti-influenza A m2 MAb. Electron microscopy showed that chimeric proteins made in plants assembled into small VLPs of approximately 30 nm in diameter. This study further confirms the utility of human papillomavirus particles as carriers of heterologous epitopes and their potential as a plant-made vaccine platform.