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

Human papillomavirus 16 (HPV 16) L1 self-assembles into pentamers or virus-like particles (VLPs) that are highly immunogenic. We investigated the potential for using HPV-16 L1 as a carrier of heterologous epitopes of influenza A virus: (i) M2e, ectodomain of the M2 protein, highly conserved among all influenza A isolates and (ii) M2e8, a shorter version including the N-terminal highly conserved SLLTEVET epitope, common for both M1 and M2 proteins. Structural models available for HPV-16 L1 showing that helix 4 (h4) and the coil region linking h4 and ?-sheet J are projected outwards in each of the five L1 monomers led us to choose these two positions for inserting the epitopes. A synthetic HPV-16 L1 gene optimized with human codon usage and fused to a calreticulin-endoplasmic reticulum targeting signal was used as a master gene to create four chimeric sequences: ChiM2e_h4, ChiM2e_c (M2e epitope into h4 and coil, respectively), ChiM2e8_h4, and ChiM2e8_c (M2e8 epitope into h4 and coil, respectively). All chimeric constructs transiently expressed in plants using the Cowpea mosaic virus-derived vector pEAQ-HT (Sainsbury et al., 2009) were recognized by a panel of linear and conformational-specific anti HPV-16 L1 MAbs. ChiM2e_h4 appeared to be the most highly expressed construct. This chimeric protein also strongly reacted with an anti-influenza A M2 MAb. Electron microscopy showed that plant-made chimeric proteins assembled into small VLPs of approximately 30 nm in diameter. This study further confirms the use of papillomavirus particles as carriers of heterologous epitopes and their potential as a plant-made vaccine platform. Work supported by EU FP7 Grant Agreement No. KBBE-227056 (PLAPROVA).