Bulk production of the antiviral lectin griffithsin

Fuqua, Joshua L.; Hamorsky, Krystal; Khalsa, Guruatma; Matoba, Nobuyuki; Palmer, Kenneth E.

Our experience with manufacturing-scale production of GRFT primarily has used dual-subgenomic promoter rTMV as the transient expression system (Fuqua et al., 2015; McCormick et al., 1999; O'Keefe et al., 2009; Shivprasad et al., 1999) ; it has proven to be scalable with few process-related issues. Transient expression of GRFT in N. benthamiana using rTMV can be done using naked infectious RNA transcribed from the plasmid or using first passage virus particles isolated from RNA-infected plants. At scale, the most cost-effective and reproducible method is to use first passage virions. Production of TMV virions for manufacturing requires qualification of the origin-plasmid containing the genome of the rTMV and continued assessment of the plasmid and virion for the duration of the manufacturing process. We have developed a program to monitor the stability and quality of the virion over the life of GRFT production, which will provide stability and activity data after long-term storage. The originplasmid DNA contains a dual-subgenomic promoter of infectious TMVU1 sequences upstream of the coat protein promoter behind which a N. benthamiana codon-optimized sequence of GRFT was cloned. The rTMV vector, designed by Shivprasad et al., used the heterologous tobacco mild green mosaic virus (U5) 3 0 sequences, coat protein cistron and 3 0 UTR ( Figure S1 ). The origin-plasmid was sequenced in both 5 0 and 3 0 directions, and a large amount of origin-plasmid DNA was purified using an endotoxin-free plasmid prep. The resulting purified plasmid was aliquoted and deemed the 'plasmid bank'. The plasmid bank was qualified by double-strand sequencing and comparison to the origin-plasmid. Ability to produce virion particles and GRFT from plasmid bank expression is assessed in planta by transcribing the DNA to RNA using an RNA transcription kit and subsequently infecting plants with the naked RNA (McCormick et al., 1999 (McCormick et al., , 2003 O'Keefe et al., 2009) . Infected plants are inspected for TMV-related symptoms at the specified harvest time with the qualification metric requiring 90% of inoculated plants to show visible symptoms (Figure 1a and b). Plants are harvested, and a gp120-capture enzyme-linked immunosorbent assay (gp120 ELISA) was used to quantify the produced GRFT protein in the green juice homogenate (O'Keefe et al., 2009) . For qualification of the inoculum, the infected plants have to be producing GRFT in excess of 200 mg/kg of biomass. Failure of plasmid to match the origin sequence or failure of the transcribed plasmid to induce significant symptomology in inoculated plants or failure to reach the target expression concentration of GRFT disqualified the plasmid bank in its entirety, and requires re-creation of the bank.