Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells

Abstract

The malaria asexual blood-stage antigen PfRipr and its most immunogenic fragment PfRipr5 have recently risen as promising vaccine candidates against this infectious disease. Continued development of high-yielding, scalable production platforms is essential to advance the malaria vaccine research. Insect cells have supplied the production of numerous vaccine antigens in a fast and cost-effective manner; improving this platform further could prove key to its wider use. In this study, insect (Sf9 and High Five) and human (HEK293) cell hosts as well as process-optimizing strategies (new baculovirus construct designs and a culture temperature shift to hypothermic conditions) were employed to improve the production of the malaria asexual blood-stage vaccine candidate PfRipr5. Protein expression was maximized using High Five cells at CCI of 2 × 106 cell/mL and MOI of 0.1 pfu/cell (production yield = 0.49 mg/ml), with high-purity PfRipr5 binding to a conformational anti-PfRipr monoclonal antibody known to hold GIA activity and parasite PfRipr staining capacity. Further improvements in the PfRipr5 expression were achieved by designing novel expression vector sequences and performing a culture temperature shift to hypothermic culture conditions. Addition of one alanine (A) amino acid residue adjacent to the signal peptide cleavage site and a glycine-serine linker (GGSGG) between the PfRipr5 sequence and the purification tag (His6) induced a 2.2-fold increase in the expression of secreted PfRipr5 over using the expression vector with none of these additions. Performing a culture temperature shift from the standard 27–22°C at the time of infection improved the PfRipr5 expression by up to 1.7 fold. Notably, a synergistic effect was attained when combining both strategies, enabling to increase production yield post-purification by 5.2 fold, with similar protein quality (i.e., purity and binding to anti-PfRipr monoclonal antibody). This work highlights the potential of insect cells to produce the PfRipr5 malaria vaccine candidate and the importance of optimizing the expression vector and culture conditions to boost the expression of secreted proteins.

A novel asexual blood-stage malaria vaccine candidate: PfRipr5 formulated with human-use adjuvants induces potent growth inhibitory antibodies

“…PfRipr5 recombinant protein was produced in a 50 L stirred-tank bioreactor (Sartorius, Göttingen, Germany) by infecting insect High Five cells (Invitrogen, Carlsbad, CA) at 2 ×10 6 cell/mL with a recombinant baculovirus encoding pfripr5 nucleotide sequence and His 6 -tag for purification, using a multiplicity of infection of 0.1 virus per cell, as described elsewhere ( 27 ). Cells were expanded by sub-culturing at 0.3-0.5 ×10 6 cell/mL every 2-3 days when cell density reached 2-3 ×10 6 cell/mL in Insect-XPRESS™ (Sartorius) and at 27°C, using shake-flasks (Corning, Corning, NY) of 500 mL (N-4 stage) and 2000 mL (N-3 stage), and stirred tank-bioreactors (Sartorius) of 2 L (N-2 stage), 10 L (N-1 stage) and 50 L (production stage, N).…”

“…Purification of secreted PfRipr5 was carried out on ÄKTA Explorer 100 systems (Cytiva, Tokyo, Japan) as described elsewhere ( 27 ). In brief, cell culture bulk was clarified using a Sartopore 2 30’’ 0.45 µm + 0.2 µm filter (Sartorius), loaded on a Histrap HP column (Cytiva), and protein was eluted with a linear Imidazole gradient.…”

“…Surface plasmon resonance (SPR) was carried out in Biacore X100 (Cytiva) as we previously described ( 27 ).…”

“…In addition, the interaction of the PfRipr5 antigen with Alhydrogel ® or CAF ® 01 was further evaluated by mixing the adjuvant with 100 µg/mL or 400 µg/mL of the PfRipr5 protein, centrifuging at 14,000 × g for 15 minutes (Alhydrogel ® formulations) or 137,400 × g for 30 minutes (CAF ® 01 formulations), and measuring non-adsorbed protein in the supernatant using bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). In the case of GLA-SE formulation, antigen stability was assessed by sandwich-ELISA established in our recent study using functional mouse anti-PfRipr mAb (clone 29B11) as capture antibody ( 27 ). Because we have previously reported the binding of PfRipr5 with mouse anti-PfRipr5 mAb 29B11, shown to have a potent GIA activity ( 27 ), thereby being used as proxy for predicting its biological activity.…”

“…In the case of GLA-SE formulation, antigen stability was assessed by sandwich-ELISA established in our recent study using functional mouse anti-PfRipr mAb (clone 29B11) as capture antibody ( 27 ). Because we have previously reported the binding of PfRipr5 with mouse anti-PfRipr5 mAb 29B11, shown to have a potent GIA activity ( 27 ), thereby being used as proxy for predicting its biological activity.…”