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

Ricardo Correia; Bárbara Fernandes; Rute Castro; Hikaru Nagaoka; Eizo Takashima; Takafumi Tsuboi; Akihisa Fukushima; Nicola K Viebig; Hilde Depraetere; Paula M Alves; António Roldão

doi:10.3389/fbioe.2022.908509

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

Front Bioeng Biotechnol. 2022 Jun 30:10:908509. doi: 10.3389/fbioe.2022.908509. eCollection 2022.

Authors

Ricardo Correia^{1

2}, Bárbara Fernandes^{1

2}, Rute Castro¹, Hikaru Nagaoka³, Eizo Takashima³, Takafumi Tsuboi³, Akihisa Fukushima⁴, Nicola K Viebig⁵, Hilde Depraetere⁵, Paula M Alves^{1

2}, António Roldão^{1

2}

Affiliations

¹ IBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
² ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
³ Division of Malaria Research, Proteo-Science Center, Ehime University, Matsuyama, Japan.
⁴ Sumitomo Pharma Co., Ltd., Osaka, Japan.
⁵ European Vaccine Initiative, UniversitätsKlinikum Heidelberg, Heidelberg, Germany.

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.

Keywords: PfRipr5; baculovirus expression vector system; improved production; insect cells; low temperature; malaria asexual blood-stage vaccine; vector design.