Insect Sf-9 (Invitrogen) and High Five (Invitrogen) cells were routinely subcultured at 0.4–1 × 10⁶ cell/mL every 3–4 days when cell density reached 2–3x10⁶cell/mL, as described elsewhere (Fernandes et al., 2020). Insect-XPRESS^TM (Sartorius) and Sf-900^TM II SFM (Thermo Fisher Scientific) media were used to culture Sf-9 and High Five cells, respectively. Human HEK293-E6 cells (NRC) (Durocher, 2002) were routinely subcultured to 0.5–0.6 × 10⁶ cells/mL every 3–4 days when cell density reached 2–3 × 10⁶ cells/mL in 125- or 500-mL shake flasks (20% working volume, w/v) in an Innova 44R incubator (orbital motion diameter of 2.54 cm Eppendorf) at 37°C with 5% CO₂ and stirring rates of 75 or 90 rpm. FreeStyle F17 (Thermo Fisher Scientific) media, supplemented with 4 mM GlutaMAX™ (Thermo Fisher Scientific) and 0.1% of Pluronic™ F-68 (Life Technologies), and 25 µg/mL of Geneticin were used to culture the HEK293 cells.

For expression in HEK293 cells, the PfCyRPA nucleotide sequence (Favuzza et al., 2017) was synthetically synthesized with the bee venom melittin (BVM) signal sequence to allow PfCyRPA secretion into the culture medium, and a C-terminal tag (His₆-tag) to allow purification, and cloned into the pTT5 vector, resulting in the pTT5-hCyRPA-6His plasmid. For expression in insect cells, the PfCyRPA nucleotide sequence (Favuzza et al., 2017) was synthetically synthesized with the BVM signal sequence and a C-terminal tag (His₆-tag, His₄-tag, or C-tag) for purification, and cloned into the pOET3 vector, resulting in three different expression plasmids: pOET3-iCyRPA-His₆, pOET3-iCyRPA-His₄, and pOET3-iCyRPA-Ctag. All plasmids were synthetically synthesized by GenScript.

Recombinant baculovirus (rBac) containing pOET3-iCyRPA-His₆, pOET3-iCyRPA-His₄, or pOET3-iCyRPA-Ctag plasmids, from now on named “rBac-CyRPA-His6,” “rBac-CyRPA-His4,” and “rBac-CyRPA-Ctag,” respectively, were generated using the flashback ULTRA^TM system (Oxford Expression Technologies) in accordance with manufacturer’s instruction. The amplification of baculovirus stocks was performed as described elsewhere (Vieira et al., 2005). Briefly, Sf-9 cells were infected at a cell concentration of 1 × 10⁶ cells/mL with 0.01–0.1 infectious baculovirus per cell (pfu/cell). When cell viability reached 80–85%, cultures were harvested and centrifuged at ×200g for 10 min at 4°C. The pellet was discarded, and the supernatant was centrifuged at ×2000g for 20 min at 4°C. The resulting supernatant was stored at 4°C until further use.

Purification of PfCyRPA was carried out on an ÄKTA Explorer 100 System (Cytiva). Cell culture bulk was harvested, filtered through 0.45- and 0.22-μm Sartopore 2 Midicap Filter Cartridges 10 (Sartorius), and concentrated with a Sartocon disposable PES membrane 2 × 0.1 m², 10 kDa (Sartorius). The concentrated sample was filtered through a Nalgene cup of 0.2 μm (Thermo Scientific). PfCyRPA His₆- and His₄-tagged proteins were purified by immobilized metal ion affinity chromatography on a Histrap HP column (5 mL volume; Cytiva), while PfCyRPA C-tag tagged protein was purified on a CaptureSelect™ C-tagaffinity matrix chromatography column (5 mL volume; Thermo Fisher). Column eluates were concentrated using an AmiconUltra 15 Centrifugal Filter Unit 10 kDa (Merck Millipore), filtered through 0.2 μm, and applied to a HiLoad 26/60 Superdex 75 gel permeation column (GE Healthcare). The eluates were concentrated using an AmiconUltra 15 Centrifugal Filter Unit 10 kDa (Merck Millipore) and filtered through 0.2-μm filter. The final sample was stored in 50 mM Tris–HCl, pH 7.4 and 150 mM NaCl buffer, aliquoted, and stored at −80°C.

The purified PfCyRPA was lipidated by chemical conjugation to 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleinimidomethyl) cyclohexane-carboxamide)] (N-MCC-DPPE, Corden Pharma, Liestal, Switzerland) after limited modification of lysine residues with 2-iminothiolane (Merck & Cie, Schaffhausen, Switzerland) to introduce free thiol groups. The lipidated PfCyRPA antigens were subsequently inserted into the virosome lipid membrane, as previously described (Amacker et al., 2020). Virosomes were prepared by Mymetics from inactivated and purified influenza virus [A/Brisbane/59/2007 (H1N1), Seqirus, Australia] with membrane-integrated 3M-052 adjuvant as a TLR7/8 agonist (3M company, St. Paul, United States). The final liquid virosome-PfCyRPA contained approximately 40 μg/mL of PfCyRPA, 6 μg/mL of hemagglutinin (HA), and 15 μg/mL of 3M–052 adjuvant and was supplied in 50 mM HEPES buffer pH 7.4, 142.5 mM NaCl. Quality controls were conducted using ELISA methods for determining the concentration (μg/mL) of PfCyRPA, HPLC for 3M-052, and single radial immunodiffusion assay (SRID) for HA concentration (μg/mL). Dynamic light scattering (DLS) for determining the virosome particle size and homogeneity (polydispersity index, PDI) was performed on a Malvern Zetasizer Nano S (Malvern Instruments, Worcestershire, United Kingdom). Each sample was analyzed in triplicate at 25°C, and each replicate was measured six times to obtain the average particle size.

New Zealand rabbits were immunized and bled at Kaneka Eurogentec S.A. (Belgium). The animals (n = 2) were given three subcutaneous immunizations (30 μg antigen per dose) in intervals of 4 weeks. Blood was collected before each immunization and 9 days after the final injection. Total serum IgG was purified from rabbit sera using protein A columns (Cytiva) and assessed for activity in a single-cycle in vitro GIA with the Pf 3D7 strain.

Culture supplements and temperature were selected as factors for the screening DoE. A full factorial design with two levels and five factors was used to evaluate the effect of each parameter and their interactions with each other (Supplementary Table S1). This design allowed the determination of the main effects and all two-factor interactions without co-founding. A total of nineteen experiments were performed with three duplicates of the center point. For optimization of DoE, a central composite circumscribed design composed of a full or fractional factorial design and star points was implemented for optimization of conditions selected from the screening DoE (Supplementary Table S2). A total of eleven experiments were performed with three duplicates of the center point. The response for both screening and optimization DoEs was defined as the fold improvement in the PfCyRPA titer (assessed by densitometry analysis of Western blot images and for cell viabilities between 30 and 40%) of each experiment when compared to standard culture conditions (i.e., 27°C without supplementation). MODDE® 12.1 Pro software (Sartorius) was used to determine significant effects, interactions, and distribution of the data.

Data were expressed as mean ± standard deviation. Differences were tested by one-way ANOVA with post hoc Tukey’s multiple comparison analysis methods and Dunnett’s multiple comparison test (adjusted p-value < 0.05 was considered statistically significant) and were tested by t-test unpaired Gaussian distribution (adjusted p-value < 0.05 was considered statistically significant).

The sensitive nature of some of the reagents used in this study (e.g., cell lines, plasmids, baculoviruses, virosomes, and antibodies) means that they are only readily available internally to the author’s institutions staff for R&D purposes. For external researchers, approval of reagent requests may be obtained via email addressed to the corresponding author.

Sf9 and High Five cells were infected with rBac-CyRPA-His6 at different CCIs (1 and 2 × 10⁶ cell/mL) and MOIs (0.1 and 1 pfu/cell), and their growth and expression kinetics were assessed in small-scale shake flasks (SF) (Supplementary Figure S1 and Figure 1 ). The highest PfCyRPA titer was achieved in High Five cells when infected at CCI = 2 × 10⁶ cell/mL with MOI = 1 pfu/cell; thus, this cell line and infection strategy were used for subsequent experiments.

The feasibility of producing PfCyRPA in High Five cells was demonstrated in controlled, scalable 2-L stirred tank bioreactors (STB) and compared to HEK293 cells; small-scale SF was used to assess scalability.

Cell growth and viability kinetics in STB and SF were similar for both cells. With regression coefficients (b) and Pearson’s correlations (r) close to 1 (Figure 2A). Likewise, PfCyRPA titers (estimated by densitometry analysis of Western blot images, Supplementary Figure S2), achieved in STB and SF, are comparable (Figure 2B). The intracellular PfCyRPA content at the time of harvest was also assessed (only for STB runs), and results have shown that 91–97% of total PfCyRPA produced was secreted, with no apparent difference between High Five and HEK293 cells.

High Five and HEK293-derived PfCyRPA produced in STB were purified, and similar recovery yields were achieved (Table 2). Both proteins present purities above 90% (Figure 2C), similar melting temperatures (Tm, ≈ 61°C) (Table 2), and identical dimensions (Figure 2D). In addition, mass spectrometry analysis of proteolytic fragments from both PfCyRPA proteins revealed similar sequence coverage and the same disulfide bond numbers (Figure 2E).

The results obtained confirm the feasibility and scalability of producing PfCyRPA in insect High Five cells.

Sets of anti-PfCyRPA mAbs have been grouped into six epitope groups (Favuzza et al., 2017) by determining competition for antigen binding and reactivity patterns with overlapping fragments of PfCyRPA as described elsewhere (Dreyer et al., 2012; Favuzza et al., 2016). No differences were observed, when the reactivity of representatives of these mAbs specific for the epitope groups A-F was tested with purified PfCyRPA produced using insect or mammalian cells (Figure 3A). Noteworthy, the immunization of rabbits with a lipid-based particle formulation of purified PfCyRPA expressed using High Five cells led to the generation of antibody responses that prevented the multiplication of P. falciparum in a parasite GIA (Figure 3B ).

The impact of culture temperature and medium supplementation on PfCyRPA expression was assessed in small-scale SF using ) a full factorial DoE to evaluate the effect of each parameter and their interaction with each other (screening DoE), followed by ) a central composite DoE for optimization of the previous conditions selected (optimization DoE). The culture temperatures explored were 21, 27, 31.5, and 36 C. The culture medium supplements evaluated were lipids, polyamines, antioxidants, and α-ketoglutarate (Table 1). The cultures at 27°C without supplements were used as the control.

Supplementation of antioxidants at the time of infection to cultures at 27°C (Supplementary Table S1) enhanced PfCyRPA expression when compared to control cultures (Figure 4A—Western blot images of these experiments are reported in Supplementary Figure S3A), and thus, antioxidants were selected for further optimization experiments.

A central composite experimental design was used to optimize the concentration of antioxidants and the culture temperature. The experimental design matrix is shown in Supplementary Table S2. Based on the adjusted response surface model, it was possible to predict the best condition to maximize PfCyRPA expression: cultures at 27°C and supplemented with 0.26 × antioxidants at the time of infection (Figure 4B–Western blot images of these experiments are reported in Supplementary Figure S3B). This optimal condition was tested in small-scale SF, with an approximately 45% increase in PfCyRPA titer observed when compared to the control culture (Figure 4C, Western blot images of these experiments are reported in Supplementary Figure S4).

The impact of different affinity tags (i.e., 6×-His, 4×-His, and C-tag) on PfCyRPA recovery yields was assessed in controlled, scalable 2-L STB. The optimal culture conditions identified before were used: CCI = 2 × 10⁶ cell/mL, MOI = 0.1 pfu/cell, culture temperature of 27°C, and culture supplementation with 0.26 x antioxidants.

Cell growth and viability kinetics were similar in all STB production runs (Figure 5A), with no apparent impact of affinity tag on PfCyRPA expression (Supplementary Figure S4). Noteworthy, PfCyRPA recovery yields were higher when using histidine tags than C-tag, with 4×-His slightly outperforming 6×-His (Table 2), thus resulting in a higher final yield (Figure 5B). Independently of the affinity tag used, purified PfCyRPA proteins showed high purity (Figure 5C), similar melting temperatures (Table 2) and size (Figure 5D), and comparable sequence coverage with the same disulfide bonds detected (Figure 5E).

These results confirm the feasibility of the strategy herein proposed (i.e. insect High Five cells cultured at 27°C with supplementation with 0.26 × antioxidants at time of infection, and 4×-His affinity tag) for the production of PfCyRPA protein.