Adaptive Laboratory Evolution to Improve Recombinant Protein Production Using Insect Cells.

Adaptive laboratory evolution (ALE) is a powerful tool for enhancing the fitness of cell lines in specific applications, including recombinant protein production. Through adaptation to nonstandard culture conditions, cells can develop specific traits that make them high producers. Despite being widely used for microorganisms and, to lesser extent, for mammalian cells, ALE has been poorly leveraged for insect cells. Here, we describe a method for adapting insect High Five and Sf9 cells to nonstandard culture conditions via an ALE approach. Aiming to demonstrate the potential of ALE to improve productivity of insect cells, two case studies are demonstrated. In the first, we adapted insect High Five cells from their standard pH (6.2) to neutral pH (7.0); this adaptation allowed to improve production of influenza virus-like particles (VLPs) by threefold, using the transient baculovirus expression vector system. In the second, we adapted insect Sf9 cells from their standard culture temperature (27 °C) to hypothermic growth (22 °C); this adaptation allowed to improve production of influenza VLPs by sixfold, using stable cell lines. These examples demonstrate the potential of ALE for enhancing productivity within distinct insect cell hosts and expression systems by manipulating different culture conditions.

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming.

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom-polyclonal antibodies isolated from the plasma of hyperimmunised animals-which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.

Vaccine-induced neutralizing antibody responses to seasonal influenza virus H1N1 strains are not enhanced during subsequent pandemic H1N1 infection.

The first exposure to influenza is presumed to shape the B-cell antibody repertoire, leading to preferential enhancement of the initially formed responses during subsequent exposure to viral variants. Here, we investigated whether this principle remains applicable when there are large genetic and antigenic differences between primary and secondary influenza virus antigens. Because humans usually have a complex history of influenza virus exposure, we conducted this investigation in influenza-naive cynomolgus macaques. Two groups of six macaques were immunized four times with influenza virus-like particles (VLPs) displaying either one (monovalent) or five (pentavalent) different hemagglutinin (HA) antigens derived from seasonal H1N1 (H1N1) strains. Four weeks after the final immunization, animals were challenged with pandemic H1N1 (H1N1pdm09). Although immunization resulted in robust virus-neutralizing responses to all VLP-based vaccine strains, there were no cross-neutralization responses to H1N1pdm09, and all animals became infected. No reductions in viral load in the nose or throat were detected in either vaccine group. After infection, strong virus-neutralizing responses to H1N1pdm09 were induced. However, there were no increases in virus-neutralizing titers against four of the five H1N1 vaccine strains; and only a mild increase was observed in virus-neutralizing titer against the influenza A/Texas/36/91 vaccine strain. After H1N1pdm09 infection, both vaccine groups showed higher virus-neutralizing titers against two H1N1 strains of intermediate antigenic distance between the H1N1 vaccine strains and H1N1pdm09, compared with the naive control group. Furthermore, both vaccine groups had higher HA-stem antibodies early after infection than the control group. In conclusion, immunization with VLPs displaying HA from antigenically distinct H1N1 variants increased the breadth of the immune response during subsequent H1N1pdm09 challenge, although this phenomenon was limited to intermediate antigenic variants.

Towards a scalable bioprocess for rAAV production using a HeLa stable cell line.

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.

Dissecting insect cell heterogeneity during influenza VLP production using single-cell transcriptomics.

The insect cell-baculovirus expression vector system (IC-BEVS) has been widely used to produce recombinant protein at high titers, including complex virus-like particles (VPLs). However, cell-to-cell variability upon infection is yet one of the least understood phenomena in virology, and little is known about its impact on production of therapeutic proteins. This study aimed at dissecting insect cell population heterogeneity during production of influenza VLPs in IC-BEVS using single-cell RNA-seq (scRNA-seq). High Five cell population was shown to be heterogeneous even before infection, with cell cycle being one of the factors contributing for this variation. In addition, infected insect cells were clustered according to the timing and level of baculovirus genes expression, with each cluster reporting similar influenza VLPs transgenes (i.e., hemagglutinin and M1) transcript counts. Trajectory analysis enabled to track infection progression throughout pseudotime. Specific pathways such as translation machinery, protein folding, sorting and degradation, endocytosis and energy metabolism were identified as being those which vary the most during insect cell infection and production of Influenza VLPs. Overall, this study lays the ground for the application of scRNA-seq in IC-BEVS processes to isolate relevant biological mechanisms during recombinant protein expression towards its further optimization.

Impact of dual-baculovirus infection on the Sf9 insect cell transcriptome during rAAV production using single-cell RNA-seq.

The insect cell-baculovirus expression vector system (IC-BEVS) has shown to be a powerful platform to produce complex biopharmaceutical products, such as recombinant proteins and virus-like particles. More recently, IC-BEVS has also been used as an alternative to produce recombinant adeno-associated virus (rAAV). However, little is known about the variability of insect cell populations and the potential effect of heterogeneity (e.g., stochastic infection process and differences in infection kinetics) on product titer and/or quality. In this study, transcriptomics analysis of Sf9 insect cells during the production of rAAV of serotype 2 (rAAV2) using a low multiplicity of infection, dual-baculovirus system was performed via single-cell RNA-sequencing (scRNA-seq). Before infection, the principal source of variability in Sf9 insect cells was associated with the cell cycle. Over the course of infection, an increase in transcriptional heterogeneity was detected, which was linked to the expression of baculovirus genes as well as to differences in rAAV transgenes (rep, cap and gfp) expression. Noteworthy, at 24 h post-infection, only 29.4% of cells enclosed all three necessary rAAV transgenes to produce packed rAAV2 particles, indicating limitations of the dual-baculovirus system. In addition, the trajectory analysis herein performed highlighted that biological processes such as protein folding, metabolic processes, translation, and stress response have been significantly altered upon infection. Overall, this work reports the first application of scRNA-seq to the IC-BEVS and highlights significant variations in individual cells within the population, providing insight into the rational cell and process engineering toward improved rAAV2 production in IC-BEVS.

Transcriptome analysis of Sf9 insect cells during production of recombinant Adeno-associated virus.

The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as an alternative time- and cost-efficient production platform for recombinant Adeno-associated virus (AAV) for gene therapy. However, a better understanding of the underlying biological mechanisms of IC-BEVS is fundamental to further optimize this expression system toward increased product titer and quality. Here, gene expression of Sf9 insect cells producing recombinant AAV through a dual baculovirus expression system, with low multiplicity of infection (MOI), was profiled by RNA-seq. An 8-fold increase in reads mapping to either baculovirus or AAV transgene sequences was observed between 24 and 48 h post-infection (hpi), confirming a take-over of the host cell transcriptome by the baculovirus. A total of 336 and 4784 genes were identified as differentially expressed at 24 hpi (vs non-infected cells) and at 48 hpi (vs. infected cells at 24 hpi), respectively, including dronc, birc5/iap5, and prp1. Functional annotation found biological processes such as cell cycle, cell growth, protein folding, and cellular amino acid metabolic processes enriched along infection. This work uncovers transcriptional changes in Sf9 in response to baculovirus infection, which provide new insights into cell and/or metabolic engineering targets that can be leveraged for rational bioprocess engineering of IC-BEVS for AAV production.

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

PfRipr is a highly conserved asexual-blood stage malaria vaccine candidate against Plasmodium falciparum. PfRipr5, a protein fragment of PfRipr inducing the most potent inhibitory antibodies, is a promising candidate for the development of next-generation malaria vaccines, requiring validation of its potential when formulated with adjuvants already approved for human use. In this study, PfRipr5 antigen was efficiently produced in a tank bioreactor using insect High Five cells and the baculovirus expression vector system; purified PfRipr5 was thermally stable in its monomeric form, had high purity and binding capacity to functional monoclonal anti-PfRipr antibody. The formulation of purified PfRipr5 with Alhydrogel®, GLA-SE or CAF®01 adjuvants accepted for human use showed acceptable compatibility. Rabbits immunized with these formulations induced comparable levels of anti-PfRipr5 antibodies, and significantly higher than the control group immunized with PfRipr5 alone. To investigate the efficacy of the antibodies, we used an in vitro parasite growth inhibition assay (GIA). The highest average GIA activity amongst all groups was attained with antibodies induced by immunization with PfRipr5 formulated with CAF®01. Overall, this study validates the potential of adjuvanted PfRipr5 as an asexual blood-stage malaria vaccine candidate, with PfRipr5/CAF®01 being a promising formulation for subsequent pre-clinical and clinical development.

AAV process intensification by perfusion bioreaction and integrated clarification.

Adeno-associated viruses (AAVs) demand for clinical trials and approved therapeutic applications is increasing due to this vector's overall success and potential. The high doses associated with administration strategies challenges bioprocess engineers to develop more efficient technologies and innovative strategies capable of increasing volumetric productivity. In this study, alternating tangential flow (ATF) and Tangential Flow Depth filtration (TFDF) techniques were compared as to their potential for 1) implementing a high-cell-density perfusion process to produce AAV8 using mammalian HEK293 cells and transient transfection, and 2) integrating AAV harvest and clarification units into a single step. On the first topic, the results obtained demonstrate that AAV expression improves with a medium exchange strategy. This was evidenced firstly in the small-scale perfusion-mocking study and later verified in the 2 L bioreactor operated in perfusion mode. Fine-tuning the shear rate in ATF and TFDF proved instrumental in maintaining high cell viabilities and, most importantly, enhancing AAV-specific titers (7.6 × 104 VG/cell), i.e., up to 4-fold compared to non-optimized perfusion cultures and 2-fold compared with batch operation mode. Regarding the second objective, TFDF enabled the highest recovery yields during perfusion-based continuous harvest of extracellular virus and lysate clarification. This study demonstrates that ATF and TFDF techniques have the potential to support the production and continuous harvest of AAV, and enable an integrated clarification procedure, contributing to the simplification of operations and improving manufacturing efficiency.

Gene Expression Analysis of Adapted Insect Cells during Influenza VLP Production Using RNA-Sequencing.

Adaptive laboratory evolution has been used to improve production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs) in insect cells. However, little is known about the underlying biological mechanisms promoting higher HA-VLP expression in such adapted cell lines. In this article, we present a study of gene expression patterns associated with high-producer insect High Five cells adapted to neutral pH, in comparison to non-adapted cells, during expression of influenza HA-VLPs. RNA-seq shows a decrease in the amount of reads mapping to host cell genomes along infection, and an increase in those mapping to baculovirus and transgenes. A total of 1742 host cell genes were found differentially expressed between adapted and non-adapted cells throughout infection, 474 of those being either up- or down-regulated at both time points evaluated (12 and 24 h post-infection). Interestingly, while host cell genes were found up- and down-regulated in an approximately 1:1 ratio, all differentially expressed baculovirus genes were found to be down-regulated in infected adapted cells. Pathway analysis of differentially expressed genes revealed enrichment of ribosome biosynthesis and carbohydrate, amino acid, and lipid metabolism. In addition, oxidative phosphorylation and protein folding, sorting and degradation pathways were also found to be overrepresented. These findings contribute to our knowledge of biological mechanisms of insect cells during baculovirus-mediated transient expression and will assist the identification of potential engineering targets to increase recombinant protein production in the future.

Efficacies of insecticide dog collars against visceral leishmaniasis in low and high-income areas and the effects for non-collared neighbor dogs.

Previous studies demonstrated that insecticide collars are highly effective in reducing canine visceral leishmaniasis (CVL); however, it is unclear if the efficacy differs by socioeconomic conditions across diverse communities. This study aimed fourfold: (i) to evaluate the protection of 4% impregnated deltamethrin collared (DMC) dogs in different areas of an endemic city for visceral leishmaniasis (VL); (ii) to analyze socioeconomic variables with the seroconversion rates; (iii) to analyze the indirect effect of DMC on untreated dogs in areas of intervention; and, (iv) to evaluate the potential transmission to other dogs in the same household when one positive dog is present. The study employed the municipality of Bauru, São Paulo, Brazil, as the area of interest and used Geographic Information System tools to fit binary logistic regression models.  Dogs were divided into three cohort studies: intervention with DMC (I), indirect effect of DMC (IE), and control (C). Pre-intervention, lower mean income was associated with higher rates of CVL and a 142% increase in the odds of transmission (OR = 1.42, p-value = 0.001, CI 1.14,1.77). Post-intervention, lower-income areas depicted greater efficacy (76%) than higher-income areas (45%). The overall efficacy of DMC in preventing CVL was 63%; however, seroconversion rates were higher for IE (6.02%) than C (3.78%), revealing the failure of the indirect protection of DMC to manage the spread of the disease among the general non-wearing DMC canine population living in the same area. The protected dogs may repel the vectors, and non-protected dogs attract them, creating a higher transmission rate for non-protected dogs. Greater seroconversion was observed for living with an infected dog (10.20% in IE and 8.75% in C) than for the indirect effect of DMC, demonstrating the social burden of CVL. Overall, uncollared dogs have three times higher odds of being infected with CVL than DMC dogs (p < 0.005), and uncollared dogs living with (an) infected dog(s) in the same household can reach 3.5 times higher odds than those living with negative ones (p < 0.005). The results may assist in enhancing public policies and minimizing inequality in low and middle-income countries that suffer from neglected diseases such as VL.

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

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.

Intensifying Continuous Production of Gag-HA VLPs at High Cell Density Using Stable Insect Cells Adapted to Low Culture Temperature.

Protein production processes based on stable insect cell lines require intensification to be competitive with the insect cell-baculovirus expression vector system (IC-BEVS). High cell density (HCD) cultures operate continuously, capable of maintaining specific production rates for extended periods of time which may lead to significant improvements in production yields. However, setting up such processes is challenging (e.g., selection of cell retention device and optimization of dilution rate), often demanding the manipulation of large volumes of culture medium with associated high cost. In this study, we developed a process for continuous production of Gag virus-like particles (VLP) pseudotyped with a model membrane protein (influenza hemagglutinin, HA) at HCD using stable insect cells adapted to low culture temperature. The impact of the cell retention device (ATF vs. TFF) and cell-specific perfusion rate (CSPR) on cell growth and protein expression kinetics was evaluated. Continuous production of Gag-HA VLPs was possible using both retention devices and CSPR of 0.04 nL/cell.d; TFF induces higher cell lysis when compared to ATF at later stages of the process (kD = 0.009 vs. 0.005 h-1, for TFF and ATF, respectively). Reducing CSPR to 0.01-0.02 nL/cell.d using ATF had a negligible impact on specific production rates (rHA = 72-68 titer/109 cell.h and rp24 = 12-11 pg/106 cell.h in all CSPR) and on particle morphology (round-shaped structures displaying HA spikes on their surface) and size distribution profile (peaks at approximately 100 nm). Notably, at these CSPRs, the amount of p24 or HA formed per volume of culture medium consumed per unit of process time increases by up to 3-fold when compared to batch and perfusion operation modes. Overall, this work demonstrates the potential of manipulating CSPRs to intensify the continuous production of Gag-HA VLPs at HCD using stable insect cells to make them an attractive alternative platform to IC-BEVS.

Scalable Process for High-Yield Production of PfCyRPA Using Insect Cells for Inclusion in a Malaria Virosome-Based Vaccine Candidate.

Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield PfCyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest PfCyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, PfCyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti-PfCyRPA antibodies that impeded the multiplication of P. falciparum in vitro. This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant PfCyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels.

Insect Cells for High-Yield Production of SARS-CoV-2 Spike Protein: Building a Virosome-Based COVID-19 Vaccine Candidate.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) homotrimeric spike (S) protein is responsible for mediating host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thus being a key viral antigen to target in a coronavirus disease 19 (COVID-19) vaccine. Despite the availability of COVID-19 vaccines, low vaccine coverage as well as unvaccinated and immune compromised subjects are contributing to the emergence of SARS-CoV-2 variants of concern. Therefore, continued development of novel and/or updated vaccines is essential for protecting against such new variants. In this study, we developed a scalable bioprocess using the insect cells-baculovirus expression vector system (IC-BEVS) to produce high-quality S protein, stabilized in its pre-fusion conformation, for inclusion in a virosome-based COVID-19 vaccine candidate. By exploring different bioprocess engineering strategies (i.e., signal peptides, baculovirus transfer vectors, cell lines, infection strategies and formulation buffers), we were able to obtain ~4 mg/L of purified S protein, which, to the best of our knowledge, is the highest value achieved to date using insect cells. In addition, the insect cell-derived S protein exhibited glycan processing similar to mammalian cells and mid-term stability upon storage (up to 90 days at -80 and 4 °C or after 5 freeze-thaw cycles). Noteworthy, antigenicity of S protein, either as single antigen or displayed on the surface of virosomes, was confirmed by ELISA, with binding of ACE2 receptor, pan-SARS antibody CR3022 and neutralizing antibodies to the various epitope clusters on the S protein. Binding capacity was also maintained on virosomes-S stored at 4 °C for 1 month. This work demonstrates the potential of using IC-BEVS to produce the highly glycosylated and complex S protein, without compromising its integrity and antigenicity, to be included in a virosome-based COVID-19 vaccine candidate.

Bioanalytics for Influenza Virus-Like Particle Characterization and Process Monitoring.

Virus-like particles (VLPs) are excellent platforms for the development of influenza vaccine candidates. Nonetheless, their characterization is challenging due to VLPs' unique biophysical and biochemical properties. To cope with such complexity, multiple analytical techniques have been developed to date (e.g., single-particle analysis, thermal stability, or quantification assays), most of which are rarely used or have been successfully demonstrated for being applicable for virus particle characterization. In this study, several biophysical and biochemical methods have been evaluated for thorough characterization of monovalent and pentavalent influenza VLPs from diverse groups (A and B) and subtypes (H1 and H3) produced in insect cells using the baculovirus expression vector system (IC-BEVS). Particle size distribution and purity profiles were monitored during the purification process using two complementary technologies - nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). VLP surface charge at the selected process pH was also assessed by this last technique. The morphology of the VLP (size, shape, and presence of hemagglutinin spikes) was evaluated using transmission electron microscopy. Circular dichroism was used to assess VLPs' thermal stability. Total protein, DNA, and baculovirus content were also assessed. All VLPs analyzed exhibited similar size ranges (90-115 nm for NTA and 129-141 nm for TRPS), surface charges (average of -20.4 mV), and morphology (pleomorphic particles resembling influenza virus) exhibiting the presence of HA molecules (spikes) uniformly displayed on M1 protein scaffold. Our data shows that HA titers and purification efficiency in terms of impurity removal and thermal stability were observed to be particle dependent. This study shows robustness and generic applicability of the tools and methods evaluated, independent of VLP valency and group/subtype. Thus, they are most valuable to assist process development and enhance product characterization.

Functionalizing Ferritin Nanoparticles for Vaccine Development.

In the last decade, the interest in ferritin-based vaccines has been increasing due to their safety and immunogenicity. Candidates against a wide range of pathogens are now on Phase I clinical trials namely for influenza, Epstein-Barr, and SARS-CoV-2 viruses. Manufacturing challenges related to particle heterogeneity, improper folding of fused antigens, and antigen interference with intersubunit interactions still need to be overcome. In addition, protocols need to be standardized so that the production bioprocess becomes reproducible, allowing ferritin-based therapeutics to become readily available. In this review, the building blocks that enable the formulation of ferritin-based vaccines at an experimental stage, including design, production, and purification are presented. Novel bioengineering strategies of functionalizing ferritin nanoparticles based on modular assembly, allowing the challenges associated with genetic fusion to be circumvented, are discussed. Distinct up/down-stream approaches to produce ferritin-based vaccines and their impact on production yield and vaccine efficacy are compared. Finally, ferritin nanoparticles currently used in vaccine development and clinical trials are summarized.

Insect High Five™ cell line development using site-specific flipase recombination technology.

Insect Trichoplusia ni High Five™ (Hi5) cells have been widely explored for production of heterologous proteins, traditionally mostly using the lytic baculovirus expression vector system (BEVS), and more recently using virus-free transient gene expression systems. Stable expression in such host cells would circumvent the drawbacks associated with both systems when it comes to scale-up and implementation of more efficient high-cell density process modes for the manufacturing of biologics. In this study, we combined Flipase (Flp) recombinase-mediated cassette exchange (RMCE) with fluorescence-activated cell sorting (FACS) for generating a stable master clonal Hi5 cell line with the flexibility to express single or multiple proteins of interest from a tagged genomic locus. The 3-step protocol herein implemented consisted of (i) introducing the RMCE docking cassette into the cell genome by random integration followed by selection in Hygromycin B and FACS (Hi5-tagging population), (ii) eliminating cells tagged in loci with low recombination efficiency by transfecting the tagging population with an eGFP-containing target cassette followed by selection in G418 and FACS (Hi5-RMCE population), and (iii) isolation of pure eGFP-expressing cells by FACS and expansion to suspension cultures (Hi5-RMCE master clone). Exchangeability of the locus in the master clone was demonstrated in small-scale suspension cultures by replacing the target cassette by one containing a single protein (i.e., iCherry, as an intracellular protein model) or two proteins (i.e., influenza HA and M1 for virus-like particles production, as an extracellular protein model). Overall, the stable insect Hi5 cell platform herein assembled has the potential to assist and accelerate biologics development.