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.

Efficient adeno-associated virus serotype 5 capture with affinity functionalized nanofiber adsorbents.

Adeno-associated viruses (AAVs) are one of the most promising tools for gene therapy applications. These vectors are purified using affinity and ion exchange chromatography, typically using packed beds of resin adsorbents. This leads to diffusion and pressure drop limitations that affect process productivity. Due to their high surface area and porosity, electrospun nanofiber adsorbents offer mass transfer and flow rate advantages over conventional chromatographic media. The present work investigated the use of affinity cellulose-based nanofiber adsorbents for adeno-associated virus serotype 5 (AAV5) capture, evaluating dynamic binding capacity, pressure drop, and AAV5 recovery at residence times (RT) less than 5 s. The dynamic binding capacity was found to be residence time-dependent, but nevertheless higher than 1.0 × 1014 TP mL-1 (RT = 1.6 s), with a pressure drop variation of 0.14 MPa obtained after loading more than 2,000 column volumes of clarified AAV5 feedstock. The single affinity chromatography purification step using these new affinity adsorbents resulted in 80% virus recovery, with the removal of impurities comparable to that of bead-based affinity adsorbents. The high binding capacity, virus recovery and reduced pressure drop observed at residence times in the sub-minute range can potentially eliminate the need for prior concentration steps, thereby reducing the overall number of unit operations, process time and costs.

Potential business model for a European vaccine R&D infrastructure and its estimated socio-economic impact.

Background: Research infrastructures are facilities or resources that have proven fundamental for supporting scientific research and innovation. However, they are also known to be very expensive in their establishment, operation and maintenance. As by far the biggest share of these costs is always borne by public funders, there is a strong interest and indeed a necessity to develop alternative business models for such infrastructures that allow them to function in a more sustainable manner that is less dependent on public financing. Methods: In this article, we describe a feasibility study we have undertaken to develop a potentially sustainable business model for a vaccine research and development (R&D) infrastructure. The model we have developed integrates two different types of business models that would provide the infrastructure with two different types of revenue streams which would facilitate its establishment and would be a measure of risk reduction. For the business model we are proposing, we have undertaken an ex ante impact assessment that estimates the expected impact for a vaccine R&D infrastructure based on the proposed models along three different dimensions: health, society and economy. Results: Our impact assessment demonstrates that such a vaccine R&D infrastructure could achieve a very significant socio-economic impact, and so its establishment is therefore considered worthwhile pursuing. Conclusions: The business model we have developed, the impact assessment and the overall process we have followed might also be of interest to other research infrastructure initiatives in the biomedical field.

Development of versatile affinity-based system for one step purification process: Case of Group A Streptococcus vaccine.

Affinity capture is one of the most attractive strategies for simplifying downstream processing. Although it is a key mainstream approach for antibody purification, the same is not true for other biologics such as vaccines, mainly due to the lack of suitable affinity material. In this study, a novel custom affinity system is introduced permitting widespread adoption of affinity capture for the purification of biologics beyond antibodies. This is illustrated here by the development of a one-step purification process of a mutant form of streptolysin O (SLO), a vaccine candidate against Streptococcus pyogenes infection. The system consists of the association of custom ligands based on the Nanofitin protein scaffold, with Eshmuno® industry-grade chromatography medium. The Nanofitins were selected for their specificity to the target product. The newly developed affinity medium was used at different column sizes to monitor scalability from process development (1 ml) and robustness verification (5 ml) to pilot (133 ml) and technical (469 ml) runs. The single-step affinity purification consistently delivered high purity product (above > 90%) and improved performances compared with the current three-step process: reduced process time and footprint (3 to 1 step) and increased product yields (0.31 g vs. 0.04 g of SLO per kg of harvest broth). The custom affinity system herein described can potentially be applied to any biologic for which a specific Nanofitin is identified, thus establishing a platform with a strong impact on the manufacturing of vaccines and other biological targets.

Continuous Affinity Purification of Adeno-Associated Virus Using Periodic Counter-Current Chromatography.

Replacing batch unit operations of biopharmaceuticals by continuous manufacturing is a maturing concept, with periodic counter-current chromatography (PCC) favoured to replace batch chromatography. Continuous affinity capture of adeno-associated virus (AAV) using PCC has the potential to cope with the high doses required for AAV therapies thanks to its inherent high throughput. The implementation of continuous AAV affinity capture using a four-column PCC process is described herein. First, elution buffer screening was used to optimize virus recovery. Second, breakthrough curves were generated and described using a mechanistic model, which was later used to characterize the loading zone of the PCC. The experimental runs achieved a stable cyclic steady state yielding virus recoveries in line with the optimized batch process (>82%), with almost a three-fold improvement in productivity. The PCC affinity capture process developed here can bolster further improvements to process economics and manufacturing footprint, thereby contributing to the integrated continuous manufacturing concept.

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.

Improved storage of influenza HA-VLPs using a trehalose-glycerol natural deep eutectic solvent system.

Vaccines are typically stored under refrigerated conditions (2-8 °C) to limit potency and efficacy loss. Maintaining the cold chain is costly and prone to vaccine wastage due to unexpected changes in the storage conditions. The development of formulations conferring enhanced stability can extend vaccine shelf-life and facilitate storage under non-refrigerated conditions, thus simplifying the distribution process and reducing vaccine wastage. In this study, the suitability of a natural deep eutectic system (NADES) consisting of trehalose and glycerol (TGly) for storage of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs) was successfully demonstrated. TGly was efficient in maintaining the activity and physical integrity of HA-VLPs for up to 4 h at 50 °C (accelerated stability study), with half-life values ≈ 20-34 h for the best TGly formulations. Importantly, improved storage is achieved at increasingly higher TGly concentrations, hence confirming the importance of TGly content in its protective capacity. In addition, HA-VLPs were stable in TGly for over one month at room temperature (short-term stability study), with no impact on HA titer or particle size distribution. This work highlights the potential of NADES to improve stability of VLP-based vaccines, showing promising protective capacity under non-refrigerated conditions and short-term thermal stress, and thus having a notable impact on vaccine's cold chain.

Oncolytic virus purification with periodic counter-current chromatography.

Virus-based biologicals are one of the most promising biopharmaceuticals of the 21st century medicine and play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. Oncolytic virus manufacturing scale can range from 5 L in research and development up to 50 L for clinical studies and reach hundreds of liters for commercial scale. The inherent productivity and high integration potential of periodic counter-current chromatography (PCC) offer a transversal solution to decrease equipment footprint and the reduction of several non-value-added unit operations. We report on the design of an efficient PCC process applied to the intermediate purification of oncolytic adenovirus. The developed ion-exchange chromatographic purification method was carried out using a four-column setup for three different scenarios: (i) variation in the feedstock, (ii) potential use of a post-load washing step to improve virus recovery, and (iii) stability during extended operation. Obtained virus recoveries (57%-86%) and impurity reductions (>80% DNA, and >70% total protein) match or overcome batch purification. Regarding process stability and automation, our results show that not only the dynamic control strategy used is able to suppress perturbations in the sample inlet but also allows for unattended operation in the case of ion exchange capture.

Integrating high cell density cultures with adapted laboratory evolution for improved Gag-HA virus-like particles production in stable insect cell lines.

Stable insect cell lines are emerging as an alternative to the insect cell-baculovirus expression vector system (IC-BEVS) for protein expression, benefiting from being a virus-free, nonlytic system. Still, the titers achieved are considerably lower. In this study, stable insect (Sf-9 and High Five) cells producing Gag virus-like particles (VLPs) were first adapted to grow under hypothermic culture conditions (22°C instead of standard 27°C), and then pseudotyped with a model membrane protein (influenza hemagglutinin [HA]) for expression of Gag-HA VLPs. Adaptation to lower temperature led to an increase in protein titers of up to 12-fold for p24 (as proxy for Gag-VLP) and sixfold for HA, with adapted Sf-9 cells outperforming High Five cells. Resulting Gag-HA VLPs producer Sf-9 cells were cultured to high cell densities, that is, 100 × 106 cell/ml, using perfusion (ATF® 2) in 1 L stirred-tank bioreactors. Specific p24 and HA production rates were similar to those of batch culture, enabling to increase volumetric titers by 7-8-fold without compromising the assembly of Gag-HA VLPs. Importantly, the antigen (HA) quantity in VLPs generated using stable adapted cells in perfusion was ≈5-fold higher than that from IC-BEVS, with the added benefit of being a baculovirus-free system. This study demonstrates the potential of combining stable expression in insect cells adapted to hypothermic culture conditions with perfusion for improving Gag-HA VLPs production.

Advances in Lentivirus Purification.

Lentiviral vectors (LVs) have been increasingly used as a tool for gene and cell therapies since they can stably integrate the genome in dividing and nondividing cells. LV production and purification processes have evolved substantially over the last decades. However, the increasing demands for higher quantities with more restrictive purity requirements are stimulating the development of novel materials and strategies to supply the market with LV in a cost-effective manner. A detailed review of each downstream process unit operation is performed, limitations, strengths, and potential outcomes being covered. Currently, the majority of large-scale LV manufacturing processes are still based on adherent cell culture, although it is known that the industry is migrating fast to suspension cultures. Regarding the purification strategy, it consists of batch chromatography and membrane technology. Nevertheless, new solutions are being created to improve the current production schemes and expand its clinical use.

Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution.

The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2-0.3), and cells were maintained at each pH value for 2-3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells.

Unveiling dynamic metabolic signatures in human induced pluripotent and neural stem cells.

Metabolism plays an essential role in cell fate decisions. However, the methods used for metabolic characterization and for finding potential metabolic regulators are still based on characterizing cellular metabolic steady-state which is dependent on the extracellular environment. In this work, we hypothesized that the response dynamics of intracellular metabolic pools to extracellular stimuli is controlled in a cell type-specific manner. We applied principles of process dynamics and control to human induced pluripotent stem cells (hiPSC) and human neural stem cells (hNSC) subjected to a sudden extracellular glutamine step. The fold-changes of steady-states and the transient profiles of metabolic pools revealed that dynamic responses were reproducible and cell type-specific. Importantly, many amino acids had conserved dynamics and readjusted their steady state concentration in response to the increased glutamine influx. Overall, we propose a novel methodology for systematic metabolic characterization and identification of potential metabolic regulators.

Establishing Suspension Cell Cultures for Improved Manufacturing of Oncolytic Adenovirus.

Recent clinical trials have shown the potential of oncolytic adenoviruses as a cancer immunotherapy. A successful transition of oncolytic adenovirus to clinical applications requires efficient and good manufacturing practice compatible production and purification bioprocesses. Suspension cultures are preferable for virus production as they can reduce process costs and increase product quality and consistency. This work describes the adaptation of the A549 cell line to suspension culture in serum-reduced medium validated by oncolytic adenovirus production in stirred tank bioreactor. Cell concentrations up to 3 × 106 cells mL-1 are obtained during the production process. At harvest 1.4 × 1010 infectious particles mL-1 and 6.9 ± 1.1 × 1010 viral genome mL-1 are obtained corresponding to a viral genome: infectious particles ratio of 5.2 (± 1.9): 1 confirming the virus quality. Overall, the suspension characteristics of these A549 cells support an easily scalable, less time-consuming, and more cost-effective process for expanded success in the use of oncolytic viruses for cancer therapy.

Adaptive laboratory evolution of stable insect cell lines for improved HIV-Gag VLPs production.

Adaptive laboratory evolution (ALE) has been extensively used to modulate the phenotype of industrial model organisms (e.g. Escherichia. coli and Saccharomyces cerevisae) towards a specific trait. Nevertheless, its application to animal cells, and in particular to insect cell lines, has been very limited. In this study, we describe employing an ALE method to improve the production of HIV-Gag virus-like particles (VLPs) in stable Sf-9 and High Five cell lines. Serial batch transfer was used for evolution experiments. During the ALE process, cells were cultured under controlled hypothermic conditions (22 °C instead of standard 27 °C) for a prolonged period of time (over 3 months), which allowed the selection of a population of cells with improved phenotype. Adapted cells expressed up to 26-fold (Sf-9 cells) and 10-fold (High Five cells) more Gag-VLPs than non-adapted cells cultured at standard conditions. The production of HIV Gag-VLPs in adapted, stable insect Sf-9 cell lines was successfully demonstrated at bioreactor scale. The Gag-VLPs produced at 22 °C and 27 °C were comparable, both in size and morphology, thus confirming the null impact of adaptation process and hypothermic culture conditions on VLP's quality. This work demonstrates the suitability of ALE as a powerful method for improving yields in stable insect cell lines producing VLPs.

Continuous Chromatography Purification of Virus-Based Biopharmaceuticals: A Shortcut Design Method.

Novel biopharmaceutical products, such as vaccines and viral vectors, play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. However, several challenges are posed when manufacturing these products. The diversity of cell lines and the different physical and chemical properties of these biologicals require the use of different production and processing technologies. Alternative purification strategies that can improve the purification yield, such as continuous chromatography, are regarded nowadays as enabling technologies to overcome some of the bottlenecks in biomanufacturing. This chapter offers a shortcut approach to implement a semi-continuous chromatography purification of hepatitis C virus-like particles produced in insect cells with recombinant baculovirus. Although the purification is based on ion exchange chromatography, the present methodology can be extended to other types of chromatography.

Current challenges in biotherapeutic particles manufacturing.

Introduction: The development of novel complex biotherapeutics led to new challenges in biopharmaceutical industry. The potential of these particles has been demonstrated by the approval of several products, in the different fields of gene therapy, oncolytic therapy, and tumor vaccines. However, their manufacturing still presents challenges related to the high dosages and purity required.Areas covered: The main challenges that biopharmaceutical industry faces today and the most recent developments in the manufacturing of different biotherapeutic particles are reported here. Several unit operations and downstream trains to purify virus, virus-like particles and extracellular vesicles are described. Innovations on the different purification steps are also highlighted with an eye on the implementation of continuous and integrated processes.Expert opinion: Manufacturing platforms that consist of a low number of unit operations, with higher-yielding processes and reduced costs will be highly appreciated by the industry. The pipeline of complex therapeutic particles is expanding and there is a clear need for advanced tools and manufacturing capacity. The use of single-use technologies, as well as continuous integrated operations, are gaining ground in the biopharmaceutical industry and should be supported by more accurate and faster analytical methods.

Improved GaLV-TR Glycoproteins to Pseudotype Lentiviral Vectors: Impact of Viral Protease Activity in the Production of LV Pseudotypes.

Lentiviral vectors (LVs) are excellent tools for gene transfer into mammalian cells. It is noteworthy that the first gene therapy treatment using LVs was approved for commercialization in 2017. The G glycoprotein from rhabdovirus vesicular stomatitis virus (VSV-G) is the glycoprotein most used to pseudotype LVs, due to its high efficiency in transducing several cell types and its resistance to viral vector purification and storage conditions. However, VSV-G expression induces cytotoxicity, which limits LV production to short periods. As alternative to VSV-G, γ-retrovirus glycoproteins (4070A derived, GaLV derived, and RD114 derived) have been used to pseudotype both γ-retroviral vectors (RVs) and LVs. These glycoproteins do not induce cytotoxicity, allowing the development of stable LV producer cells. Additionally, these LV pseudotypes present higher transduction efficiencies of hematopoietic stem cells when compared to VSV-G. Here, new 4070A-, RD114-TR-, and GaLV-TR-derived glycoproteins were developed with the aim of improving its cytoplasmic tail R-peptide cleavage and thus increase LV infectious titers. The new glycoproteins were tested in transient LV production using the wild-type or the less active T26S HIV-1 protease. The GaLV-TR-derived glycoproteins were able to overcome titer differences observed between LV production using wild-type and T26S protease. Additionally, these glycoproteins were even able to increase LV titers, evidencing its potential as an alternative glycoprotein to pseudotype LVs.

Flexible 3D Cell-Based Platforms for the Discovery and Profiling of Novel Drugs Targeting Plasmodium Hepatic Infection.

The restricted pipeline of drugs targeting the liver stage of Plasmodium infection reflects the scarcity of cell models that mimic the human hepatic phenotype and drug metabolism, as well as Plasmodium hepatic infection. Using stirred-tank culture systems, spheroids of human hepatic cell lines were generated, sustaining a stable hepatic phenotype over 4 weeks of culture. Spheroids were employed in the establishment of 3D Plasmodium berghei infection platforms that relied on static or dynamic culture conditions. P. berghei invasion and development were recapitulated in the hepatic spheroids, yielding blood-infective merozoites. The translational potential of the 3D platforms was demonstrated by comparing the in vitro minimum inhibitory concentration of M5717, a compound under clinical development, with in vivo plasma concentrations that clear liver stage P. berghei in mice. Our results show that the 3D platforms are flexible and scalable and can predict the efficacy of antiplasmodial therapies, constituting a powerful tool for integration in drug discovery programs.

Enabling PAT in insect cell bioprocesses: In situ monitoring of recombinant adeno-associated virus production by fluorescence spectroscopy.

The process analytical technology (PAT) initiative shifted the bioprocess development mindset towards real-time monitoring and control tools to measure relevant process variables online, and acting accordingly when undesirable deviations occur. Online monitoring is especially important in lytic production systems in which released proteases and changes in cell physiology are likely to affect product quality attributes, as is the case of the insect cell-baculovirus expression vector system (IC-BEVS), a well-established system for production of viral vectors and vaccines. Here, we applied fluorescence spectroscopy as a real-time monitoring tool for recombinant adeno-associated virus (rAAV) production in the IC-BEVS. Fluorescence spectroscopy is simple, yet sensitive and informative. To overcome the strong fluorescence background of the culture medium and improve predictive ability, we combined artificial neural network models with a genetic algorithm-based approach to optimize spectra preprocessing. We obtained predictive models for rAAV titer, cell viability and cell concentration with normalized root mean squared errors of 7%, 4%, and 7%, respectively, for leave-one-batch-out cross-validation. Our approach shows fluorescence spectroscopy allows real-time determination of the best time of harvest to maintain rAAV infectivity, an important quality attribute, and detection of deviations from the golden batch profile. This methodology can be applied to other biopharmaceuticals produced in the IC-BEVS, supporting the use of fluorescence spectroscopy as a versatile PAT tool.

Membrane-Based Approach for the Downstream Processing of Influenza Virus-Like Particles.

Currently, marketed influenza vaccines are only efficient against homologous viruses, thus requiring a seasonal update based on circulating subtypes. This constant reformulation adds several challenges to manufacturing, particularly in purification due to the variation of the physicochemical properties of the vaccine product. A universal platform approach capable of handling such variation is therefore of utmost importance. In this work, a filtration-based approach is explored to purify influenza virus-like particles. Switching from adsorptive separation to size-based purification allows overcoming the differences in retention observed for different influenza strains. The proposed process employs a cascade of ultrafiltration and diafiltration steps, followed by a sterile filtration step. Different process parameters are assessed in terms of product recovery and impurities' removal. Membrane chemistry, pore size, operation modes, critical flux, transmembrane pressure, and permeate control strategies are evaluated. After membrane selection and parameter optimization, concentration factors and diafiltration volumes are also defined. By optimizing the filtration mode of operation, it is possible to achieve product recoveries of approximately 80%. Overall, the process time is decreased by 30%, its scalability is improved, and the costs are reduced due to the removal of chromatography and associated buffer consumptions, cleaning, and its validation steps.