VLP Production Research Articles

Inline Raman spectroscopy as process analytical technology for SARS-CoV-2 VLP production.

The present work focused on inline Raman spectroscopy monitoring of SARS-CoV-2 VLP production using two culture media by fitting chemometric models for biochemical parameters (viable cell density, cell viability, glucose, lactate, glutamine, glutamate, ammonium, and viral titer). For that purpose, linear, partial least square (PLS), and nonlinear approaches, artificial neural network (ANN), were used as correlation techniques to build the models for each variable. ANN approach resulted in better fitting for most parameters, except for viable cell density and glucose, whose PLS presented more suitable models. Both were statistically similar for ammonium. The mean absolute error of the best models, within the quantified value range for viable cell density (375,000-1,287,500 cell/mL), cell viability (29.76-100.00%), glucose (8.700-10.500g/), lactate (0.019-0.400g/L), glutamine (0.925-1.520g/L), glutamate (0.552-1.610g/L), viral titer (no virus quantified-7.505 log10 PFU/mL) and ammonium (0.0074-0.0478g/L) were, respectively, 41,533 ± 45,273cell/mL (PLS), 1.63 ± 1.54% (ANN), 0.058 ± 0.065g/L (PLS), 0.007 ± 0.007g/L (ANN), 0.007 ± 0.006g/L (ANN), 0.006 ± 0.006g/L (ANN), 0.211 ± 0.221 log10 PFU/mL (ANN), and 0.0026 ± 0.0026g/L (PLS) or 0.0027 ± 0.0034g/L (ANN). The correlation accuracy, errors, and best models obtained are in accord with studies, both online and offline approaches while using the same insect cell/baculovirus expression system or different cell host. Besides, the biochemical tracking throughout bioreactor runs using the models showed suitable profiles, even using two different culture media.

Laser wavelength and sample conditioning effects on biochemical monitoring of SARS-CoV-2 VLP production upstream stage by Raman spectroscopy

This work assessed the impact of laser wavelength and sample conditioning on offline monitoring (viable cell density, cell viability, virus titer, glucose, lactate, glutamine, glutamate, and ammonium) of SARS-CoV-2 virus-like particles production upstream stage by Raman spectroscopy. The evaluated chemometrics techniques were Partial Least Squares (PLS) and Artificial Neural Networks (ANN), and different spectral filtering approaches were also considered. ANN showed better prediction capacity for most of the parameters, but ammonium and lactate, better predicted by PLS, and glutamine, no difference between modeling techniques was detected. For cell growth parameters and virus titer, samples without cells measured at 785 nm Raman laser wavelength originated better-adjusted models. This laser wavelength was also more appropriate for the remaining monitored experimental parameters except for glucose, in which the best model came from the spectral database acquired at 1064 nm wavelength. Cell remotion significantly increased the accuracy of viable cell density, cell viability, glutamate, and virus titer models. However, glucose, lactate, and ammonium models showed better prediction capacity for samples containing cells. Thus, it was demonstrated that laser wavelength, sample conditioning, spectral preprocessing, and chemometric modeling techniques need to be tailored for each experimental parameter to improve accuracy.

Expression of dengue virus and Zika virus NS2B-NS3pro constructs alter cellular fatty acids, but co-expression with a Zika virus virus-like particle is detrimental to virus-like particle expression

ObjectiveStudies have shown that Flavivirus infection remodels the host cell to favour viral replication. In particular, the host cell lipid profile is altered, and it has been proposed that this process alters membrane fluidity to allow wrapping of the outer structural proteins around the viral nucleocapsid. We investigated whether expression of the Zika virus (ZIKV) and dengue virus (DENV) protease induced alterations in the cellular lipid profile, and subsequently whether co-expression of these proteases with VLP constructs was able to improve VLP yield.ResultsOur results showed that both ZIKV and DENV proteases induced alterations in the lipid profile, but that both active and inactive proteases induced many of the same changes. Neither co-transfection of protease and VLP constructs nor bicistronic vectors allowing expression of both protease and VLP separated by a cell cleavable linker improved VLP yield, and indeed many of the constructs showed significantly reduced VLP production. Further work in developing improved VLP expression platforms is required.

Comparison of chemometric models using Raman spectroscopy for offline biochemical monitoring throughout the VLP-making upstream process

This work aimed to compare the predictive capacity of linear (Partial Least Squares; Principal Component Regression) and non-linear (Artificial Neural Network, ANN; Support Vector Machine, SVM) chemometric models from Raman spectroscopy spectra acquired offline with (unclarified samples) and without cells (clarified samples), and biochemical parameters (Viable cell density, cell viability, glucose, lactate, glutamine, glutamate, ammonium, and potassium) quantified by reference methods in a rabies VLP production bioprocess. The nonlinear models, ANN and SVM were the more suitable models with the lowest absolute errors. No effective differences in accuracy were detected for models from spectral data with and without cells. The mean absolute error of the best models within the assessed parameter ranges for viable cell density (0.01–8.83 × 106 cells/mL), cell viability (1.3–100.0%), glucose (5.22–10.93 g L−1), lactate (18.6–152.7 mg L−1), glutamine (158–1761 mg L−1), glutamate (807.6–2159.7 mg L−1), ammonium (62.8–117.8 mg L−1), and potassium (531.6–685. 3 mg L−1) were 0.17 ± 0.13 × 106 cells/mL, 6.8 ± 7.1%, 0.24 ± 0.14 g L− 1, 3.3 ± 3.5 mg L− 1, 61 ± 52 mg L− 1, 52 ± 51 mg L− 1, 2.1 ± 1.9 mg L− 1 and 8.6 ± 8.6 mg L− 1, respectively. The errors achieved were like those for bioprocesses using a highlighted animal cell host (CHO cell) in similar ranges.

Virus-like particles based on rotavarus A recombinant VP2/VP6 proteins for assessment the antibody immune response by ELISA

Rotavirus infection is one of the main concerns in infectious pathology in humans, mammals and birds. Newborn piglets or rodents are usually being used as a laboratory model for the evaluation of immunogenicity and efficacy for all types of vaccines against rotavirus A (RVA), and the use of ELISA for the detection of virus-specific antibodies of specific isotype is an essential step of this evaluation. Development of indirect solid-phase ELISA with VP2/VP6 rotavirus VLP as an antigen to detect and assess the distribution of RVA-specific IgG, IgM and IgA in the immune response to rotavirus A. VP2/VP6 rotavirus VLP production and purification, electron microscopy, PAGE, immunoblotting, ELISA, virus neutralization assay. The study presents the results of development of a recombinant baculovirus with RVA genes VP2-eGFP/VP6, assessment of its infectious activity and using it for VLP production. The morphology of the VP2/VP6 rotavirus VLPs was assessed, the structural composition was determined, and the high antigenic activity of the VLP was established. VLP-based ELISA assay was developed and here we report results for RVA-specific antibody detection in sera of different animals. The developed ELISA based on VP2/VP6 rotavirus VLP as a universal antigen makes it possible to detect separately IgG, IgM and IgA antibodies to rotavirus A, outlining its scientific and practical importance for the evaluation of immunogenicity and efficacy of traditional vaccines against rotavirus A and those under development.

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.

HBsAg Production in Methanol Controlled <i>P. pastoris</i> GS115 Mut<sup>S</sup> Bioreactor Process

When producing recombinant proteins with Pichia pastoris, cultivation parameters, such as induction temperature, dissolved oxygen level and residual methanol concentration play a crucial role in product biosynthesis and subsequent purification, therefore to maximize protein yields, the optimization of these parameters is imperative. Two different Pichia pastoris cultivation strategies for HBsAg VLP production in a 5 L stirred-tank bioreactor and the influence of different cultivation parameters on product yield were investigated. Residual methanol concentrations were controlled at low (>0.01 g/L), medium (1.5-2.0 g/L) and high (5.0-6.0 g/L) levels using a PI-based feed rate control algorithm based on the online methanol sensor signal. Product was purified using a novel and rapid purification method including steps of ammonium sulfate precipitation, size-exclusion chromatography and hydrophobic interaction chromatography. Employing an in-situ methanol sensor probe, the PI-based methanol feed rate control algorithm provided residual methanol concentration control with an average deviation of ±0.4 g/L from set-point value. Employing a cultivation protocol with an increased methanol concentration controlled at 6.0 g/L and a reduced DO level below 10 %, resulting in a final dry cell biomass concentration of 140 g/L and purified HBsAg VLPs yield of 186 mg/L. Developed purification method proved advantageous to other described methods, as it did not include time consuming extraction and centrifugation steps.

Characterization of alfalfa mosaic virus capsid protein using Cryo-EM

VLPs are virus-like particles that comprise viral capsid proteins that can self-assemble and mimic the shape and size of real viral particles; however, because they do not contain genetic material they cannot infect host cells. VLPs have great potential as safe drug/vehicle candidates; therefore, they are gaining popularity in the field of preventive medicine and therapeutics. Indeed, extensive studies are underway to examine their role as carriers for immunization and as vehicles for delivery of therapeutic agents. Here, we examined the possibility of developing VLP-utilizing technology based on an efficient VLP production process and high-resolution structural analysis. Nicotiana benthamiana was used as an expression platform to produce the coat protein of the alfalfa mosaic virus (AMV-CP). About 250 mg/kg of rAMV-CP was produced from Nicotiana benthamiana leaves. Structural analysis revealed that the oligomeric status of rAMV-CP changed according to the composition and pH of the buffer. Size exclusion chromatography and electron microscopy analysis confirmed the optimal conditions for rAMV-CP VLP formation, and a 2.4 Å resolution structure was confirmed by cryo-EM analysis. Based on the efficient protein production, VLP manufacturing technology, and high-resolution structure presented herein, we suggest that rAMV-CP VLP is a useful platform for development of various new drugs.

Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles.

Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites via GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.

Production of GP64-free virus-like particles from baculovirus-infected insect cells

The retroviral Gag protein is frequently used to generate 'virus-like particles' (VLPs) for a variety of applications. Retroviral Gag proteins self-assemble and bud at the plasma membrane to form enveloped VLPs that resemble natural retrovirus virions, but contain no viral genome. The baculovirus expression vector system has been used to express high levels of the retroviral Gag protein to produce VLPs. However, VLP preparations produced from baculovirus-infected insect cells typically contain relatively large concentrations of baculovirus budded virus (BV) particles, which are similar in size and density to VLPs, and thus may be difficult to separate when purifying VLPs. Additionally, these enveloped VLPs may have substantial quantities of the baculovirus-encoded GP64 envelope protein in the VLP envelope. Since VLPs are frequently produced for vaccine development, the presence of the GP64 envelope protein in VLPs, and the presence of Autographa californica multicapsid nucleopolyhedrovirus BVs in VLP preparations, is undesirable. In the current studies, we developed a strategy for reducing BVs and eliminating GP64 in the production of VLPs, by expressing the human immunodeficiency virus type 1 gag gene in the absence of the baculovirus gp64 gene. Using a GP64null recombinant baculovirus, we demonstrate Gag-mediated VLP production and an absence of GP64 in VLPs, in the context of reduced BV production. Thus, this approach represents a substantially improved method for producing VLPs in insect cells.

Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production.

Large-scale transient transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality attributes to perform accelerated in vitro evaluations and has the potential to support preclinical or even clinical studies. Here we describe the methodology to produce VLPs in a 3L bioreactor, using suspension HEK 293 cells and PEIPro as a transfection reagent. Cells are grown in the bioreactor to 1×106 cells/mL and transfected with a plasmid DNA-PEI complex at a ratio of 1:2. Dissolved oxygen and pH are controlled and are online monitored during the production phase and cell growth and viability can be measured off line taking samples from the bioreactor. If the product is labeled with a fluorescent marker, transfection efficiency can be also assessed using flow cytometry analysis. Typically, the production phase lasts between 48 and 96h until the product is harvested.

Different applications of virus-like particles in biology and medicine: Vaccination and delivery systems.

ABSTRACTVirus‐like particles (VLPs) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. VLPs can elicit efficient protective immunity as direct immunogens compared to soluble antigens co‐administered with adjuvants in several booster injections. Up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and E. coli were used to express recombinant proteins, especially for VLP production. Recent studies are also generating VLPs in plants using different transient expression vectors for edible vaccines. VLPs and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. Herein, we describe VLP production in different systems as well as its applications in biology and medicine. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 113–132, 2016.

Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures.

The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%.

PRODUCTION OF POLYOMAVIRUS-LIKE PARTICLES IN A Klgal80 KNOCKOUT STRAIN OF THE YEAST Kluyveromyces lactis

VP1, the major coat protein of polyomavirus, assembles intracellularly to virus-like particles if expressed in eukaryotes. Here, the nonconventional yeast Kluyveromyces lactis was used for production of virus-like particles of murine polyomavirus. The heterologous gene of VP1 was integrated in the LAC4 locus of the GAL/LAC genes. Consequently the expression of VP1 is regulated by the interplay of the activator KlGal4p and inhibitor KlGal80p. This cloning strategy couples the production of VP1 to that of the enzyme β-galactosidase, allowing a fast alternative for monitoring the course of recombinant protein production by measuring the β-galactosidase activity. A Klgal80 knockout strain was generated for a constitutive expression of VP1 and a continuous VLP production. High-cell-density fermentation showed that (1) Kluyveromyces lactis is generally suitable for VLP production and (2) the Klgal80 knockout strain produces higher amounts of recombinant VP1. Furthermore, VLPs could be purified chromatographically to 87% (w/w) of total protein, and showed a homogeneous species of 45-nm particles and a high resistance against proteolysis compared to conventional in vitro assembled VLPs. This demonstrates the superior stability of virus-like particles produced in yeast. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

A Specific Domain of the Chikungunya Virus E2 Protein Regulates Particle Formation in Human Cells: Implications for Alphavirus Vaccine Design

Virus-like particles (VLPs) can be generated from Chikungunya virus (CHIKV), but different strains yield variable quantities of particles. Here, we define the genetic basis for these differences and show that amino acid 234 in E2 substantially affects VLP production. This site is located within the acid-sensitive region (ASR) known to initiate a major conformational change in E1/E2. Selected other mutations in the ASR, or changes in pH, also increased VLP yield. These results demonstrate that the ASR of E2 plays an important role in regulating particle generation.

Simultaneous expression of recombinant proteins in the insect cell-baculovirus system: Production of virus-like particles

The insect cell-baculovirus system (IC-BEVS) is widely used for the production of recombinant viral proteins for vaccine applications. It is especially suitable for the production of virus-like particles, which often require the simultaneous production of several recombinant proteins. Here, the available tools and process requirements for the simultaneous production of several recombinant proteins using the IC-BEVS are discussed. The production of double-layered rotavirus like particles is used as a specific example for the simultaneous production of two recombinant proteins. Methods to quantify VLP in small samples are described. The multiplicity and time of infection are presented as tools to manipulate protein concentration, and the effect on protein concentration ratios on the assembly efficiency of double-layered rotavirus like particles is discussed. It was found that not only the ratio between the recombinant proteins is determinant of VLP assembly efficiency, but also that assembly efficiency is related to the characteristics of the assembled proteins. This is the first time that kinetics of VLP production are followed during cultures, and that the assembly efficiency is quantitatively determined.

Identification of cellular enhancing and restricting factors of dengue virus egress

Dengue has emerged as the most important life-threatening illness in the world, especially in Asian countries around Hong Kong, where the incidence of dengue hemorrhagic fever (DHF) is much greater than other continents. However, little is known about molecular and cellular processes sustaining egress of dengue virus in the host cell. To better understand the viral and cellular determinants of dengue virus egress, we have established a dengue VLP producing stable cell line (HeLa-prME) and demonstrated that dengue VLP was able to mimic the budding and egress process of dengue viruses so that it constitutes a safe and convenient tool for the study of egress of dengue virus. Under the support of RFCID grant, HeLa-prME cells were used to screen a siRNA library that included 122 cellular membrane trafficking genes. Our screen results revealed that knockdown of ADP-ribosylation factor (ARF) 1 and ARF6 had significant effects on VLP production by HeLa-prME cell. Experiments with other ARF proteins, which were not included in the siRNA library, showed that the ARF4/ARF5 double knockdown could inhibit VLP production but had no effect on secretion of other proteins such as soluble dengue E protein, suggesting the specificity of their involvement in VLP production. Further experiments using real virus showed that the depletion of ARF4/5 by siRNA could significantly reduced the replication of dengue 1 virus, dengue 4 virus and yellow fever virus, confirming the important role of ARF4 and ARF5 for not only dengue viruses but also other flaviviruses. Our study uncovered the importance of class II ARFs in the egress of dengue virus. Results from this project provided information on mechanism of dengue virus assembly and its dependence on cellular machineries.

Efficient particle production by minimal Gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain.

The human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55(gag) by itself is capable of assembling into retrovirus-like particles (VLP). In the present study, we attempted to identify the minimal Gag sequences required for the formation of VLP. Our results show that about 80% of Pr55(gag) can be either deleted or replaced by heterologous sequences without significantly compromising VLP production. The smallest chimeric molecule still able to efficiently form VLP was only about 16 kDa. This minimal Gag construct contained the leucine zipper domain of the yeast transcription factor GCN4 to substitute for the assembly function of nucleocapsid (NC), followed by a P-P-P-P-Y motif to provide late budding (L) domain function, and retained only the myristylation signal and the C-terminal capsid-p2 domain of Pr55(gag). We also show that the L domain function of HIV-1 p6(gag) is not dependent on the presence of an active viral protease and that the NC domain of Pr55(gag) is dispensable for the incorporation of Vpr into VLP.