Characterization of residual microRNAs in AAV vector batches produced in HEK293 mammalian cells and Sf9 insect cells.

With more than 130 clinical trials and 8 approved gene therapy products, adeno-associated virus (AAV) stands as one of the most popular vehicles to deliver therapeutic DNA in vivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as microRNAs (miRNAs), has not been investigated previously. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the recombinant AAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04-0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72-144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.

Membrane Protein Production in Insect Cells.

Membrane proteins are an essential part of the machinery of life. They connect the interior and exterior of cells, play an important role in cell signaling and are responsible for the influx and efflux of nutrients and metabolites. For their structural and functional analysis high yields of correctly folded and modified protein are needed. Insect cells, such as Sf9 cells, have been one of the major expression hosts for eukaryotic membrane proteins in structural investigations during the last decade, as they are easier to handle than mammalian cells and provide more natural posttranslational modifications than microbial systems. Here we describe general techniques for establishing and maintaining insect cell cultures, the generation and amplification of recombinant baculovirus stocks using the flashBAC™ or Bac-to-Bac™ systems, membrane protein production, as well as the production of membrane preparations for extraction and purification experiments.

Baculovirus-derived influenza virus-like particle confers complete protection against lethal H7N9 avian influenza virus challenge in chickens and mice.

Currently, highly pathogenic avian influenza (HPAI) H7N9 viruses still pose a potential pandemic threat. Influenza virus-like particle (VLP) is one of the most promising vaccine strategies to complement traditional egg-dependent vaccines. Here, we generated a H7N9 VLP vaccine candidate by baculovirus expression system and evaluated its efficacy in chickens and mice. The H7N9 VLP was produced through co-infection of Sf9 insect cells with three recombinant baculoviruses expressing individual HA, NA and M1 gene of the HPAI H7N9 virus A/chicken/Guangdong/GD15/2016. Intramuscular immunization of the H7N9 VLP elicited robust antibody immune responses and conferred complete clinical protection against lethal H7N9 virus challenge both in chickens and mice. Meanwhile, H7N9 VLP significantly restrained virus shedding and dramatically alleviated pulmonary lesions caused by H7N9 virus infection in birds and mice. Interestingly, chicken antibodies induced by the H7N9 VLP also had a good cross-reactivity with H7N9 field strains isolated in different years. In addition, vaccination with the H7N9 VLP elicited high T cell immunity in mouse lung, evidenced by significantly upregulated expression of IL-2, IL-4 and IFN-γ. Furthermore, the H7N9 VLP significantly decreased the expression of some key inflammatory cytokines, such as IL6, RANTES and TNF-α in mouse lung, which may partially account for its contribution to alleviate lung pathology. Therefore, our study describes the good efficacy of the HA + NA + M1-containing H7N9 VLP both in chicken and mice models, highlighting the potential of VLP-based vaccine as a critical alternative of traditional egg-based vaccine for control of H7N9 influenza virus in both humans and poultry.

Enhancing the Yield and Quality of Influenza Virus-like Particles (VLPs) Produced in Insect Cells by Inhibiting Cytopathic Effects of Matrix Protein M2.

To provide broader protection and eliminate the need for annual update of influenza vaccines, biomolecular engineering of influenza virus-like particles (VLPs) to display more conserved influenza proteins such as the matrix protein M2 has been explored. However, achieving high surface density of full-length M2 in influenza VLPs has been left unrealized. In this study, we show that the ion channel activity of M2 induces significant cytopathic effects in Spodoptera frugiperda (Sf9) insect cells when expressed using M2-encoding baculovirus. These effects include altered Sf9 cell morphology and reduced baculovirus replication, resulting in impaired influenza protein expression and thus VLP production. On the basis of the function of M2, we hypothesized that blocking its ion channel activity could potentially relieve these cytopathic effects, and thus restore influenza protein expression to improve VLP production. The use of the M2 inhibitor amantadine indeed improves Sf9 cellular expression not only of M2 (∼3-fold), but also of hemagglutinin (HA) (∼7-fold) and of matrix protein M1 (∼3-fold) when coexpressed to produce influenza VLPs. This increased cellular expression of all three influenza proteins further leads to ∼2-fold greater VLP yield. More importantly, the quality of the resulting influenza VLPs is significantly improved, as demonstrated by the ∼2-fold, ∼50-fold, and ∼2-fold increase in the antigen density to approximately 53 HA, 48 M1, and 156 M2 per influenza VLP, respectively. Taken together, this study represents a novel approach to enable the efficient incorporation of full-length M2 while enhancing both the yield and quality of influenza VLPs produced by Sf9 cells.

Expression of full-length HER2 protein in Sf9 insect cells and its presentation on the surface of budded virus-like particles.

Biomarkers of cancer are often glycosylated membrane receptor proteins present on the cellular surface. In order to develop new antibodies for cancer diagnostics or treatment, it is a main pre-requisite that these target proteins are available in a native conformation. However, membrane receptor proteins are notoriously difficult to produce due to their hydrophobic nature and complex architecture. Here, we used the baculovirus-insect cell expression system to produce budded virus-like particles (VLPs) as the scaffold for the presentation of complex membrane proteins. Since the human epidermal growth factor receptor 2 (HER2) is known to be overexpressed in a number of cancers it was chosen as model for a tumor antigen. VLPs displaying full-length HER2 on the surface were produced in Spodoptera frugiperda 9 (Sf9) insect cells and purified by sucrose gradient ultracentrifugation. The number of secreted particles was quantified by nanoparticle tracking analysis. To confirm the presence of HER2 protein on the surface, VLPs were labeled with gold-conjugated antibodies and analyzed by transmission electron microscopy. Functionality of displayed HER2 was investigated by ELISA and a newly established biolayer interferometry based technique. Detection was accomplished using the specific monoclonal antibody Herceptin and filamentous phages displaying a single-chain variable fragment of an anti-HER2 antibody. Significant stronger binding of Herceptin and anti-HER2 phages to HER2-displaying VLPs as compared to control VLPs was demonstrated. Thus, we suggest that Sf9 insect cells are highly feasible for the fast and easy production of various budded VLPs that serve as a platform for full-length membrane receptor presentation.

Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutants in the Sf9 Cell Expression System.

A large problem of histamine receptor research is data heterogeneity. Various experimental approaches, the complex signaling pathways of mammalian cells, and the use of different species orthologues render it difficult to compare and interpret the published results. Thus, the four human histamine receptor subtypes were analyzed side-by-side in the Sf9 insect cell expression system, using radioligand binding assays as well as functional readouts proximal to the receptor activation event (steady-state GTPase assays and [35S]GTPγS assays). The human H1R was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hH1R and insect cell Gαq. By contrast, functional expression of the hH2R required the generation of an hH2R-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hH2R to the long (GsαL) or short (GsαS) splice variant of Gαs resulted in comparable constitutive hH2R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH1R/hH2R and their guinea pig orthologues gpH1R/gpH2R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH3R with Gαi1, Gαi2, Gαi3, and Gαi/o in Sf9 cells revealed high constitutive activity and comparable interaction efficiency with all G protein isoforms. A comparison of various cations (Li+, Na+, K+) and anions (Cl-, Br-, I-) revealed that anions with large radii most efficiently stabilize the inactive hH3R state. Potential sodium binding sites in the hH3R protein were analyzed by expressing specific hH3R mutants in Sf9 cells. In contrast to the hH3R, the hH4R preferentially couples to co-expressed Gαi2 in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hH4R shows structural instability and adopts a G protein-independent high-affinity state. A detailed characterization of affinity and activity of a series of hH4R antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hH4R. In summary, the Sf9 cell system permitted a successful side-by-side comparison of all four human histamine receptor subtypes. This chapter summarizes the results of pharmacological as well as medicinal chemistry/molecular modeling approaches and demonstrates that these data are not only important for a deeper understanding of HxR pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general.

The role of HCV e2 protein glycosylation in functioning of virus envelope proteins in insect and Mammalian cells.

The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.

Expression of TAT recombinant Oct4, Sox2, Lin28, and Nanog proteins from baculovirus-infected Sf9 insect cells.

Somatic cell reprogramming has generated enormous interest, following the first report of generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, but the integration of viral transgenes into the genome is unlikely to be accepted. Given these safety considerations, a method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed. Here, we expressed transactivator of transcription (TAT)-fused proteins, Sox2, Oct4, Lin28, and Nanog in Sf9 cells using the baculovirus expression vector system (BEVS). The molecular weights of the TAT-Sox2, TAT-Oct4, TAT-Lin28, and TAT-Nanog fusion proteins were 36kD, 40kD, 24kD, and 36kD, respectively. Further investigation indicated that most of the recombinant proteins remained in the nuclei of the Sf9 insect cells and were therefore unavailable for purification and cellular reprogramming. Once this problem has been solved, it seems likely that protein expressed from baculovirus-infected Sf9 insect cells will be the method of choice for cellular reprogramming.

Biochemical and preliminary X-ray characterization of the tumor-associated calcium signal transducer 2 (Trop2) ectodomain.

Trop2 is a stem/progenitor cell marker, which is also upregulated in several human carcinomas. The largest part of the molecule, recognized by several monoclonal antibodies, is represented by the extracellular part (ectodomain) and is composed of three modules. The aim of our work was to prepare the ectodomain of Trop2 in quantities sufficient for structural and functional studies. We used the Spodoptera frugiperda (Sf9) insect cell expression system to prepare the Trop2 ectodomain (Trop2EC) in two forms - wt glycosylated (gTrop2EC) and mutant non-glycosylated form (Trop2EC(Δ/N)). Recombinant protein was purified from cell culture supernatants using two subsequent nickel ion-affinity chromatographies with a final yield of 15-17mg of purified recombinant protein per liter of culture. Size-exclusion chromatography together with MALS and chemical crosslinking were used to demonstrate for the first time that the Trop2 ectodomain forms a dimer. Both gTrop2EC and Trop2EC(Δ/N) exhibit similar biochemical properties, however the solubility of Trop2EC(Δ/N) is much lower (less than 1mg/ml). For the purpose of structural studies, we crystallized the glycosylated form gTrop2EC. The native dataset was collected with a resolution of 2.94Å and will be used in ongoing work for phasing and structure solution to further understand the role of Trop2 and the structure-function relation between Trop2 and the epithelial cell adhesion molecule (EpCAM).