Construction of Recombinant Baculoviruses.

The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.

Development of an immunoassay for the simultaneous detection of GADA and ZnT8A in autoimmune diabetes using a ZnT8/GAD65 chimeric molecule.

Introduction: The combined presence of autoantibodies to the 65 kDa isoform of glutamic acid decarboxylase (GADA) and to the islet-specific cation efflux transporter ZnT8 (ZnT8A) in serum is the best predictive sign of the loss of immune tolerance and the clinical manifestation of autoimmune diabetes mellitus (DM). The screening of GADA and ZnT8A could help to reach to a correct diagnosis and to start an early and adequate treatment. The aim of the study was to develop an immunoassay for the simultaneous detection of these autoantibodies using a chimera molecule that includes the immunodominant regions of ZnT8 and GAD65, expressed by baculovirus-insect cells system. Materials and Methods: ZnT8/GAD65 was expressed using the Bac to Bac™ baculovirus expression system. The recombinant chimera was purified by an His6-tag and identified by SDS-PAGE and western blot analysis, and by an indirect ELISA using specific antibodies against ZnT8 and GAD65. A fraction of ZnT8/GAD65 was biotinylated. A bridge ELISA (b-ELISA) was developed using ZnT8/GAD65 immobilized in polystyrene microplates, human sera samples from healthy individuals (n = 51) and diabetic patients (n = 49) were then incubated, and afterwards ZnT8/GAD65-biotin was added. Immune complexes were revealed with Streptavidin-Horseradish Peroxidase. Results were calculated as specific absorbance and expressed as standard deviation scores: SDs. Results: ZnT8/GAD65 was efficiently produced, yielding 30 mg/L culture medium, 80% pure. This recombinant chimera retains the immunoreactive conformation of the epitopes that are recognized by their specific antibodies, so it was used for the development of a high sensitivity (75.51%) and specificity (98.04%) b-ELISA for the detection of ZnT8A and/or GADA, in a one-step screening assay. The ROC curves demonstrated that this method had high accuracy to distinguish between samples from healthy individuals and diabetic patients (AUC = 0.9488); the cut-off value was stablished at 2 SDs. Conclusions: This immunoassay is useful either to confirm autoimmune diabetes or for detection in routine screening of individuals at risk of autoimmune DM. As DM is a slow progress disease, remaining asymptomatic for a long preclinical period, serological testing is of importance to establish a preventive treatment.

Zika virus-like particles (VLPs) produced in insect cells.

Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins' conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Results and discussion: Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.

Zika virus-like particles (VLPs) produced in insect cells

Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins’ conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Results and discussion: Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.

Improved Expression of SARS-CoV-2 Spike RBD Using the Insect Cell-Baculovirus System.

Insect cell-baculovirus expression vector system is one of the most established platforms to produce biological products, and it plays a fundamental role in the context of COVID-19 emergency, providing recombinant proteins for treatment, diagnosis, and prevention. SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host's cellular receptor. As RBD is required for many applications, in the context of pandemic it is important to meet the challenge of producing a high amount of recombinant RBD (rRBD). For this reason, in the present study, we developed a process based on Sf9 insect cells to improve rRBD yield. rRBD was recovered from the supernatant of infected cells and easily purified by metal ion affinity chromatography, with a yield of 82% and purity higher than 95%. Expressed under a novel chimeric promoter (polh-pSeL), the yield of rRBD after purification was 21.1 ± 3.7 mg/L, which is the highest performance described in Sf9 cell lines. Finally, rRBD was successfully used in an assay to detect specific antibodies in COVID-19 serum samples. The efficient strategy herein described has the potential to produce high-quality rRBD in Sf9 cell line for diagnostic purpose.

Production of Rabies VLPs in Insect Cells by Two Monocistronic Baculoviruses Approach.

Rabies is an ancient zoonotic disease that still causes the death of over 59,000 people worldwide each year. The rabies lyssavirus encodes five proteins, including the envelope glycoprotein and the matrix protein. RVGP is the only protein exposed on the surface of viral particle, and it can induce immune response with neutralizing antibody formation. RVM has the ability to assist with production process of virus-like particles. VLPs were produced in recombinant baculovirus system. In this work, two recombinant baculoviruses carrying the RVGP and RVM genes were constructed. From the infection and coinfection assays, we standardized the best multiplicity of infection and the best harvest time. Cell supernatants were collected, concentrated, and purified by sucrose gradient. Each step was used for protein detection through immunoassays. Sucrose gradient analysis enabled to verify the separation of VLPs from rBV. Through the negative contrast technique, we visualized structures resembling rabies VLPs produced in insect cells and rBV in the different fractions of the sucrose gradient. Using ELISA to measure total RVGP, the recovery efficiency of VLPs at each stage of the purification process was verified. Thus, these results encourage further studies to confirm whether rabies VLPs are a promising candidate for a veterinary rabies vaccine.

Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology.

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.

Production of rabies VLPs in insect cells by two monocistronic baculoviruses approach

Rabies is an ancient zoonotic disease that still causes the death of over 59,000 people worldwide each year. The rabies lyssavirus encodes five proteins, including the envelope glycoprotein and the matrix protein. RVGP is the only protein exposed on the surface of viral particle, and it can induce immune response with neutralizing antibody formation. RVM has the ability to assist with production process of virus-like particles. VLPs were produced in recombinant baculovirus system. In this work, two recombinant baculoviruses carrying the RVGP and RVM genes were constructed. From the infection and coinfection assays, we standardized the best multiplicity of infection and the best harvest time. Cell supernatants were collected, concentrated, and purified by sucrose gradient. Each step was used for protein detection through immunoassays. Sucrose gradient analysis enabled to verify the separation of VLPs from rBV. Through the negative contrast technique, we visualized structures resembling rabies VLPs produced in insect cells and rBV in the different fractions of the sucrose gradient. Using ELISA to measure total RVGP, the recovery efficiency of VLPs at each stage of the purification process was verified. Thus, these results encourage further studies to confirm whether rabies VLPs are a promising candidate for a veterinary rabies vaccine.

Dengue and Zika virus multi-epitope antigen expression in insect cells.

Dengue virus and Zika virus are arthropod-borne flaviviruses that cause millions of infections worldwide. The co-circulation of both viruses makes serological diagnosis difficult as they share high amino acid similarities in viral proteins. Antigens are one of the key reagents in the differential diagnosis of these viruses through the detection of IgG antibodies in serological assays during the convalescent-phase of infections. Here, we report the expression of Dengue virus (DENV) and Zika virus (ZIKV) antigens containing non-conserved and immunodominant amino acid sequences using the baculovirus expression vector system in insect cells. We designed DENV and ZIKV antigens based on the domain III of the E protein (EDIII) after analyzing previously reported epitopes and by multiple alignment of the most important flaviviruses. The ZIKV and DENV multi-epitope genes were designed as tandem repeats or impaired repeats separated by tetra- or hexa-glycine linkers. The biochemical analyses revealed adequate expression of the antigens. Then, the obtained multi-epitope antigens were semi-purified in a sucrose gradient and tested using patients' sera collected during the convalescent-phase that were previously diagnosed positive for anti-DENV and -ZIKV IgG antibodies. The optimal serum dilution was 1:200, and the mean absorbance values in the preliminary tests show that multi-epitope antigens have been recognized by human sera. The production of both antigens using the multi-epitope strategy in the eukaryotic system and based on the EDIII regions provide a proof of concept for the use of antigens in the differentiation between DENV and ZIKV.

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method.

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: an efficient immunoaffinity method

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades recombinant Rabies Virus Glycoprotein (RVGP) produced in several expression systems, has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post‐translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.

Spin label EPR suggests the presence of cholesterol rich domains in cultured insect cell membranes.

Different spin labels were incorporated to the membranes of cultured insect UFL-AG-286 cells in order to characterize their physical properties by Electron Paramagnetic Resonance spectroscopy (EPR). The spectrum of the spin label 12-SASL incorporated to cell membranes was similar as those obtained in membrane model systems composed of eggPC/cholesterol. However, the spectrum of the spin label CSL, chemically related to cholesterol, was drastically different in the two systems. Interestingly, when cell cholesterol content was reduced using methyl beta cyclodextrin, an EPR spectrum similar to those of model membranes was obtained. The analysis of these experiments suggests the existence of cholesterol rich regions in UFL-AG-286 cell membranes.

IgG Responses to the Plasmodium falciparum Antigen VAR2CSA in Colombia Are Restricted to Pregnancy and Are Not Induced by Exposure to Plasmodium vivax.

Clinical immunity to malaria is associated with the acquisition of IgG specific for members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens on the surface of infected erythrocytes (IEs). The VAR2CSA subtype of PfEMP1 mediates IE binding in the placenta. VAR2CSA-specific IgG is normally acquired only after exposure to placental parasites. However, it was recently reported that men and children from Colombia often have high levels of functional VAR2CSA-specific IgG. This potentially undermines the current understanding of malaria immunity in pregnant women, and we thus conducted a study to assess further the levels of VAR2CSA-specific IgG in pregnant and nonpregnant Colombians. Plasma IgG against two full-length recombinant PfEMP1 proteins (one of the VAR2CSA type and one not) produced in baculovirus-transfected insect cells was detected frequently among Colombian men, children, and pregnant women with acute or previous malaria exposure. In contrast, IgG reactivity to a homologous full-length VAR2CSA-type protein expressed in Chinese hamster ovary (CHO) cells was low and infrequent among the Colombian plasma samples, as was reactivity to both corresponding native PfEMP1 proteins. Moreover, human and rabbit antibodies specific for Plasmodium vivax Duffy-binding protein (PvDBP), a protein with some homology to PfEMP1, did not react with VAR2CSA-type recombinant or native proteins, although the mouse monoclonal and PvDBP-specific antibody 3D10 was weakly reactive with recombinant proteins expressed in baculovirus-transfected insect cells. Our data indicate that the previously reported Colombian IgG reactivity to recombinant VAR2CSA is not malaria specific and that the acquisition of VAR2CSA-specific IgG is restricted to pregnancy, in Colombia and elsewhere.

Platforms for Recombinant Therapeutic Glycoprotein Production.

The majority of FDA-approved biology-derived products are recombinant glycoproteins. These proteins have been used for the treatment of several diseases, with numerous products currently approved for clinical use. The choice of the expression system is a key step toward a successful functional protein production, since glycosylation influences yield, pharmacokinetics, biological activity, and immunogenicity. This chapter covers the general aspects of therapeutic recombinant glycoproteins and the platforms that are being employed for their production.

Production of Recombinant Rabies Virus Glycoprotein by Insect Cells in a Single-Use Fixed-Bed Bioreactor.

A single-use fixed-bed bioreactor (iCELLis nano) can be used for cultivating non adherent insect cells, which can be then recovered for scaling up or for harvesting a membrane-associated viral glycoprotein with high quality in terms of preserved protein structure and biological function. Here, we describe the procedures for establishing genetically modified Drosophila melanogaster Schneider 2 (S2) cell cultures in the iCELLis nano bioreactor and for quantifying by ELISA the recombinant rabies virus glycoprotein (rRVGP) synthesized. By using the described protocol of production, the following performance can be regularly achieved: 1.7 ± 0.6 × 1E10 total cells; 2.4 ± 0.8 × 1E7 cells/mL and 1.2 ± 0.9 µg of rRVGP/1E7 cells; 1.5 ± 0.8 mg of total rRVGP.

Real-time imaging reveals unique heterogeneous population features in insect cell cultures.

Heterogeneity of cellular populations has been frequently observed. We used live cell imaging to follow Sf9 insect cells before and after infection with baculovirus, to understand population dynamics. It was possible to identify in real time cells with distinctive phenotypes. Mobile cells with an elongated bipolar shape were observed. They extended pseudopods and actively moved about the culture surface. The presence of actively moving elongated cells increased when cultures were subjected to oxygen limiting or excessive conditions, suggesting that stress triggered differentiation of cells to the mobile phenotype. A dual reporter baculovirus (DRBac), coding for two fluorescent proteins under promoters with different temporality, was designed to follow sequential phenomena through infection. Oxygen limitation reduced the number of cells that expressed the reporter proteins, possibly because it reduced the efficiency of baculovirus infection. Elongated cells did not show signs of infection. To our knowledge, this is the first time that actively moving cells are observed in real time in Sf9 cultures, which had distinctive responses towards infection. Anoxia was identified as a factor that modulates baculovirus infection. Results open a new approach for understanding the insect-cell baculovirus system. Particular cellular phenotypes with unique traits can be isolated for specific applications.

Expression of Viral Envelope Glycoproteins in Drosophila melanogaster S2 Cells.

The expression of recombinant viral envelope glycoproteins in S2 (Drosophila melanogaster) has been performed with good results. This chapter contains protocols for the utilization of this system for the expression and analysis of proteins presented in cell plasma membrane.

Insect cell entrapment, growth and recovering using a single-use fixed-bed bioreactor. Scaling up and recombinant protein production.

Insect cells are largely used for industrial production of vaccines, viral vectors and recombinant proteins as well as in research and development as an important tool for biology and bioprocess studies. They grow in suspension and are semi-adherent cells. Among the cell culture systems enabling scalable bioprocess the single-use fixed-bed iCELLis(®) bioreactors offer great advantages. We have established the conditions for Drosophila melanogaster Schneider 2 (S2) and Spodoptera frugiperda (Sf9) cells entrapment into the fixed-bed, cell growth and recover from the fixed-bed once high cell densities were attained. Our established protocol allowed these cells, at a cell seeding of 2×1E5 cells/microfiber carriers (MC) (3.5×1E6cells/mL; 1.7×1E4cells/cm(2)), to grow inside a 4m(2)/200mL fixed-bed attaining a concentration of 5.3×1E6 cells/MC (9.5×1E7cells/mL; 4.7×1E5 cells/cm(2)) for S2 cells or 4.6×1E6 cells/MC (8×1E7cells/mL; 4.1×1E5cells/cm(2)) for Sf9 cells. By washing the fixed-bed, entrapped cells could then be recovered from the fixed-bed at a high rate (>85%) with high viability (>95%) by increasing the agitation to 1200/1500rpm. Although the cell yields in the fixed-bed bioreactor were comparable to those obtained in a stirred tank (respectively, 1.3×1E10 and 2.5×1E10 total cells), S2 cells stably transfected with a cDNA coding for the rabies virus glycoprotein (RVGP) showed a 30% higher preserved rRVGP production (2.5±0.1 and 1.9±0.1µg/1E7 cells), as evidenced by a conformational ELISA evaluation. These findings demonstrate not only the possibility to entrap, cultivate to high densities and recover insect cells using a single-use fixed-bed bioreactor, but also that this system provides suitable physiological conditions for the entrapped cells to produce a cell membrane associated recombinant protein with higher specific biological activity as compared to classical suspension cell cultures.

Equine arteritis virus gP5 protein induces apoptosis in cultured insect cells.

Equine Arteritis Virus (EAV) has been shown to induce apoptosis in vitro but the induction of this mechanism has not been previously associated with any viral gene product. In this work, we found a cytotoxicity effect of the EAV gP5 protein on baculovirus-insect cells and a low yield of protein recovery. Besides, different morphological features by electron transmission microscopy, DNA fragmentation in agarose gel, TUNEL analysis and caspase 3 activity were found. All these findings indicate that the EAV gP5 protein induces apoptosis in insect cells.