Porcine Deltacoronavirus-like Particles Produced by a Single Recombinant Baculovirus Elicit Virus-Specific Immune Responses in Mice.

Porcine deltacoronavirus (PDCoV) causes diarrhea and vomiting in neonatal piglets worldwide and has the potential for cross-species transmission. Therefore, virus-like particles (VLPs) are promising vaccine candidates because of their safety and strong immunogenicity. To the best of our knowledge, the present study reported for the first time the generation of PDCoV VLPs using a baculovirus expression vector system, and electron micrograph analyses revealed that PDCoV VLPs appeared as spherical particles with a diameter similar to that of the native virions. Furthermore, PDCoV VLPs effectively induced mice to produce PDCoV-specific IgG and neutralizing antibodies. In addition, VLPs could stimulate mouse splenocytes to produce high levels of cytokines IL-4 and IFN-γ. Moreover, the combination of PDCoV VLPs and Freund's adjuvant could improve the level of the immune response. Together, these data showed that PDCoV VLPs could effectively elicit humoral and cellular immunity in mice, laying a solid foundation for developing VLP-based vaccines to prevent PDCoV infections.

The Magic Staff: A Comprehensive Overview of Baculovirus-Based Technologies Applied to Human and Animal Health.

Baculoviruses are enveloped, insect-specific viruses with large double-stranded DNA genomes. Among all the baculovirus species, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most studied. Due to its characteristics regarding biosafety, narrow host range and the availability of different platforms for modifying its genome, AcMNPV has become a powerful biotechnological tool. In this review, we will address the most widespread technological applications of baculoviruses. We will begin by summarizing their natural cycle both in larvae and in cell culture and how it can be exploited. Secondly, we will explore the different baculovirus-based protein expression systems (BEVS) and their multiple applications in the pharmaceutical and biotechnological industry. We will focus particularly on the production of vaccines, many of which are either currently commercialized or in advanced stages of development (e.g., Novavax, COVID-19 vaccine). In addition, recombinant baculoviruses can be used as efficient gene transduction and protein expression vectors in vertebrate cells (e.g., BacMam). Finally, we will extensively describe various gene therapy strategies based on baculoviruses applied to the treatment of different diseases. The main objective of this work is to provide an extensive up-to-date summary of the different biotechnological applications of baculoviruses, emphasizing the genetic modification strategies used in each field.

Baculoviral COVID-19 Delta DNA vaccine cross-protects against SARS-CoV2 variants in K18-ACE2 transgenic mice.

After severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) made the world tremble with a global pandemic, SARS-CoV2 vaccines were developed. However, due to the coronavirus's intrinsic nature, new variants emerged, such as Delta and Omicron, refractory to the vaccines derived using the original Wuhan strain. We developed an HERV-enveloped recombinant baculoviral DNA vaccine against SARS-CoV2 (AcHERV-COVID19S). A non-replicating recombinant baculovirus that delivers the SARS-CoV2 spike gene showed a protective effect against the homologous challenge in a K18-hACE2 Tg mice model; however, it offered only a 50 % survival rate against the SARS-CoV2 Delta variant. Therefore, we further developed the AcHERV-COVID19 Delta vaccine (AcHERV-COVID19D). The AcHERV-COVID19D induced higher neutralizing antibodies against the Delta variant than the prototype or Omicron variant. On the other hand, cellular immunity was similarly high for all three SARS-CoV2 viruses. Cross-protection experiments revealed that mice vaccinated with the AcHERV-COVID19D showed 100 % survival upon challenge with Delta and Omicron variants and 71.4 % survival against prototype SARS-CoV2. These results support the potential of the viral vector vaccine, AcHERV-COVID19D, in preventing the spread of coronavirus variants such as Omicron and SARS-CoV2 variants.

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.

A gold nanoparticles-based lateral flow assay utilizing baculovirus expressed recombinant nucleocapsid and receptor binding domain proteins for serodetection of IgG and IgM against SARS-CoV-2.

Serological assays for SARS-CoV-2 are being utilized at an exponential rate for surveillance programs. This enterprise was designed to develop and validate a qualitative immunochromatographic test, via the Lateral Flow Assay (LFA), for detection of immunoglobulins M and G (IgM and IgG) against both nucleocapsid (N) and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Both targeted proteins were cloned and expressed in baculovirus expression system utilizing insect cells Sf9. The recombinant RBD and N proteins were purified and conjugated with gold nanoparticles (AuNPs) to set up the coating antigens pad. Both anti-human IgG and IgM were dispensed on nitrocellulose membrane to capture human antibodies in serum samples. A home-made dispensing system was developed to draw identical test and control lines. The validity of the developed LFA was verified by testing serum samples from 103 convalescent COVID-19 patients who were PCR positive for SARS-CoV-2 along with 28 control serum samples. The developed strips showed distinctive bands for IgM and IgG of both proteins (RBD and N) in positive samples. The sensitivity of RBD-based LFA was 70.9% and 39.8% for IgG and IgM, respectively, with a specificity of 100% for both. The N-based LFA exhibited a sensitivity of 73.8% and 35.9% for IgG and IgM, respectively, while its specificity was 75% and 100% for IgG and IgM, respectively. Our developed LFA could afford a tool for surveillance programs in low-resource countries. Moreover, it might be functional for rapid and inexpensive monitoring of the anti-SARS-CoV-2 antibodies in the sera of vaccinated individuals.

Baculovirus-Free SARS-CoV-2 Virus-like Particle Production in Insect Cells for Rapid Neutralization Assessment.

Virus-like particles (VLPs) resemble authentic virus while not containing any genomic information. Here, we present a fast and powerful method for the production of SARS-CoV-2 VLP in insect cells and the application of these VLPs to evaluate the inhibition capacity of monoclonal antibodies and sera of vaccinated donors. Our method avoids the baculovirus-based approaches commonly used in insect cells by employing direct plasmid transfection to co-express SARS-CoV-2 envelope, membrane, and spike protein that self-assemble into VLPs. After optimization of the expression plasmids and vector ratios, VLPs with an ~145 nm diameter and the typical "Corona" aura were obtained, as confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Fusion of the membrane protein to GFP allowed direct quantification of binding inhibition to angiotensin II-converting enzyme 2 (ACE2) on cells by therapeutic antibody candidates or sera from vaccinated individuals. Neither VLP purification nor fluorescent labeling by secondary antibodies are required to perform these flow cytometric assays.

Mamestra brassicae NIAS-Mb-32 cell strain 2g2 enables high-yield recombinant protein production in baculovirus-free and baculovirus-based insect cell expression.

The production of recombinant proteins using insect cells has been widely used for over 30 years, which contributing to life science research and biotechnology. Insect cells exhibiting enhanced N-glycosylation and recombinant protein productivity enhance the productivity of the baculovirus-insect cell system (BICS). A new highly proliferative insect cell strain, 2g2, was established from the Mamestra brassicae pupa ovary cell strain NIAS-MB-32 (RCB0413) to address the problem of Sf-rhabdovirus and to explore the newly available possibilities in BICS as well as Sf9, such as increased protein production and recombinant baculovirus amplification. The high-growth cell strain 2g2 was examined for its recombinant protein production ability and baculovirus productivity; moreover, the activity of the produced recombinant proteins was examined using Sf9 as a benchmark. Recombinant protein productivity and virus production by BICS in 2g2 was confirmed as equivalent to that of Sf9. Furthermore, we produced the severe acute respiratory syndrome coronavirus 2 spike protein in a baculovirus-free system and compared its productivity, binding activity with human angiotensin-converting enzyme 2, and N-glycosylation. The productivity and bioactivity were found to be equal to or better than that of Sf9. Moreover, N-glycosylation analysis revealed that the glycans derived from the 2g2-produced glycoproteins were mostly of the high mannose type as Sf9. Therefore, 2g2 may have the same N-glycosylation ability as Sf9. Finally, the Sf-rhabdovirus was confirmed to be negative in 2g2. Our results demonstrated that the novel insect cell strain 2g2 can serve as a protein production tool in scientific research and industrial biotechnology.

The baculovirus expression vector system: a modern technology for the future of influenza vaccine manufacturing.

INTRODUCTION: Influenza is a vaccine-preventable disease. Due to the evolving nature of influenza viruses, the composition of vaccines has to be updated annually. Most of the current influenza vaccines are still produced in embryonated chicken eggs, a well-established process with some limitations. AREA COVERED: This review focuses on the recombinant DNA technology using baculovirus expression vector system a modern method of manufacturing licensed influenza vaccines. The speed, scalability, biosafety and flexibility of the process, together with the reliability of the hemagglutinin in the vaccine, represent a significant advance toward new platforms for vaccine production. EXPERT OPINION: The scenario of vaccine production in the next years seems to be particularly interesting, involving a transition from the current egg-based production to new technologies, such as the cell culture platform, the RNA technology, the plant-based system, and the DNA vaccine. This latter offers great advantages over egg- and cell-based influenza vaccine production. The universal vaccine remains the goal of researchers and ideally would avoid the need for annual reformulation and re-administration of seasonal vaccines. The lesson learned from the COVID-19 pandemic highlights the importance of having different technologies available and able to promptly respond to a great demand of vaccines worldwide.

SARS-CoV-2 Virus-like Particles Produced by a Single Recombinant Baculovirus Generate Anti-S Antibody and Protect against Variant Challenge.

Coronavirus Disease 2019 (COVID-19), caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has highlighted the need for the rapid generation of efficient vaccines for emerging disease. Virus-like particles, VLPs, are an established vaccine technology that produces virus-like mimics, based on expression of the structural proteins of a target virus. SARS-CoV-2 is a coronavirus where the basis of VLP formation has been shown to be the co-expression of the spike, membrane and envelope structural proteins. Here we describe the generation of SARS-CoV-2 VLPs by the co-expression of the salient structural proteins in insect cells using the established baculovirus expression system. VLPs were heterologous ~100 nm diameter enveloped particles with a distinct fringe that reacted strongly with SARS-CoV-2 convalescent sera. In a Syrian hamster challenge model, non-adjuvanted VLPs induced neutralizing antibodies to the VLP-associated Wuhan S protein and reduced virus shedding and protected against disease associated weight loss following a virulent challenge with SARS-CoV-2 (B.1.1.7 variant). Immunized animals showed reduced lung pathology and lower challenge virus replication than the non-immunized controls. Our data suggest SARS-CoV-2 VLPs offer an efficient vaccine that mitigates against virus load and prevents severe disease.

Transduction of HEK293 Cells with BacMam Baculovirus Is an Efficient System for the Production of HIV-1 Virus-like Particles.

Gag virus-like particles (VLPs) are promising vaccine candidates against infectious diseases. VLPs are generally produced using the insect cell/baculovirus expression vector system (BEVS), or in mammalian cells by plasmid DNA transient gene expression (TGE). However, VLPs produced with the insect cell/BEVS are difficult to purify and might not display the appropriate post-translational modifications, whereas plasmid DNA TGE approaches are expensive and have a limited scale-up capability. In this study, the production of Gag VLPs with the BacMam expression system in a suspension culture of HEK293 cells is addressed. The optimal conditions of multiplicity of infection (MOI), viable cell density (VCD) at infection, and butyric acid (BA) concentration that maximize cell transduction and VLP production are determined. In these conditions, a maximum cell transduction efficiency of 91.5 ± 1.1%, and a VLP titer of 2.8 ± 0.1 × 109 VLPs/mL are achieved. Successful VLP generation in transduced HEK293 cells is validated using super-resolution fluorescence microscopy, with VLPs produced resembling immature HIV-1 virions and with an average size comprised in the 100-200 nm range. Additionally, evidence that BacMam transduction occurs via different pathways including dynamin-mediated endocytosis and macropinocytosis is provided. This work puts the basis for future studies aiming at scaling up the BacMam baculovirus system as an alternative strategy for VLP production.

Harnessing coronavirus spike proteins' binding affinity to ACE2 receptor through a novel baculovirus surface display system.

Coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerging infectious disease currently spreading across the world. The spike (S) protein plays a key role in the receptor recognition and cell membrane fusion, making it an important target for developing vaccines, therapeutic antibodies and diagnosis. In this study, we constructed a baculovirus surface display system that efficiently presents both SARS-CoV and SARS-CoV-2 S proteins (including ectodomain, S1 subunit and receptor-binding-domain, RBD) on the surface of recombinant baculoviruses, utilizing transmembrane anchors from gp64 (signal peptide) and vesicular stomatitis virus (VSV). These recombinant baculoviruses were capable of transducing engineered HEK 293T cells overexpressing ACE2 receptors with significantly higher transduction efficiencies, indicating that S proteins displayed on baculovirus surface have antigenicity and can recognize and bind ACE2 receptors. Additionally, the transduction of SARS-CoV-2 S proteins can be inhibited by an antibody against the SARS-CoV-2 RBD. These results demonstrate that this baculovirus surface display system is a promising tool for developing antibodies, vaccines and recombinant protein production.

[Production of antiviral vaccines using a silkworm-baculovirus expression system].

The outbreak of the new coronavirus (SARS-2) infection (COVID-19) in Wuhan, China, has become a global outbreak and continues to cause many deaths. Not only COVID-19, but the risk of pandemic continues to increase due to global environmental changes and globalization of human exchange and logistics. On the other hand, we are inadequately prepared for such unknown emerging infectious diseases, and dealing with viral infections has become one of the most important issues facing humanity. Vaccine based disease prevention is considered an ideal disease control strategy, especially for viral diseases for which there is no or little cure. New nucleic acid vaccines (mRNA and viral vector vaccines) are leading the way in the fight against COVID-19, but even today, the mainstream of vaccines are inactivated vaccines and live-attenuated vaccines. Subunit vaccines, in which some specific viral proteins of the pathogen are produced as recombinant proteins and used as vaccine antigens, have also been developed. On the other hand, many antigens that are difficult to produce in large quantities as recombinant proteins, such as the spike protein antigen of COVID-19 (the same antigen is also targeted by nucleic acid vaccines), have not yet been produced and developed. In this paper, we describe the development of a recombinant protein vaccine based on the silkworm insect factory, which has an advantage in the production of such difficult-to-express vaccine antigens.

An Integrated Platform for Serological Detection and Vaccination of COVID-19.

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is an ongoing pandemic. Detection and vaccination are essential for disease control, but they are distinct and complex operations that require significant improvements. Here, we developed an integrated detection and vaccination system to greatly simplify these efforts. We constructed recombinant baculoviruses to separately display the nucleocapsid (N) and spike (S) proteins of SARS-CoV-2. Insect cells infected by the recombinant baculoviruses were used to generate a cell-based system to accurately detect patient serum. Notably, although well-recognized by our newly developed detection system in which S-displaying insect cells acted as antigen, anti-S antibodies from many patients were barely detectable by Western blot, evidencing that COVID-19 patients primarily produce conformation-dependent anti-S antibodies. Furthermore, the same baculovirus constructs can display N (N-Bac) or S (S-Bac) on the baculovirus envelope and serve as vector vaccines. Animal experiments show that S-Bac or N-Bac immunization in mice elicited a strong and specific antibody response, and S-Bac in particular stimulated effective neutralizing antibodies without the need for adjuvant. Our integrated system maintains antigen conformation and membrane structure to facilitate serum detection and antibody stimulation. Thus, compared with currently available technologies, our system represents a simplified and efficient platform for better SARS-CoV-2 detection and vaccination.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2.

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.