Search Results (86)

Background: Feline panleukopenia virus (FPV) causes acute and frequently fatal disease in cats, underscoring the urgent need for safe, rapidly effective, and scalable vaccines. While virus-like particle (VLP) vaccines are inherently safe and immunogenic, their development is constrained by low yields of recombinant protein in insect cell expression systems. Methods: An optimized baculovirus expression vector system (BEVS) incorporating the hr1-p6.9-p10 transcriptional enhancer and the Ac-ie-01 anti-apoptotic gene was employed to enhance recombinant protein production. VP2 expression levels, viral titers, and hemagglutination activity were quantified using qPCR, SDS-PAGE/Western blotting, transmission electron microscopy (TEM), and functional assays. Immunogenicity and protective efficacy were assessed in both mice and cats through serological analysis, neutralizing antibody detection, and post-challenge clinical monitoring. Results: The optimized BEVS enhanced recombinant protein transcription by 1.5-fold, viral titers by 3.7-fold, and hemagglutination activity by 15-fold. The purified protein self-assembled into uniform 25 nm virus-like particles (VLPs). Immunization elicited earlier responses compared to commercial vaccines. Vaccinated cats maintained normal body temperature, stable leukocyte counts, and minimal viral shedding following FPV challenge. Conclusions: This study validates an enhanced BEVS that effectively overcomes VP2 yield constraints and generates highly immunogenic FPV VLPs. The platform enables rapid-onset protection and offers a scalable strategy for next-generation FPV vaccine development. Full article

Recombinant baculovirus vectors are recognized as effective gene delivery systems for mammalian cells in vitro. However, their application in vivo has been limited due to inactivation by the host’s complement system. We developed a recombinant baculoviral vector derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV), incorporating both CMV-IE and polyhedron promoter-driven green fluorescence protein (EGFP) (vAcMBac-CMV-IE-EGFP). We then evaluated the transduction efficiency and safety of vAcMBac-CMV-IE-EGFP at a high multiplicity of infection (MOI) across five distinct cell lines and in Sprague Dawley (SD) rats. In vitro, Sf9, HepG2, and Vero E6 cells showed high transduction rates (95.52 ± 4.86%, 80.53 ± 3.31%, and 80.87 ± 2.50%, respectively), significantly outperforming the other cell types tested, and cell viability remained largely unaffected even at an MOI of 1000. In vivo, EGFP expression was observed in the heart, liver, spleen, lungs, and kidneys of SD rats after tail vein injection. Direct injection of vAcMBac-CMV-IE-EGFP into the rat striatum also resulted in strong EGFP signals in neural tissues. These results demonstrate that a high-MOI baculovirus infection can serve as a remarkably efficient and versatile platform for gene delivery across diverse mammalian cell types as well as in various organs and neural tissues in animal models. This robust method might hold significant promise for future gene therapy applications. Full article

Epizootic hemorrhagic disease (EHD) is an important livestock disease caused by Epizootic hemorrhagic disease virus (EHDV). The recent incursion and wide distribution of EHDV in Europe have increased the need for effective vaccine candidates. Background/Objectives: The VP2 protein of EHDV forms the outer capsid layer of the virion and is essential for viral assembly and host cell entry. Owing to its antigenic properties, VP2 represents a major target for vaccine development. However, the recombinant production of VP2 is limited by low stability and poor yields, representing a significant barrier for the generation of safe and effective subunit vaccines. Methods: To overcome these limitations, the VP2 protein from EHDV serotype 8 (EHDV-8) was rationally engineered with targeted modifications at both the amino and carboxyl termini of its coding sequence. Recombinant expression was performed using a baculovirus vector-mediated system in Trichoplusia ni pupae (CrisBio^® technology), employed as living biofactories. Results: The engineering of VP2 resulted in up to a tenfold increase in protein yields compared with the wild-type sequence, while maintaining the trimeric structural integrity of the recombinant protein. Both wild-type and engineered VP2 protein variants were formulated and used to immunize IFNAR(−/−) mice, a model susceptible to EHDV infection. Both engineered and wild-type VP2 formulations elicited comparable neutralizing antibody responses in vaccinated animals. Furthermore, immunization with either formulation conferred full protection against lethal EHDV-8 challenge. Conclusions: In this work, we demonstrated that the rational engineering of the VP2 protein significantly improved recombinant expression yields in a baculovirus-based system without compromising structural integrity or immunogenicity. These findings additionally demonstrate the feasibility of producing high-quality VP2 antigens in T. ni pupae using CrisBio^® technology and support their potential application in the development of subunit vaccines against EHDV. Full article

African swine fever (ASF), caused by the African swine fever virus (ASFV), is an acute, febrile, highly contagious, and lethal disease that poses a severe threat to the global pig farming industry. Currently, no globally recognized, safe, and effective commercial ASF vaccine has been developed, making vaccination a crucial strategy for outbreak control. The ASFV structural proteins p72, p30, and p54 are key targets for vaccine development. In this study, we developed a novel baculovirus vector-based system for surface display of a recombinant protein comprising epitopes from p72, p30, and p54. Upon infection, the recombinant protein was expressed and anchored on the plasma membrane of Sf-9 cells. Purified virus analysis revealed that the Bac-recombinant protein enhanced gene delivery and transgene expression in mammalian cells compared to the Bac-Wild Type (Bac-WT). In a murine model, the Bac-recombinant protein induced significantly higher IFN-γ and IL-4 levels than Bac-p30 and the negative control. However, further evaluation in swine models is required to confirm its protective potential against ASFV. Furthermore, it also elicited a robust antibody response, generating high-titer Bac-recombinant protein-specific antibodies. Therefore, these findings suggest that the ASFV Bac-recombinant protein is a promising candidate for a vector-based vaccine. Full article

Background/Objectives: Virus-like particle (VLP) vaccines based on human papillomavirus (HPV) L1 proteins have high efficacy for preventing cervical cancer and other HPV-associated diseases. The production yields of commercial HPV VLPs remain suboptimal. We aimed to improve HPV VLP production efficiency using a hyper-expression vector system for the expression of L1 proteins of four major HPV serotypes—HPV 6, 11, 16, and 18. Methods: HPV L1 proteins were expressed in Trichoplusia ni (Hi5) insect cells via a hyper-expression baculovirus vector system. Following cell lysis using a microfluidizer, VLPs were purified through a two-step chromatographic process. Particle morphology was characterized using transmission electron microscopy and dynamic light scattering. Immunogenicity was evaluated using a murine model; mice received three intramuscular injections of the purified quadrivalent VLPs. The resulting IgG and neutralizing antibody responses were compared with those elicited by the commercial quadrivalent vaccine, Gardasil. Results: The L1 proteins from HPV 6, 11, 16, and 18 were successfully expressed at high levels in Hi5 cells, forming uniformly sized VLPs with hydrodynamic diameters of 50–60 nm. The average production yield of the quadrivalent VLPs exceeded 40 mg/L, an improvement over conventional yields. The candidate VLPs elicited strong HPV-specific IgG and neutralizing antibody responses in mice, comparable to those induced by Gardasil. Conclusions: The hyper-expression baculovirus vector system enables high-yield production of HPV L1 VLPs with desirable structural and immunogenic properties. This approach holds promise for the cost-effective and scalable manufacturing of next-generation HPV VLP vaccines, facilitating broader global access to HPV immunization. Full article

Background: Rabbit hemorrhagic disease (RHD) is an acute, hemorrhagic and highly lethal infectious disease caused by rabbit hemorrhagic disease virus (RHDV), which causes huge economic losses to the rabbit breeding industry. Moreover, there is limited cross-protection between the two different serotypes of classic RHDV (GI.1) and RHDV2 (GI.2). The shortcomings of traditional inactivated vaccines have led to the development of novel subunit vaccines that can protect against both strains, and the VP60 capsid protein is the ideal antigenic protein. This study focused on developing a bivalent RHDV vaccine that can prevent infection with both GI.1 and GI.2 strains. Methodology: Baculovirus vectors containing classic RHDV and RHDV2 VP60 were co-transfected with linearized baculovirus into sf9 cells and transferred to baculovirus via homologous recombination of the VP60 gene. Infected sf9 cells were lysed, and after purification via Ni-NTA chromatography, VLPs were observed using transmission electron microscopy (TEM). In order to evaluate the immunogenicity of the chimeric RHDV VLP vaccine in rabbits, the RHDV VP60-specific antibody, IL-4, IFN-γ and neutralizing antibody titers were analyzed in serum using ELISA and HI. Results: The recombinant baculovirus system successfully expressed chimeric RHDV VLPs with a diameter of 32–40 nm. After immunization, it could produce specific antibodies, IL-4 and IFN-γ. Following the second immunization, neutralizing antibodies, determined using hemagglutination inhibition (HI) assays, were elicited. Conclusions: These data show that the chimeric RHDV VLP bivalent vaccine for immunized New Zealand rabbits can induce humoral immunity and cellular immunity in vivo, and the immunization effect of the high-dose group is similar to that of the current commercial vaccine. Full article

The Baculovirus Expression Vector System (BEVS) is an important protein and complex biologics production platform. The baculovirus GP64 protein is the major envelope glycoprotein that aids in virus entry and is required for cell-to-cell transmission in cell culture. Several studies have developed strategies around gp64 gene disruption in an attempt to minimize baculovirus co-production. Here, we investigate the result of transiently targeting the baculovirus gp64 gene with CRISPR-Cas9 during infection. Because not all genomes are effectively disrupted, we describe a variant calling methodology that allows the detection of the targeted mutations in gp64 even though these mutations are not the dominant sequences. Using a transfection-infection assay (T-I assay), the AcMNPV gp64 gene was targeted at six different locations to evaluate the effects of single and multiple targeting sites, and we demonstrated a reduction in the levels of baculovirus vectors while maintaining or enhancing foreign protein production when protein was driven by a p6.9 promoter. Viral genomes were subsequently isolated from the supernatant and cell pellet fractions, and our sequencing pipeline successfully detected indel mutations within gp64 for most of the single-guide RNA (sgRNA) targets. We also observed that 68.8% of variants found in the virus stock were conserved upon virus propagation in cell culture, thus indicating that they are not detrimental to viral fitness. This work provides a comprehensive assessment of CRISPR-Cas9 genome editing of baculovirus vectors, with potential applications in enhancing the efficiency of the BEVS. Full article

Insect cell lines are a cornerstone of recombinant protein production, providing a versatile platform for biopharmaceutical and research applications. In the early 20th century, scientists first attempted to culture insect cells in vitro, developing continuous cell lines to produce the first insect cell-derived recombinant protein, IFN-β. Initial successes, along with advancements in the use of insect cells for recombinant protein manufacturing, primarily relied on baculovirus expression vector systems (BEVSs), which enable heterologous gene expression in infected cells. Today, growing attention is focused on baculovirus-free systems based on the transfection of insect cells with plasmid DNA. This approach simplifies the final product purification process and facilitates the development of stable monoclonal cell lines that produce recombinant proteins or protein complexes, particularly virus-like particles (VLPs). Thanks to advancements in genetic engineering and the application of adaptive laboratory evolution (ALE) methods, significant strides have been made in overcoming many limitations associated with insect cell BEVSs, ultimately enhancing the reliability, yield, and quality of the biomanufacturing process. Our manuscript discusses the history of developing insect cell lines, presents various recombinant protein production systems utilizing these cells, and summarizes modifications aimed at improving insect cell lines for recombinant protein biomanufacturing. Finally, we explore their implications in pharmaceutical production, particularly on Nuvaxovid^®/Covovax, which is the latest approved vaccine developed using insect cell BEVSs for protection against SARS-CoV-2. Full article

Feline panleukopenia (FPL), caused by the feline panleukopenia virus (FPLV), is a severe and highly contagious viral disease with high morbidity and mortality. Vaccination remains the gold standard for preventing and controlling this debilitating condition. The viral protein VP2 serves as the major immunogen of FPLV and represents the key target antigen in the development of a novel FPLV vaccine. Virus-like particle (VLP)-based vaccines have emerged as next-generation vaccine candidates due to their high immunogenicity and safe profile. In this study, a baculovirus expression vector system (BEVS) was employed to generate FPLV-VLPs through recombinant expression of the VP2 protein of a Chinese epidemic strain (Ala91Ser, Ile101Thr) of FPLV. The resulting FPLV-VLPs demonstrated markedly enhanced antigenicity and hemagglutination activity, achieving a hemagglutination titer of up to 1:2¹⁶. Following vaccination, immunized cats developed high titers of anti-FPLV hemagglutination inhibition (HI) antibodies (1:2¹⁶) and exhibited 100% protection against challenge with a virulent epidemic FPLV variant (Ala91Ser, Ile101Thr). These findings demonstrate that FPLV-VLPs hold strong potential as candidates for a novel subunit vaccine against FPLV infection. Full article

Rabbit hemorrhagic disease virus (RHDV) is a very virulent virus of the genus Lagovirus causing severe and fatal hepatitis in the European rabbit (Oryctolagus cuniculus). RHDV has two distinct genotypes: GI.1 (RHDV) and GI.2 (RHDV2). The first RHDV2/GI.2 strain was identified as a recombinant virus between a non-pathogenic (GI.3P) and a pathogenic (GI.2) lagovirus, and the recombination is thought to have been a key mechanism in the emergence and evolution of RHDV2. Here, a new variant of RHDV2 was identified affecting domestic rabbits on Chinese farms, with a mortality rate of 70–80%. Phylogenetic analysis indicated that the nonstructural portion of this newly identified strain’s genome clustered with the GI.1a variants. In contrast, the capsid gene shared the highest nucleotide identity of 97.9% with the North American GI.2 strains, suggesting a possible introduction in China of North American strains and recombination with the GI.1a strains circulating in China. We have produced a recombinant vaccine using the first Chinese GI.2 strain, SC2020/0401, by cloning the vp60 gene into a baculovirus expression vector. Virus-like particles (VLPs) were then produced in Sf9 insect cells, and a challenge study was performed. Rabbits immunized with the VLP vaccine survived 7 d after being challenged with the new virus. The results indicate that commercial vaccines are urgently required in China to control the circulation of RHDV2 variants. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major threat to the global swine industry, causing significant economic losses. To address this, we developed a scalable recombinant adeno-associated virus (rAAV)-based strategy for the delivery of soluble viral receptors (SVRs) to treat and potentially eliminate PRRSV infections. This strategy involves fusing the virus-binding domains of two key cellular receptors, sialoadhesin (Sn4D) and CD163 (SRCR5-9), with an Fc fragment. We then used an insect cell–baculovirus expression vector system to produce the rAAV-SRCR59-Fc/Sn4D-Fc vector. Through a series of optimizations, we determined the best conditions for rAAV production, including a baculovirus co-infection ratio of 0.5:1.0, an initial insect cell density of 2.0 × 10⁶ cells/mL, a fetal bovine serum concentration of 2%, and a culture temperature of 30 °C. Under these optimized conditions, we achieved a high titer of rAAV-SRCR59-Fc/Sn4D-Fc in a 2 L bioreactor, reaching 5.4 ± 0.9 × 10⁹ infectious viral particles (IVPs)/mL. Notably, in vitro neutralization assays using a Transwell co-culture system demonstrated a 4.3 log reduction in viral titers across genetically diverse PRRSV-2 strains, including VR2332, JXA1, JS07, and SH1705. Collectively, this study provides a robust platform for large-scale rAAV production and highlights the potential of SVR-based gene therapy to address the antigenic diversity of PRRSV-2. Full article

The alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most commonly used virus in the Baculovirus Expression Vector System (BEVS) and has been utilized for the production of many human and veterinary biologics. AcMNPV has a large dsDNA genome that remains understudied, and relatively unmodified from the wild-type, especially considering how extensively utilized it is as an expression vector. Previously, our group utilized CRISPR-Cas9 genome engineering that revealed phenotypic changes when baculovirus genes are targeted using either co-expressed sgRNA or transfected sgRNA into a stable insect cell line that produced the Cas9 protein. Here, we describe a pipeline to sequence the recombinant AcMNPV expression vectors using shotgun sequencing, provide a set of primers for tiled-amplicon sequencing, show that untargeted baculovirus vector genomes remain relatively unchanged when amplified in Sf9-Cas9 cells, and confirm that AcMNPV gp64 gene disruption can minimize baculovirus contamination in cell cultures. Our findings provide a robust baseline for analyzing in process genome editing of baculoviruses. Full article

The Spodoptera frugiperda Sf9 insect cell line is used in the baculovirus expression vector system for the development of various viral vaccines and some gene therapy products. Early studies indicated that Sf9 cells produced a reverse transcriptase (RT) activity that was detected using a sensitive PCR-enhanced reverse transcriptase (PERT) assay. Since RT is generally associated with retrovirus particles, we undertook the investigation of the physical properties and infectious nature of the extracellular RT activity that was constitutively expressed from Sf9 cells or induced after the chemical treatment of the cells with drugs known to activate endogenous retroviruses. A density gradient analysis indicated that the peak RT activity corresponded to a low buoyant density of about 1.08 g/mL. Ultracentrifugation and size filtration of cell-free Sf9 supernatant indicated that different particle sizes were associated with the RT activity. This was confirmed by transmission electron microscopy and cryoEM, which revealed a diversity in particle size and type, including viral-like and extracellular vesicles. The treatment of Sf9 cells with 5-iodo-2′-deoxyuridine (IUdR) induced a 33-fold higher RT activity with a similar low buoyant density compared to untreated cells. Infectivity studies using various target cells (human A204, A549, MRC-5, and Raji, and African green monkey Vero cells) inoculated with cell-free supernatant from untreated and IUdR-treated Sf9 cells showed the absence of a replicating retrovirus by PERT-testing of cell-free supernatant during the 30 day-culturing period. Additionally, there was no evidence of virus entry by whole genome analysis of inoculated MRC-5 cells using high-throughput sequencing. This is the first study to identify extracellular retroviral-like particles in Spodoptera. Full article

Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is an efficient protein expression platform, which is famous for its high-level expression of complex protein in insect cells. The system is based on baculoviruses such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and the expression efficiency of the target proteins has been significantly improved by optimizing the viral vectors and cell lines. In recent years, IC-BEVS have shown great potential for Adeno-Associated Virus (AAV) production, particularly excelling in AAV structural protein expression and recombinant AAV production. The system not only improves the yield and purity of AAV, but also shortens the production cycle, providing an efficient and reliable tool for gene therapy. However, the system also has some challenges, including protein modification differences, limitations in expression levels, and production costs. This paper reviews the development of the insect baculovirus expression system, its application in AAV production, and its prospects in gene therapy, aiming to provide a systematic reference and outlook for research in related fields. Full article

Nipah virus (NiV) is known to be a highly pathogenic zoonotic virus, which is included in the World Health Organization Research & Development Blueprint list of priority diseases with up to 70% mortality rate. Due to its high pathogenicity and outbreak potency, a therapeutic countermeasure against NiV is urgently needed. As NiV needs to be handled within a Biological Safety Level (BSL) 4 facility, we had developed a safe drug screening platform utilizing a baculovirus expression vector system (BEVS) based on a NiV-induced syncytium formation that could be handled within a BSL-1 facility. To reconstruct the NiV-induced syncytium formation in BEVS, two baculoviruses were generated to express recombinant proteins that are responsible for inducing the syncytium formation, including one baculovirus exhibiting co-expressed NiV fusion protein (NiV-F) and NiV attachment glycoprotein (NiV-G) and another exhibiting human EphrinB2 protein. Interestingly, syncytium formation was observed in infected insect cells when the medium was modified to have a lower pH level and supplemented with cholesterol. Fusion inhibitory properties of several compounds, such as phytochemicals and a polysulfonated naphthylamine compound, were evaluated using this platform. Among these compounds, suramin showed the highest fusion inhibitory activity against NiV-induced syncytium in the baculovirus expression system. Moreover, our in silico results provide a molecular-level glimpse of suramin’s interaction with NiV-G’s central hole and EphrinB2’s G-H loop, which could be the possible reason for its fusion inhibitory activity. Full article