Search Results (71)

Background/Objectives: Porcine circovirus type 2 (PCV2) infection causes porcine circovirus disease (PCVD), a global immunosuppressive disease in pigs. Its clinical manifestations include post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS), which cause significant economic losses to the swine industry. The Cap protein, which is the major protective antigen of PCV2, can self-assemble to form virus-like particles (VLPs) in the insect baculovirus expression system. Few studies have compared the expression of Cap proteins in different baculovirus expression systems. Methods: In this study, we compared two commonly commercialized baculovirus construction systems with the Cap protein expression in various insect cells. Results: The results demonstrate that the flashBAC system expressed the Cap protein at higher levels than the Bac-to-Bac system. Notably, when expressing four copies of the Cap protein, the flashBAC system achieved the highest protein yield in High Five cells, where it reached 432 μg/mL at 5 days post-infection (dpi) with 27 °C cultivation. Animal experiments confirmed that the purified Cap protein effectively induced specific antibody production in mice and swine. Conclusions: This study provides critical data for optimizing the production of the PCV2 Cap protein, which is of great significance for reducing the production cost of PCV2 vaccines and improving the industrial production efficiency. Full article

The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering. Full article

Background/Objectives: Feline panleukopenia, caused by FPV, is a highly contagious disease in cats. Current vaccines face challenges including complex production, high cost, and safety risks. Developing safer, more efficient alternatives is crucial. This study aimed to produce FPV virus-like particles (VLPs) using a recombinant baculovirus system expressing the VP2 gene and evaluate their immunogenicity and protective efficacy in cats. Methods: Sf9 insect cells were infected with recombinant baculovirus to express VP2 protein. The VP2 protein was purified using ultrafiltration and size-exclusion chromatography (SEC). Dynamic light scattering (DLS) and transmission electron microscopy (TEM) confirmed the assembly of VLPs. Twenty healthy cats were randomly divided into four groups; three groups received different doses (5 μg, 15 μg, and 45 μg) of FPV VLP vaccine, while the fourth group served as the control group immunized with PBS. Blood samples were collected on day 21 to measure hemagglutination inhibition (HI) and virus-neutralizing (VN) antibody responses. Cats in the 15 μg dose group were challenged with virulent FPV strain 708 on day 21, and clinical signs and white blood cell counts were monitored for 10 days. Results: Immunized cats exhibited significantly higher HI and VN antibody titers compared to controls. After challenge, vaccinated cats showed no clinical signs of disease, and their white blood cell counts remained stable. In contrast, control cats developed severe symptoms and experienced significant leukopenia. Conclusions: The FPV VLP vaccine generated in this study are highly immunogenic and provide effective protection against virulent FPV challenge, demonstrating their potential as a safer vaccine candidate for feline panleukopenia. 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

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

The development of effective vaccines necessitates a delicate balance between maximizing immunogenicity and minimizing safety concerns. Subunit vaccines, while generally considered safe, often fail to elicit robust and durable immune responses. Nanotechnology presents a promising approach to address this dilemma, enabling subunit antigens to mimic critical aspects of native pathogens, such as nanoscale dimensions, geometry, and highly repetitive antigen display. Various expression systems, including Escherichia coli (E. coli), yeast, baculovirus/insect cells, and Chinese hamster ovary (CHO) cells, have been explored for the production of nanoparticle vaccines. Among these, E. coli stands out due to its cost-effectiveness, scalability, rapid production cycle, and high yields. However, the E. coli manufacturing platform faces challenges related to its unfavorable redox environment for disulfide bond formation, lack of post-translational modifications, and difficulties in achieving proper protein folding. This review focuses on molecular and protein engineering strategies to enhance protein solubility in E. coli and facilitate the in vitro reassembly of virus-like particles (VLPs). We also discuss approaches for antigen display on nanocarrier surfaces and methods to stabilize these carriers. These bioengineering approaches, in combination with advanced nanocarrier design, hold significant potential for developing highly effective and affordable E. coli-derived nanovaccines, paving the way for improved protection against a wide range of infectious diseases. Full article

Moschus chrysogaster viral hemorrhagic disease (McVHD), caused by the Moschus chrysogaster hemorrhagic disease virus (McHDV), is an acute and highly fatal infectious disease of musk deer. At present, there is no prevention or treatment for this disease. In this study, we constructed a recombinant bacmid containing the McHDV VP60 gene and obtained the recombinant baculovirus rBac-McHDV VP60 by transfection into Sf9 (Spodoptera frugiperda) insect cells. The McHDV VP60 protein was successfully expressed in the insect cell-baculovirus expression system; furthermore, it was released in the supernatant of infected insect cells and spontaneously assembled to form virus-like particles (VLPs), which were structurally and immunologically indistinguishable from the Moschus chrysogaster viral hemorrhagic disease virion. Hypodermic vaccination of rabbits with the VLPs conferred complete protection in 14 days; this protection was found to be effective from the seventh day after VLP injection and was accompanied by a strong humoral response. This study is the first attempt to express the VP60 gene of McHDV using an insect baculovirus system, which provides an experimental basis for the virus-like particle vaccine of McVHD. 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

The evolution of the H5 highly pathogenic avian influenza (HPAI) viruses has led to the emergence of distinct groups with genetically similar clusters of hemagglutinin (HA) sequences. In this study, a consensus H5 HA sequence was cloned into the baculovirus expression system. The HA protein was expressed in baculovirus-infected insect cells and utilized as the antigen for the production of an oil emulsion-based H5 avian influenza vaccine (rBacH5Con5Mut). Twenty-one-day-old SPF chickens were immunized with this vaccine and then challenged at 21 days post-vaccination with clade 2.3.2.1, clade 2.3.4.4, and clade 7.2 of H5 HPAI viruses. The sera of vaccinated chickens exhibited high hemagglutination inhibition (HI) titers against the rBacH5 vaccine antigen, while lower HI titers were observed against the different challenge virus H5 hemagglutinins. Furthermore, the rBacH5Con5Mut vaccine provided 100% protection from mortality and clinical signs. Virus isolation results showed that oropharyngeal and cloacal shedding was prevented in 100% of the vaccinated chickens when challenged with clade 2.3.2.1 and clade 2.3.4.4 H5 viruses. When the rBacH5Con5Mut vaccine candidate was administrated at one day of age, 100% protection was demonstrated against the challenge of clade 2.3.4.4 virus at three weeks of age, indicating the potential of this vaccine for hatchery vaccination. Overall, A single immunization of rBacH5Con5Mut vaccine candidate with a consensus HA antigen can protect chickens against different clades of H5 HPAI viruses throughout the rearing period of broiler chickens without a boost, thus fulfilling the criteria for an efficacious broad-spectrum H5 avian influenza vaccine. Full article

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (β-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid. Full article

Carrion’s disease, caused by Bartonella bacilliformis, is a neglected tropical disease prevalent in the Andean region of South America. Without antimicrobial treatment, this disease has a mortality rate of up to 88% in infected patients. The most common method for diagnosing B. bacilliformis infection is serological testing. However, the current serological assays are limited in sensitivity and specificity, underscoring the need for the development of novel and more accurate diagnostic tools. Recombinant proteins have emerged as promising candidates to improve the serological diagnosis of Carrion’s disease. So, we focused on evaluating the conditions for producing two previously predicted proteins of B. bacilliformis using the baculovirus–insect cell expression system, mainly the flashBAC ULTRA technology. We assessed various parameters to identify the conditions that yield the highest protein production, including cell lines, temperature, and hours post-infection (hpi). The results showed that the expression conditions for achieving the highest yields of the Prot_689 and Prot_504 proteins were obtained using High Five™ cells at 21 °C and harvesting at 120 hpi. Subsequently, the seroreactivity of recombinant proteins was evaluated using positive sera from patients diagnosed with Carrion’s disease. These findings offer valuable insights into the production conditions of B. bacilliformis recombinant proteins using the baculovirus system, which could significantly contribute to developing more precise diagnostic tools for Carrion’s disease. Therefore, this research provides implications for improving diagnostics and potentially developing therapeutic strategies. Full article

The hemagglutination inhibition (HI) assay is a traditional laboratory procedure for detection and quantitation of serum antibodies of hemagglutinating viruses containing the hemagglutinin (HA) gene. The current study aimed to investigate the novel use of virus like particles (VLP) as an antigen for the HI assay. VLPs were prepared from a strain of H5N1 using a baculovirus expression system. The VLPs were characterized using the hemagglutination test, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and transmission electron microscopy. The comparative HI assay was performed using three different seed antigens: A/chicken/Mexico/232/94 (H5N2), A/chicken/Egypt/18-H/09(H5N1) and A/goose/Guangdong/1/1996(H5N1). The HI assay of serum antibody titrations using homologous antigens to these vaccinal seeds were compared to the VLP’s antigens for the same serum. The HI titers were logically relevant to the similarity between VLP antigens and vaccinal seeds, indicating the VLPs behave similarly to the standard HI assay which uses inactivated whole virus as an antigen. VLPs could be considered as an alternative to the HI assay antigen as they show a relatedness between the similarity with vaccinal seed and serum antibodies. Compared to typical entire H5N1 viral antigen prepared in SPF eggs that require proper inactivation to avoid any public health risk, VLPs prepared in tissue culture, plants or insect cells are a safe, inexpensive and scalable alternative to inactivated whole virus antigen. Full article