Baculovirus Expression Vector Research Articles

RNAi-Mediated Silencing in the Insect Cell-Baculovirus Expression System.

RNA interference (RNAi) serves as an indispensable tool for gene function studies and has been substantiated through extensive research for its practical applications in the baculovirus expression vector system (BEVS). This chapter expands the RNAi toolkit in insect cell culture by including small interfering RNA (siRNA) in the protocol, in addition to the conventional use of double-stranded RNA (dsRNA). This chapter also brings attention to key design and reporting considerations, based on Minimum Information About an RNAi Experiment (MIARE) guidelines. Recommendations regarding online tools for dsRNA and siRNA design are provided, along with guidance on choosing suitable methods for measuring silencing outcomes.

Scarless Baculovirus Genome Editing Using Lambda-Red Recombineering in E. coli.

Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.

A Time and Cost-Effective Pipeline for Expression Screening and Protein Production in Insect Cells Based on the HR-Bac Toolbox to Generate Recombinant Baculoviruses.

The baculovirus expression vector system (BEVS) is recognized as a powerful platform for producing challenging proteins and multiprotein complexes both in academia and industry. Since a baculovirus was first used to produce heterologous human IFN-β protein in insect cells, the BEVS has continuously been developed and its applications expanded. We have recently established a multigene expression toolbox (HR-bac) composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. Unlike platforms that rely on Tn7-medidated transposition for the construction of baculoviruses, HR-bac relies on homologous recombination, which allows to evaluate expression constructs in 2weeks and is thus perfectly adapted to parallel expression screening. In this chapter, we detail our standard operating procedures for the preparation of the reagents, the construction and evaluation of baculoviruses, and the optimization of protein production for both intracellularly expressed and secreted proteins.

Construction and immunogenicity of Senecavirus A Virus-Like Particle Vaccine with adjuvant

Senecavirus A (SVA) is constantly associated with vesicular disease in pigs, and the clinical symptoms of pig infection with SVA are indistinguishable from other porcine vesicular diseases. Vaccine is one of the best methods to eliminate and control the spread of SVA. Virus-like particles (VLPs) can play important roles in prevention for infectious diseases. Here, the SVA VLPs was assembled by the baculovirus expression vector system, and the immunogenicity of the SVA VLPs mixed with different adjuvants were evaluated in mice and pigs. Two recombinant baculoviruses (rPFBD-VP1-VP3 and rPFBD-VP2-VP4) were constructed, which co-infected with Sf9 suspension cells to assemble SVA VLPs successfully. SVA VLPs mixed with ISA201 adjuvant and ISA201+Poly(I:C) adjuvant produced higher levels of neutralizing antibody, specific antibody (total IgG, IgG1, IgG2a and IgG2b) and cytokines in the T cells. And there was no significant difference between SVA VLPs+201 group and SVA VLPs+Poly(I:C)+201 group. Pigs immunized with high dose of SVA VLPs mixed with ISA201 adjuvant could produce higher titers of neutralizing antibody and SVA-specific antibody. Furthermore, the protection rates of SVA VLPs-H and SVA VLPs-L were 100% and 80%, and the viral load of SVA VLPs-H group is the lowest in all SVA VLPs groups. It is the first time to develop the SVA VLPs using the baculovirus expression vector system, which may lay the foundation for the research and development of SVA vaccine.

A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios.

The baculovirus expression vector (BEV) system is an efficient, cost-effective, and scalable method to produce recombinant adeno-associated virus (rAAV) gene therapy vectors. Most BEV designs emulate the wild-type AAV transcriptome and translate the AAV capsid proteins, VP1, VP2, and VP3, from a single mRNA transcript with three overlapping open reading frames (ORFs). Non-canonical translation initiation codons for VP1 and VP2 reduce their abundances relative to VP3. Changing capsid ratios to improve rAAV vector efficacy requires a theoretical modification of the translational context. We have developed a Lac repressor-inducible system to empirically regulate the expression of VP1 and VP2 proteins relative to VP3 in the context of the BEV. We demonstrate the use of this system to tune the abundance, titer, and potency of a neurospecific rAAV9 serotype derivative. VP1:VP2:VP3 ratios of 1:1:8 gave optimal potency for this rAAV. It was discovered that the ratios of capsid proteins expressed were different than the ratios that ultimately were in purified capsids. Overexpressed VP1 did not become incorporated into capsids, while overexpressed VP2 did. Overabundance of VP2 correlated with reduced rAAV titers. This work demonstrates a novel technology for controlling the production of rAAV in the BEV system and shows a new perspective on the biology of rAAV capsid assembly.

Development of an Optimized Process for Functional Recombinant SARS-CoV-2 Spike S1 Receptor-Binding Domain Protein Produced in the Baculovirus Expression Vector System.

To map the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and evaluate immune response variations against this virus, it is essential to set up efficient serological tests locally. The SARS-CoV-2 immunogenic proteins were very expensive and not affordable for lower- middle-income countries (LMICs). For this purpose, the commonly used antigen, receptor-binding domain (RBD) of spike S1 protein (S1RBD), was produced using the baculovirus expression vector system (BEVS). In the current study, the expression of S1RBD was monitored using Western blot under different culture conditions. Different parameters were studied: the multiplicity of infection (MOI), cell density at infection, and harvest time. Hence, optimal conditions for efficient S1RBD production were identified: MOI 3; cell density at infection 2-3 × 106 cells/mL; and time post-infection (tPI or harvest time) of 72 h and 72-96 h, successively, for expression in shake flasks and a 7L bioreactor. A high production yield of S1RBD varying between 4 mg and 70 mg per liter of crude cell culture supernatant was achieved, respectively, in the shake flasks and 7L bioreactor. Moreover, the produced S1RBD showed an excellent antigenicity potential against COVID-19 (Wuhan strain) patient sera evaluated by Western blot. Thus, additional serological assays, such as in-house ELISA and seroprevalence studies based on the purified S1RDB, were developed.

Virus-like particle vaccines with epitopes from porcine epidemic virus and transmissible gastroenteritis virus incorporated into self-assembling ADDomer platform provide clinical immune responses in piglets.

Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are major intestinal coronaviruses that cause vomiting, diarrhea, dehydration, and mortality in piglets. These viruses coexist and lead to significant economic losses in the swine industry. Virus-like particles (VLPs) have emerged as promising alternatives to conventional inactivated vaccines due to their exceptional safety, efficacy, and ability to provide multi-disease protection with a single dose. Our study focused on specific antigenic epitopes from the PEDV S protein (SS2 and 2C10 regions) and the TGEV S protein (A and D sites) as target candidates. These epitopes were integrated into the ADDomer framework, and we successfully generated recombinant proteins AD, AD-P, AD-T, and AD-PT using the baculovirus expression vector system (BEVS). By meticulously optimizing conditions in High Five cells, we successfully expressed and purified the recombinant proteins. Subsequently, we developed the recombinant ADDomer-VLP vaccine and conducted a comprehensive evaluation of its efficacy in piglets. Following ultrafiltration concentration and sucrose gradient centrifugation purification, the recombinant proteins self-assembled into VLPs as observed by transmission electron microscopy (TEM). Administration of the vaccine did not result in any adverse reactions in the immunized piglets. Additionally, no significant instances of fever were detected in any of the experimental groups, and there were no notable changes in average daily weight gain compared to the control group that received PBS. The recombinant ADDomer-VLP vaccines demonstrated strong immunogenicity, effectively stimulating the production of neutralizing antibodies against both PEDV and TGEV. Moreover, the recombinant ADDomer-VLP vaccine induced elevated levels of IFN-γ, IL-2, and IL-4, and enhanced cytotoxic T lymphocyte (CTL) activity in the peripheral blood of piglets. These recombinant VLPs have demonstrated the ability to induce strong cellular and humoral immune responses in piglets, making them an incredibly promising platform for the rapid and simplified development of epitope vaccines.

Insect cell‐derived human CD200‐Fc increases zonula occludens‐1 tight junction protein in urothelial carcinoma cells

AbstractCD200 is a ligand that interacts with the CD200 receptor 1, with an anti‐inflammatory effect. CD200 inhibits the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) pathway, attenuating the inflammatory response and maintaining barrier function. In this study, human CD200 was fused with the fragment crystallizable (Fc) region of human immunoglobulin G (IgG) to generate the recombinant CD200‐Fc protein, produced in insect cells using the baculovirus expression vector system. The CD200‐Fc gene was cloned under the control of the polyhedrin promoter in the pFastBac‐1 vector of the baculovirus expression vector system. Immunoblot analysis confirmed the expression of CD200‐Fc in insect cells. The CD200‐Fc protein was successfully purified using protein A affinity chromatography. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay revealed that purified CD200‐Fc protein inhibited the proliferation of TCCSUP cells, a bladder epithelial cancer cell expressing CD200 receptor 1, at concentrations of 1, 10 and 100 ng/mL. Quantitative real‐time reverse transcription PCR (qRT‐PCR) and immunoblot analyses confirmed that the mRNA and protein levels of zonula occludens‐1, a tight junction protein for barrier protection in epithelial tissues, were increased in TCCSUP cells treated with insect cell‐derived CD200‐Fc. These results suggest that insect cell‐derived CD200‐Fc could alter zonula occludens‐1 expression in bladder epithelial cancer cells, enhancing the function of the cell barrier protein, which could inhibit cancer metastasis. Taken together, the baculovirus expression vector system can be applied to express antitumor therapeutic CD200‐Fc protein for the inhibition of bladder cancer.

Identification of optimal flow rate for culture media, cell density, and oxygen toward maximization of virus production in a fed-batch baculovirus-insect cell system.

In recent times, it has been realized that novel vaccines are required to combat emerging disease outbreaks, and faster optimization is required to respond to global vaccine demands. Although, fed-batch operations offer better productivity, experiment-based optimization of a new fed-batch process remains expensive and time-consuming. In this context, we propose a novel computational framework that can be used for process optimization and control of a fed-batch baculovirus-insect cell system. Since the baculovirus expression vector system (BEVS) is known to be widely used platforms for recombinant protein/vaccine production, we chose this system to demonstrate the identification of optimal profile. Toward this, first, we constructed a mathematical model that captures the time course of cell and virus growth in a baculovirus-insect cell system. Second, the proposed model was used for numerical analysis to determine the optimal operating profiles of control variables such as culture media, cell density, and oxygen based on a multiobjective optimal control formulation. Third, a detailed comparison between batch and fed-batch culture was perfromed along with a comparison between various alternatives of fed-batch operation. Finally, we demonstrate that a model-based quantification of controlled feed addition in fed-batch culture is capable of providing better productivity as compared to a batch culture. The proposed framework can be utilized for the estimation of optimal operating regions of different control variables to achieve maximum infected cell density and virus yield while minimizing the substrate/media, uninfected cell, and oxygen consumption.

A Baculovirus Expression Vector Derived Entirely from Non-Templated, Chemically Synthesized DNA Parts.

Baculovirus expression system1s are a widely used tool in recombinant protein and biologics production. To enable the possibility of genome modifications unconstrained through low-throughput and bespoke classical genome manipulation techniques, we set out to construct a baculovirus vector (>130 kb dsDNA) built from modular, chemically synthesized DNA parts. We constructed a synthetic version of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) through two steps of hierarchical Golden Gate assembly. Over 140 restriction endonuclease sites were removed to enable the discrimination of the synthetic genome from native baculovirus genomes. A head-to-head comparison of our modular, synthetic AcMNPV genome with native baculovirus vectors showed no significant difference in baculovirus growth kinetics or recombinant adeno-associated virus production-suggesting that neither baculovirus replication nor very-late gene expression were compromised by our design or assembly method. With unprecedented control over the AcMNPV genome at the single-nucleotide level, we hope to ambitiously explore novel AcMNPV vectors streamlined for biologics production and development.

Design of a novel multiepitope vaccine against Chlamydia pneumoniae using the extracellular protein as a target

Chlamydia pneumoniae (C. pneumoniae) infection in humans is universal and causes various respiratory infectious diseases, making a safe and effective preventive vaccine essential. In this study, a multi-epitope vaccine with CTLA-4 extracellular structure was constructed by an immunoinformatics approach. Since MOMP protein is the major extracellular protein in C. pneumoniae and has good immunogenicity and high conservation, we selected the MOMP protein of C. pneumoniae as the antigen target, predicted the T and B cell epitopes of the MOMP protein and then connected the CTLA-4 extracellular structure with the predicted dominant epitopes by various linkers to construct a multi-epitope vaccine. The biochemical characterization of the multi-epitope vaccine showed its immunogenicity and anti-allergic properties. The tertiary structure of this vaccine, along with molecular docking, molecular dynamics simulation, and principal component analysis, showed that the multi-epitope vaccine structure interacted with B7 (B7-1, B7-2) and toll-like receptors (TLR-2, TLR-4). Ultimately, the vaccine was cloned and effectively expressed in silico on an insect baculovirus expression vector (pFastBac1). These analyses showed that the designed vaccine could potentially target antigen-presenting cells and was immune to C. pneumoniae, which provided novel strategies for developing the vaccine.

End-to-End Semi-automated Mid-scale Protein Screening Platform for Drug Discovery Research.

The drug discovery landscape is ever-evolving and constantly demands revolutionary technology advancements in protein expression and production laboratories. We have built a higher-throughput mid-scale semi-automated protein expression and screening platform to accelerate drug discovery research. The workflow described here enables comprehensive expression and purification screening assessment of challenging or difficult-to-express recombinant proteins in a fast and efficient manner by delivering small but sufficient amounts of high-quality proteins. The platform has been implemented for a wide range of applications that include identification of optimal constructs and chaperones for poorly expressing proteins, assessment of co-expression partners for expressing stable multiprotein complexes, and suitable buffer/additive screening for insoluble or aggregation-prone proteins. The approach allows parallel expression, purification, and characterization of 24 different samples using co-infection or a polycistronic approach in insect cells and enables parallel testing of multiple parameters to improve protein yields. The strategy has been successfully adopted for screening intracellular and secreted proteins in Escherichia coli, mammalian transient expression, and baculovirus expression vector systems. Proteins purified from this platform are used for several structural and functional screens, such as negative staining, biochemical activity assays, mass spectrometry, surface plasmon resonance, and DNA-encoded chemical library screens. In this article, for simplicity, we have focused on detailed expression and purification screening of intracellular protein complexes from insect cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Baculovirus generation via homologous recombination Support Protocol 1: Anti-glycoprotein 64 antibody assay Basic Protocol 2: Generation of insect cell biomass expressing target protein(s) Basic Protocol 3: Mid-scale affinity purification Support Protocol 2: Automated method for affinity purification on Hamilton STAR Basic Protocol 4: Size exclusion chromatography Support Protocol 3: Chromeleon 7 operation on Vanquish Duo.

Standardizing In Vitro β-Lactam Antibiotic Allergy Testing with Synthetic IgE.

The global prevalence of β-lactam allergy poses a major challenge in administering first-line antibiotics, such as penicillins, to a significant portion of the population. The lack of β-lactam IgE antibody pools with defined selectivity hampers the standardization and validation of in vitro assays for β-lactam allergy testing. To address this limitation, this study introduces a synthetic IgE specific to β-lactam antibiotics as a valuable tool for drug allergy research and diagnostic tests. Using phage display technology, we constructed a library of human single-chain antibody fragments (scFv) to target the primary determinant of amoxicillin, a widely used β-lactam antibiotic. Subsequently, we produced a complete human synthetic IgE molecule using the highly efficient baculovirus expression vector system. This synthetic IgE molecule served as a standard in an in vitro chemiluminescence immunoassay for β-lactam antibiotic allergy testing. Our results demonstrated a detection limit of 0.05 IU/mL (0.63 pM), excellent specificity (100%), and a four-fold higher clinical sensitivity (73%) compared to the in vitro reference assay when testing a cohort of 150 serum samples. These findings have significant implications for reliable interlaboratory comparison studies, accurate labeling of allergic patients, and combating the global public health threat of antimicrobial resistance. Furthermore, by serving as a valuable trueness control material, the synthetic IgE facilitates the standardization of diagnostic tests for β-lactam allergy and demonstrates the potential of utilizing this synthetic strategy as a promising approach for generating reference materials in drug allergy research and diagnostics.

Application of Baculovirus Expression Vector System (BEVS) in Vaccine Development.

Vaccination is one of the most effective strategies to control epidemics. With the deepening of people's awareness of vaccination, there is a high demand for vaccination. Hence, a flexible, rapid, and cost-effective vaccine platform is urgently needed. The baculovirus expression vector system (BEVS) has emerged as a promising technology for vaccine production due to its high safety, rapid production, flexible product design, and scalability. In this review, we introduced the development history of BEVS and the procedures for preparing recombinant protein vaccines using the BEVS platform and summarized the features and limitations of this platform. Furthermore, we highlighted the progress of the BEVS platform-related research, especially in the field of vaccine. Finally, we provided a new prospect for BEVS in future vaccine manufacturing, which may pave the way for future BEVS-derived vaccine development.

Mechanistic modeling explains the production dynamics of recombinant adeno-associated virus with the baculovirus expression vector system

Current manufacturing processes for recombinant adeno-associated viruses (rAAVs) have less-than-desired yields and produce significant amounts of empty capsids. The increasing demandand the high cost of goods for rAAV-based gene therapies motivate development of more efficient manufacturing processes. Recently, the US Food and Drug Administration (FDA) approved the first rAAV-based gene therapy product manufactured in the baculovirus expression vector system (BEVS), a technology that demonstrated production of high titers of full capsids. This work presents a first mechanistic model describing the key extracellular and intracellular phenomena occurring during baculovirus infection and rAAV maturation in the BEVS. The model predictions are successfully validated for in-house and literature experimental measurements of the vector genome and of structural and non-structural proteins collected during rAAV manufacturing in the BEVS with the TwoBac and ThreeBac constructs. A model-based analysis of the process is carried out to identify the bottlenecks that limit full capsid formation. Vector genome amplification is found to be the limiting step for rAAV production in Sf9 cells using either the TwoBac or ThreeBac system. In turn, vector genome amplification is hindered by limiting Rep78 levels. Transgene and non-essential baculovirus protein expression in the insect cell during rAAV manufacturing also negatively influences the rAAV production yields.

Optimization of genetic code expansion in the baculovirus expression vector system (BEVS)

The production of recombinant proteins containing unnatural amino acids, commonly known as genetic code expansion (GCE), represents a breakthrough in protein engineering that allows for the creation of proteins having novel designed properties. The naturally occurring orthogonal pyrrolysine tRNA/aminoacyl-tRNApyl synthetase pair (tRNApyl/PylRS) found in Methanosarcinaceae species has provided a rich platform for protein engineers to build a library of amino acid derivatives suitable for the introduction of novel chemical functionalities. While reports of the production of such recombinant proteins utilizing the tRNApyl/PylRS pair, or mutants thereof, is commonplace in Escherichia coli and mammalian cell expression systems, there has only been a single such report of GCE in the other stalwart of recombinant protein production, the baculovirus expression vector system (BEVS). However, that report formulates protein production within the designs of the MultiBac expression system [1]. The current study frames protein production within the strategies of the more commonplace Bac-to-Bac system of recombinant baculovirus production, via the development of novel baculovirus transfer vectors that harbor the tRNApyl/PylRS pair. The production of recombinant proteins harboring an unnatural amino acid(s) was examined using both an in cis and an in trans arrangement of the tRNApyl/PylRS pair relative to the target protein ORF i.e. the latter resides, respectively, on either the same vector as the tRNApyl/PylRS pair, or on a separate vector and deployed in a viral co-infection experiment. Aspects of the transfer vector designs and the viral infection conditions were investigated.

A sensitive assay for scrutiny of Autographa californica multiple nucleopolyhedrovirus genes using CRISPR-Cas9.

Baculoviruses have very large genomes and previous studies have demonstrated improvements in recombinant protein production and genome stability through the removal of some nonessential sequences. However, recombinant baculovirus expression vectors (rBEVs) in widespread use remain virtually unmodified. Traditional approaches for generating knockout viruses (KOVs) require several experimental steps to remove the target gene prior to the generation of the virus. In order to optimize rBEV genomes by removing nonessential sequences, more efficient techniques for establishing and evaluating KOVs are required. Here, we have developed a sensitive assay utilizing CRISPR-Cas9-mediated gene targeting to examine the phenotypic impact of disruption of endogenous Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes. For validation, 13 AcMNPV genes were targeted for disruption and evaluated for the production of GFP and progeny virus - traits that are essential for their use as vectors for recombinant protein production. The assay involves transfection of sgRNA into a Cas9-expressing Sf9 cell line followed by infection with a baculovirus vector carrying the gfp gene under the p10 or p6.9 promoters. This assay represents an efficient strategy for scrutinizing AcMNPV gene function through targeted disruption, and represents a valuable tool for developing an optimized rBEV genome. KEY POINTS: [Formula: see text] A method to scrutinize the essentiality of baculovirus genes was developed. [Formula: see text] The method uses Sf9-Cas9 cells, a targeting plasmid carrying a sgRNA, and a rBEV-GFP. [Formula: see text] The method allows scrutiny by only needing to modify the targeting sgRNA plasmid.

YBac: A novel and simple baculovirus system for heterologous protein production in Spodoptera frugiperda 9 (Sf9) cells.

The baculovirus expression vector system (BEVS) has been widely used for heterologous protein expression due to its powerful functionality and easy access to commercial expression vectors. Currently, most laboratories prefer two strategies for protein production using BEVS. One is recombinant bacmid based on transposition in Escherichia coli (e.g., Bac-to-Bac), and another is homologous recombination in insect cells (e.g., flashBac). In this manuscript, a rapid and simple YBac system was established. This novel system uses an Ac99KO bacmid as a virus vector and co-transfected into Spodoptera frugiperda 9 (Sf9) cells with a donor plasmid capable of recombination into Ac42 loci that carry the genes of interest (GOIs) along with the complete Ac99 fragment. Based on the intracellular homologous recombination system, the production of foreign proteins was achieved by the complementation of the Ac99 gene together with the insertion of GOIs. In this study, the human thyroid peroxidase (hTPO) and porcine epidemic diarrhea virus-like particles (PEDV VLPs) were successfully expressed using the YBac system. The entire process was shortened to 10 days, and the components involved in the system could be easily prepared in the laboratory, suggesting that the YBac system may have great potential in the production of heterologous proteins.

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.

Identification, Baculoviral Expression, and Biochemical Characterization of a Novel Cholinesterase of Amblyomma americanum (Acari: Ixodidae).

A cDNA encoding a novel cholinesterase (ChE, EC 3.1.1.8) from the larvae of Amblyomma americanum (Linnaeus) was identified, sequenced, and expressed in Sf21 insect cell culture using the baculoviral expression vector pBlueBac4.5/V5-His. The open reading frame (1746 nucleotides) of the cDNA encoded 581 amino acids beginning with the initiation codon. Identical cDNA sequences were amplified from the total RNA of adult tick synganglion and salivary gland, strongly suggesting expression in both tick synganglion and saliva. The recombinant enzyme (rAaChE1) was highly sensitive to eserine and BW284c51, relatively insensitive to tetraisopropyl pyrophosphoramide (iso-OMPA) and ethopropazine, and hydrolyzed butyrylthiocholine (BuTCh) 5.7 times as fast as acetylthiocholine (ATCh) at 120 µM, with calculated KM values for acetylthiocholine (ATCh) and butyrylthiocholine of 6.39 µM and 14.18 µM, respectively. The recombinant enzyme was highly sensitive to inhibition by malaoxon, paraoxon, and coroxon in either substrate. Western blots using polyclonal rabbit antibody produced by immunization with a peptide specific for rAaChE1 exhibited reactivity in salivary and synganglial extract blots, indicating the presence of AaChE1 antigenic protein. Total cholinesterase activities of synganglial or salivary gland extracts from adult ticks exhibited biochemical properties very different from the expressed rAaACh1 enzyme, evidencing the substantial presence of additional cholinesterase activities in tick synganglion and saliva. The biological function of AaChE1 remains to be elucidated, but its presence in tick saliva is suggestive of functions in hydrolysis of cholinergic substrates present in the large blood mean and potential involvement in the modulation of host immune responses to tick feeding and introduced pathogens.