Baculovirus Expression System Research Articles

Amsacta moorei entomopoxvirus encodes a protein kinase with dual activity and a broad substrate spectrum including two putative cellular substrates.

Amsacta moorei entomopoxvirus (AMEV) is a poxvirus that can only infect insects. This virus is an attractive research material because it is similar to smallpox virus. AMEV is one of many viruses that encode protein kinases that drive the host's cellular mechanisms, modifying immune responses to it, and regulating viral protein activity. We report here the functional characterization of a serine/threonine (Ser/Thr) protein kinase (PK) gene (ORF AMV197) of AMEV. Expression of the AMV197 gene in baculovirus expression system yielded a ~ 35.5kDa protein. PK activity of expressed AMV197 was shown by standard PK assay. Substrate profiling of AMV197 protein by peptide microarray indicated that the expressed protein phosphorylated 81 of 624 substrates which belong to 28 families of PK substrates. While the hypothetical AMV197 protein phosphorylates Ser/Thr only, we demonstrated that the expressed PK also phosphorylates probes with tyrosine residues on the array which is a rare property among PKs. Pull-down assay of the AMV197 protein with the subcellular protein fractionations of Ld652 cells showed that it is using two cellular proteins (18 and 42kDa) as novel putative substrates. Our results suggest that AMEV can regulate cellular mechanisms by phosphorylating cellular proteins through AMV197 PK. However, further experiments are needed to identify the exact role of this PK in the replication of AMEV.

Multi-Gene Recombinant Baculovirus Expression Systems: From Inception to Contemporary Applications.

Many protein expression systems are primarily utilised to produce a single, specific recombinant protein. In contrast, most biological processes such as virus assembly rely upon a complex of several interacting proteins rather than the activity of a sole protein. The high complexity of the baculovirus genome, coupled with a multiphase replication cycle incorporating distinct transcriptional steps, made it the ideal system to manipulate for high-level expression of a single, or co-expression of multiple, foreign proteins within a single cell. We have developed and utilised a series of recombinant baculovirus systems to unravel the sequential assembly process of a complex non-enveloped model virus, bluetongue virus (BTV). The high protein yields expressed by the baculovirus system not only facilitated structure-function analysis of each viral protein but were also advantageous to crystallography studies and supported the first atomic-level resolution of a recombinant viral protein, the major BTV capsid protein. Further, the formation of recombinant double-shelled virus-like particles (VLPs) provided insights into the structure-function relationships among the four major structural proteins of the BTV whilst also representing a potential candidate for a viral vaccine. The baculovirus multi-gene expression system facilitated the study of structurally complex viruses (both non-enveloped and enveloped viruses) and heralded a new generation of viral vaccines.

Serological characterisation of Lagovirus virus-like particles originating from native and mutated VP60 of rabbit haemorrhagic disease virus 2 and European brown hare syndrome virus

Abstract Introduction Since lagoviruses cannot be cultivated in vitro, using expression systems is an alternative and promising way of producing diagnostic viral antigens. It opens up their use as active immunogens for vaccine production. Material and Methods Virus-like particles (VLPs) were produced in a baculovirus expression system in Spodoptera frugiperda 9 (Sf9) insect cells based on wild-type and mutated variants of the virus capsid VP60 protein from a Polish strain of European brown hare syndrome virus (EBHSV) and wild-type and mutated versions of this protein from a Polish strain of rabbit haemorrhagic disease virus 2 (RHDV2). The mutations were the substitution of an arginylglycylaspartic acid (Arg-Gly-Asp/RGD) motif in the P2 subdomain and, in the S or P2 domain, the substitution of three lysines. The VLPs were purified with sucrose gradient ultracentrifugation. Results Protein production was confirmed by Western blot analysis using rabbit or hare sera and ELISA tests with different types of monoclonal antibody. The haemagglutination properties of some VLPs were also evaluated. Electron microscopy of wild-type EBHSV, wild-type RHDV2 and the four VP60 variants produced in this experiment revealed the formation of characteristic VLP structures. Conclusion For the first time, mutated VLPs of RHDV2 with an RGD motif in the VP60 sequence were obtained, which could potentially be used to deliver cargo to eukaryotic cells. Virus-like particles based on the VP60 proteins of EBHSV and RHDV with a three-lysine substitution in the S or P2 domains were also obtained. Potential exists for VLPs of EBHSV and RHDV2 as vaccine candidates.

A SARS-CoV-2 Nanobody Displayed on the Surface of Human Ferritin with High Neutralization Activity.

COVID-19 is rampant throughout the world, which has caused great damage to human lives and seriously hindered the development of the global economy. Aiming at the treatment of SARS-CoV-2, in this study, we proposed a novel fenobody strategy based on ferritin (Fe) self-assembly technology. The neutralizing nanobody H11-D4 of SARS-CoV-2 fused to the C-terminus of end-modified human ferritin was expressed in E. coli and silkworm baculovirus expression systems. A large number of nanoparticles were successfully self-assembled in silkworms, while relatively few nanoparticles can be observed in the treated products from E. coli by electron microscopy. Subsequently, the fenobody's expression level and neutralizing activity were then evaluated. The results showed that the IC50 of H11-D4 and fenobody Fe-H11-D4 expressed in E. coli were 171.1 nmol L-1 and 20.87 nmol L-1, respectively. However, the IC50 of Fe-HD11-D4 expressed in silkworms was 1.46 nmol L-1 showing better neutralization activity. Therefore, fenobodies can be well self-assembled in silkworm baculovirus expression system, and ferritin self-assembly technology can effectively improve nanobody neutralization activity.

A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.

Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

Characterization of cleavage patterns and assembly of N-terminally modified GII.6 norovirus VP1 proteins.

When expressed in vitro, the major capsid protein VP1 of a norovirus (NoV) can self-assemble into virus-like particles (VLPs), and its N-terminus can tolerate foreign sequences without the assembly being affected. We explored the effects of adding an N-terminal sequence to the VP1 of a GII.6 NoV strain on its cleavage and assembly. Sequences of varying lengths derived from the minor capsid protein VP2 were added to the VP1 N-terminus. Using a recombinant baculovirus expression system, the fusion proteins were expressed, and their cleavage patterns and assembly were analyzed using mass spectrometry and transmission electron microscopy, respectively. All of the fusion proteins were successfully expressed and exhibited varying degrees of enzyme cleavage, most probably at the N-terminus. LC-MS results revealed that similar fragments were obtained for wild-type VP1 and fusion proteins, indicating that the cleavage sites were conserved. EM analysis indicated that VLPs of different sizes were successfully assembled for certain fusion proteins. The study data demonstrate that NoV VP1 can tolerate foreign sequences of a certain length at its N-terminus and that a conserved cleavage pattern exists, which might facilitate further investigation of the assembly and cleavage mechanisms of NoV.

Comparing ATPase activity of ATP-binding cassette subfamily C member 4, lamprey CFTR, and human CFTR using an antimony-phosphomolybdate assay.

Introduction: ATP-binding cassette (ABC) transporters use the hydrolysis of ATP to power the active transport of molecules, but paradoxically the cystic fibrosis transmembrane regulator (CFTR, ABCC7) forms an ion channel. We previously showed that ATP-binding cassette subfamily C member 4 (ABCC4) is the closest mammalian paralog to CFTR, compared to other ABC transporters. In addition, Lamprey CFTR (Lp-CFTR) is the oldest known CFTR ortholog and has unique structural and functional features compared to human CFTR (hCFTR). The availability of these evolutionarily distant orthologs gives us the opportunity to study the changes in ATPase activity that may be related to their disparate functions. Methods: We utilized the baculovirus expression system with Sf9 insect cells and made use of the highly sensitive antimony-phosphomolybdate assay for testing the ATPase activity of human ABCC4 (hABCC4), Lp-CFTR, and hCFTR under similar experimental conditions. This assay measures the production of inorganic phosphate (Pi) in the nanomolar range. Results: Crude plasma membranes were purified, and protein concentration, determined semi-quantitatively, of hABCC4, Lp-CFTR, and hCFTR ranged from 0.01 to 0.36μg/μL. No significant difference in expression level was found although hABCC4 trended toward the highest level. hABCC4 was activated by ATP with the equilibrium constant (Kd) 0.55 ± 0.28mM (n = 8). Estimated maximum ATPase rate (Vmax) for hABCC4 was about 0.2nmol/μg/min when the protein was activated with 1mM ATP at 37°C (n = 7). Estimated maximum ATPase rate for PKA-phosphorylated Lp-CFTR reached about half of hCFTR levels in the same conditions. Vmax for both Lp-CFTR and hCFTR were significantly increased in high PKA conditions compared to low PKA conditions. Maximum intrinsic ATPase rate of hABCC4 in the absence of substrate was twice that of hCFTR when activated in 1mM ATP. Conclusion: The findings here suggest that while both ABCC4 and hCFTR bear one consensus and one degenerate ATPase site, the hCFTR exhibited a reduced intrinsic ATPase activity. In addition, ATPase activity in the CFTR lineage increased from Lp-CFTR to hCFTR. Finally, the studies pave the way to purify hABCC4, Lp-CFTR, and hCFTR from Sf9 cells for their structural investigation, including by cryo-EM, and for studies of evolution in the ABC transporter superfamily.

Porcine interferon-α linked to the porcine IgG-Fc induces prolonged and broad-spectrum antiviral effects against foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is an economically important disease, and the FMD virus (FMDV) can spread rapidly in susceptible animals. FMD is usually controlled through vaccination. However, commercial FMD vaccines are only effective 4–7 days after vaccination. Furthermore, FMDV comprises seven serotypes and various topotypes, and these aspects should be considered when selecting a vaccine. Antiviral agents could provide rapid and broad protection against FMDV. Therefore, this study aimed to develop a fusion protein of consensus porcine interferon-α and Fc portion of porcine antibody IgG (poIFN-α-Fc) using a baculovirus expression system to develop a novel antiviral agent against FMDV. We measured the antiviral effects of the poIFN-α-Fc protein against FMDV and the enhanced duration in vitro and in vivo. The broad-spectrum antiviral effects were tested against seven FMDV serotypes, porcine reproductive and respiratory syndrome virus (PRRSV), and bovine enterovirus (BEV). Furthermore, the early protective effects and neutralizing antibody levels were tested by co-injecting poIFN-α-Fc and an FMD-inactivated vaccine into mice or pigs. Sustained antiviral effects in pig sera and mice were observed, and pigs injected with a combination of the poIFN-α-Fc and an inactivated FMD vaccine were protected against FMDV in a dose-dependent manner at 2- and 4-days post-vaccination. In addition, combined with the inactivated FMD vaccine, poIFN-α-Fc increased the neutralizing antibody levels in mice. Therefore, poIFN-α-Fc is a potential broad-spectrum antiviral and adjuvant candidate that can be used with inactivated FMD vaccines to protect pigs against FMDV.

A porcine circovirus type 2d-based virus-like particle vaccine induces humoral and cellular immune responses and effectively protects pigs against PCV2d challenge.

The pathogenic porcine circovirus type 2 (PCV2) leads to significant economic losses in pig production. PCV2d is currently the dominant genotype causing porcine circovirus-associated disease (PCVAD) worldwide. Therefore, development of a recombinant PCV2d-based vaccine is required to elicit complete protection against PCV2d infection. In this study, we generated virus-like particles of PCV2d-based capsid protein (Bac-2dCP) using a baculovirus expression system and evaluated its protective efficacy against PCV2d infection in specific pathogen-free (SPF) pigs. Three-week-old SPF miniature pigs were intramuscularly immunized with purified Bac-2dCP and intranasally challenged with PCV2d at 4 weeks post-vaccination. The Bac-2dCP group showed significantly higher IgG levels and neutralizing antibodies against PCV2b and PCV2d genotypes, as well as increased interferon-γ levels, and increased body weight and average daily weight gain compared with positive (challenged) and negative (unchallenged) controls. In particular, the Bac-2dCP group showed almost complete absence of PCV2d DNA in serum, nasal, and rectal swabs and in lung, lymph node, and kidney tissue samples. However, the positive control group exhibited low levels of neutralizing antibody, and high levels of PCV2 DNA in serum, swab, and tissue samples, resulting in PCV2-associated pathological lesions. The results of this study demonstrated that a recombinant Bac-2dCP vaccine conferred complete protection against a PCV2d challenge in SPF miniature pigs.

Expression of recombinant Florida clade 2 hemagglutinin in baculovirus expression system: A step for subunit vaccine development against H3N8 equine influenza virus.

Equine influenza (EI) is a transmissible viral respiratory sickness of the Equidae family. Two viruses, H7N7 and H3N8 caused EI; however, H7N7 has not been detected for decades. H3N8 has circulated and bifurcated into Eurasian and American lineages. The latter subsequently diversified into Kentucky, South America, and Florida sub-lineages. Florida clade 1 (FC1) and Florida clade 2 (FC2) strains are the only circulating EI viruses (EIVs) in the meantime. Immunization is considered the major means for the prevention and control of EI infection. Using disparate technologies and platforms, several vaccines have been developed and commercialized. According to the recommendations of the World Organization for Animal Health (WOAH), all commercial vaccines shall comprise representatives of both FC1 and FC2 strains. Unfortunately, most of the commercially available vaccines were not updated to incorporate a representative of FC2 strains. The purpose of this research was to develop a new EI vaccine candidate that incorporates the hemagglutinin (HA) antigen from the currently circulating FC2. In this study, we report the expression of the full-length recombinant HA gene of FC2 in the baculovirus expression system. The HA recombinant protein has been proven to maintain its biological characteristics by hemadsorption (HAD) and hemagglutination tests. Moreover, using a reference-specific serum, the specificity of the HA has been confirmed through the implementation of immunoperoxidase and western immunoblotting assays. In conclusion, we report the expression of specific biologically active recombinant HA of FC2, which would act as a foundation for the generation of an updated EI subunit or virus vector vaccine candidates.

Overexpression of Endoplasmic Reticulum Proteins from Arabidopsis thaliana in Baculovirus.

The overproduction of proteins of the endoplasmic reticulum (ER) of plant cells in prokaryotic heterologous gene expression system remains a technical challenge. Recent advances in genetically modified insect cell technology and virus engineering methods have paved the way to produce recombinant ER plant proteins, including those harboring posttranslational modifications, and therefore, to yield ER plant proteins that are natively folded and fully functional. The present contribution focuses on the baculovirus-expression system flashBAC, which overcomes certain technical hurdles found in other insect cell-based expression systems such as the generation of a bacmid and the negative selection of recombinant clones.

Exploring the surface epitope and nuclear localization analysis of porcine circovirus type 3 capsid protein

Porcine circovirus 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, impacting global pig populations. Porcine circoviruses contain two major open reading frames (ORFs), and the ORF2 encodes the viral capsid protein (Cap). Cap is the most antigenic structural protein and an ideal candidate for the development of vaccines and diagnostic reagents. This study generated a monoclonal antibody (MAb) specific to PCV3 Cap, MAb CCC160, for diagnosis and pathogenesis studies of this novel virus. The MAb specifically recognized PCV3-infected swine lymph node tissue in an immunohistochemical analysis confirming its clinical diagnostic potential. In addition, a novel linear B-cell epitope recognized by MAb CCC160 was identified at the amino acid region 120–134 of Cap. Nuclear localization analysis of PCV3 Cap revealed a potential nuclear localization signal (NLS) in the middle region (aa 131–143) in addition to the dominant N-terminal NLS that is already known. A cell viability assay further demonstrated that the cytotoxicity of PCV3 Cap is correlated with its nuclear localization, indicating a crucial role of Cap in the pathogenic mechanism of PCV3. A full-length construct of PCV3 Cap was successfully expressed using a baculovirus expression system and purified recombinant proteins self-assembled into virus-like particles (VLPs). The protein constitution of the VLPs was confirmed by MAb CCC160 recognition, indicating the correct conformation and specificity of VLP and exhibiting the linear epitope aa 120–134 on the VLP surface. These results provide insights for developing diagnostic tools and potential VLP vaccines for PCV3, revealing its pathogenesis and antigenic properties.

Protective effects of a novel chimeric virus–like particle vaccine against virulent NDV and IBDV challenge

Newcastle disease (ND) and infectious bursal disease (IBD) pose significant threats to the chicken industry, causing substantial economic losses. Currently, immunization through vaccination is the most effective strategy to prevent ND and IBD but currently used traditional vaccines, including inactivated or attenuated vaccines, face challenges in achieving a balance between immunogenicity and safety. To develop a green and efficient novel vaccine for ND and IBD, we developed a bivalent chimeric virus-like particle vaccine (ND-IBD cVLPs) displaying the ND virus (NDV) HN protein and the IBD virus (IBDV) VP2 protein based on the ND VLPs carrier platform and insect baculovirus expression system. This study aimed to evaluate the immunogenicity and protective efficacy of ND-IBD cVLPs in specific pathogen-free chickens. Chickens were immunized with 50 µg of purified ND-IBD cVLPs at 7 days old, boosted at 21 days old, and challenged at 42 days old. The results demonstrated that ND-IBD cVLPs stimulated highly effective hemagglutination inhibition antibody levels against NDV HN protein and enzyme-linked immunosorbent assay antibody levels against the IBDV VP2 protein. Furthermore, ND-IBD cVLPs provided complete protection against virulent NDV and IBDV challenges and mitigated pathological damage to the lung caused by NDV infection and the bursa of Fabricius caused by IBDV infection. These findings suggest that ND-IBD cVLPs hold promise as a safe and efficient novel vaccine candidate for the effective prevention of ND and IBD, extending the development of a foreign protein delivery platform of ND VLPs.

Protective Humoral Immune Response Induced by Recombinant Virus-like Particle Vaccine Expressing Leishmania donovani Surface Antigen.

To date, Leishmania spp. vaccine studies have mainly focused on cellular immunity induction, which plays a crucial role in host protection. In contrast, vaccine-induced humoral immunity is largely neglected. Virus-like particle (VLP) vaccines generated using the baculovirus expression system are well-known inducers of humoral immunity and would serve as a suitable platform for evaluating humoral immunity-mediated protection against visceral Leishmaniasis. In this study, we investigated the humoral immunity evoked through VLPs expressing the L. donovani promastigote surface antigen (PSA-VLPs) and assessed their contribution to protection in mice. PSA-VLPs vaccines were generated using the baculovirus expression system and used for mouse immunizations. Mice were intramuscularly immunized twice with PSA-VLPs and challenged with L. donovani to confirm vaccine-induced protective immunity. PSA-VLP immunization elicited parasite-specific antibody responses in the sera of mice, which were induced in a dose-dependent manner. B cell, germinal center B cell, and memory B cell responses in the spleen were found to be higher in vaccinated mice compared to unimmunized controls. PSA-VLP immunization diminished the production of pro-inflammatory cytokines IFN-γ and IL-6 in the liver. Overall, the PSA-VLPs conferred protection against L. donovani challenge infection by reducing the total parasite burden within the internal organs. These results suggest that PSA-VLPs induced protective immunity against the L. donovani challenge infection.

A streamlined, automated workflow to screen and triage large numbers of baculoviruses for protein expression

The baculovirus expression system is a powerful and widely used method to generate large quantities of recombinant protein. However, challenges exist in workflows utilizing either liquid baculovirus stocks or the Titerless Infected-Cells Preservation and Scale-Up (TIPS) method, including the time and effort to generate baculoviruses, screen for protein expression and store large numbers of baculovirus stocks. To mitigate these challenges, we have developed a streamlined, hybrid workflow which utilizes high titer liquid virus stocks for rapid plate-based protein expression screening, followed by a TIPS-based scale-up for larger protein production efforts. Additionally, we have automated each step in this screening workflow using a custom robotic system. With these process improvements, we have significantly reduced the time, effort and resources required to manage large baculovirus generation and expression screening campaigns.

A blocking ELISA based on virus-like nanoparticles chimerized with an antigenic epitope of ASFV P54 for detecting ASFV antibodies

African swine fever virus (ASFV) is a highly lethal pathogen of domestic and wild pigs. Due to no vaccines or drugs available, early accurate diagnosis and eradication of infected animals are the most important measures for ASFV prevention and control. Bluetongue virus (BTV) core-like particles (CLPs) are non-infectious hollow nanoparticles assembled from the BTV VP3 and VP7 proteins, which could be used as a platform for presenting foreign epitopes. In this study, the secondary structure of BTV VP7 protein was analyzed and predicted using the IEDB Analysis resource. Based on the prediction results of the VP7 protein, the chimeric CLPs with an ASFV P54 epitope were successfully prepared through the BAC-to-BAC baculovirus expression system and sucrose gradient centrifugation. Based on the chimeric CLPs and mAb 2E4 against AFSV P54 epitope, a blocking ELISA for detecting AFSV antibodies was established, and its reaction conditions were optimized. Through comprehensive evaluation of the method, the results showed the chimeric CLPs-based blocking ELISA displayed the best detection performance, with an AUC of 0.9961, a sensitivity of 97.65%, and a specificity of 95.24% in ROC analysis. Compared with western blot and a commercial c-ELISA for detecting anti-ASFV antibodies, this method had an excellent agreement of 96.35% (kappa value = 0.911) and 97.76% (kappa value = 0.946) with the other tests, respectively. This ELISA also had high repeatability, with CV < 10%, and no cross-reaction with the serum antibodies against other swine viruses or Orbivirus. In brief, this was the first report on developing a blocking ELISA based on virus-like nanoparticles chimerized with an antigenic epitope of ASFV P54 for serological diagnosis of ASFV.

UV-C irradiation as an effective tool for sterilization of porcine chimeric VP1-PCV2bCap recombinant vaccine

Ultraviolet irradiation is an effective method of virus and bacteria inactivation. The dose of UV-C light necessary for baculovirus inactivation by measurement of fluorescent GFP protein produced by baculovirus expression system after the irradiation of baculovirus culture in doses ranging from 3.5 to 42 J/m2 was determined. At a dose of 36.8 J/m2, only 0.5% of GFP-expressing cells were detected by flow cytometry and confocal microscopy. The stability of purified VP1-PCV2bCap protein produced by baculovirus expression system was analyzed after the irradiation at doses ranging from 3.5 to 19.3 J/m2. Up to the dose of 11 J/m2, no significant effect of UV-C light on the stability of VP1-PCV2bCap was detected. We observed a dose-dependent increase in VP1-PCV2bCap-specific immune response in BALB/c mice immunized by recombinant protein sterilized by irradiation in dose 11 J/m2 with no significant difference between vaccines sterilized by UV-C light and filtration. A substantial difference in the production of VP1-PCV2bCap specific IgG was observed in piglets immunized with VP1-PCV2bCap sterilized by UV-C in comparison with protein sterilized by filtration in combination with the inactivation of baculovirus by binary ethylenimine. UV-C irradiation represents an effective method for vaccine sterilization, where commonly used methods of sterilization are not possible.

Gene Polymorphisms and Drug-Drug Interactions Determine the Metabolic Profile of Blonanserin.

This study aimed to evaluate the effects of cytochrome P450 3A4 (CYP3A4) gene polymorphism and drug interaction on the metabolism of blonanserin. Human recombinant CYP3A4 was prepared using the Bac-to-Bac baculovirus expression system. A microsomal enzyme reaction system was established, and drug-drug interactions were evaluated using Sprague-Dawley rats. Ultra-performance liquid chromatography-tandem mass spectrometry was used to detect the concentrations of blonanserin and its metabolite. Compared with wild type CYP34A, the relative clearance of blonanserin by CYP3A4.29 significantly increased to 251.3%, while it decreased notably with CYP3A4.4, 5, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 23, 24, 28, 31, 33, and 34, ranging from 6.09% to 63.34%. Among 153 tested drugs, nimodipine, felodipine, and amlodipine were found to potently inhibit the metabolism of blonanserin. Moreover, the inhibitory potency of nimodipine, felodipine, and amlodipine varied with different CYP3A4 variants. The half-maximal inhibitory concentration and enzymatic kinetics assay demonstrated that the metabolism of blonanserin was noncompetitively inhibited by nimodipine in rat liver microsomes and was inhibited in a mixed manner by felodipine and amlodipine in both rat liver microsomes and human liver microsomes. When nimodipine and felodipine were coadministered with blonanserin, the area under the blood concentration-time curve (AUC)(0-t), AUC(0-∞), and C max of blonanserin increased. When amlodipine and blonanserin were combined, the C max of blonanserin C increased remarkably. The vast majority of CYP3A4 variants have a low ability to catalyze blonanserin. With combined administration of nimodipine, felodipine, and amlodipine, the elimination of blonanserin was inhibited. This study provides the basis for individualized clinical use of blonanserin. SIGNIFICANCE STATEMENT: The enzyme kinetics of novel CYP3A4 enzymes for metabolizing blonanserin were investigated. Clearance of blonanserin by CYP3A4.4, 5, 7-10, 12-14, 16-18, 23-24, 28, 31, 33, and 34 decreased notably, but increased with CYP3A4.29. Additionally, we established a drug interaction spectrum for blonanserin, in which nimodipine, felodipine, and amlodipine kinetics exhibited mixed inhibition. Moreover, their inhibitory potencies decreased with CYP3A4.4 and 5 compared to CYP3A4.1. This study provides essential data for personalized clinical use of blonanserin.

Development and application of classical swine fever virus monoclonal antibodies derived from single B cells

Vaccination with E2 subunit vaccines is currently the main measure to control classical swine fever virus (CSFV), which is an endemic disease, and detection of antibodies against CSFV E2 is the most effective way to evaluate herd immunity. In the present study, the E2 protein was expressed by a baculovirus expression system, and two monoclonal antibodies (mAbs), namely, 3A9 and 4F7, were successfully produced using techniques for the isolation of single B cells from splenocytes from mice immunized with the E2 protein. Moreover, two linear B-cell epitopes, 25GLTTTWKEYSHDLQL39 and 259GNTTVKVHASDERGP273, reactive to 3A9 and 4F7, respectively, were identified using epitope mapping of the E2 protein. In addition, the diagnostic performance of the two mAbs was evaluated using blocking enzyme-linked immunosorbent assay (bELISA), and the results showed that the two mAbs had high diagnostic specificity (96.08%, 94.38%) and diagnostic sensitivity (97.49%, 95.97%). Together, these findings identify two ideal candidate peptides and matching mAbs for a new method of CSFV diagnosis, which will contribute to the control and eradication of classical swine fever.

Involvement of transcriptional co-activator p300 in upregulated expression of HSP70 by aquareovirus non-structural protein NS31

Members of Aquareovirus genus, including grass carp reovirus (GCRV), contribute to a serious threat to aquaculture animals accompanied by stress response. Our previous reports revealed that GCRV nonstructural protein NS31 serves as a potent contributor for virus selectively up-regulating specific heat shock protein 70-kd gene（HSP70），however，the mechanism by which inducing HSP70 gene expression is unknown. In this study, we further found that either the N- or C-terminal domain of GCRV NS31 is responsible for enhancing fish HSP70 promoter transcription, and recombinant NS31 protein purified from baculovirus expression system seems to not directly bind HSP70 basic promoter in vitro by an electrophoretic mobility shift assay. However, the transcriptional co-activator p300 was identified as a potential interacting partner for NS31 by pull-down assay. Moreover, knock-down of p300 or addition of p300 inhibitor resulted in obviously reduced HSP70 expression by NS31 or GCRV infection suggesting that the well-characterized heat-shock-responsive HSF1/p300 transcriptional complex might involve in the induction of HSP70. These results collectively reveal this aquareovirus generates cell stress response through its nonstructural protein NS31 recruiting transcriptional co-activator p300.