Preparation and immunogenicity evaluation of recombinant poliomyelitis type 2 virus-like particles / 中华微生物学和免疫学杂志

Objective:To express virus-like particles of poliovirus type 2 (PV2-VLP) in insect cells using a recombinant baculovirus expressing P1 and 3CD and to preliminarily evaluate its immunogenicity.Methods:Based on the codon preference of High 5 cells, the sequences of P1 gene and 3CD gene of PV2 were optimized and inserted into pUC57-Amp to construct pUC57-PV2-P1 and pUC57-PV2-3CD. UC57-PV2-P1s mutant that carried P1 gene mutation affecting thermostability was then constructed. Recombinant baculovirus strains of rBac-PV2-P1s-3CD and rBac-PV2-P1-3CD (wild type) were constructed using homologous recombination. The expression of target proteins was detected by Western blot. PV2-VLP was purified by ion exchange chromatography. The structure of VLP was observed under transmission electron microscopy to evaluate the assembly efficiency. The immunogenicity of PV2-VLP was assessed in a rat model.Results:The recombinant baculovirus with stable expression of P1s and 3CD proteins was successfully constructed. Western blot results showed that the yield of VLP was higher after thermostability mutation than that of the wild type. A three-dimensional structure with a diameter of about 30 nm was observed under electron microscopy, indicating that the VLP was successfully assembled. Animal experiment showed that the recombinant PV2-VLP had immunogenicity and could effectively induce the production of neutralizing antibodies.Conclusions:Effective VLP vaccines could be successfully prepared using the insect cell-baculovirus expression system, which provided reference for the development of polio VLP vaccine.

Vaccine containing G protein fragment and recombinant baculovirus expressing M2 protein induces protective immunity to respiratory syncytial virus

PURPOSE: Respiratory syncytial virus (RSV) can cause serious respiratory illnesses such as pneumonia, asthma, and bronchiolitis in infants and elderly or immunocompromised individuals. An RSV vaccine has yet to be developed; only prophylactic anti-RSV antibody is commercially available. So, we investigated whether our vaccine candidate is able to induce type 1 CD4+ T helper (Th1), CD8+ T-cell responses, and protective immunity without vaccine-enhanced disease (VED) against RSV. MATERIALS AND METHODS: We used RSV G protein fragment (Gcf A) with recombinant baculovirus capable of expressing the RSV M2 protein (Bac M2) as a vaccine candidate, and injected this vaccine (Gcf A/Bac M2) intramuscularly, and challenged with RSV intranasally into mice. Enzyme-linked immunosorbent assay, flow cytometry, plaque assay, and weight measurement were performed to confirm humoral immunity, cellular immunity, and protective immunity. RESULTS: The Gcf A/Bac M2 formulation induced a stronger IgG response to Gcf A than Gcf A inoculation alone, and the ratio of IgG1/IgG2a indicated that the responses shifted predominantly to Th1. In addition, both RSV G-specific Th1 responses and RSV M2-specific CD8+ T-cell responses were induced, and G protein-associated eosinophilic infiltration was suppressed compared to the control group. Moreover, the Gcf A/Bac M2 group showed effective protection after an RSV challenge. CONCLUSION: Bac M2 could serve as a vaccine with intrinsic adjuvant activity, and the Gcf A/Bac M2 shows promise as a vaccine candidate for inducing protective immunity without inciting VED.

Indirect ELISA for the Detection of Rabies Virus Antibodies in Dog Sera

Rabies is known as the most fatal disease in all warm-blooded animals, including dogs. Among animals that transmit rabies, dogs are mainly responsible for transmitting animal rabies in Asian countries. Detection of rabies virus (RABV) antibodies in dogs is performed by fluorescent antibody virus neutralization (FAVN) test or rapid fluorescent focus inhibition test. These standard assays are difficult to carry out in diagnostic laboratories without sufficient instruments, designated RABV, and cell culture systems. An alternative assay that is easy to conduct and time efficient is required for rapid sero-surveillance following vaccination. Recombinant baculovirus expressing RABV nucleoprotein (RVN) was constructed and the recombinant protein was purified using Ni-NTA and fast protein liquid column chromatography. We developed and evaluated an indirect enzyme-linked immunosorbent assay (I-ELISA) with recombinant RVN for the detection of RABV antibodies in 122 dog serum samples. The I-ELISA results obtained from these samples were compared with FAVN results. The sensitivity, specificity, and accuracy of I-ELISA were 88.1%, 92.5%, and 91.0%, respectively, compared with FAVN. Results of I-ELISA were significantly correlated with that of FAVN (r = 0.81). These results suggest that I-ELISA with recombinant RVN is useful for sero-surveillance of RABV in dog sera.

Indirect ELISA for the Detection of Rabies Virus Antibodies in Dog Sera

Rabies is known as the most fatal disease in all warm-blooded animals, including dogs. Among animals that transmit rabies, dogs are mainly responsible for transmitting animal rabies in Asian countries. Detection of rabies virus (RABV) antibodies in dogs is performed by fluorescent antibody virus neutralization (FAVN) test or rapid fluorescent focus inhibition test. These standard assays are difficult to carry out in diagnostic laboratories without sufficient instruments, designated RABV, and cell culture systems. An alternative assay that is easy to conduct and time efficient is required for rapid sero-surveillance following vaccination. Recombinant baculovirus expressing RABV nucleoprotein (RVN) was constructed and the recombinant protein was purified using Ni-NTA and fast protein liquid column chromatography. We developed and evaluated an indirect enzyme-linked immunosorbent assay (I-ELISA) with recombinant RVN for the detection of RABV antibodies in 122 dog serum samples. The I-ELISA results obtained from these samples were compared with FAVN results. The sensitivity, specificity, and accuracy of I-ELISA were 88.1%, 92.5%, and 91.0%, respectively, compared with FAVN. Results of I-ELISA were significantly correlated with that of FAVN (r = 0.81). These results suggest that I-ELISA with recombinant RVN is useful for sero-surveillance of RABV in dog sera.

Baculovirus-based Vaccine Displaying Respiratory Syncytial Virus Glycoprotein Induces Protective Immunity against RSV Infection without Vaccine-Enhanced Disease

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract diseases in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV yet. The attachment glycoprotein (G) of RSV is a potentially important target for protective antiviral immune responses. Recombinant baculovirus has been recently emerged as a new vaccine vector, since it has intrinsic immunostimulatory properties and good bio-safety profile. METHODS: We have constructed a recombinant baculovirus-based RSV vaccine, Bac-RSV/G, displaying G glycoprotein, and evaluated immunogenicity and protective efficacy by intranasal immunization of BALB/c mice with Bac-RSV/G. RESULTS: Bac-RSV/G efficiently provides protective immunity against RSV challenge. Strong serum IgG and mucosal IgA responses were induced by intranasal immunization with Bac-RSV/G. In addition to humoral immunity, G-specific Th17- as well as Th1-type T-cell responses were detected in the lungs of Bac-RSV/G-immune mice upon RSV challenge. Neither lung eosinophilia nor vaccine-induced weight loss was observed upon Bac-RSV/G immunization and subsequent RSV infection. CONCLUSION: Our data demonstrate that intranasal administration of baculovirus-based Bac-RSV/G vaccine is efficient for the induction of protection against RSV and represents a promising prophylactic vaccination regimen.

Sequence Analysis and Expression of ORF5 in Korean Isolate of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of reproductive failure and respiratory disorders in pigs. The viral genome consists of eight overlapping open reading frames (ORFs). ORF5 encodes one of the major glycoproteins and is known as an immunologically important structural protein associated with virus neutralization. The ORF5 gene of the Korean PRRSV isolate, CNV-1, was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide and amino acid sequences of CNV-1 ORF5 shared 91% and 83% identity, respectively, with the American isolate (VR2332 strain) and 57% and 49% identity with the European isolate. For the expression and easy purification of ORF5, the cDNA containing the complete ORF5 sequence fused in-frame with sequence encoding glutathione S-transferase (GST) was cloned into a baculovirus transfer vector and transfected into Sf9 cells. The GST-ORF5 fusion protein produced in Sf9 cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Sequencing results confirmed that the recombinant baculovirus from Sf9 cells contains the complete ORF5 gene. Further studies in this direction will address whether ORF5 can be a good candidate for a subunit vaccine against PRRSV in Korea.

Identification of recombinant baculovirus and determination of virus titer with fluorescence quantitative PCR assay / 中国病理生理杂志

AIM: To develop a real - time PCR assay based on TaqMan technology for the identification of recombinant baculovirus and determination of virus physical titers in Bac - to - Bac system. METHODS: The recombinant baculovirus containing human IL- 18 gene was produced using Bac -to- Bac system. A 10 -fold serially diluted primary viral stock was used for plaque assay and DNA extraction. Bacmid (baculovirus plasmid) was 10 -fold serially diluted and served as standards. Real - time PCR amplification of the IL - 18 gene was performed in triplicate for each diluted recombinant virus. At the same time, plaque assays were performed using overlay agarose method. RESULTS: The standard linear range (101 to 108 copies) for quantitation was achieved with the standard curve. We also find that the"vg/mL"titer value is generally about 10 times than"pfu/mL"titer of the same recombinant virus stock. CONCLUSION: A TaqMan real -time PCR method is established to identify the recombinant baculovirus and determine the"vg/mL"titer of virus. The method is rapid and quantitative over a wide range of virus titers.

Study on HBV Gene Replication and Expression of Experimental Hepatitis B Model Using Baculovirus Gene Delivery System

PURPOSE: The lack of reliable in vitro infection systems or convenient animal models has hindered the progress of hepatitis B virus (HBV) research and the development of new treatment options. We established an in vitro model of hepatitis B, using recombinant HBV encoding baculovirus, which provided HBV replication and antigens expression in HepG2 cells. The objectives of this study were to characterize the magnitude of HBV expression and the level of replication obtainable in HepG2 cells, to establish the optimum infection and culture conditions of HBV expression and replication. METHODS: Replication of a competent HBV genome encoding the baculovirus, RC-HBV-Bac, was generated for delivering the HBV genome to HepG2 cells. HBV replication and antigens expression were determined in relation to the infection and culture conditions. RESULTS: In RC-HBV-Bac infected HepG2 cells, HBsAg, HBeAg and HBcAg were expressed in the cytoplasm and nuclei, and secreted into the medium. HBV replication was evidenced by the presence of a replication complex and covalently closed circular (ccc) DNA in the cytoplasmic fraction of infected cells. The level of HBV expression was directly proportional to the multiplication of RC-HBV-Bac infection. Polyethylene glycol was able to enhance the infection efficiency of the baculovirus to HepG2 cells. High levels of HBV replication were achieved under culture conditions supplemented with dimethyl sulfoxide and a low serum concentration. CONCLUSION: This in vitro model of hepatitis B, generated by baculovirus gene delivery, represents a simple and flexible system for the study of HBV replication and drug testing.

Expression of Recombinant Baculovirus Carrying Schistosoma japonicum 26 ku GST in Mammalian Cells / 华中科技大学学报（医学）（英德文版）

In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into Tvector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac,then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24 × 108. Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.

Cloning and Expression of Hemagglutinin-Neuraminidase Gene of a Thermostable Isolate of Newcastle Disease Virus by Baculovirus Recombinants

The hemagglutinin-neuraminidase (HN) gene of a thermostable Newcastle disease virus isolated from the diseased pheasants in Korea was cloned using Baculovirus transfer vector system, constructing pVL-NDHN inserted with HN gene (1.75 kbp). The HN recombinant baculovirus was generated in Sf-9 cells by co-transfection with pVL-NDHN and linearized baculovirus DNA. The Sf-9 cells infected with the recombinant baculovirus showed the hemagglutinating activity for chicken erythrocytes, and specific positive reactions in indirect immunofluorescence and indirect dot immunoassay. By SDS-PAGE and Western blot analysis, the expressed HN protein with the size of 74 kDa was detected in the cells infected with the recombinant baculovirus. To evaluate the immunogenicity of expressed HN protein, the chicken inoculated with the lysates of the Sf-9 cells were examined by hemagglutination inhibition and ELISA tests. The substantial levels of antibody responses were detected in both assays. The HN protein expressed in baculovirus recombinant system could be utilized for the development of diagnostic measures for Newcastle disease in poultry, and these results on HN recombinant baculovirus will expedite the development of recombinant ND vaccines.

Identification of recombination baculovirus and determination of virus titer with fluorescence quantitative PCR assay / 中国免疫学杂志

Objective:To develop a real-time PCR assays based on TaqMan chemistry for the identification of recombinant baculovirus and determination of virus physical titers in Bac-to-Bac system.Methods:The recombinant baculovirus containing human IL-18 gene was produced using Bac-to-Bac system.A 10-fold series diluted primary viral stocks were used for plaque assay and DNA extraction.Bacmid(baculovirus plasmid) was 10-fold series diluted and served as standards.Real-time PCR amplification of the IL-18 gene was performed in triplicate for each diluted recombinant virus.At the same time,plaque assays were performed using overlay agarose method.Results:The standard linear(101 to 108 copies) from quantitation was achieved with the standard curve.We also find that the "vg/ml" titer value is generally about 10 times than "pfu/ml" titer of the same recombinant virus stock.Conclusion:A TaqMan real-time PCR method is established to identify the recombinant baculovirus and determine the "vg/ml" titer of virus.The method is rapid and quantitative over a wide range of virus titers.