To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.
Animals ; Antigens, Viral/genetics* ; Biological Assay ; Chickens/immunology* ; Escherichia coli/genetics* ; Infectious bursal disease virus/immunology* ; Poultry Diseases ; Vaccines, Synthetic/isolation & purification* ; Viral Structural Proteins/immunology* ; Viral Vaccines/immunology*

Rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV), are two pathogens that have harmful effect on rabbit breeding and population decline of European rabbits in their native range, causing rabbit haemorrhagic disease (rabbit fever) and myxomatosis, respectively. The capsid protein VP60 of the RHDV represents the major antigenic protein. To develop a recombinant bivalent vaccine candidate that can simultaneously prevent these two diseases, we used the nonessential gene TK (thymidine kinase) of MYXV as the insertion site to construct a recombinant shuttle vector p7.5-VP60-GFP expressing the RHDV major capsid protein (VP60) and the selectable marker GFP. Then the shuttle vector p7.5-VP60-GFP was transfected into rabbit kidney cell line RK13 which was previously infected with MYXV. After homologous recombination, the recombinant virus expressing GFP was screened under a fluorescence microscope and named as rMV-VP60-GFP. Finally, the specific gene-knock in and expression verification of the vp60 and gfp genes of the recombinant virus was confirmed by PCR and Western blotting. The results showed that these two genes were readily knocked into the MYXV genome and also successfully expressed, indicating that the recombinant MYXV expressing the vp60 of RHDV was generated. Protection against MYXV challenge showed that the recombinant virus induced detectable antibodies against MYXV which would shed light on development of the effective vaccine.
Animals ; Blotting, Western ; Caliciviridae Infections/veterinary* ; Hemorrhagic Disease Virus, Rabbit/immunology* ; Rabbits ; Vaccines, Synthetic/immunology* ; Viral Structural Proteins/genetics*

To reveal the innate immunity of mast cells against recombinant VP1-VP4 protein of foot-and-mouth disease virus (FMDV), mouse peritoneal mast cells (PMCs) were pulsed with recombinant VP1-VP4 protein. The supernatants harvested from PMCs cultures were applied to the high throughput ELISA array. Our results show that the expression levels of CCL19, L-selectin, CCL17, and TNF alpha released from PMCs pulsed with recombinant VP1-VP4 were significantly down-regulated compared with PMCs alone (P<0.001). Surprisingly, in comparison with PMCs alone, the expression levels of CCL19, IL-15, IL-9, G-CSF, and Galectin-1 in PMCs with the mannose receptor (MR) inhibitor were significantly up-regulated (Plt;0.01), and the expression level of IL-10 was also remarkably up-regulated (Plt;0.05). Importantly, the protein expression levels in PMCs treated with MR inhibitor were higher than PMCs pulsed with VP1-VP4, including IL-10, IL-17, CCL20, IL-15, IL-9, L-selectin, CCL17, TNF alpha, and CCL19 (Plt;0.01) as well as CCL21, and G-CSF (Plt;0.05). Differential expression analysis in bioinformatics shows that both L-selectin and CCL17 were recognized as differentially expressed protein molecules (Log2(ratio)≤-1) when compared with PMCs alone. Furthermore, the up-regulation of the expression levels of CCL20, CCL19, L-selectin, and IL-15 in PMCs treated with MR inhibitor was defined as differential expression (Log2(ratio)≥1). These data indicate that PMCs are capable of secreting CCL19, L-selectin, CCL17, and TNF alpha spontaneously and the recombinant VP1-VP4 has an inhibitive potential to PMCs during their performance of innate immune response. Given the protein expression levels from PMCs pre-treated with MR inhibitor were significantly increased, it can be deduced that immunosuppression of FMDV is presumably initiated by the VP1 recognition of MR on mast cells.
Animals ; Capsid Proteins ; immunology ; Cells, Cultured ; Cytokines ; immunology ; Enzyme-Linked Immunosorbent Assay ; Foot-and-Mouth Disease ; Foot-and-Mouth Disease Virus ; Interleukins ; immunology ; Mast Cells ; immunology ; Mice ; Proteome ; immunology ; Recombinant Proteins ; immunology ; Viral Structural Proteins ; immunology

To prepare monoclonal antibodies (mAbs) against structural proteins of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), BALB/c mice were immunized using purified inactivated SFTSV virions as the antigens. Subsequently, hybridoma cell lines that secreted monoclonal antibodies against nucleoprotein (NP) and glycoproteins (GP) were obtained using a hybridoma technique. The antigen specificities of prepared mAbs were examined by indirect immunofluorescence and immunoprecipitation assays. Functional analyses were then performed,including the detection of IFA antibody titers,the levels of neutralizing activity and antibody affinities. After cell fusion and cloning,13 hybridoma cell lines secreted mAbs specifically against SFTSV-GP and 7 hybridoma cell lines secreted mAbs specifically against SFTSV-NP. Immunofluorescence and immunoprecipitation assays showed that the mAbs had high levels of antigen specificity. Among the 13 anti-SFTSV-GP mAbs,6 recognized Gn,whereas the others reacted with Gc. IFA titers of most anti-SFTSV-GP mAbs were between 1,280 and 20,480, and four anti-SFTSV-Gn mAbs showed neutralizing activity. Seven of the obtained anti-SFTSV-NP mAbs reacted specifically with NP,of which the IFA titers ranged from 5,120 to 20,480 with no observed neutralizing activity. Furthermore, two anti-SFTSV-GP mAbs, 1C8 and 1G8, showed high levels of affinity via a non-competitive ELISA. Our study lays the foundation for the development of further diagnostic assays and basic research into SFTSV.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Specificity ; Bunyaviridae Infections ; immunology ; virology ; Female ; Humans ; Hybridomas ; immunology ; Mice ; Mice, Inbred BALB C ; Phlebovirus ; immunology ; Viral Structural Proteins ; immunology

In order to determine immunogenicity and protective effect in chickens, we used the IBDV (Infectious bursal disease virus)-Vp2/Lactobacillus casei as antigen transfer system. First, the immunized and control chickens were challenged by IBDV/DQ at lethal dose to determine the protective ratio. Second, chickens were orallyand intranasally vaccinated twice with 10(9) CFU/mL pLA-VP2/L. casei, pLA/L. casei and PBS as negativecontrol and commercial vaccine as positive control. The bursa injury and the lesion score wererecorded post challenge. The level of specific IgG and sIgA in pLA-VP2/L. casei and positive control groups was significantly higher than that in negativecontrol groups. The protection efficacy in pLA-VP2/L. casei oral group was higher than that inintranasal group. The SI. of pLA-VP2/L. casei oral group was significant higher than other groups. The lesion score indicated the pLA-VP2/L. casei was safer than commercial vaccine for bursa. Collectively, the pLA-VP2/L. casei could be a vaccine candidate for IBDV.
Animals ; Antibodies, Viral ; blood ; Antibody Formation ; Birnaviridae Infections ; prevention & control ; veterinary ; Chickens ; Infectious bursal disease virus ; Lactobacillus casei ; Poultry Diseases ; prevention & control ; Recombinant Proteins ; immunology ; Viral Structural Proteins ; immunology ; Viral Vaccines ; immunology

Infectious bursal disease virus (IBDV) VP4 plays an important role in immunosuppression of host. In order to develop monoclonal antibodies (McAbs) against VP4, we vaccinated BALB/c mice with His-VP4, screened and subcloned positive clones. We established 4 hybridoma cell lines that stably secreted McAbs against VP4 and named these cell lines 3B3, 3H11, 4C8 and 4G6, respectively. We tested the dissociation constant (Kd) of these McAbs, and found that their K(d)s were 4.61 x 10(-11), 1.71 x 10(-10), 4.26 x 10(-11), 5.02 x 10(-11), respectively. The isotypes of these McAbs were determined to be IgG1, IgG1, IgG2b and IgG1. These McAbs specifically bound to VP4 in IBDV infected DF-1 cells as demonstrated by Western blotting analysis and fluorescence antibody assay. These McAbs would help to detect IBDV infection and to analyze the biological activities of IBDV VP4.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Blotting, Western ; Cell Line ; Fluorescent Antibody Technique ; Hybridomas ; Infectious bursal disease virus ; Mice ; Mice, Inbred BALB C ; Viral Structural Proteins ; immunology

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.
Animals ; Antigens, Viral/genetics/metabolism ; Caliciviridae Infections/prevention & control/*veterinary/virology ; Capsid Proteins/*genetics/metabolism ; Cell Culture Techniques/*methods ; Codon/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/veterinary ; *Gene Expression Regulation, Viral ; Hemorrhagic Disease Virus, Rabbit/*genetics/immunology ; *Rabbits ; Recombinant Proteins/genetics/metabolism ; Sf9 Cells ; Spodoptera ; Viral Structural Proteins/*genetics/metabolism ; Viral Vaccines/genetics/immunology

A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry.
Animals ; Antibodies, Viral ; Birnaviridae Infections/prevention & control/*veterinary ; Cells, Cultured ; Chick Embryo ; Chickens ; Fibroblasts/metabolism ; Infectious bursal disease virus/*immunology ; Poultry Diseases/*prevention & control/virology ; Specific Pathogen-Free Organisms ; Vaccinia virus/*genetics/immunology/metabolism ; Viral Structural Proteins/genetics/*immunology/metabolism ; Viral Vaccines/*immunology

To develop a method based on immunoreactions for detection of Ectropis obliqua Nucleopolyhedrovirus (EoNPV), the polyhedra of the virus were purified and used to immunize the mouse BALB/c. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. A hybridoma cell line which can stably secrete the monoclonal antibody against EoNPV was achieved by using indirect ELISA screening and cloning methods, and was named as 7D3. Meanwhile, the polyhedrin gene was cloned from EoNPV and expressed in E. coli. Western blotting analysis showed that the monoclonal antibody prepared from 7D3 could specifically react with the recombinant polyhedrin. An indirect ELISA method based on this monoclonal antibody for detecting EoNPV in infected tea looper was developed.
Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hybridomas ; secretion ; Lepidoptera ; growth & development ; virology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; immunology

Protective immune response of the available IBD vaccine is insufficient to fully protect against the prevailing strain of the infectious bursal disease virus (IBDV). Such a vaccination escape IBDV field isolate idenfied from Anhui province of China in December 2007, where IBD broke out at 2 weeks post vaccination. The IBDV vp2 gene was cloned into pFastBacHTA donor plasmid, followed by generation of the recombinant bacmid DNA pBac-VP2. The latter was used to transfect insect cell Sf9 with Lipofectamine to produce recombinant baculovirus vBac-VP2. The Sf9 cells infected with vBac-VP2 were stained positive against IBDV antibody using the indirect immunofluorescence assay (IFA), which was also confirmed by the detection of IBDV Vp2 protein in the infected Sf9 cells by IBDV sandwich ELISA. Western blotting revealed that the calculated protein of approximately 53 kDa was in the expressed in the insect cells. Moreover, virus-like particles (VLPs) and "inclusion body-like"structure in the infected Sf9 cells were observed under electron microscopy. We further developed an indirect ELISA for the detection of the IBDV antibodies, which was specific and sensitive. In addition, the lysates of vBac-VP2 infected cells was used to immunize 2-week-old SPF chickens, followed by challenging with the virulent IBDV, the survival rate was 30% at 14 days post primary immunization, however, the survival rate was 100% at 14 d after the booster vaccination. The ELISA antibody titers was up to 3.2 x 10(3) and neutralization antibody titer was 2536, significantly higher than those of one-shot vaccination, 8 x 10(2) and 1106, respectively. The immunized chickens did not show any clinical signs and histopathological changes of infection in 7-days trial time. The bursa/body-weight ratios were higher than those of the unimmunized control (P < 0.05). The virus-like-particle recombinant Vp2 protein expressed in insect cells promises to be a novel subunit vaccine and diagnostic reagent candidate for IBDV.
Animals ; Baculoviridae ; genetics ; Cell Line ; Chickens ; Infectious bursal disease virus ; immunology ; Insecta ; genetics ; metabolism ; Poultry Diseases ; prevention & control ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology