Hepatocytes are the primary targets of the hepatitis C virus (HCV). While immunosuppressive roles of HCV core protein have been found in several studies, it remains uncertain whether core protein expressed in hepatocytes rather than in immune cells affects the CD8+ T cell response. In order to transduce genes selectively into hepatocytes, we developed a baculoviral vector system that enabled primary hepatocytes to express a target epitope for CD8+ T cells, derived from ovalbumin (OVA), with or without HCV core protein. Culture of OVA-specific CD8+ T cells with hepatocytes infected with these baculoviral vectors revealed that core protein has no effect on proliferation or apoptosis of CD8+ T cells. Our results suggest that HCV core protein does not exert its suppressive role on the CD8+ T cell immune response through expression in hepatocytes.
Viral Core Proteins/*metabolism ; Ovalbumin/genetics/immunology ; Mice ; Hepatocytes/cytology/*virology ; Green Fluorescent Proteins/genetics ; Genetic Vectors ; Cell Proliferation ; CD8-Positive T-Lymphocytes/*immunology ; Baculoviridae/genetics ; Apoptosis ; Animals

IL-17 Receptor D (IL-17 RD) is a cytokine receptor that mediates IL-17 signaling and plays an important role in responding to the invasion of extracellular pathogens and many inflammatory and autoimmune diseases such as rheumatoid arthritis. In this study we report the generation of a mouse monoclonal antibody against human IL-17 RD. The recombinant human IL-17RD extracellular domain (hIL-17RD-ECD) was produced in the baculovirus expression system and purified from culture medium of sf9 insect cells. The purified protein was used as a T-dependent antigen to immune Balb/C mice. B cells from the spleen of immunized mice were fused with murine cell SP2/0. Hybridoma cell lines were screened for the production of the monoclonal antibody against hIL-17-RD-ECD using ELISA. A hybridoma cell line 1F8 was found to have a high production of the antibody, which was further confirmed for the specificity by both western blot and ELISA analyses. The monoclonal antibody obtained from hybridoma 1F8 was characterized to be IgG1+Kappa subclass. This study provided a base for the further therapeutic application of the antibody on the autoimmune disease including rheumatoid arthritis.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Baculoviridae ; Humans ; Insecta ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Receptors, Interleukin-17 ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.

METHODSThe heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.

RESULTSThe baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.

CONCLUSIONThe recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

Antibodies, Monoclonal ; biosynthesis ; immunology ; Baculoviridae ; genetics ; Hepatitis A virus ; immunology ; Hepatitis Antibodies ; biosynthesis ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; immunology ; Immunoglobulin G ; biosynthesis ; immunology ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Recombinant Proteins ; biosynthesis ; immunology

IL-18, as a polyphonic cytokine, is important in immune response and physiologic function. We designed one pair of primers, amplified the porcine IL-18 gene fused with a C-terminal 6xHistidine tag, and then subcloned into the pFastBacDual of Baculovirus transfer vector and transformed into DH10Bac containing a shuttle vector of Bacmid. After co-transfecting the recombinant plasmid into insect cells, the 18 kDa expressed protein of porcine IL-18 was detected by SDS-PAGE; the specificity of expressed protein was confirmed by Western blotting. The purified porcine IL-18 protein induced obvious proliferation of porcine T lymphocytes in vitro, which indicated that the expression of IL-18 had high biological activity.
Animals ; Baculoviridae ; genetics ; metabolism ; Cells, Cultured ; Genetic Vectors ; genetics ; Insecta ; genetics ; metabolism ; Interleukin-18 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transfection

OBJECTIVETo clone and express avian influenza A virus [A/Hong Kong/482/97(H5N1)] H5 subtype hemagglutinin in baculovirus-insect cell expression system and investigate the antigenicity and bioactivity of the recombinant protein.

METHODSH5 gene of influenza A virus was amplified by PCR. The recombinant bacmid was obtained by cloning the gene to the donor plasmid of pFastBacHTB and transformed into DH10Bac competent cells. The recombinant baculovirus stock was prepared by transfecting the recombinant bacmid DNA into the insect cell line for protein expression after amplification. Immunofluorescene assay (IFA) and Western blotting were performed to identify the antigenicity of the recombinant protein, and hemagglutination assay was used to identify its bioactivity.

RESULTSThe recombinant his-H5 protein was expressed in the insect cells with a relative molecular mass of 64,000, which showed erythrocyte-agglutinating activities with the red blood cells of guinea pig. Western blotting and IFA demonstrated that the recombinant his-H5 could be recognized and bound by standard anti-H5 sera.

CONCLUSIONThe recombinant his-H5 with a post-translation modification is successfully obtained in insect cells, which may provide a potential source for further study of the antigen's biological function and for production of the subunit vaccine or monoclonal antibodies.

Animals ; Baculoviridae ; genetics ; Cell Line ; Erythrocytes ; cytology ; immunology ; Genetic Vectors ; genetics ; Guinea Pigs ; Hemagglutination Tests ; Hemagglutinin Glycoproteins, Influenza Virus ; biosynthesis ; genetics ; immunology ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; immunology ; Spodoptera ; Transfection

The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid. The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion. After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid (bacmid) in DH10Bac competent cells, the colonies were identified by antibiotics and blue-white selection. The rBac-mid-M1-HA was verified by PCR and transfected into S f9 cells to produce recombinant baculovirus (rBac-M1-HA). Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot, demonstrating M1 and HA were co-expressed successfully. This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines.
Animals ; Baculoviridae ; genetics ; metabolism ; Cell Line ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; Spodoptera ; Transfection ; Viral Matrix Proteins ; genetics ; immunology

Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program.
Animals ; Baculoviridae/genetics/*immunology ; Chickens/*virology ; DNA Primers ; Gene Amplification ; HN Protein/genetics/*therapeutic use ; Korea ; Marek Disease/immunology/prevention & control ; Newcastle Disease/immunology/*prevention & control ; Spodoptera/virology ; Vaccines, Synthetic/genetics/therapeutic use ; Viral Vaccines/genetics/therapeutic use

In this study, Recombinant baculoviruses rBac-NF and rBac-NG were generated for expressing F and G proteins Nipah virus (NiV) . The expression of recommbinant G (rNG) and F (rNF) protein in rBac-NF and rBac-NG infected cells were confirmed by western-blot. Both rNG and rNF showed sensitive and specific antigenic reaction to rabbit serum anti-Nipah virus in indirect immunofluorescence detection and indirect ELISA. Immunization with rBac-NF and rBac-NG infected insect cells elicited G ad F protein specific antibody responses in mice. Furthermore, the G ad F specific antibodies could neutralize the infectivity of the VSVdeltaG* F/G, the NiV F and G envelope glycoproteins psudotyped recombinant Vesicular Stomatitis Virus expressing green fluorescence protein. The results demonstrated F and G protein expressed by the recombinant baculoviruses could be safe economic diagnostic antigens for the surveillance and monitoring of NiV and promising subunit vaccines for the prevention of NiV.
Animals ; Antigens, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Nipah Virus ; chemistry ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Recombination, Genetic ; Viral Envelope 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

Hemagglutinin (HA) contains a head domain with a high degree of variability and a relatively conserved stem region. HA is the major viral antigen on the surface of the influenza virus. To define the biologic activities of chimeric HA bearing different head domains and stem regions or their potential use, a HA chimeric gene containing the head domain of the H7 subtype virus and stem region of the H3 subtype virus was modified and expressed using a baculovirus expression vector. Then, the secreted protein was purified and its biologic activities characterized. Approximately 1.4 mg/mL cH7/3 HA could be obtained, and its molecular weight was ≈ 70 kD. The trimer form of cH7/3 protein had hemagglutination activity and could be recognized by specific antibodies. The method described here can be used for further studies on the screening of HA stem-reactive antibodies or the development of vaccines with conserved epitopes.
Antibodies, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Hemagglutination ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Humans ; Influenza Vaccines ; genetics ; immunology ; Influenza, Human ; prevention & control ; virology