OBJECTIVETo observe the regulating effect of hepatitis C virus (HCV) envelop (E) 2 gene immunization-induced immune responses by adenovirus mediated interleukin 12 (IL-12).

METHODSHCV E2 protein was expressed and purified from NIH 3T3 and then used as an antigen to detect antibodies against HCV E2. With 51Cr release, SP2/0 expressing HCV E2 was used as target cell to detect specific cytotoxic T lymphocytes (CTL) response; adenovirus recombined IL-12 was propagated by 293 cell. HCV E2 recombinant and adenovirus recombined IL-12 were injected into the quadriceps femoris muscles and abdominal cavities of 6-8 weeks old BALB/C mice. Sera were collected at 2, 3, and 4 weeks and detected for antibodies for E2. Spleen cells isolated at 4 weeks were analyzed for specific CTL response.

RESULTSIt was found that expression of IL-12 at an undetectable level did enhance HCV E2 gene immunization-induced CTL activity and there was no effect on its hormonal immune response.

CONCLUSIONUsing adenovirus to express interleukin 12 was helpful for regulation of HCV E2 gene immunization-induced immune response. Combined HCV E2 and IL-12 can render a strong anti-HCV CTL activity and may be of use in the development of HCV gene vaccine in the future.

Adenoviridae ; physiology ; Interleukin-12 ; biosynthesis ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; Viral Envelope Proteins ; genetics ; immunology

In this study, three expression vectors encoding unmodified glycoproteins E1 and E2 from H77 (1a), Hebei (1b) and JFH1 (2a) strains were constructed to form pVRC-H77-E1E2, pVRC-HeBei-E1E2 and pVRC-JFH1-E1E2 expressing constructs. The protein expression was confirmed by immunofluorescene assay(IFA) and Western blot. The Lentiviral vector has the ability to package the cellular membrane into pseudo-particles. The plasmid expressing HCV E1-E2 glycoproteins in native form was co-transfected into 293FT cells with a lentiviral packaging plasmid (pHR'CMV delta R8.2)and a self-inactivated (SIN) transfer plasmid (pCS-CG) containing a reporter EGFP gene to produce infectious HCV pseudo-particles(pp). Flow cytometry assays showed that the HCVpp could infect Huh7 and Huh7-CD81, and the infectivity in Huh7-CD81 was about 2-3 times higher than that in Huh7 cells. Meanwhile, HCVpp could neither infect non-liver cells, for example, the 293 cells, nor HepG2 cell . Titration of HCVpp by p24 ELISA assay or infection assay showed that this HCVpp may contain 5-25 ng/mL p24 or 10(4)-10(5) TU (transducing unit)/ ml. An in vitro HCV neutralizing assays based on HCVpp (1a, 1b, 2a) were then established using AP33, a monoclone antibody with cross-neutralizing ability to different HCV strains. The neutralizing ability of the antibodies from HCV infected patients was further studied with this HCVpp system. In summary, three kinds of HCVpp (1a, 1b, 2a subtype) were successfully developed; In vitro HCV neutralizing assays based on HCVpp and SIN lentiviral system were established. This system paves a way for characterization of early steps of HCV infection (host tropisms, receptor binding, membrane fusion, et al. ) or screening anti-HCV drugs (such as inhibitor to virus entry). This system can be further applied to assess the human immune responses in HCV patients or evaluate HCV vaccine candidates.
Hepacivirus ; immunology ; Hepatitis C Antibodies ; immunology ; Hepatitis C, Chronic ; immunology ; Humans ; Neutralization Tests ; Viral Envelope Proteins ; immunology ; Virion ; immunology

E2 gene of classical swine fever virus (CSFV) was cloned into secretory pPIC9K Pichia pastoris expression vector. After being linearized by digestion, the vector was transformed into Pichia pastoris by electroporation to integrate with the genome, the transformants with high copies were screened by G418 and were induced to express with methonal. The results of SDS-PAGE and Western blot demonstrated that the supernatant of the induced P. pastoris culture contained protein E2. The results of the study on the immunological activity indicated that the protein E2 expressed in P. pastoris can elicit animal bodies to produce antibodies against protein E2.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Classical swine fever virus ; genetics ; immunology ; Cloning, Molecular ; Gene Expression ; Pichia ; Rabbits ; Swine ; Viral Envelope Proteins ; genetics ; immunology

Hepatitis C ; diagnosis ; Hepatitis C Antibodies ; blood ; Humans ; Serologic Tests ; Viral Envelope Proteins ; immunology

Rubella virus (RV), one of pathogens in TORCH syndrome, can lead to anisotropy of the fetus, and therefore it is of great significance to screen RV infection among women at child-bearing age. At the present, the screening of RV infection is mainly based on the ELISA method, the specificity of RV antigens is very important for ELISA tests. The antigenicity of RV is closely associated with its structural proteins, including capsid protein C, and envelope glycoproteins E1 and E2, which are important surface antigens of RV. This paper reviews the advances in the studies on the structure features, immunogenicity and recombinant proteins of the three structural proteins.
Antibodies, Viral ; analysis ; Enzyme-Linked Immunosorbent Assay ; Recombinant Proteins ; immunology ; Rubella virus ; genetics ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural Proteins ; genetics ; immunology

Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans and there are no effective therapeutic or prophylactic treatments available. The glycoprotein (GP) of EBOV is a transmembrane envelope protein known to play multiple functions including virus attachment and entry, cell rounding and cytotoxicity, down-regulation of host surface proteins, and enhancement of virus assembly and budding. GP is the primary target of protective immunity and the key target for developing neutralizing antibodies. In this paper, the research progress on genetic structure, pathogenesis and immunogenicity of EBOV GP in the last 5 years is reviewed.
Animals ; Antibodies, Viral ; immunology ; Ebolavirus ; genetics ; immunology ; physiology ; Glycoproteins ; genetics ; immunology ; metabolism ; Hemorrhagic Fever, Ebola ; immunology ; virology ; Humans ; Viral Envelope Proteins ; genetics ; immunology ; metabolism ; Virus Assembly

OBJECTIVETo express a multi-copy specific epitope recombinant protein of herpes simplex virus type 1 (HSV-1) with immuno-reactivity.

METHODSMulti-copy genes with a specific epitope of HSV-1-glycoprotein G 112-127 were constructed by DNA recombination and cloned in E. coli JM109 pGEM-5Zf. The positive recombinants were determined by SDS-PAGE and Western blotting.

RESULTSThe recombinants with 4, 8, 16 and 32 copies of gG 112-127 were obtained. The 8-copy recombinant was expressed by 17.5%, mainly as inclusion body. And it reacted with antiserum HSV-1, but not with antiserum HSV-2.

CONCLUSIONThe HSV-1-gG112-127 recombinant could be used to distinguish HSV-1 and HSV-2 in ELISA.

Antigen-Antibody Reactions ; DNA, Recombinant ; Epitopes ; biosynthesis ; immunology ; Herpesvirus 1, Human ; immunology ; Herpesvirus 2, Human ; immunology ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; Viral Envelope Proteins ; biosynthesis ; immunology

Six recombinant plasmids co-expressing the wild-type GP5 gene or the codon-optimized GP5 gene (containing pan-DR epitope) of porcine reproductive and respiratory syndrome virus (PRRSV) and the E2 gene of classical swine fever virus (CSFV) or the E2 fused with the UL49 of pseudorabies virus (PrV) were constructed based on the suicidal DNA vaccine pSFV1CS-E2 described previously. Expression of GP5 and E2 was confirmed by indirect immunofluorescence assay. The immunogenicity of six plasmids was evaluated in BALB/c mouse model. For the six plasmids, low-level of E2 and GP5 protein specific antibodies could be detected in the sera of the immunized mice. Specific lymphoproliferative responses to the PRRSV or CSFV stimulation were induced in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. Antigen specific IFN-gamma and L-4 secretion was detected in the splenocytes of some immunized mice by cytokine ELSIA. Fusion with the PrV UL49 in the suicidal vaccines induced significantly higher lymphoproliferative responses and cytokine secretion. Taken together, the suicidal DNA vaccines co-expressing GP5 and E2 could induce PRRSV and CSFV specific humoral and cell-mediated immune responses.
Animals ; Antibodies, Viral ; blood ; Antibody Formation ; Cytokines ; blood ; Female ; Immunity, Cellular ; Lymphocytes ; immunology ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Vaccines, DNA ; biosynthesis ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural Proteins ; genetics ; immunology ; Viral Vaccines ; biosynthesis ; immunology

BACKGROUNDTo observe the features of serum specific IgA, IgG, IgM antibodies in the acute phase of hemorrhagic fever renal syndrome (HFRS).

METHODSThe nucleocapsid (NP) protein and glycoproteins (GP) of Hantavirus were expressed by recombinant baculovirus, and used as ELISA antigens to test 61 serial sera of 14 acute phase HFRS patients.

RESULTSSeoul like virus RNA were detected from 11 of 14 patients. An early and strong IgA, IgG and IgM antibody response to recombinant NP (rNP) was observed in almost all HFRS cases. The titers of antibody to rNP was apparently higher than that to Rgp. In the early stage, titer of IgG antibody elevated most drastically among all the three classes of antibodies to rNP, followed by IgM and IgA antibody responses. The elevation trend of IgM and IgA antibodies to rNP stayed nearly at the same level, but the IgA titers to rNP were apparently higher than that of IgM. Among the antibodies to rGP, IgA changed distinctly greater than IgG. The elevation trend of IgM could be found during first week after the onset, and the titers dropped gradually after the second week. IgM antibodies of one case who was viral RNA positive were not detected at early stage, but IgA titers were high. The only severe case of the 14 patients kept the lower IgA, IgG and IgM during the whole acute phase.

CONCLUSIONHFRS patients kept an early and strong humoral response to NP and GPs in acute phase of HFRS.IgA could be used together with IgM to improve the diagnostic accuracy.

Acute Disease ; Adult ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Female ; Hantavirus ; genetics ; immunology ; Hemorrhagic Fever with Renal Syndrome ; immunology ; virology ; Humans ; Immunoglobulin Isotypes ; blood ; Male ; Viral Core Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology

FMDV 2A peptide was introduced as a linker between GP5 and M protein of porcine reproduction and respiratory syndrome virus (PRRSV) to allow automatic self-cleavage the polyproteins. This strategy simultaneously displayed the neutralizing action of GP5 protein and cell-mediated immunity of M protein. We put them into the expression cassette of adenovirus vector. The results of RT-PCR, IFA and Western blotting showed that GP5 and M protein were not only expressed correctly, but also self-cleavaged and assemble heterodimers formation. To detect the advantages of rAd-GP5-2A-M, we also constructed some other recombinant adenoviruses (rAd-GP5, rAd-M and rAd-GP5-M) as control. After inoculated subcutaneously into BALB/c mice, the four recombinant adenoviruses can induce PRRSV-specific antibodies and cell-mediated immune response, but the level of humoral and cell-mediated immune response against PRRSV induced by rAd-GP5-2A-M is the strongest among the four recombinant adenoviruses. All of these suggested that it is possible to develop one multi-gene engineering vaccine utilizing FMDV 2A peptide, and also provided a novel strategy for developing other viral disease vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; Female ; Immunization ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Swine ; Vaccines, Synthetic ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; metabolism ; Viral Matrix Proteins ; genetics ; immunology ; metabolism ; Viral Vaccines ; immunology