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

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

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

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

To enhance the immuogenicity of DNA vaccines expressing the GP5 protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the tegument protein VP22 (encoded by VP22 gene) of Bovine Herpesvirus 1 (BHV-1), which has been demonstrated to exhibit the unusual protein transduction property, was fused to N-terminus of GP5 of DNA vaccine construct pCI-ORF5M, resulting in pCI-VP22-ORF5M expressing VP22-GP5 fusion protein. The expression of VP22-GP5 fusion protein was confirmed by both indirect immunofluorescence assay (IFA) and Western blot. To investigate its immunogenicity, BALB/c mice were immunized with the fusion expression plasmid pCI-VP22-ORF5M and non-fusion expression plasmid pCI-ORF5M, respectively. The GP5-specific ELISA antibodies, neutralizing antibodies and lymphocyte proliferative responses were evaluated at various time points after primary immunization. The results showed that GP5-specific ELISA antibodies, neutralizing antibodies, and lymphocyte proliferative responses induced by DNA vaccine pCI-VP22-ORF5M were higher significantly than those of DNA vaccine pCI-ORF5M, indicating that fusion expression with BHV-1 VP22 significantly enhances the immuogenicity of DNA vaccine expressing the PRRSV GP5 protein, and that this strategy may also be useful to develop more efficient DNA vaccines against other pathogens.
Animals ; Antigens, Viral ; genetics ; immunology ; Artificial Gene Fusion ; Female ; Mice ; Mice, Inbred BALB C ; Porcine Reproductive and Respiratory Syndrome ; prevention & control ; Random Allocation ; Vaccines, DNA ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural Proteins ; genetics ; Viral Vaccines ; 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

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

The major antigen region of E2 gene of Hog Cholera Prevalent Strain (Guangxi Yuling Strain) and Chinese Hog Cholera Lapinised Virus (C-strain) derived from hog and rabbit spleen tissue, was amplified by reverse transcription polymerase chain reaction(RT-PCR) and the nested Polymerase Chain Reaction (nPCR). After the amplified fragments were cloned into the expression vector pPROEX-HTb, the recombinant plasmids pPROEX-GXYL and pPROEX-C were obtained. The insert position, the size and the reading frame were right by PCR, restriction digestion and the sequence analysis. SDS-PAGE indicated that both of the reciepient germs transducted and induced by the recombinant plasmids pPROEX-GXYL and pPROEX-C could express the major antigen region of E2 gene. Western-blot indicated that the expressed antigen protein could be recognized by the positive serum of CSFV.
Blotting, Western ; Cloning, Molecular ; Escherichia coli ; genetics ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology

Pseudorabies virus (PRV), an alpha-herpesvirus, has been used as a vector for live-viral animal vaccines. The recombinant PRV TK- / gE- / GP5+, which expressing GP5 of PRRSV, is developed based on the PRV genetic-depleting vaccine-virus strain, TK- / gE- /LacZ+. However, this strain stimulated poorly the vaccinated animals to produce neutralizing antibodies against PRRSV. In order to develop a booster specific immunized response of the PRV recombinant, the ORF5 gene of PRRSV TK- / gE- / LacZ+ was substituted by a modified ORF5 gene, ORF5m. The resultant recombinant PRV, TK- /gE- / GP5m+, was verified by PCR, Southern blotting and Western blotting. TK- / gE- / GP5m+ and TK- / gE- / GP5+ expressed GP5 proteins were inoculated into balb/c mice to evaluate their immunogenicity. The results demonstrated that the amount of neutralization antibodies and cell-immunity responses induced by TK- / gE- /GP5m+ against PRRSV were higher than that of TK- / gE- / GP5+. This study indicated that the new recombinant PRV expressing the modified GP5m protein is a candidate for the development of bivalent genetic engineering vaccines against PRRSV and PRV.
Animals ; Genetic Vectors ; Herpesvirus 1, Suid ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Viral Envelope Proteins ; biosynthesis ; genetics ; Viral Vaccines ; genetics ; immunology

OBJECTIVETo provide experimental evidence for development of human cytomegalovirus (HCMV) nucleic acid vaccine, HCMV surface protein (gB), membrane protein (pp150), and gB-pp150 fused gene eukaryotic expression vector were constructed.

METHODSgB and pp150 genes were amplified and fused into gB-pp150, then were cloned into pcDNA 3.1 (+) to obtain recombinant expression plasmids pcDNA 3.1 (+) -gB, pcDNA 3.1 (+) -pp150 and pcDNA 3.1 (+) -gB-pp150, which were encapsulated with chitosan. Mouse were vaccinated and the humoral and cell immune response were determined by ELISA, specific proliferative response of plenic lymphocytes.

RESULTSThe gB, pp150 and gB-pp150 fusion gene eukaryotic expression vector were successfully constructed. The antibodies A value induced by pcDNA3.1(+) -gB or pcDNA3.1 (+) -gB-pp150 were much higher than that of pcDNA3.1 (+) (P < 0.01). The IFN-gamma levels induced by pcDNA3.1 (+) -pp150 and pcDNA3.1 (+) -gB-pp150 were significantly higher than that of pcDNA3.1 (+). There are significant diference between the stimulating indexes of pcDNA3.1(+) -pp150 or pcDNA3.1 (+) -gB-pp150 immunized and normal mice.

CONCLUSIONThe DNA vaccine pcDNA3.1 (+) -gB can induce significant humoral immunity response, and pcDNA3.1 (+) -pp150 can induce high cellular immune response, whereas pcDNA3.1 (+) -gB-pp150 can induce both humoral and cellar immune responses in BALB/c mice.

Animals ; Cytomegalovirus ; genetics ; immunology ; Humans ; Immunity, Cellular ; immunology ; Immunization ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; immunology ; Vaccination ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; immunology