Escherichia coli ; genetics ; Humans ; Recombinant Fusion Proteins ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

Cloning, Molecular ; Escherichia coli ; genetics ; Hepacivirus ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

The respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and subfamily Pneumovirinae. The RSV can cause acute infections of the lower respiratory tract in infants. The F gene of the RSV is a conservative gene and varies only slightly in its expression. Few studies focusing on the variability of the F gene have been carried out. F protein (fusion glycoprotein) is a transmembrane glycoprotein that mediates fusion and penetration between the virus and host cells. Neutralizing antibody against the F protein can protect against infection by RSV subtypes A and B. Hence, F protein has become the main target for the development of a monoclonal antibody and vaccine against the RSV. An effective vaccine is not available, so a monoclonal antibody against F protein is now the most important method to reduce the morbidity and severity associated with RSV infection in high-risk children. However, a monoclonal antibody can lead to the production of drug-resistant strains of the RSV. This review focuses on genetic variation of the F gene of the RSV as well as progress in the development of a monoclonal antibody against F protein and a vaccine in the last decade.
Animals ; Antibodies, Monoclonal ; immunology ; Humans ; Respiratory Syncytial Virus Infections ; immunology ; prevention & control ; virology ; Respiratory Syncytial Viruses ; genetics ; immunology ; Viral Fusion Proteins ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

BACKGROUNDTo observe the LMP2 specific cellular and humoral immune responses after immunization with recombinant adenovirus Ad-LMP2 in rhesus.

METHODSThe rhesuses were immunized with Ad-LMP2 through intra muscular injection in three groups, high dosage (4.5 x 10(11) VP/kg), medium dosage (1.5 x 10(11) VP/kg) and low dosage (0.5 x 10(11) VP/kg) groups. They were totally immunized six times at intervals of 5 days. The specific cellular immune responses were tested during the 7th week by ELISPOT after immunization. And the titers of anti-LMP2 antibody were tested by EIA throughout the period of immunization.

RESULTSLMP2 induced specific cellular and humoral immune responses in all three dosage group. The potency of immune responses was related with the dosage of immunization. Higher dosage elicited more potent immune response. Both the neutralizing antibody to adenovirus and anti-LMP2 antibody could be detected from 2 weeks after immunization. They would reach the peak during 3-4 weeks after immunization, then declined during the 7th week after immunization.

CONCLUSIONThe recombinant adenovirus LMP2 could induce specific cellular and humoral responses in rhesus after immunization.

Adenoviridae ; genetics ; Animals ; Antibodies, Viral ; blood ; Antibody Formation ; immunology ; Female ; Immunization ; methods ; Macaca mulatta ; Male ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, DNA ; genetics ; immunology ; Viral Matrix Proteins ; genetics ; immunology ; Viral Vaccines ; 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

In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.
Animals ; Antibodies, Viral ; biosynthesis ; blood ; Chickens ; immunology ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vaccines, Subunit ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; 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

LTB gene fragment was amplified by PCR from plasmid pMDTLT, and a recombinant plasmid pETLTBVP1 was constructed by inserting LTB gene fragment into VP1 gene expression plasmid pETVP1 constructed previously. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The recombinant protein existed in the inclusion body and its molecular weight was about 39 kD proved by SDS-PAGE analysis. Western blotting showed that the fusion protein could be reacted with both anti-FMDV and anti-cholera toxin serum demonstrating the immunoactivity of the fusion protein. Strong immune responses can be induced in mice inoculated with the fusion protein intraperitoneally, and the serum antibody level is higher than that of commercial foot-and-mouth disease vaccines.
Animals ; Antibodies, Viral ; blood ; Bacterial Toxins ; genetics ; immunology ; metabolism ; Capsid Proteins ; genetics ; immunology ; metabolism ; Enterotoxins ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; Female ; Gene Fusion ; genetics ; Mice ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

To construct plasmid of recombinant protein candidate vaccine of respiratory syncytial virus, express it in E. coli, and to investigate its immunogenicity and protective efficacy. A CD8+ T cell epitope from respiratory syncytial virus (RSV) M2 protein F/M2:81 - 95 and the G:125-225 (G1) gene fragments from RSV-G protein containing B cell epitopes were amplified by PCR method and then inserted into the prokaryotic expression vector pET-DsbA after bonding to a linker. The fusion protein DsbA-G1-Linker-F/M2:81-95 (D-G1LF/M2) was expressed successfully in E. coli BL21 (DE3). The product was proved to be RSV-specific by Western-blot. After purified by affinity chromatography on Ni+ Sepharose and renatured by gradient dialysis. D-G1LF/M2 was used to immune BALB/c mice. D-G1LF/M2 induced high anti-D-G1LF/M2 IgG, anti-RSV IgG and neutralizing antibody titers in serum and lung of BALB/c mice, and elicied RSV-specific CTL responses. The IgG subclass distribution revealed that IgG1/IgG2a ratio was 2.66. Viral titration indicated that D-G1LF/M2 could protect BALB/c mice against RSV challenge in lung.
Animals ; Antibodies, Viral ; blood ; immunology ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin G ; blood ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Respiratory Syncytial Virus Infections ; prevention & control ; Respiratory Syncytial Virus Vaccines ; biosynthesis ; genetics ; immunology ; Respiratory Syncytial Virus, Human ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; Viral Fusion Proteins ; genetics ; Viral Proteins ; genetics