Re-engineering of bicistronic plasmid pGPD/IFN to construct fusion gene co-expressing Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of Edwardsiella tarda and Interferon-gamma (IFN-?) gene of Labeo rohita (Hamilton) and its in vitro functional analysis

Edwardsiella septicemia disease in the cultured Indian major carps is caused by the fish pathogen Edwardsiella tarda and it is preventable by DNA vaccination. Here, we tried to develop a bicistronic DNA vaccine pGPD/IFN expressing the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of Edwardsiella tarda and Interferon-gamma (IFN-?) gene of Labeo rohita. The vaccine showed high protective efficiency in our previous studies; however as a limitation of bicistronic construct the expression of gene cloned in second frame (B) is poor. To overcome this limitation we re-engineered the construct and designed a fusion gene co-expressing the GAPDH and IFN-? genes as one frame with an aim to get the optimum expression of both the genes. For this purpose, a fusion insert comprising GAPDH and IFN-? coding sequences was cloned in to pcDNA3.1(+) plasmid vector. The fusion genes' in vitro expression was confirmed in the striped snakehead fish cell line (SSN-1). Successful expression of the re-engineered fusion gene DNA vaccine in the cell line was achieved at 48h post-transfection, which was confirmed by amplifying the expression transcripts of GAPDH and IFN-? genes. Thus, the study concludes that the re-engineered fusion vaccine pcGPD/IFN (pcDNA3.1(+) plasmid having fusion GPD/IFN) is functional and can be effectively utilized to vaccinate rohu (Labeo rohita) as it contains the species-specific immune gene (IFN-?) as an adjuvant

Effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the anti-fertility efficacy of canine zona pellucida glycoprotein 3 (CZP3) DNA vaccine / 中华微生物学和免疫学杂志

Objective To investigate the effects of GM-CSF (granulocyte-macrophage colony-stimulating factor) as an immune adjuvant on the anti-fertility efficacy of canine zona pellucida glycoprotein 3 (CZP3) DNA vaccine.Methods RT-PCR and quantitative real-time PCR were performed to analyze the effects of GM-CSF on the maturation of antigen presenting cells (APCs) and the enrichment of APCs at injection site.Female BALB/c mice were immunized with CZP3 DNA vaccine pcDNA3-CZP3 alone or in combination with genetic adjuvant pcDNA3-GM-CSF by using the method of electrical impulses.ELISA (enzyme-linked immunosorbent assay) was used to detect the levels of IgG and sIgA (secretory IgA) antibodies in serum samples and genital tracts and the levels of IL-4 and IFN-γ in serum samples.MTT method was used to analyze the proliferation of spleen T cells in mice.The binding ability of serum anti-CZP3 antibody to native mouse oocytes was analyzed by indirect immunofluorescence assay.All of the female BALB/c mice were coupled with male mice of the same age six weeks after the first-dose vaccination.Litter size at birth in each group was counted and the differences between different groups were comparatively analyzed.ResultsImmunizing the mice with pcDNA3-CZP3 and GM-CSF significantly promoted the expression of CD80, CD83 and CD86 (P<0.05) and increased the sIgA antibody level in genital tract and IgG level in serum (P<0.01).Moreover, the levels of IL-4 and IFN-γ in serum samples were significantly up-regulated (P<0.05) and the proliferation of spleen T cells was significantly enhanced (P<0.05).Results of the indirect immunofluorescence assay showed that the fluorescence intensity and density on mouse egg surface were positively correlated with the level of antibody in serum.Results of the anti-fertility test suggested that GM-CSF significantly reduced the litter size in mice immunized with pcDNA3-CZP3 vaccine (P<0.05).Conclusion GM-CSF could be used as an effective adjuvant to enhance the anti-fertility efficacy of CZP3 DNA vaccine.

Lectin-Like Oxidized LDL Receptor 1 Mediates the Uptake of the C-Terminal Domain of Hsp70 (A Promising Immune Adjuvant Molecule) and Antigen Peptide Complexes.

Extracellular heat shock protein 70 (Hsp70) is an adjuvant molecule that stimulates the immune system. The C-terminal domain of Hsp70 (C70), without the ATPase domain, is sufficient for antigen cross-presentation. However, the mechanism by which the receptor mediates the uptake of C70–peptide complex remains unclear. We therefore aimed to determine the process by which the receptor mediates the uptake of antigenic peptide-bound C70. Methodology: Hsp70 and C70 individually cloned into pET28a were expressed in Escherichia coli BL21 (DE3) and were purified on Ni-NTA agarose and MonoQ HR5/5. Hsp70 and C70 were labeled with Alexa 555 and Alexa 633, respectively, to detect cellular binding. HEK293 cells stably expressing lectin-like oxidized LDL receptor-1 (LOX 1) and KG-1 human dendritic-like cells were incubated with Alexa-labeled Hsp70 and C70 individually or with C70 and antigenic complexes and were observed using fluorescence microscopy. The affinity of LOX-1 toward Hsp70 and C70 was analyzed by chip assay using surface plasmon resonance, which immobilized LOX-1 ligand recognition domain. Results: HEK293 cells stably expressing LOX-1 and KG-1 cells accepted the C70– peptide and Hsp70–peptide complexes. Anti-LOX-1-neutralizing antibody inhibited the uptake of the C70–peptide complexes by KG-1 cells. The dissociation constant (KD) of C70 toward the LOX-1 extracellular domain, measured by surface plasmon resonance, was 4.02 × 10−7 M and that of the C70–peptide complex was 6.6 × 10−8 M. C70 increased the LOX-1 affinity by forming a complex with the antigen peptide. Conclusion: Our findings suggest that LOX-1 is the primary receptor for the C70–peptide and the Hsp70–peptide complexes. C70 is a promising adjuvant molecule that is internalized via LOX-1. In addition, it is convenient to prepare C70 using an E. coli expression system and C70 is more stable than full-length Hsp70.

Study on Adjuvant Immunoactivity of Polysaccharides from Panax Japonicus / 中国中医药信息杂志

Objective To study the adjuvant immunoactivity of polysaccharides from Panax japonicus by alcohol of different concentrations;To discuss its part with the strongest adjuvant immunoactivity. Methods Polysaccharides from Panax japonicus was sunk with alcohol of different concentrations, and 30%alcohol compound, 60%and 90%alcohol polysaccharide were obtained. Different segments of polysaccharide and OVA protein were injected to mice once a week for three times for immunity. Five days after the last immunity, the mice were executed to collect blood, and the antibody titer was determined. The three parts of alcohol compound were scanned by infrared spectrum to determine the type of polysaccharide preliminarily. Results Compared with the control group, the antibody titer of different segments of polysaccharide obviously increased, especially the polysaccharide sunk by 60%alcohol. Infrared spectrum analysis showed that polysaccharides from Panax japonicus contained pyranose ring structure. Conclusion Polysaccharides from Panax japonicus has significant adjuvant immunoactivity, and polysaccharide sunk by 60%alcohol has the strongest adjuvant effects.

Effect of Dendritic Cell Vaccine Coupled with Immune Adjuvant on the Inhibition of Murine Breast Carcinoma / 中国康复理论与实践

@#ObjectiveTo investigate whether immune adjuvant can enhance the immunity of dendritic cell vaccine against murine breast cancer. Methods4 groups of mice with tumor are injected saline, immume adjuvant, dendritic cell (DC) vaccine and DC vaccine coupled with immune vaccine, respectively. Tumor volume and weight are measured 21 d later.ResultsThe tumor size in the DC vaccine coupled with immune vaccine group was significantly small compared with control group (P=0.001) and the DC vaccine group (P=0.047).ConclusionImmune adjuvant can enhance the immunity of dendritic cell vaccine against murine breast cancer.

Induction of immune responses against rotavirus with liposome coated rotavirus DNA vaccine / 中国免疫学杂志

Objective:To search for the efficient immune adjuvant for rotavirus DNA vaccine by evaluating the efficacy in modifying immune responses to rotavirus.Methods:BALB/c mice were immunized by intramuscular and intranasal routes with 3 doses of naked DNA and liposome-coated DNA vaccine at two-week intervals. Titers of serum IgG and IgA were measured by ELISA.Results:Serum titers of rotavirus specific IgG in naked DNA and liposome-coated DNA vaccine-immunized mice were significantly higher than that of the unimmunized controls. Interestingly, only those mice administrated with liposome-coated DNA vaccine by intramuscular route generated serum rotavirus IgA,and neither by naked plasmid nor by intranasal immunization with DNA-liposome complex.Conclusion:These results indicate that liposome can act as both the vector and adjuvant for DNA vaccine against rotavirus by intramuscular injection,which induces rotavirus-specific IgA in the sera,and may provide partial protection against rotavirus infection.