Analysis of immunogenicity and protective effect of a bivalent DNA vaccine expressing interleukin-2 and an outer membrane protein of Treponema pallidum(Gpd) / 中华皮肤科杂志

Objective To investigate the immune response to and protective effect of a bivalent DNA vaccine expressing interleukin-2(IL-2)and Gpd proteins in New Zealand rabbits.Methods Seventy-two male New Zealand white rabbits were equally and randomly divided into 4 groups to be immunized with recombinant plasmids pcDNA3.1(+)/Gpd-IL-2(pcD/Gpd-IL-2),pcDNA3.1(+)/Gpd(pcD/Gpd),empty plasmid pcDNA3.1(+)(pcD)and phosphate buffered saline(PBS),respectively.Immunization was carried out by intramuscular injection at multiple sites with a 2-week interval for 3 times.On week 10 after the initial immunization,the rabbits were challenged intradermally with T.pallidum(Nichols strain).Enzyme-linked immunosorbent assay(ELISA)was used to quantify the serum level of anti-Gpd antibodies in the rabbits and the level of IL-2 and interferon(IFN-γ)in the supernatant of Gpd protein-stimulated spleen cells from the rabbits at different time pionts.MTT assay was conducted to detect the proliferation response of spleen cells collected from the rabbits on day 0,14,28,140 and 168 after the challenge.Results Compared with pcD and PBS,both the vaccines pcD/Gpd and pcD/Gpd-IL-2 elicited significantly higher levels of anti-Gpd IgG antibodies in rabbits at different time points during the vaccination and infection period,with the titers peaking at 1 ∶ 1024 and 1∶4096,respectively(both P ＜ 0.01).There were also significant differences in the serum levels of anti-Gpd IgG antibodies between the pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits at different time points(all P ＜0.01).The levels of IL-2 in the supematant of spleen cells from pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits on week 8 after the immunization were 110 ± 12.6 and 167 ± 15.7 μg/L respectively,and those of IFN-γwere 225 ± 17.6 and 447 ± 22.4 μg/L respectively,significantly higher than those in that from the other two groups of rabbits(all P ＜ 0.01).Furthermore,an apparent proliferation response was observed in spleen cells from pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits with a higher stimulation index compared with pcD-and PBS-immunized rabbits(all P ＜ 0.01).Dark-field microscopic examination of early-stage infected lesions revealed that pcD/Gpd-IL-2-immunized rabbits had a lower detection rate(17.5％)of Tp from lesions,occurrence of ulcerative lesions(15％)and shorter curing time compared with pcD/Gpd-immunized rabbits.Conclusion The recombinant plasmid pcDNA3.1(+)/Gpd-IL-2 could induce protective humoral and cellular immune response more efficiently in rabbits.

Modulation of protective immunity against herpes simplex virus via mucosal genetic co-transfer of DNA vaccine with beta2-adrenergic agonist

Cholera toxin, which has been frequently used as mucosal adjuvant, leads to an irreversible activation of adenylyl cyclase, thereby accumulating cAMP in target cells. Here, it was assumed that beta2-adrenergic agonist salbutamol may have modulatory functions of immunity induced by DNA vaccine, since beta2-adrenergic agonists induce a temporary cAMP accumulation. To test this assumption, the present study evaluated the modulatory functions of salbutamol co-administered with DNA vaccine expressing gB of herpes simplex virus (HSV) via intranasal (i.n.) route. We found that the i.n. co-administration of salbutamol enhanced gB-specific IgG and IgA responses in both systemic and mucosal tissues, but optimal dosages of co-administered salbutamol were required to induce maximal immune responses. Moreover, the mucosal co-delivery of salbutamol with HSV DNA vaccine induced Th2-biased immunity against HSV antigen, as evidenced by IgG isotypes and Th1/Th2-type cytokine production. The enhanced immune responses caused by co-administration of salbutamol provided effective and rapid responses to HSV mucosal challenge, thereby conferring prolonged survival and reduced inflammation against viral infection. Therefore, these results suggest that salbutamol may be an attractive adjuvant for mucosal genetic transfer of DNA vaccine.

The influence of HBV replication regulator on the immune response induced by HBV DNA vaccine / 中华传染病杂志

Objective To study the influence of HBV replication regulator,enhancer I and Pre- S2,on the immune response of HBV DNA vaccine.Methods DNA fragments of HBsAg,PreS2 HBsAg,HBsAg-enhancer I and PreS2-HBsAg-enhancer I region of HBV were amplified by PCR using the complete genome DNA of HBV adr subtype,and inserted into VR1012 vectors,respective- ly.The recombinant plasmids were transfected into HepG2 cells,and injected into Balb/C mice.The expression of HepG2 cells and the cellular and humoral immune response of mice were tested by cell immuno-chemistry,ELISA and ELISPOT.Results The target protein were expressed by transfected HepG2 cells,enhancer I and Pre-S2 can promote the expression of HBsAg in transfected cells.The HBsAb and the HBsAg specific CTL in inoculated mice were found in the second week after injection, PreS2 but not enhancer I can promote the immune response in inoculated mice.Conclusions When inserted into HBV DNA vaccine,enhancer I and PreS2 can promote the expression of HBsAg in transfected HepG2 cells,PreS2 can promote the immune response in inoculated Balb/C mice.

Determination of host bacterium genomic DNA content in DNA vaccine with digoxigenin-labeled probe / 第二军医大学学报

The ribosomal DNA fragment was amplified with PCR utilizing synthetic primers with host bacteria chromosomal DNA as template. The resultant probes were labeled with Digoxigenin and were applied in the dot blot of DNA vaccine samples. The host bacteria genomic DNA in DNA vaccine against hepatitis B was less than 15 ng/?g. Digoxigenin labeled probes proved sensitive and reliable in determining genomic DNA.

Construction of bicistronic DNA vaccine expressing prostate-specific membrane antigen and granulocyte-macrophage colony-stimulating factor and determination of its activity / 第二军医大学学报

Objective:To construct DNA vaccines expressing prostate-specific membrane antigen(PSMA) and/or granulocyte-macrophage colony-stimulating factor(GM-CSF) and to determine their immunoactivity.Methods: Recombinant plasmids pIRES-PSMA-mGM-CSF,pIRES-PSMA,and pIRES-mGM-CSF were constructed with DNA vaccine vector pIRES.After identified by endonuclease digestion,the above 3 plasmids and blank pIRES vector were used to immunize C56BL/6 mice(n=15).LDH release assay was used to exam the cytotoxicity of cytolytic T lymphocytes in each group.Results: We successfully constructed the above mentioned recombinant plasmids.Mice in pIRES-PSMA-mGM-CSF immunized group had the highest specific cytotoxicity,followed by pIRES-PSMA and pIRES-mGM-CSF immunized groups.The blank pIRES group had the lowest cytotoxicity(P

RESEARCH ADVANCES ON PSEUDORABIES NEW-TYPE VACCINES / 微生物学通报

Pseudorabies is an important infectious disease for many kinds of livestock and wild animals, and causes important economics losses for pig industry. Many kinds of vaccines including attenuated live viruses or inactivated are widely used for vaccination of pigs and other animals. In the present review, research advances on pseudorabies new-type vaccines such as subunit vaccine, DNA vaccine, recombination vaccine, deletion-mutant vaccine is presented and point out the further development of the vaccine.

HBV DNA-based immunization inducing humoral immune response in nomal and HBV tr ansgenic mice / 第二军医大学学报

Objective:To investigate the feasibility of treating chronic HBV infection by inducing spe cific humoral immune response to HBV middle envelope protiens in normal and HBV transgenic mice with HBV DNA based immunization. Methods: The eukaryotic expression vector pCMV S2.S (PS) containing HBV S2.S gene and pc DNA3.0 were used respectively to immunize 5 C57BL/6 normal mice and HBV transgen ic mice. Each mouse was injected intramuscularly with one of those plasmids at t he same dose (100 ?g). Sera of mice were detected for anti HBs, anti preS2, HBsAg and HBeAg with ELISA. Pathological changes of transgenic mice liver were o bserved by microscopy. Results:PS can stimulate immune respons es of anti HBs and anti preS2 in normal and transgenic mice.The appearance of a nti preS2 was 1 2 weeks earlier than that of anti HBs. HBsAg and HBeAg in se ra turned negative 8 weeks after immunization. At the 8th week, hepatocytes show ed extensive granular degeneration and hydropic degeneration. There was no obvious difference in the amount of mononuclear lymphocytes between pre and post gen e immunization. Conclusion: It is showed that specific humoral immune response can be effectively induced by PS after DNA based immunization, and PS seems to be responsible for the disapearance of HBsAg and HBeAg in sera o f HBV transgenic mice. The results provide an evidence for furth er investigation of genetic vaccine in the treatment of chronic HBV infection.