Intranasal Immunization With Plasmid PcD85B Elicits Mucosal Immunity In A Murine Model

Objective We aimed to construct a DNA vaccine encoding Mtb Ag85B, then appliedto the C57BL/6 murine model by two different strategies. One is in aplasmid-lipid complex delivered by the intranasal route; the other is aBCG/plasmid-lipid complex prime/boost regime. It would lay the groundworkfor a new tuberculosis vaccine and provide data for a further study atmucosal immunogenicity. Methods 1. The eukaryotic expression plasmid pcD85B was constructed. The recombinantplsmid was transfected into COS-7 cells and the expressed target protein was tested byRT-PCR, ELISA and dot blotting. The C57BL/6 mice were immunized with pcD85B,then the total serum IgG, the proliferation of splenocytes and IFN-γlevel were detectedby MTT and ELISA to analyze the specific immune responses induced by DNAvaccination. 2. C57BL/6 mice were intranasally immunized with pcD85B, then the total serumIgG,IgA,IgG2a,the BALF sIgA and IFN-γ level ,IL-4 level and cytoxicity ofCD8+ T cells were tested by ELISA to analyze the specific immune responsesinduced by DNA vaccination. 3. C57BL/6 mice were vaccined s.c. with 4×105 CFU of BCG, after 14 wk,micewere boosted i.n. with pcD85B-lipid complex. Ten days later, serum was detectedfor the total IgG and IgA and IgG2a isotype, BALF for the sIgA; supernants of spleencells were assayed for the IFN-γ,IL-4 level and cytoxicity response. All of thesementioned were done by ELISA. Results 1. pcD85B was constructed and the inserted target gene was confirmed byrestriction enzyme analysis as well as DNA sequencing. The supernatant of COS-7 cellcultures transfected with pcD85B, showed positive reaction to rAg85B antibodies byELISA. The antibody titer against PPD, the proliferation of antigen-specific lymphocyteand IFN-γ production in DNA vaccination group were higher than those of the controlgroups(P<0.05). 2.Among the intranasally immunized C57BL/6 mice ,group of plasmid-lipidcomplex ①induced local mucosal immunity, the level of BALF antibody sIgA is thehighest(P<0.05).②induced Th1 type cell immunity, IFN-γ production was similar toi.m. group,while was lower than the positive control(P<0.05); IL-4 level under thelower detection limitation(P<0.05) ; the level of serum antibody IgG2a is thehighest(P<0.05).③ total serum antibody IgG level lower than i.m. group and thepositive control(P<0.05). ④ a controversial cytotoxicity responses. Perhaps themethod for this test is unmature,no variance was observed between the vector controland the plasmid groups,only the cytotoxicity responses in the positive control wasvaluable . 3. The BCG/ plasmid-lipid complex prime/boost group was induced a higherantibody titer of BALF sIgA and serum antibody isotype IgA,IgG2a than the BCGcontrol(P<0.05); while the serum antibody IgG was lower; IFN-γ production waslower than the BCG control(P<0.05); IL-4 level under the lower detectionlimitation(P<0.05) ; spleen cells'cytotoxicity responses was a little lower than theBCG control.Conclusions1. Recombinant pcD85B was constructed correctly. Target protein wasexpressed in COS-7 cells. The plasmid could induce specific humoral andcellular immune responses in mice after DNA vaccination.2. pcD85B -lipid complex delivered by the intranasal route could inducemucosal immunity, which is an advantage compared to the i.m. group.but itstotal serum IgG and Th1 type cell immunity couldn't transcend the BCGgroup .3. BCG/ plasmid-lipid complex prime/boost strategy was superior to s.c.BCG, i.m. and i.n. pcD85B regimes;it has a promising future.