D for 3 min at 2000 rpm and were washed twice with staining-buffer.

D for 3 min at 2000 rpm and were washed twice with staining-buffer. Baseline fluorescence was measured with unstained cells.vaccine co-formulated with PA-MSHA, and Group 8?4 were three-inoculation strategies as delineated in Table 1. Two weeks following the last immunization, mice were euthanized and spleens were collected for analysis of cell-mediated immune responses by ELISPOT assay. HIV Env-specific antibody titers and antibody avidity were measured by ELISA.ELISPOT assayThe ELISPOT assay for HIV-1 Env-specific T-cell responses was ��-Sitosterol ��-D-glucoside biological activity carried out according to the protocol provided by the manufacturer with minor modifications (BD ELISPOT Mouse IFN-c ELISPOT Set and IL-2 ELISPOT Set, BD, San Diego, CA). Briefly, 96-well plates were coated at 4uC overnight with 10 mg/ml of anti-mouse IFN-c or IL-2 in sterile PBS. The plates were washed four times with 200 ml/well phosphate buffered saline Tween-20 solution (PBST) and blocked with RPMI-1640 containing 10 fetal bovine serum (FBS) at room temperature for 2 h. Splenocytes were seeded into wells with at 56105 cells/well with 100 ml of envelope (env) peptide (at final concentration of 5 mg/ml) (peptide sequence: C0604200005: CKEVHNVWATHACVPTDPNP, C060420006: SELYKYKVVEIKPLGIAPTA, C0604200007: QQSNLLRAIEAQQHLLQLTV) and incubated in a humidified 5 CO2 incubator at 37uC for 24 h. After incubation, ELISPOT plates were developed according to manufacturer’s instructions. Finally, plates were air-dried, and the spot-forming cells (SFC) were quantified with a Bioreader-4000 automated ELISPOT reader (BioSys, Karben, Germany) and normalized for 106 splenocytes.BMDC endocytosis activityThe BMDC maturation was detected at the endocytosis activity by the take of dextran-FITC (Sigma). 1317923 BMDC was stimulated for 24 h in the absence or presence of PA-MSHA. Then, BMDCs were suspended in staining buffer (1 fetal bovine serum in PBS) with 200 mg/ml FITC-Dextran and incubated in the dark at 4uC for 1 h to assess non-specific binding or at 37uC to assess specific uptake, after which cells were washed extensively with PBS and analyzed by flow cytometry.Immunization of miceThe pGP1455m vaccine was co-formulated with PA-MSHA. Briefly, different concentrations of PA-MSHA (from 102 to 108 CFU/mouse) were premixed with 50 mg DNA vaccine to a final volume of 100 ml each (50 ml for each tibialis anterior muscle) and injected directly. Table 1 shows the immunization timeline and strategies. Six- to eight-week-old female BALB/c mice (Vital River Laboratories) were randomly divided into 14 groups with six mice in each group. Vaccinations for groups 1? were administered intramuscularly twice at a 3-week interval with pGP1455m DNAELISA assay96-well flat-bottom plates (Salmon calcitonin site Costar, Corning, NY) were coated with purified recombinant gp120 protein (final concentration 0.5 mg/ml) in coating buffer (0.012 M Na2CO3 and 0.038 M NaHCO3, pH 9.6) at 4uC overnight. The gp120 (a recombinant protein of HIV-1 CN54 strain) was expressed in 293T cells and purified to 95 purity. Plates were washed five times with phosphate buffered saline Tween-20 solution (PBST), and blocked with 3 bovine serum albumin (BSA) in PBST at 37uC for 1 h. Sera from each mouse group were sequentially diluted (two-fold) with PBST with starting concentration of 1:100, and a 100 ml aliquot of the diluted sera was added to each well. After 2 h incubation at 37uC, the plates were washed five times with PBST and then incubated for 1 h with 1:10000 diluted HRP-labeled goat ant.D for 3 min at 2000 rpm and were washed twice with staining-buffer. Baseline fluorescence was measured with unstained cells.vaccine co-formulated with PA-MSHA, and Group 8?4 were three-inoculation strategies as delineated in Table 1. Two weeks following the last immunization, mice were euthanized and spleens were collected for analysis of cell-mediated immune responses by ELISPOT assay. HIV Env-specific antibody titers and antibody avidity were measured by ELISA.ELISPOT assayThe ELISPOT assay for HIV-1 Env-specific T-cell responses was carried out according to the protocol provided by the manufacturer with minor modifications (BD ELISPOT Mouse IFN-c ELISPOT Set and IL-2 ELISPOT Set, BD, San Diego, CA). Briefly, 96-well plates were coated at 4uC overnight with 10 mg/ml of anti-mouse IFN-c or IL-2 in sterile PBS. The plates were washed four times with 200 ml/well phosphate buffered saline Tween-20 solution (PBST) and blocked with RPMI-1640 containing 10 fetal bovine serum (FBS) at room temperature for 2 h. Splenocytes were seeded into wells with at 56105 cells/well with 100 ml of envelope (env) peptide (at final concentration of 5 mg/ml) (peptide sequence: C0604200005: CKEVHNVWATHACVPTDPNP, C060420006: SELYKYKVVEIKPLGIAPTA, C0604200007: QQSNLLRAIEAQQHLLQLTV) and incubated in a humidified 5 CO2 incubator at 37uC for 24 h. After incubation, ELISPOT plates were developed according to manufacturer’s instructions. Finally, plates were air-dried, and the spot-forming cells (SFC) were quantified with a Bioreader-4000 automated ELISPOT reader (BioSys, Karben, Germany) and normalized for 106 splenocytes.BMDC endocytosis activityThe BMDC maturation was detected at the endocytosis activity by the take of dextran-FITC (Sigma). 1317923 BMDC was stimulated for 24 h in the absence or presence of PA-MSHA. Then, BMDCs were suspended in staining buffer (1 fetal bovine serum in PBS) with 200 mg/ml FITC-Dextran and incubated in the dark at 4uC for 1 h to assess non-specific binding or at 37uC to assess specific uptake, after which cells were washed extensively with PBS and analyzed by flow cytometry.Immunization of miceThe pGP1455m vaccine was co-formulated with PA-MSHA. Briefly, different concentrations of PA-MSHA (from 102 to 108 CFU/mouse) were premixed with 50 mg DNA vaccine to a final volume of 100 ml each (50 ml for each tibialis anterior muscle) and injected directly. Table 1 shows the immunization timeline and strategies. Six- to eight-week-old female BALB/c mice (Vital River Laboratories) were randomly divided into 14 groups with six mice in each group. Vaccinations for groups 1? were administered intramuscularly twice at a 3-week interval with pGP1455m DNAELISA assay96-well flat-bottom plates (Costar, Corning, NY) were coated with purified recombinant gp120 protein (final concentration 0.5 mg/ml) in coating buffer (0.012 M Na2CO3 and 0.038 M NaHCO3, pH 9.6) at 4uC overnight. The gp120 (a recombinant protein of HIV-1 CN54 strain) was expressed in 293T cells and purified to 95 purity. Plates were washed five times with phosphate buffered saline Tween-20 solution (PBST), and blocked with 3 bovine serum albumin (BSA) in PBST at 37uC for 1 h. Sera from each mouse group were sequentially diluted (two-fold) with PBST with starting concentration of 1:100, and a 100 ml aliquot of the diluted sera was added to each well. After 2 h incubation at 37uC, the plates were washed five times with PBST and then incubated for 1 h with 1:10000 diluted HRP-labeled goat ant.