Figure 1.
Anchoring of VP1 on the surface of infected Sf9 cells.
(a) Confirmation of the anchoring of VP1 on the plasma membrane of Sf9-II cells infected with Bac-VP1. (b) No anchoring of VP1 was observed on the plasma membrane of Sf9- II cells infected with Bac-wt. The cells were cultured on sterile cover slips and infected at an MOI of 0.1. Cells were fixed with 4% PFA and blocked with 2% bovine serum albumin for 30 min at 37°C. VP1 was detected by a polyclonal primary antibody against bacterially expressed VP1 raised in guinea pig (1∶300 dilution; in house production) followed by a secondary FITC-conjugated rabbit anti-guinea pig mAb (1∶100 dilution; Dako). Plasma membrane staining was done using the CellMask™ Orange plasma membrane stain (Invitrogen).
Figure 2.
Confirmation of VP1 expression in purified baculoviruses by Coomassie staining and Western blotting.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of purified baculoviruses, showing the presence of VP1. Lane 1, prestained protein marker; lane 2, Coomassie staining; lane 3, Western blot using anti-VP1 guinea pig polyclonal antibody and respective secondary antibody (a) Bac-VP1, (b) Bac-wt.
Figure 3.
Negative staining of Bac-VP1 associated with bilosomes.
(a) Bac-VP1 associated with bilosomes, (b) Inactive Bac-VP1 associated with bilosomes (c) Bilosomes only. Imaging was carried out using a transmission electron microscope JEM-1230 (Jeol). Arrows pointing to the successful association of Bac-VP1 with bilosomes.
Figure 4.
Measurement of VP1 specific IgG antibody response in the serum of immunized mice by indirect ELISA.
(A) Orally immunized mice: Groups of mice (n = 10) were orally vaccinated three times on days 0, 7, and 21 with 200 µl containing live or inactivated recombinant baculovirus associated or non-associated with bilosomes. Bac-wt and PBS were given as negative controls. Each point represents the arithmetic mean value (n = 6) ±SD. *, P<0.05; **, P<0.01; ***, P<0.001 between live Bac-VP1 associated with bilosomes and live Bac-VP1 alone (a) or ***, P<0.001 between live Bac-VP1 associated with bilosomes and inactive Bac-VP1 associated with bilosomes (b) or ***, P<0.001 between live Bac-VP1 and inactive Bac-VP1 (c). (B) Subcutaneously immunized mice: Groups of mice (n = 10) were subcutaneously vaccinated two times on days 0 and 21 with 200 µl of live Bac-VP1 or inactivated EV71 or Bac-wt and PBS as negative controls. VP1-specific IgG antibody levels were determined by indirect ELISA. Each point represents the arithmetic mean value (n = 6) ± SD. *, P<0.05 between inactive EV71 and live Bac-VP1.
Figure 5.
Measurement of VP1 specific mucosal IgA antibody response in orally immunized mice by indirect ELISA.
Groups of mice (n = 10) were orally vaccinated three times on days 0, 7, and 21 with 200 µl containing live or inactivated recombinant baculovirus associated or non-associated with bilosomes or Bac-wt. Each point represents the arithmetic mean value (n = 6) ± SD. **, P<0.01 between live Bac-VP1 associated with bilosomes and live Bac-VP1 alone or ***, P<0.001 between live Bac-VP1 and inactive Bac-VP1 or ***, P<0.001 between bilosomes associated live Bac-VP1 and bilosomes associated inactive Bac-VP1.
Figure 6.
Neutralization antibody titers of vaccinated sera against homologous EV71 strain.
Sera were collected from mice on days 0, 14, 28, 42, and 56. 25 µl of serial two-fold dilutions of sera were then mixed with 25 µl of 100 TCID50 of virus (Fuyang EV71, C4 virus) and incubated at 37°C for 2 h to neutralize infectious virus. The mixtures were then transferred to 96-well plates with more than 90% confluent monolayers of RD cells grown in DMEM containing 5% FBS. After incubation for 5 days at 37°C, the neutralizing antibody titers were read as the highest dilution of sera that completely inhibited virus growth. (A) Neutralization titers of orally vaccinated mice candidate’s sera. Each point represents the arithmetic mean value (n = 6) ±SD. **, P<0.01; ***, P<0.001 between live Bac-VP1 associated with bilosomes and live Bac-VP1 alone (a) or ***, P<0.001 between bilosomes associated live Bac-VP1 and bilosomes associated inactive Bac-VP1 (b) or ***, P<0.001 between live Bac-VP1 and inactive Bac-VP1 (c). (B) Neutralization titers of subcutaneously vaccinated mice candidate’s sera. Each point represents the arithmetic mean value (n = 6) ± SD. **, P<0.01; ***P<0.001 between inactive EV71 and live Bac-VP1.
Figure 7.
Neutralization antibody titers of vaccinated sera against heterologous EV71 strains.
Sera were collected from mice on day 56. 25 µl of serial two-fold dilutions of sera were then mixed with 25 µl of 100 TCID50 of virus, and incubated at 37°C for 2 h to neutralize infectious virus. The mixtures were then transferred to 96-well plates with more than 90% confluent monolayers of RD cells grown in DMEM containing 5% FBS. After incubation for 5 days at 37°C, the neutralizing antibody titers were read as the highest dilution of sera that completely inhibited virus growth. (a) Neutralization titers of orally vaccinated mice candidate’s sera. (b) Neutralization titers of subcutaneously vaccinated mice candidate’s sera.
Table 1.
EV71 genotypes used in the in vitro microneutralization assay.
Figure 8.
Passive protection of neonatal mice by vaccinated mice sera against mouse adapted lethal HEV71 infection.
Six day old BALB/c mice were inoculated i.p. with mouse adapted EV71-B4 strain at a dose of 5 MLD50 per mouse. 24 h later, each mouse was passively immunized with sera obtained from the vaccinated adult mice. The control groups received sera from Bac-wt and PBS immunized mice. Mortality was monitored until 21 days post infection. (a) Passive protection study using sera from orally vaccinated mice (b) Passive protection study using sera from subcutaneously vaccinated mice.
Figure 9.
Measurement of viral copies in brain by real-time PCR.
Mice were treated with orally or subcutaneously vaccinated sera on day 1 post challenge with 5 MLD50 (mouse lethal dose) of mouse adapted EV71 B4 strain HFM 41 virus. The viral loads were measured in the brain of dead and survived animals. (a) Mice treated with orally vaccinated mice sera (b) Mice treated with subcutaneously vaccinated mice sera. Each column represents the mean of triplicate assays with standard deviation.