Table 1.
Plasmids and strains used in this study.
Fig 1.
Construction of the expression vector for production of the H2 antigen fused to the Lp_1261 lipoprotein anchor.
SalI and HindIII restriction sites enabled easy gene exchange. The gene fragment encoding the Hirep2-DC antigen (darker gray) was introduced into a plasmid harboring the lipoprotein anchor derived from L. plantarum protein Lp_1261 (light gray). The complete gene construct was translationally fused to the inducible promoter that is indicated by the thin arrow.
Table 2.
Experimental groups and immunization protocol.
Fig 2.
Detection of anchor-fused H2 antigen (A) and cell growth of L. plantarum producing the H2 antigen (B). Panel A shows Western blot analysis of a cell-free protein extract from Lp_H2 (lane 2) and a molecular mass standard (1). The predicted molecular mass of the Lp_1261H2-DC protein is 37 kDa. Panel B shows the growth rate of Lp_H2 and Lp_Ev, used as a control. The OD600 was measured at the induction point (black bars) and 3 h after induction (gray bars) for both strains. The data are presented as means of triplicates ± SEM.
Fig 3.
Presence of the H2 antigen at the L. plantarum surface detected by flow cytometry (A) and fluorescence microscopy (B). Cells were probed with an anti-H2 antibody. Panel A shows flow cytometry analysis for Lp_H2 (black histogram) and Lp_Ev used as a negative control (gray histogram). Panel B shows indirect immunofluorescence microscopy of the indicated strains. The data presented are from one representative experiment. Each experiment was performed three independent times, giving similar results.
Fig 4.
Estimation of the total amount of antigen by semi-quantitative Western blotting.
Serial dilutions of bacterial cell lysate from 1 x 109 CFU of induced Lp_H2 and dilutions of purified Hirep1 with known concentration were subjected to Western blotting and proteins were detected using the anti-H2 antibody (which also binds to Hirep1 protein). Panel A shows a typical Western blot: wells 1–5, standard protein (20 ng, 10 ng, 7.5 ng, 5 ng and 2.5 ng, respectively); wells 6–9, lysate of Lp_H2, diluted 20-fold, 30-fold, 40-fold and 50-fold, respectively. Signal intensities of different dilutions of the standard protein were used to make a standard curve that is presented in panel B. The marked points on the standard curve correspond with the amount of standard protein used: (1) - 20 ng, (2) - 10 ng, (3)– 7.5 ng, (4) - 5 ng, (5) - 2.5 ng.
Fig 5.
Immune responses induced by the booster Lp_H2 vaccine.
Panel A presents the cellular response measured by H2-specific IFN-γ production in splenocytes. Cells were purified from individual mice and stimulated with H2 for 72 h. Produced IFN-γ was measured in harvested supernatants, by ELISA. Each point represents an individual mouse and the overall results per group are presented as a mean ± SEM (n = 8 for naive, H2 and H2/Lp_H2 groups, n = 7 for Lp_H2). Statistical significance was determined using one-way ANOVA with Tukey's post hoc test and is indicated as follows: **, p < 0.01. Panels B—E show antibody responses: H2-specific IgG in plasma (B) and H2-specific IgA in plasma (C), vaginal washes (D) and lung fluids (E). The samples from individual mice were serially diluted and added to H2-coated plates and specific IgG and IgA levels were measured by ELISA. The individual points represent the average OD450 values per group ± SEM (n = 8 for naive, H2 and H2/Lp_H2 groups, n = 7 for Lp_H2) at each dilution. Panels F and G show H2-specific IgA in 10-fold diluted plasma samples (F) and 5-fold diluted vaginal samples (G). Each point represents an individual mouse and the overall results per group are presented as a mean ± SEM (n = 8 for naive, H2 and H2/Lp_H2 groups, n = 7 for Lp_H2). Statistical significance was determined using one-way ANOVA with post hoc Dunnett's test and is shown as follows: *, p < 0.05; **, p < 0.01.