Fig 1.
SDS-PAGE-Coomassie staining and Western Blot of HPV18L1–45RG1 VLP.
Purified and dialyzed 18L1–45RG1 VLP (lane 1), HPV18 wt L1 VLP (lane 2) or crude Sf9 lysate (lane 3) were separated by SDS-PAGE followed by Coomassie staining (A). The L1-RG1 fusion protein migrated at a molecular weight of about 50kDa, slightly slower than wt HPV18 L1, for which smaller degradation products are also visible. Insertion of the RG1 peptide into the L1 protein and its antigenicity were verified by Western Blot using an anti-HPV16 L2 aa 11–200 serum (B), or Camvir-1 reacting to HPV18 L1 (C). For both Western Blots, HPV18L1–45RG1 fusion proteins show a molecular weight of about 50kDa (Fig. 1B lane 1; Fig. 1C lane 1), with smaller bands representing proteolytic degradation products. As controls HPV18 wt L1 VLP (Fig. 1B lane 2; 1C lane 2), HPV16L1L2 VLP (Fig. 1B lane 4) and Sf9 cells only (Fig. 1B lane 3; Fig. 1C lane 3) were used.
Fig 2.
Transmission electron microscopy of chimeric HPV18L1–45RG1 VLP.
VLP were gradient-purified, negatively stained and visualized at 30,000-fold magnification using a JEOL 1010 electron microscope. The size of the bar indicates 200nm.
Fig 3.
Characterization of chimeric HPV18L1–45RG1 by ELISA.
Chimeric 18L1–45RG1 VLP (left) or wt HPV18L1 VLP (right) (50ng per well) were used as antigens and attached to 96 well plates under native conditions. ELISA was performed in triplicates with fourfold serial antibody endpoint dilutions ranging from 1:200–1:204,800 using HPV18-specific conformational-dependent neutralizing mAb H18.G10 and H18.J4, or non-neutralizing mAb Camvir-1 directed to a linear L1 epitope. Results indicate that RG1 epitope insertion into HPV18L1–45RG1 VLP disrupted (or sterically hindered) the recognition site of H18.J4, whereas the sites for H18.G10 and Camvir-1 remained intact. Data are shown as mean OD ± standard deviation (SD).
Fig 4.
ELISA to characterize VLP surface-display of RG1-peptide and to detect antibodies to RG1 induced by 18L1–45RG1 VLP vaccination.
(A) Native or denatured HPV18L1–45RG1 VLP were attached to 96-well ELISA plate and contacted with serial dilutions of anti-HPV16 L2 polyclonal serum recognizing the RG1 epitope. The anti-L2 serum recognized both native and denatured chimeric VLP, indicating RG1 display on the surface of VLP. As a control, binding of mAb Camvir-1 was assessed, which is directed to a linear L1 epitope hidden in the assembled protein. (B) Biotinylated peptides representing HPV45 RG1 were attached to 96-well Streptavidin plates. ELISA was performed in triplicates using either immune sera raised to HPV18L1–45RG1 VLP or HPV18L1 VLP, or pre-immune sera, with dilutions ranging from 1:200 to 1:51,200. HPV18L1–45RG1 immune sera, but not HPV18L1 VLP sera or pre-immune sera, bound to the RG1 peptide at titers of 12,800 to 51,200 (serum #2 and #1, respectively). Data are shown as mean OD ± standard deviation (SD) and titers reported as mean values 3 SD above background signals (pre-immune values).
Table 1.
In vitro cross-neutralization of α7 mucosal hr and lr HPV by antisera raised against 18L1–45RG1 VLP using L1-based pseudovirion neutralization assays.
Table 2.
In vitro cross-neutralization of α7 mucosal hr and lr HPV by antisera raised against 18L1–45RG1 VLP using L2-based pseudovirion neutralization assay.
Fig 5.
Th1 cell—mediated immune response induced by HPV18L1–45RG1 VLP vaccination by measuring IFN-γ by Elispot.
Groups of C57BL/6 mice (n = 3) were s.c. immunized twice (day 0 and 10) with 2μg of either wt HPV18L1 VLP, chimeric HPV18L1–45RG1 VLP, or PBS as mock control. On day 20 spleens were harvested, groups pooled and splenocytes isolated. 106 splenocytes were plated onto Elispot plates, and stimulated with either wt HPV18L1 VLP, or HPV45 RG1 synthetic peptide, or a combination of both. The graphs show that HPV18L1 VLP, but not the RG1 peptide, induced IFN-γ production in splenocytes of HPV18L1–45RG1 or wt HPV18L1 VLP pre-sensitized mice. Shown are mean values ± SD of triplicate cultures.
Fig 6.
HPV18L1-45RG1 VLP vaccine efficacy against experimental genital challenge with hr α7 HPV18, 39, 45, 59, 68 pseudovirions in mice.
Progesterone synchronized groups of mice were passively immunized by intravenous transfer of 20μl of pre-immune or immune sera raised to HPV18L1–45RG1 VLP, 18L1 wt VLP, or a type-specific immune serum. 24 hours later, the vaginal epithelium was mechanically disrupted followed by intravaginal installation of indicated pseudovirions (HPV18, 39, 45, 59, 68 or 16) enclosing a luciferase gene. Three days later infection was detected by a bioluminescence imager (IVIS). Rabbit antiserum to HPV18L1–45RG1 VLP conferred levels of protection similar to type specific L1 sera for HPV18, 39, 45 and 68 (p-values of 0.0151; 0.0003; 0.0001 and 0.0038, respectively, using One-way ANOVA), but not to HPV59 or HPV16 PsV (p-values of 0.0099 and 0.1114). In contrast, HPV18L1 VLP sera conferred mostly type-restricted protection to HPV18 and cross-protection against HPV45 only (t-test p-values of 0.0311 and 0.0044). Luciferase activity was measured as p/s/cm2/sr (average radiance) and results are shown after subtraction of background luminescence (unvaccinated mice challenged with CMC only). P-values for significant differences between pre-immune versus HPV18L1–45RG1 VLP groups (t-test) are shown or indicated not significant (n.s.).
Table 3.
RG1 amino acid sequence homology.