ELISA to characterize VLP surface-display of RG1-peptide and to detect antibodies to RG1 induced by 18L1–45RG1 VLP vaccination.

<p>(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).</p

Chimeric L2-Based Virus-Like Particle (VLP) Vaccines Targeting Cutaneous Human Papillomaviruses (HPV)

<div><p>Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17–36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. <i>In vivo</i> cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous HPV infections.</p></div

Th1 cell—mediated immune response induced by HPV18L1–45RG1 VLP vaccination by measuring IFN-γ by Elispot.

<p>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.</p

Characterization of chimeric fusion proteins by SDS-PAGE / Coomassie staining and Western Blot.

<p><b>(A)</b> VLP purified on density gradients were separated by SDS-PAGE, followed by Coomassie blue staining and verified by Western blot using <b>(B)</b> an antiserum raised against HPV5 L1 VLP <b>(C)</b> mAb Camvir-1 <b>(D)</b> or antiserum raised against HPV16 L2 aa11-200. As indicated, non-infected Sf9 cell extract, HPV5 PsV, wt HPV16L1, HPV16 L1+L2, or HPV18 L1 VLP were used as controls. Recombinant chimeric fusion proteins migrating ~50 kDa are marked with (*).</p

Evaluation of HPV16L1-5L2(aa53-72) and HPV18L1-5L2(aa53-72)-mediated immune response by L1- and L2-based PBNA.

<p>Evaluation of HPV16L1-5L2(aa53-72) and HPV18L1-5L2(aa53-72)-mediated immune response by L1- and L2-based PBNA.</p

Evaluation of antigenicity and L2 epitope presentation of L1-5L2(aa53-72) and HPV1L1-4RG1 VLP by ELISA.

<p><b>(A)</b> Native HPV16L1-5L2(aa53-72) VLP were compared to native wt HPV16 L1+L2 VLP using an antiserum to HPV16 L2 aa11-200, non-neutralizing mAb Camvir-1 and four indicated neutralizing mAb. <b>(B)</b> HPV18L1-5L2(aa53-72) VLP were compared to wt HPV18 L1+L2 VLP under native conditions using the anti-L2 serum, Camvir-1 and neutralizing mAb H18.J4. <b>(C)</b> Immunogenicity of L2 epitopes presented by either 5L2 aa53-72-based chimeric VLP and <b>(D)</b> HPV1L1-4RG1 VLP were evaluated by indicated L2 peptide ELISA using antisera raised against the respective chimeric VLP. Serum titers were graded positive for mean OD values greater than OD of pre-immune sera + 3 SD.</p

Characterization of L1 and L2 proteins of novel beta PsV by Western Blot.

<p>Pseudovirions were generated in 293TT cells and purified preparations analysed by SDS-PAGE and Western Blot. <b>(A)</b> MAb Camvir-1, <b>(B)</b> mAb AU1, <b>(C)</b> or polyclonal antisera raised against PsV20, <b>(D)</b> PsV38, <b>(E)</b> HPV92 L1 VLP or <b>(F)</b> HPV16 L2 aa1-88 were used to detect respective L1 or L2 proteins as indicated. HPV16L1 VLP, BPV1 L1 VLP, RG1 VLP and 293TT producer cells were used as controls.</p

Transmission electron microscopy of chimeric HPV18L1–45RG1 VLP.

<p>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.</p

HPV18L1-45RG1 VLP vaccine efficacy against experimental genital challenge with hr α7 HPV18, 39, 45, 59, 68 pseudovirions in mice.

<p>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.).</p

SDS-PAGE-Coomassie staining and Western Blot of HPV18L1–45RG1 VLP.

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120152#pone.0120152.g001" target="_blank">Fig. 1B</a> lane 1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120152#pone.0120152.g001" target="_blank">Fig. 1C</a> lane 1), with smaller bands representing proteolytic degradation products. As controls HPV18 wt L1 VLP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120152#pone.0120152.g001" target="_blank">Fig. 1B</a> lane 2; 1C lane 2), HPV16L1L2 VLP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120152#pone.0120152.g001" target="_blank">Fig. 1B</a> lane 4) and Sf9 cells only (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120152#pone.0120152.g001" target="_blank">Fig. 1B</a> lane 3; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120152#pone.0120152.g001" target="_blank">Fig. 1C</a> lane 3) were used.</p