Analysis of the structural characteristics of hHPV16 and cHPV16 VLP.

<p>To analyze the structural integrity of the two VLP types, the HPV16 VLPs were fractionated on Optiprep density gradients (A). Eight fractions were collected in experiment A (0.5 ml each). The results of TEM analysis are presented in panel B. Magnification is 41,000× (bars 50 nm).</p

Camvir-1, H16.V5 and H16.E70 reactivity towards scHPV16 VLPs and schHPV16 VLPs.

<p>The amounts of L1 proteins contained in scHPV16 VLP and schHPV16 VLP were confirmed by SDS-PAGE and Western blotting prior to performing ELISAs (A). In panel A, loading amount indicates protein amount loaded for SDS-PAGE and Western blot. L1 amount indicates L1 protein amount contained in the loading sample. The L1 protein amount was confirmed by L1 band intensities on SDS-PAGE and Western blot. M indicates the molecular weight marker. The SDS-PAGE and western blot are representatives of duplicate assays. The Camvir-1, H16.V5 and H16.E70 reactivity towards schHPV16 VLPs and scHPV16 VLPs were determined by direct ELISA and are presented in B, C and D, respectively. The ODs of hHPV16 VLPs after reaction with 1 µg/ml of Camvir-1, 0.25 µg/ml of H16.V5 and 0.25 µg/ml of H16.E70 were set at 100% in B, C and D, respectively. The ELISA values are the means ± SD of two independent assays.</p

Camvir-1, H16.V5 and H16.E70 reactivity towards hHPV16 VLPs and cHPV16 VLPs.

<p>The HPV16 L1 protein residues recognized by H16.V5, H16.E70 and Camvir-1 are displayed graphically in (A). BC, CD, DE, EF, FG and HI indicate the loop structures in HPV16 L1. The numbers refer to the amino acid residues as counted from the N-terminus, the black boxes indicate loops covering the solvent-exposed face of the capsid, and the white box (CD) indicates an internal loop. The gray box (EF) indicates a loop partly located on the outside of the capsid. The hHPV16 VLP and cHPV16 VLP concentrations were confirmed by SDS-PAGE prior to running ELISAs (B). The protein concentration of each VLP preparation was determined by Bradford protein assay, and 500 to 62 ng of proteins were loaded for SDS-PAGE analysis. M indicates the molecular weight marker. Camvir-1, H16.V5 and H16.E70 reactivity towards hHPV16 VLPs and cHPV16 VLPs was determined by direct ELISA. The ELISA results are presented in C, D and E, respectively. The ODs of the hHPV16 VLPs after reaction with 1 µg/ml of Camvir-1, 0.25 µg/ml of H16.V5 and 0.25 µg/ml of H16.E70 were set at 100% in C, D and E, respectively. The ELISA values are the means ± SD of two independent assays.</p

Immunization Protocol-1 Anti-HPV16 L1 IgG titer and neutralization activity resulting.

<p>Mice were immunized according to immunization protocol-1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035893#pone-0035893-t002" target="_blank">Table 2</a>). The mice were immunized four times with 100 µl of PBS, 8 ng of hHPV16 VLP or 8 ng of cHPV16 VLP at two-week intervals, in the absence of adjuvant. Ten days after the 3^rd and 4^th immunization, the mice sera were obtained and analyzed. Panels A and B present the anti-HPV16 L1 IgG titer and neutralization activity after the 3^rd immunization, respectively. Panels C and D present the anti-HPV16 L1 IgG titer and neutralization activity after the 4^th immunization, respectively. The horizontal bars are median values of the IgG titer and neutralization activity (PBS, n = 6; hHPV16 VLP, n = 15; cHPV16 VLP, n = 15).</p

Particle size distributions of hHPV16 VLP and cHPV16 VLP.

<p>The HPV16 VLP populations along with their associated hydrodynamic diameters were analyzed by DLS as described in the Materials and Methods section. Each HPV16 VLP was prepared in 25 mM MOPS containing 75 mM NaCl pH 7.0 and adjusted to 40 µg/ml. Panel A and B are representatives of duplicate measurements of the hHPV16 VLP and cHPV16 VLP populations, respectively.</p

Mouse immunization protocols used in this study.

<p>Mouse immunization protocols used in this study.</p

Procedures used to purify the HPV16 VLPs used in this study.

<p>All the HPV16 VLP preparations were finally dialyzed against 0.325 M NaCl in phosphate buffer pH 7.2.</p>a<p>The scHPV16 VLP was further separated by heparin chromatography.</p

Immunization Protocol-2 Anti-HPV16 L1 IgG and neutralization antibody titers.

<p>The anti-HPV16 L1 IgG and anti-HPV16 neutralizing antibody titers of the PBS-, hHPV16 VLP- and cHPV16 VLP-immunization groups are presented in panels A and B, respectively. For the immunizations, the mice were subcutaneously injected three times with 100 µl of PBS, 1000 ng of hHPV16 VLP or 1000 ng of cHPV16 VLP at two-week intervals (protocol-2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035893#pone-0035893-t002" target="_blank">Table 2</a>). The anti-HPV16 L1 IgG and anti-HPV16 neutralizing antibody titers were determined by ELISA and SEAP-based neutralization assays, respectively. The data values of seven individual mice (n = 7) are represented with dots. The horizontal bars indicate the median titers of the anti-HPV16 L1 IgG and neutralization antibodies.</p