Identification of a human papillomavirus type 16-specific epitope on the C-terminal arm of the major capsid protein L1

Abstract

To characterize epitopes on human papillomavirus (HPV) virus-like particles (VLPs), a panel of mutated HPV-16 VLPs was created. Each mutated VLP had residues substituted from HPV-31 or HPV-52 L1 sequences to the HPV-16 L1 backbone. Mutations were created on the HPV-31 and -52 L1 proteins to determine if HPV-16 type-specific recognition could be transferred. Correct folding of the mutated proteins was verified by resistance to trypsin digestion and by binding to one or more conformation-dependent monoclonal antibodies. Several of the antibodies tested were found to bind to regions already identified as being important for HPV VLP recognition (loops DE, EF, FG, and HI). Sequences at both ends of the long FG loop (amino acids 260 to 290) were required for both H16.V5 and H16.E70 reactivity. A new antibody-binding site was discovered on the C-terminal arm of L1 between positions 427 and 445. Recognition of these residues by the H16.U4 antibody suggests that this region is surface exposed and supports a recently proposed molecular model of HPV VLPs.

FIG. 1.

Identification of poorly conserved regions of HPV L1s. The members of the A9 group of HPVs (types 16, 31, 33, 35, 52, 58, and 67) were aligned by Clustal W and the percent identity within a five-residue window between the types determined. The regions indicated on top were defined by Chen et al. (3). Arrows indicate regions of greatest variability. Filled arrows indicate the regions mutated in this study.

FIG. 2.

Stability of VLPs in the presence of trypsin. VLPs were treated with increasing concentrations of trypsin and incubated for 1 h. Following trypsin treatment the proteins were run on polyacrylamide gels and immunoblotted with an anti-L1 antibody (CAMVIR-1). The left hand lane of each panel corresponds to samples that received no trypsin.

FIG. 3.

Binding of 16 and 52 specific MAbs to VLPs with mutations of the BC loop. VLPs 16:wt (▪), 16:BC (□), 52:wt (•), and 52:BC (○) were titrated across a plate and reacted with CAMVIR-1, H16.V5, H16.E70, H52.B9, and H52.D12 in an ELISA.

FIG. 4.

Binding of MAbs to HPV-16/31 hybrid VLPs. MAbs were tested by ELISA for reactivity to HPV-16 and HPV-31 wild-type VLPs and mutated VLPs that are indicated on the x-Axis. The y-axis are normalized optical density values (see text) for H16.V5 (solid), H16.E70 (striped), H16.U4 (cross-hatched), and H31.A6 (opposite striped) binding. The error bars show standard deviations.

FIG. 5.

EF loop is necessary and sufficient for H31.A6 binding. VLPs were serially diluted across microtiter plates and ELISAs performed with CAMVIR-1, H16.V5 or H31.A6 as indicated. The VLPs were HPV-16:wt (▪), HPV-31:wt (•), 16:EF (□), and 31:EF (○).

FIG. 6.

Identification of residues on the FG loop important for MAb binding. The sequence of HPV-16 and HPV-31 L1 are compared (A). Residues that are predicted to be >45% surface accessible are indicated with arrows and bold type is used to indicate nonconserved residues. CAMVIR-1, H16.V5, H16.E70, and H16.U4 were tested for binding to VLPs that were serially diluted prior to testing. The normalized binding values are presented (B), with each MAb represented by bars, as described in the legend to Fig. 4 (error bars show standard deviations).

FIG. 7.

Position of mutations made on the C-terminal arm of HPV-16 L1. The upper panel compares the sequence of HPV-16 and HPV-31 with the differences emphasized with bold type. The arrows indicate residues predicted by Modis et al. ¹⁸ to be potentially immunogenic. In the lower panel the carbon backbone of HPV-16 is shown based on the Modis model showing the C-terminal arm from a neighboring capsomer (in yellow) as it wraps around and inserts into a capsomer (gray). The mutations in this region are indicated as larger red balls. The view is from the side.