The DE and FG loops of the HPV major capsid protein contribute to the epitopes of vaccine-induced cross-neutralising antibodies

Abstract

The human papillomavirus (HPV) vaccines consist of major capsid protein (L1) virus-like particles (VLP) and are highly efficacious against the development of cervical cancer precursors attributable to oncogenic genotypes, HPV16 and HPV18. A degree of vaccine-induced cross-protection has also been demonstrated against genetically-related genotypes in the Alpha-7 (HPV18-like) and Alpha-9 (HPV16-like) species groups which is coincident with the detection of L1 cross-neutralising antibodies. In this study the L1 domains recognised by inter-genotype cross-neutralising antibodies were delineated. L1 crystallographic homology models predicted a degree of structural diversity between the L1 loops of HPV16 and the non-vaccine Alpha-9 genotypes. These structural predictions informed the design of chimeric pseudovirions with inter-genotype loop swaps which demonstrated that the L1 domains recognised by inter-genotype cross-neutralising antibodies comprise residues within the DE loop and the late region of the FG loop. These data contribute to our understanding of the L1 domains recognised by vaccine-induced cross-neutralising antibodies. Such specificities may play a critical role in vaccine-induced cross-protection.

Figure 1. Predicted differences in L1 loop structure between homology models representing HPV16 and non-vaccine Alpha-9 genotypes.

(a) Top view of HPV16 L1 surface-filled model with the BC, DE, EF, FG and HI loops highlighted. Expanded view of predicted structural differences in the BC (b), DE (c), EF (d), FG (e) and HI (f) with loops colour-coded by genotype. (g) Heatmap represents the predicted distance (mean Å) between the L1 loops of the homology models representing HPV16 and the non-vaccine Alpha-9 genotypes. Key indicates Å distance from HPV16 with (-) indicating that it was not possible to resolve any potential differences between loops.

Figure 2

Chimeric L1L2 PsV (a) Top view of L1 capsomer with variable positons between HPV31 and HPV35 within the DE (pink), FG (orange) and HI (green) loops highlighted. (b) EM images of HPV31, HPV35 and chimeric L1L2 PsV with preparations characterised for the median (IQR) particle diameter, infectivity, L1 concentration and the resultant particle-to-infectivity ratio. TCID₅₀, 50% Tissue Culture Infectious Dose.

Figure 3. Neutralisation sensitivity of chimeric L1L2 PsV to HPV vaccine-induced antibodies.

Colour indicates L1L2 PsV constructs: HPV31 (white), HPV35 (grey), HPV31 L1 backbone with HPV35 loop switches (blue) and HPV35 L1 backbone with HPV31 loop switches (red). Bar graph represents the neutralisation GMT of n = 36 HPV vaccine sera (Cervarix^® n = 19; Gardasil^® n = 17), all of which were tested against HPV31, HPV35 and the chimeric L1L2 PsV. Error bars represent GMT neutralisation titre 95% CI. p values obtained using the Wilcoxon paired signed-rank test represent differences in neutralisation titre between either the HPV31 PsV and the chimeric PsV with a HPV31 backbone or the HPV35 PsV and the chimeric PsV with a HPV35 backbone.

Figure 4

Chimeric L1L2 PsV with DE or FG loop amino acid switches (a) Amino acid alignment of HPV16, HPV31 and HPV35 DE and FG loops. Amino acid positions within the DE and FG loops selected for site-directed mutagenesis are highlighted with identical residues between HPV16 and HPV31 but different from HPV35 boxed in grey. (b) Top view of L1 capsomer with the DE (pink) and FG (orange) loops highlighted alongside amino acid positions identified for site-directed mutagenesis (blue). (c) EM images of chimeric L1L2 PsV preparations with a HPV35 backbone and HPV31 DE and FG loops with amino acid switches characterised for the median (IQR) particle diameter, infectivity, L1 concentration and the resultant particle-to-infectivity ratio.

Figure 5. Fine-mapping of L1 epitope footprint of cross-neutralising antibodies.

(a) Neutralisation sensitivity of chimeric L1L2 PsV with a HPV35 backbone and HPV31 DE and FG loops with amino acid switches to HPV vaccine-induced antibodies. Colour indicates L1L2 PsV constructs: HPV35 (grey), HPV35 backbone and HPV31 DE and FG loops (red), HPV35 L1 backbone with HPV31 DE and FG loop switches with DE or FG amino acids switches (green). Bar graph represents the neutralisation GMT of n = 24 HPV vaccine sera (Cervarix^® n = 12; Gardasil^® n = 12), all of which were tested against HPV35 and the chimeric L1L2 PsV. Error bars represent GMT neutralisation titre 95% CI. p values obtained using the Wilcoxon paired signed-rank test represent differences in neutralisation titre between the chimeric PsV with a HPV35 backbone and HPV31 DE and FG loops and the chimeric PsV with a HPV35 backbone and HPV31 DE and FG loops with DE or FG amino acid switches. (b) Close up view on HPV31 L1 homology model of residues identified for site-directed mutagenesis (blue) within the DE (pink) and FG (orange) loops. The mutation of positions indicated with an asterisk resulted in a ≥4-fold drop in cross-neutralisation titre compared to the chimeric PsV with a HPV35 backbone and HPV31 DE and FG loops.