Functional assessment and structural basis of antibody binding to human papillomavirus capsid

Abstract

Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus-like particle (VLP)-based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)-based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV-based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D-2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher-resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In-depth understanding of epitope composition would ensure the development of function-indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type-specific epitopes would enable rational design of cross-type neutralization via epitope re-engineering or epitope grafting in hybrid VLPs.

Figure 1. HBsAg based hepatitis B vaccines (1981 – 1986, plasma derived; 1986-present, yeast derived recombinant VLP based).

(a) Two-dimensional cryoEM class average of yeast-derived recombinant HBsAg VLPs, from Ref. [27]. (b) 3-D image reconstruction at 15 Å resolution from cryoEM of the yeast-derived VLPs, from Ref. [27]. Segmentation of the map revealed regions of high density, presumed to be protein (gray), surrounded by regions of lesser density, presumed to be lipid (yellow). (c) CryoEM image reconstruction at 12-Å resolution of the plasma-derived HBsAg particles, from Ref. [69]. The particles are 20-23 nm in diameter and have octahedral symmetry. (d) Measurement of recombinant HBsAg VLP binding to neutralizing antibodies RF1 and A1.2 by surface plasmon resonance. Binding affinities were interpreted as “relative antigenicity”, with plasma-derived HBsAg particles as the reference [27].

Figure 2. Structure, assembly and antigenic determinants of human papillomavirus (HPV) VLPs.

Atomic force microscopy (AFM) images of HPV16, (a) before and (b) after dis- and reassembly treatment, from Ref. [20, 53]. (c) CryoEM structure of HPV16 [21]. (d) Atomic model of HPV16 generated from crystal structure of HPV16 L1 [66] and cryoEM structures of bovine papillomavirus [61, 64]. (e) The surface of the atomic model of HPV16 shown in panel (d) with the pentavalent capsomere colored in blue, the hexavalent capsomeres in grey and a full-length IgG crystal structure in cartoon representation drawn to scale, with the Fab moieties pointing in the general direction of epitopes. (f) Binding affinities of various neutralizing antibodies to pre- (black stripes) and post- (red) dis- and reassembly VLPs, from Ref. [21]. (g) Close-up of a capsomere from the atomic model of HPV16 shown in panel (d), with each key antibody epitopes in a different color [21].

Figure 3. Structural and functional analysis of the hepatitis E vaccine.

(a) Negatively-stained transmission EM of p239 VLPs (in bulk from Hecolin® vaccine). (b) Sedimentation velocity analytical ultracentrifugation (SV-AUC) profiles of the protrusion domain of E2s, the neutralizing antibody 8C11 Fab, and the E2s-8C11 Fab complex. The sedimentation profiles show that E2s binds 8c11 Fab with a 1:1 stoichiometry, or one E2s dimer to two Fabs. (c) The association/dissociation curve of E2 protein binding with the HEV neutralizing antibody 8C11 and the binding constant were calculated by surface plasmon resonance (Biacore). (d) Crystal structure of the E2s-Fab 8C11 immune complex (PDB code 3RKD), revealing an immunodominant neutralizing epitope of the HEV capsid. (e) A p239 “shrunken VLP” model constructed by in silico truncation of the icosahedral T = 1 E2 VLP (PDB code 2ZTN) [26]. (f) Crystal structure of T = 1 VLPs (PDB code 2ZTN). (g) CryoEM structure of T = 3 VLPs (PDB code 3IYO). (h) Schematic profile of HEV ORF2 functional domains and some historic constructs.