Naturally Occurring Capsid Protein Variants of Human Papillomavirus Genotype 31 Represent a Single L1 Serotype

Abstract

We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of oncogenic human papillomavirus (HPV) genotype 31 (HPV31) to determine the impact on capsid antigenicity. L1L2 pseudoviruses (PsVs) representing the three HPV31 variant lineages, variant lineages A, B, and C, exhibited comparable particle-to-infectivity ratios and morphologies. Lineage-specific L1L2 PsVs demonstrated subtle differences in susceptibility to neutralization by antibodies elicited following vaccination or preclinical L1 virus-like particle (VLP) immunization or by monoclonal antibodies; however, these differences were generally of a low magnitude. These data indicate that the diagnostic lineage-specific single nucleotide polymorphisms within the HPV31 capsid genes have a limited effect on L1 antibody-mediated neutralization and that the three HPV31 variant lineages belong to a single L1 serotype. These data contribute to our understanding of HPV L1 variant antigenicity.

Importance: The virus coat (capsid) of the human papillomavirus contains major (L1) and minor (L2) capsid proteins. These proteins facilitate host cell attachment and viral infectivity and are the targets for antibodies which interfere with these events. In this study, we investigated the impact of naturally occurring variation within these proteins upon susceptibility to viral neutralization by antibodies induced by L1 VLP immunization. We demonstrate that HPV31 L1 and L2 variants exhibit similar susceptibility to antibody-mediated neutralization and that for the purposes of L1 VLP-based vaccines, these variant lineages represent a single serotype.

FIG 1

HPV31 L1 and L2 variation. (A) Phylogenetic tree constructed from concatenated L1 and L2 nucleotide sequences, including representative sequences from lineages A (J04353), B (HQ537677), and C (HQ537682). (51) and bootstrap values of >95%. (B) Site-specific amino acid (aa) covariation within the L1 and L2 proteins. N, number of sequences in the phylogenetic tree represented by each L1 and L2 combination.

FIG 2

HPV31 L1 and L2 variants. (A) Graphical representation of L1 and L2 variant protein combinations. Site-specific amino acid alterations from the reference (HPV31 A; top sequence, open squares) are indicated using the residue position and resulting amino acid sequence code (filled squares). (B) L1L2 pseudovirus preparation characterized for the median (IQR) particle dimension, infectivity, and L1 concentration. (C) Median (IQR) L1 VLP dimensions (for particles >40 nm in diameter) and L1 concentrations.

FIG 3

Heat maps representing the potential of serum from mice immunized with variant HPV31 L1 VLPs to neutralize variant HPV31 L1L2 PsVs. The log₁₀ neutralization titers of sera from BALB/c mice (n = 10) following variant HPV31 VLP immunization carried out over three separate schedules are presented as the averages for two data sets per sample. The key on the right indicates the log₁₀ heat map gradient. P values, obtained using the Wilcoxon paired signed-rank test, represent differences in median neutralization titers from homologous variant VLP and PsV pairs. *, P < 0.05; **, P < 0.01; NS, no significant difference (P > 0.05).

FIG 4

Crystal model surface highlighting HPV31 FG loop variant residue locations. (A) Linear amino acid epitope footprint of HPV31 FG loop MAbs. (B) Side view highlighting loops in close proximity to FG loop variant residues 267 and 274. (C and D) Top view of loop ribbons. Circled areas indicate regions within a 10-Å radius of residues 267 (C) and 274 (D), as determined by the Swiss-PDP viewer algorithm. Blue, lysine residues at positions 279 and 362; orange, FG loop of monomer 1 (FG₁); black, residues 267 and 274. Neighboring loops on the same monomer (dark pink, DE₁; red, EF₁) or adjacent monomers (dark green, HI₄; yellow, BC₅; light pink, DE₅; light green, HI₅) are indicated. The remaining surface-exposed regions of the capsomer are colored in light gray, and core regions are colored in dark gray.