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. IMPORTANCEThe 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. Human papillomaviruses (HPVs) have a double-stranded DNA genome of approximately 8 kb which is replicated via host cell polymerases with an error rate of ca. 2 ϫ 10 Ϫ8 base substitutions per site per year (1), substantially lower than that found in the majority of single-stranded RNA viruses (ca. 1 ϫ 10 Ϫ3 base substitutions per site per year) (2). Despite the low evolutionary rate of the HPV genome, variants have arisen over time, leading to the generation of distinct intragenotype lineages classified by a sequence difference of 1 to 10% across the whole genome (3). The single nucleotide polymorphisms (SNPs) that allow segregation of these variants into distinct lineages can be found in each gene/ region, with the highest number accumulating in the noncoding regions (NCR1, NCR2, and URR) and the lowest number accumulating in structural (L1 and L2) genes (4).The HPV structural genes encode the major (L1) and minor (L2) proteins that form the nonenveloped icosahedral viral capsid, which comprises 72 pentameric L1 capsomers, and each capsomer has an upper estimate of one L2 protein (5). The L1 protein mediates attachment to host cells (6), while the L2 protein is essential for subsequent viral infectivity (7).The humoral immune response following natural HPV infection predominately targets conformational epitopes on the surface-exposed loop regions of the L1 protein (8, 9). Seroconvers...

Sensitivity of Human Papillomavirus (HPV) Lineage and Sublineage Variant Pseudoviruses to Neutralization by Nonavalent Vaccine Antibodies

“…Codon-optimized L1 and L2 genes representing consensus lineage and sublineage variant sequences (Supplementary Figure 1) were synthesized (GeneArt; Thermo Fisher Scientific) with additional site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit, Agilent Technologies) as required [8–12]. Inserts were confirmed by Sanger sequencing.…”

“…For each genotype, a single serial dilution series of each serum was tested against all relevant variant PsVs in parallel. The titer of each serum against each variant PsV was compared to that generated against the consensus A/A1 variant PsV and a fold difference metric was generated to allow normalization and intra-/intergenotype comparisons [8–12].…”

“…The biological consequences of HPV genome variants are unclear. Studies examining the potential impact of natural variation on L1 antigenicity have examined variants of HPV16 [6, 7], HPV31 [8], HPV33 [9], HPV45 [10], HPV52 [11], and HPV58 [12] and demonstrated, in some cases, variant-specific differences in sensitivity to monovalent, bivalent, and quadrivalent VLP immune sera, natural infection sera, and monoclonal antibodies (MAbs). Here, we evaluate the sensitivity of lineage and sublineage variants to neutralization by nonavalent HPV vaccine antibodies.…”

Naturally Occurring Major and Minor Capsid Protein Variants of Human Papillomavirus 45 (HPV45): Differential Recognition by Cross-Neutralizing Antibodies Generated by HPV Vaccines

“…1D). All HPV45 variant PsVs displayed similar particle sizes (median, 55 nm; interquartile range [ IQR], 53 to 57 nm) and particle-to-infectivity ratios (27) (median, 2.9 ϫ 10 2 ; IQR, 2.0 ϫ 10 2 to 4.7 ϫ 10 2 ). Sera from 12-to 15-year-old girls, collected following three doses of Cervarix or Gardasil HPV vaccine (22), were used to assess HPV45 sublineage variant sensitivity to cross-neutralizing antibodies ( Fig.…”

“…Single-cycle replication-incompetent pseudoviruses (PsVs) representing HPV16 L1, but not L2, variants (26) appear to exhibit little difference in their susceptibilities to type-specific antibodies elicited by HPV16 virus-like particles (VLP). We recently demonstrated that although PsVs incorporating HPV31 L1 and L2 lineage variants (A, B, and C) were susceptible to cross-neutralizing antibodies elicited by the Cervarix and Gardasil HPV vaccines, there were lineage-specific differences in sensitivity (27). Here we examine the potential impact of lineage-specific L1 and L2 HPV45 variation on sensitivity to cross-neutralizing antibodies elicited by the Cervarix and Gardasil HPV vaccines.…”