Identification of B-cell epitopes on virus-like particles of cutaneous alpha-human papillomaviruses

Abstract

Human papillomavirus (PV) (HPV) types 2, 27, and 57 are closely related and, hence, represent a promising model system to study the correlation of phylogenetic relationship and immunological distinctiveness of PVs. These HPV types cause a large fraction of cutaneous warts occurring in immunocompromised patients. Therefore, they constitute a target for the development of virus-like particle (VLP)-based vaccines. However, the immunogenic structure of HPV type 2, 27, and 57 capsids has not been studied yet. Here we provide, for the first time, a characterization of the B-cell epitopes on VLPs of cutaneous alpha-HPVs using a panel of 94 monoclonal antibodies (MAbs) generated upon immunization with capsids from HPV types 2, 27, and 57. The MAbs generated were characterized regarding their reactivities with glutathione S-transferase-L1 fusion proteins from 18 different PV types, the nature of their recognized epitopes, their isotypes, and their ability to neutralize HPV type 2, 27, 57, or 16. In total, 33 of the 94 MAbs (35%) showed type-specific reactivity. All type-specific MAbs recognize linear epitopes, most of which map to the hypervariable surface loop regions of the L1 amino acid sequence. Four of the generated MAbs neutralized pseudovirions of the inoculated HPV type efficiently. All four MAbs recognized epitopes within the BC loop, which is required and sufficient for their neutralizing activity. Our data highlight the immunological distinctiveness of individual HPV types, even in comparison to their closest relatives, and they provide a basis for the development of VLP-based vaccines against cutaneous alpha-HPVs.

FIG. 1.

Number of MAbs recognizing different antigens. Reactivities of MAbs with VLPs, capsomeres, the GST-L1 fusion protein, or denatured L1 proteins of the respective immunogen HPV types were analyzed. The distribution of MAbs generated after immunization with VLPs from HPV types 2, 27, and 57 (black, dark gray, and light gray columns, respectively) according to their recognition of epitopes on the corresponding antigens is illustrated.

FIG. 2.

Four MAbs neutralize HPV types 27 and 57 type specifically. Data for the neutralization of HPV types 2 (A), 27 (B), 57 (C), and 16 (D) by MAbs TS27-30, TS57-1, TS57-5, and TS57-6 or a serum pool from three mice immunized with VLPs of the respective HPV type emulsified in Freund's adjuvant are shown. Pseudovirions were incubated with serial dilutions of MAbs or the mouse serum pool prior to inoculation of the cells. As a control, cells were inoculated with virus-free medium (not infected). The IgG content of the MAbs and the mouse serum pool was quantified, and neutralization was determined based on the reduction of the reporter signal. Samples were assayed in duplicates, and mean IC₅₀s with standard deviations are displayed.

FIG. 3.

Map of linear epitopes recognized by MAbs and L1 sequence identity between HPV types 2, 27, and 57. (Top) Linear epitopes recognized by MAbs were mapped by using synthetic 20-mer peptides with 10-amino-acid overlaps. The positions of these epitopes within the L1 protein are illustrated, with BC, DE, EF, FG, and HI surface loop regions as references. Black arrows indicate epitopes to cross-reactive MAbs, gray arrows indicate epitopes to type-specific MAbs, and asterisks mark epitopes to type-specific and neutralizing MAbs. (Bottom) For an analysis of the L1 amino acid sequence identity between HPV types 2, 27, and 57 over the length of the protein, the L1 sequences were aligned using the T-Coffee algorithm, and sequence conservation at any position was scored for 5-amino-acid intervals.

FIG. 4.

BC loop regions are necessary and sufficient for neutralization by MAbs. MAbs TS27-30 (A) and TS57-1, TS57-5, and TS57-6 (B) were analyzed for the neutralization of their respective immunogen HPV types and HPV type 16 as well as for neutralization of mutant pseudovirions chimeric for the BC loop region of L1. Their capacity to neutralize the respective pseudovirions was quantified as mean IC₅₀s of duplicates with standard deviations.

FIG. 5.

The BC loop region is not necessary but is sufficient for neutralization by sera of VLP-immunized mice. The BC loop region of the L1 protein in HPV pseudovirions was replaced by the corresponding amino acid sequence of a different HPV type. Neutralization of the chimeric and nonchimeric pseudovirions by serum pools from two mice immunized with VLPs of the indicated HPV type was analyzed (note that a different pseudovirion dilution than that used for experiments presented in Fig. 2 and 4 was used). Data are displayed as mean IC₅₀s of duplicates with standard deviations.