Identification of two cross-neutralizing linear epitopes within the L1 major capsid protein of human papillomaviruses

Abstract

The neutralizing activities of polyclonal antibodies and monoclonal antibodies (MAbs) obtained by immunization of mice with L1 virus-like particles (VLPs) were investigated by using pseudovirion infectivity assays for human papillomavirus type 16 (HPV-16), HPV-31, HPV-33, HPV-45, HPV-58, and HPV-59 to obtain a better definition of cross-neutralization between high-risk HPVs. In this study, we confirmed and extended previous studies indicating that most genital HPV genotypes represent separate serotypes, and the results suggest that the classification of serotypes is similar to that of genotypes. In addition, three cross-neutralizing MAbs were identified (HPV-16.J4, HPV-16.I23, and HPV-33.E12). MAb HPV-16.J4 recognized a conserved linear epitope located within the FG loop of the L1 protein, and HPV-16.I23 recognized another located within the DE loop. The results suggested that reactivity of MAb HPV-16.I23 to L1 protein is lost when leucine 152 of the HPV-16 L1 protein is replaced by phenylalanine. This confirmed the existence of linear epitopes within the L1 protein that induce neutralizing antibodies, and this is the first evidence that such linear epitopes induce cross-neutralization. However, the cross-neutralization induced by L1 VLPs represents less than 1% of the neutralizing activity induced by the dominant conformational epitopes, and it is questionable whether this is sufficient to offer cross-protection in vivo.

FIG. 1.

Gene transfer of HPV L1 pseudovirion types 16, 31, and 59 and of HPV L1-L2 pseudovirion types 33 and 58. Data were obtained using 10 μg of VLPs per well.

FIG. 2.

Gene transfer inhibition assay. Gene transfer inhibition was done with two HPV-16 MAbs (V5 and J4) and with mouse antisera obtained by immunization with HPV-16 or HPV-31 VLPs.

FIG. 3.

In vitro neutralization of HPV pseudovirion types 16, 31, 33, 45, 58, and 59 with antisera to HPV VLPs.

FIG. 4.

Inhibition of J4 neutralizing MAbs and anti-HPV-31 VLP antibody with L1 peptides (amino acids 267 to 281 and 473 to 492).

FIG. 5.

Alignment of amino acid sequences corresponding to the HPV-16 linear epitope recognized by MAb HPV16.I23.