Characterization of a major neutralizing epitope on human papillomavirus type 16 L1

Persistent infection with human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Neutralizing epitopes present on the major coat protein, L1, have not been well characterized, al though three neutralizing monoclonal antibodies (MAbs) had been identified by using HPV-16 pseudovirions (R. B. Roden et al., J. Virol. 71:6247-6252, 1997). Here, two of these MAbs (H16.V5 and H16.E70) were demonstrated to ne utralize authentic HPV-16 in vitro, while the third (H16.U4) did not. Bindi ng studies were conducted with the three MAbs and virus-like particles (VLP s) composed of the reference L1 sequence (114K) and three variant L1 sequen ces: Rochester-lk (derived from viral stock DNA), GU-1 (derived from cervic al biopsy DNA), and GU-2 (derived from biopsy DNA, but containing some sequ ence changes likely to be artifactual). While all three MAbs bound to 114K and Rochester-lk VLPs, GU-1 VLPs were not recognized by H16.E70, and both H 16.E70 and H16.V5 failed to bind to GU-2 VLPs. Site-directed mutagenesis wa s used to replace disparate amino acids in the GU-2 L1 with those found in the 114K L1. Alteration of the amino acid at position 50, from L to F, comp letely restored H16.V5 binding and partially restored H16.E70 binding, whil e complete restoration of H16.E70 binding occurred with GU-2 VLPs containin g both L50F and T266A alterations. Immunization of mice with L1 variant VLP s revealed that GU-2 VLPs were poorly Immunogenic. The L50F mutant of GU-2 L1, in which the H16.V5 epitope was restored, elicited HPV-16 antibody resp onses comparable to those obtained with 114K VLPs. These results demonstrat e the importance of the H16.V5 epitope in the generation of potent HPV-16 n eutralizing antibody responses.