Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies

Abstract

Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies. Therefore, we screened a total of 1,078 serum samples for HPV16 L2 reactivity, and these were obtained from four prior clinical studies: a population-based (n = 880) surveillance study with a high-risk HPV DNA prevalence of 10.8%, a cohort study of women (n = 160) with high-grade cervical intraepithelial neoplasia (CIN), and two phase II trials in women with high-grade vulvar intraepithelial neoplasia (VIN) receiving imiquimod therapy combined with either photodynamic therapy (PDT) (n = 19) or vaccination with a fusion protein comprising HPV16 L2, E7, and E6 (TA-CIN) (n = 19). Sera were screened sequentially by HPV16 L2 enzyme-linked immunosorbent assay (ELISA) and then Western blot. Seven of the 1,078 serum samples tested had L2-specific antibodies, but none were detectably neutralizing for HPV16. To develop a standard, we substituted human IgG1 sequences into conserved regions of two rodent monoclonal antibodies (MAbs) specific for neutralizing epitopes at HPV16 L2 residues 17 to 36 and 58 to 64, creating JWW-1 and JWW-2, respectively. These chimeric MAbs retained neutralizing activity and together reacted with 33/34 clinically relevant HPV types tested. In conclusion, our inability to identify an HPV16 L2-specific neutralizing antibody response even in the sera of patients with active genital HPV disease suggests the subdominance of L2 protective epitopes and the value of the chimeric MAbs JWW-1 and JWW-2 as standards for immunoassays to measure L2-specific human antibodies.

FIG 1

Screening of patient sera for HPV16 L2-specific antibody. Optical density values plotted for HPV16 L2 ELISA measurements of the serological responses of previously unscreened CATCH study patients (n = 880), of which 10.3% were positive for high-risk HPV (A), high-grade CIN patients (n = 160) with 1 to 4 visits (n = 298) (B), high-grade HPV⁺ VIN patients (n = 19) who underwent 10 weeks of topical imiquimod therapy, which activates TLR7-dependent innate responses (IMQ) before being treated with photodynamic therapy (PDT) in a phase II study (34) (C), and high-grade HPV⁺ VIN patients (n = 19) who underwent 10 weeks of topical imiquimod therapy (INN), followed by three vaccinations with 125 μg of TA-CIN at monthly intervals in a prior phase II study prior to serum collection (35) (D). The absorbance values to the right of the arrow in panels A and B (mean + 3 SD cutoff) were considered putative positive responses and rescreened in a Western blot assay. (A) Eighteen data points corresponding to 18 different patients were above the cutoff (arrow). Representative Western blot analyses from 4 out of 6 of these 18 high-risk HPV patients were HPV16 L2 ELISA positive (E). (B) The 4 data points larger than the cutoff (arrow) correspond to four serum samples collected at different visits of a single HPV16⁺ CIN2 patient, and each was also reactive for HPV16 L2 by Western blotting (F). These results are summarized in Table 2.

FIG 2

Reactivity of JWW-1 and JWW-2 human chimeric monoclonal antibodies with L2 by ELISA. (A) Coomassie blue-stained SDS-PAGE gel analysis of JWW-1 and JWW-2 monoclonal antibodies. (B) L2α 11-88x5 multimer ELISA using purified JWW-1 (closed squares), rat monoclonal WW-1 (open squares), WHO standard (STD) HPV16 serum 05/134 (closed circles), and serum from an HPV16⁺ CIN2 patient (visit 1, open circles) containing HPV16 L2-reactive antibodies. (C) L2α 11-88x5 multimer ELISA using purified JWW-2 (closed squares), mouse monoclonal MAb24b (open squares), WHO standard HPV16 serum 05/134 (closed circles), and serum from an HPV16⁺ CIN2 patient (visit 1, open circles) containing HPV16 L2-reactive antibodies. (B and C) ELISAs were tested with anti-human IgG secondary antibodies to test for human specificity of the chimeric MAbs JWW-1 and JWW-2 (P < 0.001) versus their parental antibodies (WW-1 or MAb24), which were used as negative controls.

FIG 3

Reactivity of JWW-1 and JWW-2 human chimeric monoclonal antibodies with L2 of diverse HPV, as determined by Western blotting. Pseudovirion preparations of 34 clinically relevant alpha- and beta-HPV types were separated by SDS-PAGE and transferred to a membrane. The reactivity of JWW-1 or JWW-2 against L2 of each type was determined by probing with peroxidase-linked anti-human IgG for JWW-1 and JWW-2 or peroxidase-linked anti-rabbit IgG for the L2α 11-88x8 antiserum. After washing, these Western blots were developed by chemiluminescence.