A protective and broadly cross-neutralizing epitope of human papillomavirus L2

Abstract

We generated a monoclonal antibody, RG-1, that binds to highly conserved L2 residues 17 to 36 and neutralizes human papillomavirus 16 (HPV16) and HPV18. Passive immunotherapy with RG-1 was protective in mice. Antiserum to the HPV16 L2 peptide comprising residues 17 to 36 (peptide 17-36) neutralized pseudoviruses HPV5, HPV6, HPV16, HPV 18, HPV31, HPV 45, HPV 52, HPV 58, bovine papillomavirus 1, and HPV11 native virions. Depletion of HPV16 L2 peptide 17-36-reactive antibodies from cross-neutralizing rabbit and human L2-specific sera abolished cross-neutralization and drastically reduced neutralization of the cognate type. This cross-neutralization of diverse HPVs associated with cervical cancer, genital warts, and epidermodysplasia verruciformis suggests the possibility of a broadly protective, peptide-based vaccine.

FIG. 1.

The RG-1 neutralizing MAb recognizes the evolutionarily conserved L2 17-36 motif and provides passive immunity. (A) CLUSTAL W homology comparison of residues 17 to 36 of HPV16 L2 peptide and L2 sequences from different papillomavirus types. The HPV16 L2 sequence comprising amino acids 17 to 36 is highly conserved among different types and exhibits 78% identity with the L2 sequences from HPV2 (skin type), HPV5 (EV related), and HPV45; 80% identity with HPV6 and HPV11 L2 (benign types); and 84% identity with HPV18 (high-risk type). In contrast, L2 as a whole exhibits only ∼25% conservation among these types. This sequence was conserved even in BPV1, which is evolutionarily distant from high-risk HPV. (B) Peptide ELISA using monoclonal antibody and polyclonal antiserum to HPV16 L2 peptide 17-36. Wells were coated with synthetic peptide 1 (ASATQLYKVVKQAGTCPPD), comprising HPV16 L2 residues 13 to 31 in which residues 21 and 22 are both changed to valine (in bold); 2 (TASADPCPKQLYKCQATGT), comprising HPV16 L2 residues 13 to 31 in which the amino acid order is scrambled; 3 (ASATQLYKTCKQAGTCPPD), comprising wild-type HPV16 L2 residues 13 to 31; or 4 (ASATQAAKTCKQAGTCPPD) comprising HPV16 L2 residues 13 to 31 in which residues 18 and 19 are both replaced with alanine (in bold). Plates were probed with RG-1, MAb C9, an irrelevant isotype-matched MAb, or rabbit antiserum to HPV16 L2 peptide 17-36 as indicated. (C) Blockade of RG-1 neutralization of HPV16 pseudovirus by titrations of synthetic peptide comprising wild-type HPV16 L2 residues 17 to 36 and the peptides described above for panel B. (D) Passive transfer of RG-1 (n = 5), but not an irrelevant isotype-matched control MAb (n = 6) or phosphate-buffered saline (PBS) (n = 5), protects mice from cutaneous challenge with HPV16 pseudovirus (see http://home.ccr.cancer.gov/lco/ for plasmid maps and production methods). A patch on the belly of each anesthetized BALB/c mouse was shaved with an electric razor without traumatizing the epithelium. MAb was injected (100 μg intraperitoneally) 5 h prior to challenge by application to the shaved skin of 3 × 10⁹ HPV16 pseudovirions (100 ng) in 10 μl of 0.6% carboxymethylcellulose (Sigma C5013) containing L1 and L2 (or L1 alone for background determination) and carrying an encapsidated luciferase reporter construct. Three days later, the mice were anesthetized and injected with luciferin (100 μl at 7 mg/ml), and their images were acquired for 10 min with a Xenogen IVIS 200. Equally sized areas encompassing the site of inoculation were analyzed using Living Image 2.20 software, and background was determined by challenge with noninfectious HPV pseudovirions lacking L2. A representative image is shown in panel D, and the bioluminescence data for each group are plotted in panel E. OD, optical density.

FIG. 2.

Depletion of anti-HPV16 L2 peptide 17-36 antibodies from L2 immune serum abolishes cross-neutralization. Rabbit antisera to BPV1 L2 peptide 1-88 (A to C) or HPV16 L2 peptide 11-200 (D and E) and serum of an HPV16-positive AGIN patient (no. 201) who had been vaccinated with HPV16 L2E7E6 L2 (F and G) were depleted of HPV16 L2 peptide 17-36-specific antibodies by use of a peptide column. Antibodies bound to the column were recovered by elution at low pH and brought back to neutral pH. The sera, both before and after depletion, as well as the recovered antibodies were tested for neutralizing titers for BPV1, HPV16, or HPV18 pseudovirions, as indicated. The serum dilutions for the antibody recovered from the column are not corrected for the dilution that occurs during their elution and return to neutral pH. Neutralization of BPV1 pseudovirions by BPV1 L2 peptide 1-88 antiserum (A), HPV16 pseudovirions by BPV1 L2 peptide 1-88 antiserum (B), HPV18 pseudovirions by BPV1 L2 peptide 1-88 antiserum (C), HPV16 pseudovirions by HPV16 L2 peptide 11-200 antiserum (D), HPV18 pseudovirions by HPV16 L2 peptide 11-200 antiserum (E), HPV16 pseudovirions by immune serum from a patient vaccinated with HPV16 L2E7E6 (F), and HPV18 pseudovirions by immune serum from a patient vaccinated with HPV16 L2E7E6 (G) is shown. OD, optical density.