Two of the four other nonneutralizing, capsid-reactive MAbs recognized HPV16 L2 residues 89 to 100; one acknowledged residues 73 to 84 and another acknowledged residues 33 to 52. virus-like-particle (VLP) vaccines to obtain broad protection against oncogenic human papillomaviruses (HPVs) (16). Vaccination with Rostafuroxin (PST-2238) L2 as a full-length protein or as polypeptides protects animals against homologous-type viral difficulties at both cutaneous and mucosal sites (2-4,6,12). Protection is not mediated by cellular immunity, suggesting the importance of neutralizing antibodies (5,7). L2 is usually subdominant in the context of L1/L2 VLPs (19), but antibodies elicited by recombinant L2 immunogens are able to neutralize a remarkably broad range of HPV genotypes (15). This suggests that neutralizing epitopes of L2 may be conserved across HPV types due to some crucial viral function (13). Furthermore, it Rostafuroxin (PST-2238) raises the possibility that a single L2 protein- or peptide-based vaccine might provide comprehensive protection against the HPV types causing genital malignancy and genital warts and possibly even those associated with cutaneous warts and epidermodysplasia verruciformis (EV). == Identification of neutralizing epitopes within HPV16 L2. == The rational design of a broadly protective L2-based preventive vaccine requires knowledge of the relevant neutralizing epitopes. To identify the neutralizing epitopes in L2, we vaccinated BALB/c mice with full-length six-His-tagged HPV16 L2 protein and produced hybridomas by using standard procedures (18). Of the 100 supernatants reactive with L2 protein, only 45 reacted with HPV16 L1/L2 pseudovirions, and only one (RG-1) neutralized HPV16 pseudovirus and was cloned. The RG-1 supernatant exhibited a Rabbit Polyclonal to GPR34 neutralizing titer of 1 1,280 and also reacted with HPV16 L1/L2 pseudivirions by an enzyme-linked immunosorbent assay (ELISA). RG-1 and another four monoclonal antibodies (MAbs) that showed the highest ELISA reactivities with HPV16 pseudovirions were all the immunoglobulin G1() [IgG1()] isotype and reacted with HPV16 L2 protein by Western blotting (Table1). == TABLE 1. == Capsid surface reactivity and neutralizing activity of HPV16 L2 MAbsa Undiluted hybridoma supernatants from five clones were tested by L2 protein ELISA and by L1/L2 pseudovirus ELISA (results given as optical densities at 405 nm [OD405] after background subtraction), for L2 reactivity by Western blotting (shown as yes/no), for antibody isotype, and for their ability to neutralize HPV16 pseudovirus. The epitopes recognized by each monoclonal antibody were defined by peptide ELISA using 56 20-mer peptides derived from HPV16 L2, each overlapping by 12 amino acids (aa). Where adjacent peptides reacted, the overlapping sequence is given. Each MAb was screened for reactivity with 56 20-mer peptides of HPV16 L2 that overlapped each other by 12 amino acids (Table1). The neutralizing MAb RG-1 reacted with a peptide comprising residues 17 to 36 of HPV 16 L2 (peptide 17-36) (Fig.1A) but not the overlapping peptides 9-28 and 25-44. Two of the four other nonneutralizing, capsid-reactive MAbs acknowledged HPV16 L2 residues 89 to 100; one acknowledged residues 73 to 84 and another acknowledged residues 33 to 52. RG-1 ascites exhibited a titer of 1 1,024,000 in an HPV16 L1/L2 VLP ELISA and neutralized both HPV16 and HPV18 pseudovirions with titers of 204,800 and 25,600, respectively, but failed to neutralize HPV5, HPV6, HPV45, HPV52 or HPV58 or bovine papillomavirus 1 (BPV1) pseudovirions at a titer of 40. Sequence comparison suggests that RG-1 recognizes lysine at residue 20, which is usually conserved in HPV16 and HPV18 but different among other types that were not neutralized (R or Q). == FIG. 1. == Rostafuroxin (PST-2238) 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 usually highly Rostafuroxin (PST-2238) 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 usually 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.