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.Minor capsid antigen L2 is a possible alternative to highly multivalent L1 virus-like-particle (VLP) vaccines to obtain broad protection against oncogenic human papillomaviruses (HPVs) (16). Vaccination with L2 as a full-length protein or as polypeptides protects animals against homologous-type viral challenges 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 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 critical viral function (13). Furthermore, it raises the possibility that a single L2 protein-or peptide-based vaccine might provide comprehensive protection against the HPV types causing genital cancer 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 neutralizing titer of 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 (Table 1).Each MAb was screened for reactivity with 56 20-mer peptides of HPV16 L2 that overlapped each other by 12 amino acids ( Table 1). 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 ...

BackgroundVaccination with minor capsid protein L2 induces antibodies that cross-neutralize diverse papillomavirus types. However, neutralizing antibody titers against the papillomavirus type from which the L2 vaccine was derived are generally higher than the titers against heterologous types, which could limit effectiveness against heterologous types. We hypothesized that vaccination with concatenated multitype L2 fusion proteins derived from known cross-protective epitopes of several divergent human papillomavirus (HPV) types might enhance immunity across clinically relevant HPV genotypes.MethodsAntibody responses of mice (n = 120) and rabbits (n = 23) to vaccination with HPV-16 amino-terminal L2 polypeptides or multitype L2 fusion proteins, namely, 11-200 × 3 (HPV types 6, 16, 18), 11-88 × 5 (HPV types 1, 5, 6, 16, 18), or 17-36 × 22 (five cutaneous, two mucosal low-risk, and 15 oncogenic types), that were formulated alone or in GPI-0100, alum, or 1018 ISS adjuvants were compared with vaccination with L1 virus-like particles (VLPs), including Gardasil, a licensed quadrivalent HPV L1 vaccine, and a negative control. Mice were challenged with HPV-16 pseudovirions 4 months after vaccination. Statistical tests were two-sided.ResultsThe HPV-16 L2 polypeptides generated robust HPV-16–neutralizing antibody responses, albeit lower than those to HPV-16 L1 VLPs, and lower responses against other HPVs. In contrast, vaccination with the multitype L2 fusion proteins 11-200 x 3 and 11-88 x 5 induced high serum neutralizing antibody titers against all heterologous HPVs tested. 11-200 × 3 formulated in GPI-0100 adjuvant or alum with 1018 ISS protected mice against HPV-16 challenge (reduction in HPV-16 infection vs phosphate-buffered saline control, P < .001) 4 months after vaccination as well as HPV-16 L1 VLPs, but 11-200 × 3 alone or formulated with either alum or 1018 ISS was less effective (reduction in HPV-16 infection, P < .001).ConclusionConcatenated multitype L2 proteins in adjuvant have potential as pan-oncogenic HPV vaccines.

Persistent infection with the high-risk subset of genitotropic human papillomavirus (HPV) genotypes is a necessary cause of cervical cancer. Given the global burden of cervical cancer, a low-cost, broadly protective vaccine is needed. RG-1 is a crossneutralizing and protective monoclonal antibody that recognizes residues 17-36 of HPV16 minor capsid protein L2. Because this epitope is highly conserved in divergent HPV types, we determined whether vaccination with HPV16 L2 17-36 peptide is broadly protective. The peptide was administered to BALB/c mice three times at monthly intervals, either alone or in the context of a synthetic lipopeptide vaccine candidate (P25-P2C-HPV) produced by linkage of the HPV peptide with a broadly recognized T helper epitope (P25) and the Toll-like receptor-2 (TLR2) ligand dipalmitoyl-S-glyceryl cysteine (P2C). In contrast to vaccination with the L2 17-36 peptide or P25-P2C alone, a potent L2-specific antibody response was generated to the P25-P2C-HPV lipopeptide when delivered either s.c. or intranasally. Sera from mice vaccinated with the P25-P2C-HPV lipopeptide neutralized not only HPV16 pseudovirions but also other evolutionarily divergent oncogenic genital (HPV18, HPV45) and cutaneous (HPV5, BPV1) types. The L2-specific antibody response depended on MHC class II, CD40, and MyD88 signaling. Additionally, vaccination with the P25-P2C-HPV lipopeptide protected mice from homologous challenge with HPV16 pseudovirions at cutaneous and genital sites and heterologous challenge with HPV45 pseudovirions. If provided in the appropriate context, therefore, HPV16 L2 17-36 might be used in a totally synthetic cross-protective HPV vaccine. G enitotropic human papillomavirus (HPV) infections are considered the most common sexually transmitted infection in the United States (1). The major manifestations of anogenital HPV include genital warts (condyloma acuminatum) and anogenital intraepithelial neoplasia. If left untreated, a small fraction of persistent high-risk HPV infections progresses to cancer. The presence of HPV DNA has been reported in 99.7% of cervical carcinomas worldwide, indicating that HPV infection is a necessary cause of this cancer and that this disease can be prevented by prophylactic HPV vaccination (2).Approximately 35 of the Ͼ100 subtypes of HPV are specific for the anogenital epithelium and have varying potentials for malignant transformation (3). Of the 15 oncogenic genital HPV types, HPV16 is the most common, followed by HPV18 and HPV45 (contributing Ϸ50%, 20%, and 10%, respectively, of cervical cancer cases worldwide). Public health efforts have successfully reduced the incidence and mortality of cervical cancer with the implementation of cervical cytology screening programs. Women who do not undergo regular screening account for most of the patients with invasive cancers (4), and cervical cancer remains the second most common cause of cancer death in women worldwide and the most prevalent cancer in women of sub-Saharan Africa, Central America, south-central Asia, and Me...

Summary Using a human papillomavirus (HPV) cervicovaginal murine challenge model, we microscopically examined the in vivo mechanisms of L1 virus-like particle (VLP) and L2 vaccine-induced inhibition of infection. In vivo HPV infection requires an initial association with the acellular basement membrane (BM) to induce conformational changes in the virion that permit its association with the keratinocyte cell surface. By passive transfer of immune serum, we determined that anti-L1 antibodies can interfere with infection at two stages. Similarly to active VLP immunization, transfer of high L1 antibody concentrations prevented BM binding. In the presence of low concentrations of anti-L1 antibodies, virions associated with the BM, however binding to the epithelial cell surface was not detected. Regardless of the concentration, L2 vaccine-induced antibodies allow BM association, but prevent association with the cell surface. This is the first study to examine the mechanisms of vaccine-induced inhibition of virus infection in vivo.

Current L1 virus-like particle (VLP) vaccines provide type-restricted protection against a small subset of the human papillomavirus (HPV) genotypes associated with cervical cancer, necessitating continued cytologic screening of vaccinees. Cervical cancer is most problematic in countries that lack the resources for screening or highly multivalent HPV VLP vaccines, suggesting the need for a low-cost, broadly protective vaccinogen. Here, N-terminal L2 polypeptides comprising residues 1 to 88 or 11 to 200 derived from HPV16, bovine papillomavirus type 1 (BPV1), or cottontail rabbit papillomavirus (CRPV) were produced in bacteria. Rabbits were immunized with these N-terminal L2 polypeptides and concurrently challenged with CRPV and rabbit oral papillomavirus (ROPV). Vaccination with either N-terminal L2 polypeptides of CRPV effectively protected rabbits from CRPV challenge but not from papillomas induced by cutaneous challenge with CRPV genomic DNA. Furthermore, papillomas induced by CRPV genomic DNA deficient for L2 expression grew at the same rate as those induced by wild-type CRPV genomic DNA, further suggesting that the L2 polypeptide vaccines lack therapeutic activity. Neutralizing serum antibody titers of >15 correlated with protection (P < 0.001), a finding consistent with neutralizing antibody-mediated protection. Surprisingly, a remarkable degree of protection against heterologous papillomavirus types was observed after vaccination with N-terminal L2 polypeptides. Notably, vaccination with HPV16 L2 11-200 protected against cutaneous and mucosal challenge with CRPV and ROPV, respectively, papillomaviruses that are evolutionarily divergent from HPV16. Further, vaccination with HPV16 L2 11-200 generates broadly cross-neutralizing serum antibody, suggesting the potential of L2 as a second-generation preventive HPV vaccine antigen.The recognition that persistent infection with high-risk human papillomavirus (HPV) types is a necessary cause of cervical cancer has driven the development of prophylactic vaccines based upon the capsid proteins L1 and L2 (41). Vaccination with L1 virus-like particles (VLPs) (19,25,36) or capsomers (37), but not denatured L1, elicits high-titer but type-restricted neutralizing antibodies (8,19,32,33,35). Studies in dogs challenged with canine oral papillomavirus and rabbits challenged with cottontail rabbit papillomavirus (CRPV) demonstrate that neutralizing antibody induced by L1 VLP vaccination provides immunity from infection against the cognate papillomavirus type from which the vaccinogen was derived (1, 38). Recent clinical studies showed protection against the acquisition of persistent infection and clinical disease related to the HPV types used to derive the monovalent (21, 23), bivalent (13,14), and tetravalent L1 VLP vaccines (39). Papanicolaou (PAP) cytologic screening and intervention in the United States is estimated to have reduced the incidence of cervical cancer by ϳ80% (31), but at a cost of Ͼ$6 billion annually. Elimination of these expensive screening programs necess...