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A peptide derived from the human papillomavirus type 16 (HPV-16) minor capsid protein, L2, has previously been reported to induce cross-neutralizing antibodies in mice. In this report, four HPV L2 peptides, including the HPV-16 peptide and its HPV type 6 and 11 homologues, along with extended peptides containing a conserved set of amino acids, were used to immunize rabbits and mice. Antibody responses were evaluated for specificity and ability to neutralize viral infection in vitro with a quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) assay. All peptide immunizations resulted in cognate and cross-peptide reactivity, but this did not translate equally into recognition of full-length protein, VLP, or neutralization of virus in vitro. This report provides the first evidence of cross-neutralization of authentic HPV by antiserum to L2 peptides. Comparison of the anti-peptide serum reactivity, especially with regard to neutralization of virus, indicates that the extended peptides may offer more potential to induce adequate responses for cross-protective immunity.

HPV-40 is a rare HPV type that has been detected only in genital mucosal tissues. This HPV type is very closely related to HPV-7, which has a predominantly cutaneous tissue tropism. We have shown, previously, that an isolate of HPV-40 (described here as HPV-40(Hershey) or HPV-40(H)) productively infected genital tissues. In this study, HPV-40(H) was tested for productive infection of cutaneous tissue. Fetal hand skin fragments were incubated with infectious HPV-40(H) and implanted subrenally into athymic mice. After 120 days, xenografts showed morphological changes consistent with HPV-40(H) infection and were HPV-40 DNA in situ positive and capsid antigen positive. The results demonstrated that hand skin can support HPV-40(H) infection thereby indicating that this viral type has the capacity to infect both genital mucosal and cutaneous tissues.

Human papillomavirus (HPV) hybrid virus-like particles (VLPs) were prepared using complementary regions of the major capsid L1 proteins of HPV-11 and -16. These hybrid L1 proteins were tested for assembly into VLPs, for presentation and mapping of conformational neutralizing epitopes, and as immunogens in rabbits and mice. Two small noncontiguous hypervariable regions of HPV-16 L1, when replaced into the HPV-11 L1 backbone, produced an assembly-positive hybrid L1 which was recognized by the type-specific, conformationally dependent HPV-16 neutralizing monoclonal antibody (N-MAb) H16.V5. Several new N-MAbs that were generated following immunization of mice with wild-type HPV-16 L1 VLPs also recognized this reconstructed VLP, demonstrating that these two hypervariable regions collectively constituted an immunodominant epitope. When a set of hybrid VLPs was tested as immunogens in rabbits, antibodies to both HPV-11 and -16 wild-type L1 VLPs were obtained. One of the hybrid VLPs containing hypervariable FG and HI loops of HPV-16 L1 replaced into an HPV-11 L1 background provoked neutralizing activity against both HPV-11 and HPV-16. In addition, conformationally dependent and type-specific MAbs to both HPV-11 and HPV-16 L1 VLP were obtained from mice immunized with hybrid L1 VLPs. These data indicated that hybrid L1 proteins can be constructed that retain VLP-assembly properties, retain type-specific conformational neutralizing epitopes, can map noncontiguous regions of L1 which constitute type-specific conformational neutralizing epitopes recognized by N-MAbs, and trigger polyclonal antibodies which can neutralize antigenically unrelated HPV types., ((C)2001 Elsevier Science.)

The high-molecular-weight sulfated or sulfonated polysaccharides or polymers cellulose sulfate, dextran sulfate, and polystyrene sulfonate were tested for microbicidal activity against bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 11 (HPV-11) and type 40 (HPV-40). In vitro assays included the BPV-1-induced focus-forming assay and transient infection of human A431 cells with HPVs. The compounds were tested for microbicidal activity directly by preincubation with virus prior to addition to cell cultures and indirectly by addition of virus to compound-treated cells and to virus-coated cells to test inactivation of the virus after virus-cell binding. The data indicated that all three compounds showed direct microbicidal activity with 50% effective concentrations between 10 to 100 microg/ml. These concentrations were nontoxic to cell cultures for both assays. When a clone of C127 cells was tested for microbicidal activity, approximately 10-fold-less compound was required to achieve a 50% reduction in BPV-1-induced foci than for the uncloned parental C127 cells. Pretreatment of cells with compound prior to addition of virus also demonstrated strong microbicidal activity with dextran sulfate and polystyrene sulfonate, but cellulose sulfate required several orders of magnitude more compound for virus inactivation. Polystyrene sulfonate prevented subsequent infection of HPV-11 after virus-cell binding, and this inactivation was observed up to 4 h after addition of virus. These data indicate that the polysulfated and polysulfonated compounds may be useful nontoxic microbicidal compounds that are active against a variety of sexually transmitted disease agents including papillomaviruses.

The athymic mouse xenograft system was used to prepare infectious stocks of two additional anogenital tissue-targeting human papillomaviruses (HPVs) in a manner similar to that for the development of infectious stocks of HPV-11. An anal condyloma from a transplant patient was used as material for extraction of infectious virus, and human foreskin fragments were incubated with the virus suspension and transplanted subrenally into athymic mice. Partial viral sequencing indicated that two rare HPV types (HPV-40 and HPVLVX82/MM7) were concurrently present in both the patient condyloma and the foreskin xenografts, and passage of both types was achieved as a mixed infection with HPV-40 predominating. Xenografts that developed from simultaneous infection of human foreskin fragments with HPV-11, -40, and -LVX82/MM7 virions produced regionally separate areas of HPV-11 and -40 infection as determined by in situ hybridization. In addition, in situ hybridization with HPV-40 and HPVLVX82/MM7 DNA probes demonstrated that both of these HPV types were present as adjacent but separate infections within the same anal condyloma of the transplant patient. These studies indicate that multiple HPV types can simultaneously infect genital tissue and that each HPV type predominantly maintains regional separation within the same papilloma.

A set of 13 monoclonal antibodies (MAbs) was generated against HPV-6 L1 virus-like particles (VLPs), screened for reactivity to HPV-6 and HPV-11 L1 VLPs by ELISA, and tested for neutralization of HPV-11 infection. Both cross-reactive and type-specific epitopes were detected such that 4 of 13 MAbs reacted to surface conformational sites on HPV-6 L1 VLPs and the remaining 9 MAbs were cross-reactive to both HPV-6 and HPV-11 L1 VLPs. four of the 9 cross-reactive MAbs were able to neutralize HPV-11 infectivity, and 3 of 4 of these neutralizing MAbs (N-MAbs) identified shared surface conformational sites. One N-MAb therefore recognized a surface linear epitope as judged by positive binding to L1 in a Western blot assay. The neutralization status of these cross-reactive MAbs with regard to HPV-6 could not be assayed. These results demonstrated that the closely related HPV types 6 and 11 contain both type-specific and shared neutralizing epitopes.

A panel of 24 monoclonal antibodies (MAbs) was generated against human papillomavirus (HPV) types 16 and 18 L1 virus-like particles (VLPs). The MAbs were screened for reactivity to a variety of VLPs prepared from HPV-6, -11, -16, -18, -31, -33, -35, and -45, cottontail rabbit papillomavirus, bovine papillomavirus type 1, and a set of 35 overlapping 20-amino-acid peptides spanning the entire HPV-16 L1 gene. Type-specific linear and conformational surface epitopes were detected as well as several cross-reactive linear epitopes that showed various levels of cross-reactivity between different genital HPV and animal papillomavirus L1s. Most of the linear epitopes were mapped using synthetic peptides, and the epitopes were identified as being either surface or buried within the VLP as defined by the pattern of reactivity in ELISA using intact and disrupted VLP antigen. These MAbs may be useful reagents to help define neutralizing epitopes of HPV-16 and -18 when infectivity assays become available, and to define the regions of L1 that are exposed on the surface or buried within the assembled capsid.

Anti-idiotypic antibodies (anti-Ids) were generated against three mouse monoclonal antibodies (MAbs) which neutralized three different papillomaviruses. The neutralizing MAbs (N-MAbs) were generated against infectious human papillomavirus type 11 (HPV-11), cottontail rabbit papillomavirus (CRPV), and bovine papillomavirus type 1 (BPV-1), and all recognized surface conformational epitopes that were lost upon denaturation of the virions. The polyclonal anti-Ids were screened in an ELISA using a panel of 10 N-MAbs (4 HPV-11 neutralizing, 5 CRPV neutralizing, and 1 BPV-1 neutralizing), and cross-reactive idiotypic reactivity was observed. Affinity-purified anti-H11.B2 (HPV-11 N-MAb) cross-reacted with the 3 other HPV-11 N-MAbs, was unreactive to a nonneutralizing HPV-11 MAb and all 5 CRPV N-MAbs, but surprisingly was reactive to the BPV-1 N-MAb. In contrast, affinity-purified anti-CRPV-1A (CRPV N-MAb) reacted with all 5 CRPV N-MAbs, but not with any other MAb. Affinity-purified anti-B1.A1 (BPV-1 N-MAb) also showed unexpected cross-reactivity with the 4 HPV-11 N-MAbs, but not with any of the CRPV N-MAbs. Two of the three polyclonal rabbit anti-Ids induced strong anti-viral antibody responses (including virus-neutralizing antibodies) in BALB/c mice following immunization. The results indicate that within a mouse strain, papillomavirus-neutralizing antibodies share cross-reactive idiotopes that group the N-MAbs into several subsets. One group includes the CRPV N-MAbs, and another group includes N-MAbs directed to apparently unrelated papillomaviruses, including HPV-11, BPV-1, and possibly HPV-6.

Postattachment neutralization of papillomaviruses (PVs) was analyzed in three PV-infectivity models: (i) the BPV-1-induced focus-forming assay using C127 cells; (ii) in vitro abortive infection of rabbit RK-13 and Sf1Ep cells with CRPV; and (iii) HPV-11-induced morphological transformation of human foreskin chips in the athymic mouse xenograft system. In each assay system, aliquots of infectious virus were added to the appropriate target cells and incubated at 37 degrees, followed at various postinfection time intervals with neutralizing monoclonal antibodies (N-MAbs) that target surface conformational epitopes. In all three model systems, the N-MAbs were able to neutralize PV infection when added as late as 8 hr after addition of infectious PV to host cells. These results imply that papillomaviruses attach to but do not penetrate inside host cells for a significant period of time and that the bound virus is thus still susceptible to neutralization by neutralizing antibodies.

Baculovirus-expressed human papillomavirus type 11 (HPV-11) major capsid protein (L1) virus-like particles (VLPs) were produced in insect cells and purified on CsCl density gradients. The VLPs retained conformational neutralizing epitopes that were detected by a series of HPV-11-neutralizing monoclonal antibodies. Electron microscopy determined that the HPV-11 L1 VLPs were variable in size with a surface topography similar to that of infectious HPV-11. The VLPs were very antigenic, and induced high titres of neutralizing antibodies in rabbits and mice when used as an immunogen without commercial preparations of adjuvant. These VLP reagents may be effective vaccines for protection against HPV infections.