Combination treatment with intralesional cidofovir and viral-DNA vaccination cures large cottontail rabbit papillomavirus-induced papillomas and reduces recurrences.

We used the cottontail rabbit papillomavirus (CRPV) New Zealand White rabbit model to test a combination treatment of large established papillomas with intralesional cidofovir and DNA vaccination to cure sites and reduce recurrences. Intralesional 1% (wt/vol) (0.036 M) cidofovir treatment of rabbit papillomas led to elimination, or "cure," of the papillomas over a 6- to 8-week treatment period (N. D. Christenson, M. D. Pickel, L. R. Budgeon, and J. W. Kreider, Antivir. Res. 48:131-142, 2000). However, recurrences at periods from 1 to 8 weeks after treatment cessation were observed at approximately 50% of cured sites. DNA vaccinations with CRPV E1, E2, E6, and E7 were initiated either after or at the time of intralesional treatments, and the recurrence rates were observed. When DNA vaccinations were started after intralesional cures, recurrence rates were similar to those of vector-vaccinated rabbits. A small proportion of recurrent sites subsequently regressed (4 out of 10, or 40%) in the vaccinated group versus no regression of recurrences in the vector-immunized group (0 out of 19, or 0%), indicating partial effectiveness. In contrast, when DNA vaccinations were conducted during intralesional treatments, a significant reduction of recurrences (from 10 out of 19, or 53%, of sites in vector-immunized rabbits to 3 out of 20, or 15%, of sites in viral-DNA-immunized rabbits) was observed. DNA vaccination without intralesional treatments had a minimal effect on preexisting papillomas. These data indicated that treatment with a combination of antiviral compounds and specific immune stimulation may lead to long-term cures of lesions without the ensuing problem of papilloma recurrence.

Hybrid papillomavirus L1 molecules assemble into virus-like particles that reconstitute conformational epitopes and induce neutralizing antibodies to distinct HPV types.

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.

DNA vaccination prevents and/or delays carcinoma development of papillomavirus-induced skin papillomas on rabbits.

Malignant progression is a life-threatening consequence of human papillomavirus-associated lesions. In this study, we tested the efficacy of papillomavirus early-gene-based vaccines for prevention of carcinoma development of papillomavirus-induced skin papillomas on rabbits. Rabbit skin papillomas were initiated by infection with cottontail rabbit papillomavirus (CRPV). The papillomas were allowed to grow for 3 months without any treatment intervention. Rabbits were then immunized by gene gun-mediated intracutaneous administration of four DNA plasmids encoding CRPV E1, E2, E6, and E7 genes, respectively. All eight control rabbits receiving vector alone developed invasive carcinoma within 8 to 13 months. In contrast, only two of eight vaccinated rabbits developed carcinoma at 12 and 15 months, respectively. Papilloma growth was suppressed in the majority of vaccinated rabbits but not completely eradicated. These results indicate that gene gun-mediated immunization with papillomavirus early genes may be a promising strategy for prevention of malignant progression of human papillomavirus-associated lesions in humans.

Immunization of rabbits with cottontail rabbit papillomavirus E1 and E2 genes: protective immunity induced by gene gun-mediated intracutaneous delivery but not by intramuscular injection.

We previously demonstrated that gene gun-based intracutaneous vaccination of rabbits with a combination of, but not with individual papillomavirus E1, E2, E6 and E7 genes provided complete protection against cottontail rabbit papillomavirus (CRPV) infection. In the present study, we tested whether vaccination of inbred and outbred rabbits with a combination of CRPV E1 and E2 genes could provide complete protection against virus infection. In the first experiment, gene gun-based intracutaneous vaccination with E1 and E2 genes prevented papilloma formation in the majority of inbred rabbits and promoted systemic papilloma regression in one non-protected rabbit. In contrast, needle-mediated intramuscular injection of E1 and E2 genes did not prevent papilloma formation nor promoted systemic papilloma regression, indicating an absence of strong protective immunity. In the second experiment, six outbred rabbits were immunized by gene gun-based intracutaneous administration of the E1 and E2 genes. Prevention of papilloma formation or systemic papilloma regression was observed in three vaccinated rabbits. Papillomas persisted on the remaining three rabbits, but were significantly smaller than that on control rabbits. These results suggested that gene gun-based intracutaneous vaccination with the combination of papillomavirus E1 and E2 genes induced strong protective antivirus immunity but may be insufficient for complete protection in an outbred population.

Rabbit oral papillomavirus complete genome sequence and immunity following genital infection.

Rabbit oral papillomavirus (ROPV) infects mucosal tissues of domestic rabbits. The viral genomic sequence has been determined and the most related papillomavirus type was the cutaneous cottontail rabbit papillomavirus (CRPV). Homologies between the open reading frames (ORFs) of ROPV and CRPV, however, ranged from 68% amino acid identity for L1 to only 23% identity for E4. Shared features unique to the two rabbit viruses included a large E6 ORF and a small E8 ORF that overlapped the E6 ORF. Serological responses to ROPV L1 viruslike particles (VLPs) were detected in rabbits infected at either the genital or oral mucosa with ROPV. The antibody response was specific to intact ROPV L1 VLP antigen, was first detected at the time of late regression, and persisted at high levels for several months after complete regression. Both oral and genital lesions regressed spontaneously, accompanied by a heavy infiltrate of lymphocytes. ROPV infection of rabbit genital mucosa is a useful model to study host immunological responses to genital papillomavirus infections.

Protection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cottontail rabbit papillomavirus E1, E2, E6, and E7 genes.

In this study, cottontail rabbit papillomavirus infection of domestic rabbits was used as an animal model to develop papillomavirus early gene-based vaccines. Groups of rabbits were intracutaneously vaccinated with single papillomavirus early genes E1, E2, E6, and E7 or with a combination of these four genes. Only a fraction of rabbits were protected from subsequent viral challenge when vaccinated with the E1 or E6 gene. Viral tumor growth in those rabbits vaccinated with the E1 or E2 gene was suppressed compared to that in controls. In contrast, seven of nine rabbits vaccinated with the combination of the E1, E2, E6, and E7 genes were completely protected against viral challenge. These data indicated that intracutaneous genetic vaccination with the combination of the E1, E2, E6, and E7 genes can be an effective strategy for immunoprophylaxis of papillomavirus infection.

Intramuscular injection of plasmid DNA encoding cottontail rabbit papillomavirus E1, E2, E6 and E7 induces T cell-mediated but not humoral immune responses in rabbits.

To test the efficacy of genetic vaccination against papillomavirus infection, plasmid DNA encoding cottontail rabbit papillomavirus (CRPV) E1, E2, E6, E7 or without insert were intramuscularly injected into five groups of rabbits. Peripheral blood mononuclear cells (PBMCs) showed specific proliferation upon in vitro stimulation with E1, E2, E6 or E7 proteins in a majority of vaccinated rabbits but Western blot analysis did not detect antibodies specific for these viral proteins in rabbit serum. All rabbits grew papillomas after virus challenge and none of the rabbits showed systemic papilloma regression. These observations showed that intramuscular injection of plasmid DNA encoding CRPV E1, E2, E6 or E7 induced CD4+ T cell-mediated but not humoral immune responses, and did not result in the protection of rabbits from virus infection.

Characterization of transformation function of cottontail rabbit papillomavirus E5 and E8 genes.

Cottontail rabbit papillomavirus (CRPV) induces rabbit skin papillomas, which progress to invasive carcinoma in some animals. Two early genes, E7 and E6, have been demonstrated previously to be oncogenes. In this study, we identified two additional transforming genes, E8 and E5. Both E8 and E5 stimulated C127 and BALB/c A31 (A31) cell proliferation and affected cell cycle transition. The E8 and E5 transfectants lost cell contact inhibition, reaching a high saturation density when cultured up to 2 weeks. E8-C127 transfectants formed colonies in soft agar in the presence of platelet-derived growth factor (PDGF) while E5-C127 transfectants formed colonies without the requirement for PDGF. E8-C127 transfectants were highly tumorigenic whereas E5-C127 transfectants showed a weak tumorigenicity in nude mice. Both E8 and E5 A31 transfectants failed to form colonies in soft agar even in the presence of platelet-derived growth factor (PDGF) and did not develop tumors in nude mice. These results clearly showed that CRPVE8 and E5 are oncogenes and that the PDGF beta-receptor signaling pathway may be involved in E8-mediated C127 cell transformation. The difference in colony formation in soft agar and tumorigenicity in nude mice between C127 and A31 cell lines indicates that additional alterations in cellular gene expression are needed for E5- and E8-transfected cells to acquire a malignant phenotype.

Rabbit genital tissue is susceptible to infection by rabbit oral papillomavirus: an animal model for a genital tissue-targeting papillomavirus.

Rabbit oral papillomavirus (ROPV) is a mucosatropic papillomavirus which naturally infects oral mucosal sites of domestic rabbits. In this study, we tested the hypothesis that rabbit genital mucosa is also susceptible to ROPV infection by using the athymic mouse xenograft system and adult immunocompetent rabbits. Subrenal xenografts of ROPV-infected rabbit vulvar and penile sheath tissues were strongly positive for ROPV infection by histologic, in situ hybridization, and Southern analyses. Direct inoculation of adult rabbit penises with infectious ROPV produced small raised lesions of approximately 1 by 1 by 1 mm that were ROPV positive by both in situ hybridization and Southern analyses and were also viral capsid antigen positive by immunohistological staining. Infection of rabbit genital tissues with ROPV may be a useful animal model for the study of genital tissue-targeting papillomaviruses.

Coinfection of human foreskin fragments with multiple human papillomavirus types (HPV-11, -40, and -LVX82/MM7) produces regionally separate HPV infections within the same athymic mouse xenograft.

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.

Monoclonal antibodies to HPV-6 L1 virus-like particles identify conformational and linear neutralizing epitopes on HPV-11 in addition to type-specific epitopes on HPV-6.

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.

Surface conformational and linear epitopes on HPV-16 and HPV-18 L1 virus-like particles as defined by monoclonal antibodies.

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.

Laboratory production of infectious stocks of rabbit oral papillomavirus.

Several small, raised lesions from the underside of the tongue of domestic rabbits were isolated, and an extract prepared and tested for the presence of rabbit oral papillomavirus (ROPV). Two weeks after inoculation of this extract into the underside of rabbit tongues, multiple small discrete, grey-white nodules were observed that reached a maximum size of 2 mm in diameter by 5 weeks. These lesions showed typical ROPV pathology, and nuclei stained positive for papillomavirus (PV) group-specific antigen (GSA) by immunocytochemistry. Tissue fragments from rabbit tongues were incubated with a suspension of ROPV and placed subrenally into athymic mice. After 60 days, cysts were removed, sections cut for histology, and a virus stock prepared. GSA staining and in situ hybridization demonstrated that the xenografts were morphologically transformed with areas showing strong nuclear staining for viral capsid antigen and ROPV DNA. Extracts prepared from the pooled xenografts contained infectious ROPV as demonstrated by inoculation into the undersurface of tongues of nonimmune New Zealand White rabbits. The results demonstrated that stocks of infectious ROPV can be prepared in the athymic mouse xenograft system for use in studies on the experimental transmission of a mucosal-targeting animal papillomavirus.

Conserved features in papillomavirus and polyomavirus capsids.

Capsids of papilloma and polyoma viruses (papovavirus family) are composed of 72 pentameric capsomeres arranged on a skewed icosahedral lattice (triangulation number of seven, T = 7). Cottontail rabbit papillomavirus (CRPV) was reported previously to be a T = 7laevo (left-handed) structure, whereas human wart virus, simian virus 40, and murine polyomavirus were shown to be T = 7dextro (right-handed). The CRPV structure determined by cryoelectron microscopy and image reconstruction was similar to previously determined structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1). CRPV capsids were observed in closed (compact) and open (swollen) forms. Both forms have star-shaped capsomeres, as do BPV-1 and HPV-1, but the open CRPV capsids are approximately 2 nm larger in radius. The lattice hands of all papillomaviruses examined in this study were found to be T = 7dextro. In the region of maximum contact, papillomavirus capsomeres interact in a manner similar to that found in polyomaviruses. Although papilloma and polyoma viruses have differences in capsid size (approximately 60 versus approximately 50 nm), capsomere morphology (11 to 12 nm star-shaped versus 8 nm barrel-shaped), and intercapsomere interactions (slightly different contacts between capsomeres), papovavirus capsids have a conserved, 72-pentamer, T = 7dextro structure. These features are conserved despite significant differences in amino acid sequences of the major capsid proteins. The conserved features may be a consequence of stable contacts that occur within capsomeres and flexible links that form among capsomeres.

Immunization with viruslike particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus.

Rabbits were immunized with recombinant baculovirus-produced virus-like particles (VLPs) of cottontail rabbit papillomavirus (CRPV) to determine whether these antigens could induce long-term protection against experimental challenge with CRPV. Infectious CRPV and human papillomavirus type 11 L1 VLPs were used as positive and negative control immunogens, respectively. Three groups of immunized animals were challenged with 10-fold serial dilutions of infectious CRPV at 2 weeks, 6 months, and 12 months after immunizations. Antibody titers in serum reached 1:10,000 immediately after the final booster immunization and then decayed to 1:150 at 6 months and 1:100 at 12 months in unchallenged rabbits. Serum neutralization titers followed similar kinetics. Papillomas grew on control-immunized rabbits at sites challenged with 10(-1) (100% of sites), 10(-2) (96% of sites), 10(-3) (63% of sites), and 10(-4) (13% of sites) dilutions of virus. At 2 weeks after CRPV L1 VLP immunizations, the rabbits were completely protected against virus challenge. At both 6 and 12 months after CRPV L1 VLP immunizations, strong protection was also observed. In the last two groups, three of seven rabbits were completely protected and only 4 of 14 or 29% of sites challenged with 10(-1 dilution of virus grew papillomas. Papillomas growing at these four sites were also reduced in size (3.5 +/- 0.7 mm) at 50 days postchallenge compared with sites challenged with 10(-1) dilution on control-immunized rabbits (13.2 +/- 4.2 mm). The results demonstrate that strong and long-lasting protection against experimental challenge with papillomaviruses can be achieved with VLP immunogens.

High efficiency induction of papillomas in vivo using recombinant cottontail rabbit papillomavirus DNA.

Plasmids containing cottontail rabbit papillomavirus (CRPV) DNA can induce papillomas in vivo, but efficiency has been low. The aim of the present investigation was to explore some of the technical variables involved in inoculation of rabbits with recombinant CRPV DNA in attempts to improve both yield and consistency of papilloma induction. It was found that induction of epidermal hyperplasia, with either a mixture of turpentine and acetone or phorbol esters, produced a marked increase in papilloma yield. An additional powerful factor was the use of very vigorous, cutaneous scarification, sufficient to penetrate the papillary dermis and produce bleeding. When used in combination, papilloma yields were consistent and often reached 90-100% of inoculated sites. A number of other variables which did not consistently affect papilloma yield were tested. These included bleb and puncture injections, plasmid dose, vector type, occlusive dressings, lipofection reagent, carrier DNA, and different methods for plasmid DNA extraction and purification. It is concluded that the most important variables in improving papilloma yields were prior induction of epidermal hyperplasia and vigorous cutaneous scarification.

Induction of neutralizing antibodies to papillomaviruses by anti-idiotypic antibodies.

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 by monoclonal and polyclonal antibodies.

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.

Assembled baculovirus-expressed human papillomavirus type 11 L1 capsid protein virus-like particles are recognized by neutralizing monoclonal antibodies and induce high titres of 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.

Podofilox-induced regression of Shope papillomas may be independent of host immunity.

We tested the hypothesis that infiltrating leukocytes might contribute to papilloma destruction following podofilox treatment. New Zealand White (NZW) rabbits were inoculated with cottontail rabbit papillomavirus (CRPV) onto abraded areas of the dorsal skin. At 21 d after viral inoculation, 5.0% podofilox solution was applied to some papillomas, whereas others were used as controls. Three rabbits were sacrificed at each of three different periods after treatment initiation (1, 4, and 7 d). Four monoclonal antibodies (MoAbs), RG-16 (for B cells), L11/135 (specific for T cells), 2C4 (specific for class II antigen), and Ki67 (specific for proliferating cells), were used in an immunohistochemical study. All positive cells and total cells in the field were counted with an ocular grid. After 1 d of treatment, proliferation of papilloma cells was strongly suppressed in treated papillomas, but leukocytic infiltration was not altered. At 4 d and 7 d of treatment, there were substantial increases (about two to three times) in the numbers of B and T cells and class II-expressing leukocytes. The upper layers of the papillomas were highly necrotic and cell proliferation was absent in all layers. These data support the view that podofilox has a direct toxic effect on papilloma tissue. Leukocyte infiltration is not strongly associated with papilloma tissue and may not contribute to papilloma destruction.