Modeling HPV-Associated Disease and Cancer Using the Cottontail Rabbit Papillomavirus.

Approximately 5% of all human cancers are attributable to human papillomavirus (HPV) infections. HPV-associated diseases and cancers remain a substantial public health and economic burden worldwide despite the availability of prophylactic HPV vaccines. Current diagnosis and treatments for HPV-associated diseases and cancers are predominantly based on cell/tissue morphological examination and/or testing for the presence of high-risk HPV types. There is a lack of robust targets/markers to improve the accuracy of diagnosis and treatments. Several naturally occurring animal papillomavirus models have been established as surrogates to study HPV pathogenesis. Among them, the Cottontail rabbit papillomavirus (CRPV) model has become known as the gold standard. This model has played a pivotal role in the successful development of vaccines now available to prevent HPV infections. Over the past eighty years, the CRPV model has been widely applied to study HPV carcinogenesis. Taking advantage of a large panel of functional mutant CRPV genomes with distinct, reproducible, and predictable phenotypes, we have gained a deeper understanding of viral-host interaction during tumor progression. In recent years, the application of genome-wide RNA-seq analysis to the CRPV model has allowed us to learn and validate changes that parallel those reported in HPV-associated cancers. In addition, we have established a selection of gene-modified rabbit lines to facilitate mechanistic studies and the development of novel therapeutic strategies. In the current review, we summarize some significant findings that have advanced our understanding of HPV pathogenesis and highlight the implication of the development of novel gene-modified rabbits to future mechanistic studies.

Cottontail rabbit papillomavirus E6 proteins: Interaction with MAML1 and modulation of the Notch signaling pathway.

Animal models are necessary to study how cutaneous human papillomaviruses (HPVs) are associated with carcinogenesis. The cottontail rabbit papillomavirus (CRPV) induces papilloma in the -cutaneous skin of rabbits and serves as an established animal model for HPVlinked carcinogenesis where viral E6 proteins play crucial roles. Several studies have reported the dysregulation of the Notch signaling pathway by cutaneous beta HPV, bovine PV and mouse PV E6 via their association with Mastermind-like 1 protein (MAML1), thus interfering with cell proliferation and differentiation. However, the CRPV E6 gene encodes an elongated E6 protein (long E6, LE6) and an N-terminally truncated product (short E6, SE6) making it unique from other E6 proteins. Here, we describe the interaction between both CRPV E6 proteins and MAML1 and their ability to downregulate the Notch signaling pathway which could be a way CRPV infection induces carcinogenesis similar to beta HPV.

Orf Virus-Based Therapeutic Vaccine for Treatment of Papillomavirus-Induced Tumors.

Orf virus (ORFV) represents a suitable vector for the generation of efficient, prophylactic antiviral vaccines against different pathogens. The present study investigated for the first time the therapeutic application of ORFV vector-based vaccines against tumors induced by cottontail rabbit papillomavirus (CRPV). ORFV-CRPV recombinants were constructed expressing the early CRPV gene E1, E2, E7, or LE6. In two independent experiments we used in total 23 rabbits which were immunized with a mixture of the four ORFV-CRPV recombinants or empty ORFV vector as a control 5 weeks after the appearance of skin tumors. For the determination of the therapeutic efficacy, the subsequent growth of the tumors was recorded. In the first experiment, we could demonstrate that three immunizations of rabbits with high tumor burden with the combined four ORFV-CRPV recombinants resulted in significant growth retardation of the tumors compared to the control. A second experiment was performed to test the therapeutic effect of 5 doses of the combined vaccine in rabbits with a lower tumor burden than in nonimmunized rabbits. Tumor growth was significantly reduced after immunization, and one vaccinated rabbit even displayed complete tumor regression until the end of the observation period at 26 weeks. Results of delayed-type hypersensitivity (DTH) skin tests suggest the induction of a cellular immune response mediated by the ORFV-CRPV vaccine. The data presented show for the first time a therapeutic potential of the ORFV vector platform and encourage further studies for the development of a therapeutic vaccine against virus-induced tumors.IMPORTANCE Viral vectors are widely used for the development of therapeutic vaccines for the treatment of tumors. In our study we have used Orf virus (ORFV) strain D1701-V for the generation of recombinant vaccines expressing cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, LE6, and E7. The therapeutic efficacy of the ORFV-CRPV vaccines was evaluated in two independent experiments using the outbred CRPV rabbit model. In both experiments the immunization achieved significant suppression of tumor growth. In total, 84.6% of all outbred animals benefited from the ORFV-CRPV vaccination, showing reduction in tumor size and significant tumor growth inhibition, including one animal with complete tumor regression without recurrence.

Cottontail Rabbit Papillomavirus E1 and E2 Proteins Mutually Influence Their Subcellular Localizations.

The papillomavirus (PV) E2 protein is a nuclear, sequence-specific DNA-binding protein that regulates transcription and nuclear retention of viral genomes. E2 also interacts with the viral E1 protein to replicate the viral genome. E2 residue K111 is highly conserved among PV and has been implicated in contributing to nuclear transport, transcription, and replication. Cottontail rabbit (Sylvilagus floridanus) PV (CRPV or SfPV1) E2 K111R, A, or Q mutations are transcription deficient and localized to the cytoplasm, comparable to other PV types. The addition of a nuclear localization signal (NLS) resulted in nuclear E2 K111 mutant proteins but did not restore transcriptional activation, and this is most likely due to an impaired binding to the cellular Brd4 protein. Surprisingly, coexpression of E1 with E2 K111 mutations resulted in their nuclear localization and, for K111A and R mutations, the activation of an E1/E2-dependent reporter construct. Interestingly, the nuclear localization of E2 K111Q mutant protein was independent from the presence of the conserved bipartite NLS in E1 and the direct interaction between E1 and E2. On the other hand, the cytoplasmic E1 NLS mutation could be targeted to the nucleus by wild-type E2, and this was dependent upon an interaction between E1 and E2. In summary, our studies have uncovered that E1 and E2 control each other's subcellular localization: direct binding of E2 to E1 can direct E1 to the nucleus independently from the E1 NLS, and E1 can direct E2 to the nucleus without an intact NLS or direct binding to E2.IMPORTANCE Papillomaviruses encode the DNA-binding E1 and E2 proteins, which form a complex and are essential for genome replication. Both proteins are targeted to the nucleus via nuclear localization signals. Our studies have uncovered that cytoplasmic mutant E1 or E2 proteins can be localized to the nucleus when E1 or E2 is also present. An interaction between E1 and E2 is necessary to target cytoplasmic E1 mutant proteins to the nucleus, but cytoplasmic E2 mutant proteins can be targeted to the nucleus without a direct interaction, which points to a novel function of E1.

Recent advances in preclinical model systems for papillomaviruses.

Preclinical model systems to study multiple features of the papillomavirus life cycle have greatly aided our understanding of Human Papillomavirus (HPV) biology, disease progression and treatments. The challenge to studying HPV in hosts is that HPV along with most PVs are both species and tissue restricted. Thus, fundamental properties of HPV viral proteins can be assessed in specialized cell culture systems but host responses that involve innate immunity and host restriction factors requires preclinical surrogate models. Fortunately, there are several well-characterized and new animal models of papillomavirus infections that are available to the PV research community. Old models that continue to have value include canine, bovine and rabbit PV models and new rodent models are in place to better assess host-virus interactions. Questions arise as to the strengths and weaknesses of animal PV models for HPV disease and how accurately these preclinical models predict malignant progression, vaccine efficacy and therapeutic control of HPV-associated disease. In this review, we examine current preclinical models and highlight the strengths and weaknesses of the various models as well as provide an update on new opportunities to study the numerous unknowns that persist in the HPV research field.

RNA sequencing analysis identifies novel spliced transcripts but does not indicate quantitative or qualitative changes of viral transcripts during progression of cottontail rabbit papillomavirus-induced tumours.

Persistent infections with high-risk human papillomaviruses (HPVs) can result in the development of cancer of the cervix uteri and other malignancies. The underlying molecular mechanisms leading to the progression of HPV-induced lesions are, however, not well understood. Cottontail rabbit papillomavirus (CRPV) induces papillomas in domestic rabbits which progress at a very high rate to cancer. Using this model, we compared the transcriptional patterns of CRPV in papillomas and carcinomas by RNA sequencing (RNA-seq). The most abundant transcripts can encode E7, short E6 and E1^E4, followed by full-length E6, E2, E1 and E9^E2C. In addition, we identified two rare, novel splice junctions 7810/3714 and 1751/3065 in both papillomas and carcinomas which have been described for other papillomaviruses. Neither RNA-seq nor quantitative real-time PCR-based assays identified qualitative or quantitative changes of viral transcription between papillomas and carcinomas. In summary, our analyses confirmed that papillomaviruses have highly similar transcriptional patterns, but they do not suggest that changes in these patterns contribute to the progression of CRPV-induced tumours.

A Century of Shope Papillomavirus in Museum Rabbit Specimens.

Sylvilagus floridanus Papillomavirus (SfPV) causes growth of large horn-like tumors on rabbits. SfPV was described in cottontail rabbits (probably Sylvilagus floridanus) from Kansas and Iowa by Richard Shope in 1933, and detected in S. audubonii in 2011. It is known almost exclusively from the US Midwest. We explored the University of Kansas Natural History Museum for historical museum specimens infected with SfPV, using molecular techniques, to assess if additional wild species host SfPV, and whether SfPV occurs throughout the host range, or just in the Midwest. Secondary aims were to detect distinct strains, and evidence for strain spatio-temporal specificity. We found 20 of 1395 rabbits in the KU collection SfPV symptomatic. Three of 17 lagomorph species (S. nuttallii, and the two known hosts) were symptomatic, while Brachylagus, Lepus and eight additional Sylvilagus species were not. 13 symptomatic individuals were positive by molecular testing, including the first S. nuttallii detection. Prevalence of symptomatic individuals was significantly higher in Sylvilagus (1.8%) than Lepus. Half of these specimens came from Kansas, though new molecular detections were obtained from Jalisco-Mexico's first-and Nebraska, Nevada, New Mexico, and Texas, USA. We document the oldest lab-confirmed case (Kansas, 1915), pre-dating Shope's first case. SfPV amplification was possible from 63.2% of symptomatic museum specimens. Using multiple methodologies, rolling circle amplification and, multiple isothermal displacement amplification in addition to PCR, greatly improved detection rates. Short sequences were obtained from six individuals for two genes. L1 gene sequences were identical to all previously detected sequences; E7 gene sequences, were more variable, yielding five distinct SfPV1 strains that differing by less than 2% from strains circulating in the Midwest and Mexico, between 1915 and 2005. Our results do not clarify whether strains are host species specific, though they are consistent with SfPV specificity to genus Sylvilagus.

Evaluation of positron emission tomographic tracers for imaging of papillomavirus-induced tumors in rabbits.

In this study, simultaneous positron emission tomography (PET)/magnetic resonance (MR) imaging was employed to evaluate the feasibility of the PET tracers 2-deoxy-2-18F-fluoro-d-glucose (18F-FDG), 11C-choline, and 18F-fluorothymidine (18F-FLT) to detect papillomavirus-induced tumors in an established rabbit model system. The combined PET/MR allowed the analysis of tracer uptake of the tumors using the morphologic information acquired by MR. New Zealand White rabbits were infected with cottontail rabbit papillomavirus genomes and were imaged for up to 10 months with a simultaneous PET/MR system during the course of infection. The uptake characteristics of the PET tracers 11C-choline and 18F-FLT of tumors and reference tissues were examined relative to the clinical standard, 18F-FDG. Tracer biodistribution of various organs was measured by gamma-counting after the last PET scan and compared to the in vivo PET/MR 18F-FDG uptake. Increased tracer uptake was found 2 months postinfection in primary tumors with 18F-FDG and 11C-choline, whereas 18F-FLT failed to detect the tumors at all measured time points. Our data show that the PET tracer 18F-FDG is superior for imaging papillomavirus-induced tumors in rabbits compared to 11C-choline and 18F-FLT. However, 11C-choline imaging, which has previously been applied to detect various tumor entities in patients, appears to be an alternative to 18F-FDG.

Synonymous codon changes in the oncogenes of the cottontail rabbit papillomavirus lead to increased oncogenicity and immunogenicity of the virus.

Papillomaviruses use rare codons with respect to the host. The reasons for this are incompletely understood but among the hypotheses is the concept that rare codons result in low protein production and this allows the virus to escape immune surveillance. We changed rare codons in the oncogenes E6 and E7 of the cottontail rabbit papillomavirus to make them more mammalian-like and tested the mutant genomes in our in vivo animal model. While the amino acid sequences of the proteins remained unchanged, the oncogenic potential of some of the altered genomes increased dramatically. In addition, increased immunogenicity, as measured by spontaneous regression, was observed as the numbers of codon changes increased. This work suggests that codon usage may modify protein production in ways that influence disease outcome and that evaluation of synonymous codons should be included in the analysis of genetic variants of infectious agents and their association with disease.

A recombinant cottontail rabbit papillomavirus genome for ectopic expression of genes in cells infected with virus in vivo.

The objective of this study was to construct a cottontail rabbit papillomavirus (CRPV) genome that would co-express a gene of choice and the viral genome simultaneously. Using this construct, the effects of the ectopic expression of diverse viral or cellular genes on PV-infected cells can be examined to elucidate which genes are essential for tumor formation. CRPV-pLAIIdelXba1, which lacks the major portion of L2 (designated the XbaI fragment), has been previously shown to fully retain the ability to induce tumors, and this ability was confirmed in this study. Insertion of the XbaI fragment in an antisense orientation did not change the efficiency of tumor induction. An SV40 overexpression cassette that originated from pSG5 and contains a more diverse multiple cloning site (MCS) was cloned into CRPV-Xba1-mcs, a CRPV genome based on CRPV-pLAIIdelXba1 that contains an additional MCS inserted via XbaI digestion. Additionally, the L1 ATG initiation codon of this construct, designated CRPV-Xba1-oe-WT, was mutated to avoid unnecessary L1 protein expression, which produced the CRPV-Xba1-oe-L1mut construct. Injection of these constructs into two New Zealand White rabbits and monitoring of tumor growth for two to six months showed that CRPV-Xba1-oe-WT induced tumors at 1/10 and 1/10 of the injection sites in two animals, while the control injections in each rabbit induced tumors at 3/10 and 4/10 injection sites, respectively. However, CRPV-Xba1-oe-L1mut induced tumors at 3/10, 6/10, 7/12 and 11/12 sites in four injected animals, and the control injections induced tumor growth in these animals at 6/10, 10/10, 12/12 and 12/12 of the injected sites, respectively. Thus, CRPV-Xba1-oe-L1mut could potentially be used to conduct overexpression experiments in vivo that can be used to measure the negative or positive influences of ectopically expressed foreign or HPV genes on tumor growth.

Vaccine generated immunity targets an HPV16 E7 HLA-A2.1-restricted CD8(+) T cell epitope relocated to an early gene or a late gene of the cottontail rabbit papillomavirus (CRPV) genome in HLA-A2.1 transgenic rabbits.

The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well known and computer-predicted A2-restricted epitopes. In the current study we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope that has been relocated to positions within the CRPV E7 gene and the CRPV L2 gene. Epitope expression from both the E7 protein and the L2 protein resulted in increased protection against viral DNA challenge of the HLA-A2.1 transgenic rabbits as compared to control-vaccinated rabbit groups. These data indicate that proteins expressed at both early and late time points during a natural papillomavirus infection can be targeted by epitope-specific immunity and indicate this immunity is increased to early rather than late expressed proteins of papillomaviruses. This study also highlights the broad utility of the HLAA2.1 transgenic rabbit model for testing numerous immunological factors involved in vaccine generated protective immunity.

Papillomavirus infection requires gamma secretase.

The mechanism by which papillomaviruses breach cellular membranes to deliver their genomic cargo to the nucleus is poorly understood. Here, we show that infection by a broad range of papillomavirus types requires the intramembrane protease γ secretase. The γ-secretase inhibitor (S,S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide (compound XXI) inhibits infection in vitro by all types of papillomavirus pseudovirions tested, with a 50% inhibitory concentration (IC(50)) of 130 to 1,000 pM, regardless of reporter construct and without impacting cellular viability. Conversely, XXI does not inhibit in vitro infection by adenovirus or pseudovirions derived from the BK or Merkel cell polyomaviruses. Vaginal application of XXI prevents infection of the mouse genital tract by human papillomavirus type 16 (HPV16) pseudovirions. Nicastrin and presenilin-1 are essential components of the γ-secretase complex, and mouse embryo fibroblasts deficient in any one of these components were not infected by HPV16, whereas wild-type and ß-secretase (BACE1)-deficient cells were susceptible. Neither the uptake of HPV16 into Lamp-1-positive perinuclear vesicles nor the disassembly of capsid to reveal both internal L1 and L2 epitopes and bromodeoxyuridine (BrdU)-labeled encapsidated DNA is dependent upon γ-secretase activity. However, blockade of γ-secretase activity by XXI prevents the BrdU-labeled DNA encapsidated by HPV16 from reaching the ND10 subnuclear domains. Since prior studies indicate that L2 is critical for endosomal escape and targeting of the viral DNA to ND10 and that γ secretase is located in endosomal membranes, our findings suggest that either L2 or an intracellular receptor are cleaved by γ secretase as papillomavirus escapes the endosome.

Papillomavirus DNA complementation in vivo.

Recent phylogenic studies indicate that DNA recombination could have occurred in ancient papillomavirus types. However, no experimental data are available to demonstrate this event because of the lack of human papillomavirus infection models. We have used the cottontail rabbit papillomavirus (CRPV)/rabbit model to study pathogenesis and immunogenicity of different mutant genomes in vivo. Although the domestic rabbit is not a natural host for CRPV infection, it is possible to initiate infection with naked CRPV DNA cloned into a plasmid and monitor papilloma outgrowth on these animals. Taking advantage of a large panel of mutants based on a CRPV strain (Hershey CRPV), we tested the hypothesis that two non-viable mutant genomes could induce papillomas by either recombination or complementation. We found that co-infection with a dysfunctional mutant with an E2 transactivation domain mutation and another mutant with an E7 ATG knock out generated papillomas in rabbits. DNA extracted from these papillomas contained genotypes from both parental genomes. Three additional pairs of dysfunctional mutants also showed similar results. Individual wild type genes were also shown to rescue the function of corresponding dysfunctional mutants. Therefore, we suggest that complementation occurred between these two non-viable mutant PV genomes in vivo.

AP-1 and ERK1 but not p38 nor JNK is required for CRPV early protein 2-dependent MMP-9 promoter activation in rabbit epithelial cells.

Human papillomaviruses (HPV) are the known cause for a variety of cancers including cervical and epithelial cancers. The cottontail-rabbit papillomavirus (CRPV) serves as a suitable animal model to study the development of these cancers in vivo. We have previously demonstrated that CRPV-induced skin carcinomas express high levels of MMP-9, a metalloproteinase contributing to cancer progression by extracellular matrix remodelling. Based on our previously reported finding that CRPV early protein 2 can activate a truncated human 670bp MMP-9 promoter fragment, we hypothesized that MMP-9 expression in the rabbit carcinomas is a consequence of activation of the rabbit MMP-9 promoter in-trans by CRPV early protein 2. Further elucidation of the mechanism revealed the requirement for both a proximal and distal AP-1 transcription factor binding site in the rabbit MMP-9 promoter and the AP-1 complex as demonstrated by the inhibitory effect of TAM67, a trans-activation deficient c-jun mutant. The characterization of signal-transduction requirements revealed predominantly ERK1 to be required for CRPV early protein 2-dependent MMP-9 promoter activation, but not JNK nor p38. In summary CRPV early protein 2 activates the expression of MMP-9 in-trans through AP-1 and ERK1 and may contribute to cancer development and progression via this mechanism within the animal model.

CRPV genomes with synonymous codon optimizations in the CRPV E7 gene show phenotypic differences in growth and altered immunity upon E7 vaccination.

Papillomaviruses use rare codons relative to their hosts. Recent studies have demonstrated that synonymous codon changes in viral genes can lead to increased protein production when the codons are matched to those of cells in which the protein is being expressed. We theorized that the immunogenicity of the virus would be enhanced by matching codons of selected viral genes to those of the host. We report here that synonymous codon changes in the E7 oncogene are tolerated in the context of the cottontail rabbit papillomavirus (CRPV) genome. Papilloma growth rates differ depending upon the changes made indicating that synonymous codons are not necessarily neutral. Immunization with wild type E7 DNA yielded significant protection from subsequent challenge by both wild type and codon-modified genomes. The reduction in growth was most dramatic with the genome containing the greatest number of synonymous codon changes.

The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP.

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19(ARF) induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53.

Persistent conjunctival papilloma due to oral papillomavirus infection in a rabbit in New Zealand.

A 3-yr-old female Flemish Giant pet rabbit developed a papilloma on the right nictitating membrane. Although the papilloma was excised surgically, it promptly recurred. Examination of the eye 10 wk after surgery revealed that in addition to the initial mass, 2 smaller papillomas were present on the lower eyelid. All 3 masses were excised, and histology revealed papillomatous hyperplasia of the conjunctival epithelium, koilocytosis, and intranuclear viral inclusions. Polymerase chain reaction amplified papillomaviral DNA from the largest papilloma. Sequencing of the amplicon revealed 99.3% homology with rabbit oral papillomavirus (ROPV). All 3 masses recurred after removal. In addition, the rabbit was noted to be losing weight. Weight loss continued until the rabbit died 3 mo later. All 3 papillomas persisted until death. This article provides the fi rst description of ROPV causing conjunctival papillomas and is the fi rst report of ROPV from the southern hemisphere. The persistence of the papillomas in this case is also unusual and may suggest that ROPV-induced conjunctival papillomas are less likely than oral papillomas to spontaneously regress. Alternatively, the death of this rabbit may indicate a compromised immune system that allowed papillomaviral persistence.

Therapeutic vaccination of rabbits with a ubiquitin-fused papillomavirus E1, E2, E6 and E7 DNA vaccine.

Previously, we showed that intracutaneous vaccination of rabbits with DNA vectors encoding ubiquitin-fused versions of the cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, E6 and E7 protected against subsequent challenge with CRPV. Here, we tested the immunotherapeutic activity of a vaccine composed of the four CRPV DNA vectors (designated UbE1267) in rabbits. The results show that the UbE1267 DNA vaccine, relative to empty vector DNA, virtually eliminated papilloma growth in rabbits with subclinical infection and greatly reduced papilloma volumes in rabbits bearing papillomas at the time of vaccination. These results in a physiologically relevant animal model of high-risk human papillomavirus (HPV) infection indicate that DNA vaccines targeting the early papillomavirus proteins may have a role in the treatment of HPV-associated lesions in humans.

Rabbits immunised with recombinant BCG expressing the cottontail rabbit papillomavirus (CRPV) L2E7E2 genes induces regression of established papillomas.

We previously demonstrated in a cottontail rabbit papillomavirus (CRPV) challenge model that recombinant Bacille Calmette-Guerin (rBCG) could potentially be used as a prophylactic vaccine vehicle to deliver papillomavirus proteins. In this study we investigated whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with rBCG expressing CRPVL2, CRPVE2, CRPVE7 or CRPVL2E7E2 proteins. Rabbits immunised with rBCG/CRPVL2E7E2 had papillomas that were largely suppressed and were significantly smaller compared to the rBCG negative control group (P

Impact of genetic changes to the CRPV genome and their application to the study of pathogenesis in vivo.

The cottontail rabbit papillomavirus (CRPV)/rabbit model has been used to study oncogenicity and immunogenicity of different antigens from the papillomavirus genome and has therefore served as a preclinical model for the development of preventive and therapeutic vaccines against papillomavirus infections. One unique property of the CRPV model is that infection can be initiated using viral DNA. This property allows for the functional testing of viral mutants in vivo. We have introduced point mutations, insertions and deletions into all of the different coding and non-coding regions of the CRPV genome and have tested their infectivity in this model. We found that the majority of the mutant genomes retained viability and could induce papillomas in domestic rabbits. These data indicated that the CRPV genome is tolerant of many modifications without compromising its ability to initiate skin papillomas. In combination with our recently established HLA-A2.1 transgenic rabbit model, this plasticity allows us to extend the utility of the CRPV/rabbit model to the screening of HLA-A2.1 restricted epitopes from other human viral and tumor antigens.