Cottontail Rabbit Papillomavirus Research Articles

Cottontail Rabbit Papillomavirus (CRPV) Related Animal Models for Head and Neck Cancer Research: A Comprehensive Review of the Literature

Having suitable animal models is crucial to mimic human disease states and for the successful transfer of experimental data into clinical practice. In the field of papillomavirus research, the domestic rabbit (Oryctolagus cuniculus) has served as an indispensable model organism for almost 100 years. The identification and characterization of the first papillomaviruses in rabbits, their carcinogenic potential and their immunogenicity have contributed significantly to the state of knowledge on the genetics and life cycle of papillomaviruses in general, as well as the development of antiviral strategies such as vaccination procedures. Due to the high species specificity of papillomaviruses, only rabbit papillomaviruses (RPVs) can be used for animal studies on papilloma-based tumor diseases in the rabbit. The major focus of this article is on cottontail rabbit papillomavirus (CRPV)-related rabbit squamous cell carcinoma (RSCC). A brief history outlines the discovery and generation of experimentally used RSCC tumors. A comprehensive overview of the current CRPV-associated VX2 carcinoma-based tumor models with a major focus on human head and neck squamous cell carcinoma (HNSCC) tumor models is provided, and their strengths in terms of transferability to human HNSCC are discussed.

The full transcription map of cottontail rabbit papillomavirus in tumor tissues.

Cottontail rabbit papillomavirus (CRPV), the first papillomavirus associated with tumor development, has been used as a powerful model to study papillomavirus pathogenesis for more than 90 years. However, lack of a comprehensive analysis of the CRPV transcriptome has impeded the understanding of CRPV biology and molecular pathogenesis. Here, we report the construction of a complete CRPV transcription map from Hershey CRPV-induced skin tumor tissues. By using RNA-seq in combination with long-reads PacBio Iso-seq, 5' and 3' RACE, primer-walking RT-PCR, Northern blot, and RNA in situ hybridization, we demonstrated that the CRPV genome transcribes its early and late RNA transcripts unidirectionally from at least five distinct major promoters (P) and polyadenylates its transcripts at two major polyadenylation (pA) sites. The viral early transcripts are primarily transcribed from three "early" promoters, P90, P156, and P907 and polyadenylated at nt 4368 by using an early polyadenylation signal (PAS) at nt 4351. Like other low-risk human papillomaviruses and animal papillomaviruses, CRPV E6 and E7 transcripts are transcribed from three separate early promoters. Transcripts from two "late" promoters, P7525, and P1225, utilize either an early PAS for E1^E4 or a late PAS at 7399 for L2 and L1 RNA polyadenylation at nt 7415 to express capsid L2 and L1 proteins respectively. By using the mapped four 5' splice sites and three 3' splice sites, CRPV RNA transcripts undergo extensive alternative splicing to produce more than 33 viral RNA isoforms for production of at least 12 viral proteins, some of which without codon optimization are expressible in rabbit RK13 and human HEK293T cells. The constructed full CRPV transcription map in this study for the first time will enhance our understanding of the structures and expressions of CRPV genes and their contribution to molecular pathogenesis and tumorigenesis.

RNAi-Mediated Knockdown of Cottontail Rabbit Papillomavirus Oncogenes Using Low-Toxicity Lipopolyplexes as a Paradigm to Treat Papillomavirus-Associated Cancers.

The cottontail rabbit papillomavirus (CRPV)-associated VX2 carcinoma of the New Zealand White rabbit serves as a model system for human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCCs). The aim of this study was to evaluate the tumor-inhibiting effect of RNAi-mediated knockdown of the CRPV oncogenes, E6 and E7, using siRNA-loaded lipopolyplexes (LPPs). VX2-carcinoma-derived cells were cultured for up to 150 passages. In addition, CRPV E6 and E7 oncogenes were transiently expressed in COS-7 cells. Efficiency and safety of LPPs were evaluated in both VX2 cells and the COS-7 cell line. Both of these in vitro CRPV systems were validated and characterized by fluorescence microscopy, Western blot, and RT-qPCR. Efficient knockdown of CRPV E6 and E7 was achieved in VX2 cells and COS-7 cells pretransfected with CRPV E6 and E7 expression vectors. Knockdown of CRPV oncogenes in VX2 cells resulted in reduced viability, migration, and proliferation and led to a G0/G1 block in the cell cycle. CRPV E6 and E7 siRNA-loaded LPPs could represent promising therapeutic agents serving as a paradigm for the treatment of papillomavirus-positive cancers and could be of value for the treatment of CRPV-associated diseases in the rabbit such as papillomas and cancers of the skin.

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.

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.

A Comparative Study on Delivery of Externally Attached DNA by Papillomavirus VLPs and Pseudoviruses.

Human papillomavirus (HPV) 16 capsids have been chosen as a DNA delivery vehicle in many studies. Our preliminary studies suggest that HPV58 capsids could be better vehicles than HPV16 capsids to deliver encapsidated DNA in vitro and in vivo. In the current study, we compared HPV16, HPV58, and the cottontail rabbit papillomavirus (CRPV) capsids either as L1/L2 VLPs or pseudoviruses (PSVs) to deliver externally attached GFP-expressing DNA. Both rabbit and human cells were used to test whether there was a species-specific effect. DNA delivery efficiency was determined by quantifying either GFP-expressing cell populations or mean fluorescent intensities (MFI) by flow cytometry. Interestingly, CRPV and 58-VLPs and PSVs were significantly more efficient at delivering attached DNA when compared to 16-VLPs and PSVs. A capsid/DNA ratio of 2:1 showed the highest efficiency for delivering external DNA. The PSVs with papillomavirus DNA genomes also showed higher efficiency than those with irrelevant plasmid DNA. HPV16L1/58L2 hybrid VLPs displayed increased efficiency compared to HPV58L1/16L2 VLPs, suggesting that L2 may play a critical role in the delivery of attached DNA. Additionally, we demonstrated that VLPs increased in vivo infectivity of CRPV DNA in rabbits. We conclude that choosing CRPV or 58 capsids to deliver external DNA could improve DNA uptake in in vitro and in vivo models.

Not Just Uterine Adenocarcinoma—Neoplastic and Non-Neoplastic Masses in Domestic Pet Rabbits (Oryctolagus cuniculus): A Review

With increasing numbers of pet rabbits living out their natural lifespan, rabbit oncology is stepping more and more into the limelight. On the other hand, rabbit tumors are less covered in recent editions of textbooks of veterinary pathology than before. We present 1238 cases with neoplastic and non-neoplastic masses in rabbit tissue, submitted from 2008 to 2019, supplemented by a review of the literature on neoplasms in rabbits. Cutaneous masses comprised 47% of submissions. Trichoblastoma was by far the most common skin neoplasm, and nodular suppurative panniculitis was the second most frequent skin nodule in this series. Epithelial as well as mesenchymal cutaneous neoplasms can be virally induced in rabbits (eg, Shope papilloma, myxomatosis) but were infrequent in the current cases. Mammary neoplasms comprised 21% of submitted masses and 94% of these had histologic features of malignancy. Tumors of the female reproductive tract were responsible for 9% of biopsies and were predominantly uterine adenocarcinoma. Polypoid proliferation of rectal mucosa was the most common lesion in the alimentary tract. A broad spectrum of other neoplasms was described, including sarcomas at vaccination sites and ocular posttraumatic sarcomas, comparable to lesions described in cats.

High resolution cryo EM analysis of HPV16 identifies minor structural protein L2 and describes capsid flexibility

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. HPV is epitheliotropic and its replication is tightly associated with terminal keratinocyte differentiation making production and purification of high titer virus preparations for research problematic, therefore alternative HPV production methods have been developed for virological and structural studies. In this study we use HPV16 quasivirus, composed of HPV16 L1/L2 capsid proteins with a packaged cottontail rabbit papillomavirus genome. We have achieved the first high resolution, 3.1 Å, structure of HPV16 by using a local subvolume refinement approach. The high resolution enabled us to build L1 unambiguously and identify L2 protein strands. The L2 density is incorporated adjacent to conserved L1 residues on the interior of the capsid. Further interpretation with our own software for Icosahedral Subvolume Extraction and Correlated Classification revealed flexibility, on the whole-particle level through diameter analysis and local movement with inter-capsomer analysis. Inter-capsomer expansion or contraction, governed by the connecting arms, showed no bias in the magnitude or direction of capsomer movement. We propose that papillomavirus capsids are dynamic and capsomers move as rigid bodies connected by flexible linkers. The resulting virus structure will provide a framework for continuing biochemical, genetic and biophysical research for papillomaviruses. Furthermore, our approach has allowed insight into the resolution barrier that has previously been a limitation in papillomavirus structural studies.

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.

Abstract LB-200: A cGMP-grade chimeric papillomavirus candidate vaccine (HPV16 RG1-VLP) confers long term cross-protection compared to a nonavalent hpv vaccine in a pre-clinical papillomavirus animal model

Abstract Background: The National Cancer Institute (NCI) PREVENT Cancer Program (PREVENT) is a peer-reviewed R&D pipeline with the core emphasis on preclinical development and clinical translation of novel cancer preventive interventions. One of the latest PREVENT-supported projects include cGMP production and IND enabling studies of a broad spectrum experimental human papillomavirus (HPV) vaccine- HPV16 RG1-VLP. This monovalent chimeric virus-like particle (VLP) displays 360 copies of the highly conserved epitope RG1 (aa 17-36 of minor capsid protein HPV16 L2) in the DE loop of HPV16L1 VLP backbone, and is capable of eliciting broadly neutralizing antibodies that target several clinically relevant HPV genotypes. RG-1 induced cross-neutralizing titers are typically lower than anti-L1 antibodies generated by currently licensed HPV vaccines. Hence, durability of protection has been cited as a cause of concern. Here, using engineering-run cGMP grade HPV16-RG1VLPs formulated with alhdyrogel®, the durability of protection 6 month post-vaccination of HPV16 RG1-VLPs against Gardasil-9®, a licensed HPV vaccine was evaluated using an established papillomavirus disease model. Methods: New Zealand white rabbits (n=15 per treatment group) were administered three intra-muscular vaccinations of HPV16-RG1 (80 µg), human doses of Gardasil-9®, or no vaccine (adjuvant only). Following vaccination, in vivo protection was assessed against 8 high-risk oncogenic HPVs utilizing methods from an established cottontail rabbit papillomavirus (CRPV) disease model. Within each treatment group, 5 rabbits were challenged two weeks post-final vaccination (at peak serum ELISA titer), while another 5 were challenged six months post-final vaccination to assess durability of protection. The remainder 5 rabbits will be challenged one year post final vaccination. Results: During the peak-titer period, rabbits vaccinated with monovalent HPV16-RG1VLP were protected from disease development, which was comparable to the protection afforded by Gardasil-9®. Six months after final vaccination, despite lower serum titers, HPV16-RG1VLP immunized rabbits were still protected from disease development with vaccine efficacy comparable to that of Gardasil-9®. And, in some instances, HPV16-RG1VLP vaccine demonstrated a superior cross-protection. Conclusions: Even as a monovalent formulation, HPV16 RG1 VLP vaccination was able to provide comparable protection against a number of high-risk oncogenic HPV types, including types not covered by Gardasil-9®, even after six months post-vaccination. As a monovalent VLP, HPV16 RG1 VLP holds promise as a broad-spectrum preventative vaccine candidate that could potentially provide broader protection at lower production costs. Studies evaluating protection one-year-post vaccination is currently in progress. Citation Format: Jiafen Hu, Karla Balogh, Ken Matsui, Huimin Tan, Pola Olczak, George Buchman, Brian Howard, Jonathan White, Michelle Kennedy, Shizuko Sei, Elizabeth Glaze, Sarah Brendle, Christina Schellenbacher, Reinhard Kirnbauer, Richard Roden, Robert Shoemaker, Neil Christensen, Joshua Weiyuan Wang. A cGMP-grade chimeric papillomavirus candidate vaccine (HPV16 RG1-VLP) confers long term cross-protection compared to a nonavalent hpv vaccine in a pre-clinical papillomavirus animal model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-200.

The rabbit papillomavirus model: a valuable tool to study viral-host interactions.

Cottontail rabbit papillomavirus (CRPV) was the first DNA virus shown to be tumorigenic. The virus has since been renamed and is officially known as Sylvilagus floridanus papillomavirus 1 (SfPV1). Since its inception as a surrogate preclinical model for high-risk human papillomavirus (HPV) infections, the SfPV1/rabbit model has been widely used to study viral-host interactions and has played a pivotal role in the successful development of three prophylactic virus-like particle vaccines. In this review, we will focus on the use of the model to gain a better understanding of viral pathogenesis, gene function and host immune responses to viral infections. We will discuss the application of the model in HPV-associated vaccine testing, in therapeutic vaccine development (using our novel HLA-A2.1 transgenic rabbits) and in the development and validation of novel anti-viral and anti-tumour compounds. Our goal is to demonstrate the role the SfPV1/rabbit model has played, and continues to play, in helping to unravel the intricacies of papillomavirus infections and to develop tools to thwart the disease. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.

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.

Ozone therapy prevents the onset of dysplasia in HPV16-transgenic mice—A pre-clinical efficacy and safety analysis

Infection with high-risk human papillomavirus (HPV), most often HPV16, is associated with the development of anogenital and oropharyngeal cancers. Recently, ozone therapy was reported to have considerable efficacy against rabbit VX2 tumors, induced by the cottontail rabbit papillomavirus. The present study aims to determine whether similar results can be obtained in HPV16-transgenic mice, possibly paving the way for new therapeutic options against HPV-induced cancers.HPV16-transgenic and wild-type, female, 20 weeks-old mice were injected intraperitoneally with medical O3/O2 (80░mL/kg, at O3 50░μg/mL), once a day, for 5 consecutive days. The animals were sacrificed at 25 weeks-old, and skin samples were analyzed histologically to study tumour progression. Blood and internal organ samples were used to study toxicological parameters.85.7% of untreated transgenic mice showed dysplastic skin lesions, compared with 28.6% of O3-treated mice. This was associated with a marked reduction of dermal inflammation associated with those lesions. No significant changes were observed in any toxicological parameters.These preliminary results support the hypothesis that O3 therapy is effective against papillomavirus-induced lesions, particularly against those induced by the most common high-risk virus, HPV16. Further studies are needed to confirm the mechanisms underlying these effects.

The contribution of SP100 to cottontail rabbit papillomavirus transcription and replication.

SP100 proteins are components of nuclear domain 10 structures and have been implicated as inhibitors of human papillomavirus (HPV) replication. In this study, we have addressed the role of SP100 in tumour formation by the cottontail rabbit (Sylvilagus floridanus) papillomavirus (CRPV or SfPV1) in a rabbit model. Tissue culture studies using rabbit keratinocyte lines indicated that rabbit SP100 is an interferon-beta-inducible gene similar to its human counterpart. Stable knockdown of SP100 by shRNA in a cell line harbouring CRPV genomes resulted in a decrease of viral early transcripts. In contrast, infection of domestic rabbits with recombinant CRPV genomes expressing short hairpin (sh)RNAs directed against SP100 did not reveal changes in tumour formation rate, tumour size or early viral transcript levels. However, late viral transcript levels and viral genome copies were consistently lower in CRPV/shSP100-induced tumours than in the control, but these differences did not reach statistical significance. In summary, this study suggests that rabbit SP100 is not an inhibitor but an activator of CRPV replication and transcription.

Spatiotemporal Optoacoustic Mapping of Tumor Hemodynamics in a Clinically Relevant Orthotopic Rabbit Model of Head and Neck Cancer

The purpose of this study was to investigate the usefulness of photoacoustic imaging (PAI) for spatiotemporal mapping of tumor hemodynamics in a rabbit model of head and neck carcinoma. Shope cottontail rabbit papilloma virus associated VX2 carcinomas were established in adult male New Zealand White rabbits (n = 9) by surgical transplantation of tumor tissue in the neck. Noninvasive PAI with co-registered ultrasound (US) was performed to longitudinally monitor tumor growth, oxygen saturation (%sO2), and hemoglobin concentration (HbT). PAI findings were validated with Doppler sonography measures of percent vascularity (PV). Differences in tumor volumes, %sO2, HbT, and PV values over time were analyzed using repeated-measures analysis of variance with multiple comparisons. Two-tailed Spearman correlation analysis was performed to determine the correlation coefficient (r) for comparisons between %sO2, HbT, and tumor volume. US revealed a significant (P < .0001) increase in tumor volume over the 3-week period from 549 ± 260 mm3 on day 7 to 5055 ± 438 mm3 at 21 days postimplantation. Consistent with this aggressive tumor growth, PAI revealed a significant (P < .05) and progressive reduction in %sO2 from day 7 (37.6 ± 7.4%) to day 21 (9.5 ± 2.1%). Corresponding Doppler images also showed a decrease in PV over time. PAI revealed considerable intratumoral spatial heterogeneity with the tumor rim showing two- to three-fold higher %sO2 values compared to the core. Noninvasive PAI based on endogenous contrast provides a label-free method for longitudinal monitoring of temporal changes and spatial heterogeneity in thick head and neck tumors.

Incorporation of RG1 epitope concatemers into a self-adjuvanting Flagellin-L2 vaccine broaden durable protection against cutaneous challenge with diverse human papillomavirus genotypes.

Aim:To achieve durable and broad protection against human papillomaviruses by vaccination with multimers of minor capsid antigen L2 using self-adjuvanting fusions with the toll-like receptor-5 (TLR5) ligand bacterial flagellin (Fla) instead of co-formulation with alum.Methods:Fla fusions with L2 protective epitopes comprising residues 11–200, 11–88 and/or 17–38 of a single or multiple HPV types were produced in E. coli and their capacity to activate TLR5 signaling was assessed. Immunogenicity was evaluated serially following administration of 3 intramuscular doses of Fla-L2 multimer without exogenous adjuvant, followed by challenge 1, 3, 6 or 12 months later, and efficacy compared to vaccination with human doses of L1 VLP vaccines (Gardasil and Cervarix) or L2 multimer formulated in alum. Serum antibody responses were assessed by peptide ELISA, in vitro neutralization assays and passive transfer to naïve rabbits in which End-Point Protection Titers (EPPT) were determined using serial dilutions of pooled immune sera collected 1, 3, 6 or 12 months after completing active immunization. Efficacy was assessed by determining wart volume following concurrent challenge at different sites with HPV6/16/18/31/45/58 ‘quasivirions’ containing cottontail rabbit papillomavirus (CRPV) genomes.Results:Vaccination in the absence of exogenous adjuvant with Fla-HPV16 L2 11–200 fusion protein elicited durable protection against HPV16, but limited cross-protection against other HPV types. Peptide mapping data suggested the importance of the 17–38 aa region in conferring immunity. Indeed, addition of L2 residues 17–38 of HPV6/18/31/39/52 to a Fla-HPV16 L2 11–200 or 11–88 elicited broader protection via active or passive immunization, similar to that seen with vaccination with an alum-adjuvanted L2 multimer comprising the aa 11–88 peptides of five or eight genital HPV types.Conclusions:Vaccination with flagellin fused L2 multimers provided lasting (>1 year) immunity without the need for an exogenous adjuvant. Inclusion of the L2 amino acid 17–38 region in such multi-HPV type fusions expanded the spectrum of protection.

P.2.a.009 - Visuospatial planning in unmedicated major depressive and bipolar disorder: distinct and common neural correlates

This study analyzed in vivo antiviral cellular immune reactions in the Shope rabbit papilloma – carcinoma model. Antigens studied in experimentally infected domestic rabbits were cottontail rabbit papillomavirus particles produced with the athymic (nu/nu) mouse xenograft system and bacterial fusion proteins containing the major or minor capsid protein. Recall reactions to antigens were tested by classic intracutaneous tests. Positive reactions had a biphasic course. Histopathology of skin test biopsy specimens showed infiltrating polymorphonuclear cells during the early stages. Later they were replaced by predominantly perivascular infiltrates composed of mononuclear cells. Time course of swelling and infiltrates resembled a delayed-type hypersensitivity reaction. Ten of 11 regressor rabbits (p = 0.00006) and 10 of 20 progressors (p = 0.009) had positive skin tests with intact and/or denaturated virus particles and individual capsid proteins also could elicit specific skin reactions. Skin reactivity to the cottontail rabbit papillomavirus particles was also greater (p = 0.042) in regressor rabbits (8 of 11) when compared to progressors (7 of 20). Recall reactions remained detectable at post-regression times, ranging from several months up to more than 2 years. We conclude that specific skin reactions against the cottontail rabbit papillomavirus in infected domestic rabbits exist, and are strongly positive to intact particles of this papillomavirus in animals (regressors) clinically free of disease.

Host genetic influence on papillomavirus-induced tumors in the horse.

The common equine skin tumors known as sarcoids have been causally associated with infection by bovine papillomavirus (BPV). Additionally, there is evidence for host genetic susceptibility to sarcoids. We investigated the genetic basis of susceptibility to sarcoid tumors on a cohort of 82 affected horses and 270 controls genotyped on a genome-wide platform and two custom panels. A Genome Wide Association Study (GWAS) identified candidate regions on six chromosomes. Bayesian probability analysis of the same dataset verified only the regions on equine chromosomes (ECA) 20 and 22. Fine mapping using custom-produced SNP arrays for ECA20 and ECA22 regions identified two marker loci with high levels of significance: SNP BIEC2-530826 (map position 32,787,619) on ECA20 in an intron of the DQA1 gene in the Major Histocompatibility Complex (MHC) class II region (p = 4.6e-06), and SNP BIEC2-589604 (map position 25,951,536) on ECA22 in a 200 kb region containing four candidate genes: PROCR, EDEM2, EIF6 and MMP24 (p = 2.14e-06). The marker loci yielded odds ratios of 5.05 and 4.02 for ECA20 and ECA22, respectively. Associations between genetic MHC class II variants and papillomavirus-induced tumors have been reported for human papillomavirus and cottontail rabbit papillomavirus infections. This suggests a common mechanism for susceptibility to tumor progression that may involve subversion of the host immune response. This study also identified a genomic region other than MHC that influenced papillomavirus-induced tumor development in the studied population.

536 In vitro and in vivo efficacy of nitric oxide-releasing antiviral therapeutic agents

536 In vitro and in vivo efficacy of nitric oxide-releasing antiviral therapeutic agents KA McHale, K Balogh, H Wang, S Hollenbach, N Christensen, L Chow, T Broker and N Stasko 1 Novan, Inc, Durham, NC, 2 Department of Experimental Pathology, Pennsylvania State University at Hershey Park, Hershey, PA and 3 Department of Biochemistry & Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL The efficacy of topical nitric oxide-releasing therapeutics to inhibit viral replication was demonstrated in vitro and in vivo. NVN1000 or NVN4000 drug substance, dissolved in phosphate buffered saline, was applied 1 hour/day for 6 days to organotypic cultures of primary human keratinocytes containing HPV-18 genomic replicons. Preliminary results indicate a dose responsive reduction of viral DNA copy number to less than 20% of the untreated cultures. At the highest concentrations applied (1.5 mg or 2 mg/ml), no cytotoxicity was detected, as judged from hematoxylin and eosin staining of normal or HPV-18 containing raft cultures. The NVN1000 drug substance was formulated into SB206 gel and the ability of a daily topical drug product (5 doses/week for 8 weeks) to inhibit papilloma growth in vivo was assessed in the Cottontail Rabbit Papillomavirus (CRPV) model. Dosing of SB206 gel was initiated two weeks after inoculation of CRPV viral DNA into the scarified skin sites. Topical SB206 treatment inhibited papilloma formation in a dose dependent fashion with the highest dose, 10% SB206 gel, achieving 82% inhibition compared to the Vehicle control group. Blinded histological analysis of biopsies from 10% SB206-treated animals exhibited mild hyperplasias, with an absence of viral infection as evidenced by a lack of intra-nuclear basophilic inclusions. Qualitative assessment of inflammation and quantitative cytokine gene expression was similar across all dose groups, suggesting immune activation did not account for the efficacy observed in the CRPV model and supports a direct anti-viral effect of Novan’s nitric oxide releasing therapeutic agents. SB206 Gel is currently being evaluated as a topical therapy for external genital warts in a Phase 2 clinical trial. 537 Topical application of chitosan-based nanoformulated green tea polyphenol EGCG ameliorates imiquimod-induced psoriasis-like skin lesion in mice J Chamcheu, I Siddiqui, V Adhami, S Dodwad, D Bharali, G Wood, S Mousa and H Mukhtar Dermatology, University of Wisconsin Madison, Madison, WI Psoriasis is a chronic and currently incurable inflammatory skin disease characterized by hyperproliferation, aberrant differentiation and inflammation, leading to disrupted skin barrier function. The use of natural agents that possess the ability to abrogate these effects could be useful for the treatment of psoriasis. Earlier studies have shown that treatment of keratinocytes and mouse skin with the green tea polyphenol, epigallocatechin-3-gallate (EGCG), increased the expression of caspase-14 that is involved in epidermal differentiation and cornification. However, dose and bioavailability restrict the therapeutic development and application of EGCG in the treatment of psoriasis. To overcome these limitations, we developed fully characterized, chitosan-based nanocarrier to encapsulate EGCG (hereafter termed as nanoEGCG) suitable for topical delivery. Here, we assessed the efficacy of nanoEGCG (48mg/ animal) using an IMQ-induced mouse psoriasis-like skin model and compared the effects to free EGCG (1mg/amimal). Treatment with nanoEGCG resulted in significant (p<0.01) amelioration of pathological markers of psoriasiform lesions including reduction in i) ear and skin thickness, erytherma and scales, ii) proliferation (Ki-67), iii) infiltratory immune cells (mast cells, neutrophils, macrophages and CD4+ T cells), iv) angiogenesis (CD31), and v) increase in the protein expression of caspase-14, early (K1 and k10) and late (filaggrin and loricrin) differentiation markers and AP-1 factors (JunB and cJun), and (v) modulates several psoriasis-related inflammatory cytokines and chemokines analyzed compared to high dose of free EGCG (p<0.05). Taken together, topical application of nanoEGCG was found to ameliorate IMQ-induced Balb/c mouse psoriasis-like lesions with greater than 25-fold dose advantage over free EGCG. Our observations warrant further in vivo efficacy studies of this unique nanoEGCG formulation in robust inducible and genetic models of psoriasis.

Model systems of human papillomavirus-associated disease.

Human papillomaviruses (HPVs) cause a range of serious diseases, including the vast majority of cervical cancers, most anal cancers and around half of head and neck cancers. They are also responsible for troublesome benign epithelial lesions, including genital warts and laryngeal papillomas, and in some individuals HPVs lead to recurrent respiratory papillomatosis and other difficult-to-manage diseases. As a result, there is a great need for model systems that accurately mimic papillomavirus infections in humans. This is complicated by the diverse variety of HPVs, which now number over 200 types, and the different strategies they have evolved to persist in the population. The most well-developed models involve the culture of HPV-containing keratinocytes in organotypic raft culture, an approach which appears to accurately mimic the life cycle of several of the high-risk cancer-associated HPV types. Included amongst these are HPV16 and 18, which cause the majority of cervical cancers. The low-risk HPV types persist less well in tissue-culture models, and our ability to study the productive life cycle of these viruses is more limited. Although ongoing research is likely to improve this situation, animal models of papillomavirus disease can provide considerable basic information as to how lesions form, regress and can be controlled by the immune system. The best studied are cottontail rabbit papillomavirus, rabbit oral papillomavirus and, more recently, mouse papillomavirus (MmuPV), the last of which is providing exciting new insights into viral tropisms and immune control. In addition, transgenic models of disease have helped us to understand the consequences of persistent viral gene expression and the importance of co-factors such as hormones and UV irradiation in the development of neoplasia and cancer. It is hoped that such disease models will eventually lead us to better understanding and better treatments for human disease.