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.

Passive Immunization with a Single Monoclonal Neutralizing Antibody Protects against Cutaneous and Mucosal Mouse Papillomavirus Infections.

We have established a mouse papillomavirus (MmuPV1) model that induces both cutaneous and mucosal infections and cancers. In the current study, we use this model to test our hypothesis that passive immunization using a single neutralizing monoclonal antibody can protect both cutaneous and mucosal sites at different time points after viral inoculation. We conducted a series of experiments involving the administration of either a neutralizing monoclonal antibody, MPV.A4, or control monoclonal antibodies to both outbred and inbred athymic mice. Three clinically relevant mucosal sites (lower genital tract for females and anus and tongue for both males and females) and two cutaneous sites (muzzle and tail) were tested. At the termination of the experiments, all tested tissues were harvested for virological analyses. Significantly lower levels of viral signals were detected in the MPV.A4-treated female mice up to 6 h post-viral inoculation compared to those in the isotype control. Interestingly, males displayed partial protection when they received MPV.A4 at the time of viral inoculation, even though they were completely protected when receiving MPV.A4 at 24 h before viral inoculation. We detected MPV.A4 in the blood starting at 1 h and up to 8 weeks postadministration in some mice. Parallel to these in vivo studies, we conducted in vitro neutralization using a mouse keratinocyte cell line and observed complete neutralization up to 8 h post-viral inoculation. Thus, passive immunization with a monoclonal neutralizing antibody can protect against papillomavirus infection at both cutaneous and mucosal sites and is time dependent. IMPORTANCE This is the first study testing a single monoclonal neutralizing antibody (MPV.A4) by passive immunization against papillomavirus infections at both cutaneous and mucosal sites in the same host in the mouse papillomavirus model. We demonstrated that MPV.A4 administered before viral inoculation can protect both male and female athymic mice against MmuPV1 infections at cutaneous and mucosal sites. MPV.A4 also offers partial protection at 6 h post-viral inoculation in female mice. MPV.A4 can be detected in the blood from 1 h to 8 weeks after intraperitoneal (i.p.) injection. Interestingly, males were only partially protected when they received MPV.A4 at the time of viral inoculation. The failed protection in males was due to the absence of neutralizing MPV.A4 at the infected sites. Our findings suggest passive immunization with a single monoclonal neutralizing antibody can protect against diverse papillomavirus infections in a time-dependent manner in mice.

Depo Medroxyprogesterone (DMPA) Promotes Papillomavirus Infections but Does Not Accelerate Disease Progression in the Anogenital Tract of a Mouse Model.

Contraceptives such as Depo-medroxyprogesterone (DMPA) are used by an estimated 34 million women worldwide. DMPA has been associated with increased risk of several viral infections including Herpes simplex virus-2 (HSV-2) and Human immunodeficiency virus (HIV). In the current study, we used the mouse papillomavirus (MmuPV1) anogenital infection model to test two hypotheses: (1) contraceptives such as DMPA increase the susceptibility of the anogenital tract to viral infection and (2) long-term contraceptive administration induces more advanced disease at the anogenital tract. DMPA treatments of both athymic nude mice and heterozygous NU/J (Foxn1nu/+) but ovariectomized mice led to a significantly increased viral load at the anogenital tract, suggesting that endogenous sex hormones were involved in increased viral susceptibility by DMPA treatment. Consistent with previous reports, DMPA treatment suppressed host anti-viral activities at the lower genital tract. To test the impact of long-term contraceptive treatment on the MmuPV1-infected lower genital tract, we included two other treatments in addition to DMPA: 17ß-estradiol and a non-hormone based contraceptive Cilostazol (CLZ, Pletal). Viral infections were monitored monthly up to nine months post infection by qPCR. The infected vaginal and anal tissues were harvested and further examined by histological, virological, and immunological analyses. Surprisingly, we did not detect a significantly higher grade of histology in animals in the long-term DMPA and 17ß-estradiol treated groups when compared to the control groups in the athymic mice we tested. Therefore, although DMPA promotes initial papillomavirus infections in the lower genital tract, the chronic administration of DMPA does not promote cancer development in the infected tissues in our mouse model.

Mouse Papillomavirus L1 and L2 Are Dispensable for Viral Infection and Persistence at Both Cutaneous and Mucosal Tissues.

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.

Papillomavirus can be transmitted through the blood and produce infections in blood recipients: Evidence from two animal models.

Human papillomaviruses (HPV) contribute to most cervical cancers and are considered to be sexually transmitted. However, papillomaviruses are often found in cancers of internal organs, including the stomach, raising the question as to how the viruses gain access to these sites. A possible connection between blood transfusion and HPV-associated disease has not received much attention. Here we show, in rabbit and mouse models, that blood infected with papillomavirus yields infections at permissive sites with detectable viral DNA, RNA transcripts, and protein products. The rabbit skin tumours induced via blood infection displayed decreased expression of SLN, TAC1, MYH8, PGAM2, and APOBEC2 and increased expression of SDRC7, KRT16, S100A9, IL36G, and FABP9, as seen in tumours induced by local infections. Furthermore, we demonstrate that blood from infected mice can transmit the infection to uninfected animals. Finally, we demonstrate the presence of papillomavirus infections and virus-induced hyperplasia in the stomach tissues of animals infected via the blood. These results indicate that blood transmission could be another route for papillomavirus infection, implying that the human blood supply, which is not screened for papillomaviruses, could be a potential source of HPV infection as well as subsequent cancers in tissues not normally associated with the viruses.

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'.

Mouse papillomavirus infection persists in mucosal tissues of an immunocompetent mouse strain and progresses to cancer.

Mouse papillomavirus has shown broad tissue tropism in nude mice. Previous studies have tested cutaneous infections in different immunocompromised and immunocompetent mouse strains. In the current study, we examined mucosal infection in several immunocompetent and immunocompromised mouse strains. Viral DNA was monitored periodically by Q-PCR of lavage samples. Immunohistochemistry and in situ hybridization were used to determine viral capsid protein and viral DNA respectively. All athymic nude mouse strains showed active infections at both cutaneous and mucosal sites. Interestingly, NOD/SCID mice, which have a deficiency in T, B, and NK cells, showed minimal disease at cutaneous sites but developed persistent infection at the mucosal sites including those of the anogenital region and the oral cavity. Three strains of immunocompetent mice supported mucosal infections. Infections of the lower genital tract in heterozygous (immunocompetent) mice of the NU/J strain progressed to high grade dysplasia and to carcinoma in situ. Anti-MmuPV1 neutralizing antibodies were detected in the sera of all immunocompetent animals. Our findings demonstrate that the mucosae may be the preferred sites for this virus in mice. The mouse model is expected to be a valuable model for the study of mucosal papillomavirus disease, progression, and host immune control.

Mouse papillomavirus infections spread to cutaneous sites with progression to malignancy.

We report secondary cutaneous infections in the mouse papillomavirus (MmuPV1)/mouse model. Our previous study demonstrated that cutaneous MmuPV1 infection could spread to mucosal sites. Recently, we observed that mucosal infections could also spread to various cutaneous sites including the back, tail, muzzle and mammary tissues. The secondary site lesions were positive for viral DNA, viral capsid protein and viral particles as determined by in situ hybridization, immunohistochemistry and transmission electron microscopy analyses, respectively. We also demonstrated differential viral production and tumour growth at different secondarily infected skin sites. For example, fewer viral particles were detected in the least susceptible back tissues when compared with those in the infected muzzle and tail, although similar amounts of viral DNA were detected. Follow-up studies demonstrated that significantly lower amounts of viral DNA were packaged in the back lesions. Lavages harvested from the oral cavity and lower genital tracts were equally infectious at both cutaneous and mucosal sites, supporting the broad tissue tropism of this papillomavirus. Importantly, two secondary skin lesions on the forearms of two mice displayed a malignant phenotype at about 9.5 months post-primary infection. Therefore, MmuPV1 induces not only dysplasia at mucosal sites such as the vagina, anus and oral cavity but also skin carcinoma at cutaneous sites. These findings demonstrate that MmuPV1 mucosal infection can be spread to cutaneous sites and suggest that the model could serve a useful role in the study of the viral life cycle and pathogenesis of papillomavirus.

The Mouse Papillomavirus Infection Model.

The mouse papillomavirus (MmuPV1) was first reported in 2011 and has since become a powerful research tool. Through collective efforts from different groups, significant progress has been made in the understanding of molecular, virological, and immunological mechanisms of MmuPV1 infections in both immunocompromised and immunocompetent hosts. This mouse papillomavirus provides, for the first time, the opportunity to study papillomavirus infections in the context of a small common laboratory animal for which abundant reagents are available and for which many strains exist. The model is a major step forward in the study of papillomavirus disease and pathology. In this review, we summarize studies using MmuPV1 over the past six years and share our perspectives on the value of this unique model system. Specifically, we discuss viral pathogenesis in cutaneous and mucosal tissues as well as in different mouse strains, immune responses to the virus, and local host-restricted factors that may be involved in MmuPV1 infections and associated disease progression.

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.

Mouse papillomavirus MmuPV1 infects oral mucosa and preferentially targets the base of the tongue.

In 2010, a new mouse papillomavirus, MmuPV1, was discovered in a colony of NMRI- Foxn1(nu)/Foxn1(nu) athymic mice in India. This finding was significant because it was the first papillomavirus to be found in a laboratory mouse. In this paper we report successful infections of both dorsal and ventral surfaces of the rostral tongues of outbred athymic nude mice. We also report the observation that the base of the tongue, the area of the tongue often targeted by cancer-associated high-risk papillomavirus infections in humans, is especially susceptible to infection. A suitable animal model for the study of oral papillomavirus infections, co-infections, and cancers has long been sought. The work presented here suggests that such a model is now at hand.

Tracking vaginal, anal and oral infection in a mouse papillomavirus infection model.

Noninvasive and practical techniques to longitudinally track viral infection are sought after in clinical practice. We report a proof-of-principle study to monitor the viral DNA copy number using a newly established mouse papillomavirus (MmuPV1) mucosal infection model. We hypothesized that viral presence could be identified and quantified by collecting lavage samples from cervicovaginal, anal and oral sites. Nude mice infected at these sites with infectious MmuPV1 were tracked for up to 23 weeks starting at 6 weeks post-infection. Viral DNA copy number was determined by SYBR Green Q-PCR analysis. In addition, we tracked viral DNA load through three complete oestrous cycles to pinpoint whether there was a correlation between the DNA load and the four stages of the oestrous cycle. Our results showed that high viral DNA copy number was reproducibly detected from both anal and cervicovaginal lavage samples. The infection and disease progression were further confirmed by histology, cytology, in situ hybridization, immunohistochemistry and transmission electron microscopy. Interestingly, the viral copy number fluctuated over the oestrous cycle, with the highest level at the oestrus stage, implying that multiple sampling might be necessary to provide a reliable diagnosis. Virus DNA was detected in oral lavage samples at a later time after infection. Lower viral DNA load was found in oral samples when compared with those in anal and vaginal tracts. To our knowledge, our study is the first in vivo study to sequentially monitor papillomavirus infection from mucosal anal, oral and vaginal tracts in a preclinical model.

A novel pre-clinical murine model to study the life cycle and progression of cervical and anal papillomavirus infections.

BACKGROUND: Papillomavirus disease and associated cancers remain a significant health burden in much of the world. The current protective vaccines, Gardasil and Cervarix, are expensive and not readily available to the underprivileged. In addition, the vaccines have not gained wide acceptance in the United States nor do they provide therapeutic value. Papillomaviruses are strictly species specific and thus human viruses cannot be studied in an animal host. An appropriate model for mucosal disease has long been sought. We chose to investigate whether the newly discovered mouse papillomavirus, MmuPV1, could infect mucosal tissues in Foxn1nu/Foxn1nu mice. METHODS: The vaginal and anal canals of Foxn1nu/Foxn1nu mice were gently abraded using Nonoxynol-9 and "Doctor's BrushPicks" and MmuPV1 was delivered into the vaginal tract or the anal canal. RESULTS: Productive vaginal, cervical and anal infections developed in all mice. Vaginal/cervical infections could be monitored by vaginal lavage. Dysplasias were evident in all animals. CONCLUSIONS: Anogenital tissues of a common laboratory mouse can be infected with a papillomavirus unique to that animal. This observation will pave the way for fundamental virological and immunological studies that have been challenging to carry out heretofore due to lack of a suitable model system.

Long-peptide therapeutic vaccination against CRPV-induced papillomas in HLA-A2.1 transgenic rabbits.

Long peptide immunization is a promising strategy to clear established tumors. In the current study, we investigated the therapeutic effect of a naturally existing long peptide that contained two HLA-A2.1 restricted epitopes (CRPVE1/149-157 and CRPVE1/161-169) from cottontail rabbit papillomavirus (CRPV) E1 using our CRPV/HLA-A2.1 transgenic rabbit model. A universal Tetanus Toxin helper motif (TT helper) was tagged at either the N-terminus or the carboxyl-terminus of this long peptide and designated as TT-E1 peptide and E1 peptide-TT respectively. Four groups of HLA-A2.1 transgenic rabbits were infected with wild type CRPV DNA. Three weeks post-infection, the rabbits were immunized four times with TT-E1 peptide, E1peptide only, E1 peptide -TT or TT-control peptide with two-week intervals between immunizations. Tumor outgrowth was monitored and recorded weekly. After the third booster immunization, tumors on two of the four E1 peptide-TT immunized rabbits began to shrink. One animal from this group was free of tumors at the termination of the study. The mean papilloma size of E1 peptide-TT immunized rabbits was significantly smaller when compared with that of the three other groups (P<0.05, one way ANOVA analysis). It is interesting that E1 peptide-TT vaccination not only stimulated stronger T cell mediate immune responses but also stronger antibody generations. We conclude that the location of a TT helper motif tagged at the long peptide vaccine is critical for the outcome of therapeutic responses to persistent tumors in our HLA-A2.1 transgenic rabbit model.

Formulation of cidofovir improves the anti-papillomaviral activity of topical treatments in the CRPV/rabbit model.

Current topical treatments for papillomas use ablative, cytotoxic and immunomodulating strategies and reagents. However, the effectiveness of topical treatments using different formulations has not been examined in preclinical models or clinical trials. The purpose of this study was to determine whether formulation of the small molecule acyclic nucleoside, cidofovir (CDV), could lead to improved therapeutic endpoints following topical treatment of papillomas using the cottontail rabbit papillomavirus (CRPV)/rabbit model. Different formulations with a set dose of 1% cidofovir were tested to establish comparative data. The results demonstrated that anti-papilloma treatments with topical CDV were greatly enhanced when formulated versus unformulated. Best results were obtained with CDV formulated in cremophor, then in Carbomer 940, and then in DMSO. Further studies indicated that effective formulations led to complete cures of papillomas at dilutions less than 0.3% CDV. These studies together with previous observations demonstrated that unformulated CDV under the same treatment regime required doses of 2% to achieve cures demonstrating that much less compound can be used when properly formulated.

Secondary infections, expanded tissue tropism, and evidence for malignant potential in immunocompromised mice infected with Mus musculus papillomavirus 1 DNA and virus.

Papillomavirus disease poses a special challenge to people with compromised immune systems. Appropriate models to study infections in these individuals are lacking. We report here the development of a model that will help to address these deficiencies. The MmuPV1 genome was synthesized and used successfully to produce virus from DNA infections in immunocompromised mice. In these early studies, we have demonstrated both primary and secondary infections, expanded tissue tropism, and extensive dysplasia.

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.

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.

Human alpha and beta papillomaviruses use different synonymous codon profiles.

Human papillomaviruses use rare codons relative to their hosts. It has been theorized that this is a mechanism to allow the virus to escape immune surveillance. In the present study, we examined the codings of four major genes of 21 human alpha (mucosatropic) viruses and 16 human beta (cutaneous-tropic) viruses. We compared the codon usage of different genes from a given papillomavirus and also the same genes from different papillomaviruses. Our data showed that codon usage was not always uniform between two genes of a given papillomavirus or between the same genes of papillomaviruses from different genera. We speculate as to why this might be and conclude that codon usage in the papillomaviruses may not only play a role in facilitating escape from immune surveillance but may also underlie some of the unanswered questions in the papillomavirus field.

Differences in methodology, but not differences in viral strain, account for variable experimental outcomes in laboratories utilizing the cottontail rabbit papillomavirus model.

The cottontail rabbit papillomavirus (CRPV) animal model is used in several laboratories worldwide to investigate immunogenicity, carcinogenicity and life cycle aspects of papillomaviruses. It is the only animal model in which the full life cycle of the virus from initiation of infection to malignant progression can be studied. A major strength of the model is that the viral DNA is infectious. This feature allows for the study of mutant genomes without the need to create infectious mutant virus. Results from laboratory to laboratory have not always been consistent. Different laboratories use different methods for creating infections from DNA and it was postulated that the different challenge methods could play a role in the differential outcomes. Because different laboratories use different strains of CRPV, it was also desirable to test if the difference in CRPV genomes contributed to the differential outcomes. In this study, three of the CRPV strains used most widely (Washington B, Orth CRPV and Hershey CRPV) were cloned into PUC19; the E8 ATG ko mutants for each strain were also generated. We employed the infection technique reported previously in which scarification is done first and is followed with delivery of DNA by pipette 3 days later. The papilloma outgrowth generated by these three wild type constructs and their E8 ATG ko mutants was compared. No significant difference was found among the three strains or their E8 ATG ko mutants. E8 ATG ko mutants induced significantly smaller but persistent papillomas when compared to their respective wild type CRPVs. The gene gun was also used to create infections with both Hershey CRPV DNA and the corresponding E8 ATG ko and was found to lead to less vigorous growth as well as some regressions. Further studies suggested that gene gun delivery might have induced an immune response which then resulted in compromised growth of papillomas. It was concluded that the E8 gene is not required for infection. We suggest that standardized infection methods should be used in laboratories so that inconsistencies in conclusions will be minimized.