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.

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.

T cell-mediated antitumor immune response eliminates skin tumors induced by mouse papillomavirus, MmuPV1.

Previous studies of naturally occurring mouse papillomavirus (PV) MmuPV1-induced tumors in B6.Cg-Foxn1nu/nu mice suggest that T cell deficiency is necessary and sufficient for the development of such tumors. To confirm this, MmuPV1-induced tumors were transplanted from T cell-deficient mice into immunocompetent congenic mice. Consequently, the tumors regressed and eventually disappeared. The elimination of MmuPV1-infected skin/tumors in immunocompetent mice was consistent with the induction of antitumor T cell immunity. This was confirmed by adoptive cell experiments using hyperimmune splenocytes collected from graft-recipient mice. In the present study, such splenocytes were injected into T cell-deficient mice infected with MmuPV1, and they eliminated both early-stage and fully formed tumors. We clearly show that anti-tumor T cell immunity activated during tumor regression in immunocompetent mice effectively eliminates tumors developing in T cell-deficient congenic mice. The results corroborate the notion that PV-induced tumors are strongly linked to the immune status of the host, and that PV antigens are major anti-tumor antigens. Successful anti-PV T cell responses should, therefore, lead to effective anti-tumor immune therapy in human PV-infected patients.

MmuPV1 infection and tumor development of T cell-deficient mice is prevented by passively transferred hyperimmune sera from normal congenic mice immunized with MmuPV1 virus-like particles (VLPs).

Infection by mouse papillomavirus (PV), MmuPV1, of T cell-deficient, B6.Cg-Foxn1(nu)/J nude mice revealed that four, distinct squamous papilloma phenotypes developed simultaneously after infection of experimental mice. Papillomas appeared on the muzzle, vagina, and tail at or about day 42days post-inoculation. The dorsal skin developed papillomas and hair follicle tumors (trichoblastomas) as early as 26days after infection. Passive transfer of hyperimmune sera from normal congenic mice immunized with MmuPV1 virus-like particles (VLPs) to T cell-deficient strains of mice prevented infection by virions of experimental mice. This study provides further evidence that T cell deficiency is critical for tumor formation by MmuPV1 infection.

Retinoic acid metabolism proteins are altered in trichoblastomas induced by mouse papillomavirus 1.

Skin cancer burden is significant as treatment costs have skyrocketed to $8.1 million annually and some forms metastasize, such as cutaneous squamous cell carcinoma (cSCC) and melanoma. cSCC is caused by altered growth factor signaling induced by chemical carcinogens, ultraviolet light (UV) exposure, and infections with papillomaviruses (PVs). One of the few options for preventing cSCC in high-risk patients is oral retinoids. While much is understood about retinoid treatments and metabolism in mouse models of chemically and UV exposure induced cSCC, little is known about the role of retinoids in PV-induced cSCC. To better understand how retinoid metabolism is altered in cSCC, we examined the expression of this pathway in the newly discovered mouse papillomavirus (MmuPV1), which produces trichoblastomas in dorsal skin but not cSCC. We found significant increases in a rate-limiting enzyme involved in retinoic acid synthesis and retinoic acid binding proteins, suggestive of increased RA synthesis, in MmuPV1-induced tumors in B6.Cg-Foxn1(nu)/J mice. Similar increases in these proteins were seen after acute UVB exposure in Crl:SKH1-Hr(hr) mice and in regressing pre-cancerous lesions in a chemically-induced mouse model, suggesting a common mechanism in limiting the progression of papillomas to full blown cSCC.

Searching for the initiating site of the major capsid protein to generate virus-like particles for a novel laboratory mouse papillomavirus.

Correctly folded virus-like particles (VLPs) of papillomavirus (PV) display conformationally dependent epitopes that are type specific, maintained on authentic virions, and induce neutralizing antibodies. Alignment of the L1 amino acid (aa) sequences of 84 PVs revealed that the lengths of their N-termini are diverse and that multiple, possible initiation methionine (met) codons exist. The L1 gene of MusPV (MmuPV1), that naturally infects immunodeficient laboratory mouse strain (NMRI-Foxn1(nu)/Foxn1(nu)), has four met codons at the 1st, 2nd, 28th, and 30th aas from its N-terminus. Of these, the 3rd and 4th mets, that are at the 28th and 30th aa position from the N-termius, respectively, are located at the position where most PVs have their first met. These two mets, located at the 9th and 11th from the YLPP conserved aas of most PVs, should be considered as consensus initiation codons of PV L1s. Three L1 proteins of MusPV, starting from the 2nd, 3rd, and 4th mets, were expressed using a baculovirus expression system and characterized for their ability to self-assemble into VLPs. While MusPV L1 proteins starting from the 2nd met expressed an L1 protein that did not fold into VLPs, the L1s starting from the 3rd and 4th mets generated correct VLPs in abundant quantities. We now conclude that the highest quantity and best quality VLPs are made from the consensus L1 met of MusPV.

Key aspects of papillomavirus infection influence the host cervicovaginal microbiome in a preclinical murine papillomavirus (MmuPV1) infection model.

Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States and are a major etiological agent of cancers in the anogenital tract and oral cavity. Growing evidence suggests changes in the host microbiome are associated with the natural history and ultimate outcome of HPV infection. We sought to define changes in the host cervicovaginal microbiome during papillomavirus infection, persistence, and pathogenesis using the murine papillomavirus (MmuPV1) cervicovaginal infection model. Cervicovaginal lavages were performed over a time course of MmuPV1 infection in immunocompetent female FVB/N mice and extracted DNA was analyzed by qPCR to track MmuPV1 viral copy number. 16S ribosomal RNA (rRNA) gene sequencing was used to determine the composition and diversity of microbial communities throughout this time course. We also sought to determine whether specific microbial communities exist across the spectrum of MmuPV1-induced neoplastic disease. We, therefore, performed laser-capture microdissection to isolate regions of disease representing all stages of neoplastic disease progression (normal, low- and high-grade dysplasia, and cancer) from female reproductive tract tissue sections from MmuPV1-infected mice and performed 16S rRNA sequencing. Consistent with other studies, we found that the natural murine cervicovaginal microbiome is highly variable across different experiments. Despite these differences in initial microbiome composition between experiments, we observed that MmuPV1 persistence, viral load, and severity of disease influenced the composition of the cervicovaginal microbiome. These studies demonstrate that papillomavirus infection can alter the cervicovaginal microbiome.IMPORTANCEHuman papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. A subset of HPVs that infect the anogenital tract (cervix, vagina, anus) and oral cavity cause at least 5% of cancers worldwide. Recent evidence indicates that the community of microbial organisms present in the human cervix and vagina, known as the cervicovaginal microbiome, plays a role in HPV-induced cervical cancer. However, the mechanisms underlying this interplay are not well-defined. In this study, we infected the female reproductive tract of mice with a murine papillomavirus (MmuPV1) and found that key aspects of papillomavirus infection and disease influence the host cervicovaginal microbiome. This is the first study to define changes in the host microbiome associated with MmuPV1 infection in a preclinical animal model of HPV-induced cervical cancer. These results pave the way for using MmuPV1 infection models to further investigate the interactions between papillomaviruses and the host microbiome.

Monitoring mouse papillomavirus-associated cancer development using longitudinal Pap smear screening.

A substantial percentage of the population remains at risk for cervical cancer due to pre-existing human papillomavirus (HPV) infections, despite prophylactic vaccines. Early diagnosis and treatment are crucial for better disease outcomes. The development of new treatments heavily relies on suitable preclinical model systems. Recently, we established a mouse papillomavirus (MmuPV1) model that is relevant to HPV genital pathogenesis. In the current study, we validated the use of Papanicolaou (Pap) smears, a valuable early diagnostic tool for detecting HPV cervical cancer, to monitor disease progression in the MmuPV1 mouse model. Biweekly cervicovaginal swabs were collected from the MmuPV1-infected mice for viral DNA quantitation and cytology assessment. The Pap smear slides were evaluated for signs of epithelial cell abnormalities using the 2014 Bethesda system criteria. Tissues from the infected mice were harvested at various times post-viral infection for additional histological and virological assays. Over time, increased viral replication was consistent with higher levels of viral DNA, and it coincided with an uptick in epithelial cell abnormalities with higher severity scores noted as early as 10 weeks after viral infection. The cytological results also correlated with the histological evaluation of tissues harvested simultaneously. Both immunocompromised and immunocompetent mice with squamous cell carcinoma (SCC) cytology also developed vaginal SCCs. Notably, samples from the MmuPV1-infected mice exhibited similar cellular abnormalities compared to the corresponding human samples at similar disease stages. Hence, Pap smear screening proves to be an effective tool for the longitudinal monitoring of disease progression in the MmuPV1 mouse model. IMPORTANCE: Papanicolaou (Pap) smear has saved millions of women's lives as a valuable early screening tool for detecting human papillomavirus (HPV) cervical precancers and cancer. However, more than 200,000 women in the United States alone remain at risk for cervical cancer due to pre-existing HPV infection-induced precancers, as there are currently no effective treatments for HPV-associated precancers and cancers other than invasive procedures including a loop electrosurgical excision procedure (LEEP) to remove abnormal tissues. In the current study, we validated the use of Pap smears to monitor disease progression in our recently established mouse papillomavirus model. To the best of our knowledge, this is the first study that provides compelling evidence of applying Pap smears from cervicovaginal swabs to monitor disease progression in mice. This HPV-relevant cytology assay will enable us to develop and test novel antiviral and anti-tumor therapies using this model to eliminate HPV-associated diseases and cancers.

Immune Responses in Oral Papillomavirus Clearance in the MmuPV1 Mouse Model.

Human papillomavirus (HPV)-induced oropharyngeal cancer now exceeds HPV-induced cervical cancer, with a noticeable sex bias. Although it is well established that women have a more proficient immune system, it remains unclear whether immune control of oral papillomavirus infections differs between sexes. In the current study, we use genetically modified mice to target CCR2 and Stat1 pathways, with the aim of investigating the role of both innate and adaptive immune responses in clearing oral papillomavirus, using our established papillomavirus (MmuPV1) infection model. Persistent oral MmuPV1 infection was detected in Rag1ko mice with T and B cell deficiencies. Meanwhile, other tested mice were susceptible to MmuPV1 infections but were able to clear the virus. We found sex differences in key myeloid cells, including macrophages, neutrophils, and dendritic cells in the infected tongues of wild type and Stat1ko mice but these differences were not observed in CCR2ko mice. Intriguingly, we also observed a sex difference in anti-MmuPV1 E4 antibody levels, especially for two IgG isotypes: IgG2b and IgG3. However, we found comparable numbers of interferon-gamma-producing CD8 T cells stimulated by E6 and E7 in both sexes. These findings suggest that males and females may use different components of innate and adaptive immune responses to control papillomavirus infections in the MmuPV1 mouse model. The observed sex difference in immune responses, especially in myeloid cells including dendritic cell (DC) subsets, may have potential diagnostic and prognostic values for HPV-associated oropharyngeal cancer.

Compromised T Cell Immunity Links Increased Cutaneous Papillomavirus Activity to Squamous Cell Carcinoma Risk.

Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer, with increased incidence in immunosuppressed patients. ß-Human papillomavirus has been proposed as a contributor to cSCC risk partly on the basis of increased ß-human papillomavirus viral load and seropositivity observed among patients with cSCC. Experimental data in mice colonized with mouse papillomavirus type 1 suggest that T cell immunity against ß-human papillomavirus suppresses skin cancer in immunocompetent hosts, and the loss of this immunity leads to the increased risk of cSCC. In this study, we show that CD8+ T cell depletion in mouse papillomavirus type 1‒colonized mice that underwent skin carcinogenesis protocol led to increased viral load in the skin and seropositivity for anti‒mouse papillomavirus type 1 antibodies. These findings provide evidence that compromised T cell immunity can be the link that connects increased ß-human papillomavirus detection to cSCC risk.

MmuPV1-Induced Cutaneous Squamous Cell Carcinoma Arises Preferentially from Lgr5+ Epithelial Progenitor Cells.

Murine papillomavirus, MmuPV1, causes natural infections in laboratory mice that can progress to squamous cell carcinoma (SCC) making it a useful preclinical model to study the role of papillomaviruses in cancer. Papillomavirus can infect cells within hair follicles, which contain multiple epithelial progenitor cell populations, including Lgr5+ progenitors, and transgenic mice expressing human papillomavirus oncogenes develop tumors derived from Lgr5 progenitors. We therefore tested the hypothesis that Lgr5+ progenitors contribute to neoplastic lesions arising in skins infected with MmuPV1 by performing lineage tracing experiments. Ears of 6-8-week-old Lgr5-eGFP-IRES-CreERT2/Rosa26LSLtdTomato mice were treated topically with 4-OH Tamoxifen to label Lgr5+ progenitor cells and their progeny with tdTomato and, 72 h later, infected with MmuPV1. Four months post-infection, tissue at the infection site was harvested for histopathological analysis and immunofluorescence to determine the percentage of tdTomato+ cells within the epithelial lesions caused by MmuPV1. Squamous cell dysplasia showed a low percentage of tdTomato+ cells (7%), indicating that it arises primarily from non-Lgr5 progenitor cells. In contrast, cutaneous SCC (cSCC) was substantially more positive for tdTomato+ cells (42%), indicating that cSCCs preferentially arise from Lgr5+ progenitors. Biomarker analyses of dysplasia vs. cSCC revealed further differences consistent with cSCC arising from LGR5+ progenitor cells.

The use of a topical protease inhibitor, Saquinavir, to alleviate mouse papillomavirus-mediated anal disease.

Select protease inhibitors (PI) have been found to be effective in decreasing human papillomavirus oncoprotein expression. This study evaluated whether the topical PI, Saquinavir (SQV), promotes viral clearance in an infectious mouse model with Mus musculus papillomavirus 1 (MmuPV1). NOD scid gamma (NSG) mice were anally infected with ∼4 × 108 viral genome equivalents of MmuPV1 and 120 days post-infection (when majority have high-grade anal dysplasia), began topical treatments: control (mock), 7,12-dimethylbenz(a)anthracene (DMBA) only, once weekly to promote carcinogenesis, 1% SQV only, daily (Monday - Friday), and SQV + DMBA. Viral MmuPV1 load was analyzed from anal lavages pre and post-treatment. Anal tissue was harvested, processed, and evaluated for drug absorption, grade of anal disease, and anal viral RNA. Results suggest that topical SQV promotes decreased viral shedding in female mice treated with SQV.

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.

Cryo EM Analysis Reveals Inherent Flexibility of Authentic Murine Papillomavirus Capsids.

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. However, studies have been hampered due to restricted tropism that makes production and purification of high titer virus problematic. This issue has been overcome by developing alternative HPV production methods such as virus-like particles (VLPs), which are devoid of a native viral genome. Structural studies have been limited in resolution due to the heterogeneity, fragility, and stability of the VLP capsids. The mouse papillomavirus (MmuPV1) presented here has provided the opportunity to study a native papillomavirus in the context of a common laboratory animal. Using cryo EM to solve the structure of MmuPV1, we achieved 3.3 Å resolution with a local symmetry refinement method that defined smaller, symmetry related subparticles. The resulting high-resolution structure allowed us to build the MmuPV1 asymmetric unit for the first time and identify putative L2 density. We also used our program ISECC to quantify capsid flexibility, which revealed that capsomers move as rigid bodies connected by flexible linkers. The MmuPV1 flexibility was comparable to that of a HPV VLP previously characterized. The resulting MmuPV1 structure is a promising step forward in the study of papillomavirus and will provide a framework for continuing biochemical, genetic, and biophysical research for papillomaviruses.

The environmental pollutant and tobacco smoke constituent dibenzo[def,p]chrysene is a co-factor for malignant progression of mouse oral papillomavirus infections.

HPV infections in the oral cavity that progress to cancer are on the increase in the USA. Model systems to study co-factors for progression of these infections are lacking as HPVs are species-restricted and cannot grow in preclinical animal models. We have recently developed a mouse papillomavirus (MmuPV1) oral mucosal infection model that provides opportunities to test, for the first time, the hypothesis that tobacco carcinogens are co-factors that can impact the progression of oral papillomas to squamous cell carcinoma (SCC). Four cohorts of mice per sex were included: (1) infected with MmuPV1 and treated orally with DMSO-saline; (2) infected with MmuPV1 and treated orally with the tobacco carcinogen, dibenzo[def,p]chrysene (DBP); (3) uninfected and treated orally with DMSO-saline, and (4) uninfected and treated orally with DBP. Oral swabs were collected monthly for subsequent assessment of viral load. Oral tissues were collected for in situ viral DNA/RNA detection, viral protein staining, and pathological assessment for hyperplasia, papillomas and SCC at study termination. We observed increased rates of SCC in oral tissue infected with MmuPV1 and treated with DBP when compared to mice treated with DBP or virus individually, each of which showed minimal disease. Virally-infected epithelium showed strong levels of viral DNA/RNA and viral protein E4/L1 staining. In contrast, areas of SCC showed reduced viral DNA staining indicative of lower viral copy per nucleus but strong RNA signals. Several host markers (p120 ctn, p53, S100A9) were also examined in the mouse oral tissues; of particular significance, p120 ctn discriminated normal un-infected epithelium from SCC or papilloma epithelium. In summary, we have confirmed that our infection model is an excellent platform to assess the impact of co-factors including tobacco carcinogens for oral PV cancerous progression. Our findings can assist in the design of novel prevention/treatment strategies for HPV positive vs. HPV negative disease.

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.

The human papillomavirus 16 E5 gene potentiates MmuPV1-Dependent pathogenesis.

The papillomavirus E5 gene contributes to transformation and tumorigenesis; however, its exact function in these processes and viral pathogenesis is unclear. While E5 is present in high-risk mucosotropic HPVs that cause anogenital and head and neck cancers, it is absent in cutaneous HPVs and the recently discovered mouse papillomavirus (MmuPV1), which causes papillomas and squamous cell carcinomas of the skin and mucosal epithelia in laboratory mice. We infected K14E5 transgenic mice, which express the high-risk mucosotropic HPV16 E5 gene in stratified epithelia, with MmuPV1 to investigate the effects of E5 on papillomavirus-induced pathogenesis. Skin lesions in MmuPV1-infected K14E5 mice had earlier onset, higher incidence, and reduced frequency of spontaneous regression compared to those in non-transgenic mice. K14E5 mice were also more susceptible to cervicovaginal cancers when infected with MmuPV1 and treated with estrogen compared to non-transgenic mice. Our studies support the hypothesis that E5 contributes to papillomavirus-induced pathogenesis.

Development of an in vivo infection model to study Mouse papillomavirus-1 (MmuPV1).

Preclinical model systems to study multiple features of the papillomavirus life cycle are extremely valuable tools to aid our understanding of Human Papillomavirus (HPV) biology, disease progression and treatments. Mouse papillomavirus (MmuPV1) is the first ever rodent papillomavirus that can infect the laboratory strain of mice and was discovered recently in 2011. This model is an attractive model to study papillomavirus pathogenesis due to the ubiquitous availability of lab mice and the fact that this mouse species is easily genetically modifiable. Several other groups, including ours, have reported that MmuPV1-induced papillomas are restricted to T-cell deficient immunosuppressed mice. In our lab we showed for the first time that MmuPV1 causes skin cancers in UVB-irradiated immunocompetent animals. In this report we describe in detail the MmuPV1-UV infection model that can be adapted to study MmuPV1 biology in immunocompetent animals.

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.

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.