[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Noninvasive and practical techniques to track viral infection longitudinally are sought after in the 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 quantitated 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 estrous cycles to pinpoint whether there was a correlation between the DNA load and the four stages of the estrous 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 estrous cycle, with the highest level at the estrus 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.
Journal of General Virology 09/2015; DOI:10.1099/jgv.0.000295 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
PLoS ONE 03/2015; 10(3):e0120128. DOI:10.1371/journal.pone.0120128 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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, E1 peptide 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 mediated 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.
Trials in Vaccinology 12/2014; 3(1):134–142. DOI:10.1016/j.trivac.2014.06.002
[Show abstract][Hide abstract] ABSTRACT: Adoptive T cell transfer (ACT) has achieved clinical success in treating established cancer, particularly in combination with lymphodepleting regimens. Our group previously demonstrated that ACT following whole-body irradiation (WBI) promotes high-level T cell accumulation, regression of established brain tumors, and long-term protection from tumor recurrence in a mouse model of SV40 T antigen-induced choroid plexus tumors. Here we asked whether an approach that can promote strong donor T-cell responses in the absence of WBI might also produce this dramatic and durable tumor elimination following ACT. Agonist anti-CD40 antibody can enhance antigen-specific CD8(+) T-cell responses and has shown clinical efficacy as a monotherapy in the setting of cancer. We show that anti-CD40 conditioning promotes rapid accumulation of tumor-specific donor CD8(+) T cells in the brain and regression of autochthonous T antigen-induced choroid plexus tumors, similar to WBI. Despite a significant increase in the lifespan, tumors eventually recurred in anti-CD40-conditioned mice coincident with loss of T-cell persistence from both the brain and lymphoid organs. Depletion of CD8(+) T cells from the peripheral lymphoid organs of WBI-conditioned recipients failed to promote tumor recurrence, but donor cells persisted in the brains long-term in CD8-depleted mice. These results demonstrate that anti-CD40 conditioning effectively enhances ACT-mediated acute elimination of autochthonous tumors, but suggest that mechanisms associated with WBI conditioning, such as the induction of long-lived T cells, may be critical for protection from tumor recurrence.
Cancer Immunology and Immunotherapy 11/2014; 64(3). DOI:10.1007/s00262-014-1635-7 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NOD.B10 Idd9.3 mice are congenic for the insulin-dependent diabetes (Idd) Idd9.3 locus, which confers significant type 1 diabetes (T1D) protection and encodes 19 genes, including microRNA (miR)-34a, from T1D-resistant C57BL/10 mice. B cells have been shown to play a critical role in the priming of autoantigen-specific CD4+ T cells in T1D pathogenesis in non-obese diabetic (NOD) mice. We show that early B-cell development is impaired in NOD.B10 Idd9.3 mice, resulting in the profound reduction of transitional and mature splenic B cells as compared with NOD mice. Molecular analysis revealed that miR-34a expression was significantly higher in B cell progenitors and marginal zone B cells from NOD.B10 Idd9.3 mice than in NOD mice. Furthermore, miR-34a expression in these cell populations inversely correlated with levels of Foxp1, an essential regulator of B-cell lymphopoiesis, which is directly repressed by miR-34a. We further show that islet-specific CD4+ T cells proliferated inefficiently when primed by NOD.B10 Idd9.3 B cells in vitro or in response to endogenous autoantigen in NOD.B10 Idd9.3 mice. Thus, Idd9.3-encoded miR-34a is a likely candidate in negatively regulating B-cell lymphopoiesis, which may contribute to inefficient expansion of islet-specific CD4+ T cells and to T1D protection in NOD.B10 Idd9.3 mice.This article is protected by copyright. All rights reserved
European Journal of Immunology 06/2014; 44(6). DOI:10.1002/eji.201344116 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 06/2013; 87(16). DOI:10.1128/JVI.00777-13 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Multiple sclerosis (MS) is an autoimmune disease that is mediated by myelin-reactive T cells resulting in CNS demyelination, however the mechanisms that control their activation are unclear. Mice that are transgenic for a myelin proteolipid protein (PLP)-specific TCR spontaneously develop experimental autoimmune encephalomyelitis (EAE), the animal model of MS. They mimic the spontaneous onset of MS and thus offer the unique opportunity to investigate the mechanisms that may contribute to the development of spontaneous CNS autoimmunity. MyD88 is an adaptor protein that mediates signal transduction by TLRs, IL-1R and IL-18R, resulting in the activation of innate immune cells, including DCs. We investigated the requirement of MyD88 in the pathogenesis of spontaneous EAE in PLP TCR transgenic SJL mice. We show that genetic loss of MyD88 does not intrinsically preclude development of spontaneous EAE and autoimmune demyelination in these mice. EAE was associated with functionally mature peripheral DCs that promoted superior PLP-specific Th1 and Th17 responses compared to those from disease-free mice. Together, our data suggest that MyD88-independent innate immune signaling critically contributes to priming of myelin-reactive T cells and development of spontaneous EAE in MyD88-deficient PLP TCR transgenic mice.
Journal of neuroimmunology 12/2012; 255(1-2). DOI:10.1016/j.jneuroim.2012.11.004 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adoptive T-cell immunotherapy has garnered wide attention, but its effective use is limited by the need of multiple ex vivo manipulations and infusions that are complex and expensive. In this study, we show how highly reactive antigen (Ag)-specific CTLs can be generated from induced pluripotent stem (iPS) cells to provide an unlimited source of functional CTLs for adoptive immunotherapy. iPS cell-derived T cells can offer the advantages of avoiding possible immune rejection and circumventing ethical and practical issues associated with other stem cell types. iPS cells can be differentiated into progenitor T cells in vitro by stimulation with the Notch ligand Delta-like 1 (DL1) overexpressed on bone marrow stromal cells, with complete maturation occurring upon adoptive transfer into Rag1-deficient mice. Here, we report that these iPS cells can be differentiated in vivo into functional CTLs after overexpression of MHC I-restricted Ag-specific T-cell receptors (TCR). In this study, we generated murine iPS cells genetically modified with ovalbumin (OVA)-specific and MHC-I restricted TCR (OT-I) by retrovirus-mediated transduction. After their adoptive transfer into recipient mice, the majority of OT-I/iPS cells underwent differentiation into CD8+ CTLs. TCR-transduced iPS cells developed in vivo responded in vitro to peptide stimulation by secreting interleukin 2 and IFN-γ. Most importantly, adoptive transfer of TCR-transduced iPS cells triggered infiltration of OVA-reactive CTLs into tumor tissues and protected animals from tumor challenge. Taken together, our findings offer proof of concept for a potentially more efficient approach to generate Ag-specific T lymphocytes for adoptive immunotherapy.
Cancer Research 05/2011; 71(14):4742-7. DOI:10.1158/0008-5472.CAN-11-0359 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent phylogenic studies indicate that DNA recombination could have occurred in ancient papillomavirus types. However, no experimental data are available to demonstrate this event because of the lack of human papillomavirus infection models. We have used the cottontail rabbit papillomavirus (CRPV)/rabbit model to study pathogenesis and immunogenicity of different mutant genomes in vivo. Although the domestic rabbit is not a natural host for CRPV infection, it is possible to initiate infection with naked CRPV DNA cloned into a plasmid and monitor papilloma outgrowth on these animals. Taking advantage of a large panel of mutants based on a CRPV strain (Hershey CRPV), we tested the hypothesis that two non-viable mutant genomes could induce papillomas by either recombination or complementation. We found that co-infection with a dysfunctional mutant with an E2 transactivation domain mutation and another mutant with an E7 ATG knock out generated papillomas in rabbits. DNA extracted from these papillomas contained genotypes from both parental genomes. Three additional pairs of dysfunctional mutants also showed similar results. Individual wild type genes were also shown to rescue the function of corresponding dysfunctional mutants. Therefore, we suggest that complementation occurred between these two non-viable mutant PV genomes in vivo.
Virus Research 05/2009; 144(1-2):117-22. DOI:10.1016/j.virusres.2009.04.006 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Shope papillomavirus or cottontail rabbit papillomavirus (CRPV) is one of the first small DNA tumour viruses to be characterized. Although the natural host for CRPV is the cottontail rabbit (Sylvilagus floridanus), CRPV can infect domestic laboratory rabbits (Oryctolagus cuniculus) and induce tumour outgrowth and cancer development. In previous studies, investigators attempted to passage CRPV in domestic rabbits, but achieved very limited success, leading to the suggestion that CRPV infection in domestic rabbits was abortive. The persistence of specific anti-L1 antibody in sera from rabbits infected with either virus or viral DNA led us to revisit the questions as to whether L1 and infectious CRPV can be produced in domestic rabbit tissues. We detected various levels of L1 protein in most papillomas from CRPV-infected rabbits using recently developed monoclonal antibodies. Sensitive in vitro infectivity assays additionally confirmed that extracts from these papillomas were infectious. These studies demonstrated that the CRPV/New Zealand White rabbit model could be used as an in vivo model to study natural virus infection and viral life cycle of CRPV and not be limited to studies on abortive infections.
Journal of General Virology 01/2008; 88(Pt 12):3286-93. DOI:10.1099/vir.0.82879-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Three transgenic rabbit lines that express a well-characterized human major histocompatibility complex class I (MHC-I) gene
(HLA-A2.1) have been established. All three lines carry the HLA-A2.1 heavy chain and are able to pass the transgene to their
offspring with both the outbred and the inbred EIII/JC genetic background. HLA-A2.1 colocalizes exclusively with rabbit MHC-I
on the cell surfaces. These HLA-A2.1 transgenic rabbits demonstrated infection patterns similar to those found after cottontail
rabbit papillomavirus (CRPV) challenge when compared with results in normal rabbits, although higher regression rates were
found in HLA-A2.1 transgenic rabbits. Because the CRPV genome can accommodate significant modifications, the CRPV/HLA-A2.1
rabbit model has the potential to be used to screen HLA-A2.1-restricted immunogenic epitopes from human papillomaviruses in
the context of in vivo papillomavirus infection.
Journal of Virology 08/2007; 81(13):7171-7. DOI:10.1128/JVI.00200-07 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cottontail rabbit papillomavirus (CRPV)/rabbit model has been used to study oncogenicity and immunogenicity of different antigens from the papillomavirus genome and has therefore served as a preclinical model for the development of preventive and therapeutic vaccines against papillomavirus infections. One unique property of the CRPV model is that infection can be initiated using viral DNA. This property allows for the functional testing of viral mutants in vivo. We have introduced point mutations, insertions and deletions into all of the different coding and non-coding regions of the CRPV genome and have tested their infectivity in this model. We found that the majority of the mutant genomes retained viability and could induce papillomas in domestic rabbits. These data indicated that the CRPV genome is tolerant of many modifications without compromising its ability to initiate skin papillomas. In combination with our recently established HLA-A2.1 transgenic rabbit model, this plasticity allows us to extend the utility of the CRPV/rabbit model to the screening of HLA-A2.1 restricted epitopes from other human viral and tumor antigens.
[Show abstract][Hide abstract] ABSTRACT: A human papillomavirus (HPV) vaccine consisting of virus-like particles (VLPs) was recently approved for human use. It is
generally assumed that VLP vaccines protect by inducing type-specific neutralizing antibodies. Preclinical animal models cannot
be used to test for protection against HPV infections due to species restriction. We developed a model using chimeric HPV
capsid/cottontail rabbit papillomavirus (CRPV) genome particles to permit the direct testing of HPV VLP vaccines in rabbits.
Animals vaccinated with CRPV, HPV type 16 (HPV-16), or HPV-11 VLPs were challenged with both homologous (CRPV capsid) and
chimeric (HPV-16 capsid) particles. Strong type-specific protection was observed, demonstrating the potential application
of this approach.
Journal of Virology 01/2007; 80(24):12393-7. DOI:10.1128/JVI.01583-06 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8(+) T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition, vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy.
The Journal of Immunology 01/2007; 177(11):8037-45. DOI:10.4049/jimmunol.177.11.8037 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Papillomaviruses (PVs) demonstrate both tissue and species tropisms. Because PVs replicate only in terminally differentiating
epithelium, the recent production of infectious PV particles in 293 cells marks an important breakthrough. In this article,
we demonstrate that infectious PV particles produced in 293TT cells can cause papillomatous growths in the natural host animal.
Moreover, we show that species-matched PV genomes can be successfully delivered in vivo by a heterologous, species-mismatched
PV capsid. Additionally, our results indicate that the addition of the simian virus 40 origin of replication to the papillomavirus
genome increases the production of infectious papillomavirus particles by increasing genome amplification in the transfected
Journal of Virology 12/2006; 80(22):11381-4. DOI:10.1128/JVI.01328-06 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human papillomaviruses (HPVs) replicate only in the terminally differentiating epithelium of the skin and mucosa. While infection
of basal keratinocytes is considered a requirement for permissive infection, it remains unclear whether virions can specifically
target basal cells for adsorption and uptake following epithelial wounding. We present evidence that HPV binds specifically
to laminin 5 (LN5), a component of the extracellular matrix (ECM) secreted by migrating and basal keratinocytes. HPV type
11 capsids colocalized with LN5 in the ECM secreted by vaginal keratinocytes. Binding of both virions and virus-like particles
to purified LN5 and to the LN5-rich ECM secreted by cultured keratinocytes was effectively blocked by pretreatment with anti-LN5
antibodies. HPV capsid binding to human cervical mucosa sections included the basement membrane which contains LN5. Cultured
keratinocytes expressing α6 integrin, a transmembrane protein known to bind LN5, were readily infected by virions preadsorbed
to LN5-containing substrates, whereas mutant keratinocytes lacking α6 integrin were relatively resistant to infection via
this route. These findings suggest a model of natural HPV infection in which proliferating keratinocytes expressing α6 integrin
at the site of epithelial wounding might be targeted by virions adsorbed transiently to LN5 secreted by migrating keratinocytes.
Journal of Virology 10/2006; 80(18):8940-50. DOI:10.1128/JVI.00724-06 · 4.44 Impact Factor