Mechanistic mathematical models: An underused platform for HPV research

Health economic modeling has become an invaluable methodology for the design and evaluation of clinical and public health interventions against the human papillomavirus (HPV) and associated diseases. At the same time, relatively little attention has been paid to a different yet complementary class of models, namely that of mechanistic mathematical models. The primary focus of mechanistic mathematical models is to better understand the intricate biologic mechanisms and dynamics of disease. Inspired by a long and successful history of mechanistic modeling in other biomedical fields, we highlight several areas of HPV research where mechanistic models have the potential to advance the field. We argue that by building quantitative bridges between biologic mechanism and population level data, mechanistic mathematical models provide a unique platform to enable collaborations between experimentalists who collect data at different physical scales of the HPV infection process. Through such collaborations, mechanistic mathematical models can accelerate and enhance the investigation of HPV and related diseases

HPV-induced field cancerisation: Transformation of adult tissue stem cell into cancer stem cell

Field cancerisation was originally described as a basis for multiple head and neck squamous cell carcinoma (HNSCC) and is a pre-malignant phenomenon that is frequently attributable to oncogenic human papillomavirus (HPV) infection. Our work on β-HPV-induced cutaneous squamous cell carcinomas identified a novel Lrig1+ hair follicle junctional zone keratinocyte stem cell population as the basis for field cancerisation. Herein, we describe the ability for HPV to infect adult tissue stem cells in order to establish persistent infection and induce their proliferation and displacement resulting in field cancerisation. By review of the HPV literature, we reveal how this mechanism is conserved as the basis of field cancerisation across many tissues. New insights have identified the capacity for HPV early region genes to dysregulate adult tissue stem cell self-renewal pathways ensuring that the expanded population preserve its stem cell characteristics beyond the stem cell niche. HPV-infected cells acquire additional transforming mutations that can give rise to intraepithelial neoplasia (IEN), from environmental factors such as sunlight or tobacco induced mutations in skin and oral cavity, respectively. With establishment of IEN, HPV viral replication is sacrificed with loss of the episome, and the tissue is predisposed to multiple cancer stem cell-driven carcinomas

The Role of Epidermal Stem/Progenitor-Like Cells In HPV-Mediated Pre-Neoplastic Transformation

The role of epidermal basal stem cells in dysplasia is a matter of great interest in the human papillomavirus (HPV)-driven cancers. To assess the relationship between “stemness” and HPV-mediated transformation, we made use of 3-D suspension culture and fluorescence activated cell sorting (FACS) to purify stem/progenitor-like cells from primary normal human keratinocyte (NHKc) cultures. We found that NHKc cells derived from multicellular keratinocyte spheroids were enriched for a basal subpopulation of epidermal stem-like cells, that could be maintained for prolonged time in culture and used to conduct transfection experiments with full-length HPV16 DNA. Thus, by using these stem cell enrichment methods, we set out to investigate in detail the effects of increased and decreased basal stem cell density on keratinocytes’ immortalization and transformation efficiencies. We hypothesized that stem cell properties of NHKc cultures established from neonatal genital skin would positively influence susceptibility to transformation by HPV16 DNA. Our findings reveal that epidermal stem cells (EpSCs) are more effectively immortalized and transformed by oncogenic HPV16 DNA, while terminally differentiated keratinocyte populations fail to successfully immortalize in culture. Tissue stem cell density may prove useful in predicting individual susceptibility to HPV16-mediated transformation in persons with persistent HPV infections, improving on current triage and follow-up measures

In situ expression of biomarkers in placenta in the context of hypertensive disorders of pregnancy

Overview: Hypertensive disorders in pregnancy (HDP), of which the most common is pre- eclampsia (PE), are a leading cause of maternal and foetal morbidity and mortality worldwide. Despite extensive research, no clear cause or mechanism for HDP has been discovered. Human papillomavirus (HPV) has transformative abilities among many cell types and alters key pathways in reproductive cells essential for embryo implantation and placental development in vitro. It has also been consistently found in the placentae, especially in those complicated by PE. These findings suggest HPV may not be only a bystander in complicated pregnancies, but has a direct role in abnormal placental development. Whether HPV genes such as E6/E7 that encode the E6 and E7 proteins, which affect trophoblast function in vitro, are expressed in vivo is unknown. Understanding gene expression profiles in placental tissue is complicated by the wide variety of cell types present and their distinct genetic origins. This study used RNAscope® to investigate whether in situ based techniques could identify HPV E6/E7 expression differences in placentas from a cohort of Japanese women. A second aspect of this study used RNAscope® to investigate if genes found to be differentially methylated in the cohort were associated with HDP. NOTCH2 and CMIP were chosen. Methods: Fifteen placenta from uncomplicated pregnancies, sixteen placenta from pregnancies complicated by HDP, and six additional cases of non-HDP pathologies, were converted to FFPE slides and examined using RNAScope®, or immunohistochemistry. RNAscope® assays to determine RNA quality, non-specific staining, HPV high-risk E6/E7, HPV low-risk E6/E7 and NOTCH2 were undertaken, along with immunohistochemistry for CMIP. Results were compared between the HDP and control groups. Results: The majority of placentas from the HDP (n=15/16, 94%) and the control (n=11/15, 73%) cohorts had sufficient RNA quality. In the HDP group, six cases (n=6/15) tested positive for high risk HPV E6/E7 (40%), and five tested positive for low risk HPV E6/E7 (33%). None of the control cases tested positive for high risk HPV (0%) or low risk HPV (0%). Expression of HPV high and low risk E6/E7 was increased in the HDP compared to the control cohort (p<0.05). The NOTCH2 assay was optimised and NOTCH2 was expressed heterogeneously between neighbouring cells in individual cases, but the number of cases tested was too low for statistical power. The CMIP assay did not work, requiring further optimisation. Conclusion: This study provides evidence that HPV high and low risk E6/E7 is expressed in the placenta of pregnancies complicated by HDP. These results add validity that HPV may affect placental function by showing HPV gene expression is active in the HDP affected placenta. Further research is needed to understand why transcriptionally active HPV E6/E7, of both high and low risk types, would be present in the developing and term placenta. In addition, RNAscope® proved viable for visualising cell types to target for further sequencing, which could be used to determine if one cell population is more differentially methylated within the heterogeneous placenta

Investigating Early Lesion Formation Following Papillomavirus Infection Using a Mouse Model and Cell Culture

Papillomaviruses (PV) are small non-enveloped double-stranded DNA tumour viruses, which are able to infect more than 80 different host species. They are a diverse group with over 400 types discovered, of which almost half infect humans. Human papillomaviruses have been linked to a myriad of diseases, including multiple cancers, recurrent respiratory papillomatosis, and genital warts. The disease burden of HPV-related conditions is severe, and there is currently no treatment that can guarantee eradication of viral infection. All PV types characterised so far have a similar genomic structure, and contain the so called 'core ORFs' – E1, E2, L1 and L2 which are essential for viral genome replication and packaging into infectious virions. PV evolution and diversification appears to have been impacted by the availability of certain epithelial niches, with co-evolution and niche adaptation allowing PVs to develop a remarkable species and tissue specificity. Consequently, the function of the PV early proteins can vary between different PV species and types, but as a group they share important organisational similarities that reflect their common requirement to infect and persist in the epithelium following infection. This has allowed the use of animal models to gain insight into the basic virus/host interactions that are targeted by this group of viruses as a whole. The mechanisms by which HPV establishes a lesion, particularly in low-risk types, are not fully understood. However, the recently identified mouse model of PV infection is a useful biological tool to study this period of PV infection in vivo. This body of work aims to expand current knowledge of early events in the PV life cycle. To further understand the mechanisms of cell persistence during PV infection, immunodeficient mouse tail samples inoculated with MmuPV1 were examined to investigate early lesion formation. Five discrete stages of lesion formation were characterised in the immunodeficient animals. In parallel studies, microlesions were rarely observed in immunocompetent C57BL/6J mice, reaching stage three of lesion formation. In-depth tissue analysis suggested a modulation of basal cell density in infected epithelium, and a delay in normal differentiation commitment in E6/E7 expressing cells. Whole genome cell culture experiments were attempted in parallel with human high-risk types, which showed a post-confluent effect of high concentration EGF on cell growth and genome copy number in cells containing HPV16 genomes. A role for MmuPV1 E6 in growth of cell populations to significantly higher densities was shown through experimentation with cells exogenously expressing viral proteins. Differentiation was also delayed in the cells expressing MmuPV1 E6, demonstrating a recapitulation of events characterised in in vivo infections. Novel use of fluorescent cell lines in tandem with confocal microscopy allowed innovative analysis of a high-density monolayer cell culture model. These experiments revealed that MmuPV1 E6 expression resulted in preferential persistence of cells in the lower layer over cells expressing control vector only. Disruption of MmuPV1 E6 binding with MAML1 protein abrogated this phenotype, suggesting that this interaction was necessary for the lower layer persistence phenotype shown by MmuPV1 E6 expressing cells. Overall, the findings of this thesis suggest that expression of MmuPV1 E6 confers a competitive advantage on infected cells in the basal layer of the epithelium, allowing expansion of the reservoir of infection. Patterns of virus gene expression suggest a related but distinct life cycle phenotype for MmuPV1, a pi papillomavirus type, when compared to alpha papillomaviruses. Wherein, amplification begins immediately upon basal layer exit as opposed to the exit and reentry phenotype suggested in high-risk lesions. Further characterisation of these phenotypes will likely provide important information on key mechanisms in early lesion formation, and it is reasonable to consider that pi papillomaviruses may serve as a better model for beta papillomaviruses than alpha types

HPV Clearance and the Neglected Role of Stochasticity

<div><p>Clearance of anogenital and oropharyngeal HPV infections is attributed primarily to a successful adaptive immune response. To date, little attention has been paid to the potential role of stochastic cell dynamics in the time it takes to clear an HPV infection. In this study, we combine mechanistic mathematical models at the cellular level with epidemiological data at the population level to disentangle the respective roles of immune capacity and cell dynamics in the clearing mechanism. Our results suggest that chance—in form of the stochastic dynamics of basal stem cells—plays a critical role in the elimination of HPV-infected cell clones. In particular, we find that in immunocompetent adolescents with cervical HPV infections, the immune response may contribute less than 20% to virus clearance—the rest is taken care of by the stochastic proliferation dynamics in the basal layer. In HIV-negative individuals, the contribution of the immune response may be negligible.</p></div