title: Microwaves: a potential new therapy for HPV-positive anogenital lesions? An investigation into the molecular mechanism of microwave treatment on human papillomavirus replication, cellular transcription and innate immunity
Creators: name: Kirk, Anna
Publisher: name: University of Glasgow
Year: 2024

description: Human papillomavirus (HPV) is the most common viral sexually transmitted infection (STI) globally with almost the entire population coming into contact with the virus within a few months to a few years of becoming sexually active. HPV infection at the mucosal epithelium of the anogenital tract is common and infection with a high-risk virus can result in anogenital lesions, which can progress into cervical and other anogenital cancers. Infection with a low-risk HPV type in the anogenital tract can result in genital warts. The incidence of many of these diseases and cancers, for example, anal and oropharyngeal cancers, has increased significantly over the last two decades. The current treatment options for pathologies caused by both high and lowrisk HPV have associated side effects including high pain, recurrence rate and risk of complications. Therefore, better treatment options are required. Hyperthermia is a well-documented adjunct to cancer therapy, which is also gaining traction in the treatment of HPV-driven pathologies. Microwaves offer a precise and targeted approach to deliver hyperthermia, and the Emblation Limited Swift® Microwave Tissue Ablation system is currently in use for clinical treatment of plantar warts: cutaneous lesions associated with HPV infection. Microwave ablation is a promising solution to overcome some of the limitations associated with the current treatment options for HPV-positive anogenital lesions but before implementation, an understanding of the molecular mechanisms behind microwave therapy, particularly how it impacts the HPV replication cycle, and the infected host cell must be carried out.

To analyse how microwave-delivered hyperthermia affected the host cell, we examined transcriptional changes following treatment. Microwave treatment rapidly altered the keratinocyte transcriptome, with 59 genes differentially expressed 4 hours post-treatment. Of these genes, 56 were upregulated and 3 were downregulated. At later time points post-treatment, 150 genes were differentially expressed and all of these were upregulated. Microwave treatment triggered a heat shock response and resulted in the upregulation of many chaperones and co-chaperones, which are important in preventing proteolysis following temperature rises. Many of these genes remained significantly upregulated at later time points. Genes involved in immune pathways, including interleukins (IL8, IL20, IL24), interferons (IFNĸ) and chemokines (CCL24, CCL26) were also upregulated following microwave treatment. Analysis of the mRNA expression of IL-6, IL-1β and TNF-α confirmed an upregulation of innate immunity following microwave treatment. The magnitude of this increase was greater in HPV-positive tissues. Microwave-treated tissues secreted a higher concentration of Th-1 cytokines (IL-1β, IL-8, IL-2, TNF-α, IFN-Ɣ) in comparison to mock-treated tissues. Th-1 mediated immunity is important for HPV clearance, so this has important implications for the recurrence of pathologies following microwave treatment.

The HPV genome is associated with histones and uses host RNA polymerase II for viral transcription. Therefore, it was hypothesised that HPV transcription may also be affected by microwave treatment. In models containing HPV16 episomes, there was a transient increase in the expression of transcripts encoding the HPV16 early (E6/E7 and E4 containing) and late (E4^L1 and L1 containing) viral transcripts. The magnitude of this increase was greater for transcripts encoding the viral late proteins. An increase in the differentiated compartment of these tissues following microwave treatment was evident through increased transcription of the late epithelial differentiation markers including transglutaminase, keratins (KRT37 and KRT75), corneodesmosomes and late cornified envelope proteins (LCE3A, LCE3C, LCE3D, LCE6A). Following the transient increase, the levels of early and late viral transcripts decreased by 72 hours post-treatment, to levels below that of untreated proteins. Spatial analysis using RNA Scope in situ hybridisation revealed this decrease in viral transcripts occurring first within the treated area and then spreading out radially into the neighbouring tissue. Microwave treatment could also inhibit viral genome replication up to 24 hours posttreatment.

Finally, we sought to investigate if microwave treatment could disrupt virion production. The L1 protein is the structural determinant of the human papillomavirus capsid but its localisation within the nucleus, and how this enables virion assembly in natural models of infection is poorly understood. Using immunofluorescent staining and confocal microscopy, the subcellular localisation of L1 within differentiated keratinocytes from organotypic rafts was examined. Over time, L1 accumulated in the nucleus, the site of virion assembly. The majority of nuclei positive for L1 showed diffuse nuclear staining but as rafts were grown in culture for extended periods, more cells contained L1 that was localised to subnuclear foci domains. This is similar to previous reports that have identified these sites as promyelocytic leukaemia nuclear bodies (PML-NBs) and proposed that L2, the minor structural protein, directs L1 to these sites once it is expressed at a high level. Electron microscopy analysis of keratinocytes grown in organotypic raft cultures revealed clusters of viral-like particles within the upper, differentiated cells of the tissues. We propose that these clusters may represent the foci of L1 staining observed by confocal microscopy, suggesting that papillomaviruses assemble in subnuclear domains. The intracellular localisation of L1 was disrupted following microwave treatment, which may restrict virion assembly. Further experiments to confirm the presence of viral-like particles with immunogold labelling will validate these findings.

Overall, this thesis builds on previous work that suggests that microwaves are a safe and effective form of treatment for HPV-associated lesions. The data suggests that microwave treatment inhibits human papillomavirus replication and induces an immune response within HPV-infected cells. This provides an argument for the extension of microwaves as a novel treatment option for HPV-positive anogenital lesions. Future research will determine the regrowth of lesions to predict the effectiveness and recurrence rates following treatment.