Viruses

Oncolytic Vaccinia Virus Expressing HSP70 shRNA Exerts Anti-Tumor Effects in Human Ovarian Cancer via Triggering the Autophagy–ROS Feedback Loop and Immune Activation

Heat shock protein 70 (HSP70) represents a promising target for cancer therapy. Oncolytic vaccinia virus (oncoVV) mediates tumor regression through direct oncolysis and immune activation. However, the anti-tumor potential of HSP70-silenced oncoVV (oncoVV-shHSP70) remains unexplored. Here, we demonstrate that oncoVV-shHSP70 achieves superior tumor regression in ovarian cancer models (cell lines, immunodeficient mice and humanized mice) via dual mechanisms including enhancing apoptosis, autophagy flux, ROS generation, and immune reprogramming. Notably, we found that oncoVV-shHSP70 triggers an autophagy–ROS feedback loop that amplifies viral replication and pro-inflammatory cytokine expression. Crucially, in humanized mice, oncoVV-shHSP70 induced spatial redistribution of cytotoxic T cells, expanding tumor-infiltrating hCD8+hGZMB+ populations. These findings position oncoVV-shHSP70 as a promising viro-immunotherapy that co-opts HSP70 silencing to potentiate both direct oncolysis and anti-tumor immunity, providing a preclinical rationale for viro-immunotherapy in solid tumors.

A Replication-Defective Myxoma Virus Inducing Pro-Inflammatory Responses as Monotherapy and an Adjuvant to Chemo- and DC Immuno-Therapy for Ovarian Cancer

Myxoma virus (MYXV), a rabbit-specific poxvirus and non-pathogenic in humans and mice, is an excellent candidate oncolytic virus for cancer therapy. MYXV also has immunotherapeutic benefits. In ovarian cancer (OC), immunosuppressive tumor-associated macrophages (TAMs) are key to inhibiting antitumor immunity while hindering therapeutic benefit by chemotherapy and dendritic cell (DC) vaccine. Because MYXV favors binding/entry of macrophages/monocytes, we examined the therapeutic potential of MYXV against TAMs. We found previously that a replication-defective MYXV with targeted deletion of an essential gene, M062R, designated ΔM062R MYXV, activated both the host DNA sensing pathway and the SAMD9 pathway. Treatment with ΔM062R confers therapeutic benefit comparable to that of wild-type replicating MYXV in preclinical models. Here we found that ΔM062R MYXV, when integrated with cisplatin and DC immunotherapy, further improved treatment benefit, likely through promoting tumor antigen-specific T cell function. Moreover, we also tested ΔM062R MYXV in targeting human immunosuppressive TAMs from OC patient ascites in a co-culture system. We found that ΔM062R treatment subverted the immunosuppressive properties of TAMs and elevated the avidity of cytokine production in tumor antigen-specific CD4+ T cells. Overall, ΔM062R presents a promising immunotherapeutic platform as a beneficial adjuvant to chemotherapy and DC vaccine.

Update on Effects of the Prophylactic HPV Vaccines on HPV Type Prevalence and Cervical Pathology

Most national prophylactic HPV vaccination programs started in approximately 2008, with either the bivalent Cervarix HPV16/18 or quadrivalent Gardasil (HPV6/11/16/18) vaccines, which were then followed by introduction of the nonavalent Gardasil 9 (HPV6/11/16/18/ 31/33/45/52/58) vaccine from 2015. Since that time, these products have demonstrated their ability to prevent infection with vaccine-covered HPV types and subsequent development of HPV-related cervical and genital pathologies. The data indicate that vaccination of young girls prior to sexual debut is more effective than vaccination of older HPV+ve women. Although some studies have shown a decline in the prevalence of vaccine-covered HPV types, there are national and regional differences in overall vaccine efficacy. Furthermore, several recently published studies show an increase in the prevalence of non-vaccine-covered HPV types in vaccinated populations, which is indicative of HPV type-replacement. It is also notable that vaccine-related changes in HPV type prevalence spread between vaccinated and unvaccinated women at the same geographical location—presumably via sexual transmission. In conclusion, it is not yet clear what effect dissemination of vaccine-associated changes in HPV type prevalence will have on vaccine efficacy and cervical pathology, particularly in mixed populations of vaccinated and unvaccinated women. However, it is very clear these observations do underscore the need for long-term continuation of cervical screening combined with regular reassessment of testing practices.

Tepilamide Fumarate as a Novel Potentiator of Virus-Based Therapy

Oncolytic virotherapy, using viruses such as vesicular stomatitis virus (VSVΔ51) and Herpes Simplex Virus-1 (HSV-1) to selectively attack cancer cells, faces challenges such as cellular resistance mediated by the interferon (IFN) response. Dimethyl fumarate (DMF) is used in the treatment of multiple sclerosis and psoriasis and is recognized for its anti-cancer properties and has been shown to enhance both VSVΔ51 and HSV-1 oncolytic activity. Tepilamide fumarate (TPF) is a DMF analog currently undergoing clinical trials for the treatment of moderate-to-severe plaque psoriasis. The aim of this study was to evaluate the potential of TPF in enhancing the effectiveness of oncolytic viruses. In vitro, TPF treatment rendered 786-0 carcinoma cells more susceptible to VSVΔ51 infection, leading to increased viral replication. It outperformed DMF in both increasing viral infection and increasing the killing of these resistant cancer cells and other cancer cell lines tested. Ex vivo studies demonstrated TPF’s selective boosting of oncolytic virus infection in cancer cells without affecting healthy tissues. Effectiveness was notably high in pancreatic and ovarian tumor samples. Our study further indicates that TPF can downregulate the IFN pathway through a similar mechanism to DMF, making resistant cancer cells more vulnerable to viral infection. Furthermore, TPF’s impact on gene therapy was assessed, revealing its ability to enhance the transduction efficiency of vectors such as lentivirus, adenovirus type 5, and adeno-associated virus type 2 across various cell lines. This data underscore TPF’s potential role in not only oncolytic virotherapy but also in the broader application of gene therapy. Collectively, these findings position TPF as a promising agent in oncolytic virotherapy, warranting further exploration of its therapeutic potential.

The Sibylline Relationship Between Human Papillomavirus and Endometrial Cancer: Scarcity of Strong Evidence Linking Both Conditions

Endometrial cancer (EC) is the fourth-most frequent cancer among the female population and a leading cause of death. Multiple factors are susceptible to causing tumorigenesis, including obesity, lack of physical activity, diabetes mellitus, high concentration of estrogen during menopause, unopposed exposure to estrogen, duration of menses, nulliparity and infertility. Human papillomavirus (HPV) is a double-stranded DNA virus, with certain genotypes exclusively human. HPV plays a major role in some cancers (cervical cancer, head and neck cancer, lung cancer, and anogenital cancers). Given the intricate correlation between HPV and cervical cancer, the scientific community conjectured that HPV may be implicated in the carcinogenesis of the endometrium. In this review, we will direct our interest towards previous studies that focused on the expression of HPV on EC samples and cover how both conditions might connect to each other.

From Infection to Malignancy: Tracing the Impact of Human Papillomavirus on Uterine Endometrial Cancer in a Nationwide Population-Based Cohort Study

Uterine endometrial cancer (EC) is the most common gynecological malignancy in Taiwan. This study aimed to investigate the association between human papillomavirus (HPV) infection and the development of uterine EC among Taiwanese women. A nationwide population cohort research approach was employed, leveraging longitudinal health insurance databases (LHID 2007 and 2015) from the National Health Insurance Research Database alongside data from the Taiwan Cancer Registry datasets. A comparative analysis examined 472,420 female patients with HPV infection and 944,840 without HPV infection. The results demonstrated that the HPV cohort exhibited a significantly elevated risk of uterine EC, as evidenced by an adjusted hazard ratio (aHR) of 1.588 (95% CI: 1.335–1.888). Furthermore, this elevated risk extended to type 1 EC with an aHR of 1.671 (95% CI: 1.376–2.029), specifically the endometrioid adenocarcinoma subtype with an aHR 1.686 (95% CI: 1.377–2.065). Importantly, these findings were statistically significant (p < 0.001). In conclusion, this research unveils a potential association between HPV infection and an increased risk of uterine EC, particularly the type 1 endometrial cancer subtype, within the Taiwanese female population. These findings have implications for preventive measures and screening programs targeting HPV infection to reduce the risk of this prevalent gynecological malignancy in Taiwan.

Plant Compounds Inhibit the Growth of W12 Cervical Precancer Cells Containing Episomal or Integrant HPV DNA; Tanshinone IIA Synergizes with Curcumin in Cervical Cancer Cells

This study explores the effects of plant compounds on human papillomavirus (HPV)-induced W12 cervical precancer cells and bioelectric signaling. The aim is to identify effective phytochemicals, both individually and in combination, that can prevent and treat HPV infection and HPV associated cervical cancer. Phytochemicals were tested using growth inhibition, combination, gene expression, RT PCR, and molecular docking assays. W12 cells, derived from a cervical precancerous lesion, contain either episomal or integrated HPV16 DNA. Several compounds, including digoxin, tanshinone IIA, dihydromethysticin and carrageenan, as well as fractions of turmeric, ginger and pomegranate inhibited the growth of W12 precancer and cervical cancer cells. Curcumin and tanshinone IIA were the most active and relatively nontoxic compounds. RT-PCR analysis showed that tanshinone IIA activated the expression of p53, while repressing the expression of HPV16 E1, E2, E4, E6, and E7 viral transcripts in W12 (type 1 and 2) integrant cells. In addition, curcumin synergized with tanshinone IIA in HeLa cells. Molecular docking studies suggested tanshinone IIA and curcumin bind to the Na+/K+-ATPase ion channel, with curcumin binding with higher affinity. Our findings highlight the potential of these multifaceted phytochemicals to prevent and treat HPV-induced cervical cancer, offering a promising approach for combinatorial therapeutic intervention.

Regulation and Deregulation of Viral Gene Expression During High-Risk HPV Infection

Cervical cancer remains a global health burden, with persistent infection by high-risk human papillomaviruses (HR-HPVs) being the primary etiological factor. HR-HPVs target stem-like cells of the cervical epithelium to establish chronic infections. Upon infection of the cervical transformation zone (TZ)—a region adjacent to the squamocolumnar junction (SCJ)—these viruses drive neoplastic transformation, which is due in part to the unique cellular composition and hormonal responsiveness of the TZ. Reserve cells, which can accumulate at the cervical crypt entrances of the TZ, are thought to be highly susceptible to HR-HPV infection because of their location beneath a single layer of columnar cells. Infection of the stratified ectocervical epithelium, in contrast, requires a wound to allow basal cell infection, replication, and the expression of early genes to adjust epithelial homeostasis while facilitating immune evasion. Persistent infection by HR-HPV types, particularly HPV16 and HPV18, can result in the deregulated expression of viral genes E6 and E7, driving cell cycle disruption, genomic instability, and subsequent viral genome integration. Differences in the microenvironment and transcriptional environment of the ectocervix compared with the TZ could explain the frequent deregulation of E6 and E7 at the latter site, which can drive disease progression towards cancer.

HTLV-1 Infection and Cervicovaginal Susceptibility to High-Risk HPV: Findings from Women Living with HTLV-1 in Salvador, Brazil

Persistent oncogenic HPV infection is strongly associated with cervical cancer. Studies have suggested a higher prevalence of HPV in women living with HTLV-1. This study aimed to determine whether HTLV-1 infection is associated with cervicovaginal HPV infection and to characterize HPV types according to oncogenic risk. Vaginal fluid samples were subjected to HPV diagnosis via PCR, and positive samples were subjected to Sanger sequencing and massive sequencing. Papanicolaou smears were examined using light microscopy to identify cell abnormalities. Among the 155 women screened, 79 were HTLV-1-infected and 76 were uninfected. HPV PCR identified 23 positive samples (15/79 vs. 8/76; p = 0.13). Twenty-three HPV types were identified, of which only types 31, 54, and 58 were present in both groups. When the number of HPV58 infections in each group was compared, women with HTLV-1 had a higher prevalence (8/79 versus 1/76; p = 0.03). In total, 61.9% of HTLV-1-infected women had at least one high-risk or probable high-risk HPV type (p = 0.12). Cytopathological findings were not significantly different between the groups. Further research is needed to determine whether HTLV-1 infection affects HPV progression and cervical cancer development and to assess the potential benefits of vaccination for women living with HTLV-1.

Elucidating Alterations in Viral and Human Gene Expression Due to Human Papillomavirus Integration by Using Multimodal RNA Sequencing

Human papillomavirus (HPV) infection is a primary driver of cervical cancer. Integration of HPV into the human genome causes persistent expression of viral oncogenes E6 and E7, which promote carcinogenesis and disrupt host genomic function. However, the impact of integration on host gene expression remains incompletely understood. We used multimodal RNA sequencing, combining total RNA-seq and Cap Analysis of Gene Expression (CAGE), to clarify virus–host interactions after HPV integration. HPV-derived transcripts were detected in 17 of 20 clinical samples. In most specimens, transcriptional start sites (TSSs) showed predominant early promoter usage, and transcript patterns differed with detectable E4 RNA region. Notably, the high RNA expressions of E4 region and viral-human chimeric RNAs were mutually exclusive. Chimeric RNAs were identified in 13 of 17 samples, revealing 16 viral integration sites (ISs). CAGE data revealed two patterns of TSS upregulation centered on the ISs: a two-sided pattern (43.8%) and a one-sided pattern (31.3%). Total RNA-seq showed upregulation of 12 putative cancer-related genes near ISs, including MAGI1-AS1, HAS3, CASC8, BIRC2, and MMP12. These findings indicate that HPV integration drives transcriptional activation near ISs, enhancing expression of adjacent oncogenes. Our study deepens understanding of HPV-induced carcinogenesis and informs precision medicine strategies for cervical cancer.

Susceptibility of HPV-18 Cancer Cells to HIV Protease Inhibitors

Cervical cancer cases continue to rise despite all the advanced screening and preventative measures put in place, which include human papillomavirus (HPV) vaccination. These soaring numbers can be attributed to the lack of effective anticancer drugs against cervical cancer; thus, repurposing the human immunodeficiency virus protease inhibitors is an attractive innovation. Therefore, this work was aimed at evaluating the potential anticancer activities of HIV-PIs against cervical cancer cells. The MTT viability assay was used to evaluate the effect of HIV protease inhibitors on the viability of cervical cancer cells (HeLa) and non-cancerous cells (HEK-293). Further confirmation of the MTT assay was performed by confirming the IC50s of these HIV protease inhibitors on cervical cancer cells and non-cancerous cells using the Muse™ Count and Viability assay. To confirm the mode of death induced by HIV protease inhibitors in the HPV-associated cervical cancer cell line, apoptosis was performed using Annexin V assay. In addition, the Muse™ Cell Cycle assay was used to check whether the HIV protease inhibitors promote or halt cell cycle progression in cervical cancer cells. HIV protease inhibitors did not affect the viability of non-cancerous cells (HEK-293), but they decreased the viability of HeLa cervical cancer cells in a dose-dependent manner. HIV protease inhibitors induced apoptosis in HPV-related cervical cancer cells. Furthermore, they also induced cell cycle arrest, thus halting cell cycle progression. Therefore, the use of HIV drugs, particularly HIV-1 protease inhibitors, as potential cancer therapeutics represents a promising strategy. This is supported by our study demonstrating their anticancer properties, notably in HPV-associated cervical cancer cell line.

CTLA4 Haplotype Structures and −318 C>T (rs5742909) Genetic Variant Contribute to the Susceptibility of HPV Infection and Cervical Cancer

High-risk Human Papillomavirus (HPV) infection is the main etiological factor for cervical carcinogenesis, although genetic cofactors also play a role. Single-nucleotide variants (SNVs) in the CTLA4 gene can alter the gene expression and immune response against HPV, influencing cervical malignancy progression. This study analyzed the association of the alleles, genotypes, and haplotypes of the CTLA4 SNVs rs5742909 (−318 C>T), rs231775 (+49 A>G), and rs3087243 (+6230 G>A) with HPV infection, the development of low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs), and cervical cancer in 445 women treated by the public health service of Paraná, Brazil. Peripheral blood and cervical secretion samples were collected for genomic DNA extraction, CTLA4 SNV genotyping, and HPV detection via PCR. Statistical analyses used p < 0.05. The HPV-negative control group included 181 women, while the HPV-positive group included 264 women. The HPV-positive group was divided into no lesion (n = 84), LSILs (n = 19), HSILs (n = 56), and cervical cancer (n = 105). The T allele of −318 C>T and the TAG haplotype were associated with increased susceptibility to HPV infection, HSILs, and cervical cancer. These findings suggest that the T allele of −318 C>T and the TAG haplotype may serve as potential molecular biomarkers for HPV susceptibility and worse prognosis.

HPV and HCMV in Cervical Cancer: A Review of Their Co-Occurrence in Premalignant and Malignant Lesions

Cervical cancer remains a significant global health concern, particularly in low- and middle-income countries. While persistent infection with high-risk human papillomavirus (HR-HPV) is essential for cervical cancer development, it is not sufficient on its own, suggesting the involvement of additional cofactors. The human cytomegalovirus (HCMV) is a widespread β-herpesvirus known for its ability to establish lifelong latency and reactivate under certain conditions, often contributing to chronic inflammation and immune modulation. Emerging evidence suggests that HCMV may play a role in various cancers, including cervical cancer, through its potential to influence oncogenic pathways and disrupt host immune responses. This review explores clinical evidence regarding the co-presence of HR-HPV and HCMV in premalignant lesions and cervical cancer. The literature reviewed indicates that HCMV is frequently detected in cervical lesions, particularly in those co-infected with HPV, suggesting a potential synergistic interaction that could enhance HPV’s oncogenic effects, thereby facilitating the progression from low-grade squamous intraepithelial lesions (LSIL) to high-grade squamous intraepithelial lesions (HSIL) and invasive cancer. Although the precise molecular mechanisms were not thoroughly investigated in this review, the clinical evidence suggests the importance of considering HCMV alongside HPV in the management of cervical lesions. A better understanding of the interaction between HR-HPV and HCMV may lead to improved diagnostic, therapeutic, and preventive strategies for cervical cancer.

HPV-Associated Sexually Transmitted Infections in Cervical Cancer Screening: A Prospective Cohort Study

High-risk human papillomavirus (HR-HPV) and other sexually transmitted infections (STIs-O) are promoters to the development of cervical cancer (CC), especially when they co-exist. This study aims to determine the prevalence of the major STIs-O and the rate of co-infection in women previously diagnosed with HR-HPV infection. For this observational study, 254 women aged 25–65 years who were being followed up for HR-HPV infection (without a CC history) were recruited at a hospital’s Gynaecology Department from February 2024 to November 2024. Their endocervical specimens were collected and processed for HR-HPV, Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis detection by RT-PCR using commercially available reagents and equipment. The overall rate of infection was 38.6% for HPV and 4.3% for ITSs-O (3.8% in HPV-negative women and 5.1% in HPV-positive women). The presence of ITSs-O in women aged 25–34 was higher in those with a persistent positive result for HR-HPV (20.0% vs. 4.2%). Diverse multiple co-infections were found in HPV-positive women, whilst some single STIs-O were found in HPV-negative women. These results support the benefits of STI-O screening beyond an HR-HPV positive result, especially in those women under 35 years old.

Prevalence, Characteristics, and Distribution of Human Papillomavirus According to Age and HIV Status in Women of Eastern Cape Province, South Africa

Human papillomavirus (HPV) is a sexually transmitted infection associated with the development of cervical cancer. This study investigated cervical HPV prevalence, characteristics, and distribution according to age and human immunodeficiency virus (HIV) status among women attending a public community health facility in the Eastern Cape Province of South Africa. A total of 325 participants (aged 18 to 60) visiting a community health facility for any reason were recruited. Cervical HPV infection was detected using the Seegene Anyplex™ II HPV28 assay (Seegene Inc., Seoul, South Korea). Overall HPV prevalence was 65.2% (95% CI: 59.9–70.2%), with the highest prevalence of 80.9% (95% CI: 67.2–89.8%) observed in the 18–25-year-old age group and the lowest prevalence of 46.3% (95% CI: 35.8–57.1%) in the 46–60-year-old age group. HR-HPV infection was found to decrease with increasing age (p < 0.001) in the overall population and according to HIV status. In contrast, LR-HPV infection was found to significantly decrease with age among HIV-negative women (p = 0.001) but not for the overall population and HIV-positive women. A proportion of 12.9% were infected with one or more HPV types covered by the Cervarix® HPV vaccine (HPV-16 and/or -18), 18.8% (by those covered by Gardasil®4 (HPV-6, -11, -16 and/or -18), and 42.2% by those covered by Gardasil®9 (HPV-6, -11, -16, -18, -31, -33, -45, -52 and/or -58). The alpha-9 HPV species was the most dominant species (40.6%), followed by the alpha-7 species (29.8%). High overall HPV, HR-HPV, and alpha-9 species prevalence were observed among the women attending the public health facility. These findings contribute to the limited HPV distribution data among the Eastern Cape women, which could be used to improve HPV-related policy and assess the effectiveness of the HPV vaccination.

Genome-Wide Analysis of p53 Targets Reveals SCN2A as a Novel Player in p53-Induced Cell Arrest in HPV-Positive Cells

The host transcription factor p53 is a critical tumor suppressor in HPV-induced carcinogenesis, regulating target genes involved in cell cycle arrest and apoptosis. However, the p53 targets have not been thoroughly analyzed in HPV-infected cells. In this study, p53 signaling in HPV16 and HPV18 cells was activated by depleting the viral oncoprotein E6. Subsequently, p53-regulated genes were identified by comparing them with genes altered in p53-silenced cells. True p53 targets were defined as genes with at least one overlapping p53 binding site and ChIP peak near their locus. Our analysis revealed that while some p53 targets were common to both the HPV16 and HPV18 cells, the majority of the targets differed between these two types, potentially contributing to the varying prevalence of HPV16 and HPV18 in cervical cancer. Additionally, we identified SCN2A as a novel p53 target involved in p53-induced cell cycle arrest in HPV-related carcinogenesis. This study provides new insights into the mechanisms by which p53 inhibits HPV-induced carcinogenesis.

Looking Back, Moving Forward: Challenges and Opportunities for Global Cervical Cancer Prevention and Control

Despite the introduction of Pap testing for screening to prevent cervical cancer in the mid-20th century, cervical cancer remains a common cause of cancer-related mortality and morbidity globally. This is primarily due to differences in access to screening and care between low-income and high-income resource settings, resulting in cervical cancer being one of the cancers with the greatest health disparity. The discovery of human papillomavirus (HPV) as the near-obligate viral cause of cervical cancer can revolutionize how it can be prevented: HPV vaccination against infection for prophylaxis and HPV testing-based screening for the detection and treatment of cervical pre-cancers for interception. As a result of this progress, the World Health Organization has championed the elimination of cervical cancer as a global health problem. However, unless research, investments, and actions are taken to ensure equitable global access to these highly effective preventive interventions, there is a real threat to exacerbating the current health inequities in cervical cancer. In this review, the progress to date and the challenges and opportunities for fulfilling the potential of HPV-targeted prevention for global cervical cancer control are discussed.

The Rare Condition of a Double Cervix: Results from the High-Risk Human Papillomavirus-Based Cervical Cancer Screening Program in the Lazio Region

Precancerous and cancerous lesions of the uterine cervix are known to be associated with Human Papillomavirus (HPV) infection. The screening of high-risk (HR)-HPV infection in the female population has led to the discovery of several cases of a double cervix, a congenital malformation that is very rare. The purpose of this study was to evaluate HR-HPV infections in women with a double cervix within the National Cervical Cancer Screening program of the Lazio region (Italy). From June 2021 to March 2024, a total of 142,437 samples were analyzed by Seegene’s Anyplex TM II HR-HPV method, which identifies 14 HR-HPV genotypes. For each woman identified with a double cervix, two separate samples were taken from both cervices and analyzed separately. Twenty-seven women with a double cervix were identified (0.019%): 23 women were tested as negative for both cervices, while the remaining four (namely A, B, C, and D) resulted positive. By genotyping, the following results were obtained: (A) Both samples showed genotype 31; (B) one cervix was negative while the other showed genotype 58; (C) one cervix was positive for HPV 18 and 31 while for 18, 31, and 33 in the other; and (D) one cervix showed genotype 66 while the other carried the 66 and 68 genotypes. Double cervix is a very rare condition where the presence of HR-HPV genotypes is not homogeneous. As already described, our study confirms that different genotypes can be detected in double cervix malformation, suggesting the need to perform HPV screening on brushing samples from both cervices.

Identification of HPV16 Lineages in South African and Mozambican Women with Normal and Abnormal Cervical Cytology

Background: Human papillomavirus 16 (HPV16) is an oncogenic virus responsible for the majority of invasive cervical cancer cases worldwide. Due to genetic modifications, some variants are more oncogenic than others. We analysed the HPV16 phylogeny in HPV16-positive cervical Desoxyribonucleic Acid (DNA) samples collected from South African and Mozambican women to detect the circulating lineages. Methods: Polymerase chain reaction (PCR) amplification of the long control region (LCR) and 300 nucleotides of the E6 region was performed using HPV16-specific primers on HPV16-positive cervical samples collected in women from South Africa and Mozambique. HPV16 sequences were obtained through Next Generation Sequencing (NGS) methods. Geneious prime and MEGA 11 software were used to align the sequences to 16 HPV16 reference sequences, gathering the A, B, C, and D lineages and generating the phylogenetic tree. Single nucleotide polymorphisms (SNPs) in the LCR and E6 regions were analysed and the phylogenetic tree was generated using Geneious Prime software. Results: Fifty-eight sequences were analysed. Of these sequences, 79% (46/58) were from women who had abnormal cervical cytology. Fifteen SNPs in the LCR and eight in the E6 region were found to be the most common in all sequences. The phylogenetic analysis determined that 45% of the isolates belonged to the A1 sublineage (European variant), 34% belonged to the C1 sublineage (African 1 variant), 16% belonged to the B1 and B2 sublineage (African 2 variant), two isolates belonged to the D1–3 sublineages (Asian-American variant), and one to the North American variant. Conclusions: The African and European HPV16 variants were the most common circulating lineages in South African and Mozambican women. A high-grade squamous intraepithelial lesion (HSIL) was the most common cervical abnormality observed and linked to European and African lineages. These findings may contribute to understanding molecular HPV16 epidemiology in South Africa and Mozambique.

Papillomavirus-Induced Oncogenesis: Bridging Molecular Mechanisms, Diagnostics, and Global Prevention Strategies

Human papillomavirus (HPV) has long been recognized for causing benign lesions such as warts, but its pivotal role in cancer was established in the late 20th century, culminating in the 2008 Nobel Prize awarded to Harald zur Hausen for linking HPV to cervical cancer [...]

Vaccine and Non-Vaccine HPV Types Presence in Adolescents with Vertically Acquired HIV Five Years Post Gardasil Quadrivalent Vaccination: The ZIMGARD Cohort

Background: Human papillomavirus (HPV) vaccination programs are a key intervention in protecting individuals against HPV-related disease. HIV1-infected individuals are at increased risk of HPV-associated cancers. This study was conducted to evaluate the potential role of prophylactic HPV vaccines in preventing new HPV infections among participants with perinatally acquired HIV who received the quadrivalent HPV vaccine at least five years before this study. Methods: This cross-sectional study was conducted at Newlands Clinic, Harare, Zimbabwe. The clinic provided the Gardasil quadrivalent HPV vaccine (4vHPV) to 624 adolescents living with HIV starting in December 2015. Vaginal and penile swabs were collected and tested for HPV types from the study participants who had received the 4vHPV vaccine 5–6 years before enrolment. Results: We present the results of 98 participants (44.6% female) vaccinated at a median age of 15 years (IQR 12–16). The mean amount of time since vaccination was 6 years (SD: ±0.4). The HPV-positive rate amongst the analyzed swabs was 69% (68/98). Among 30/98 (31%) HPV-positive participants, 13/98 (13%) had low-risk HPV types, and 17/98 (17%) had high-risk HPV types. Twelve participants tested positive for HPV18, only one participant tested positive for HPV16, and an additional four (4.3%) tested positive for either type 6 or 11, with respect to vaccine-preventable low-risk HPV types. Conclusion: The Gardasil quadrivalent HPV vaccine (4vHPV) was expected to protect against infection with HPV types 16, 18, 6, and 11. We demonstrated a possible waning of immunity to HPV18 in 17% of the participants, and an associated loss in cross-protection against HPV45. We observed a relatively high prevalence of ‘opportunistic non-vaccine HPV types’ or ‘ecological niche occupiers’ in this cohort, and suggest further research on the involvement of these types in cervical and other genital cancers. Our study is one of the few, if not the first, to report on HPV vaccine immunoprotection among people living with HIV (PLWH), thereby setting a baseline for further studies on HPV vaccine effectiveness among PLWH.

HPV16 Intratypic Variants in Head and Neck Cancers: A North American Perspective

Human papillomavirus (HPV) is the major causative agent for cervical and many head and neck cancers (HNCs). HPVs randomly acquire single nucleotide polymorphisms (SNPs) that may become established via positive selection. Within an HPV type, viral isolates differing by <2% in the L1 region are termed “variants” and classified based on combinations of SNPs. Studies in cervical cancer demonstrate clear differences between HPV16 intratypic variants in terms of persistence of infection, tumor histology, cancer risk, and death. Much less is known about the frequency of HPV16 variants in HNC, and their effects on clinical outcomes. We combined HPV16 positive (HPV16+) HNC samples from a local Southwestern Ontario, Canada cohort with those from the Cancer Genome Atlas to create a larger North American cohort of 149 cases with clinical data and determined the distribution of intratypic variants and their impact on clinical outcomes. Most isolates were lineage A, sublineage A1, or A2, with roughly half exhibiting the T350G polymorphism in E6. Univariable analysis identified significant differences between 350T and 350G intratypic variants in clinical T, N, and O staging, as well as disease-free survival. Multivariable analysis failed to identify any clinical factor as a statistically significant covariate for disease-free survival differences between 350T and 350G. Significant differences in several measures of B-cell mediated immune response were also observed between 350T and 350G intratypic variants. We suggest that HPV genetic variation may be associated with HNC clinical characteristics and may have prognostic value.

Emerging Prognostic and Predictive Significance of Stress Keratin 17 in HPV-Associated and Non HPV-Associated Human Cancers: A Scoping Review

A growing body of literature suggests that the expression of cytokeratin 17 (K17) correlates with inferior clinical outcomes across various cancer types. In this scoping review, we aimed to review and map the available clinical evidence of the prognostic and predictive value of K17 in human cancers. PubMed, Web of Science, Embase (via Scopus), Cochrane Central Register of Controlled Trials, and Google Scholar were searched for studies of K17 expression in human cancers. Eligible studies were peer-reviewed, published in English, presented original data, and directly evaluated the association between K17 and clinical outcomes in human cancers. Of the 1705 studies identified in our search, 58 studies met criteria for inclusion. Studies assessed the prognostic significance (n = 54), predictive significance (n = 2), or both the prognostic and predictive significance (n = 2). Altogether, 11 studies (19.0%) investigated the clinical relevance of K17 in cancers with a known etiologic association to HPV; of those, 8 (13.8%) were focused on head and neck squamous cell carcinoma (HNSCC), and 3 (5.1%) were focused on cervical squamous cell carcinoma (SCC). To date, HNSCC, as well as triple-negative breast cancer (TNBC) and pancreatic cancer, were the most frequently studied cancer types. K17 had prognostic significance in 16/17 investigated cancer types and 43/56 studies. Our analysis suggests that K17 is a negative prognostic factor in the majority of studied cancer types, including HPV-associated types such as HNSCC and cervical cancer (13/17), and a positive prognostic factor in 2/17 studied cancer types (urothelial carcinoma of the upper urinary tract and breast cancer). In three out of four predictive studies, K17 was a negative predictive factor for chemotherapy and immune checkpoint blockade therapy response.

HPV16 E6 Oncogene Contributes to Cancer Immune Evasion by Regulating PD-L1 Expression through a miR-143/HIF-1a Pathway

Human Papillomaviruses have been associated with the occurrence of cervical cancer, the fourth most common cancer that affects women globally, while 70% of cases are caused by infection with the high-risk types HPV16 and HPV18. The integration of these viruses’ oncogenes E6 and E7 into the host’s genome affects a multitude of cellular functions and alters the expression of molecules. The aim of this study was to investigate how these oncogenes contribute to the expression of immune system control molecules, using cell lines with integrated HPV16 genome, before and after knocking out E6 viral gene using the CRISPR/Cas9 system, delivered with a lentiviral vector. The molecules studied are the T-cell inactivating protein PD-L1, its transcription factor HIF-1a and the latter’s negative regulator, miR-143. According to our results, in the E6 knock out (E6KO) cell lines an increased expression of miR-143 was recorded, while a decrease in the expression of HIF-1a and PD-L1 was exhibited. These findings indicate that E6 protein probably plays a significant role in enabling cervical cancer cells to evade the immune system, while we propose a molecular pathway in cervical cancer, where PD-L1’s expression is regulated by E6 protein through a miR-143/HIF-1a axis.

Human Papillomavirus Genotype Landscape Across Cervical Cytology Grades and Impact of HIV Among Women of Eastern Cape Province, South Africa

Continuous surveillance of human papillomavirus (HPV) prevalence and genotype distribution in different cervical cytology grades is necessary for cervical cancer prevention and monitoring. This study investigated the distribution of HPV genotypes and associated factors, stratified by cervical cytology grades and human immunodeficiency (HIV) status, among women in the Eastern Cape Province, South Africa. A total of 540 women were recruited from a community health facility and a referral hospital in the OR Tambo District Municipality in Eastern Cape Province. HPV detection and genotyping in cervical cells were performed using the Seegene AllplexTM and AnyplexTM II HPV28 assays. HPV prevalence was 60.6% among women with normal cervical cytology, 93.8% among atypical squamous cells of undetermined significance (ASC-US), 100.0% among low-grade squamous intraepithelial lesions (LSILs), 95.2% among atypical squamous cells cannot exclude high-grade lesion (ASC-H), 93.7% among high-grade squamous intraepithelial lesions (HSILs), and 92.5% among women with cervical cancer. HPV types targeted by Gardasil-9® were detected in 36.0% of women with normal cervical cytology, 83.0% of those with HSIL, and 81.0% of those with cervical cancer. Among women with normal cervical cytology, HPV58, 35, and 68 were the most dominant types, HPV16, 33, and 35 in HSIL, and HPV16, 18, and 35 in cervical cancer. Differences were observed in the prevailing HPV genotype patterns when stratified by HIV infection status. This study highlights the high HPV prevalence, which further increased among women with abnormal cervical cytology. While HPV prevalence did not significantly increase with HIV co-infection, distinct differences were observed in the HPV genotype patterns when stratified by HIV status. The dominance of non-HPV vaccine types in HSIL and cervical cancer cases underscores a critical gap in current prevention strategies.

Chemical Peeling Therapy Using Phenol for the Cervico-Vaginal Intraepithelial Neoplasia

Objective: This study aimed to validate the use of liquid phenol-based chemical peeling therapy for cervical and vaginal intraepithelial neoplasia (CIN and VaIN, respectively), with the goal of circumventing obstetric complications associated with surgical treatment and to determine the factors associated with treatment resistance. Methods: A total of 483 eligible women diagnosed with CIN, VaIN, or both, participated in this study. Participants underwent phenol-based chemical peeling therapy every 4 weeks until disease clearance. Disease clearance was determined by negative Pap tests for four consecutive weeks or by colposcopy. HPV genotyping was conducted at the onset of the study and after disease clearance in select cases. Our preliminary analysis compared the recurrence and persistence rates between 294 individuals who received phenol-based chemical peeling therapy and 189 untreated patients. Results: At 2 years following diagnosis, persistent disease was observed in 18%, 60%, and 88% of untreated patients with CIN1–3, respectively, and <2% of patients with CIN who received phenol-based chemical peeling therapy. Among 483 participants, 10 immune-suppressed patients required multiple treatments to achieve disease clearance, and 7 were diagnosed with cervical cancer. Of the 466 participants, except those with cancer or immune suppression, the number of treatment sessions until CIN/VaIN clearance ranged from 2 to 42 (average: 9.2 sessions). In total, 43 participants (9.2%) underwent surgical treatment. Six patients (1.3%) experienced recurrence of CIN2 or worse, suggesting that treatment failed in 46 patients (9.9%). No obstetrical complications were noted among the 98 pregnancies following this therapy. Factors associated with resistance to this therapy include immune suppression, ages 35–39 years, higher-grade lesions, and multiple HPV-type infections. Conclusions: Phenol-based therapy is safe and effective for CINs and VaINs. Women aged < 35 years and with persistent CIN1 or CIN2 with a single HPV-type infection are suitable candidates for phenol-based chemical peeling therapy. However, this therapy requires multiple lengthy sessions.

Intra-Patient Genomic Variations of Human Papillomavirus Type 31 in Cervical Cancer and Precancer

Human papillomavirus type 31 (HPV31) is detected less frequently in cervical cancer than two major causative types, HPV16 and HPV18. Here, we report a comprehensive analysis of HPV31 genome sequences in cervical lesions collected from Japanese women. Of 52 HPV31-positive cervical specimens analyzed by deep sequencing, 43 samples yielded complete genome sequences of around 7900 base pairs and 9 samples yielded partially deleted genome sequences. Phylogenetic analysis showed that HPV31 variant distribution was lineage A in 19 samples (36.5%), lineage B in 28 samples (53.8%), and lineage C in 5 samples (9.6%), indicating that lineage B variants are dominant among HPV31 infections in Japan. Deletions in the viral genome were found in the region from the E1 to L1 genes, but all the deleted genomes retained the E6/E7 genes. Among intra-patient nucleotide variations relative to a consensus genome sequence in each sample, C-to-T substitutions were most frequently detected, followed by T-to-C and C-to-A substitutions. High-frequency, intra-patient mutations (>10%) in cervical cancer samples were found in the E1, E2, and E7 genes, and all of them were nonsynonymous substitutions. The enrichment of high-frequency nonsynonymous substitutions strongly suggests that these intra-patient mutations are positively selected during the development of cervical cancer/precancer.

Trend of HPV Molecular Epidemiology in the Post-Vaccine Era: A 10-Year Study

Cervical cancer, a major health concern among women worldwide, is closely linked to human papillomavirus (HPV) infection. This study explores the evolving landscape of HPV molecular epidemiology in Taiwan over a decade (2010–2020), where prophylactic HPV vaccination has been implemented since 2007. Analyzing data from 40,561 vaginal swab samples, with 42.0% testing positive for HPV, we reveal shifting trends in HPV genotype distribution and infection patterns. The 12 high-risk genotypes, in order of decreasing percentage, were HPV 52, 58, 16, 18, 51, 56, 39, 59, 33, 31, 45, and 35. The predominant genotypes were HPV 52, 58, and 16, accounting for over 70% of cases annually. The proportions of high-risk and non-high-risk HPV infections varied across age groups. High-risk infections predominated in sexually active individuals aged 30-50 and were mixed-type infections. The composition of high-risk HPV genotypes was generally stable over time; however, HPV31, 33, 39, and 51 significantly decreased over the decade. Of the strains, HPV31 and 33 are shielded by the nonavalent HPV vaccine. However, no reduction was noted for the other seven genotypes. This study offers valuable insights into the post-vaccine HPV epidemiology. Future investigations should delve into HPV vaccines’ effects and their implications for cervical cancer prevention strategies. These findings underscore the need for continued surveillance and research to guide effective public health interventions targeting HPV-associated diseases.

Efficiency of CIN2+ Detection by Thyrotropin-Releasing Hormone (TRH) Site-Specific Methylation

Cervical cancer screening typically involves a Pap smear combined with high-risk human papillomavirus (hr-HPV) detection. Women with hr-HPV positivity but normal cytology, as well as those with precancerous abnormal cytology, such as low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL), are referred for colposcopy and histology examination to identify abnormal lesions, such as cervical intraepithelial neoplasia (CIN) and cervical cancer. However, in order to enhance the accuracy of detection, bioinformatics analysis of a microarray database was performed, which identified cg01009664, a methylation marker of the thyrotropin-releasing hormone (TRH). Consequently, a real-time PCR assay was developed to distinguish CIN2+ (CIN2, CIN3, and cervical cancer) from CIN2- (CIN1 and normal cervical epithelia). The real-time PCR assay utilized specific primers targeting methylated cg01009664 sites, whereas an unmethylated reaction was used to check the DNA quality. A cut-off value for the methylated reaction of Ct < 33 was established, resulting in improved precision in identifying CIN2+. In the first cohort group, the assay demonstrated a sensitivity of 93.7% and a specificity of 98.6%. In the cytology samples identified as atypical squamous cells of undetermined significance (ASC-US) and LSIL, the sensitivity and specificity for detecting CIN2+ were 95.0% and 98.9%, respectively. However, when self-collected samples from women with confirmed histology were tested, the sensitivity for CIN2+ detection dropped to 49.15%, while maintaining a specificity of 100%. Notably, the use of clinician-collected samples increased the sensitivity of TRH methylation testing. TRH methylation analysis can effectively identify women who require referral for colposcopy examinations, aiding in the detection of CIN2+.

The High-Risk Human Papillomavirus Type Influences the Tissue Microenvironment in Cervical Intraepithelial Neoplasia Grade 2

High-risk, cancer-causing human papillomavirus (HPV) types are associated with cervical precancer and cancer. A high proportion of high-risk HPV precancer lesions undergo immune-mediated regression. The purpose of this study was to determine if the tissue microenvironment of HPV16 and 18 (HPV16/18) cervical intraepithelial neoplasia grade 2 lesions differed from other high-risk types (HPV ‘other’). Consistent with other studies, we found that progression to higher-grade disease was more frequent in HPV16/18 lesions when compared with HPV ‘other’ lesions. HPV16/18 lesions were significantly more likely to be indoleamine 2,3,-dioxygenase 1 (IDO1)-positive and were associated with reduced CD8 and FoxP3 T cells in the lesion. In the stroma, reduced Tbet- and CD32-positive cells and increased Blimp1-positive cells were significantly associated with HPV16/18 lesions when compared with HPV ‘other’ types. On analysis of the IDO1-positive tissues, lesional IDO1 was associated with significantly decreased numbers of CD4-, CD8-, and FoxP3-positive cells in the stroma compared with IDO1-negative tissues. These data suggest that IDO1 expression may impair infiltration of CD4, CD8, and FoxP3 cells into the stroma beneath the precancer lesion. Increased expression of IDO1 may contribute to immune avoidance and an increased frequency of disease progression in HPV16- and 18-positive lesions.

Burden of HPV-Related Hospitalization in Germany from 2000 to 2021

HPV has been linked to the development of precancerous and cancerous lesions. The aim of this study was to evaluate the burden of HPV-related hospitalization in Germany from 2000 to 2021 and the potential impact of the COVID-19 pandemic on it. Methods: We performed a retrospective query using data from the German Statistical Office from 2000 to 2021, including hospital admission, inpatient mortality and hospital stay length data on cervical cancer/dysplasia, female genitourinary tract, anal, penile, head and neck cancers. Results: The HPV-attributable hospitalization rate per 100,000 inhabitants in Germany has decreased over time, from 89 cases in 2000 to 60 in 2021, with an average annual percent change (AAPC) of −1.93 (CI −2.08–−1.79, p < 0.05). The same trend was observed for the average hospital stay, which declined from 9 to 7 days, with an AAPC of −1.33 (CI −1.52–−1.21, p < 0.05). An undulating but overall slightly declining pattern was observed for the inpatient mortality (AAPC −0.92, CI −1.21–−0.64, p < 0.05). We observed a reduction in the hospitalization rates for invasive and non-invasive cervical cancer, which was observed in almost all age groups and in all German federal states. Conclusion: Our study provides a comprehensive analysis of the trends in HPV-related hospitalizations over the past two decades. The decline in hospitalization rates for cervical cancer and dysplasia suggests the potential efficacy of the HPV vaccination and screening programs.

Genetic Variability in the E6/E7 Region of Human Papillomavirus 16 in Women from Ecuador

Human Papillomavirus (HPV) infection is associated with intraepithelial neoplasia and cervical cancer (CC). Ecuador has a high prevalence of cervical cancer, with more than 1600 new cases diagnosed annually. This study aimed to analyze oncogenes E6 and E7 of HPV16 in samples collected from women with cancerous and precancerous cervical lesions from the Ecuadorian coast. Twenty-nine women, including six with ASCUS, three with LSIL, thirteen with HSIL, and seven with Cacu, were analyzed. The most common SNPs were E6 350G or L83V (82.6%) and E6 145T/286A/289G/335T/350G or Q14H/F78Y/L83V (17.4%). Both variants are reported to be associated with an increased risk of cervical cancer in worldwide studies. In contrast, all E7 genes have conserved amino-acid positions. Phylogenetic trees showed the circulation of the D (26.1%) and A (73.9) lineages. The frequency of D was higher than that reported in other comparable studies in Ecuador and Latin America, and may be related to the ethnic composition of the studied populations. This study contributes to the characterization of the potential risk factors for cervical carcinogenesis associated with Ecuadorian women infected with HPV16.

Special Issue “HPV in the Head and Neck Region 2.0”

Members of the human papillomavirus (HPV) family have been known for causing cancers and condylomas in the anogenital tract for some time, as reflected by the Nobel Prize in Medicine given to Professor Harald zur Hausen 2008 [...]

Immunostaining of βA-Activin and Follistatin Is Decreased in HPV(+) Cervical Pre-Neoplastic and Neoplastic Lesions

The activin–follistatin system regulates several cellular processes, including differentiation and tumorigenesis. We hypothesized that the immunostaining of βA-activin and follistatin varies in neoplastic cervical lesions. Cervical paraffin-embedded tissues from 162 patients sorted in control (n = 15), cervical intraepithelial neoplasia (CIN) grade 1 (n = 38), CIN2 (n = 37), CIN3 (n = 39), and squamous cell carcinoma (SCC; n = 33) groups were examined for βA-activin and follistatin immunostaining. Human papillomavirus (HPV) detection and genotyping were performed by PCR and immunohistochemistry. Sixteen samples were inconclusive for HPV detection. In total, 93% of the specimens exhibited HPV positivity, which increased with patient age. The most detected high-risk (HR)-HPV type was HPV16 (41.2%) followed by HPV18 (16%). The immunostaining of cytoplasmatic βA-activin and follistatin was higher than nuclear immunostaining in all cervical epithelium layers of the CIN1, CIN2, CIN3, and SCC groups. A significant decrease (p < 0.05) in the cytoplasmic and nuclear immunostaining of βA-activin was detected in all cervical epithelial layers from the control to the CIN1, CIN2, CIN3, and SCC groups. Only nuclear follistatin immunostaining exhibited a significant reduction (p < 0.05) in specific epithelial layers of cervical tissues from CIN1, CIN2, CIN3, and SCC compared to the control. Decreased immunostaining of cervical βA-activin and follistatin at specific stages of CIN progression suggests that the activin–follistatin system participates in the loss of the differentiation control of pre-neoplastic and neoplastic cervical specimens predominantly positive for HPV.

Non-Metabolic Functions of PKM2 Contribute to Cervical Cancer Cell Proliferation Induced by the HPV16 E7 Oncoprotein

Pyruvate kinase M2 (PKM2) mainly catalyzes glycolysis, but it also exerts non-glycolytic functions in several cancers. While it has been shown to interact with the human papillomavirus 16 (HPV16) E7 oncoprotein, the functional significance of PKM2 in HPV-associated cervical cancer has been elusive. Here, we show that HPV16 E7 increased the expression of PKM2 in cervical cancer cells. TCGA data analyses revealed a higher level of PKM2 in HPV+ than HPV− cervical cancers and a worse prognosis for patients with high PKM2 expression. Functionally, we demonstrate that shRNA-mediated PKM2 knockdown decreased the proliferation of HPV+ SiHa cervical cancer cells. PKM2 knockdown also inhibited the E7-induced proliferation of cervical cancer cells. ML265 activating the pyruvate kinase function of PKM2 inhibited cell cycle progression and colony formation. ML265 treatments decreased phosphorylation of PKM2 at the Y105 position that has been associated with non-glycolytic functions. On the contrary, HPV16 E7 increased the PKM2 phosphorylation. Our results indicate that E7 increases PKM2 expression and activates a non-glycolytic function of PKM2 to promote cervical cancer cell proliferation.

Short Communication: Understanding the Barriers to Cervical Cancer Prevention and HPV Vaccination in Saudi Arabia

Cervical cancer, along with other sexual and reproductive health and rights (SRHR) conditions, poses a significant burden in the Kingdom of Saudi Arabia (KSA). Despite the availability of effective preventive methods such as vaccinations, particularly against the Human Papillomavirus (HPV), awareness about such preventive methods and HPV vaccination remains alarmingly low in the KSA, even with governmental effort and support. While many women are aware of the risks, the uptake of the HPV vaccine remains below 10% (7.6%) at the country level. This highlights the urgent need for Knowledge, Attitude, and Practice (KAP) at the community level to raise awareness, dispel misconceptions, and empower women to embrace vaccinations. Additionally, there is a need to revitalize the cancer registry system to better track and monitor cervical cancer cases. This short communication aims to map these barriers while identifying opportunities for impactful research. Drawing from the scientific literature, government reports, and expert insights, we highlight the challenges surrounding the tackling of HPV. By exploring diverse sources of knowledge, this paper not only highlights current obstacles but also proposes actionable solutions for future interventions.

Mapping the HPV Landscape in South African Women: A Systematic Review and Meta-Analysis of Viral Genotypes, Microbiota, and Immune Signals

This systematic review and meta-analysis evaluate human papillomavirus (HPV) prevalence, genotype distribution, and associations with cervicovaginal microbiota and cytokine profiles among South African women, where cervical cancer ranks as the second most common cancer. PubMed, SCOPUS, and Web of Science were searched for studies on HPV infection up to 21 September 2024. The pooled prevalence was estimated using a random-effects model, with subgroup analyses by province, sample type, and HIV status. Publication bias was evaluated using funnel plots and Egger’s test. Of the 19,765 studies screened, 120 met the inclusion criteria, comprising 83,266 participants. Results indicate a high HPV burden, with a pooled prevalence of 58% (95% CI: 52–64%), varying regionally from 53% (95% CI: 41–65%) to 64% (95% CI: 55–73%), with some regions under-researched. Cervical samples had the highest HPV prevalence (60% (95% CI: 54–66%)), while non-genital samples were less studied. High-risk (HR) HPV types, notably HPV 16 (7.5%), HPV 35 (4.1%), and HPV 18 (3.9%), were prominent, with HPV 35 emphasizing the need for expanded vaccine coverage. HIV-positive women had a higher pooled HPV prevalence (63% (95% CI: 55–71%)). Funnel plot analysis and Egger’s test suggested a potential publication bias (p = 0.047). HPV-positive women exhibited lower Lactobacillus levels and an increase in Bacterial Vaginosis (BV)-associated species like Gardnerella, potentially supporting HPV persistence. Cytokine analysis showed elevated MIP-1α and MIP-1β in HPV infections, though cytokine profiles may depend on HPV genotypes. These findings underscore the need for research on HPV–microbiome-immune interactions and call for comprehensive HPV-prevention strategies, including vaccines targeting regional HPV types and tailored interventions for HIV-positive populations.

HPV and Cervical Cancer: Molecular and Immunological Aspects, Epidemiology and Effect of Vaccination in Latin American Women

Cervical cancer is primarily caused by Human Papillomavirus (HPV) infection and remains a significant public health concern, particularly in Latin American regions. This comprehensive narrative review addresses the relationship between Human Papillomavirus (HPV) and cervical cancer, focusing on Latin American women. It explores molecular and immunological aspects of HPV infection, its role in cervical cancer development, and the epidemiology in this region, highlighting the prevalence and diversity of HPV genotypes. The impact of vaccination initiatives on cervical cancer rates in Latin America is critically evaluated. The advent of HPV vaccines has presented a significant tool in combating the burden of this malignancy, with notable successes observed in various countries, the latter due to their impact on immune responses. The review synthesizes current knowledge, emphasizes the importance of continued research and strategies for cervical cancer prevention, and underscores the need for ongoing efforts in this field.

Effects of the Prophylactic HPV Vaccines on HPV Type Prevalence and Cervical Pathology

Vaccination programs with the current prophylactic HPV vaccines started in most countries around 2008 with introduction of the bivalent Cervarix HPV16/18 vaccine, rapidly followed by Gardasil (HPV6/11/16/18) and, finally, Gardasil 9 (HPV6/11/16/18/31/33/45/52/58), from 2015. Many studies have now confirmed their ability to prevent infection with vaccine-covered HPV types, and the subsequent development of either genital warts and/or cervical neoplasia, although this is clearly more effective in younger women vaccinated prior to sexual debut. Most notably, reductions in the prevalence of vaccine-covered HPV types were also observed in unvaccinated women at the same geographical location, presumably by sexual dissemination of these changes, between vaccinated and unvaccinated women. Furthermore, there are several studies that have demonstrated vaccine-associated HPV type-replacement, where vaccine-covered, high-risk HPV types are replaced by high-risk HPV types not covered by the vaccines, and these changes were also observed in vaccinated and unvaccinated women in the same study population. In light of these observations, it is not entirely clear what effects vaccine-associated HPV type-replacement will have, particularly in older, unvaccinated women.

HPV16 Phylogenetic Variants in Anogenital and Head and Neck Cancers: State of the Art and Perspectives

HPV16 is responsible for approximately 60% and 90% of global HPV–induced cervical and oropharyngeal cancers, respectively. HPV16 intratype variants have been identified by HPV genome sequencing and classified into four phylogenetic lineages (A–D). Our understanding of HPV16 variants mostly derives from epidemiological studies on cervical cancer (CC) in which HPV16 B, C, and D lineages (previously named “non-European” variants) were mainly associated with high-grade cervical lesions and cancer. Although a predominance of HPV16 lineage A (previously named “European variants”) has been observed in head and neck squamous cell carcinoma (HNSCC), epidemiological and in vitro biological studies are still limited for this tumor site. Next Generation Sequencing (NGS) of the entire HPV genome has deepened our knowledge of the prevalence and distribution of HPV variants in CC and HNSCC. Research on cervical cancer has shown that certain HPV16 sublineages, such as D2, D3, A3, and A4, are associated with an increased risk of cervical cancer, and sublineages A4, D2, and D3 are linked to a higher risk of developing adenocarcinomas. Additionally, lineage C and sublineages D2 or D3 of HPV16 show an elevated risk of developing premalignant cervical lesions. However, it is still crucial to conduct large-scale studies on HPV16 variants in different HPV–related tumor sites to deeply evaluate their association with disease development and outcomes. This review discusses the current knowledge and updates on HPV16 phylogenetic variants distribution in HPV–driven anogenital and head and neck cancers.

Monitoring HPV Prevalence and Risk Cofactors for Abnormal Cytology in the Post-Vaccination Period among Croatian Women

The incidence and mortality rate of cervical cancer in Croatia remains a health challenge despite screening efforts. Besides the persistent infection with HPV, the development of cancer is also associated with some cofactors. The goal of this study was to assess circulating HPV genotypes and risk factors for the development of cervical precancer after almost 16 years from the onset of HPV vaccination in Croatia. In this study, a total of 321 women attending gynecological care were evaluated. Relevant medical and demographic information, including cytology, were collected. HPV genotyping was performed by PCR. Comparing the HPV types found in circulation in the pre-vaccination (1999–2015) and post-vaccination periods (2020–2023), a statistically significant reduction in HPV 31 was noted, while the overall prevalence increased in the post-vaccination period. Besides the expected HPV positivity as a risk factor, the history of smoking was associated with LSIL or worse cytology at enrollment. For the first time, this population study revealed a statistically significant shift in the HPV genotype in the post-vaccination period, as well as the confirmation of risk factors for the development of abnormal cytology among Croatian women.

Findings and Challenges in Replacing Traditional Uterine Cervical Cancer Diagnosis with Molecular Tools in Private Gynecological Practice in Mexico

We have been encouraging practicing gynecologists to adopt molecular diagnostics tests, PCR, and cancer biomarkers, as alternatives enabled by these platforms, to traditional Papanicolaou and colposcopy tests, respectively. An aliquot of liquid-based cytology was used for the molecular test [high-risk HPV types, (HR HPV)], another for the PAP test, and one more for p16/Ki67 dual-stain cytology. A total of 4499 laboratory samples were evaluated, and we found that 25.1% of low-grade samples and 47.9% of high-grade samples after PAP testing had a negative HR HPV-PCR result. In those cases, reported as Pap-negative, 22.1% had a positive HR HPV-PCR result. Dual staining with p16/Ki67 biomarkers in samples was positive for HR HPV, and 31.7% were also positive for these markers. Out of the PCR results that were positive for any of these HR HPV subtypes, n 68.3%, we did not find evidence for the presence of cancerous cells, highlighting the importance of performing dual staining with p16/Ki67 after PCR to avoid unnecessary colposcopies. The encountered challenges are a deep-rooted social reluctance in Mexico to abandon traditional Pap smears and the opinion of many specialists. Therefore, we still believe that colposcopy continues to be a preferred procedure over the dual-staining protocol.

Accuracy of Human Papillomavirus (HPV) Testing on Urine and Vaginal Self-Samples Compared to Clinician-Collected Cervical Sample in Women Referred to Colposcopy

In the context of cervical cancer prevention, where human papillomavirus (HPV) infection is pivotal, HPV testing is replacing Pap Smear in primary screening. This transition offers an opportunity for integrating self-sampling to enhance coverage. We evaluated the accuracy of HPV testing using self-collected urine and vaginal samples, comparing them to physician-collected cervical swabs. From a cohort of 245 women with abnormal cytology, we collected self-sampled vaginal, urine, and clinician-administered cervical specimens. Employing Anyplex™II HPV28 assay, outcomes revealed HPV positivity rates of 75.1% (cervical), 78.4% (vaginal), and 77.1% (urine). Significant, hr-HPV detection concordance was observed between self-taken cervical samples and clinical counterparts (k = 0.898 for vaginal; k = 0.715 for urine). This study extends beyond accuracy, highlighting self-collected sample efficacy in detecting high-grade cervical lesions. The insight underscores self-sampling’s role in bolstering participation and aligns with WHO’s goal to eliminate cervical cancer by 2030.

Recent Developments in Human Papillomavirus (HPV) Vaccinology

Human papillomavirus (HPV) is causally associated with 5% of cancers, including cancers of the cervix, penis, vulva, vagina, anus and oropharynx. The most carcinogenic HPV is HPV-16, which dominates the types causing cancer. There is also sufficient evidence that HPV types 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 cause cervical cancer. The L1 protein, which, when assembled into virus-like particles, induces HPV-type-specific neutralising antibodies, forms the basis of all commercial HPV vaccines. There are six licensed prophylactic HPV vaccines: three bivalent, two quadrivalent and one nonavalent vaccine. The bivalent vaccines protect from HPV types 16 and 18, which are associated with more than 70% of cervical cancers. Prophylactic vaccination targets children before sexual debut, but there are now catch-up campaigns, which have also been shown to be beneficial in reducing HPV infection and disease. HPV vaccination of adults after treatment for cervical lesions or recurrent respiratory papillomatosis has impacted recurrence. Gender-neutral vaccination will improve herd immunity and prevent infection in men and women. HPV vaccines are immunogenic in people living with HIV, but more research is needed on the long-term impact of vaccination and to determine whether further boosters are required.

Manipulation of JAK/STAT Signalling by High-Risk HPVs: Potential Therapeutic Targets for HPV-Associated Malignancies

Human papillomaviruses (HPVs) are small, DNA viruses that cause around 5% of all cancers in humans, including almost all cervical cancer cases and a significant proportion of anogenital and oral cancers. The HPV oncoproteins E5, E6 and E7 manipulate cellular signalling pathways to evade the immune response and promote virus persistence. The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has emerged as a key mediator in a wide range of important biological signalling pathways, including cell proliferation, cell survival and the immune response. While STAT1 and STAT2 primarily drive immune signalling initiated by interferons, STAT3 and STAT5 have widely been linked to the survival and proliferative potential of a number of cancers. As such, the inhibition of STAT3 and STAT5 may offer a therapeutic benefit in HPV-associated cancers. In this review, we will discuss how HPV manipulates JAK/STAT signalling to evade the immune system and promote cell proliferation, enabling viral persistence and driving cancer development. We also discuss approaches to inhibit the JAK/STAT pathway and how these could potentially be used in the treatment of HPV-associated disease.

Preparation, Characterization and Diagnostic Valuation of Two Novel Anti-HPV16 E7 Oncoprotein Monoclonal Antibodies

At present, the clinical detection method of human papillomavirus (HPV) is mainly based on the PCR method. However, this method can only be used to detect HPV DNA and HPV types, and cannot be used to accurately predict cervical cancer. HPV16 E7 is an oncoprotein selectively expressed in cervical cancers. In this study, we prepared an HPV16 E7-histidine (HIS) fusion oncoprotein by using a prokaryotic expression and gained several mouse anti-HPV16 E7-HIS fusion oncoprotein monoclonal antibodies (mAbs) by using hybridoma technology. Two mAbs, 69E2 (IgG2a) and 79A11 (IgM), were identified. Immunocytochemistry, immunofluorescence, immunohistochemistry, and Western blot were used to characterize the specificity of these mAbs. The sequences of the nucleotide bases and predicted amino acids of the 69E2 and 79A11 antibodies showed that they were novel antibodies. Indirect enzyme-linked immunosorbent assay (ELISA) with overlapping peptides, indirect competitive ELISA, and 3D structural modeling showed that mAbs 69E2 and 79A11 specifically bound to the three exposed peptides of the HPV16 E7 (HPV16 E749–66, HPV16 E773–85, and HPV16 E791–97). We used these two antibodies (79A11 as a capture antibody and 69E2 as a detection antibody) to establish a double-antibody sandwich ELISA based on a horseradish peroxidase (HRP)-labeled mAb and tetramethylbenzidine (TMB) detection system for quantitative detection of the HPV16 E7-HIS fusion oncoprotein, however, it was not ideal. Then we established a chemiluminescence immunoassay based on a labeled streptavidin-biotin (LSAB)-ELISA method and luminol detection system—this was sufficient for quantitative detection of the HPV16 E7-HIS fusion oncogenic protein in ng levels and was suitable for the detection of HPV16-positive cervical carcinoma tissues. Collectively, we obtained two novel mouse anti-HPV16 E7 oncoprotein mAbs and established an LSAB-lumino-dual-antibody sandwich ELISA method for the detection of the HPV16 E7-HIS fusion oncogenic protein, which might be a promising method for the diagnosis of HPV16-type cervical cancers in the early stage.

Extracellular Vesicles from Human Papilloma Virus-Infected Cervical Cancer Cells Enhance HIV-1 Replication in Differentiated U1 Cell Line

In the current study, we hypothesized that extracellular vesicles (EVs) secreted from human papilloma virus (HPV)-infected cervical cancer cells exacerbate human immunodeficiency virus (HIV)-1 replication in differentiated U1 cell line through an oxidative stress pathway. To test the hypothesis, we treated an HIV-1-infected macrophage cell line (U1) with HPV-infected Caski cell culture supernatant (CCS). We observed a significant increase in HIV-1 replication, which was associated with an increase in the expression of cytochrome P450 (CYPs 1A1 and 2A6) in the CCS-treated U1 cells. Furthermore, we isolated EVs from CCS (CCS-EVs), which showed the presence of CYPs (1A1, 2A6), superoxide dismutase 1 (SOD1), and HPV oncoproteins HPV16 E6. CCS-EVs when exposed to the U1 cells also significantly increased HIV-1 replication. Treatment of antioxidant, CYP1A1 and CYP2A6 inhibitors, and chemodietary agents with antioxidant properties significantly reduced the CCS and CCS-EVs mediated HIV-1 replication in U1 cells. Altogether, we demonstrate that cervical cancer cells exacerbate HIV-1 replication in differentiated U1 cell line via transferring CYPs and HPV oncoproteins through EVs. We also show that the viral replication occurs via CYP and oxidative stress pathways, and the viral replication is also reduced by chemodietary agents. This study provides important information regarding biological interactions between HPV and HIV-1 via EVs leading to enhanced HIV-1 replication.

Uptake of Human Papillomavirus Vaccine and Intention to Vaccinate among Healthy Pregnant Women in Serbia: A Cross-Sectional Study on Awareness, Knowledge, and Attitudes

We aimed to assess awareness, knowledge, and attitudes of healthy pregnant women towards human papillomavirus (HPV), to estimate factors associated with a positive attitude towards HPV immunization and to assess the uptake of the vaccine among their children. A cross-sectional study was conducted at the University Clinic of Gynecology and Obstetrics, Belgrade, Serbia among pregnant women attending their regular gynecological check-ups at the 12th gestational week. Knowledge about HPV and HPV vaccine was assessed using a specifically designed 12-item and 5-item questionnaires. Out of total 265 included women, 79.3% had heard of HPV, and 37.5% knew that HPV vaccine exists. HPV vaccine knowledge score was associated with higher odds for a positive attitude towards vaccination of both female (OR = 4.10, 95% CI 1.50–11.29) and male (OR = 3.71, 95% CI 1.52–9.01) child. The number of children (OR = 1.32, 95% CI 1.04–1.67) and high vaccine knowledge score (OR = 1.64 95% CI 1.13–2.39) were independent predictors associated with willingness to vaccinate child against HPV. The gynecologist was the preferable point of reference for information seeking about the HPV vaccine. Despite relatively high HPV awareness and knowledge among pregnant women in Serbia, about one-third of them are HPV vaccine aware, and are willing to vaccinate their children against HPV.

Betapapillomaviruses in p16-Negative Vulvar Intraepithelial Lesions Associated with Squamous Cell Carcinoma

Approximately 40% of vulvar squamous cell carcinoma (vSCC) cases are etiologically associated with high-risk human papillomaviruses (HPVs) of the alpha genera (α-HPV) that cause other anogenital cancers; however, the etiology of α-HPV-negative vSCC is poorly understood. HPVs of the beta genera (β-HPV) are risk factors for cutaneous squamous cell carcinoma (cSCC) and may be related to carcinomas originating in other cutaneous sites such as the vulva. In this study, we investigate the presence of β-HPVs, with an emphasis on p16-negative squamous lesions adjacent to vSCC. We subjected 28 vulvar squamous intraepithelial lesions adjacent to vSCC for comprehensive HPV genotyping, p16 and p53 immunohistochemistry, and consensus morphology review. Selected cases were subjected to qPCR and RNA in situ hybridization. Clinical data were obtained from medical records. β-HPV DNA was detected in eight of ten p16-negative lesions and three of fourteen p16-positive high-grade squamous intraepithelial lesions. The HPV DNA loads in vulvar squamous intraepithelial lesions ranged between less than 1 HPV DNA copy per cell to more than 100 HPV DNA copies per cell. This is, to the best of our knowledge, the first report of the association of p16-negative vulvar intraepithelial squamous lesions with detection of β-HPVs. These findings expand possible etiologic mechanisms that may contribute to p16-negative lesions of the vulva.

The First Human Vulvar Intraepithelial Neoplasia Cell Line with Naturally Infected Episomal HPV18 Genome

Persistent infection with high-risk HPV leads to cervical cancers and other anogenital cancers and head and neck carcinomas in both men and women. There is no effective drug fortreating HPV infection and HPV-associated carcinomas, largely due to a lack of models of natural HPV infection and the complexity of the HPV life cycle. There are no available cell lines from vulvar, anal, or penile lesions and cancers in the field. In this study, we established the first human cell line from vulvar intraepithelial neoplasia (VIN) with naturally infected HPV18 by conditional reprogramming (CR) method. Our data demonstrated that VIN cells possessed different biological characteristics and diploid karyotypes from HPV18-positive cancer cells (HeLa). Then, we determined that VIN cells contained episomal HPV18 using approaches including the ratio of HPV E2copy/E7copy, rolling cycle amplification, and sequencing. The VIN cells expressed squamous epithelium-specific markers that are different from HeLa cells, a cervical adenocarcinoma cell line. When cultured under 3D air–liquid interface (ALI) system, we observed the expression of both early and late differentiation markers involucrin and filaggrin. Most importantly, we were able to detect the expression of viral late gene L1 in the cornified layer of ALI 3D culture derived from VIN cells, suggesting quite different HPV genomic status from cancer cells. We also observed progeny viral particles under transmission electron microscopy (TEM) in ALI 3D cultures, confirming the episomal HPV18 genome and active viral life cycle in the new cell line. To our knowledge, this is the first human VIN cell line with naturally infected HPV18 genome and provides a valuable model for HPV biology studies, HPV-associated cancer initiation and progression, and drug-screening platforms.

Phylogenomic Analysis of Human Papillomavirus Type 31 and Cervical Carcinogenesis: A Study of 2093 Viral Genomes

Human papillomavirus (HPV) type 31 (HPV31) is closely related to the most carcinogenic type, HPV16, but only accounts for 4% of cervical cancer cases worldwide. Viral genetic and epigenetic variations have been associated with carcinogenesis for other high-risk HPV types, but little is known about HPV31. We sequenced 2093 HPV31 viral whole genomes from two large studies, one from the U.S. and one international. In addition, we investigated CpG methylation in a subset of 175 samples. We evaluated the association of HPV31 lineages/sublineages, single nucleotide polymorphisms (SNPs) and viral methylation with cervical carcinogenesis. HPV31 A/B clade was >1.8-fold more associated with cervical intraepithelial neoplasia grade 3 and cancer (CIN3+) compared to the most common C lineage. Lineage/sublineage distribution varied by race/ethnicity and geographic region. A viral genome-wide association analysis identified SNPs within the A/B clade associated with CIN3+, including H23Y (C626T) (odds ratio = 1.60, confidence intervals = 1.17–2.19) located in the pRb CR2 binding-site within the E7 oncogene. Viral CpG methylation was higher in lineage B, compared to the other lineages, and was most elevated in CIN3+. In conclusion, these data support the increased oncogenicity of the A/B lineages and suggest variation of E7 as a contributing risk factor.

Genomic Signatures in HPV-Associated Tumors

Papillomaviruses dysregulate the G1/S cell cycle transition in order to promote DNA synthesis in S phase, which is a requirement for viral replication. The human papillomaviruses (HPV) E6 and E7 oncoproteins mediate degradation of the cell cycle regulators p53 and Rb, which are two of the most universally disrupted tumor-suppressor genes in all of cancer. The G1/S checkpoint is activated in normal cells to allow sufficient time for DNA repair in G1 before proceeding to replicate DNA and risk propagating unrepaired errors. The TP53 pathway suppresses a variety of such errors, including translocation, copy number alterations, and aneuploidy, which are thus found in HPV-associated tumors similarly to HPV-negative tumors with other mechanisms of TP53 disruption. However, E6 and E7 maintain a variety of other virus–host interactions that directly disrupt a growing list of other DNA repair and chromatin remodeling factors, implying HPV-specific repair deficiencies. In addition, HPV-associated squamous cell carcinomas tumors clinically respond differently to DNA damaging agents compared to their HPV negative counterparts. The focus of this review is to integrate three categories of observations: (1) pre-clinical understanding as to the effect of HPV on DNA repair, (2) genomic signatures of DNA repair in HPV-associated tumor genomes, and (3) clinical responses of HPV-associated tumors to DNA damaging agents. The goals are to try to explain why HPV-associated tumors respond so well to DNA damaging agents, identify missing pieces, and suggest clinical strategies could be used to further improve treatment of these cancers.

Nanog, in Cooperation with AP1, Increases the Expression of E6/E7 Oncogenes from HPV Types 16/18

Persistent infections with some types of human papillomavirus (HPV) constitute the major etiological factor for cervical cancer development. Nanog, a stem cell transcription factor has been shown to increase during cancer progression. We wanted to determine whether Nanog could modulate transcription of E6 and E7 oncogenes. We used luciferase reporters under the regulation of the long control region (LCR) of HPV types 16 and 18 (HPV16/18) and performed RT-qPCR. We found that Nanog increases activity of both viral regulatory regions and elevates endogenous E6/E7 mRNA levels in cervical cancer-derived cells. We demonstrated by in vitro mutagenesis that changes at Nanog-binding sites found in the HPV18 LCR significantly inhibit transcriptional activation. Chromatin immunoprecipitation (ChIP) assays showed that Nanog binds in vivo to the HPV18 LCR, and its overexpression increases its binding as well as that of c-Jun. Surprisingly, we observed that mutation of AP1-binding sites also affect Nanog’s ability to activate transcription, suggesting cooperation between the two factors. We searched for putative Nanog-binding sites in the LCR of several HPVs and surprisingly found them only in those types associated with cancer development. Our study shows, for the first time, a role for Nanog in the regulation of E6/E7 transcription of HPV16/18.

Multiple Human Papilloma Virus (HPV) Infections Are Associated with HSIL and Persistent HPV Infection Status in Korean Patients

Infections with multiple human papilloma virus (HPV) types have been reported, but their role in cervical carcinogenesis has not been fully elucidated. In this study, 236 cases with multiple HPV infection were examined and compared to 180 cases with single HPV infection. HPV genotyping was performed with cervico-vaginal swab specimens using multiplex (real-time) polymerase chain reaction (PCR). In multiple HPV infection, the most prevalent HPV genotype was HPV 53, followed by HPV 16, 58, 52, and 68. HPV 33, 35, 39, 51, 52, 53, 58, and 68 were high-risk-HPV (HR-HPV) genotypes that were more frequently detected in multiple HPV infection compared to that in single HPV infection. The association between multiple HPV infection and high-grade SIL (HSIL) was significantly stronger compared to that of single HPV infection and HSIL (p = 0.002). Patients with multiple HPV infection displayed persistent and longer duration of the HPV infection compared to patients with single HPV infection. Multiple HPV infections have distinct clinicopathologic characteristics. Since it is associated with persistent HPV infection, HSIL, and different HR-HPV strains in contrast to single HPV infection, the presence of multiple HPV infection should be reported; close follow up is warranted.

Human Papilloma Virus Vaccination

Human papilloma virus (HPV) is the most common sexually transmitted infection worldwide causing a variety of benign and malignant conditions. A significant portion of the global population is infected with HPV, with the virus attributed to causing up to 5% of cancers worldwide. Bivalent, quadrivalent, and nine-valent vaccinations exist to aid in the prevention of these diseases and have been proven to be effective at preventing both benign and malignant disease. While vaccination is readily accessible in more developed countries, barriers exist to worldwide distribution and acceptance of vaccination. Vaccination and screening of HPV infection when used in combination are proven and predicted to decrease HPV related pathology. Improvements in vaccination formulations, for treatment as well as prevention, are actively being sought from a variety of mechanisms. Despite these advancements, and the data supporting their efficacy, there has been substantial delay in obtaining adequate vaccination coverage. In reviewing these challenges and looking forward to new vaccine development—especially within the current pandemic—it is clear from the challenges of HPV we require methods to more effectively encourage vaccination, ways to dispel vaccination myths as they occur, and implement better processes for vaccine distribution globally.

Mixed Bacteriophage MS2-L2 VLPs Elicit Long-Lasting Protective Antibodies against HPV Pseudovirus 51

Three prophylactic vaccines are approved to protect against HPV infections. These vaccines are highly immunogenic. The most recent HPV vaccine, Gardasil-9, protects against HPV types associated with ~90% of cervical cancer (worldwide). Thus, ~10% of HPV-associated cancers are not protected by Gardasil-9. Although this is not a large percentage overall, the HPV types associated with 10% of cervical cancer not protected by the current vaccine are significantly important, especially in HIV/AIDS patients who are infected with multiple HPV types. To broaden the spectrum of protection against HPV infections, we developed mixed MS2-L2 VLPs (MS2-31L2/16L2 VLPs and MS2-consL2 (69-86) VLPs) in a previous study. Immunization with the VLPs neutralized/protected mice against infection with eleven high-risk HPV types associated with ~95% of cervical cancer and against one low-risk HPV type associated with ~36% of genital warts & up to 32% of recurrent respiratory papillomatosis. Here, we report that the mixed MS2-L2 VLPs can protect mice from three additional HPV types: HPV51, which is associated with ~0.8% of cervical cancer; HPV6, which is associated with up to 60% of genital warts; HPV5, which is associated with skin cancers in patients with epidermodysplasia verruciformis (EV). Overall, mixed MS2-L2 VLPs can protect against twelve HPV types associated with ~95.8% of cervical cancers and against two HPV types associated with ~90% of genital warts and >90% recurrent respiratory papillomatosis. Additionally, the VLPs protect against one of two HPV types associated with ~90% of HPV-associated skin cancers in patients with EV. More importantly, we observed that mixed MS2-L2 VLPs elicit protective antibodies that last over 9 months. Furthermore, a spray-freeze-dried formulation of the VLPs is stable, immunogenic, and protective at room temperature and 37 °C.

Distribution of Human Papillomavirus (HPV) Genotypes in HIV-Negative and HIV-Positive Women with Cervical Intraepithelial Lesions in the Eastern Cape Province, South Africa

South African women have a high rate of cervical cancer cases, but there are limited data on human papillomavirus (HPV) genotypes in cervical intraepithelial neoplasia (CIN) in the Eastern Cape province, South Africa. A total of 193 cervical specimens with confirmed CIN from women aged 18 years or older, recruited from a referral hospital, were tested for HPV infection. The cervical specimens, smeared onto FTA cards, were screened for 36 HPV types using an HPV direct flow kit. HPV prevalence was 93.5% (43/46) in CIN2 and 96.6% (142/147) in CIN3. HIV-positive women had a significantly higher HPV prevalence than HIV-negative women (98.0% vs. 89.1%, p = 0.012). The prevalence of multiple types was significantly higher in HIV-positive than HIV-negative women (p = 0.034). The frequently detected genotypes were HPV35 (23.9%), HPV58 (23.9%), HPV45 (19.6%), and HPV16 (17.3%) in CIN2 cases, while in CIN3, HPV35 (22.5%), HPV16 (21.8%), HPV33 (15.6%), and HPV58 (14.3%) were the most common identified HPV types, independent of HIV status. The prevalence of HPV types targeted by the nonavalent HPV vaccine was 60.9% and 68.7% among women with CIN2 and CIN3, respectively, indicating that vaccination would have an impact both in HIV-negative and HIV-positive South African women, although it will not provide full protection in preventing HPV infection and cervical cancer lesions.

Leptomeningeal Carcinomatosis of a Poorly Differentiated Cervical Carcinoma Caused by Human Papillomavirus Type 18

Cervical cancer is caused by a persistent infection with high-risk types of Papillomaviruses (hrHPV); HPV16 and HPV18 are associated with about 70% of the cases. In the last decades the introduction of a cervical cancer screening has allowed a decrease in cervical cancer incidence and mortality; regular adhesion to the screening procedures, by pap test or HPV test, and colposcopy, according to the international guidelines, prevents cancer development and allows for diagnosis at the early stages. Nowadays, in industrialized countries, it is not common to diagnose this pathology in advanced stages, and this occurrence is frequently associated with patient’s unattendance of cervical screening programs. We describe a case of delayed diagnosis of cervical cancer, posed only after the onset of the neurological symptoms caused by leptomeningeal metastases, despite a two-year history of abnormal cytology. The endocervical mass was analyzed by immunohistochemistry, and search and typing of HPV sequences was performed by PCR in the meningeal carcinomatous cells. A poorly differentiated squamous cell carcinoma was diagnosed, and HPV18 sequences were detected. This rapidly fatal case highlights the importance of following the evidence-based recommended protocols and the preventive role of the population-based cervical cancer screening programs.

Changes in the Cervical Microbiota of Women with Different High-Risk Human Papillomavirus Loads

The cervical microbiota is essential in female sexual health, and its altered states seem to have a central role in the dynamic of high-risk papillomavirus (hrHPV) infections. This study aimed to evaluate the variation in bacterial communities’ compositions according to hrHPV. We collected two samples per woman, with a difference of 12 ± 1 months between them, and performed a follow-up on 66 of these women. The viral load (VL) of the hrHPV was estimated by quantitative PCR (qPCR), then it was normalized (using the HMBS gene as reference) and transformed to the Log10 scale to facilitate the interpretation. The VL was categorized as Negative, without hrHPV copies; Low, less than 100 hrHPV copies; Medium, between 100 to 102 hrHPV copies; and High, >102 hrHPV copies. The microbiota composition was described through the Illumina Novaseq PE250 platform. The diversity analyses revealed changes regarding the hrHPV VL, where women with low VL (<100 hrHPV copies) presented high diversity. The community state type (CST) IV was the most common. However, in women with high VL, a lower association with Lactobacillus depletion was found. Lactobacillus gallinarum and L. iners were the most abundant species in women with high VL, whereas women with low VL had a 6.06 greater probability of exhibiting Lactobacillus dominance. We identified conspicuous differences in the abundance of 78 bacterial genera between women with low and high VL, where 26 were depleted (e.g., Gardnerella) and 52 increased (e.g., Mycoplasma). A multilevel mixed-effects linear regression showed changes in the diversity due to the interaction between the measurement time and the VL, with a decrease in diversity in the second follow-up in women with low VL (Coeff. = 0.47), whereas the women with medium VL displayed an increase in diversity (Coeff. = 0.58). Here, we report for the first time that the cervical microbiota is influenced by the number of copies of hrHPV, where a decrease in the abundance of Lactobacillus, greater diversity, and enrichment of bacterial taxa is relevant in women with low VL.

High-Risk Human Papillomavirus Detection via Cobas® 4800 and REBA HPV-ID® Assays

Persistent infection with high-risk human papillomaviruses (HR-HPVs), particularly HPV16 and 18, has long been known to induce cervical cancer progression. However, given that a minority of HPV-infected women develop cancer, analysis of HR-HPV-infected women could help to predict who is at risk of acquiring cervical cancer. Therefore, to improve HR-HPVs detection, we used the FDA-approved cobas® 4800 HPV and REBA HPV-ID® HPV assays to detect HR-HPVs in colposcopy-derived cervical cells from 303 patients, detecting 72.28% (219) and 71.62% (217) of HR-HPVs positive cases, with HPV16 detection rates of 35.64% (108) and 30.69% (93), respectively. Of the HPV16-positive cases, cobas® 4800 and REBA HPV-ID® identified 28.81% (51) and 25.42% (45) of the CIN1 cases, and 55% (33) and 50% (30) of the 60 CIN2/3 cases, respectively. HPV-diagnostic concordance was 82.17% overall (kappa = 0.488), 87.45% for HR-HPVs (kappa = 0.689), and 88.33% for CIN2/3 (kappa = 0.51). The HR-HPVs detection rates of these assays were comparable. Our findings reveal that the FDA-approved HR-HPVs detection assay is appropriate for screening women with HR-HPVs infection, and for predicting increased risk of cervical cancer progression. REBA HPV-ID® can be used to detect low risk-HPV types in high-grade cervical lesions that are HR-HPV negative as well as in the distribution of HPV types.

High APOBEC3B mRNA Expression Is Associated with Human Papillomavirus Type 18 Infection in Cervical Cancer

The APOBEC3 (A3) proteins are cytidine deaminases that exhibit the ability to insert mutations in DNA and/or RNA sequences. APOBEC3B (A3B) has been evidenced as a DNA mutagen with consistent high expression in several cancer types. Data concerning the A3B influence on HPV infection and cervical cancer are limited and controversial. We investigated the role of A3B expression levels in cervical cancer in affected women positive for infection by different HPV types. Tumor biopsies from cancerous uterine cervix were collected from 216 women registered at Hospital do Câncer II of Instituto Nacional de Câncer, and infecting HPV was typed. A3B expression levels were quantified from RNA samples extracted from cervical biopsies using real-time quantitative PCR. Median A3B expression levels were higher among HPV18+ samples when compared to HPV16+ counterparts and were also increased compared to samples positive for other HPV types. In squamous cell carcinoma, HPV18+ samples also showed increased median A3B expression when compared to HPV Alpha-9 species or only to HPV16+ samples. Our findings suggest that A3B expression is differentially upregulated in cervical cancer samples infected with HPV18. A3B could be potentially used as a biomarker for HPV infection and as a prognostic tool for clinical outcomes in the context of cervical cancer.

Molecular Mechanisms of MmuPV1 E6 and E7 and Implications for Human Disease

Human papillomaviruses (HPVs) cause a substantial amount of human disease from benign disease such as warts to malignant cancers including cervical carcinoma, head and neck cancer, and non-melanoma skin cancer. Our ability to model HPV-induced malignant disease has been impeded by species specific barriers and pre-clinical animal models have been challenging to develop. The recent discovery of a murine papillomavirus, MmuPV1, that infects laboratory mice and causes the same range of malignancies caused by HPVs provides the papillomavirus field the opportunity to test mechanistic hypotheses in a genetically manipulatable laboratory animal species in the context of natural infections. The E6 and E7 proteins encoded by high-risk HPVs, which are the HPV genotypes associated with human cancers, are multifunctional proteins that contribute to HPV-induced cancers in multiple ways. In this review, we describe the known activities of the MmuPV1-encoded E6 and E7 proteins and how those activities relate to the activities of HPV E6 and E7 oncoproteins encoded by mucosal and cutaneous high-risk HPV genotypes.

Is the Physiological Composition of the Vaginal Microbiome Altered in High-Risk HPV Infection of the Uterine Cervix?

Background: Cervical cancer is the fourth most common malignancy and fourth leading cause of cancer death in women worldwide. More than 99.7% of cases are caused by human papillomavirus (HPV), while HPV types 16 and 18 cause over 70% of all cervical cancer cases. In this preliminary study, we aimed to investigate the presence of HPV infection and diversity of bacteria associated with bacterial vaginosis. Methods: Cervical swabs (n = 21) taken from women aged 21–47 years, in seventeen cases, with different degrees of cervical abnormality, and from four healthy women, were tested for the presence of HPV DNA, as well as the bacterial strains associated with bacterial vaginosis, using the real-time PCR method. Results: HPV16 was the dominant genotype in 53% (9/17) of patients with confirmed precancerous lesions (ASCUS, LSIL, and HSIL). In specimens with confirmed cytological abnormalities and hrHPV infection, we detected a wide diversity of microbes, while the most common species were Gardnerella vaginalis, Atopobium vaginae, Prevotella bivia, Ureaplasma parvum, Ureaplasma urealyticum, Leptotrichia amnionii, Bacteroides ureolyticus, and Sneathia sanguinegens. The presence of pathogens did not differ, depending on the degree of precancerous lesions or HPV type. Conclusion: In our work, HPV16 dominated in patients with cervical precancerous lesions. We also suggest an increased bacterial diversity of the vaginal microbiome in patients with cervical lesions, for which the HPV virus is largely responsible.

The Prevalence, Genotype Distribution and Risk Factors of Human Papillomavirus in Tunisia: A National-Based Study

There are limited national population-based studies on HPV genotypes distribution in Tunisia, thus making difficult an assessment of the burden of vaccine-preventable cervical cancer. In this context, we conducted a national survey to determine the HPV prevalence and genotypes distribution and the risk factors for HPV infections in Tunisian women. This is a cross-sectional study performed between December 2012 and December 2014. A liquid-based Pap smear sample was obtained from all women and samples’ DNAs were extracted. Only women with betaglobin-positive PCR were further analysed for HPV detection and typing by a nested-PCR of the L1 region followed by next-generation sequencing. A multiple logistic regression model was used for the analysis of associations between the variables. A total of 1517 women were enrolled in this study, and 1229 out of the 1517 cervical samples were positive for the betaglobin control PCR and tested for HPV. Overall HPV infection prevalence was measured to be 7.8% (96/1229), with significant differences between the grand regions, ranging from 2% in the North to 13.1% in Grand Tunis. High-risk HPV genotypes accounted for 5% of the infections. The most prevalent genotypes were HPV 31 (1%), 16 (0.9%), 59 (0.7%). HPV18 was detected only in four cases of the study population. Potential risk factors were living in Grand Tunis region (OR: 7.94 [2.74–22.99]), married status (OR: 2.74 [1.23–6.13]), smoking habit (OR: 2.73 [1.35–5.51]), occupation (OR: 1.81 [1.09–3.01]) and women with multiple sexual partners (OR: 1.91 [1.07–3.39]). These findings underscore the need to evaluate the cost effectiveness of HPV vaccine implementation, contribute to the evidence on the burden of HPV infections, the critical role of sexual behaviour and socioeconomic status, and call for increased support to the preventive program of cervical cancer in Tunisia.

Detection of HPV RNA in Extracellular Vesicles from Neuroendocrine Cervical Cancer Cells

Background: Neuroendocrine carcinoma of the cervix (NECC) is an aggressive and rare type of cervical cancer. The five-year overall survival is low at 30% and there is no standardized therapy based on controlled trials for this type of tumour. Most are locally advanced or metastasized at the time of the diagnosis. Extracellular vesicles (EVs) could be a carrier of viral DNA/RNA, given their vital role in cellular communication. The content of EV derived from NECC cells has not been investigated due to the lack of cell line, and it is not known whether they contain human papillomaviruses (HPV) DNA/RNA or not. Methods: The presence of viral E7 DNA/RNA in EVs purified from a culture of a recently established NECC cell line, GUMC-395, was evaluated by using droplet digital polymerase chain reaction (ddPCR). These EVs were characterized using nanoparticle tracking analysis (NTA) for size distribution, transmission electron microscopy (TEM) for morphology, Western blot for CD63, and bioanalyser for RNA quantity and quality. Results: HPV16 viral-RNA, but not DNA, was detected in EVs from GUMC-395 using ddPCR. NTA identified EVs with a mean diameter of 105.0 nm, TEM confirmed normal morphological shape and size, and Western blot analysis confirmed the presence of EV-associated proteins CD63. The EVs were found to be enriched with small RNAs using a bioanalyser. Conclusions: HPV16 RNA is found in EVs from a neuroendocrine cervical cancer and could be involved in the pathogenesis of the disease and used as a diagnostic biomarker.

Human Virome in Cervix Controlled by the Domination of Human Papillomavirus

Although other co-viral infections could also be considered influencing factors, cervical human papillomavirus (HPV) infection is the main cause of cervical cancer. Metagenomics have been employed in the NGS era to study the microbial community in each habitat. Thus, in this investigation, virome capture sequencing was used to examine the virome composition in the HPV-infected cervix. Based on the amount of HPV present in each sample, the results revealed that the cervical virome of HPV-infected individuals could be split into two categories: HPV-dominated (HD; ≥60%) and non-HPV-dominated (NHD; <60%). Cervical samples contained traces of several human viral species, including the molluscum contagiosum virus (MCV), human herpesvirus 4 (HHV4), torque teno virus (TTV), and influenza A virus. When compared to the HD group, the NHD group had a higher abundance of several viruses. Human viral diversity appears to be influenced by HPV dominance. This is the first proof that the diversity of human viruses in the cervix is impacted by HPV abundance. However, more research is required to determine whether human viral variety and the emergence of cancer are related.

CIGB-300 Peptide Targets the CK2 Phospho-Acceptor Domain on Human Papillomavirus E7 and Disrupts the Retinoblastoma (RB) Complex in Cervical Cancer Cells

CIGB-300 is a clinical-grade anti-Protein Kinase CK2 peptide, binding both its substrate’s phospho-acceptor site and the CK2α catalytic subunit. The cyclic p15 inhibitory domain of CIGB-300 was initially selected in a phage display library screen for its ability to bind the CK2 phospho-acceptor domain ofHPV-16 E7. However, the actual role of this targeting in CIGB-300 antitumoral mechanism remains unexplored. Here, we investigated the physical interaction of CIGB-300 with HPV-E7 and its impact on CK2-mediated phosphorylation. Hence, we studied the relevance of targeting E7 phosphorylation for the cytotoxic effect induced by CIGB-300. Finally, co-immunoprecipitation experiments followed by western blotting were performed to study the impact of the peptide on the E7–pRB interaction. Interestingly, we found a clear binding of CIGB-300 to the N terminal region of E7 proteins of the HPV-16 type. Accordingly, the in vivo physical interaction of the peptide with HPV-16 E7 reduced CK2-mediated phosphorylation of E7, as well as its binding to the tumor suppressor pRB. However, the targeting of E7 phosphorylation by CIGB-300 seemed to be dispensable for the induction of cell death in HPV-18 cervical cancer-derived C4-1 cells. These findings unveil novel molecular clues to the means by which CIGB-300 triggers cell death in cervical cancer cells.

Clinical Validation of the Fully Automated NeuMoDx HPV Assay for Cervical Cancer Screening

The NeuMoDx HPV assay is a novel fully automated, real-time PCR-based assay for the qualitative detection of high-risk human papillomavirus (HPV) DNA in cervical specimens. The assay specifically identifies HPV16 and HPV18 and concurrently detects 13 other high-risk HPV types at clinically relevant infection levels. Following the international guidelines, the clinical performance of the NeuMoDx HPV assay for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) against the reference standard Hybrid Capture 2, as well as intra- and inter-laboratory reproducibility were assessed on PreservCyt samples. The clinical accuracy of the assay was additionally evaluated against the clinically validated Alinity m HR HPV and COBAS 4800 HPV Test on PreservCyt samples, and against the clinically validated HPV-Risk assay on SurePath samples. The NeuMoDx HPV assay performance for CIN2+ was non-inferior to the reference methods on both sample types (all p < 0.05), and showed excellent intra- and inter-laboratory reproducibility (95.7%; 95% CI: 93.9–97.3; kappa value 0.90 (95% CI: 0.86–0.94); and 94.5%; 95% CI: 92.6–96.2; kappa value 0.87 (95% CI: 0.82–0.92), respectively). In conclusion, the NeuMoDx HPV assay meets international guideline criteria for cross-sectional accuracy and reproducibility, and performs equally well on cervical screening specimens collected in two widely used collection media. The NeuMoDx HPV assay fulfils the requirements to be used for primary cervical screening.

Ancient Evolutionary History of Human Papillomavirus Type 16, 18 and 58 Variants Prevalent Exclusively in Japan

Human papillomavirus (HPV) is a sexually transmitted virus with an approximately 8-kilo base DNA genome, which establishes long-term persistent infection in anogenital tissues. High levels of genetic variations, including viral genotypes and intra-type variants, have been described for HPV genomes, together with geographical differences in the distribution of genotypes and variants. Here, by employing a maximum likelihood method, we performed phylogenetic analyses of the complete genome sequences of HPV16, HPV18 and HPV58 available from GenBank (n = 627, 146 and 157, respectively). We found several characteristic clusters that exclusively contain HPV genomes from Japan: two for HPV16 (sublineages A4 and A5), one for HPV18 (sublineage A1) and two for HPV58 (sublineages A1 and A2). Bayesian phylogenetic analyses of concatenated viral gene sequences showed that divergence of the most recent common ancestor of these Japan-specific clades was estimated to have occurred ~98,000 years before present (YBP) for HPV16 A4, ~39,000 YBP for HPV16 A5, ~38,000 YBP for HPV18 A1, ~26,000 for HPV58 A1 and ~25,000 YBP for HPV58 A2. This estimated timeframe for the divergence of the Japan-specific clades suggests that the introduction of these HPV variants into the Japanese archipelago dates back to at least ~25,000 YBP and provides a scenario of virus co-migration with ancestral Japanese populations from continental Asia during the Upper Paleolithic period.

Therapeutic DNA Vaccines against HPV-Related Malignancies: Promising Leads from Clinical Trials

In 2014 and 2021, two nucleic-acid vaccine candidates named MAV E2 and VGX-3100 completed phase III clinical trials in Mexico and U.S., respectively, for patients with human papillomavirus (HPV)-related, high-grade squamous intraepithelial lesions (HSIL). These well-tolerated but still unlicensed vaccines encode distinct HPV antigens (E2 versus E6+E7) to elicit cell-mediated immune responses; their clinical efficacy, as measured by HSIL regression or cure, was modest when compared with placebo or surgery (conization), but both proved highly effective in clearing HPV infection, which should help further optimize strategies for enhancing vaccine immunogenicity, toward an ultimate goal of preventing malignancies in millions of patients who are living with persistent, oncogenic HPV infection but are not expected to benefit from current, prophylactic vaccines. The major roadblocks to a highly efficacious and practical product remain challenging and can be classified into five categories: (i) getting the vaccines into the right cells for efficient expression and presentation of HPV antigens (fusion proteins or epitopes); (ii) having adequate coverage of oncogenic HPV types, beyond the current focus on HPV-16 and -18; (iii) directing immune protection to various epithelial niches, especially anogenital mucosa and upper aerodigestive tract where HPV-transformed cells wreak havoc; (iv) establishing the time window and vaccination regimen, including dosage, interval and even combination therapy, for achieving maximum efficacy; and (v) validating therapeutic efficacy in patients with poor prognosis because of advanced, recurrent or non-resectable malignancies. Overall, the room for improvements is still large enough that continuing efforts for research and development will very likely extend into the next decade.