mBio

The vaginal microbiome, papillomavirus infection, and cervical cancer: established associations in search of model systems and mechanistic answers

ABSTRACT High-risk human papillomavirus (HPV) infection is the causative factor for approximately 5% of all human cancers and the leading cause of cervical cancer. High-risk HPV-associated cervical cancer still claims more than 340,000 women’s lives globally each year despite the availability of prophylactic HPV vaccines. Currently, there is no medical treatment for HPV infections and associated lesions except invasive surgical procedures. For more than a decade, numerous studies have demonstrated a correlation between certain community state types (CSTs) of the vaginal microbiome and HPV-associated infection and cancer. This review aims to provide a general overview of the most recent studies on this topic, focusing primarily on clinical data linking a Lactobacillus -depleted vaginal microbiome (i.e., bacterial vaginosis and CST-IV) and HPV but also describing the limited mechanistic findings in the field. Finally, a novel mouse model addressing the causative effect of the vaginal microbiome on papillomavirus-associated disease progression and cancer development is proposed.

Immune responses, therapeutic anti-tumor effects, and tolerability upon therapeutic HPV16/18 E6/E7 DNA vaccination via needle-free biojector

ABSTRACT Intramuscular vaccination of mice with the naked pBI-11 DNA plasmid targeting E6 and E7 of HPV16 and HPV18 via a conventional syringe and needle generates human papillomavirus (HPV) antigen-specific CD8+ T cell-mediated immune responses and therapeutic effects against the TC-1 tumor model. However, delivery of DNA vaccines by this method is less effective in patients, likely due to poor transduction of host tissues. Needle-free biojectors show great promise for DNA vaccination because of their simplicity of administration and high patient acceptability and also, we hypothesize, because of greater efficiency of cell transduction in host tissues. Here, we compared the kinetics of transgene expression from a plasmid DNA using intramuscular injection with a conventional needle administration to intradermal or intramuscular delivery with a customized Tropis biojector. Delivery using the customized Tropis biojector leads to enhanced transgene expression compared to intramuscular needle injection. In addition, we characterized the HPV antigen-specific CD8+ T cell-mediated immune responses and anti-tumor effects generated by pBI-11 DNA vaccination by each route of administration, as well as by split-dose multi-site injection. Intradermal, but not intramuscular, vaccination with pBI-11 DNA vaccine via customized Tropis biojector enhanced HPV antigen-specific CD8+ T cell-mediated immune responses over needle injection. Intradermal, but not intramuscular, vaccination via customized needle-free Tropis biojector elicited greater HPV antigen-specific CD8+ T cell-mediated immune responses as well as anti-tumor effects compared to intramuscular injection of pBI-11 with a needle. Good manufacturing practices (GMP) grade pBI-11 DNA vaccine delivered intradermally or intramuscularly via customized Tropis biojector was well tolerated by mice. IMPORTANCE Respectively, HPV16 and HPV18 cause 50% and 20% of cervical cancer cases globally. Viral proteins E6 and E7 are obligate drivers of oncogenic transformation. We recently developed a candidate therapeutic DNA vaccine, pBI-11, that targets HPV16 and HPV18 E6 and E7. Single-site intramuscular delivery of pBI-11 via a needle elicited therapeutic anti-tumor effects in mice and is now being tested in high-risk human papillomavirus+ head and neck cancer patients (NCT05799144). Needle-free biojectors such as the Tropis device show promise due to ease of administration, high patient acceptability, and the possibility of improved delivery. For example, vaccination of patients with the ZyCoV-D DNA vaccine using the Tropis device is effective against COVID19, well tolerated, and licensed. Here we show that split-dose, multi-site administration and intradermal delivery via the Tropis biojector increase the delivery of pBI-11 DNA vaccine, enhance HPV antigen-specific CD8+ T-cell responses, and improve anti-tumor therapeutic effects, suggesting its translational potential to treat HPV16/18 infection and disease.

Expression of the Long Noncoding RNA DINO in Human Papillomavirus-Positive Cervical Cancer Cells Reactivates the Dormant TP53 Tumor Suppressor through ATM/CHK2 Signaling

Functional restoration of the TP53 tumor suppressor holds great promise for anticancer therapy. Current strategies are focused on modulating TP53 regulatory proteins. Long noncoding RNAs (lncRNAs) have emerged as important regulators of TP53 as well as modulators of downstream tumor-suppressive transcriptional responses. Unlike many other cancer types, human papillomavirus (HPV)-positive cancer cells retain wild-type TP53 that is rendered dysfunctional by the viral E6 protein. We show that acute expression of the damage-induced long noncoding RNA, DINO, a known TP53 transcriptional target and functional modulator, causes TP53 reactivation in HPV-positive cervical cancer cells. This causes increased vulnerability to standard chemotherapeutics as well as biguanide compounds that cause metabolic stress. Hence, strategies that target DINO may be useful for restoring TP53 tumor suppressor activity in HPV-positive cancers and other tumor types that retain wild-type TP53.

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

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

HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer

ABSTRACT The integration of HPV DNA into human chromosomes plays a pivotal role in the onset of papillomavirus-related cancers. HPV DNA integration often occurs by linearizing the viral DNA in the E1/E2 region, resulting in the loss of a critical viral early polyadenylation signal (PAS), which is essential for the polyadenylation of the E6E7 bicistronic transcripts and for the expression of the viral E6 and E7 oncogenes. Here, we provide compelling evidence that, despite the presence of numerous integrated viral DNA copies, virus-host fusion transcripts originate from only a single integrated HPV DNA in HPV16 and HPV18 cervical cancers and cervical cancer-derived cell lines. The host genomic elements neighboring the integrated HPV DNA are critical for the efficient expression of the viral oncogenes that leads to clonal cell expansion. The fusion RNAs that are produced use a host RNA polyadenylation signal downstream of the integration site, and almost all involve splicing to host sequences. In cell culture, siRNAs specifically targeting the host portion of the virus-host fusion transcripts effectively silenced viral E6 and E7 expression. This, in turn, inhibited cell growth and promoted cell senescence in HPV16+ CaSki and HPV18+ HeLa cells. Showing that HPV E6 and E7 expression from a single integration site is instrumental in clonal cell expansion sheds new light on the mechanisms of HPV-induced carcinogenesis and could be used for the development of precision medicine tailored to combat HPV-related malignancies. IMPORTANCE Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.

Development of DNA Vaccine Targeting E6 and E7 Proteins of Human Papillomavirus 16 (HPV16) and HPV18 for Immunotherapy in Combination with Recombinant Vaccinia Boost and PD-1 Antibody

Persistent expression of high-risk human papillomavirus (HPV) E6 and E7 is an obligate driver for several human malignancies, including cervical cancer, wherein HPV16 and HPV18 are the most common types. PD-1 antibody immunotherapy helps a subset of cervical cancer patients, and its efficacy might be improved by combination with active vaccination against E6 and/or E7.

ZER1 Contributes to the Carcinogenic Activity of High-Risk HPV E7 Proteins

HPV16 is highly carcinogenic and is the most predominant HPV genotype associated with human cancers. The mechanisms that underlie differences between high-risk HPV genotypes are currently unknown, but many of these differences are likely attributable to the activities of the oncogenic HPV proteins, including E7.

Assessing the Cervicovaginal Microbiota in the Context of hrHPV Infections: Temporal Dynamics and Therapeutic Strategies

Cervical cancer is the third leading cause of female cancers globally, resulting in more than 300,000 deaths every year. The majority of all cervical cancers are caused by persistent infections with high-risk human papillomaviruses (hrHPV) that can progress to cancer via a series of premalignant lesions.

Human Papillomavirus Type 16 Circular RNA Is Barely Detectable for the Claimed Biological Activity

RNA back-splicing is a rare splicing event accounting for <1% of canonical RNA splicing and, thus, is thought to have little or no biological significance. Recently, circular RNAs (circRNAs) from RNA back-splicing have been found widely in cells and tissues and may have a role in modulating RNA transcription, splicing, and interference and antiviral innate immunity.