It is noteworthy that HR-HPV infection may persist in certain individuals known as “people at risk”. These include people with sexually-transmissible diseases (AIDS-HIV, Chlamydia, and Gonorrhea), organ-transplant receivers, and the elderly; in short, people with an innate or acquired immune deficiency [2]. However, besides these people qualified as people at risk, it has been newly discovered that healthy women with a disrupted cervicovaginal microbiota (CVM) have a high risk for developing cervical intra-epithelial neoplasia grade 1 or 2 (CIN1/2) and invasive CC. Indeed, these studies described that the unbalanced bacterial population in the cervicovaginal area, caused by an increase or decrease of certain bacterial populations, is a major factor for HR-HPV acquisition and persistence of the infection towards high-grade lesions and cancer development [2, 8, 9].

A healthy CVM, which consists of a balanced variable population of bacteria, is dominated by Lactobacillus spp. [9, 10] and plays a crucial role in vaginal area protection against STIs and diseases [2, 8, 9]. Its disruption has been associated with several vaginal infections and diseases [8, 9]. Bacterial vaginosis (BV), for instance, which is a vaginal pathological condition, is characterized by a decreased amount of vaginal Lactobacillus spp. and an abundance of polymicrobial anaerobic bacteria, including Gardnerella vaginalis, Prevotella spp., Mobiluncus spp., Ureaplasma urealyticum, and Mycoplasma hominis [9]. Also, BV has been found to be a strong predictor of Neisseria gonorrhoeae and Chlamydia trachomatis infections [11], which are among the risk factors for HPV acquisition and infection persistence, the occurrence of CIN1/2, and malignancies, leading to the hypothesis that changes in CVM homeostasis have a crucial implication in the occurrence of cancerous lesions (CINs) [12]. Other studies have evidenced the strong causal link between genital immune mediators and the decrease in Lactobacillus spp. in cervical carcinogenesis [13, 14]. Several studies further confirmed this hypothesis (reviewed in [2]). More precisely, in their study, Usyk et al. [15] demonstrated that Lactobacillus spp. depletion in vaginal microbiota along with the abundance of strictly anaerobic bacteria, including Gardnerella vaginalis, Prevotella spp., Mobiluncus spp., Atopobium vaginae, and Mycoplasma hominis, is associated with a high risk of persistence of HR-HPV infections an occurrence of CIN2/3. Moreover, Norenhag et al. [10] confirmed that a CVM dominated by other bacterial populations than Lactobacillus spp. was associated with up to five times higher risk of contracting any HPVs and up to three times higher for HR-HPVs, which may induce invasive CC. Taken together, this demonstrates that—the abundance of—strictly anaerobic bacteria, but especially Gardnerella spp., are associated with the persistence of HR-HPVs infection and occurrence of malignancies, making “abnormal Gardnerella spp. increase” a point of control for monitoring HPV infection persistence (reviewed in [2]).

Controversially, while BV (or imbalanced CVM) has been since no long ago taught to be among the risk factors for HR-HPV infection and related carcinogenesis, Lebeau et al. [16] claimed in their recent study that it is instead the HR-HPV infection that leads to CVM alteration, making their causal relationship again questionable. Given that a healthy CVM consists of a low microbial diversity largely dominated ($>$90%) by a few Lactobacillus spp. (mainly L. crispatus, L. jensenii and L. iners) [9, 10, 16, 17, 18], the authors showed that, in the course of HPV infection, HR-HPV oncoproteins (E6 but mainly E7) down-regulate expression of the host innate peptides that are required for survival and sustainability of Lactobacillus spp., and antimicrobial activity on Gardnerella vaginalis in healthy vaginal mucosa [16]. Thus, inhibition of these host peptides, including defensin family (H$\beta$D1-4, HD5, HD6) and antimicrobial defensin-like family (SLPI, S100A7, Elafin, and LL-37) peptides, through the interaction of HPV E7 with NEMO, CK1, and $\beta$-TrCP of both NF-$\kappa$B and Wnt/$\beta$-catenin signaling pathways, leads to BV (or dysbiosis), characterized by changes of the normally dominant lactic acid bacteria as described above [16, 17, 18] by a high diversity of bacterial population predominated by Gardnerella vaginalis and other anaerobic bacteria including Atopobium vaginae, Prevotella spp., Mobiluncus spp., Sneathia spp., Ureaplasma urealyticum, and Mycoplasma hominis [9, 16].

Overall, it should be retained that while unbalanced CVM (where Gardnerella spp. is high and Lactobacillus spp. is low) is now a well-characterized risk factor for HPV acquisition and infection persistence, HR-HPV itself participates in CVM alteration through molecular interactions, which weaken the bacterial cervicovaginal protection and enhance the occurrence of CC. Hence, an unbalanced CVM is a microbial marker that should alert clinicians to precancerous lesion risk and should be considered with high priority for treatment.

Additionally, to the negative effect of HPV infection on CVM (and/or vis-versa) leading to HPV-associated disorders, the cervicovaginal metabolic profile (CVMP) might constitute another point of control for monitoring HPV infection risk and persistence of HR-HPV infection, but preferably for monitoring cervicovaginal cancers, because as metabolic diseases, cancers target and modify metabolome composition at the local environment [19, 20]. Studies in women with CC showed unique and typical CVMPs defined by a specific composition from amino acids to nucleic acids in CC tissues, which was completely absent from cancer-free/healthy women [21]. In the context of HPV-related CC, this typical CVM change is indirectly induced by HR-HPV infection through inhibition of host innate peptide [16] and cytokine [22] production that subsequently alters bacterial dynamics and antimicrobial activity in the cervicovaginal microenvironment and causes BV as described hereinbefore. For instance, changes from a healthy Lactobacillus spp.-dominated CVM towards an increased Gardnerella spp.-dominated anaerobic and microaerophilic poly-bacterial communities are strongly associated with increased amino acid catabolites and polyamines [23, 24]. More precisely, in a study profiling the cervicovaginal metabolism in HPV-infected women, with PCLs, CIN1/2/3, and CC, it was reported that for each woman and each specific grade of HPV-infection severity, the CVMP was typical and specifically different with a huge variation in vaginal pH and genital inflammation (Fig. 1) [25]. The CVMP in HPV-positive women with PCLs and CIN1/2/3 showed a decrease of amino acids metabolites and depletion of dipeptides characterized by HPV-positive women with CIN3. However, in HPV-positive women diagnosed with CC, the CVMP profile is characterized by increased xenobiotics and lipids from multiple macromolecule sources [25]. Hence, any increase of xenobiotics (mainly anti-inflammatory and analgesic drugs) and lipids (mainly 3-hydroxybutyrate, eicosenoate, and oleate/vaccinate) in an HPV-infected woman should be a suspicion of cancer development [25, 26], because many others studies evidenced that high level of 3-hydroxybutyrate, eicosenoate, and oleate/vaccinate augment are associated with tumor development and growth [27, 28], specifically CC [26, 29].

Fig. 1.

Effect of human papillomavirus (HPV) on cervicovaginal microbiota and metabolome profile and implication in carcinogenesis. The background color circled on the female’s body indicates the health state of the cervicovaginal microbiota: healthy and balanced cytomegalovirus (CMV) (colorless), slightly unbalanced cervicovaginal microbiota (CVM) (yellow), moderately unbalanced CVM (pink), severely unbalanced CVM (red). The color of the arrow bar (yellow [acid] to blue [basic]) indicates the vaginal pH. CIN1/2/3: cervical intra-epithelial neoplasia grade 1, 2 or 3.

Cervical cancer is a preventable disease as it lies on infection by HPV. The currently available HPV prophylaxis, including Cevarix, Gardasil, and Gardasil-9, are made from one of the main HPV proteins involved in the first steps of the infection, the major capsid L1 protein, obtained in the form of virus-like particles [55, 56]. Targeting this protein blocks further HPV interactions with host cells to initiate the early steps and prevents infection progression, which explains the proven efficacy of these three vaccines [52, 57] and long-term preventive immunity [58, 59, 60]. However, these vaccines have limitations, including limited protection against some but not all HR-HPVs.

Screening the molecular and intracellular trafficking of the HPV infection steps provides molecular insights for developing alternative drugs against HPV infections and CC (reviewed in [61]). Specifically, three main molecular target sites in the early steps of HPV infection, including (i) the heparan sulfate binding site, (ii) the HPV cellular internalization site, and (iii) the intracellular trafficking molecules, are promising in preventing HPV entry into cells and replication [61]. Of interest, targeting these therapeutic sites could allow the development of broad-spectrum prophylactics against all HPV types and, to a lesser extent, other viruses, as the antiviral mechanism lies on inhibiting the viral attachment to the cell surface or preventing the interactions between viral particles and intracellular components, by either binding to viral particles or interacting with intra- or extracellular receptors [61].

Specifically, heparan sulfate proteoglycans (HSPGs) are required cell receptors for the initial attachment of HPV and many other viruses, including coronaviruses [62, 63], herpes simplex viruses 1 and 2 (HSV-1/2), Dengue virus (DENV), echoviruses 5 and 6, enterovirus, human cytomegalovirus (HCMV), HIV, polyomaviruses [64], and hepatitis viruses [64, 65]. Thus, potential inhibitors that target the HSPG-depending early attachment might be promising against broad-spectrum viral infections [61, 64]. Interestingly, several molecules mimicking or interacting with HSPGs, including heparin, dendrimers, and carrageenan, have proven to be effective against HPV, HSV-1/2, HCMV, SARS-CoV-2, and DENV [61, 62, 64]. Hence, deeper in vivo studies to assess the protective efficacy and safety of these molecules are highly encouraged.

Likewise, during infection, HPVs use similar downstream intracellular trafficking with other viruses, which may constitute therapeutic targets against a broad range of viruses [61]. Several reports describe the promising use of molecules to prevent infection by HPVs and the occurrence of CC. Zhang et al. [66] designed a short peptide that can bind to retromer (an intracellular virus interacting protein that binds to HPV L2 protein [67]) and potentially compete with HPV16 as an antiviral strategy. They demonstrated that the synthetic peptide can enter cells, sequester retromer by occupying the HPV-16 L2 retromer-binding site and inhibit HPV infection [66]. Such a drug could prevent the occurrence of CC.

Moreover, endoplasmic reticulum (ER) chaperones have been identified as common proteins involved and required in intracellular viral trafficking, from entry and viral replication to the assembly and release of virions. To develop broad-spectrum antiviral therapeutics, several studies have proposed potential inhibitors of ER chaperones that can be used against HPV, HIV, hepatitis viruses, SARS-CoV-2, Zika virus, DENV, Ebola virus, HCMV, and many others [68]. ER chaperone inhibitors, including AR-12 (OSU-03012), EGCG (polyphenol), PU-WS13, Migitol, Indonlizidine derivatives, Juniferdin, PACMA 31, 16F16, Isoquecetin, Bacitracin, Origamicin, LOC14, and Tizoxanide, have proven to block the progression of viral infection [68]. Similarly, myosin motors are proteins that bind to actin filaments to generate force, mobility, and shape for cell structure and dynamics [69]. They play a central role in intracellular trafficking, exo- and endocytosis, and transcription. Thus, viruses, including HPVs, use or take advantage of these proteins of the cytoskeleton, which are necessary for HPV infection, specifically for viral internalization, packaging, and release [70, 71]. Therefore, the specific interactions between HPVs and myosin motors serve as a basis for developing antiviral drugs that could compromise such interactions. Overall, these studies might help develop broad-spectrum antiviral prophylactics that target common intracellular virus trafficking.

For effective CC prevention, sexually active women are recommended to undergo cervicovaginal screening to identify those who are at risk of cervical pre-cancer (CIN1, 2, or 3). Currently, several diagnostic tests are available on the market. The well-known of these precancerous lesion screening methods include the Papanicolaou test, also known as the Pap smear or smear test, a cervicovaginal cytological test designed to detect abnormal cervical cells [72, 73, 74, 75]. Although wildly considered an effective method for detecting and preventing CC, the Pap smear test sensitivity is low (ranging from 30% to 87%) [72, 73, 76, 77, 78]. Hence, it was highly recommended that the Pap smear must be performed yearly for a better follow-up [73]. Besides, it was also proposed that the Pap smear may be replaced by liquid-based cytology (LBC) (which is slightly more sensitive than the Pap smear [72]) or associated with colposcopy-directed biopsy [73] to reduce the rate of undiagnosed cervical dysplasia. Therefore, it has been finally adopted that colposcopy-directed biopsy should be done in cases of macroscopic cervical architectural changes, abnormal findings, or positivity of the Pap smear cytology result [54, 73, 79]. Appropriate treatment is then determined after a biopsy of suspicious lesions for histological diagnosis. This approach has been proven effective in developed countries where cytology-based CC screening programs are well-implemented and strictly followed, yielding a five-time reduce fatalities from CC [80]. This observation is unfortunately not the case in developing countries [81], where access to health care and availability of standard care facilities are still major public health concerns. This implies that governments from low- and middle-income countries should make available financial resources to set up or strengthen screening programs, to reduce the CC-related death rate.

In the last decade, researchers have developed improved diagnostic/screening tests in order to reach the WHO’s goal by 2030. These screening tests include visual inspection with acetic acid (VIA) and molecular tests, specifically HR-HPV DNA detection-based tests by PCR [54], which are more suitable to use in any laboratory setting (Table 1, Ref. [54, 72, 74, 75, 82, 83, 84, 85]). More interestingly, it was recently reported that newer cervicovaginal screening tests had been made available for diagnosing HR-HPV infection and precancerous lesions, among which molecular tests, specifically directed to the detection of HPV mRNA, HPV-E6/E7, and HPV-DNA methylation. Besides that, more conclusive screening tests performed on cytological samples (p16/Ki-67 dual staining) and highly technology-based advanced visual inspection screening tests based on artificial intelligence or machine learning platforms (automated visual evaluation of digital images) have been launched (Table 1) [54, 82, 83, 84, 85, 86].

There are two regulation approaches for screening and treatment of precancerous lesions and preventing CC occurrence, categorized into seven possible combinations or algorithms, which include (i) the screen-and-treat approach, for which the treatment is only based on a positive primary screening test (2 algorithms), and (ii) the screen-triage-and-treat approach for which the adequate treatment is based on and follows a positive primary test followed by a positive secondary test [54] (Fig. 2).

Fig. 2.

The seven algorithms considered for screening and treatment of cervical pre-cancer lesions and cervical cancer (CC) prevention. HPV, human papillomavirus; VIA, visual inspection with acetic acid; Cyt., cytology; +, positive; -, negative; Susp, suspicion of positive cytology test.

Specifically, in the screen-and-treat approach, women positive for HR-HPV and precancerous lesions after the primary screening test are directly eligible for the appropriate treatment. Secondary screening is not needed in this approach to start the treatment. Precancerous cell ablation and excision are the main treatments for women to prevent CC development. Once the primary screening is positive, the appropriate treatment is done on either the same visit of the screening test (single-visit approach) or another day (multiple-visit approach) [2, 54].

In a screen-triage-and-treat approach, women who are positive for HR-HPV and precancerous lesions after the primary screening test undergo a secondary screening test. If this later is confirmed positive, the appropriate treatment is determined by prime colposcopy with biopsy and histopathological examination. However, if the secondary screening test is negative, only an appropriate follow-up evaluation is required [2, 54].