DNA vaccination represents a useful approach for human papillomavirus (HPV) cancer therapy. The therapeutic potential of plant-based natural compounds for control of HPV- associated cancers has been also widely explored. Genetic vaccines for HPV-associated tumors that include plant protein-encoding gene sequences, used alone or in combinations with plant metabolites, are being investigated but are still in their infancy. Main focus of this paper is to provide an overview of the current state of novel therapeutic strategies employing genetic vaccines along with plant-derived compounds and genes. We highlight the importance of multimodality treatment regimen such as combining immunotherapy with plant-derived agents.Antigen-specific immunotherapy and, in particular, DNA vaccination provides an established approach for tackling human papillomavirus (HPV) cancers at different stages. DNA vaccines are stable and have a cost-effective production. Their intrinsic low immunogenicity has been improved by several strategies with some success, including fusion of HPV antigens with plant gene sequences. Another approach for the control of HPV cancers is the use of natural immunomodulatory agents like those derived from plants, that are able to interfere in carcinogenesis by modulating many different cellular pathways and, in some instances, to reduce chemo- and radiotherapy resistance of tumors. Indeed, plant-derived compounds represent, in many cases, an abundantly available, cost-effective source of molecules that can be either harvested directly in nature or obtained from plant cell cultures. In this review, an overview of the most relevant data reported in literature on the use of plant natural compounds and genetic vaccines that include plant-derived sequences against HPV tumors is provided. The purpose is also to highlight the still under-explored potential of multimodal treatments implying DNA vaccination along with plant-derived agents. Cancer is considered the leading cause of death in wealthy countries, and 15–20% of all human cancers are associated with viral infections ,4. The iNicotiana benthamiana, containing ectopically expressed HPV 16 E7 protein, induced a cell-mediated immune response able to protect vaccinated mice from tumor challenge, notably without any adjuvant [In the past, we described that a plant extract of adjuvant ,11.Chlamydomonas reinhardtii [This extract induced maturation of human dendritic cells (DCs) that became able to prime in vitro human blood-derived T lymphocytes from healthy individuals to induce HPV 16 E7-specific cytotoxic response . A similnhardtii . A commoMeanwhile, it was established that enhanced release of HPV antigens from tumor cells pretreated with chemo/radiotherapy can be modulated by plant-derived chemotherapeutic agents. Indeed, chemotherapeutic agents such as Apigenin  and EpigBeside plant molecules, plant viruses and gene sequences encoding plant proteins  have been employed to improve HPV therapeutic genetic vaccine. Fusion of HPV 16 E7 gene to the Potato Virus X coat protein (PVX-CP) gene increased the rate of proteasomal degradation in transfected cells and, as a consequence, vaccine efficacy . Indeed,In this review, we focus on the most relevant aspects of plant-derived compounds and genetic vaccines that might be decisive for the future development of cost-effective HPV vaccines.The high-risk (HR) HPV types  are considered to be the main etiological agents of genital tract cancers, such as cervical, vulvar, vaginal, penile, and anal cancers, and of a subset of head and neck cancers. Among the 15 most frequent oncogenic HPV types associated with these cancers, HPV 16 is the most common and associated with the highest risk of progression to cancer ,23,24.The primary evidence of cancer development is HPV integration into the host genome in malignant tumors. HPV integration can take place in multiple non-recurrent regions of amplification and in flanked regions where deletions occur. There is a robust association between HPV insertional breakpoints and genomic structural alterations, which ultimately results in genomic instability, a peculiar sign of HPV-positive tumors. HPV alone is necessary but not sufficient to induce tumors, while it holds an important role in cancer maintenance. However, other genetic and epigenetic events are required for cancer development. This phenomenon may partly explain the latency period that occurs in tumor development and how persistent infection with HR HPVs is necessary for progression to high-grade lesions or cancer ,26.Other risk factors for progression to high-grade dysplasia and cancer include age over 30 years, infection with multiple HPV types, immunosuppression, and tobacco use ,28.HR HPV E6 and E7 are oncoproteins that bind and promote degradation of tumor suppressor proteins, p53 and retinoblastoma (pRb), respectively. However, HPVs interact with many other cancer-relevant pathways, even in a p53- and/or pRb-independent way. In addition, these oncoproteins may deregulate intracellular microRNA (miRNA) networks, and many HPV-altered miRNAs have been linked to carcinogenesis . E6/E7 oIdentifying HPV as an etiological factor of cervical cancer and other HPV-associated malignancies helped in the development of immunization strategies to prevent infection and associated diseases caused by HPV. Since 2013, HPV vaccines  have been included in the national immunization programs of at least 66 nations, including North America and Western Europe, primarily . RecombiPreventive HPV vaccines aim to prevent HPV infection by inducing a neutralizing antibody response. Improved understanding of protective humoral immune response against primary HPV infection has led to the development of preventive HPV vaccines targeting L1 and/or L2 viral capsid proteins . BecauseDevelopment of this technology led to the production of current VLP-based preventive vaccines targeting L1 in order to generate neutralizing antibodies against HPV. Commercially available efficacious prophylactic vaccines include the bivalent Cervarix   as well More than 10 years have elapsed since HPV vaccination was implemented, and a systematic review of HPV vaccination programs in 14 countries that included over 60 million people vaccinated presented evidence of vaccine efficacy . In compHowever, there is still an urgent need for the development of therapeutic HPV vaccines to tackle existing HPV infections, prevent the development of cancer, and act as immunotherapies for HPV-associated malignancies.Many different technologies have been utilized to develop therapeutic vaccines and most of them target E6 and/or E7 oncoproteins of HR HPV because they are constantly expressed in HPV-associated cancer . It is wProtein- or peptide-based vaccines have been also evaluated and tested in clinical trials with some interesting outcomes for synthetic long peptide-vaccine in early stages of HPV carcinogenesis ,43,44. MNowadays, nucleic acid therapeutics accounts as promising alternatives to conventional vaccine approaches. Once a DNA vaccine has reached the nucleus of a myocyte, a primary keratinocyte, or a resident antigen presenting cell (APC), the expressed antigen gene is processed by cell machinery. Cross or direct priming of DC produces the presentation of the antigen within the class I or II major histocompatibility complex (MHC) on their surface  for immuIn addition, DNA vaccines are characterized by ease of production and high stability. Their safety and use in different administrations without losing their efficacy make them the ideal treatment for the control of HPV infections and associated diseases ,52. DNA Many different HPV DNA vaccines have been constructed and proven to be active in pre-clinical models and few of them are in clinical trials (for review see ). In parVGX-3100 has the potential to be the first approved treatment for HPV infection of the cervix and the first non-surgical treatment for precancerous cervical lesions. VGX-3100 works by stimulating cellular and humoral responses against HPV 16 and HPV 18 E6/E7 oncogenes.Another DNA vaccine with potential clinical use is the GX-188E . This vaccine consists of a tissue plasminogen activator (tpa) signal sequence, an FMS-like tyrosine kinase 3 ligand (Flt3L), and shuffled E6 and E7 genes of HPV type 16/18. Flt3L and tpa are included in the fusion gene to promote antigen presentation and trafficking of the fused protein to the secretory pathway, respectively . Recentl® vaccine. CureVac’s RNActive® cancer vaccines (CV9103 and CV9104) have successfully completed Phase I/IIa clinical studies in prostate cancer and non-small cell lung cancer [The use of mRNA has several beneficial features: (i) Safety: there is no potential risk of infection or insertional mutagenesis; (ii) efficacy: some adjustments make mRNA stable and highly translatable; (iii) production: mRNA vaccines have the potential for fast, cheap, and scalable production, essentially because of the high yields of in vitro transcription reactions . RNA-basg cancer ,60.Recently, this technology has been used to develop prophylactic vaccines for infectious diseases such as COVID-19 due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . UnfortuThe innate immune system has a key role in triggering an active adaptive T-cell response in the initial phase of in vivo priming . Thus, mToll-like receptor (TLR) agonists, , have bBacteria- such as IFA and Montanide or virus-derived molecules are the most utilized adjuvants. Bacille Calmette-Guerin (BCG) is utilized as a therapeutic vaccine and its adjuvant activity is mediated mostly by TLRs. On the contrary, TLR independent adjuvant activity can be elicited by cytosolic nucleic acids secreted by bacteria. Cyclic dinucleotides (CDNs) may activate stimulator of interferon genes (STING) that activates TANK-binding kinase 1/interferon regulatory factor 3 (TBK1/IRF3), nuclear factor κB (NF-κB), and signal transducer and activator of transcription 6 (STAT6) signaling pathways causing type 1 Interferon (IFN 1) and proinflammatory cytokine activation in response to deviant host cells  or cytosolic double stranded DNA (dsDNA) from pathogens ,71. IndeSystemic adjuvants are represented by cytokines and monoclonal antibodies. However, conflicting results were reported in human cancer for cytokines ,74,75 orNicotiana benthamiana plant extracts as well as Chlamydomonas reinhardtii, a well-characterized unicellular alga, and hairy root cultures may display adjuvating activity in cancer vaccines significantly eliciting type 1 helper T cells (Th1) and cytotoxic T-lymphocyte (CTL) responses [® peptide, a self-assembling domain of the maize gamma-zein seed storage protein. This peptide is able to target recombinant proteins to endoplasmic reticulum and to determine their accumulation as protein bodies (PBs). These PBs induce stronger immune responses compared to the soluble recombinant proteins. Zera® peptide has been either fused or combined  to a harmless shuffled HPV 16 E7 (16E7SH) synthetic protein. Significantly higher humoral and cellular immune responses to E7 were induced either as ZERA-16E7SH fusion protein or as Zera® PBs mixture with 16E7SH compared to 16E7SH alone. This effect is supposedly determined by a more efficient antigen presentation by PBs and suggests that Zera® may act as an adjuvant [Plant extracts are emerging as new adjuvant compounds in cancer vaccines. esponses ,77,78,79esponses . Adjuvanadjuvant .Thus, different plant components may exert common potentiating activity on therapeutic vaccines, which further strengthens the plant-based platforms as useful tools for vaccine preparation.Even if significant progress was made against HPV disease through preventive vaccination, and despite the success of experimental therapeutic vaccines, early and efficient treatment of HPV cancers is still a challenging issue. For this reason, an active area of research has involved and still considers plants as a source of potential pharmaceutical agents for treatment of HPV-associated tumors. A well-known example is genital warts, that can be treated with plant-based anticancer therapies, such as vincristine, vinblastine, paclitaxel, camptothecin, and podophyllotoxin .Indeed, plant-derived compounds represent about 75% of the whole approved anti-tumor drugs, as either natural products themselves or as molecules mimicking or directly deriving from natural sources . In manyMany plant-derived compounds possess the specificities of ideal chemopreventive agents, with no effect on normal cells, bioavailability, multiple mechanisms of action, easy manner of administration and significant cellular effects in combating oncogenesis, as they may prevent carcinogens from reaching their targets, inhibit malignant cell proliferation, to modulate tumor suppressing agents and immune surveillance ,84.Moreover, some of them (like polyphenols) have a prominent role in neutralizing reactive oxygen species, that are well-established messengers in intracellular signaling inducing oncogenesis and as genotoxic damage inducers . As an eDespite the significant advantages of high specificity and low toxicity of plant-derived compounds as anti-cancer agents, main drawbacks can be the rapid catabolism and the low bioavailability at the target site. Combination with existing drugs, to reach synergistic effects, or the use of nanoformulation of polyphenols, to prevent their degradation, have showed promising results .Phytochemicals, as either purified and characterized entities , or extracts and mixtures composed by different herbal derivatives, were, indeed, the first compounds to be used in the search of tools able to tackle cervical cancer even before its etiology was discovered and since HeLa (HPV 18 positive) cells were developed in 1952 ,91.It has been shown that anti-cervical cancer drugs can be found in several ethno-botanical sources and there is a wealth of plant extracts that were described to have HPV-related effects (for a very exhaustive list see ). NevertIn the following sections, some studies describing effects of phytochemicals or extracts on HPV cancer cells in vitro and in vivo will be described. A particular mention for those directly affecting HPV E6/E7 activity or displaying adjuvant properties in combination with chemo- or radiation therapies will be deserved.Several in vitro studies mainly focused on purified phytochemicals and, in particular, on polyphenols, among which mainly flavonoids, as well as on other chemical species such as alkaloids, polysaccharides and protein-based entities, along with plant extracts .Polyphenols are a heterogeneous group of chemical substances known to have safe preventive and anticancer effects and ability to specifically target viral oncogenes . It is iAnthocyanins  are mainly encountered in berries, currants, eggplant, grapes, and black rice. The feature of anthocyanins, as many tumor cells growth inhibitors, is related to their ability to induce tumor cells apoptosis and neutralize ROS . In part(-)-Epigallocatechin-3-gallate (EGCG)  extracted from green tea, can contrast tumor cell growth interfering with tumor-related angiogenesis and propensity to metastasize. EGCG was demonstrated also to have a possible gene regulatory role. In the case of HPV, it was shown in vitro that CaSki (HPV 16 positive) cervical cancer cells are guided to apoptosis by EGCG with cell cycle arrest in the G1 phase. Furthermore, in vivo anti-HPV tumor effects of EGCG were also observed .The mechanism found for EGCG suppression of HPV-related cancer cell lines is the inhibition of HPV E6/E7, estrogen receptor α, and aromatase expression by apoptosis in a time-dependent manner ,97. IndeIn HeLa cells, EGCG abrogated HDAC1 activity, also affecting expression of retinoic acid receptor-β, cadherin 1, and death-associated protein kinase-1 , showingA genome-wide study reported that EGCG affects also DNA methylation in oral squamous cell carcinoma (CAL-27) . BesidesApigenin , the main plant-derived bioactive flavone, is abundant in common fruits and vegetables such as parsley and celery, onions, oranges, wheat sprouts, and chamomile . ApigeniCompositae family. This compound was demonstrated to inhibit the function of E6 and E7 oncogenes by impairing binding of these oncoproteins with p53 and pRb and making them non-functional [Jaceosidin  was first isolated in plants of the nctional .Luteolin  is most often found in leaves and its sources include celery, broccoli, green pepper, parsley, thyme, perilla, chamomile tea, carrots, olive oil, peppermint, rosemary, oranges, and oregano. It was demonstrated to induce apoptosis in HeLa cells . LuteoliScutellaria baicalensis, was found to promote apoptosis through suppression of E6 and E7 and increase in p53 and pRb in SiHa (HPV 16 positive) and CasKi human cervix tumor cells [Wogonin , accumulating in the plant or cells .Curcuma with a wide spectrum of pharmacological properties among which are anti-inflammatory and antioxidant activities by inhibiting lipo-oxygenase and cyclo-oxygenase [c-fos and fra-1 [Curcumin  is a hydrophobic polyphenol derived from the rhizome of xygenase . Downregxygenase ,124,125 xygenase . In HeLand fra-1 .The cytotoxic potential of diarylpentanoids, curcumin analogues, was evaluated in HeLa and CaSki cervical cancer cell lines as an improved alternative to curcumin. In particular, the MS17 analogue 1,5-Bis(2-hydroxyphenyl)-1,4-pentadiene-3-one exhibited cytotoxic, anti-proliferative, and apoptosis-inducing potential. Apoptosis was sustained by activation of caspase-3 activity in CaSki cells. Quantitative real-time PCR also detected significant down-regulation of HPV 18- and HPV 16-associated E6 and E7 oncogene expression following treatment .Salvia species, downregulates E6 and E7 and trigger apoptosis, inhibiting growth of HeLa, SiHa, CasKi, and C33A cells [Tanshinone IIA (abietane-type diterpenoid) from 3A cells .Berberis species and shows anti-inflammatory and anti-cancer properties with no apparent toxicity. Berberine interferes with of E6, E7, p53, pRb, and c-Fos expression, ultimately leading to the inhibition of cervical cancer cells growth [Berberine  mainly accumulates in plants of the s growth . Furthers growth .Withania somnifera induces apoptosis of CasKi cells through E6 and E7 repression determining inhibition of tumor growth [Withaferin A (Steroid Lactone) from r growth .Other secondary metabolites, together with polysaccharide, lectin, and protein/peptide fractions that are not described in the text are listed with their HPV-related activities in Compared to purified phytochemicals, few studies are available on the anti-carcinogenic effects of crude or partially fractionated extracts or mixtures composed by different herbal derivatives.Pinellia pedatisecta, for Bryophyllin A-rich leaf fractionated extracts from Bryophyllum pinnata, for fruit extracts from Phyllanthus emblica and for oils extracted from Brucea javanica [Inhibition of AP-1 and STAT3, known to induce cervical carcinogenesis, and specific down-regulation of viral oncogenes E6 and E7 expression have been demonstrated for rhizome extracts from javanica ,141,142.Emblica officinalis and Aloe vera extracts and Mentha citrata oil [Azadirachta indica, Emblica officinalis, and Aloe vera mixed to Mentha citrata oil, have been shown able to block transfer of HPV16 pseudovirions in HeLa cells [Two botanical formulations called, respectively, “Basant”, made of purified curcumin and saponins mixed to rata oil , and “PrLa cells ,152.Cudrania tricuspidata stem extract were evaluated in HPV-positive cervical cancer cells  and HaCaT human normal keratinocytes. This extract showed dose-dependent cytotoxic effects in cervical cancer cells with no cytotoxic effect on HaCaT keratinocytes at concentrations of 0.125–0.5 mg/mL. The extract induced apoptosis by down-regulating the E6 and E7 viral oncogenes in SiHa cervical cancer cells. Its mechanism of induction of apoptosis was exclusively based on the increase of mRNA expression of extrinsic factors  and on activation of caspase-3/caspase-8 and cleavage of polyADP-ribose polymerase. No effects on intrinsic pathway molecules such as Bcl-2, Bcl-xL, Bax, and cytochrome C were observed. These results suggest that this extract can be used as a modulating agent in cervical cancer [The anti-cancer effects of l cancer .Ficus carica fruit latex were explored onto cervical cancer CaSki and HeLa cell lines. Data show that latex inhibits rapid growth and invasion and downregulated the expression of p16 and HPV onco-proteins E6, E7 [The in vitro biological activities of s E6, E7 .Although prophylactic vaccination represents the most effective method for cervical cancer prevention, chemotherapy is still the primary invasive intervention against HPV cancer lesions. It is urgent to exploit low-toxic natural anticancer drugs on account of high cytotoxicity and side-effects of conventional agents. Moreover, the resistance of cervical tumor cells to chemo- and radiotherapy is one of the crucial problems in the treatment of cervical neoplasia, leading to decreased efficacy or failure of the therapy. In this field, the combination of natural compounds with chemotherapy and radiotherapy in the treatment of cervical cancer was reported to improve in some cases sensitization of HPV cancer cells and to minimize the toxicity of these therapies .Curcumin was demonstrated to sensitize cervical cancer cells to taxol- or cisplatin-induced apoptosis by down-regulation of NF-kB ,167. SimBupleurum falcatum), wogonin, and apigenin sensitized cervical cancer cells to cisplatin by sensitizing HPV-related cancer cells to paclitaxel-induced apoptosis through intracellular ROS accumulation [Apigenin showed synergistic effects with paclitaxel improving apoptosis rates of HeLa and SiHa cancer cells . Quercetmulation ,155,156.mulation .The combination of tea polyphenols with EGCG and bleomycin demonstrated to have therapeutic effects on cervical cancer. Bleomycin is an anti-neoplastic chemotherapeutic used in redox-related cancer, including cervical squamous cell cancer, that cause severe side effects in normal cells such as immune system damage, pneumonitis, and pulmonary fibrosis, which are mediated by redox status disturbances. The combination therapy induced stronger cancer cell apoptosis ability than treated either tea polyphenols or bleomycin alone, by activating caspase-3, -8, -9, and up-regulating the expressions of p53 and Bcl-2 .A few phytochemicals have shown radiosensitizing effects. Resveratrol , genisteClinical trials focused on the anti-HPV carcinogenic effects of natural compounds were less frequent than in vitro and in vivo studies . EGCG anThe clinical efficacy of EGCG and other green tea compounds  delivered orally ± vaginally to patients with HPV cervical lesions was evaluated. In this study, a 69% clearance rate was detected in the treated patients as compared with a 10% response rate in untreated controls, showing that green tea compounds can be effective in the treatment of HPV-related cervical lesions .Green tea extracts components  were also tested in patients with HPV mild, moderate, and severe dysplasia. One of these studies recruited 90 patients. Components were applied as either ointment or oral administration. In particular, polyphenon E was applied as a topical ointment to 27 patients twice a week. On the other hand, 200 mg of polyphenon E or EGCG were delivered orally on a daily base for eight–twelve weeks.p < 0.05) [Despite differences between ointment and capsules, giving indication of a higher efficacy of ointment with respect to capsule administration, overall, 69% of patients (35/51) showed a response to treatment with green tea components, compared to a 10% response rate (4/39) of untreated patients ( < 0.05) .Evaluation of curcumin in clinical settings was started with a phase I clinical testing of oral administration of 0.5–12 mg of this compound for 3 months. The main result of this study was to define the safety of oral administration of curcumin as up to 8 mg/day and its bioavailability and efficacy in determining histological improvements in 1 out of the 4 patients .Subsequently, a phase I/II clinical trial demonstrated that intra-vaginal administration of either curcumin capsules or Basant cream in HPV-infected women (without high grade CIN), was able to induce higher infection clearance rate  than untreated patients (73.3%) . Basant Intra-vaginal administration of Praneem for thirty days to 10 HPV-infected women with low-grade squamous intraepithelial lesions determined the clearance of HPV16 in 60% of patients. Another round of administration was able to induce HPV clearance in 50% of the patients that had not been able to eliminate HPV upon the first treatment . It was Previous sections mentioned a non-exhaustive list of plant compounds, extracts, and formulations of different origin and source tested against HPV cancers at various levels. Although many products are in the list, many of these entities need to be further clarified in terms of the role played in cytotoxicity in cancer once translated into clinic and of possible adverse effects in healthy cells. These tasks might be particularly complex to fulfill in the case of multi-component extracts. Indeed, the role of single constituents has to be investigated to evaluate the potential of the plant products as safe and effective anticancer agents.It is clearly desirable that compounds showing cytotoxic activity in vitro against HPV cancer cell lines have also anti-cancer effects and strong tumor-selective action once tested in clinical trial. Unfortunately, not all the studies demonstrating in vitro efficacy of plant substances on HPV-related cell lines  have been translated in in vivo or clinical studies, as already mentioned. The lack of clinical studies is clearly a limitation for the future clinical application of plant compounds. Despite this, it can be said that specifically HeLa and CaSki cells are models commonly used in in vitro cervical cancer research, since they contain the HR HPV types 18 and 16 viral genomes respectively, that are causing seven out of ten cases of invasive cervical cancers. Therefore, cytotoxicity of plant compounds, demonstrated by means of these cell lines in vitro, can be considered truly relevant in view of a clinical application as anti-HPV cancer agents. Moreover, plant products exerted cytotoxic effects on these cell models through specific targeting of E6, E7, and/or other hallmarks of HPV carcinogenesis. Thus, they have even more significance for a possible translation in clinical trial.Ficus carica latex was reported to inhibit the growth of CaSki and HeLa cells without a cytotoxic effect on human keratinocytes cell line (HaCaT) [Pinellia pedatisecta, an enhancement of antitumor T-cell responses by restoring tumor-associated dendritic cell activation and maturation was shown [Cudrania tricuspidata stem extract was investigated both in HPV-positive cervical cancer cells and in normal HaCaT keratinocytes. The extract showed significant dose-dependent cytotoxic effects in cervical cancer cells and no cytotoxicity on HaCaT. In addition, it was demonstrated that the extract-related apoptosis was induced by down-regulating the E6 and E7 viral oncogenes [In view of clinical administration, special attention should be paid also to the issue of possible unwanted toxicity exerted by some phyto-compounds toward normal cells. Clearly, compounds with selective cytotoxic effects on cancer cells should be preferred. Undoubtedly, literature reporting comparative data on cytotoxicity of plant compounds on cancer and healthy cells should be expanded by further studies. Nevertheless, selective cytotoxic effects and doses on cancer cells have been already demonstrated for some purified compounds and extracts. For dietary compounds such as flavonoids, general safety and selective tumor cytotoxicity have been already well established . Recentl (HaCaT) . For othas shown . Interesas shown ,112,125 ncogenes .A strategy to avoid possible adverse effects in normal cells and to improve bioavailability of plant compounds might be to induce a confined action by tumor-targeting strategies. Nanoparticles of different types, such as multi-functionalized, magnetic or solid lipid particles, dendrimers, liposomes and micelles, some of which are already FDA approved, have been used for targeted delivery of plant compounds without disturbing the physiology of normal cells . NaringeAdjuvant activity is a conceptual issue that, in a broad sense, can be applied to any procedure/compound able to improve vaccine effectiveness. Plants and derived molecule/sequences can offer some solutions based on what we already know about their anti-cancer and apoptotic activity as well as pro-immune properties. A summary of these activities is reported, highlighting the possible adjuvant effect for HPV DNA vaccines.Although not commercially available yet, genetic vaccination represents a convenient platform with respect to traditional approaches that involve the production and purification of proteins or components of the pathogen. Owing to the ease of preparation, intrinsic safety and stability, DNA vaccination is useful for the quick assay of new synthetic immunogens as well as ensuring the induction of antibodies against conformational epitopes of interest.Many strategies have been developed to enhance DNA vaccines effectiveness including codon optimization, particular methods of transfection , addition of adjuvants or genetic fusions with immune-stimulating sequences, combination with heterologous boosts, etc.Several genetic vaccines are in an advanced clinical trial phase for the treatment of HPV-associated malignancies and represent a potential additional weapon to those already available, like chemotherapy and radiotherapy. In this case, improvements of HPV genetic vaccines have focused on (1) increasing DC uptake of HPV antigens, (2) DC processing and presentation of HPV antigens, and (3) enhancing DC and T-cell interactions .The fusion of HPV antigens with immunostimulatory sequences to achieve improvement in their “visibility” to the immune system, has been an approach very often explored in the development of experimental therapeutic vaccines . NeverthIn an attempt to potentiate a genetic vaccine for HPV, the attenuated E7 gene (E7GGG) of HPV 16 was fused with the gene encoding the coat protein of a plant virus  ,177.It was subsequently shown that the inclusion of sequences deriving from plant proteins and peptides with immune-modulatory and anti-cancer activity  can poteAmong these proteins, some “ribosome inactivating protein” (RIPs) might be included. RIPs show a regulatory and defensive role against pathogens and accumulate in various organs of many plant species ,180,181.Saponaria officinalis but rendered no longer catalytically active through mutagenesis (SAP-KQ), potentiates antitumor activity against E7-expressing tumors. The anti-tumor activity was associated with enhanced antibody and cell-mediated immune responses and antigen-specific delayed-type hypersensibility (DTH) [Our results indeed support this hypothesis. We demonstrated for the first time that a DNA vaccine involving the fusion of the HPV 16 E7GGG gene with saporin (SAP), a RIP found in ty (DTH) .SAP-KQ/E7GGG fusion proteins may undergo rapid degradation via the ubiquitin-proteasome pathway, as postulated also in the case of coat protein of PVX fused to the C-terminus of E7GGG . Indeed,Then, we designed a novel genetic fusion vaccine comprising synthetic genes derived from the E7 and L2 proteins of HPV 16. Here, a signal peptide derived from a plant protein was fused upstream the N-terminal portion of the papillomavirus fusion antigen . Signal Phaseolus vulgaris, was able to target the HPV 16 E7 protein to the plant secretory compartment, enhancing the E7 protein expression level at least five-fold compared with the unfused version of the antigen [In a previous work, we had shown that the ss of the polygalacturonase-inhibiting protein (PGIPss) from  antigen . Startin antigen . Further1–200) and E7GGG of HPV 16, and cloned in a mammalian expression vector (pVAX1) (1–200-E7GGG) was delivered in C57BL/6 mice according to a prime/boost schedule, implying the use of electroporation (EP) after intra-muscular immunization.With the idea to develop a HPV preventive/therapeutic vaccine, the DNA sequence for PGIPss was fused upstream to a synthetic codon-optimized gene consisting of a cross-reactive epitope of the L2 protein  . The chi1–200 and E7 that persisted for at least six months upon immunization in the mouse model used together with a cell-mediated immune response [The immunization protocol, performed together with EP, induced a long-lasting humoral IgG immune response against L2response . Electroresponse ,191.post-mortem analysis alone, representing complementary approaches for monitoring tumor progression and treatment response in orthotopic preclinical studies. In addition, the bioluminescent AT-84 HPV-16 E7-Luc model, generates data that can be compared with those obtained by caliper measurements and allows earlier cancer detection  [Furthermore, the new DNA vaccine was able to determine an effective tumor regression in vivo in two mouse models: the TC-1 subcutaneous model in C57BL/6 mice and the AT-84 E7 orthotopic oral model in C3H/HeJ mice . The AT-ectable) .1–200-E7 DNA vaccine was delivered intra-muscle with EP in two different animal models of HP-associated tumor, resulting in a dramatic reduction of tumor growth [1–200-E7 administration induced a specific cell-mediated immune response against HPV E7 tumor antigen. Further, the ability of the PGIPss to evoke antibody response in mammalian cells was confirmed in this system [The pVAX-PGIPss-L2r growth . Antitum1–200-E7 genetic vaccine represents a promising candidate against HPV-associated cancers and opens novel perspectives in the design of vaccines for other antigens and/or for different pathogens, in particular against infections where a fast and protective immune response is required (as in the current SARS-CoV-2 pandemic). Moreover, these vaccines, which can be easily produced on an industrial scale, under good manufacturing practices (GMP) conditions, represent safer vaccines as they do not involve the production of chemo/cytokines (which could induce secondary responses) or animal antigens (which could cause autoimmune cross-reactive responses).As far as we know, this represents the first demonstration that a plant-derived signal sequence has biological activity in mammalian cells, increasing the immunogenicity of an antigen of interest. The chimeric PGIPss-L2The combination of chemotherapy with immunotherapy (including DNA vaccination) might represent a potential strategy for cancer treatment because certain chemotherapy-based cancer treatments may activate the immune system against the tumor through several molecular and cellular mechanisms and reduce tumor .It has been described that some natural molecules have the ability to increase vaccine-induced immunity against cancer. Furthermore, bulky tumor growth was inhibited by EGCG in combination with a genetic vaccine  while a Nevertheless, very few works describe the effects of combined used of natural compounds with HPV genetic vaccination.G. uralensis polysaccharides were investigated in combination with HPV DNA vaccination [G. uralensis polysaccharides injection had no side effect on mice, enhancing the immunity of mice and the antigen-specific cellular and humoral immune responses induced by HPV DNA vaccine.The immunomodulatory properties of cination . G. uralCrocus sativus L.) and its components, like monoterpene aldehyde and carotenoids derived from dry stigmas, have been suggested as favorable candidates for cancer prevention. The potential use of saffron derivatives such as extracts or purified components-carotenoids derived from dry stigmas of pure saffron alone or combined with genetic vaccination, was investigated on HPV-related experimental tumor [Saffron  DNA. Crocin, picrocrocin, and saffron extract were given orally at the time of the initial DNA immunization and for the next 16 days. While the multimodal treatment using DNA vaccine along with picrocrocin augmented the anti-tumor effects of picrocrocin, the combination of DNA vaccine with saffron extract and crocin at certain concentrations could not potentiate protective and therapeutic effects compared to mono-therapies for the control of TC-1 tumors. In particular, oral administration of crocin resulted in complete tumor regression, while it did not increase DNA vaccine-mediated anti-tumor effects. These data are apparently in contrast to those from other studies about combination of chemotherapy with EGCG , apigeniTogether with evidences of efficacy and safety, the pharmacological use of plant-derived compounds could benefit, in many cases, from their wide availability in nature or by in vitro plant cell/tissue cultures. Certainly, chemically defined and purified entities are more suited and have higher potential as therapeutic compounds against cancer (and HPV-associated pathologies are no exception). All other plant derivatives, such as extracts and mixtures, may suffer from batch to batch variation.In addition to this, studies to further investigate the encouraging promises of plant-derived compounds and formulations in advanced clinical trial phases, having large numbers of subjects, are needed. These studies should open the way to a deeper evaluation of the synergistic effects of plant compounds with canonical HPV tumor therapy approaches in combinatorial schedules, probably the most feasible and intriguing application of plant-derived compounds in this field.The use of therapeutic vaccines against HPVs has been shown to induce regression of precancerous lesions of cervix and produce some clinical benefits in cancer patients. The immunosuppressive effects of the cancerous microenvironment can be attenuated by multimodal therapeutic approaches by combining the therapeutic vaccine with radiotherapy, chemotherapy, immunomodulators, and immune checkpoint inhibitors. In fact, experimental clinical evidence has clearly highlighted a synergistic action of these combinatorial approaches .Therapeutic vaccines for pre- and neoplastic lesions of the uterine cervix are becoming a reality and the improvement of all therapeutic strategies associated with a multimodal approach opens a new scenario in the treatment not only of cervical cancer and pre-neoplastic lesions but also of other HPV-associated tumors. Despite to date, no therapeutic vaccine has been approved for clinical use in the treatment of HPV infections and related malignancies, it should be noted that at least some DNA vaccine candidates such as VGX-3100 and GX-188E or ADXS11-001 bacterial vector vaccine are in phase III clinical trials demonstrating that a therapeutic vaccine is in the near future . In addition, therapeutic vaccines could be produced with plant expression systems that make production less expensive, expanding the possibility of their use also in low-middle-income countries where the disease burden is greater. However, these types of vaccines are still in an early stage of experimentation which makes them currently unavailable for clinical translation.Even for DNA vaccines, their clinical use still requires a better knowledge of the mechanisms through which they are able to induce specific immune responses in vivo.DNA vaccines can be introduced into the body by intramuscular, intradermal, or mucosal delivery through a variety of technologies, and most of them have been shown to be safe for humans despite causing varying degrees of discomfort. Furthermore, such therapeutic vaccines should not cause the activation of the immunosuppressive population of regulatory T lymphocytes (Treg). To achieve these results, vaccines and in particular DNA vaccines need to be associated to adjuvating procedures, including modification of antigen by fusion with other molecules or modifying the fate of antigen processing or host immune response .In an effort to make easier the transfer of adjuvated DNA vaccine from pre-clinical studies to clinical trials, it should be conceivable to use compounds and technologies already used in humans for other clinical indications. In this sense, building a DNA genetic vaccine using vectors already used in humans and with few antigen modifications can have a preferential route for their use. However, it is also important to address concerns regarding the potential for oncogenicity associated with administration of oncogenes  as DNA vaccines into the body. In response to that, all oncogenes in DNA vaccine are harmless version or shuffled epitopes ,198.If we can assume that DNA vector and modified antigen sequences are relatively safe, the same cannot be said for adjuvant activities. Such activities often involve molecules interfering with multiple pathways. This makes it difficult to directly use adjuvants in human experimentation without going through long toxicology phases in animal models. In this panorama, plant-based compounds, often derived from common food products  offer an undoubted advantage regarding their non-toxicity and, in addition, they often have a millennial use in traditional medicine. Other natural products like apigenin were already tested in several clinical trials and proven to be safe in humans ,200.Therefore, the study of plant compounds with adjuvant activity  for genetic vaccines is a field of research that requires expansion in order to obtain vaccine products that can be used more quickly in humans. Finally, these studies will also be the basis for helping Regulatory Agencies dealing with drugs for human use by providing the necessary knowledge for an assessment of their human use.Many efforts have been made to find and produce DNA therapeutic vaccines against HPV-associated lesions and cancers. The most effective vaccines in pre-clinical animal models are in clinical trials and two of them (VGX-3100 DNA and ADXS11-001) are in phase III clinical trials with promising results. Nevertheless, these vaccines are more effective in pre-cancerous lesions  than in cancer indicating that there is need of adjuvants to overcome the immunosuppressive tumor microenvironment. Plant sequences and also other genes from the “green world,” such as plant-virus genes and signal sequences could be the answer considering their safety and avoidance of any autoimmune or pathogenic response.In addition, plant compounds that directly abrogate HPV E6/E7 activity are feasible candidates for a HPV phytotherapy, as they affect the major HPV “oncoplayers.” Purified phytochemicals, due to easy extraction and batch to batch consistency, will offer major perspectives to a comparatively safer alternative/combinational approach to HPV current therapy, once their efficacy in clinical trials will be confirmed. A summary of these new therapeutic strategies implying interconnections among DNA vaccines, plant compounds, and plant genes is depicted in Thus, more studies are to be performed to check the effectiveness of these new therapeutic strategies including combination with existing approaches. Nevertheless, preliminary results open new horizons for the therapy of HPV-associated cancers. In addition, other tumors, where patient-specific neoantigens are detectable as a consequence of tumor-specific mutations, might benefit from these approaches .Franconi R, Massa S, Venuti A. (2016). Plant protein signal sequence as adjuvant in DNA vaccines. Italian patent 102016000131935, PCT/IT2017/050008, WO 2018/122885 A1. European Patent Application 17826321.6, publication number EP 3,562,504 (EP’504).


The cervical cancer burden in Uganda is high amidst low uptake of HPV vaccination. Identification of individual and community factors associated with HPV vaccination are imperative for directed interventions. Conversely, in most Low and Middle Income Countries (LMICs) including Uganda this problem has not been sufficiently studied as the influence of individual and contextual determinants remains undetermined in spite of their substantial effect on HPV vaccine uptake. The aim of the study was to identify individual  and community (socioeconomic disadvantages) factors associated with HPV vaccination.Based on a modified conceptual framework for health care utilization, hierarchical modelling was used to study 6093 girls, aged 10–14 years (level 1), nested within 686 communities (level 2) in Uganda by analyzing data from the 2016 Uganda Demographic and Health Survey.Majority (78%) of the girls had not been vaccinated. A number of both individual and community factors were significantly associated with HPV vaccination. The Odds of HPV vaccination were higher among girls age; 11, 13, and 14 compared to girls age 10 years, attending school compared to girls not attending school, who were; foreigners, Iteso, Karamajong, Banyoro, Basoga, and other tribe compared to Baganda, living in families with 1–8 members compared to those living in families with 9 or more members and middle social economic status compared to poor wealth quintile.Both individual and community factors show a noticeable effect on HPV vaccination. If higher vaccination rates are to be achieved in Uganda, these factors should be addressed. Strategies aimed at reaching younger girls, street children, out of school girls, and girls with lower SES should be embraced in order to achieve high vaccination uptake. Worldwide, cervical cancer is the fourth most common type of cancer with 528,000 new cases annually, after lung cancer 583,100 cases), colorectal cancer 614304), and breast cancer  [00 cases,304, and Sub-Saharan Africa has the third highest incidence 17.5%) of cervical cancer cases after India (17.7%) and East and Central Asia 18.2%). The region shares the second largest number of global cervical cancer deaths 21.6%) after India (25.4%). It is the only region where cervical cancer is equivalent to breast cancer with each constituting a quarter of the global cancer burden [%. The re% after I.5% of ceUganda is among the five countries with the highest cervical cancer incidence rates in the world. It is the most commonly diagnosed cancer and has the highest incidence of malignancy and mortality among women . The couMajority of the cervical cancer cases are potentially preventable. World Health Organization (WHO) and Uganda’s Ministry of Health (MOH) recognize primary prevention of cervical cancer i.e. preventing the initial onset of cervical cancer by vaccinating girls aged 9–14 years before exposure to sex/ HPV as a very important factor in the prevention of cervical cancer –12. In 2In Uganda, HPV vaccines against HPV 16 and 18 have been available since 2006 . The breA number of studies have examined the predictors of HPV vaccination –23. MostThe study used secondary data from the 2016 Uganda Demographic and Health Survey (UDHS). Permission to access the UDHS data was sought from Measure DHS . The UDHThe outcome variable “HPV vaccination” was measured using the question: “has (name) ever had HPV Vaccine to prevent cancer?” (No/Yes). This question was asked eligible household respondents who were parents or guardians of girls age 10 to 14 years.Individual and community characteristics that were examined for possible associations with HPV vaccination were based on a framework with components adapted from Anderson -Newman behavioral model of health services utilization and Bandura’s social cognitive theory , 31. ThiIndividual-level variables included girls’ age –14, currThe community (cluster) was used as the primary sampling unit (PSU) of the data. The community influence on HPV vaccination was measured by considering the socioeconomic status of the community in which the girls were living. The community socio-economic disadvantage was operationalized by combining two factors: place of residence  and wealth index . These variables were obtained by combining individual answers for each question to the cluster (community) level. The Principal Component Analysis (PCA) was used to generate community wealth quintiles. A number of studies have applied community wealth quintile as a community-level determinant , 33.x2) tests to examine the independent predictors of HPV Vaccination and the level of statistical significance was set at p <  0.05. Data analysis was guided by the framework for health care utilization and the hierarchical nature of the Uganda DHS data. Thus, we used the three-step multi-variable multilevel logistic regression with the log-binomial function of the generalized linear mixed models family [P-values and 95% confidence intervals [We used frequency distributions to describe the demographic and socioeconomic characteristics of the girls. Associations of individual and community level characteristics with HPV vaccination (predicted variable) were investigated using cross-tabulations. We used Pearson’s chi-squared .All data that was used in the study were obtained from the 2016 UDHS. During data collection, written informed consent was obtained from each respondent before the interviews . We obtaThe general characteristics of the study population are shown in Table Table P-value < 0.001) variation in HPV vaccination at community-level. The study findings show that community-level determinants partly account for the total variance in HPV vaccination hence the community-level determinants were sufficiently catered for by the Multivariable Multilevel Regression Analysis (MMLRA). Our variance partition coefficient (VPC) or intra-cluster correlation (ICC) of 0.56 indicate that the communities in which the girls live contribute to 56% of the variation in HPV vaccination. This also suggests that the intra-community correlation amongst girls vis-à-vis the likelihood of HPV vaccination was 0.56.The results of multi-level analysis are presented in Table P <  0.0001). It is worthy to state that a random intercept model was considered rather than the usual single-level model due to the hierarchical nature of the data and to avoid biased associations.The estimated community variance was also presented as median odds ratios (MOR = 0.24) which means that girls from an average community in Uganda had 24% less odds of having already been vaccinated. After the decomposition of HPV vaccination in model 1, level one fixed effects  were added into the empty model to form model 2. The community-level variance increased in model 2 which means that the frequency of individual factors is different in all communities in Uganda. After considering both individual and community-level characteristics in model 3, it was observed that the community-level variance reduced marginally in model 3. This showed that the frequency of community factors is almost similar in all communities in Uganda. After the addition of both individual and community-level factors, the variation in HPV vaccination behavior among communities remained significant. The estimated ICC show that the variability (54%) in HPV vaccination was due to community differences .Table In the final model Table , we inclAccording to the study findings, uptake of the HPV vaccine among Ugandan girls aged 10 to 14 years was low (22%). Although MOH had committed itself to achieve 80% HPV vaccine coverage by 2015 , one yeaThis study established the impact of contextual factors besides individual characteristics on HPV vaccination. Our findings established that the likelihood of HPV vaccination was not solely shaped by individual characteristics, but also communities where these girls were residing. Both community and individual-level factors were significantly associated with HPV vaccination. The study results found a significant negative association of socioeconomic deprivation of communities  with HPV vaccination. The strength of deprivation is determined by those two elements of socioeconomic disadvantages though they don’t coexist together in similar proportions. There is scanty evidence in LMICs regarding the relationship between community level characteristics and HPV vaccination yet findings indicate that community level characteristics strongly predict health care utilization , 30–33. Our study indicate that older girls were more likely to be vaccinated than their one year younger counter parts. These findings are in consonance with earlier studies , 23, 42.The results of this study indicate that School attendance status was positively associated with HPV status. These findings are consistent with prior studies , 22. TheThe current study found that ethnicity was significantly associated with HPV vaccination. This finding is similar to previous studies , 25, 43.Notwithstanding the strength of this study, there were some limitations with the data. Our study was based on cross-sectional and secondary data. The dataset had no variables on mother’s characteristics to facilitate better assessment of mother’s characteristics. The dataset had no information for 9 year old girls to facilitate better assessment of vaccination coverage among girls age 9 years. Second, we combined individual responses to generate our measures at community level. It is therefore difficult to ascertain whether some girls were not classified into wrong administratively demarcated boundaries (clusters). The use of hierarchical regression models require aggregating individual responses to community level assuming that the groups are homogenous. This has potential consequences on the interpretation of results because associations at aggregated levels may not directly apply to individuals but to the group of individuals with in a given area. Nevertheless, the current study points to important programmatic areas of intervention for promoting HPV vaccination in Uganda.This study considered countrywide representative data on HPV vaccination for the 2016 Uganda demographic and health survey. The results of the study established low HPV vaccine coverage in Uganda. Both community (community socioeconomic disadvantage) and individual  level factors were found to be significantly associated with HPV vaccination. Other countries in the region with organized school-based programmes have had much higher uptake rates. If higher vaccination rates are to be achieved in Uganda, both individual and community level factors responsible for variation in HPV vaccination should be addressed. System-wide interventions should be implemented to increase vaccine coverage in Uganda. Our findings point to the need for universal basic education, creation of job opportunities, and poverty alleviation. Our findings further suggest that effort should be directed at women and rural affirmative interventions to narrow gender and type of residence inequality gaps respectively. These are vital interventions that can be implemented at community level to mitigate the effects of community socioeconomic disadvantages. Variation in HPV vaccination among different ethnic groups indicate that communication on HPV vaccine should be tailored to ethnic communities.


The rate of HPV-driven cases was significantly higher among women, never smokers, patients with T1–T2 tumors, and with nodal involvement. A trend was also observed toward an increase in HPV-driven OPSCCs among patients >61 years, women, former smokers, and patients with no nodal involvement in 2015–2019. Our findings consolidate the observation that HPV-associated OPSCCs are also increasing in Italy. Moreover, they suggest that the profile of the HPV-driven OPSCC patient might be changing.Globally, 30% of oropharyngeal carcinomas (OPSCC) are caused by Human Papillomavirus (HPV). Recently, increasing incidence trends for HPV-driven OPSCC have been reported in many countries and changes in the typical HPV-positive OPSCC patient have been recorded, with an increase in the median age and diagnoses in women. We investigated the characteristics of the OPSCC patients attending an Italian Cancer Institute from 2010 to 2019 and assessed possible changes overtime of demographic, behavioral, and clinico-pathologic variables of HPV-driven OPSCC. Overall, 339 OPSCCs were evaluated. HPV-DNA and p16 positivity were 48.7% and 55.2%, respectively, with an HPV-driven fraction  of 48.3%. We observed a significant increase overtime in the rate of HPV-associated cases (53.7% in 2015–2019 vs. 40.3% in 2010–2014,  Head and neck squamous cell carcinomas represent the sixth most common malignancy by incidence worldwide and are mostly caused by tobacco and alcohol consumption . HoweverThe role of HPV in the development of OPSCC has led to essential modifications both in the Classification  and in tIn recent decades, increasing incidence trends for HPV-driven OPSCC have been reported in many countries, particularly the USA ,20,21,22The study included all consecutive cases with a histologically confirmed diagnosis of primary and untreated OPSCC  that were tested for HPV-DNA on formalin fixed paraffin embedded (FFPE) tissue at the Pathology Department of the Regina Elena National Cancer Institute between June 2010 and December 2019.For each patient, the medical records were used to retrieve data regarding gender, age at diagnosis, history of tobacco use, primary tumor subsite, and histopathologic factors. Missing data were due to incomplete medical records. Patients provided a general consent for use of their personal and clinical data for research purposes. The study was approved by the Ethics Committee of the Regina Elena National Cancer Institute .HPV-DNA detection and genotyping were performed using the Inno-LiPA HPV Genotyping Extra or Extra II kit , for the cases diagnosed until 2014 and since 2015, respectively. Both assays are based on the SPF10 primer set used for the amplification of a 65-bp fragment within the L1 region and they detect individually, among others, the high-risk HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, the probably high-risk HPV68, and the possibly high-risk HPV66 [® Histology kit , following the manufacturer’s instructions. The immunostaining, which was independently evaluated by two investigators blinded to the HPV findings, was considered as positive when a strong nuclear expression was observed for >75% of tumor cells [Immunohistochemical staining for p16 was performed using the CINtecor cells .n = 146 OPSCCs) and January 2015–December 2019 (n = 193 OPSCCs). Analyses were performed both overall and according to the period of diagnosis. The following variables were included: age at diagnosis , gender, smoking habits , cancer subsite , tumor classification (T1–T2 vs. T3–T4) and nodal classification. In order to overcome discrepancies due to changes in pathologic classification that had taken place during the study period, we classified the nodal status in two groups: patients with  and without nodal involvement (N0). A chi-square test was used to test the relationship between each variable and the outcome of interest . The Mann-Whitney test was used for comparisons between median values. A p value < 0.05 was considered as statistically significant. Statistical analyses were conducted using the MedCalc Statistical Software v. 19.3.1 .Descriptive statistics were used to summarize the characteristics of the study population . Samples containing any of the 12 high-risk HPVs were considered positive for high-risk types. To analyze the HPV-attributable fraction, only the cases evaluated for both HPV-DNA and p16 staining were included in the analysis. They were considered as HPV-driven when positive for both markers. In order to investigate changes over time, two periods of diagnosis of approximately the same length were taken into account: June 2010–December 2014 . p = 0.054).The median age for the overall population was 61 years with no significant modification over time. In the two study periods, we evidenced no substantial modification in the distribution of cases by cancer subsite, T, N and smoking status, whereas we evidenced an increase, although marginally significant, in OPSCC diagnosis among women , while the increase of p16 positivity showed only a trend towards statistical significance (p = 0.06).Overall, the HPV-DNA and p16 positive cases were 48.7% and 55.2%, respectively, with an HPV-driven fraction of 48.3%. When we considered the HPV-DNA results or the combination of HPV-DNA and p16 over time, we observed a significant increase in the rate of positive cases in the second period compared to the first one . HPV-related cases were more prevalent in women, although the difference by sex was not significant in the first period. Moreover, overall and in each of the two periods, the HPV-driven rate was significantly associated with smoking status, the highest prevalence being observed among never smokers, with T status, being higher in smaller tumors, and with N status, being higher in those with positive lymph-node status. In addition, HPV-driven OPSCC prevalence was significantly associated with cancer subsite when taking into account all the ten-year observation period and in the most recent years, while only a trend towards statistical significance was evidenced in the previous period. Tonsils were always found to be the site with the highest prevalence of HPV-driven cases. Furthermore, we compared the prevalence of the HPV-driven fraction between the two periods according to the variables under study in order to investigate whether changes have occurred over time . The most up-to-date overview of OPSCC incidence trend in our country has reported an increasing trend over the period 1998–2010 . HoweverOPSCC diagnoses were more frequent in men than women, with a M/F ratio of 3.5, in line with the global scenario of a higher burden of this neoplasia among men. Globally, OPSCC diagnoses in men are 4 times more frequent than in women . OPSCC cConcerning T status, we found no significant difference between the two periods, even though we observed an increase in OPSCCs with T3–T4 status in 2015–2019 compared to 2010–2014. This difference may be due to the small number of cases and/or missing data especially in the first period.Overall, the median age of our OPSCC patients was 61 years, and remained similar in the two periods, with no significant changes observed. A slightly higher median age was observed for OPSCC patients diagnosed in a large clinical center in Italy, i.e., 65 years . Similarp = 0.019). These findings confirm other European [Overall, HPV-driven cases were approximately 48%. This figure is lower than those found in Northern European countries, such as Sweden and Denmark, where the HPV-attributable fraction is well over 60%, but it is still high when compared to other Southern European countries ,5,36 andEuropean ,37 and IEuropean , which iRegardless of the period of diagnosis, HPV16 represented the most frequent type detected in HPV-positive cases, with other high-risk types infrequently found, in line with the worldwide scenario for HPV-related OPSCCs . Indeed,With regard to the patients with HPV-driven OPSCC, their median age at diagnosis was 61 years, in line with other countries. In the US, HPV-driven OPSCC diagnoses peaked in those aged 60–64 years . We obseBesides the increase in the overall OPSCC diagnoses among women, we also observed that the proportion of HPV-driven cases among women was significantly higher than that among men, overall and in 2015–2019. In the latter period, for every 10 OPSCC diagnoses in men, 5 were HPV-driven, whereas among 10 diagnoses in women, nearly 7 were caused by HPV. In the international study by Castellsaguè et al., which analyzed over 1,000 OPSCC cases, the HPV-attributable fraction was almost double in women compared to men . SimilarRegarding smoking status, HPV-driven OPSCC cases were significantly more common among never smokers, both overall and in each study period. The lowest prevalence of HPV-related cases was found in current smokers, in line with other studies ,45,46. WWhen the proportions of HPV-attributable cancers in the two study periods were compared stratifying for each variable, the only significant change was found for cancer subsite. In detail, the HPV-driven cases among tonsillar cancers went from 44.6% in 2010–2014 to 66.3% in the following period. An increasing trend that did not reach statistical significance was observed for patients older than 61 years, women, former smokers, patients with T1–T2, T3–T4 and those with no nodal involvement. Specifically, we observed an increase over time in the prevalence of HPV-related OPSCCs in those over the age of 61 years (from 41.5% to 55.8% in the most recent period). The HPV-attributable fraction among women increased from 44.0% in 2010–2014 to 66.0% in 2015–2019, figures that nearly overlap those found in another Italian study, which evidenced a significant increasing trend from 46.2% (2007–2012) to 73.3% 2013–2018) . Differe–2018 . Dp = 0.05), in the HPV-attributable fraction among the patients with no nodal involvement in the second period. These data need confirmation on a prospective larger series of cases.Regarding nodal status, we found an association, stable over time, between HPV etiology and nodal involvement. Indeed, it has been reported that patients with HPV-driven OPSCCs are more likely to have cervical lymphadenopathy, but nodal involvement does not have the same bad prognostic implications observed for HPV-negative OPSCC ,48. InteThis study has some limitations. Firstly, the single-center design means we cannot generalize our findings since they may not reflect the overall Italian landscape of patients with HPV-driven OPSCC. However, the study was conducted at a National Cancer Institute which cares for patients coming from the center and also south of the country. Since Italy lacks a population-based cancer registry, case series from single institutions are important to collect data that would otherwise be lost. The second limit regards the fact that we used smoking status and not pack-years as the smoking variable, due to the absence of detailed information in this regard. In addition, information on this variable was missing for a certain proportion of patients, particularly from the first period. Finally, the sample size for each period may have limited the ability to observe significant associations or differences, resulting in only trends towards statistical significance for certain variables. Moreover, the ten-year study period may not be sufficient to observe significant differences.In conclusion, our study confirmed the established association between cancer-associated variables  and patient characteristics (smoking status) with OPSCC with HPV etiology. Moreover, a significant increase in the HPV-driven cases over time was evidenced, overall and for tonsillar cancers. Finally, in the most recent years, a trend was observed toward an increase in HPV-driven OPSCCs also among patients above 61 years of age, women, former smokers, and patients with no nodal involvement compared to the previous period. Taken together, our findings consolidate the concept that HPV-associated OPSCCs are increasing also in Italy and suggest, at least in part, a change in the profile of the HPV-driven OPSCC patient.


Accurate HPV genotyping is crucial in facilitating epidemiology studies, vaccine trials, and HPV-related cancer research. Contemporary HPV genotyping assays only detect < 25% of all known HPV genotypes and are not accurate for low-risk or mixed HPV genotypes. Current genomic HPV genotyping algorithms use a simple read-alignment and filtering strategy that has difficulty handling repeats and homology sequences. Therefore, we have developed an optimized expectation–maximization algorithm, designated HPV-EM, to address the ambiguities caused by repetitive sequencing reads. HPV-EM achieved 97–100% accuracy when benchmarked using cell line data and TCGA cervical cancer data. We also validated HPV-EM using DNA tiling data on an institutional cervical cancer cohort (96.5% accuracy). Using HPV-EM, we demonstrated HPV genotypic differences in recurrence and patient outcomes in cervical and head and neck cancers. Of these, HPV-associated cervical cancer is the most common primary site, with ~ 500,000 new cervical cancer diagnoses and ~ 300,000 cervical cancer-associated deaths worldwide annually3. Among head and neck cancers, a growing portion of cases are also attributed to HPV in the United States and other more developed countries1. In addition, HPV accounts for a substantial fraction of cancers at other anogenital sites. Currently, > 200 HPV genotypes have been identified, and ~ 15 high-risk HPV genotypes are well-established as causative agents in cancers6. Highly sensitive and accurate HPV detection and genotyping are crucial in facilitating epidemiology studies, vaccine designs, and clinical trials7.Human papillomavirus (HPV) infection is causative for ~ 630,000 new cancer cases globally each year8. Benchmarking using the Roche Linear Array that detects 37 HPV genotypes, the NCI assay achieved a high agreement rate (~ 95%) for samples with a single high-risk HPV genotype, but the agreement rates for samples with low-risk HPV genotypes and mixed HPV genotypes varied (~ 40–90%). Multiple factors could affect the accuracy of array-based HPV detection and genotyping methods, including variability in viral load, consensus primer homology, unbalanced primer sharing, and differential amplification efficiencies8. The National Institute of Allergy and Infectious Diseases (NIAID) has recently developed a papillomavirus genome database (PaVE)6, which has facilitated the creation of HPV genotyping tools using non-array-selected genomic sequencing data10. However, genomic methods that adopt a simple read-alignment and filtering strategy can easily encounter difficulties in handling repeats and homology sequences. Recently, an accurate HPV genotyping algorithm was developed using metagenomic DNA data, which uses a strategy of masking simple repeats and homology sequences10; however, this methodology does not take into account expression of HPV related proteins, including the capacity of some viral strains to express alternatively spliced isoforms of key oncogenic proteins12. To address these shortcomings of currently available HPV genotyping methods, we have focused our efforts on developing an accurate HPV detection and genotyping algorithm using whole transcriptome sequencing (RNA-seq) data.Existing HPV genotyping methods are limited in the capacity to accurately identify HPV genotypes, particularly for rare genotypes. The National Cancer Insititute (NCI) recently developed a new HPV genotyping assay for detecting 51 HPV genotypes10, we designed a novel expectation–maximization (EM) algorithm, designated HPV-EM, to calculate an optimized mapping of RNA-seq reads to HPV reference genomes based on a probability model. Genotyping using RNA-seq data has an advantage over using DNA data, as it enables us to simultaneously measure HPV gene expression. While the read masking strategy in the previous method10 would lose data and so is not suitable for estimating HPV gene expression, our HPV-EM algorithm uses all the available data and accurately estimates HPV gene expression. HPV-EM is able to evaluate HPV gene expression in samples with either single or mixed HPV genotypes based on its accurate genotyping prediction. To facilitate functional analysis for HPV induced cancers, we also implemented an automated visualization tool to show the read coverage for each HPV genotype within a sample. We benchmarked HPV-EM using cervical cancer cell line data, simulated data, and TCGA-CESC data, and validated HPV-EM using an institutional cohort of cervical cancer samples (with both RNA-seq data and DNA tiling array data). To demonstrate its broader applicability, we also applied HPV-EM to TCGA-HNSC cohort. Using this novel tool, we demonstrate HPV genotype-specific differences in recurrence and patient outcomes in both cervical and head and neck cancers. Here, we present the details of the design and implementation our accurate EM algorithm and the benchmarking experiments and applications. The HPV-EM tool is free and open source software and can be downloaded at: https://github.com/jin-wash-u/HPV-EM.To specifically address the ambiguities caused by repeats and homology6 in FASTA format (223 reference HPV genomes and 222 non-reference HPV genomes as of 01/13/2020), but can employ a user-generated set of viral genomes if so desired. Details of downloading and generating these reference genomes can be found at the website of the HPV-EM tool. As shown in Fig. 13, retaining the non-aligned (non-human) reads; (II) Filter low-complexity reads using the DUST algorithm14, retaining all other reads; (III) Align the retained reads from the previous steps to the set of HPV reference genomes using STAR213; (IV) Collect features from the aligned HPV reads, including whether a read is uniquely or ambiguously aligned, the HPV genotypes a read is aligned to, and the number of mismatches and read length; (V) Use the EM algorithm we designed (See the “The HPV-EM tool is designed to detect HPV gene expression and estimate the makeup of HPV genotypes present in a sample that is sequenced using whole transcriptome sequencing (RNA-seq). The input of HPV-EM includes RNA-seq reads in FASTQ format (which may contain both human and HPV sequences) and the path to a directory containing an indexed human reference genome. By default the application will seek reads that can be aligned to an up-to-date collection of HPV reference genomes assembled by PaVEAs discussed above, the set of RNA-seq reads aligned to one or more HPV genomes is passed to an expectation–maximization (EM) algorithm in order to produce a maximum likelihood estimate (MLE) of HPV genotypes in the sample.m reads, denoted as n HPV genotypes  from randomly generated initial values in order to maximize the chance of convergence to the global likelihood maximum, selecting the result with the highest likelihood value. HPV-EM then reports the MLE read counts and probabilities for the HPV genotypes detected within a sample, if any.Due to repetitive and homologous sequences, RNA-seq reads from one sample can often be aligned to multiple HPV genotypes at the same time. The HPV-EM algorithm calculates the maximum likelihood estimate for the proportion of each HPV genotype in a sample. If the sample only contains one HPV genotype, then the algorithm will predict the sample to be 100% of this HPV genotype. If the sample is a mixture of different HPV genotypes, the algorithm will predict the probablity of all the HPV genotypes presented. For example, Figs. 15 for STAR2 in Step III in order to obtain the set of HPV-aligned reads from an independent source. Note that these retained HPV reads still contain repeats that can be aligned to multiple HPV genotypes. We randomly selected a TCGA-CESC sample (TCGA-HM-A6W2) designated by TCGA consortium study16 as HPV-negative, which contains not only human RNA-seq reads but also all other types of reads generated by the current sequencing technology. To accurately spike-in HPV-reads to the intended HPV expression levels, RNA-seq reads in the above three data sets were trimmed uniformly to 48 nt to match the read length of TCGA-CESC samples. We spiked-in SiHa  and HeLa  reads to the HPV-negative sample, at the overall HPV Transcripts Per Million (TPM) levels 0.2, 0.5, 1, 2, 5, 10, 100, and 1,000. For each TPM level, HPV16 and HPV18 reads were randomly selected with the ratios of 0:100, 1:99, 10:90, 25:75, 50:50, 75:25, 90:10, 99:1, and 100:0. Note that some levels  represent less than 1 HPV read and therefore were omitted from the simulation. These simulated samples were processed by HPV-EM using standard settings, except that the TPM threshold was set to 0 to report the presence of HPV reads in all the above abundances . The HPV-EM estimated TPM values and HPV16:HPV18 ratios for each sample were then compared with the known true values and ratios for each simulated sample.We tested the ability of HPV-EM to accurately detect various levels of HPV gene expression and genotypic makeups for samples with single and mixed HPV genotypes by creating a series of simulated data sets using real cervical cancer cell line data. We first downloaded paired-end RNA-seq data for SiHa  and HeLa  cells from the NCBI SRA database and validated their HPV genotypes using HPV-EM. We then retained the HPV reads from the two cervical cancer cell line data sets by running Steps (I)–(III) of the HPV-EM tool .In order to assess the performance of our HPV-EM tool on real data sets, we performed HPV genotyping for the full provisional TCGA cervical cancer cohort of 304 primary tumour samples with paired-end whole transcriptome sequencing data. A subset of 178 of these patients were previously included in the TCGA-CESC consortium study, and the HPV genotypes of these patients were detected using both MassArray and RNA-seq data, with a subset also having whole genome sequencing dataN = 67) were generated in the Genome Technology Access Center (GTAC) at Washington University . HPV genotyping by DNA tiling array was also performed for an expanded cervical cancer cohort of 90 patients (including the 67 with RNA-seq data) and the relationship between HPV genotypes and patient prognosis was examined using prospectively gathered patient clinical information. The DNA tiling array experiment probes were designed for 33 HPV genotypes, covering the whole genomes of HPV16 and HPV18 and the E6 and E7 regions of the other 31 HPV genotypes, as described previously21. On average, 156,114 reads were acquired by the probes for each sample and sequenced using the Illumina HiSeq platform with paired 2 × 100 bp reads, and a threshold of 100 reads was used as the cut off to accurately call an HPV genotype. The HPV genotype results from the tiling array were compared to those generated by HPV-EM among the 67 patients with RNA-seq data. Since HPVDetector is not capable of analysing single-end sequencing data, only HPV-EM was executed and compared with DNA array data.As an additional validation, we executed HPV-EM on RNA-seq data from a cohort of cervical cancer patients treated at Washington University School of Medicine. Cervical tumour samples were collected as part of a prospective tumour banking study (201105374), and data analyses were performed as part of a retrospective analysis study with waiver of consent (202001180). Approval of both studies of Washington University Institutional Review Board (IRB) and waiver of consent of 202001180 by Protocol Review and Monitoring Committee (PRMC) of Washington University was obtained. All methods were carried out in accordance with all relevant guidelines and regulations. These patients were uniformly treated with curative-intent chemoradiation therapy and clinical outcome data were prospectively collected. RNA-seq data  of the 304 primary cervical cancer samples as HPV-positive. Within the 285 HPV-positive samples, 275 (96.5%) have 1 HPV genotype, 8 (2.8%) have 2 HPV genotypes, and only 2 (0.7%) have 3 HPV genotypes. HPVDetector identified 304 (100%) of the primary cervical cancer samples as HPV-positive. Moreover, the HPVDetector identified all 304 samples as having at least low levels of 3 or more (up to 15) HPV genotypes. In Fig. 16 showed that the majority of HPV infections are from a single HPV genotype, this result demonstrates that our EM algorithm effectively removed false HPV genotyping calls caused by repetitive sequencing reads and homologies. In Fig. N = 304), encompassing the previously identified high-risk HPV genotypes. Among these, HPV16 is the most frequent HPV genotype , and HPV18 is the second frequent HPV genotype . The remaining samples consist of HPV genotypes including HPV45, HPV33, HPV52, HPV58, HPV31, etc.  of the primary cervical cancer samples that were included in TCGA consortium study16. For these 178 samples, both RNA-seq and MassArray data were corroborated and compared using TCGA’s in-house strategies to decide the final HPV genotypes. The HPV-EM tool only used RNA-seq data to detect and estimate the HPV genotypes in the same set of samples. In the TCGA consortium study, 169 out of the 178 cervical cancer samples were estimated to be HPV-positive . While HPV-EM identified the same 169 samples as HPV-positive, we compared the HPV genotyping calls against TCGA results. HPV-EM’s prediction is 97% consistent (i.e. 164 out of 169) with the result of TCGA consortium study as a gold standard. The 5 cases of disagreement between HPV-EM and TCGA genotyping calls are presented in Table To further estimate the accuracy of HPV-EM, we identified a subset N = 78 of thep = 0.054)  of the 67 samples using RNA-seq data Table . Within p = 0.0055). However, when breaking down HPV-positive patients by genotypic group, we can see that the combined HPV33 and HPV35 group has unfavorable outcomes compared to both the HPV16 group and HPV-negative patients (p = 0.0044) . In the TCGA-HNSC consortium study, 279 of these head and neck cancer samples were included, and 36 among these samples were identifed in the study as HPV-positive. HPV-EM’s HPV genotyping predictions Table  were 100Overall, we designed and implemented a novel tool, designated HPV-EM, for highly sensitive and accurate HPV detection and genotyping using whole transcriptome sequencing data. To address the ambiguities caused by repetitiveness and homology in sequencing data, we designed and implemented a novel expectation–maximization (EM) algorithm in our tool to calculate an optimized mapping of sequencing reads to HPV reference genomes based on a probability model. We validated the sensitivity and accuracy of our tool using simulated data, gold standards in real public data, and DNA tiling array data. In addition to HPV detection and genotyping, the HPV-EM tool also provides functions to estimate and visualize HPV gene expression, which will aid researchers with their functional studies related to HPV biology. An HPV genotyping tool without limitations in HPV detection and genotyping is crucial in facilitating epidemiology studies and vaccine trials, as well as basic research in HPV-related diseases.The EM algorithm we designed in the HPV-EM tool is essential to accurately identify whether a sample contains a single or multiple HPV genotypes and to identify the proportions of each genotype. Because of repeats, homology and sequencing errors, HPV reads from a sample can be aligned to multiple HPV reference genomes, as represented in the samples shown in Fig. 26. Using HPV-EM, we accurately discovered the HPV genotypes in our institutional cervical cancer cohort treated uniformly with curative-intent chemoradiation therapy (CRT), and further demonstrated cervical cancer patients with HPV18  have worse recurrence-free survival compared to HPV16  is currently considered a prognostic marker, with HPV-positive head and neck cancers associated to favorable survival outcomes. Our study demonstated that while head and neck patients with HPV16 have favorable overall survival, patients with HPV33 and HPV35 have worse patient outcomes. The rich HPV genotypes shown in patients with cervical and head and neck cancers warrant further comparison between their genomic characteristics  to develop HPV genotype-specific treatment methods in future genomics and translational studies.Taken together, we developed an accurate HPV detection and genotyping algorithm to identify the exact HPV genotypic makeup of HPV-positive cases and demonstrated HPV genotypes as prognostic biomarkers in both cervical and head and neck cancers. These HPV genotype-specific prognostic biomarkers could be applied to modify therapies for patients  according to risks of failure after CRT and improve patient outcomes. We expect HPV-EM will be applied broadly in HPV-related cancer research and clinical settings. The algorithm also has the potential to be adapted to studies in other pathogenic microbes and metagenomic studies.Supplementary information.


It is widely acknowledged that HPV prophylactic vaccine could prevent new infections and their associated lesions among women who are predominantly HPV-naive at vaccination. Yet there still remains uncertainty about whether HPV vaccination could benefit to individuals who have undergone surgery for cervical disease.ClinicalTrials.gov, number NCT00779766) conducted in Jiangsu province, China. We evaluate the impact of HPV vaccination on preventing women from subsequent infection and cervical lesions (LSIL+ and CIN2+) after excision treatment.This post-hoc analysis intends to focus on intent-to-treat participants who underwent excision treatment at baseline and the follow-up period in a phase II/III, double-blind, randomized trial  performed excisional treatment in this clinical trial. We observed a significant effect of vaccination on acquiring 14 high-risk HPV (HR-HPV) infection after treatment . The vaccine efficacy against new infections after treatment for 14 HR-HPV infection was estimated as 32.0% , and was 41.2%  for HPV16/18 infection. The accumulative clearance rates of the vaccine group and placebo group were 88.9 and 81.6% for HPV16/18 infection (P = 0.345), 63.4, 48.7% for 14 HR-HPV infection (P = 0.062), respectively. No significant difference was observed on the persistent rate of HPV16/18, 14 HR-HPV infection and occurrence rate of LSIL+ between the two groups.One hundred sixty-eight (vaccine, No significant evidence from this study showed that HPV-16/18 AS04-adjuvanted vaccine could lead to viral faster clearance or have any effect on the rates of persistent infection among women who had excision treatment. However, the vaccine may still benefit post-treatment women with “primary prophylactic” effect. Further research is required in clarifying the effect of using the prophylactic HPV vaccine as therapeutic agents.ClinicalTrials.gov identifier: NCT00779766. Date and status of trial registration: October 24, 2008. Completed; Has Results. Persistent high-risk human papillomavirus (HR-HPV) infection is necessary for the progression of cervical cancer , 2. The Women after treatment for CIN remain at a substantially increased risk of subsequent cervical cancer –8. Both However, few studies evaluate whether the observed effects arise from: (1) “therapeutic effect” of the prophylactic vaccination in leading to faster clearance of the residual infection; or (2) “primary prophylactic” effect of the vaccination on the newly developed lesions caused by new HPV infections after treatment; or (3) “secondary prophylactic” effect of the vaccination in reducing the ability of the residual virus to infect new cells. In this study, we explore the role of a bivalent vaccine in preventing secondary lesions and provide more scientific evidence for the impact of the vaccine on the women who are treated for cervical disease.n = 3026) or aluminum hydroxide (n = 3025) as a placebo at months 0, 1 and 6. Enrolment in this trial started in October 2008 and follow-up lasted for 72 months (14 visits). The trial was conducted according to The Code of Ethics of the World Medical Association (Declaration of Helsinki) and the International Conference on Harmonisation Good Clinical Practice guidelines. The study protocol and informed consent were approved by the ethics committees of the Center for Disease Control and Prevention (CDC) Jiangsu Province and the Cancer Foundation of China. The trial was registered at the ClinicalTrials.gov (number NCT00779766) and adhered to CONSORT guidelines. Before the study-specific procedures, written informed consent was obtained from each participant. The further details of the trial have been described in previous published papers [Women included in the present evaluation were the participants who underwent excision treatment at baseline and the follow-up period in a phase II/III, double-blind, randomized trial conducted in Jiangsu province , China. In this trial, women aged 18–25 years were randomized (1:1) to receive HPV-16/18 AS04-adjuvanted vaccine. And the results were interpreted according to the Bethesda 2001 classification system . ParticiThe automatic analyzer SPF10 PCR-DEIA-LiPA25 version 1  detected HPV DNA of cervical, biopsy samples and tissue specimens including 14 high-risk HPV types  and 11 low-risk HPV types .2 or Fisher’s exact tests, as appropriate. Vaccine efficacy (VE) was calculated as 1-(incidence rate in vaccinated/incidence rate in unvaccinated) and the corresponding 95% confidence intervals (95% CIs) around vaccine efficacy were calculated by exact conditional procedure based on the observed case split between vaccine and placebo recipients, adjusted for the person time in each arm. In the squamous intraepithelial lesion (SIL) analysis, we analyzed the occurrence rate of LSIL+ and HSIL+ stratified by specific-type after excisional procedure. Results of vaccine efficacy were considered as statistically significant if the estimates and corresponding 95% CIs were above zero. Kaplan-Meier method with log-rank test was applied to compare the difference in occurrence of LSIL+ between the two groups. All statistical tests were two-sided and only p-values < 0.05 were considered statistically significant. The statistical analyses were performed using WinPEPI version 4.0 [Comparisons of enrollment characteristic, accumulative clearance rate and persistent infection rate between the two groups were conducted by Pearson χsion 4.0  or SPSS n = 87; placebo, n = 81) received excisional treatment for their first cervical lesion identified at this clinical trial. One woman in each group underwent loop electrosurgical excision procedure (LEEP) at the last visit (visit 14), and thus 166 women had been followed up and were categorized based on cytological results and HPV status at enrollment. Comparison of analytical cohort stratified by study arm revealed the balance regarding to cytology results, and HPV status (HPV type) at enrollment , corresponding to a median of 9 study visits . The median duration between enrollment and treatment was 17 months for those in the vaccine arm (IQR:4.0–30.0 months) and 17 months for women in the placebo arm (IQR:6.0–34.8 months).n = 5; placebo, n = 5) were excluded because of no follow-up after excision treatment  were included in the analysis. There was no significant difference in the distribution of baseline HPV infection between the two groups . The woman was included in this analysis from the day when the women received an excisional procedure (LEEP or cone) for a first cervical lesion to the day of their last follow-up visit, considering the fact that women could be infected by HPV or had abnormal cytological results for several times after excisional treatment. Among all the infections that treated women had, 71.06% were HR-HPV infection and of these 52.49% were the result of new infections. We observed a significant effect of vaccination on acquiring 14 HR-HPV infection , and a nonsignificant but positive vaccine efficacy estimate of 28.1%  for HPV16/18 infection after excision treatment , 81.3, 80.0% for HPV31/33/45 infection(P>0.999), 63.4, 48.7% for 14 HR-HPV infection(P = 0.062), respectively. No significant difference on the persistent infection rate of HPV16/18, HPV31/33/45,14 HR-HPV  was identified between the two groups.Then, we evaluated whether the viral clearance rates or HPV persistent infection rates differed by vaccination status. Results were shown in Fig. When LSIL+ was examined as the outcome, nonsignificant but positive vaccine efficacy was estimated at 45.5%  for 14 HR-HPV infection, and 85.1%  for the newly detected outcome after treatment, respectively. Similar patterns were observed for HPV-16/18 infection as well.n = 7; placebo, n = 8) were excluded because of no cytological results . The distribution of cytological results at baseline was similar between two groups . In this analysis, the follow-up time was defined as the duration from the day of excisional procedure to the day of ascertainment of the subsequent disease end point (LSIL+ incidence), while for women without a subsequent disease end point, until the day of their last follow-up visit. The median follow-up time was 46.0 months among women who were treated. The median follow-up time for vaccine group and placebo group were 48.5 months, 44.0 months, respectively. The vaccine efficacy for the occurrence of LSIL+ was 55.3% , irrespective of HPV DNA types. There was no significant difference between two groups , one with VAIN1, and one with CIN1 post-surgery. Of the two women in the placebo group, one developed CIN2 and one had CIN1 after treatment. The women Case 4: Fig. : Fig. 4 This is the first analysis in China to evaluate the efficacy of HPV 16/18 AS04-adjuvanted vaccination on the relapse of cervical precancerous lesions or cancers among women who underwent excisional procedures for cervical lesions after vaccination. In this study, vaccination failed to lead to viral faster clearance or have any effect on persistent HPV infection. However, the available evidence suggested that the vaccine may still benefit women after excisional treatment for cervical disease by “primary prophylactic” effect, which protects women from new infection after treatment.Former studies have indicated that the prophylactic HPV vaccines have the possibility in benefiting post-treatment women. The post-hoc analysis of the PApilloma TRIal against Cancer In young Adults (PATRICIA) showed that efficacy of the HPV 16/18 AS04-adjuvanted vaccine post-surgery (60 days or more) for the original lesion was 88.2% against CIN2+ and 42.6% against CIN1+ . FurtherThe women who were treated due to HPV-related disease were at high risk for developing subsequent HPV related disease. The persistent or recurrent rate of CIN2+ after excisional treatment was reported as 4% ~ 18% . A meta-The major advantage of our study is that we have detailed information about the margin status of excisional material for each woman treated for the first cervical lesion who subsequently developed lesions after surgery. This made it possible to confirm whether subsequent cervical lesions were associated with HPV genotype infection found in the original lesion. However, there are some limitations in our analysis. The subgroup of women who underwent excision treatment was not a randomized group, so we had limited power to evaluate post-treatment vaccine efficacy. Furthermore, because no any sexual behavior data was collected, we were not able to evaluate the sexual behavior difference between two groups or estimate the effect of vaccination by adjusting the factor. However, a good balance regarding HPV status at enrollment had been achieved between the two arms, which indicated that there may be no significant difference in sexual behavior between the two groups. And we used cytological results as an outcome in the analysis due to not all women after treatment underwent colposcopy, which may have potential bias. Based on previous study , cytologSignificant vaccine efficacy against new infections after treatment for 14 HR-HPV infection was observed. The nonsignificant effect against new infections associated with HPV-16/18 and cervical lesions (LSIL+ and CIN2+) maybe caused by the limited power with smaller number of women. However, the vaccine may still benefit post-treatment women with the “primary prophylactic” effect. Further research is required in clarifying the effect of using the prophylactic HPV vaccine as therapeutic agents.


Human Papilloma Virus (HPV) vaccination of the preadolescent (9-14 years) females is the potential solution to eradicate carcinoma cervix. Nonavalent vaccine provides wider coverage than the quadrivalent vaccine. On long-term follow-up, even after single-dose HPV vaccination, the antibody titer remains good. Herd immunity can also be achieved by HPV vaccination. Hence, mass single-dose nonavalent HPV vaccination for sexually naive preadolescent girls can provide almost 100% protections and a cost-effective approach for the developing countries. Cervical cancer is a preventable disease and whole world is now focusing on prevention of carcinoma cervix. Cervical intraepithelial neoplasia (CIN) is the forerunner of cervical cancer. HPV infection is invariably associated with CIN. In carcinoma cervix, HPV type 16 and 18 are responsible for about 70% cases and HPV type 31, 33 and 45 are responsible for another 13% cases. Hence, HPV vaccination is the key to eradicate carcinoma cervix. There are various HPV vaccines been invented so far including bivalent targeting HPV (type 16 and 18); quadrivalent targeting HPV ; and nonavalent targeting HPV . Human papillomavirus 9-valent vaccine, recombinant; 9vHPV (Gardasil 9) was approved by FDA in 2014. Since the end of 2016, only 9-valent HPV vaccine is available in the United States [In the multi-centric Indian clinical trial NCT00923702) quadrivalent HPV vaccine was given and the participants were ended up in 4 groups – 1) 3 dose group: girls vaccinated on days 1, 60 and 180 or later, 2) 2 dose group: girls vaccinated on days 1 and 180 or later, 3) 2 dose group: girls vaccinated on days 1 and 60 by default and 4) Single dose: by default because administration of vaccine was stopped in April 2010 by Govt of India 3702 quad. Forty yHPV vaccine also provides herd immunity. While comparing data of 2003–2006 to 2011-2014 it was found that, in sexually active women of 14-24 years the prevalence of HPV is reduced by 89% in vaccinated women (4vHPV) and 34% in unvaccinated women [Australia, United Kingdom and Denmark have achieved high coverage of their population by including HPV vaccination in their national immunization programme . In IndiConsidering all these facts, it would be prudent to initiate the process of including single dose nonavalent HPV vaccination for preadolescent sexually naive girls in the national immunization schedule.