HPV pathogenesis, various types of vaccines, safety concern, prophylactic and therapeutic applications to control cervical cancer, and future perspective

Over 98% of cervical cancers (CC) are caused by regular infections with "high risk" genotype of the human papilloma virus (HPV). However, this is not always the causative factor. Therefore, production of HPV vaccinations represents a significant chance to minimize the risk of CC. Phase III studies for a number of preventative HPV vaccines based on L1-virus-like particle (VLPs) have just been completed and the preliminary results are very convincing. However, there are a lot of practical concerns that need to be resolved before the use of these vaccinations. These vaccines were challenged with obvious queries such as protection time, subject receiving vaccines, time of vaccination, and how to include them into ongoing screening programs. Although these vaccines were 90% effective at preventing HPV infection as these offered only modest advantages for the removal of pre-existing infections. New advancements in the creation of therapeutic vaccinations have been explored for further improvement and post-vaccination surveillance. Therapeutic vaccines attempted to boost cell-mediated immunities and these are detrimental to the infected cell as opposed to neutralizing antibodies (different from prophylactic vaccines).


Introduction
Cervical cancer (CC) is the deadliest and life-threatening malignant health issue (fourth most common cancer) among women globally. With an expected 570,000 cases and 311,000 deaths in 2018, CC is the second most frequent malignancy in women living in developing countries (World Health Organization, WHO). In developing and less developed countries, estimated death reached to about 85% of 311,000 in 2018 [1]. As per the current report, 84% of HPVrelated cancer lesions are CC which is transmitted sexually and causes CC [2]. Despite virus, few bacteria have been known to cause cancer and these are named as oncogenic or carcinogenic bacteria. These bacteria are exemplified as Salmonella typhi (gall bladder cancer), Streptococcus bovis (colorectal cancer), Chlamydia pneuminae (lungs cancer), Mycoplasma (various cancers such as prostate cancer), H. pylori (stomach cancer). Cervical cancer and bacterial vaginosis (BV) are linked together. However, the pathogenesis of BV in cervical cancer is complicated to understand and conclude a single causative bacteria as its pathogenesis involve various bacteria (G. vaginalis, Atopobium vagina, Bacterioides, Mycoplasma, Fusobacterium, Peptostreptococci, Mobiluncus spp, Ureaplasma urealyticum) [3]. The genus sneathia is related to various cancers such as cervical, vaginal, oral, and gastrointestinal types of cancer. The genus includes two species such as S. amnii, and S. sanguinegens commonly observed in oral, intestinal, vaginal, and cervical area. In a large cohort study (736 women), about 43% of sample specimens contained Sneathia [4].
Only the epithelium's basal cells are infected with HPV. Unchecked cell proliferation and mutations cause HPV lesions, which eventually progress to cancer. It is the second main cause of women getting cancer worldwide, especially in developing nations. Interestingly, epidemiological studies in the past suggested a link between the emergence of cervical cancer cases and the metabolic syndrome (MetS). In contrast to endometrial cancer, there is a dearth of solid data that might support the association between MetS and cervical cancer. On the other hand, a number of epidemiological studies shakily connect MetS presence with increased risk of cervical cancer [5]. HPV infection is virtually always a factor in the development of cervical cancer. Over 200 distinct HPV strains have been found that infect epithelial cells [6], and about 40 of them show a preference for mucosal tissues. Depending on their ability to cause cancer, they are further categorized into low-risk and high-risk HPV (lr-HPV and hr-HPV, respectively) [7]. While hr-HPV types are linked to cervical intraepithelial neoplasia (CIN) and CC, lr-HPV types are linked to the growth of anogenital warts. Around 70% of CC cases worldwide are caused by hr-HPV, primarily HPV-16 and HPV-18 [8,9]. Although HPV infection alone is insufficient to cause CC, the emergence of persistent infection is a critical element in the development of cervical lesions and the course of the disease. Immune responses control infection, avoiding cervical lesion formation and its development into cancer in the majority of HPV-infected women [10]. As a result, only a small percentage of infected women are unable to control their infection and go on to develop CC. The progression of CIN to CC or its regression may be influenced by additional factors, according to this fact. The complex cervical milieu contains immune cells as well as a unique microbiome that controls regional immune responses [11][12][13]. Based on the types of bacteria present, the cervicovaginal microbiota was determined using 16S rRNA high-throughput sequencing (16S-HTS) data [14]. They are referred to as community state types (CSTs). Dysbiosis is a condition characterized by an unbalanced cervicovaginal microbiota composition with high variety and low Lactobacillus abundance, similar to CST IV. Dysbiosis can cause some women to experience symptoms such abnormal vaginal discharge, irritation, odor, and itching. In these cases, bacterial vaginosis is diagnosed [15]. Despite the fact that some women have symptoms, the majority of women are asymptomatic. However, women are more likely to contract HIV, HPV, and other infections when they are both symptomatic and asymptomatic [16]. There are various preventive strategies which are classified as primary, secondary, tertiary and palliative care. HPV based vaccines and chemotherapy-based treatment (and diagnosis) of cancerous lesions, are under primary and secondary prevention whereas diagnosis and treatment of invasive CC are tertiary prevention method. Moreover, palliative care involves a comprehensive CC control strategy. Preventing HPV-related disease and death successfully require both screening for pre-cancerous and cancerous lesions, and primary immunization [17]. Many nations have approved the use of three preventative vaccines. These are categorized as (a) quadrivalent-HPV vaccine, (b) bivalent-HPV vaccine, and (c) nonavalent-HPV vaccines. These three vaccinations demonstrate safety and efficacy in randomized studies and post marketing surveillance preventing 70%-90% of HPVrelated malignancies. The HPV6 and HPV11 vaccines are nonavalent and quadrivalent to provide protection against anogenital warts. The first dose of the HPV vaccines was administered to about 118 million women globally [18]. These vaccines are substantially efficient and effective to prevent HPV infections and neoplastic illnesses. As these are preventive in nature, these have limited therapeutic benefits and no effect on treating pre-existing infections. Additionally, it appears that the impact of vaccination may not lower the incidence of cancer due to the lengthy period of time needed for the development of pre-cancerous lesions [19]. HPV16 and HPV18 are two the most common HPVcaused cancer-causing viral strains causing about 70% of CC and these are the focus of the two vaccines presently under evaluation. As discussed before, these three prophylactic vaccines are available in the market (a quadrivalent HPV vaccine, a bivalent HPV vaccine, and a novel nonavalent HPV vaccine). In India, Merck and GlaxoSmithKline legally marketed Gardasil™ (quadrivalent, type 6,11, 16, and 18) for 9-45 ages and Cervarix™ (bivalent vaccine), respectively to control CC. Gardasil™-9 can protect against HPV type 6, 11, 16,18, 31, 33, 45, 52, and 58. For developing both vaccines, a recombinant DNA technology was used and created non-infectious VLPs containing HPV-L1 protein.
Effectiveness against CIN-2/3 and adenocarcinoma in-situ (AIS) using HPV based vaccines has been employed as key end points in clinical trials. The cross-protection against HPV strains (not included in the relevant vaccine) has also been studied for both vaccines. Notably, these vaccines are not effective and protective against previous infection as diagnosed in serotype diagnosis with HPV virus [20]. The main financial advantage of HPV vaccines in industrialized nations would be the cost savings from a decrease in the annual number of abnormal cervical smears found by screening that required further investigation. The main effect on developing nations would be a decrease in the actual number of CC cases and the overall cost. However, the cost of producing and delivering the current HPV VLP vaccines must be weighed against these advantages. In response to an HPV vaccine, the body produces antibodies that attach to the virus and stop it from infecting cells in subsequent contacts with the virus. The basis for the current HPV vaccines is the formation of virus-like particles (VLPs) from HPV surface elements. VLPs are not contagious since the virus's DNA is missing from them. In contrast, they resemble the natural virus and antibodies to the VLPs also work against the natural virus. According to research, the VLPs are strongly immunogenic to produce substantial antibodies, effective and safe to control CC.

Preventive measures and treatments
The dysplastic process takes times about 1-2 decades to mature and gives systematic screening programmed time to find and treat pre-invasive disease. According to estimates, cervical screening stops about 5000 deaths in the UK each year [21]. In fact, the increased incidence of HPV infection due to altered sexual behavior patterns may be an underestimation [21]. In a pilot trials study, the United Kingdom (UK) adopted a liquid-based cytology method for screening as per the National Institute for Health and Clinical Excellence (NICE) advice. Similarly, the ARTIS-TIC trial (a randomized trial of HPV-Testing in primary CC Screening) of Manchester, adopted a new HPV DNA testing method by hybrid capture and it is currently considered as a primary screening test [22]. To detect high grade cervical intraepithelial neoplasia (CIN), HPV-testing seems to be highly sensitive but less specific than cytology [23]. As it can identify numerous transitory illnesses, it lacks specificity. A potential screening paradigm includes HPV-testing as a main screening test and cytological triage for those who found as positive for HPV. It has been discovered that repeat testing after a 12-month break is just as beneficial as early colposcopy for women with normal or borderline cytology who have tested positive for HPV (Cuzick et al. 2003) [23]. This might increase the diagnosis and detection of highgrade CIN while reducing the cases of women who are recommended for colposcopy. Currently, screening aimed to find CIN and treat high grade illness. In contrast, 25% of CIN II advances to late-stage dysplasia within five years, and around one-third of CIN III progresses to late stage cancer. This might be easy to correlate with the ˃ 70% of mild dysplasia (including CIN I) being cured spontaneously. Therefore, treatment is not always necessary when CIN-1 is discovered. It is important to manage treatment strategy and diagnosis follow up. It was said that until therapy is necessary, cytological and colposcopic follow up should be accomplished (NHSCSP 2004). It might be possible that the follow up process may be restricted to high-risk HPV type CC women only. There is a chance of recurrence and this method of treatment does not always completely remove HPV infection from the upper genital tract. Pathologically, routine histological examination and cytological assessment of even high-risk HPV can be used to monitor any residual disease that develops during follow-up. Physical removal has already failed. Therefore, an ant treatment strategy capable of targeting the underlying HPV strain could be certainly preferable and ideal. A method of treatment for successful therapeutic immunization could be considered as preferable and acceptable to control pre-malignant and malignant CC whereas prevention through prophylactic vaccination could potentially eliminate the development of CC.

HPV vaccines development pipeline
Historically, vaccination has been the least expensive and the most efficient method of preventing infectious disease. There is therefore a profound volume of research on the topic of HPV vaccines and CC [24][25][26]. In this article, we focused on some of the most therapeutically pertinent findings. According to the estimates, CC mortality would decline by 88.9% by 2070 and 98.6% by 2120 if all three WHO targets for eliminating the disease by 2030 were met. Without the screening and treatment targets, achieving the 90% immunization goal would reduce mortality by 61.7% by 2070 and by 89.5% by 2120 (Fig. 1).

Evidence for achievability
Is it possible to eliminate CC to the WHO threshold? A worldwide study team used comparative modelling to estimate how long it would take to eradicate CC in the United States in order to respond to this question. According to a simulation model, the WHO incidence criterion of less than four cases per 100,000 women-years may reach by 2038 assuming the status quo assumptions for immunization and screening. In a different scenario, it assumes screening, scaling up to 90%, and predicts the eradication of CC by 2028. Interestingly, a third scenario with the assumption of 90% vaccination coverage predicted CC elimination at about the same time as the status quo scenario. So, this suggests that the most effective intervention and the fastest way for the United States to achieve the goal of CC elimination is to scale up screening and treatment, especially focusing on the under-screened and undertreated [27]. There is evidence from multiple countries to support a reduction in HPV infection after the vaccine is rolled out into public health practice. One study comparing HPV prevalence among Australian women aged 18-24 pre and post-vaccination found a large reduction in the four types of HPV (Gardasil protected). The prevalence was 22.7% (n = 88) pre-vaccination in the years 2005-2007 whereas the post-vaccination prevalence dropped to 7.3% (n = 688) from 2010 to 2012 and to 1.5% (n = 200) in 2015 [28]. What is the evidence that the vaccine will work? While the HPV vaccine is the only one part of the three-legged stool, when it is added to screening and treatment. It reduces infection in the population. In sequence over time, this reduction in infection is followed by a reduction in genital warts, reduction in cervical intraepithelial neoplasia (CIN), and a reduction in CC. There is evidence from multiple countries to support a reduction in HPV infection after the vaccine is rolled out into public health practice. In the United States, even with a relatively low vaccine dissemination, there are significant reductions in genital wart incidence, pointing to the potential benefits in public health. Flagg and Torrone observed decreases in the prevalence of genital warts in young women likely to be affected by HPV several years after licensure of the HPV vaccine [29]. Similar to the incidence pattern for genital warts, CIN 2 incidence has declined in the United States despite the low dissemination of vaccine. To evaluate the impact of HPV vaccination on the reduction of cervical pre-cancer, McClung et al. examined archived specimens from women aged 18-39 with CIN 2 + . They found that between the years 2008 and 2014, the proportion of CIN 2 + cases decreased from 51.0% to 47.3% [30]. Finally, there is evidence that oral HPV infection is also declining as a consequence of vaccination. One study in the United Kingdom examined the effect of HPV vaccination on the prevalence of oropharyngeal HPV-16 infection among girls and young adult women and compared infection levels with those in unvaccinated young males of similar age [31]. They found that the UK female-only vaccination program was associated with a significantly lower oral HPV-16 prevalence among vaccinated females compared to unvaccinated females [31].

HPV pathogenesis
Mucosal or epidermal epithelial cells are infected by HPVs. The host's immune system eliminates the majority of infections. However, benign cervical lesions were caused by chronic HPV infections that the immune system was unable to eradicate. Three stages are frequently used to categorize cervical intraepithelial neoplasia (CIN-1, CIN-2, and CIN-3). The low-grade lesion stage known as CIN-1 is seen in many HPV infections. Within few months, the immune system eradicates almost 80% of CIN1 instances. The development of CIN2/3 as high-grade CIN lesions from an untreated HPV infection, can lead to aggressive CC [32,33]. The main route by which HPVs infect the multi-layered stratified epithelium of the ectocervix and enter the basal lamina is a micro-wound [34,35]. The early promoters can be turned on in the infected basal-layer cells early in the HPV life cycle to start the E1-viral helicase expression, which interacts with E2. The viral episomes are then amplified and quickly reproduced [36]. The virus genomes maintain a small amount of intact genomes replication at this point [37]. Daughter Fig. 1 Timelines of HPV vaccines: The HPV vaccine was first developed by the University of Queensland in Australia by Professors Ian Frazer and Jian Zhou. In 1990, Frazer and Zhou began to synthesize particles that mimicked HPV, from which the vaccine would later be made. These particles are called "virus-like particles" (VLPs), and are small particles that contain proteins from the outer layer of the HPV virus. In 1991, Frazer and Zhou's findings were first presented to the scientific community. After seven years of design and testing, the first human trials for the vaccine, named Gardasil, were completed. Since then, two further vaccines have been approved: a biva-lent vaccine called Cervarix approved in 2007 that prevents two HPV types (HPV16 and 18) and a nonavalent vaccine called Gardasil 9 in 2014 that protects against nine HPV types (HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58). As of October 2019, 100 countries worldwide vaccinate against HPV as part of their regular vaccine schedule. Currently there are six licensed HPV vaccines: three bivalent, two quadrivalent, and one nonavalent vaccine. Those that have been prequalified are being marketed in countries throughout the world ("Created with BioRender.com") cells of infected basal cells gradually move to the top layers while also differentiating into distinct types of epithelium. Cell division then expedites bulk viral genome amplification, inducing delayed gene expressions, virosome assembly, and in vivo release. (Fig. 2).

HPV vaccine history
The HPV L1 main capsid protein is expressed via recombinant DNA technology in Saccharomyces cerevisiae (yeast), which self-assembles to create shells (empty) resembling viruses, known as virus-like particles (VLPs). The VLPs lack genetic material but have the same exterior L1-protein.
These VLPs are used in the vaccine as an antigen to trigger powerful immune responses that are protective in the event of exposure. Antibodies to the L1-protein will bind the HPV to stop releasing the genetic materials [38].

Vaccination as the best form of treatment strategy
Currently, the only ways to avoid genital HPV infections are lifetime mutual monogamy and abstinence. The use of physical barrier techniques (condom and contraceptive) is not the effective and conclusive proof to provide protection against HPV infections. Second, the virus has no symptoms other than genital warts [39,40]. Even in developed nations, the susceptible female population has not adhered to routine screening by periodic Pap smears, and in developing nations like India, it is challenging to implement routine screening on a wide scale.

The HPV vaccines to control CC
The primary mechanism of vaccine-induced protection is the induction of antibodies. In the event of HPV contact, the vaccination attaches to the HPV viruses and stops to infect further epithelial tissues. The amount of antibody generated during natural infection is typically not enough to stop a reinfection. There isn't secondary lymphoid tissue in the infected cervical region, which would have produced antibodies and neutralized the virus before absorption (if it did contain a lot of memory B cells) [41]. Additionally, large and extended levels of natural antibodies generated by vaccines are necessary to maintain the protective immune responses during sexually active life. Therefore, a perfect HPV vaccination should boost the immune system's defenses and protect against all high-risk HPV types as well as those forms that are probably or maybe carcinogenic. The HPV vaccines available today are based on VLPs, which lack viral DNA and are made from recombinant HPV capsid proteins. Different forms of type-specific nAbs can be induced by VLP vaccination, and these nAbs can bind to native VPs and neutralize the virus by inhibiting cellular uptake (epithelial cells).

Prophylactic HPV vaccines
As we discussed before, there are three approved commercial vaccines to control CC for prophylactic therapy. The yeast strain was used to express these proteins and manufactured the 4vHPV and 9vHPV vaccines. These proteins were further conjugated with aluminum hydroxy phosphate sulfate (AAHS) adjuvant to get an amorphous construct. The 4vHPV vaccine was recommended to prevent from genital wart, dysplasia lesion, precancerous lesion, and CC for both sexes aged between 9 to 26 years [42]. On the other hand, the 9vHPV vaccine protects from five more HPV strains in both sexes aged between 9 to 45 years and is effective to protect from other cancers such as vulvar, CC, vaginal, and anal cancers [43]. The production of the 2vHPV, which originally got permission in 2007, uses a baculovirus expression vector system and an adjuvant called AS04 (aluminum hydroxide and 3-deacylated monophosphorylate lipid A) [44]. AIS, CC, and CIN may be prevented by HPV vaccination [45,46]. These three preventative vaccinations use VLPs that were put together using L1's recombinant expression. Recombinant L1 protein has been demonstrated to self-assemble into VLPs that resemble natural virions morphologically and lack the oncogenic viral genome [47]. By inducing high and long-lasting antibodies titers that bind to the native virion and destroy the virus, HPV vaccinations can prevent HPV infection and render people immunized to subsequent viral challenges.

Prophylactic vaccine schedules and strategies
A three-dose vaccination schedule was first approved for the preventive HPV. Recent research has demonstrated that the 2 or even 1 dosage of the 2vHPV vaccination was significantly more effective than the 3-doses strategy [48]. Girls aged from 9 to 14 years who received two doses of the 2vHPV vaccine at months 0 and 6 showed superior HPV-16/18 antibody responses compared to those who received two doses of the 4vHPV vaccine at months 0 and six or three doses of the 4vHPV vaccine at months 0, 2, and 6, in an observer-blind study [49]. The 2-doses vaccination schedule appeared to be the most cost-effective assuming the vaccines could provide protection for more than 20 years [50]. The WHO has suggested that individuals between the ages of 9 and 15 receiving two doses of the HPV vaccine at least six months apart, the gap between the first and second doses should be between 6 and 15 months. A third-dose regimen is necessary for people older than 14 or immunocompromised patient regardless of age who have their two doses less than five months apart [51].

Safety concern to receive prophylactic HPV vaccination
A rough estimate of ˃ 200 million doses of the preventive HPV vaccines had been administered globally [52]. Several evidences support the vaccine's safety. However, a number of safety concerns, have surfaced including discomfort, swelling in the arm at the shot site, redness, slight fever, fatigue, nausea, headache, and muscle, and joint pain. The Vaccine Adverse Event Reporting System (VAERS), the Vaccine Safety Datalink (VSD), and the Clinical Immunization Safety Assessment (CISA) Project are three systems used by the Centers for Disease Control and Prevention (CDC) and the Food and Drug Administration (FDA) to monitor the safety aspects of vaccines. Prior to the FDA granting the license in 2014, seven studies investigated the 9vHPV vaccine's safety. According to these pre-licensure trials, the 9vHPV vaccination is equally safe as the 4vHPV vaccine. Additionally, according to statistics from the 4vHPV vaccine reports, 92% of them were not considered significant [53]. Serious adverse reactions are extremely rare after receiving an HPV vaccine, and the causes still remained unknown. While the immunizations do not appear to increase the likelihood of miscarriage, the fetal adverse events following HPV vaccination primarily involve spontaneous miscarriage. There are no risks of unfavorable pregnancy outcomes when receiving the HPV vaccine during pregnancy, postpregnancy, or right after conception [54,55]. Although, it is not advised for pregnant women to obtain the HPV vaccine. Lactating women can get the vaccine. Additionally, the safety profile of the 9vHPV vaccination was supported by preclinical investigation on rat models wherein the nonavalent HPV vaccine had no negative impact on reproductive performance, fertility, and any signs of developmental toxicity.

The new version of HPV vaccines (second generation HPV)
The aforementioned vaccines (three) are now approved and these are developed by yeast or insect cell expression systems, making their manufacturing procedures complicated and this is the main cause of the high cost. The manufacture of VLP-based vaccines has been improved via the efforts of many professionals, with notable success. In preclinical or clinical studies, HPV L1 VLP-based vaccines made in methylotrophic yeast species such as Hansenula Polymorpha, P. pastoris), S. cerevisiae, and S. cerevisiae. (Table 1) [56].

Therapeutically used HPV vaccination
The prime goal of preventative HPV vaccinations is to stimulate humoral-immunity against the target proteins (late proteins L1 and L2), which will induce antibodies production and antigens neutralization. Therapeutic vaccinations, as opposed to preventative ones, are primarily intended to treat precancerous lesions and chronic HPV infections. In HPV infection, the majority of CC, and precancerous lesions, the early proteins E6 and E7 are consistently produced (but not in normal tissues). Notably, these early proteins (E6 and E7) are the most preferred target proteins to develop antigen-specific immunotherapy and treatment therapy of HPV infections, and related disorders. Their continuous expression is crucial to induce and maintain the malignant phenotype of cancer cells. Various therapeutic vaccines to control CC using the same viral strains have been reported to target these early proteins (E6, E7), late proteins (L1 and L2), and virus antigens over the last two decades. These vaccines included nucleic acid, live vector, and acid/protein/peptide based approaches. The following Table 2 lists the characteristics of vaccination platform technologies and the development of the several therapeutic HPV vaccines available ( Table 2). For a number of reasons, developing prophylactic vaccines are considerably more difficult than developing preventive vaccinations. E6 and E7 oncoproteins are expressed in high grade CIN lesions at a low level only.

Live vector-based vaccines
Recombinant technology has been used to tailor vaccines to control CC. These are viral and bacterial vector-based vaccines used to transport HPV antigen within host cells, and elicit immune response against HPV infection. These are of two subgroups of live vector-based vaccinations. Additionally, these are capable of promoting antigen presentation via both MHC-I and MHC-II class pathways which offered significant level of immunogenic responses. The development of natural antibodies against pre-existing bacteria and viruses may restrict the use of these vectors in recurrent therapy. Another barrier to the use of live vector vaccinations is the probable immuno-dominance of the vectors rather than the HPV antigen [57,58].

Bacterial vector vaccines
An ideal vaccination vector should stimulate the infected host's innate and adaptive immune systems strongly. In order to defend the host from pathogen, attenuated bacteria can activate immunogenic responses (cellular and humoral immune). Peptidoglycans, flagellin, lipoteichoic acid, and lipopolysaccharides are examples of conserved molecular patterns that are identified by pattern recognition receptors (PRRs) and trigger innate immune responses [59]. It is efficient to create novel vaccines by delivering heterologous antigens through the bacterial vector. The bacterial vector executes several benefits over other vaccines such as (a) bacterial vectors to deliver heterologous antigen for reducing the challenges of targeting antigen purification and (b) making large-scale manufacture and inoculation relatively simple and affordable. The stimulation of adaptive and innate immune responses to antigens (pathogens) could boost attenuated bacteria to execute specific immune responses against target antigen [60]. Attenuated Lm is currently being employed for vaccine development, treatment and prevention of HPV infection, and disorders associated with it. A live attenuated Lmbased immunotherapy called ADXS11-001 (ADXS-HPV) secretes the antigen-adjuvant fusion protein Lm-LLO-E7 that targets HPV-related cancers [61]. Viral vector vaccines Viral vectors like adenoviruses (Ad), alphaviruses, adeno-associated viruses, and vaccinia viruses have been extensively investigated due to their high infection effectiveness, high antigen expression, and natural tendency for transduction of genetic material into the host for virus replications. Notably, undesired viral genes are swapped out for foreign genes responsible to encode immunogenic protein expression from pathogens. Then, recombinant viral vectors are allowed to transduce the target cell and express the encoded antigens. In order to prevent HPV16 and HPV18-related diseases, Khan et al. [62] produced two unique replication deficient vaccines called "replicationdeficient Ad26 and Ad35 vector vaccines" employing fusion proteins of HPV-E6, E26, and E7. The created vaccine vector formed a viable therapeutic active vaccine immunotherapy (active), as evidenced by their significant therapeutic efficacy in the TC-1 mouse model. In a recent clinical phase-III experiment including 180 male and 1176 female patients, Rosales et al. assessed an MVA-viral vector targeting HPV16-E2 to cure intraepithelial lesion linked to the HPV infections. Direct injections of MVA-E2 virus particles were made into the urethra, uterus, vulva, and anus. All of the male patients and 89.3% of the female patients who received MVA-E2 treatment saw the lesions completely disappear, and no adverse symptoms were noticed. In this study, a control vaccine was not used in the trial. Therefore, the actual effectiveness of the vaccine is still unknown and questionable [63].

DNA-based vaccines
For both human and animal illnesses, DNA-based vaccinations have emerged as a secure substitute for traditional live and inactivated vaccines [64]. The DNA vaccine GX-188E was created in 2014 by Kim et al. [65], expressed the HPV16/18-E6 and E7 antigens as well as the Fms-like tyrosine kinase-3 ligand (Flt3L), to provoke DCs (dendritic cells).
To improve immunization, they employed electroporation. The first most effective therapeutic DNA vaccine to date has demonstrated efficacy against CIN2/3 is VGX3100. A phase III trial is currently evaluating VGX-3100's effectiveness, safety, and tolerability in women with CIN2/3 that has been histologically confirmed to be HPV 16/18-positive (NCT03185013).

RNA-based vaccines
The foundation of RNA-based vaccines is the RNA-replicon system developed from non-pathogenic ssRNA (single stranded RNA) viruses with negative or positive polarity [66]. Various RNA viruses, including picornaviruses, measles, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, and retroviruses, have been modified to serve as vectors. These vectors can expression the targeted protein to control diseases by triggering immune responses [67]. Variety of studies have shown that self-replicating RNA-viral vectors can effectively deliver recombinant heterologous antigens and promote their expression at high levels due to their high autonomous RNA replication capability. They can also induce strong immune responses and protect against challenges from infectious agents and tumor cells. However, RNA-based vaccines are constrained by their poor durability and inability to disseminate between cells [68]. Few clinical trials for RNA-based vaccinations against illnesses linked to HPV have been conducted.

Protein and Peptide-based vaccines
The advantages of these vaccines are safety, stability, storage, and ease of manufacturing [69]. Low immunogenicity and the required MHC-specific drugs matching the patient's HLA haplotypes are major drawbacks [70]. Particular epitope (short) peptides and synthetic long peptides (SLPs) are two categories for peptide-based vaccinations. Prior to injection, the short peptides must be pre-HLA typed as these are limited to the patient's unique HLA types [71]. Seven women with highgrade CIN and HPV16-positive, underwent testing for short peptide-based therapeutic HPV vaccines (CIGB-228) adjuvant with very tiny size proteoliposomes (VSSP). Five individuals experienced lesion regression and HPV eradication as a result of the vaccine-induced IFN-associated T-cell response [72]. SLPs, as opposed to short peptides, need to be digested and presented by APCs, but patient selection with a specific MHC profile is not necessary [73]. PepCan (NCT01653249), a peptide-based vaccine made up of four synthetic HPV16 E6 peptides and a novel adjuvant called Candin, was tested on 24 patients in the dose-escalation phase I clinical research.

Dendritic cell-based vaccines
In physiology and anatomy, it is well known that there are several antigen presenting cells responsible to execute immunological reactions and responses against certain pathogens and external antigens. Among them dendritic cells (DCs) are the excellent target for immunotherapy. These cells have been identified as the most potent cells in in-vivo condition, mediating and generating both innate and adaptive immunological responses. DCs exhibit significant quantities of either co-stimulatory or co-inhibitory molecules and have a strong ability to gather and prepare antigens for presentation to T lymphocytes [74]. Additionally, DCs can act as organic adjuvants to boost the effectiveness of antigenspecific immunotherapy for the treatment of cancer [75]. The two types of DC-based vaccinations are categorized as (a) transduced with DNA or viral vectors responsible to encode foreign antigens and (b) pulsed with HPV-specific protein (or peptide) antigens [76]. Both mature and immature cells can be used to make DCs. It was found that immature types of DCs lack the full T-cell co-stimulatory ability whereas mature DCs were used in the majority of experiments [77]. Thirty-two patients with advanced cervical malignancies  [90] were treated with the PIDC pulsed with HPV16 E6 or E7 peptide by Rahma et al., [78].

Adaptive T-cell therapy
The process of adaptive T-cell treatment (ACT) entails the isolation, in vitro growth, and re-infusion of T-cells with the desired specificity and phenotype. With the use of ACT, T cells can be selected and handled appropriately in vitro for immunotherapy, which results in tenfold more antigen-specific T cells than existing vaccine regimens used alone [79]. Nine individuals who had been diagnosed with metastatic CC were investigated for ACT by Stevanovic et al. [80].

Prophylactic and therapeutic vaccines to control CC
The two vaccines designed to prevent CC are composed of empty VLPs generated by expression systems for recombinant capsid antigen L1 [91,92]. VLPs do not contain viral DNA and these are non-infectious. Recombinant L1 VLPs strongly resemble authentic papilloma virions in terms of structure and immunogenicity [93]. Recent clinical studies with L1 VLP-based vaccines have primarily revolved around the prevention of cancer associated with the female genital tract, including CC. L1 VLP vaccines may also prevent additional HPV-associated cancers. Although prophylactic vaccines against high-risk HPV types have been highly effective. Unresolved issues may shape the future of CC vaccines. Current vaccines must contain intact L1 VLPs to promote the generation of strong, type-restricted, and neutralizing IgG antibody responses. The minimum antibody titer needed for protection in human is unknown. Though, the importance of antibody responses to prophylactic VLP vaccination has been suggested by studies in preclinical models. A denatured L1 VLP does not induce protection in the cotton tail rabbit papillomavirus or canine oral papillomavirus (COPV) challenge models, suggesting the importance of L1 conformation-dependent antibody responses [94]. Increased COPV L1-specific antibody titers were associated with protection from papillomas, suggesting a certain level of humoral immunity associated with protection against papilloma viruses [95]. Prophylactic VLP-based vaccines do not eliminate pre-existing persistent infections [96]. However, therapeutic vaccination could have an immediate impact in reducing the incidence of CC. L1 and L2 late capsid proteins are not expressed in the basal cells that harbor infection in precancerous tissues or cancerous tissues, [97] suggesting that they are not good targets for therapeutic vaccines [98]. Conversely, E6 and E7 proteins are very promising target proteins for therapeutic vaccines, as these are the only viral proteins constitutively expressed in CC cells and are required to maintain the disease phenotype [99]. Table 3 gives brief description on prophylactic and therapeutic vaccines intended for CC [100]. Although prophylactic HPV vaccines are available in the market whereas HPV remained prevalent globally due to the unevenness of vaccination. Therefore, there is a great demand for effective treatment of established HPV infection, and therapeutic HPV vaccines are expected to become an effective method to treat HPV. In an attempt to eliminate existing HPV infection or lesions, therapeutic vaccines mainly trigger robust cytotoxic T lymphocyte (CTL) responses against antigens that can be continuously expressed in abnormal cells, thereby enhancing the antitumor effect.

Challenges with HPV vaccinations
CC poses a very critical public health challenge to Sub-Saharan African (SSA) countries, where it is the major cause of female cancer deaths [101]. However, implementing HPV vaccination at this region has been met with stiff challenges ranging from sociocultural belief, logistical difficulties, and lack of proper financing. Notably, the most developed countries have a high uptake of the HPV vaccine, countries like the United States still have many challenges associated with the low uptake of the HPV vaccine [102]. This challenges ranges from misconceptions about HPV and the HPV vaccine and socio-economic status. Myriads of challenges hampering the implementation of HPV vaccination also abounds in the Middle East and North Africa, where there are no organized CC screening systems in place [103]. Thus, it results in CC cases being diagnosed at advanced stages of the diseases. Other factors hampering the implementation of HPV vaccination include cultural and religious sensitivities, weak health systems and infrastructures, political instability, financial constraints, and limited public health funding amid competing priorities. In Europe, challenges with HPV vaccination uptake were associated with concerns about the long-term effectiveness and possible side effects of the vaccine [104]. Despite these myriads of issues and challenges, studies have shown that education can be used as a tool towards influencing the acceptance of the HPV vaccine. In addition, the provision of the right information to clients by healthcare workers would go a long way in helping to improv the acceptance of HPV vaccination [105]. Massive Public Health education on issues relating to CC and its causal relationship with HPV is needed towards the successful implementation of the HPV Vaccination programs among all age groups. Governments needs to invest more into this primary preventive initiative, and donor agencies and supporting partners all need to forge a formidable front at combating the menace of this deadly disease especially in Low and Middle-income countries. International efforts at mitigating the impact of this disease through the Global Alliance for Vaccine and Immunization (GAVI Alliance) are also strongly encouraged. Large pharmaceutical companies producing these vaccines could also be supported towards subsidizing the price of these vaccines to making them more affordable to poorer countries. The involvement of the men folks as active partners in the advocacy for HPV vaccination initiatives is also strongly advocated [106].

Current landscape and future perspectives
Phase III worldwide studies are being carried out by significant pharmaceutical companies, who are close to receiving regulatory approval for their preventative vaccinations. The first generation of preventive vaccinations is almost finished and there is still room for an enhanced second generation of vaccines to addresses some of the first generation's flaws (production and distribution costs CC, often referred to as the "silent killer," is a disease of unequal access. The new SAGE recommendation is supported by worries over the poor HPV vaccine access into the immunization programs and low population covered in less developed and poor countries. CC is almost entirely preventable. CC, the fourth most frequent type of cancer in women worldwide with 90% of these women living in low-and middle-income countries, is caused by sexually transmitted HPV in more than 95% of cases [107]. CC vaccine produced in India (Serum Institute of India) "Ceravac" is an in-house manufactured vaccine for CC brought on by the human papillomavirus (HPV), will cost between Rs 200 and 400 per dosage and it may be available within few months. Depending upon age, the vaccination is administered in a two-or three-dose course. Currently, there are two foreign-produced HPV vaccinations on the private market: Cervarix by Glaxo Smithkline and Gardasil by Merck. Serum India's arrival is anticipated to lower prices for HPV vaccines. 483.5 million Indian women over the age of 15 are at risk for developing CC. According to current figures, 123,907 women are given a CC diagnosis annually, and 77,348 of them passed away from the condition. In India, CC is the second most common malignancy in women. CC should be included in the National Immunization Mission, according to the standing technical subcommittee of the National Technical Advisory Group on Immunization (NTAGI) (NIM) [108]. Even while the progress being made on HPV vaccines, it is encouraging. Women who had HPV infection before vaccine administration or post vaccination, should continue identification and routine screening [109]. Additional study is required to determine the duration of protection brought on by these vaccinations, the necessity of booster shots, and the impact on occurrence and prevalence of the HPV types covered by the vaccines. Additional information regarding various HPV vaccine types for various populations, safety and pregnancy outcomes, immunogenicity of concurrent administration with other vaccines, and other information are needed to be considered. After the current common varieties of HPV, further research must be performed to investigate the effectiveness in patients (both sexes) with age ˃ 26 years, the function of regular HPV vaccination in men to prevent the genital warts using additional, and rarer HPV types. Future research is needed to determine CC screening methods, safe sexual behavior, and more economic analysis. The target group for vaccination will be prepubescent females between the ages of 9 and 10 because vaccines will be effective as a preventative before viral exposure. However, this will cause cultural and societal problems. Epidemiological research on the logistics, long-term effectiveness, and economics of widespread HPV-vaccination (women) are urgently needed in nations like India. Various vaccine delivery systems such as different routes of immunization and physical/chemical delivery methods have been used in cancer therapy with the goal to induce immunity against tumor-associated antigens. Two basic delivery approaches including physical delivery to achieve higher levels of antigen production and formulation with microparticles to target antigen-presenting cells (APCs) have demonstrated to be effective in animal models. New developments in vaccine delivery systems will improve the efficiency of clinical trials in the near future. Among them, nanoparticles (NPs) such as dendrimers, polymeric NPs, metallic NPs, magnetic NPs and quantum dots have emerged as effective vaccine adjuvants for infectious diseases and cancer therapy [110].

Conclusion
CC is the most deadly, life threatening, and lethal cancer in women globally and a serious concern for health care personnel particularly in poor or less developed countries.
These counties have poor access to drugs and vaccines. Comparing vaccines with chemotherapy, immunotherapy using specific vaccines at the right dose is relatively beneficial and patient friendly. Various approaches have been used to achieve simplicity, scalability, translation to industrial production, effectiveness and economic burden. Despite several benefits of these vaccines, there were still limitations and gaps for further investigation and research at the preclinical and clinical stage. Luckily, the developed prophylactic HPV-vaccines successfully saved lives by treating HPV infected patients in the last ten years after HPV vaccine introduction. It was estimated that about 95% of those who received the vaccine demonstrated high safety and efficacy in preventing persistent infections of precancerous lesions. The widespread use is constrained by a number of restrictions, including the limited HPV types for prevention and expensive manufacturing. Preclinical or clinical trials for a variety of HPV L2-based and second-generation preventive vaccinations are currently being conducted. Additionally, the approved HPV-vaccines are unable to execute effectively in patients already infected with HPV and develop the accompanying cancers. Therefore, therapeutic HPV-vaccines are urgently needed to minimize the incidence of CC in these infected patients. Research on the immune evasion mechanism and the cancer microenvironment is still largely undefined. In our opinion, the development of successful vaccines in the future will be facilitated by initiatives to improve vaccine design, including the use of effective adjuvants, as more knowledge about the immune mechanisms against HPV infection becomes available. Although many of the novel vaccines described in this review are reported to be immunogenic in humans, it may be necessary to continue developing novel adjuvants to establish clinical efficacy.