Safety, Efficacy, and Immunogenicity of Therapeutic Vaccines for Patients with High-Grade Cervical Intraepithelial Neoplasia (CIN 2/3) Associated with Human Papillomavirus: A Systematic Review

Simple Summary We conducted a systematic review using 16 trials (RCTs and NRCTs) across eight countries and three continents; which included 672 patients with high-grade CIN associated with HPV. To the best of our knowledge, this is the first synthesis related to the safety, efficacy, and immunogenicity of therapeutic vaccines for the treatment of patients with high-grade CIN associated with HPV. The findings from the trials revealed that therapeutic vaccines can be considered promising for these injuries. However, there are still only a few phase III RCTs, and a better understanding of the specificities of different vaccine compositions and delivery systems and pathways to prevent viral escape mechanisms are needed. Overall, the results of this systematic review indicate that the therapeutic vaccines that are being developed for the treatment of CIN 2/3 are safe and well tolerated and that most trigger only systemic symptoms classified as mild or moderate that improve spontaneously in a short period of time. Considering the inconsistent results among phase I and II trials involving different therapeutic vaccines, our findings provide some clarification and have implications for multiple stakeholders. Abstract Despite the knowledge that HPV is responsible for high-grade CIN and cervical cancer, little is known about the use of therapeutic vaccines as a treatment. We aimed to synthesize and critically evaluate the evidence from clinical trials on the safety, efficacy, and immunogenicity of therapeutic vaccines in the treatment of patients with high-grade CIN associated with HPV. A systematic review of clinical trials adhering to the PRISMA 2020 statement in MEDLINE/PubMed, Embase, CENTRAL Cochrane, Web of Science, Scopus, and LILACS was undertaken, with no data or language restrictions. Primary endpoints related to the safety, efficacy, and immunogenicity of these vaccines were assessed by reviewing the adverse/toxic effects associated with the therapeutic vaccine administration via histopathological regression of the lesion and/or regression of the lesion size and via viral clearance and through the immunological response of individuals who received treatment compared to those who did not or before and after receiving the vaccine, respectively. A total of 1184 studies were identified, and 16 met all the criteria. Overall, the therapeutic vaccines were heterogeneous regarding their formulation, dose, intervention protocol, and routes of administration, making a meta-analysis unfeasible. In most studies (n = 15), the vaccines were safe and well tolerated, with clinical efficacy regarding the lesions and histopathological regression or viral clearance. In addition, eleven studies showed favorable immunological responses against HPV, and seven studies showed a positive correlation between immunogenicity and the clinical response, indicating promising results that should be further investigated. In summary, therapeutic vaccines, although urgently needed to avoid progression of CIN 2/3 patients, still present sparse data, requiring greater investments in a well-designed phase III RCT.


Simple Summary:
We conducted a systematic review using 16 trials (RCTs and NRCTs) across eight countries and three continents; which included 672 patients with high-grade CIN associated with HPV.To the best of our knowledge, this is the first synthesis related to the safety, efficacy, and immunogenicity of therapeutic vaccines for the treatment of patients with high-grade CIN associated with HPV.The findings from the trials revealed that therapeutic vaccines can be considered promising for these injuries.However, there are still only a few phase III RCTs, and a better understanding of the specificities of different vaccine compositions and delivery systems and pathways to prevent viral escape mechanisms are needed.Overall, the results of this systematic review indicate that the therapeutic vaccines that are being developed for the treatment of CIN 2/3 are safe and well tolerated and that most trigger only systemic symptoms classified as mild or moderate that improve spontaneously in a short period of time.Considering the inconsistent results among phase I and II trials involving different therapeutic vaccines, our findings provide some clarification and have implications for multiple stakeholders.
Abstract: Despite the knowledge that HPV is responsible for high-grade CIN and cervical cancer, little is known about the use of therapeutic vaccines as a treatment.We aimed to synthesize and critically evaluate the evidence from clinical trials on the safety, efficacy, and immunogenicity of therapeutic vaccines in the treatment of patients with high-grade CIN associated with HPV.A systematic review of clinical trials adhering to the PRISMA 2020 statement in MEDLINE/PubMed, Embase, CENTRAL Cochrane, Web of Science, Scopus, and LILACS was undertaken, with no data or language restrictions.Primary endpoints related to the safety, efficacy, and immunogenicity of these vaccines were assessed by reviewing the adverse/toxic effects associated with the therapeutic vaccine administration via histopathological regression of the lesion and/or regression of the lesion size and via viral clearance and through the immunological response of individuals who received treatment compared to those who did not or before and after receiving the vaccine, respectively.A total of 1184 studies were identified, and 16 met all the criteria.Overall, the therapeutic vaccines were heterogeneous regarding their formulation, dose, intervention protocol, and routes of administration, making a meta-analysis unfeasible.In most studies (n = 15), the vaccines were safe and well tolerated, with clinical efficacy regarding the lesions and histopathological regression or viral clearance.In addition, eleven studies showed favorable immunological responses against HPV, and seven studies showed a positive correlation between immunogenicity and the clinical response, indicating promising results that should be further investigated.In summary, therapeutic vaccines, although urgently needed to

Introduction
It is estimated that 80% of sexually active people will be affected by at least one human papilloma virus (HPV) type at some point in life [1,2], indicating a high prevalence of the virus, especially in underdeveloped countries.Although the presence of the virus does not imply cancer, it is a necessary condition for high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer, constituting a major global public health problem [3,4], mainly due to the high morbidity and mortality of HPV-related diseases [5].
High-risk HPVs, especially types 16 and 18, are present in approximately 100% of cases of CIN 2 and 3 and may progress to several types of cancer, especially cervical cancer [6].The treatments currently used for CIN may result in recurrent or persistent infections because of the incomplete elimination of the virus.Additionally, conventional therapies are associated with reproductive and psychological impairments, with negative impacts on the quality of life of patients [7][8][9].Furthermore, a larger problem is also the lack of access to these vaccines in low-income countries, particularly since therapy for CIN is less widely available in low-income countries [10].
The prevention of neoplasms occurs through prophylactic vaccines, which, although safe and effective, cannot eliminate already established lesions and have no effect on already established lesions caused by HPV [10][11][12].HPV uses aggressive immune evasion strategies via the expression of oncoproteins E6 and E7, which induce the hyperproliferation of keratinocytes, making the virus less liable to an immune attack.These oncoproteins are involved in the disruption of cell cycle checkpoints and the modulation of the host immune response, blocking gene expression in these cells and favoring an immunosuppressive environment.Additionally, it interferes in the activation of adaptive immune cells and in the release of pro-inflammatory cytokines.These mechanisms allow viral replication, promoting cancer development [13].The products of oncogenic viruses cause specific T cell responses, in addition to responses by other cells of the innate and acquired immune systems; these responses function to eradicate viruses.Tumors induced by viruses are considered to be the most immunogenic because they originate from antigens that are foreign to our bodies [14].
Therapeutic vaccines aim to stimulate cell-mediated immune responses against specific antigens and promote the death of infected cells.They are used in cases where the disease is already established, originating from persistent or recurrent lesions, to promote the regression of precancerous lesions and the remission of invasive cancer [15][16][17].Currently, there are several therapeutic vaccine candidates in preclinical studies as well as in clinical trials; however, there has been no demonstration of efficacy in phase 3 studies, which support the licensing of these vaccines yet.
Despite a growing body of literature pointing to the development of therapeutic vaccines for CIN, there is still no systematic review that synthesizes the state of the art of clinical trials simultaneously taking into account the three endpoints (safety, efficacy, and immunogenicity) of these therapeutic vaccines.Hence, the purpose of this study was to synthesize and critically evaluate the evidence from clinical trials on the safety, efficacy, and immunogenicity of therapeutic vaccines for the treatment of patients with CIN 2/3 associated with HPV.  .There was no date or language restriction in the search strategy.In addition to the aforementioned databases, secondary searches were performed in other sources, such as ClinicalTrials.gov(National Institutes of Health-NIH, Bethesda, MD, USA); Brazilian Clinical Trials Registry (ReBEC); The British Library; Scientific Electronic Library Online-SciELO; and Google Scholar.The references in the included studies were manually analyzed to find additional relevant studies.All the steps of this systematic review were independently performed by 2 researchers (CAG and LCLJ).The reference manager EndNote™ was used to store, organize, and exclude duplicates to ensure a systematic and manageable search.The database searches were conducted in November 2018 and updated in August 2023.Supplementary Table S1 shows the complete search strategy for each database.
Primary studies (phase I, II, or III RCT or quasi-experimental studies, NRCT) conducted with patients with high-grade CIN 2/3 associated with HPV and no associated immunodeficiency were included.In addition, we included trials in which patients received therapeutic vaccines, regardless of the route of administration, and that evaluated the safety, efficacy, and immunogenicity endpoints through comparisons with a control group (placebo or standard treatment) or with each patient's own parameters before and after vaccine administration (for NRCT).Studies that evaluated only 1 or 2 of the 3 proposed endpoints (safety, efficacy, and immunogenicity) or evaluated the vaccine in men were excluded.
The primary endpoints evaluated were the safety, efficacy, and immunogenicity of the therapeutic vaccines.Therefore, we assessed safety by analyzing the adverse and toxic effects associated with the administration of therapeutic vaccines.Efficacy was assessed via histopathological regression of the lesion and/or regression of the lesion size as well as via viral clearance.The immunogenicity of therapeutic vaccines was assessed by comparing the immunological adaptative response in serum or in peripheral blood mononuclear cells and target tissue of individuals who received treatment with that of those who did not or by comparing factors in individuals before and after receiving the vaccine.
Initially, the selection of articles was based on information contained in the title and abstract of each study and was independently performed by 2 researchers (CAG and LCLJ).Full-text reading of the articles was independently performed by the researchers after the initial selection.Cohen's kappa coefficient was used to estimate the index of agreement between the 2 evaluators in each review phase (selection, extraction, and methodological evaluation of the included studies).Discrepancies were resolved through discussions at each stage, and a consensus was achieved, with acceptable inter-rater reliability (k = 0.93).A third researcher (RCCPS) verified the eligibility of the included studies.

Data Analysis
Two researchers (CAG and LCLJ) independently extracted the following data using pre-established and adapted tools [20][21][22][23]: (I) study characteristics (article title, country of origin of the study authors, year of publication, study host institution (hospital, university; research center, multicenter study, or study in a single institution), conflicts of interest, and funding); (II) methodological characteristics (study design, trial register, location, study objective or research question or hypotheses, sample characteristics, e.g., sample size, inclusion and exclusion criteria, ethical issues, baseline characteristics of the experimental and control groups, recruitment method, randomization, masking, intervention protocol, drop-outs, duration of follow-up, procedures for data collection, outcomes and statistical analysis); (III) main findings and implications for clinical practice; and (IV) limitations and conclusions (Supplementary Table S2).For data extraction, 2 Microsoft Excel ® (version 16.67 for Mac Book pro) spreadsheets were prepared by the researchers (CAG and LCLJ) to synthesize the data from the included studies.After this phase, the data were compiled into a single spreadsheet before proceeding with analyses.In addition, if data were missing or unclear or the nature of the intervention was unclear, we contacted the corresponding author of the publication via email for clarification.
The internal validity and risk of bias of RCT were assessed using the revised Cochrane Risk-Of-Bias tool for randomized trials (RoB 2) [24], which assesses the risk of bias in 5 domains: (1) randomization process; (2) deviations from the intended interventions; (3) missing outcome data; (4) measurement of the outcome; and (5) selection of the reported result [24].The RoB 2 classifies risk of bias as follows: (1) low risk of bias: low risk of bias for all domains; (2) some concerns: some concerns in at least 1 domain, but no high risk of bias for any domain; and (3) high risk of bias: high risk of bias in at least 1 domain or some concerns for multiple domains, substantially reducing the confidence in the result [24].To assess NRCTs, the Risk of Bias in Non-randomized Studies of Interventions (ROBINS-I) was used [25].The ROBINS-I comprises 7 chronologically arranged bias domains (preintervention, at intervention, and post-intervention), and the domain level and overall risk of bias are classified as low, moderate, serious, or critical [25].Using both tools (RoB2 and ROBINS-I), the same 2 reviewers (CAG and LCLJ) independently assessed the risk of bias for each included study.Discrepancies were resolved through a discussion at each stage, and a consensus was achieved, with acceptable inter-rater reliability (k = 0.93).A third researcher (RCCPS) verified the eligibility of the included studies.
We assessed the heterogeneity between the 2 estimates using an interaction test.The Q test was used to assess between-study heterogeneity, and the I 2 statistic, which expresses the percentage of the total observed variability due to study heterogeneity, was calculated [26].The I 2 values were set relative to zero, with values ranging from 0% to 100%, [27] where 0% indicates no heterogeneity and 25%, 50%, and 75% indicate low, moderate, and high heterogeneities, respectively [26][27][28].
It is noteworthy that the study protocol for this systematic review has been published elsewhere [29] in order to ensure transparency and methodological rigor, as recommended by the Cochrane Collaboration.

Role of the Funding Source
There was no funding source for this study.The first author and the corresponding author had full access to all the study data.

Results
The search strategy yielded 1184 studies: 960 from the databases, 35 from clinical trial records, and 189 from additional sources.After the exclusion of 81 duplicates using EndNote™, 914 studies were selected for the title and abstract selection process.Most studies were excluded (804) based on pre-established inclusion and exclusion criteria.Among the 110 studies retained, 87 were excluded because they did not address the guiding question of the review, resulting in 23 articles for full, exhaustive reading.Among the twenty-three eligible studies, seven were excluded because they did not address the three primary endpoints simultaneously (safety, efficacy, and immunogenicity).Thus, 16 studies (5 RCTs and 11 NRCTs) were selected for data extraction, methodological evaluation, and quantitative analysis (Figure 1).None of the 189 studies from the additional sources were included in this review due to them not answering the research question.
It is noteworthy that the study protocol for this systematic review has been published elsewhere [29] in order to ensure transparency and methodological rigor, as recommended by the Cochrane Collaboration.

Role of the Funding Source
There was no funding source for this study.The first author and the corresponding author had full access to all the study data.

Results
The search strategy yielded 1184 studies: 960 from the databases, 35 from clinical trial records, and 189 from additional sources.After the exclusion of 81 duplicates using EndNote™, 914 studies were selected for the title and abstract selection process.Most studies were excluded (804) based on pre-established inclusion and exclusion criteria.Among the 110 studies retained, 87 were excluded because they did not address the guiding question of the review, resulting in 23 articles for full, exhaustive reading.Among the twenty-three eligible studies, seven were excluded because they did not address the three primary endpoints simultaneously (safety, efficacy, and immunogenicity).Thus, 16 studies (5 RCTs and 11 NRCTs) were selected for data extraction, methodological evaluation, and quantitative analysis (Figure 1).None of the 189 studies from the additional sources were included in this review due to them not answering the research question.

Risk of Bias
The internal validity and risk of bias of the RCTs were assessed using the revised Cochrane RoB 2 [24] (Figure 2).Among the five RCT, two (40%) had a low risk of bias [31,42], the others two [33,38] had a high risk of bias, and one had some concerns [45].Only two RCTs [31,42] appropriately described the method to generate the randomization sequence, allocation confidentiality, and the blinding of the participants and the team involved.
The analysis of the risk of bias in non-randomized studies of intervention using the ROBINS-I tool [25] (Table 3) indicated that 10 of the 11 studies had a serious risk of bias and that only 1 study [43] had a moderate risk.The studies were classified as such mainly due to participant selection bias.In addition, all eleven trials had a moderate risk of bias because they were not randomized, and five trials had a risk of bias because they did not adequately report the dropouts and/or missing data [34,40,41,43,44], in addition to not properly describing allocation confidentiality or the blinding of the participants and the team involved.Of these studies, only two [35,39] reported blinding.
and that only 1 study [43] had a moderate risk.The studies were classified as such mainly due to participant selection bias.In addition, all eleven trials had a moderate risk of bias because they were not randomized, and five trials had a risk of bias because they did not adequately report the dropouts and/or missing data [34,40,41,43,44], in addition to not properly describing allocation confidentiality or the blinding of the participants and the team involved.Of these studies, only two [35,39] reported blinding.

Endpoints
Overall, it can be inferred that most patients who had adverse events related to the use of vaccines experienced mild or moderate events.There were no serious, i.e., grade 3 or higher, adverse events.In addition, there were no deaths associated with vaccine administration.The adverse events improved spontaneously, and in most studies, there were no losses associated with adverse events, except in two trials [38,42], one of which [38] was stopped prematurely.The symptoms were more associated with local events, such as pruritus, oedema, erythema, and pain than with the injection and included some systemic signs, such as flu symptoms, headaches, fatigue, and nausea.Most authors concluded that the vaccines were safe and well tolerated.
Most studies included patients with CIN 2/3 to evaluate the efficacy of therapeutic vaccines, except for three studies [39,40,45] that included only patients with CIN 3.All the included studies evaluated, at different time points, the histological regression of patients who received the vaccine.However, seven studies [34,35,[38][39][40]43,44] did not evaluate lesion size regression.Viral clearance was evaluated by most studies, except for two trials [35,39].Of the sixteen studies selected, four [31,42,44,45] correlated the efficacy of vaccines with the age of the patients, and only two trials [30,31] correlated the results with smoking and the use of oral contraceptives.Acronyms: * ROBINS-I, Risk of Bias In Non-randomized Studies of Intervention [41].** The global judgement of ROBINS-I is systematized and attributed as follows: Low risk of bias, in which the study is comparable to a well-designed randomized trial (the study is considered as having a low risk of bias for all domains).Moderate risk of bias: the study is consistent with a non-randomized study design, but cannot be considered comparable to a well-designed randomized study (in this case, the study is considered as having a low or moderate risk of bias for all domains).Serious risk of bias: the study has some important problems (the study is considered as having a low or moderate risk of bias for most domains, but presents a serious risk of bias in at least one of the domains).Critical risk of bias: the study is too problematic to provide any evidence (the study is considered as having a critical risk of bias in at least one domain).No information: when no information is available to provide grounds to any judgment of the risk of bias (missing information on one or more domains) [41].Two reviewers gave identical assessments in each domain in an independent manner.
Figure 3 briefly illustrates the main endpoints of therapeutic vaccines evaluated in this systematic review.
Since the therapeutic vaccines were heterogeneous regarding their formulation, dose, intervention protocol, and routes of administration, making meta-analysis unfeasible, therefore, we considered it more appropriate to present a qualitative synthesis of the data.
Figure 3 briefly illustrates the main endpoints of therapeutic vaccines evaluated in this systematic review.Since the therapeutic vaccines were heterogeneous regarding their formulation, dose, intervention protocol, and routes of administration, making meta-analysis unfeasible, therefore, we considered it more appropriate to present a qualitative synthesis of the data.

Discussion
To the best of our knowledge, this is the first systematic review to critically evaluate evidence from clinical trials simultaneously taking into account the three endpoints (safety, efficacy, and immunogenicity) of therapeutic vaccines for the treatment of patients with high-grade CIN associated with HPV.In addition, the trials investigating therapeutic vaccines reported promising results for the treatment of these lesions; however, a greater understanding of the kinetics of the immune response and of how to prevent viral escape mechanisms is required.
Overall, the trials have shown good safety and tolerability with respect to these vaccines [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45].Most patients had adverse reactions, but they were classified as mild or moderate; there were no grade 3 or more adverse reactions.There were no cases of death, and most participants did not discontinue the treatment due to adverse events.The adverse events most associated with vaccines were pruritus at the injection site and some systemic symptoms similar to flu symptoms.
Recent studies have also demonstrated the safety and tolerability of prophylactic HPV vaccines [46][47][48][49].Such vaccines generate neutralizing antibodies against the proteins that form virus capsids, preventing pathologies that may originate from these viruses.This parameter is also frequently evaluated for therapeutic vaccines.Other researchers [50] determined the toxicity, safety, immunogenicity, and efficacy of the subcutaneous HPV16 SLP vaccine in 20 patients with advanced or recurrent HPV16-induced gynecological carcinoma.The authors concluded that the vaccine was well tolerated, as determined by the absence of systemic toxicity above grade II and the presence of only transient flu-like symptoms.They associated these data with favorable immunogenicity by generating a broad T cell response associated with IFNγ, TNFα, IL-5, and/or IL-10 in 84.6% of the patients.However, the clinical response did not induce tumor regression or prevent a progressive disease, demonstrating the need to administer these vaccines combined with conventional treatments such as chemotherapy.Bagarazzi et al. [14] conducted a phase I trial with 18 women previously treated for CIN 2/3 and described promising results related to the safety, tolerability, and immunogenicity of the VGX-3100 vaccine synthesized with HPV 16/18 and administered via in vivo electroporation.In addition, the immunization was well tolerated, with reports of mild reactions at the injection site and without severe or grade 3 and 4 adverse events, and no dose-limiting toxicity was observed.In addition to providing safety data, the study performed flow cytometry analysis, where it was possible to identify the induction of HPV-specific CD8+ T cells associated with granzyme B and perforin, which exhibited complete cytolytic functionality at all the doses tested.These data indicate that this vaccine was able to generate robust immune responses to high-risk HPV antigens, favouring the elimination of infected cells and the subsequent regression of the dysplastic process [14].
The safety, efficacy, and immunogenicity of a therapeutic vaccine (ISA101, associated or not with imiquimod) were also evaluated by Van Poelgeest et al. [52] in a multicenter RCT with 43 patients with vulvar and vaginal intraepithelial neoplasia.In the trial, clinical responses induced by the vaccine, in a period of 3 months, were observed in 18 of 34 patients (53%) (95% CI: 35.1-70.2),and at 12 months after vaccination, the same parameter was observed in 15 of 29 patients (52%) (95% CI: 32.5-70.6), of whom 8 had a complete histological response.Clearance occurred in all the patients.An immune response mediated by CD8+ T cells was observed in all the patients and was significantly stronger in the patients with complete responses, indicating a correlation between the immune response with the clinical response and efficacy for the treatment of high-grade vaginal and vulvar neoplasms associated with HPV 16.However, although the efficacy and immunogenicity data are promising, the safety of the vaccine needs to be better evaluated because 18% of patients in each group had symptoms, for example, allergic reactions most likely associated with the peptide used in the study, and long-term reactions at the injection site (with oedema still present after 12 months).Ten patients experienced severe adverse events (such as the development of ulcers at the injection site, which in some cases, required special interventions).To reduce adverse events in future studies, the authors suggested the use of alternative adjuvants to replace the one used in the study (Montanide), dose-response studies, and vaccination combined with imiquimod on the lesion.
Another study that showed promising results related to the efficacy and immunogenicity of therapeutic vaccines was conducted using a peptide vaccine combined with Freund's incomplete adjuvant in 20 women with high-grade HPV-16-positive vulvar intraepithelial neoplasia [53].The results of the study [53] indicated local adverse events, such as oedema, in 100% of the patients and systemic events, such as fever, in 64% of the patients; however, none of the adverse events exceeded grade 2, and these symptoms improved within 3 months after the last vaccination.In this same period, twelve of twenty patients (60%) (95% CI: 36-81) showed clinical responses; of these, five women showed complete lesion regression, and four showed complete HPV-16 clearance.In the follow-up period, i.e., 12 months after vaccination, 15 of the 19 patients (79%) had clinical responses (95% CI: 54-94), and 9 (47%) had a complete response (95% CI: 24-71) that was maintained at 24 months of follow-up.All the patients developed vaccine-induced T cell responses.The post hoc analyses suggested that the five patients who presented a complete response at 3 months had a significantly stronger CD4+ response and a broader response to interferon-γ CD8+ T cells than those in the patients without a complete response, demonstrating that there may be a correlation between the clinical response and immunogenicity [53].
One of the most promising studies [54] regarding the development of therapeutic vaccines against HPV was a phase III RCT in which the authors evaluated the safety, efficacy, and immunogenicity of the MVA E2 recombinant vaccine to treat intraepithelial lesions associated with HPV infection.For this, the trial evaluated 1176 women and 180 men who received the vaccine directly in their uterus, urethra, vulva, or anus.The results showed that 89.3% of female patients had complete lesion elimination after treatment with MVA E2 and that another 2.4% with CIN I exhibited histopathological regression.In the men, all the lesions were eliminated.Efficacy could also be assessed according to total HPV DNA clearance after treatment in 83% of all the patients included in the study.In addition, the vaccine did not present significant adverse events, and an excellent immune response was observed through the development of antibodies against HPV and the generation of a specific cytotoxic response against cells transformed by HPV.These data suggest that therapeutic vaccination with the MVA E2 vaccine is promising to promote immunogenicity and efficacy, in addition to demonstrating safety for the synthesis of therapeutic vaccines against HPV when applied locally [54].
In a phase II RCT, 19 patients with HPV-16-associated vulvar intraepithelial neoplasia (grade 2/3) were administered imiquimod followed by TA-CIN vaccination [55].The results of that trial showed that complete histological regression occurred in 32% of the patients evaluated on week 10, increasing to 58% on week 20 and to 63% on week 52.The clearance of HPV 16 was evaluated on week 52, when it was possible to determine that 36% of the lesions had been cleared.On week 20, there was a significant increase in the local infiltration of CD8 and CD4 T cells in the responder patients who showed lesion regression.In contrast, the non-responder patients who had histological lesions showed an increase in regulatory T cells [55].
The safety, efficacy, and immunogenicity of therapeutic vaccines have also been evaluated for the treatment of cervical cancer.Reuschenbach et al. [56] evaluated these parameters using a peptide vaccine in 26 patients with advanced cancers over a period of 6 months.The study did not show severe adverse events associated with the use of the vaccine, and there were no dose-limiting toxicities.The development of CD4+ T cells was observed in 14 of the 20 patients, the presence of CD8+ T cells was detected in 5 of the 20 patients, and antibodies were detected in 14 of the 20 patients.The efficacy related to the tumor response was evaluated in 14 patients, of whom 64% had a stable disease as the best overall response, and 36% developed progressive disease.Thus, the authors [56] suggested that the vaccine induces cellular and humoral immune responses, does not cause severe toxicities, and provides promising results for the development of immunotherapy for cervical cancer.
Most of the studies reported in our systematic review evaluated immunogenicity only in peripheral blood cells and not in the tissue with the lesion.A positive association between immunogenicity and clinical efficacy was found in six studies [32,37,39,40,42,44].However, despite showing promising results related to the safety, efficacy, and immunogenicity of therapeutic vaccines against HPV, some variables relevant to the success of cancer treatment, such as smoking, oral contraceptive use, age, and number of births, were controlled and/or correlated in a few studies, and none of them associated the results with the number of partners or age when they first had sexual intercourse [30,31,35,44].This association may be relevant for the development of effective vaccines because all these factors can affect the progression of the disease as well as the immune response [57].
The assessment of immune cells is crucial in clinical research involving therapeutic vaccines to evaluate their efficacy by establishing the number and types of immune cells that infiltrated the tissue and blood circulation to determine the kinetics of these cells in the response against HPV, including the possible escape mechanisms of these viruses [39,58].It is important to consider that disease progression is not solely related to viral infection, but to several genetic and environmental factors as well, as they modulate the immunological responses, inflammatory processes, and physiopathological mechanisms of many diseases, including cancer.Toll-like receptors are important markers of the innate immune response; they start the immunological response, releasing pro-inflammatory cytokines, causing the inhibition of its expression by microorganisms such as HPV that may promote events related to carcinogenesis [59,60].Furthermore, the development of a therapeutic vaccine may be associated with several factors, such as a significant immune response, effective measures that control viral escape mechanisms, and the presence or absence of a correlation between immunogenicity and clinical efficacy and immunosuppression associated with these infections [61].
Strategies for the development of effective vaccines should be designed to overcome some limitations inherent to clinical trials, such as blocking local immunosuppression via in situ vaccination or using a therapeutic vaccine associated with antagonistic antibodies against inhibitory receptors such as CTLA-4 or agonist antibodies against costimulatory molecules such as CD137.The use of efficient adjuvants, such as electroporation, facilitates an increase in the permeability of the cell membrane and the consequent release of antigens and causes damage at the injection site by acting as an adjuvant and promoting the inflammatory response, the depletion of Tregs through the use of anti-CD25 antibody, and the use of TLR agonists and simultaneous administration via systemic and intralesional routes [62,63].The intralesional route induces a more intense recruitment of intraepithelial CD8+ T cells than other routes of administration do [43], and this association with other routes of administration, such as the intramuscular route, is beneficial for the immunogenicity of therapeutic vaccines.
Since CIN 2/3 are precursor lesions of cervical cancer that have high rates of recurrence, morbidity, and mortality, the best immunological and clinical responses are observed in patients with precursor lesions.This fact is linked to systemic and local changes associated with cancer and might be related to deleterious effects on immunocompetent T cells [64,65].Besides the strategies to produce therapeutic vaccines included in the Results section, it is noteworthy the promising results from pre-clinical trials such as those of oncolytic virus which promote tumoral reduction and significantly improve the immunological system.Such studies might also be considered as venues for effective results against cervical cancer [66][67][68].
Taken together, the studies conducted with therapeutic vaccines report promising results for the treatment of these lesions; however, a greater understanding of the kinetics of the immune response and how to prevent viral escape mechanisms and improve the associated immunogenicity and clinical response in a safe manner is required.Our study confirms the need for future RCTs, especially phase III RCTs, with a high methodological quality (i.e., larger samples, randomized, double-blind, placebo-controlled phase III, and longer follow-ups with less attrition) and standardization to enable precise comparison.We recognize some limitations of our study.First, the methodological limitations of the articles included in the analysis may have affected the outcomes, and therefore, the data should be interpreted with caution.Second, most of the studies were phase I and II trials.Third, there were significant differences in the protocols, doses, types of vaccine, definitions of clinical response, virologic clearance, and follow-up times among the studies, making meta-analysis unfeasible.Another limitation of the present systematic review is due to the use of very rigorous selection criteria, i.e., including the three endpoints (safety, efficacy and immunogenicity) simultaneously, which made us exclude large and important trials in this field from the sample, which addressed one or two of the outcomes (although such trials were addressed in the Discussion section due to their great contribution to this area of knowledge).Finally, we recommend that future systematic reviews in this field take into account at least two of the outcomes reported here in order to expand the sample of potentially included studies for evaluation.

Conclusions
In summary, we conclude that vaccines under development for the treatment of highgrade CIN (2/3) are safe and well tolerated and that most triggered only mild or moderate systemic symptoms, which spontaneously improved in a short time.The authors reported promising results for the variables related to efficacy and immunogenicity, both in the activation of T cells and development of HPV-specific antibodies and in significant results related to lesion regression, viral clearance, and/or histopathological lesion regression.These findings should be interpreted with caution because they cannot yet be considered conclusive, as most studies had a small sample size, had a low methodological quality (most of the studies were NRC and/or phase I or II RCTs), and had a relatively short follow-up period.
Thus, there is a need for future well-designed large-scale phase III RCTs with a high methodological quality, with a follow-up period of more than 1 year and with strategies to control confounding variables that may interfere with the endpoints.In addition, future studies should focus on approaches that have been underexplored, but that have yielded favorable results with respect to the other neoplasms associated with HPV.Such approaches include, for example, other routes of administration, such as administration at the lesion site, and the inclusion of other high-grade HPV types to better understand viral escape mechanisms and the development of immune responses associated with clinical efficacy.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers16030672/s1,Table S1: Search strategy; Table S2: Data extraction form; Table S3: Characteristics and protocols of therapeutic vaccines used in the studies included in the systematic review.

Figure 1 .
Figure 1.PRISMA flowchart for study selection.* Reasons for exclusion; (P) population: NIC I patients, patients with immunosuppression associated with HPV, pathologies associated with HPV other than cervical intraepithelial, studies with male subjects, and studies with animals; (I) intervention: vaccination program or prophylactic vaccine; (S) study design: reviews, specialist

Figure 1 .
Figure 1.PRISMA flowchart for study selection.* Reasons for exclusion; (P) population: NIC I patients, patients with immunosuppression associated with HPV, pathologies associated with HPV other than cervical intraepithelial, studies with male subjects, and studies with animals; (I) intervention: vaccination program or prophylactic vaccine; (S) study design: reviews, specialist opinions, theses, dissertations, and observational studies.** RCT = Randomized Controlled Trial; NCRT = Non-Randomized Controlled Trial.

Figure 2 .
Figure 2. Summary of risk-of-bias judgements of Randomized Controlled Trials included according to the revised Cochrane Risk-of-Bias tool for randomized trials (RoB 2).(A) Internal validity and risk-of-bias assessment of clinical trials according to the RoB 2. (B) Percentage of risk of bias among clinical trials according to the domains of the revised Cochrane Risk-of-Bias tool for randomized trials (RoB 2).Plus symbol (+) indicates low risk of bias; negative symbol (−) indicates some concerns; (X) indicates high risk of bias.Two reviewers gave identical assessments in each domain in an independent manner.

Figure 2 .
Figure 2. Summary of risk-of-bias judgements of Randomized Controlled Trials included according to the revised Cochrane Risk-of-Bias tool for randomized trials (RoB 2).(A) Internal validity and risk-of-bias assessment of clinical trials according to the RoB 2. (B) Percentage of risk of bias among clinical trials according to the domains of the revised Cochrane Risk-of-Bias tool for randomized trials (RoB 2).Plus symbol (+) indicates low risk of bias; negative symbol (−) indicates some concerns; (X) indicates high risk of bias.Two reviewers gave identical assessments in each domain in an independent manner.

Figure 3 .
Figure 3. Efficacy, immunogenicity, and safety of the therapeutic vaccine used in the studies.The most frequent parameters in the studies are expressed in % of patients who presented them.(A) Panel Safety compares mild/moderate and severe adverse events, considering the % of patients presenting the most frequent events of the category.(B) Panel Efficacy compares viral reduction and histological regression.(C) Panel Immunogenicity compares Tcell/IFN responses.* Data presented in the study did not allow precise percentage descriptions.** Parameter not evaluated in the study.

Figure 3 .
Figure 3. Efficacy, immunogenicity, and safety of the therapeutic vaccine used in the studies.The most frequent parameters in the studies are expressed in % of patients who presented them.(A) Panel Safety compares mild/moderate and severe adverse events, considering the % of patients presenting the most frequent events of the category.(B) Panel Efficacy compares viral reduction and histological regression.(C) Panel Immunogenicity compares Tcell/IFN responses.* Data presented in the study did not allow precise percentage descriptions.** Parameter not evaluated in the study.

Author
Contributions: C.A.G. and L.C.L.-J.contributed to the conception and design of the work.C.A.G. and L.C.L.-J.defined the concepts and search items, data extraction process as well as methodological appraisal of the studies, acquisition, or interpretation of data for the work: C.A.G., L.C.L.-J., R.C.C.P.S., P.C.M.M., A.Z. and G.P.-d.-S.Data analysis, critical appraisal, and GRADE was performed by L.C.L.-J.All authors contributed to drafting the work and revising it critically for important intellectual content and gave final approval of the version to be published.C.A.G. and L.C.L.-J., agreed on questions related to the accuracy or integrity of the work.C.A.G. and L.C.L.-J.are the guarantors and are joint first authors.The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the ITAIPU Binacional-Fundação Parque Tecnológico de Itaipu (FPTI) (Process Number: 0003/2018).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 2 .
Characteristic and primary endpoints (efficacy, safety, and immunogenicity) of the studies included in the systematic review.

Table 3 .
Risk-of-bias judgements of non-randomized studies of interventions via ROBINS-I.