HPV is an essential member of the Papillomaviridae family, encompassing a wide range of primitive DNA viruses distributed among various host species (9). Phylogenetically, HPVs are categorized into five genera: Alpha, Beta, Gamma, Mu, and Nu, based on the homology of the nucleotide sequence in the L1 gene (9, 10). Alpha and Beta HPVs are the most commonly studied and have distinctive biological properties. Beta viruses frequently infect cutaneous epithelium and are involved in non-melanoma skin cancers, whereas Alpha genera-HPVs infect both mucosal and genital epithelium and are the causes of many anogenital cancers in humans and primates (11, 12). The Alpha HPVs are further grouped into low-risk HPVs (LR-HPVs) and high-risk HPVs (HR-HPVs) depending on their capacity to cause cancer. The LR-HPVs include HPV-6/11/42/43/44. HPV 6 and 11 cause most cases of genital warts (13). HPV-16/18/31/33/35/39/45/51/52/56/58/59/68 are members of HR-HPVs (14, 15).

The HPV viral particles are composed of a single double-stranded DNA molecule, with approximately 8,000 base pairs (bp), and a protein capsid containing 72 pentameric capsomers (16). The genomes of all HPV types contain about eight open-reading frames (ORFs) that can be functionally divided into the early (E), the late (L), and the long control region (LCR) ( Figure 1 ). Specifically, the E gene encodes the early proteins (E1–E7) responsible for viral replication and oncogenesis. The L gene encodes the structural proteins (L1–L2) necessary for virion assembly. The non-coding region is thought to be the most highly variable part of the viral genome (17). The structural capsid proteins of HPVs are composed of two key proteins, major basic L1 and minor basic L2, respectively encoded by L1 and L2 genes. L1 genomes display differently amongst HPV types, while the L2 genomic sequence is relatively conserved (18). The HPV capsid is made up of 360 copies of major (L1) and 72 copies of minor (L2) capsid proteins (19). The L1 protein is self-assembled into the virus-like particles (VLP), while the L2 protein, as a minor structural molecule, is unable to form VLPs. Although the L2 protein is not responsible for binding to and entering cells during the process of infection, it plays a key role in enhancing L1 assembly into VLPs and viral genome encapsidation (20). Once entering host cells, the viral E6 and E7 oncoproteins are responsible for viral oncogenesis by affecting the function of tumor suppressors p53 and pRB (21). HPV types that cause genital warts are called low-risk, while those types that cause cervical cancer are considered high-risk (22). High-risk HPV genomes are commonly integrated into the host genome in most progressive cancer cases. Conversely, low-risk HPV genomes are commonly identified extra-chromosomally in benign and low-grade lesions, which are rarely found in tumors (23).

Currently, five commercial HPV vaccines targeting high-risk HPV types have been approved for prophylactic use. All licensed vaccines contain L1 VLP. One of the primary distinctions among these authorized HPV vaccines is their level of valency.

Cervarix^® was licensed by the European Medicines Agency (EMA) in 2007 and by the U.S. Food and Drug Administration (FDA) in 2009. Cervarix^® is effective in defending against HPV-16 and HPV-18, responsible for nearly 70% of cervical cancers (24). Cervarix^® is formulated in a proprietary AS04 adjuvant containing aluminum hydroxide combined with a Toll-like receptor 4 (TLR4) ligand, 3-O-desacyl-4’-monophosphoryl lipid A (MPL) (25, 26). It is indicated for use in females aged 10 through 25 years for the prevention of persistent infection, premalignant cervical lesions, and cervical cancer caused by HPVs in the US.

Gardasil^®, a quadrivalent HPV vaccine, is the first commercially available HPV vaccine approved in 2006 for people 9 through 26 years of age for the prevention of diseases caused by HPV infection (27). Aside from HPV-16 and 18, Gardasil^® also protects against HPV-6 and -11 infections, which cause approximately 90% of genital warts. Gardasil^® is adsorbed on an amorphous aluminum hydroxyphosphate sulfate adjuvant (28).

Gardasil 9^®, licensed by the FDA in 2014, offers broader protection by covering five additional HPV types (HPV-31, 33, 45, 53, and 58) that might account for about 20% of cervical cancer cases (29). Gardasil 9^® is indicated for females aged 9 through 45 years for the prevention of HPV-associated diseases. Additionally, Gardasil 9^® is indicated in males 9 through 45 years of age for the prevention of anal, oropharyngeal, head neck cancers, anal precancerous, dysplastic lesions, and genital warts caused by HR-HPVs (30). Gardasil 9^® is formulated with amorphous aluminum hydroxyphosphate sulfate (AAHS) as an adjuvant (31).

Recently, two more bivalent HPV vaccines (HPV-16 and 18) have been approved in China for protection against HPV infection-induced precancerous lesions or high-grade genital lesions (32–34). Clinical data revealed that the 2-valent HPV vaccine showed favorable immunogenicity, safety, and a positive seroconvertsion rate (35–37). Both bivalent vaccines contain twice the amount of HPV-16 L1 protein as the Cervarix^® vaccine and use different adjuvants and antigen expression systems. A comprehensive comparison of marketed HPV vaccines is shown in Table 1 .

The immunogenicity and efficacy of five licensed HPV vaccines have been summarized elsewhere (33, 38, 39). Briefly, all marketed HPV vaccines elicit potent humoral immune responses and exhibited long-lasting protective efficacy after vaccination (34, 40–43). Serum antibody responses are the major indicators used for evaluating immunogenicity evoked by HPV vaccines in clinical trials, and comparison of vaccine-induced antibody levels or B-cell responses between HPV vaccines have been discussed (44–46). Nevertheless, definitive correlation between humoral immune responses and efficacy of HPV vaccines has not yet been reported (47). Prophylactic HPV vaccines based on L1-VLPs trigger T-cell responses, which showed no correlation with protection, probably due to the lack of L1 expression in HPV-infected cells (48). Since the protection efficacy of HPV vaccines are characterized in clinical trials by prevention of cervical cancer and intraepithelial neoplasia, immune profiles other than serum neutralizing antibody titers should be clarified, such as characteristics of Fc-effector functions, mucosal immunity and T-cell responses against HPV early proteins. For example, a comparative study between Gardasil^® and Cervarix^® indicated that the differences in antibody Fc-effector functions might contribute to post-infection protection (49). Immune correlate of protection by HPV vaccination needs further elucidation based on more clinical data with functional characteristic of serum or mucosal antibodies, as well as cellular immune responses (50).

Despite the high efficacy against HPV infection and HPV-related disease, low vaccination coverage of prophylactic HPV vaccines still exists. Particularly, most low- and middle-income countries incompletely implement HPV vaccination programs (51). The high cost, limited supply, and delivery requirements of the cold chain make it unavailable to people in these countries (52). Additionally, the biosafety considerations played a critical role in the successful development and deployment of HPV vaccines. Based on high quality data from very large studies, HPV vaccines exhibit favorable safety profiles (53, 54). The implementation of a robust safety monitoring system for HPV vaccines, which consistently updated data and promptly disclosed findings, would enhance accurate understanding and comprehensive awareness regarding HPV vaccine safety, mitigate vaccine hesitancy, and increase vaccine acceptance and coverage rates.

There are 12 high-risk HPV types that are involved in cancer, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 (55). The Gardasil 9^® vaccine, which contains the highest number of high-risk HPV serotypes in circulation worldwide, does not fully cover all high-risk HPV types. To achieve complete protection, the development of prophylactic HPV vaccines should further enlarge their protective extent to target more life-threatening HPV types. The following section will summarize the progress in the development of prophylactic L1-VLP-based HPV vaccines currently in clinical trials ( Table 2 ).

All 9-valent HPV vaccines target the same highly carcinogenic HPV types (HPV-6/11/16/18/31/33/45/52/58). Shanghai Bovax Biotechnology Co., Ltd. has created a 9-valent HPV vaccine that exhibits high immunogenicity, good tolerance, and non-inferiority to Gardasil 4 in terms of both immunogenicity and safety (56). However, data regarding other HPV vaccines has not been made public. The 11-valent HPV vaccine, developed by the National Vaccine and Serum Institute (China), covered two additional HPV types (HPV-59/68) on top of the commonly used HPV types in 9-valent vaccines. The underlying development strategy might be linked to the findings that HPV-59 and 68 are potentially associated with genital cancerogenesis (57). SCT1000, developed by Sinocelltech Ltd, is a recombinant 14-valent L1-VLP-based HPV vaccine designed to prevent HPV infection and HPV-related cancers. It covers five additional HPV types (35, 39, 51, 56, and 59) compared with Gardasil 9^®. SCT1000 is currently being investigated in a Phase III study for HPV infection and HPV-related carcinoma (NCT06041061). A 15-valent vaccine developed by Chengda Bio targets HPV-68, in addition to the 14 HPV types covered by SCT1000, is currently in development for the prevention of HPV-infected diseases.

Prophylactic HPV vaccines scarcely eradicate preexisting infection because L1 capsid proteins are not expressed on the surface of infected basal epithelial cells (58). Therefore, the development of prophylactic HPV vaccines is not an effective treatment approach for people already infected with HPV (59). Cellular immune responses to HPV antigens play important roles in viral clearance and anticancer immune responses. Ineffective cellular immune responses to HPV-16 E2, E6, or E7 peptides involve infection persistence or disease progression in low-grade intraepithelial lesion (LSIL) patients (60). HPV-16 E6 and E7-specific cellular immune responses facilitate the regression of HPV-16-associated lesions (61). Similarly, the percentages of IFN-γ positive enzyme-linked immunospot (ELISpot) responses to HPV-16 E6 and E7 are significantly increased among women with recently resolved HPV infection versus those with persistent HPV-16 infection in the cervix (62).

Therapeutic HPV vaccines are developed to stimulate cell-mediated immunity for the clearance of infection (63, 64). Among HPV early proteins (E1, E2, E6, and E7) and late proteins (L1, L2), HPV E6 and E7 proteins are constitutively expressed in both premalignant and invasive lesions and play key roles in the development of malignancy (65). Further, E6 and E7 can activate antigen-specific CD8⁺ or CD4⁺ T cells and finally evoke cellular immune responses (66–68). Therefore, E6 and E7 proteins are considered ideal antigens for therapeutic HPV vaccines. TLR agonists alone or mixed with other components are commonly tested adjuvants for therapeutic HPV vaccines (69–72). Recently developed new adjuvants, such as α-Galactosylceramide (α-GalCer), manganese (Mn4⁺)-doped silica nanoparticles (Mn4⁺-SNPs), and very small-size proteoliposomes (VSSP) (73–75), might play roles in reinforcing the efficacy of therapeutic HPV vaccines.

Prophylactic HPV vaccines are mainly protein-based VLPs. Therapeutic HPV vaccines are primarily based on nucleic acid, bacterial/viral vector, protein/peptide or whole cells (76). To date, no therapeutic HPV vaccine has been licensed. Safety profiles and vaccine efficacy for therapeutic HPV vaccines tested in clinical trials are summarized in Table 3 . A complete regression of lesions triggered by therapeutic HPV vaccines has been reported, with a regression rate ranging from 17.4% to 89.4% in female patients (54, 78, 81, 82, 84–86, 88, 89). The clearance of HPV DNA in infected cells is considered a predictor of vaccine efficacy, as the presence of HPV DNA at the cervical site is often associated with histologic and cytological changes in cervical intraepithelial neoplasia (CIN) (77, 79–81, 83, 87, 90).

In addition, vaccine antigen-specific T-cell responses were comprehensively evaluated. In a Phase I trial, AMV002 vaccination induced E6 and/or E7 specific cellular immune response in 83.3% subjects (78). In a Phase II study of, GX-188E, 67.3% of patients showed histopathologic regression (<CIN1) and 55.8% of patients with viral clearance 36 weeks after GX-188E treatment. Importantly, E6 and E7-specific IFN-γ positive T-cell response in the patients with HPV clearance presented significant increases compared with patients without clearance (81). In a Phase I study of Candida Skin Test, a colorless extract of Candida albicans FDA-approved for use as a human adjuvant, vaccine-induced cellular immunity against HPV-16 E6 was detected in 65% of vaccine recipients. The percentages of Th1 cells and Th2 cells increased significantly, while no change was observed regarding the percentage of Tregs after two doses of vaccine administration.

Several therapeutic HPV vaccines in clinical trials indicated a correlation between vaccine efficacy and vaccine-induced cellular immune responses. The results of Phase I/II of IGMKK16E7, the first oral immunotherapeutic vaccine, showed the number of HPV-16 E7–specific interferon-γ producing cells in blood increased with response levels (stable disease, partial, and complete responses) (89). In a Phase IIb trial of VGX-3100, vaccine-induced T-cell responses to E6 were positively correlated with clinical outcomes rather than E7, while higher frequencies of HPV-specific CD8⁺ CD137⁺ T cells indicated better outcomes (82). Similarly, a recombinant MVA E2 vaccine induced antigen-specific cytotoxic T-cell responses among all vaccinated patients in a Phase III study (85). Accordingly, 90% of female and 100% of male participants exhibited complete regression of lesions 14 weeks after vaccination (85).

L1 protein is a commonly used vaccine target for prophylactic HPV vaccines (91). Preliminary data indicated that L1 protein triggers both humoral and cellular immune responses (92, 93). Indeed, L1 capsomeres induced tumor regression in mice via activation of antigen-specific cytotoxic T lymphocytes (94). In HPV-positive oropharyngeal cancer (OPC) patients, E6, E7, and L1 capsid proteins were recognized by HPV-specific CD8⁺ and CD4⁺ T cells (95). However, L1-based HPV vaccines failed to be effective treatment approaches due to the lack of constitutive expression in premalignant and invasive lesions (58). Currently, many therapeutic vaccines in clinical trials primarily target E6 and E7 proteins (96). Alternative antigenic targets, such as E2 and L2, are also in clinical tests ( Table 4 ).

HPV DNA integration and diminished E2 expression were essential for the oncogenic transition of HPV-infected cells (108). E2 controlled HPV DNA transcription and suppressed E6 and E7 expression (96). As a result, E2 was probably irrelevant to the therapeutic vaccine target. However, E2-specific T-cell responses were related to the lack of progression into high-grade intraepithelial lesions (HSILs) (60). Accordingly, more potent E2-specific T-cell responses have been reported in patients with resolved cervical dysplasia, supporting the role of E2 as a candidate for vaccine antigens in immunotherapy of pre-cancerous cervical lesions (109). E1 is continuously expressed in the basal epithelial cells after HPV infection and plays an important role in virus replication (110). E1 is crucial for carcinogenesis (111). However, E1 might not be an ideal target for vaccine antigens due to its ubiquitination and rapid degradation attributes (112, 113).

The minor capsid protein L2 is a promising candidate for an HPV vaccine since the linear neutralizing epitopes on the L2 N-terminus are well conserved across several HPV genotypes (114). In mouse models, HPV-16 L2 antigen-induced neutralizing antibodies protected against infectious HPV-16/45 challenges (115, 116). However, L2 might be less immunogenic since it is unable to form VLPs (117). Therefore, strategies to enhance the immunogenicity of L2 protein have become attractive (114, 118, 119). Notably, peptide antigens (120, 121) and HSP70-based fusion proteins are widely tested in both preclinical and clinical studies (122, 123).

Cervarix^® and Gardasil^® were formulated with AS04 and amorphous aluminum hydroxyphosphate sulfate, respectively (124, 125). Alum-based adjuvants could induce Th2-biased immune responses (126). The immunostimulant MPL, one component of the AS04 adjuvant, activates innate immune responses through Toll-like receptor 4 (TLR4) and induces a mixed Th1/Th2 immune response (127). Participants received Cervarix^® displayed stronger humoral and CD4⁺ T cell responses against HPV-16 and 18 than patients received Gardasil^® (125, 128, 129). Meanwhile, distinctive immune profiles induced by HPV vaccines formulated with an Alum-based adjuvant, or AS04, have been reported (129, 130).

Adjuvants may affect the strength and durability of antigen-specific immune responses. Exploratory studies of novel adjuvants used alone or in combination have highlighted their potential use in HPV vaccines ( Table 4 ). Bacterial-derived components, such as Beclin-1 (105), AS04 (NLX+Alum) (25), CIA06B (nontoxic derivative of lipopolysaccharide (CIA05) + Alum) (100), rough LPS (R-LPS) (98), and bacterial enzymatic combinatorial chemistry (BECC)-derived TLR4 agonists/Alum (70), are widely utilized in exploratory studies.

In addition, novel molecules with distinctive contributions to immunogenicity are being evaluated in preclinical studies of HPV vaccines, such as CpG oligodeoxynucleotide (CpG) (71, 104), polyinosinic-polycytidylic acid (Poly I:C) (104), α-Galactosylceramide (α-GalCer) (73), very small size proteoliposomes (VSSP) (75), poly di (carboxylatoethylphenoxy) phosphazene (PCEP) (99), c-di-AMP (101), S-540956 (a CpG Oligonucleotide) (72), the metal ions like manganese (Mn4⁺)-doped silica nanoparticles (Mn4⁺-SNPs) (74), plant extracts like Bai Hua She She Cao (BHSSC) (102), and mRNA-encoded STING^V155M (106), Candida skin test reagent (Extract of Candida albicans) (103).

Although many novel adjuvants have demonstrated their unique efficacy in modulating immune responses in mouse models, only a few have been licensed for use in humans to date, primarily due to safety concerns. Therefore, novel HPV vaccine adjuvants require further safety and efficacy validation in clinical trials.

HPVs employ multiple strategies to evade the host’s immune response, including interference with antigen presentation, downregulation of antigen production, inhibition of antiviral molecules, suppression of type 1 T helper (Th1) cell immune responses, and promotion of regulatory T cell (Treg) responses (84, 131). Therapeutic HPV vaccine-evoked antigen-specific T cells are the key to tumor cell elimination and rely primarily on the loss of inhibitory signals in the tumor microenvironment (TME). Thus, the combination of therapeutic HPV vaccines and immune checkpoint inhibitors may show synergistic effects in tumor regression.

Preclinical studies have demonstrated that therapeutic HPV vaccines, such as adenovirus delivery vectors carrying modified HPV-16 E6 and E7 genes, HPV-16 E7 long peptide with DCs, and the Lm-LLO fused-E6 vaccine (Lm-LLO-E6), significantly promoted tumor regression and survival rate in tumor-bearing mouse models when used with a programmed death-ligand 1 (PD-L1) inhibitor (132–134). The clinical trials of therapeutic HPV vaccines combined with ICB therapeutic modalities are mainly in Phase I/II. Preliminary data show good efficacy and safety, and further verification in later clinical trials is needed. The current status of combination trials is listed in Table 5 .

L1 VLPs-based HPV vaccines have been approved primarily for the prevention of cervical cancer in populations without preexisting HPV infection. Studies have suggested an indispensable role of HPV L1 VLP vaccines as adjuvant therapy after surgical treatment for patients suffering from HPV-related clinical diseases such as recurrent respiratory papillomatosis (RRP) (140) and cervical intraepithelial neoplasia (CIN) (141). The data reported that surgical intervention and HPV vaccination combination therapy can effectively reduce the recurrence and severity of respiratory papillomatosis (142). Likewise, another study has reported that HPV vaccination significantly prolongs the intervals between surgical procedures and reduces the number of procedures in the majority of RRP patients (143). Most CINs are caused by HPV infection (144) and might progress to cervical cancer (145). CIN 1 rarely progresses to malignancy with a high potential for regression. Instead, CIN 2 and 3, as the high-grade lesions, become highly cancerous with a lower potential for regression (146, 147). Studies have reported that vaccination with the quadrivalent HPV vaccine in patients after loop electrosurgical excision procedure (LEEP) treatment significantly prevents the recurrence of CIN2–3 related to vaccine HPV types compared with non-HPV-vaccinated patients (148). These data demonstrate that HPV vaccination plays a critical role as adjuvant therapy in HPV-related disorders other than cancer.