Currently, the research and development technology for prophylactic human papillomavirus (HPV) vaccines is becoming increasingly mature (related reading: "Current Status of Research and Application of Prophylactic Human Papillomavirus (HPV) Vaccines"). With the increase in vaccine coverage, the incidence of HPV-related diseases is gradually decreasing. However, the prognosis for patients with advanced and recurrent cervical cancer remains poor, with a high mortality rate. Therefore, the development of therapeutic HPV vaccines for the treatment of cervical intraepithelial lesions and cervical cancer caused by HPV infection has become a current research hotspot.

Therapeutic vaccines differ from preventive vaccines as they aim to induce cell-mediated immune responses, rather than producing neutralizing antibodies. HPV therapeutic vaccines are a novel class of vaccines that break the immune tolerance in chronic infections, rebuild or enhance immune responses, thus treating HPV infections, blocking HPV-induced precancerous lesions, and promoting the regression of lesion tissues. Generally speaking, therapeutic vaccines contain E6 and E7 antigens in various forms and deliver these antigens to local antigen-presenting cells (APCs), further stimulating antigen expression through MHC Class I and MHC Class II molecules, resulting in the generation of CD8+ cytotoxic T cell (CTL) or CD4+ helper T cell (Th) responses. Before being recognized by MHC Class I molecules on APCs, E6 and E7 antigens are first processed and digested by proteases in APCs into smaller peptide segments. It is important to note that not all of these peptides can successfully bind to MHC molecules and be recognized by specific T cells. Only some peptides containing specific antigenic epitopes can bind to MHC molecules with high affinity and interact with the receptors (TCR) of specific T cells that can trigger immune responses. Currently, most therapeutic HPV vaccines aim to induce immune responses against the E7 antigen, as E7 antigen immunogenicity is better than E6 antigen in preclinical models.

Current Status of Therapeutic HPV Vaccine Research

During the process of cervical intraepithelial lesions and carcinogenesis caused by chronic HPV infection, there is often persistent expression of the oncogenic proteins E6 and E7. Therefore, there are currently various therapeutic vaccines targeting the antigens E6 and E7, including live vector vaccines, protein or peptide vaccines, nucleic acid vaccines, and cell vaccines. Many of these vaccine development efforts have entered preclinical models and clinical trial stages.

1. Live Attenuated Bacterial Vector Vaccines

Live vector vaccines are classified into bacterial or viral vector vaccines. These vectors replicate within the body, promoting the dissemination of antigens. Live vector vaccines possess high immunogenicity, capable of generating strong cellular and humoral immune responses. They can also present the E6 and E7 antigens to antigen-presenting cells (APCs), facilitating the recognition of antigens by major histocompatibility complex (MHC) Class I and II molecules. However, live vector vaccines pose potential safety risks, especially in immunocompromised individuals. Additionally, the efficacy of the immune response generated upon repeated administration of the same live vector vaccine is limited.

Bacterial vectors primarily include Listeria monocytogenes (Lm), Lactobacillus species, and Salmonella, among others. Listeria is a Gram-positive intracellular pathogen that can induce innate and acquired immunity, protecting the body against viral and parasitic infections. As a vector in immunotherapy, it has the following advantages: first, it can infect macrophages without being captured, possessing the ability to deliver antigens through MHC I and MHC II pathways; second, it escapes phagosome phagocytosis by secreting listeriolysin (LLO). Lm is a widely used and representative vector, and currently, Lm-based vaccines have been tested in numerous clinical trials. ADXSⅡ-001 is an inactivated attenuated Listeria vector vaccine based on the HPV16 E7 antigen, which can be used to treat HPV-related oropharyngeal cancer, cervical cancer, and high-grade cervical intraepithelial neoplasia patients. Lactobacillus casei (L. casei) is another common bacterial vector. In the vaccine preparation process, the gene encoding HPV-E7 is transduced into L. casei to express the E7 protein, which can be recognized by APCs in the mucosa and presented to CTLs to induce cell-mediated immunity.

Viral vectors primarily include adenoviruses, adeno-associated viruses, alphaviruses, lentiviruses, and vaccinia viruses. These vectors utilize gene engineering methods to carry gene sequences encoding HPV E6/E7, enabling the delivery of target antigens. TG4001 is a vaccine based on a recombinant modified vaccinia virus Ankara (MVA) vector, which has been tested in a clinical trial for the treatment of HPV-16-related CIN2/3 patients. The preparation of RNA replicon vaccines utilizes RNA alphaviruses as vectors, including Sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), and Semliki forest virus (SFV). RNA replicons are capable of self-replication, thereby inducing sustained antigen expression and increasing immunogenicity.

2. Protein or Peptide Vaccines

Peptides and proteins in peptide and protein vaccines are isolated from HPV antigens. After being processed by dendritic cells (DCs), they are presented to MHC I or II molecules, effectively stimulating immune responses from CD8+ or CD4+ T cells. Peptide and protein vaccines are safe, stable, and easy to produce, demonstrating significant potential for application.

Protein vaccines tend to have weaker immunogenicity, primarily inducing antibody production through MHC II antigen presentation rather than generating CTL immune responses. In recent years, research has focused on protein vaccines such as TA-CIN, GTL001 (ProCervix), and TVGV-1. TACIN is an HPV-16-E6/E7/L2 fusion protein vaccine that, combined with the adjuvant GPI-0100 and cisplatin, induced therapeutic immune responses in HPV-16 E6/E7-positive mouse models. GTL001 is a therapeutic vaccine that fuses recombinant HPV-16/18-E7 protein to the catalytically inactive Bordetella pertussis CyaA. A phase I trial showed that in HPV-16/18-infected female patients with normal cytology, two treatments with 600μg GTL001 administered intradermally at a 6-week interval, combined with local application of imiquimod ointment at the injection site, effectively cleared HPV-16/18 after two treatments. TVGV-1 is a fusion protein based on the HPV-16 E7 antigen that, in preclinical trials, induced strong antigen-specific T cell immune responses when combined with adjuvants such as GPI-0100 and CpG oligodeoxynucleotides.

Peptide vaccines have weak immunogenicity and often require the assistance of immune adjuvants or liposomes to enhance their therapeutic efficacy. Peptide vaccines are MHC-specific, and the specific epitopes of the patient's antigens need to be determined before treatment. There are various types of peptide vaccines, and their core is the use of HPV-16 E6 and/or E7 peptides as antigens. Representative vaccines mainly include PepCan, HPV16-SLP, GL-0810, PDS0101, and DPX-E7. PepCan contains four synthetic peptides and adds Candida albicans extract as an adjuvant, which can be administered through intradermal injection. HPV16-SLP is a synthetic long peptide vaccine. Studies have suggested that vaccination with this vaccine after chemotherapy with carboplatin-paclitaxel can promote the T-cell response induced by the vaccine in HPV-16 E6/E7-positive cervical cancer patients. GL-0810 is an HPV-16-specific peptide vaccine prepared by adding granulocyte-macrophage colony-stimulating factor and Montanide adjuvant for subcutaneous vaccination to treat recurrent/metastatic squamous cell carcinoma of the head and neck. PDS0101 is developed based on HPV-16 E6/E7 peptides, and its safety and tolerability in the treatment of high-risk HPV infection and CIN 1 female patients are being evaluated (NCT02065973, Phase I). Additionally, DPX-E7 is also being used in clinical trials for HPV-16 E7 and HLA-A*02-positive oropharyngeal cancer, cervical cancer, and anal cancer patients (NCT02865135, Phase Ib/II).

3. Nucleic Acid Vaccines

Nucleic acid vaccines include DNA and RNA vaccines, which can be administered multiple times. DNA vaccines are advantageous in terms of safety, stability, and ease of mass production, with longer-lasting antigen expression within cells compared to RNA vaccines. They are commonly administered through intramuscular injection. However, their main disadvantage is that during intramuscular injection, they can easily be taken up by cardiomyocytes, blocking the induction of sustained immunogenicity. Additionally, the efficiency of DNA delivery and transfection in vivo is limited, resulting in weaker immunogenicity. With the development of genetic engineering techniques, auxiliary factors can be added to the vector antigen sequence to promote effective expression.

Chemokine-based antigen DNA vaccines are a novel type of DNA tumor vaccine. By fusing the HPV16 E7 gene with the chemokine SLC and the Fc fragment of IgG, SLC can attract DCs, T, and B lymphocytes. DCs can efficiently capture the fused antigen through Fc receptors, promoting antigen presentation. This DNA vaccine can induce a robust anti-tumor immune response through CD4+ and CD8+ T cell-dependent pathways.

Another vaccine, pNGVL4a-CRT/E7, consists of the pNGVL4a expression vector encoding HPV16 E7 and the Calreticulin (CRT) sequence. CRT is a member of the heat shock protein family. This vaccine has been used to treat high-grade cervical intraepithelial neoplasia. A preliminary study involving 32 HPV16-positive CIN2/3 patients showed the feasibility, safety, good tolerability, and potential clinical value of this vaccine. Furthermore, a randomized, double-blind Phase IIb clinical trial in HPV16 and HPV18-positive CIN2/3 patients demonstrated that the DNA vaccine VGX-3100, encoding HPV16 and HPV18 E6 and E7 gene fragments, is the first therapeutic vaccine effective in HPV16 and HPV18-positive CIN 2/3 patients, offering a non-surgical treatment option for CIN2/3 patients.

Compared to DNA vaccines, RNA vaccines are not integrated into the host's genome and do not induce cellular transformation, but they are less stable. Currently, there have been no reported clinical trials on the use of RNA vaccines for the treatment of HPV-related diseases.

4.Cell Vaccines

The safety and immunogenicity of dendritic cell vaccines based on HPV16/18 E7 antigens have been demonstrated in phase I clinical trials and have entered phase II clinical trials. Some scholars have administered autologous DCs sensitized with recombinant HPV16/18 E7 antigens to 10 cervical cancer patients with stage IB or IIA disease. After immunization, it was found that all subjects developed specific CD4+ T cell responses and antibody responses against E7. Additionally, adoptive cell immunotherapy techniques are also evolving, and research results have shown that after injecting tumor-infiltrating T lymphocytes into 9 patients with metastatic cervical cancer, 2 patients achieved complete regression of their lesions.

Immunotherapy using vaccines is a promising approach for cancer treatment. In patients with cervical lesions and cervical cancer, the use of therapeutic vaccines is closely related to the outcome of precancerous lesions and clinical prognosis. Compared to traditional treatments, therapeutic vaccines have significant advantages and feasibility, offering good prospects for development. However, the research progress of HPV therapeutic vaccines has been relatively slow, still in the stage of animal experiments or clinical trials. The mechanisms of therapeutic vaccines are complex. For instance, tumor patients often have weakened immune function, making it difficult for vaccines to effectively activate the immune system. Additionally, issues such as the safety of vaccines, immune evasion of tumor cells, and HPV-infected cells must also be taken into consideration.

References

[1] Feng Jie, Gao Lingjuan, Xu Haoqin, Wu Yulin, Lin Ning, Yan Linping, Zhong Tianying. Research Progress on Therapeutic Polypeptide Vaccines for HPV [J]. Chinese Journal of Family Planning, 2018, 26(01): 63-69.

[2] Zeng Ming, Deng Liehua. New Progress in Therapeutic Vaccines for HPV Infection [J]. Chinese Journal of Dermatology and Venereology, 2020, 37(04): 369-374+4.

[3] Zhao Shuang, Zhao Fanghui. Research Progress on Therapeutic Vaccines for HPV [J]. Chinese Journal of Preventive Medicine, 2018, 52(5): 5.

About the Author

Xiaomichong, a pharmaceutical quality researcher, has been committed to pharmaceutical quality research and drug analysis method validation for a long time. Currently employed by a large domestic pharmaceutical research and development company, she is engaged in drug inspection and analysis as well as method validation.