Peptide-based nanovaccines for cervical cancer treatment | IJN - Dove Medical Press

Introduction

Cervical cancer is a malignant tumor formed in the stem of the uterus.¹ Human papillomavirus (HPV) is a pathogen that causes fatal cervical cancer and related cancerous diseases; it has been detected in 99.7% of all cervical cancers.² Two viral proteins, E6 and E7, are pivotal in triggering infected cells to undergo oncogenesis with uncontrolled proliferation, unrestricted telomerase activity, and subsequently cervical cancer growth.³ HPV infection can cause cancer in the cervix, but also in the vagina, vulva, penis, and oropharynx.¹ Typically, the cervix comprises squamous cells on the outer layer and columnar gland cells on the inner side. Carcinogenesis mainly occurs at the junction of these two cell types, forming squamous cell carcinoma (>90%) or adenocarcinoma.^4,5 Specifically, the initial infection may cause dysplasia, namely, cervical intraepithelial neoplasia (CIN) or adenocarcinoma, in situ. If the infection persists and cannot be cleared by the body's immune system, it leads to an invasive cancer.⁵

The treatment of cervical cancer depends on how developed the cancer is: from cervical carcinoma in situ to stages I–IV. Primary treatments for cervical cancer include surgery, radiation therapy and/or chemotherapy. For early or small-scale carcinoma, surgery and radiation therapy can virtually eradicate cell infection and achieve recovery. For cervical cancer stages IB and IIA, interventional chemotherapy is often required for co-treatment. Commonly used chemotherapy drugs include cisplatin, carboplatin, paclitaxel (Taxol^®), and topotecan.⁵ Unfortunately, radiation and chemotherapy often cause many side effects due to their limited targeting affinity towards tumor cells. Vaccination against HPV infection and cervical cancer offers a promising new treatment option without the risk of significant adverse effects.⁶

Traditional vaccines have been based on live-attenuated or killed microorganisms derived from pathogenic viruses or bacteria, which directly trigger the immune system to secrete neutralizing antibodies against pathogenic antigens.^6–8 Importantly, currently available HPV prophylactic subunit vaccines, which induce antibody-based responses, cannot be used for effective cancer eradication.^9,10 Fortunately, new vaccines targeting cellular immune responses are being developed and can be applied for cancer treatment.^10–12 However, the use of cancer cell homogenates or cancer-associated proteins is associated with a risk of inducing autoimmune responses. Therefore, peptide-based antigens are being extensively assessed in cancer vaccine designs: several of these vaccines have reached Phase III clinical trials.^13,14

As an effective vaccine candidate platform, peptide-based vaccines have the potential to induce cellular immune responses and eliminate cervical cancer using non-autoimmunogenic antigen sequences derived from cancer-associated proteins. Peptide-based vaccines typically have increased stability during storage and transport, and they can be rapidly obtained through a convenient and straightforward preparation method.¹⁵ Nevertheless, the low immunogenicity of peptide-based vaccines and poor stability in vivo have been primary drawbacks. One of the most promising strategies to overcome these problems is the introduction of nanosized delivery systems and adjuvants (immune stimulants) to generate so-called nanovaccines.^16,17 Nanoparticles mediate the stimulatory effect on the immune system through their particle properties, such as size, charge, hydrophobicity, and surface-anchored targeting moieties.¹⁸ Optimized nanoparticles can more effectively deliver bound antigen to antigen presenting cells (APCs), thereby triggering a durable and efficient immune response.¹⁷

This review details the current global burden of cervical cancer, mechanisms of HPV infection, associated cancer formation, and immune reactions. It also summarizes recent advances in therapeutic peptide-based vaccines against cervical cancer, including the identification of major HPV vaccine antigens and epitopes, and appropriate vaccine delivery systems and adjuvants. There is a strong focus on HPV peptide-based nanovaccines. Finally, we highlight the most recent progress of preclinical and human trials of HPV peptide-based nanovaccines.

Disease Burden and Current Prophylactic Vaccines Against Cervical Cancer

Cervical cancer is a primary public health problem. The disease has the fourth-highest reported cancer incidence in females worldwide, ranking only after breast, colorectal, and lung cancers.^19,20 According to recent statistics from the World Health Organization, there was an estimated 569,847 cases of cervical cancer and 311,365 deaths in 2018; cervical cancer accounted for approximately 7.5% of all female cancer deaths globally.^21,22 The age-standardized prevalence of cervical cancer was around 13.1 per 100,000 females worldwide, although there were significant differences between countries.²⁰ The global average age at diagnosis and death of cervical cancer patients was approximately 53 years and 59 years, respectively.²⁰ It has been projected that cervical cancer will lead to more than 443,000 deaths annually by 2030, with the majority of these expected in sub-Saharan Africa.²³

Over the past 30 years, the incidence of cervical cancer in high-income nations has been half of that of low-income countries due to accurate cervical screening initiatives, effective cancer treatments, and broad availability of prophylactic HPV vaccines.²⁴ In 2020 alone, the mortality rate of cervical cancer across all 78 low- and middle-income countries was around 13.2 per 100,000 women.²⁴ Cervical cancer-related deaths primarily occurred in eastern, western, middle, and southern Africa, with the highest rates recorded in Eswatini.²⁰ It has been estimated that cervical cancer cases and associated mortality in low-income countries are 6 and 18 times higher, respectively, compared with wealthier countries.^24–27 In addition, a third of global cervical cancer burden was associated with China and India, with 106,000 and 97,000 new cases diagnosed per year, respectively.²⁰ In contrast, cervical cancer incidence and prevalence are remarkably lower in high-income nations, and the survival rate of cervical cancer patients is higher than in undeveloped and developing nations. In 2018, the American Cancer Society reported that around 13,240 women suffered from invasive cervical cancer, with approximately 4170 deaths. The one-year and five-year survival rates of cervical cancer are approximately 87% in the United States of America.^19,28 In contrast, survival rates in Uganda and Gambia's are less than 25%.¹⁹

Epigenetics and HPV strain type are two pathogenic risk factors responsible for the development of fatal cancers in the cervix. Approximately 70–75% of cervical cancer cases are caused by high-risk HPVs, such as HPV-16 and HPV-18, while five additional oncogenic HPVs (HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58) have been shown to elicit 15–30% of cervical cancer cases.^29–32

Since the discovery of HPV oncoproteins and pathogenic factors regulate the generation and maintenance of HPV-associated malignancies, with the consistent induction of cervical cancer, which significantly encourages the development of HPV vaccines, including both prophylactic and therapeutic vaccines.³³ However, the current success of HPV vaccines is available in the prophylactic vaccines. At present, the administration of prophylactic HPV vaccine, namely, GlaxoSmithKline's Cervarix^®, and Merck's Gardasil^® and Gardasil^® 9 vaccines, is regarded as the primary prevention measure for HPV infections and cervical cancer.^34,35 The bivalent HPV vaccine, Cervarix^®, targets HPV types −16 and −18; the quadrivalent HPV vaccine, Gardasil^®, targets HPV types −6, −11, −16 and −18; and the nine-valent HPV vaccine, Gardasil^® 9, targets HPV types −6, −11, −16, −18, −31, −33, −45, −52 and −58.³⁶ The three vaccines have been designed based on non-infectious virus-like particles (VLPs), which contain HPV capsid protein L1, produced in eukaryotic systems.^9,37,38 Gardasil^® and Gardasil^® 9 contain an aluminum adjuvant, whereas Cervarix^® contains monophosphoryl lipid A-based adjuvant (AS04).^39,40 Through clinical trial testing, these vaccines have proven to be highly immunogenic, safe, efficient, and capable of stimulating antibody-based responses against HPV infection.^40,41 They are able to protect up to 100% of females 9 to 26 years of age against HPV-associated cervical cancer.^41,42

However, prophylactic HPV vaccines do not confer any cross-protection against further carcinogenic HPVs, and roughly 20% to 30% of malignancies associated with these viruses are unavoidable.³⁹ Also, following vaccination with Cervarix^®, Gardasil^®, or Gardasil^® 9, a small number of users reported moderate side effects (eg, pain, inflammation, and erythema) at the injection site, diarrhea, and nausea.⁴³ While the available prophylactic vaccines have proven effective at protecting the host against HPV infection, they do not eradicate tumor cells. Hence, it is essential to develop therapeutic peptide-based vaccines that can directly kill tumor cells and reduce the number of cancer-related deaths.

Human Papillomaviruses (HPVs) and Infection

HPV Genotypes and Risks

HPV is a double-stranded, circular DNA virus within the family Papillomaviridae. The family includes five genera (α, β, γ, μ, and ν), 48 species, and 206 genotypes.^44–46 Initial experiments to detect the relationship between HPVs and cervical cancer date back to 1972.⁴⁷ HPVs genotypes can be classified into high-risk and low-risk, according to their oncogenic potential: 15 HPV genotypes (HPV-16, −18, −31, −33, −35, −39, −45, −51, −52, −56, −58, −59, −68, −73, and −82) are considered high-risk HPV infection types that can cause invasive and fatal cervical cancer in females, and three other HPV genotypes (HPV-25, −53, and −66) are considered to be possible high-risk types that might trigger cervical cancer.^39,48–50 HPV-16 (55%) and HPV-18 (12.8%) are particularly carcinogenic, accounting for up to 70% of all cervical cell lesions.^51,52 High-risk HPVs have been shown to directly modify the function of normal cell proteins, in addition to influencing gene expression.⁵³ For example, through microarray analysis, high-risk HPV-31 was reported to up- and down-regulate 178 and 150 cellular genes, respectively, affecting normal cell growth, several keratinocyte-specific genes, and/or interferon (IFN)-responsive genes.^53,54 In contrast, low-risk HPVs (HPV-6, −11, −40, −42, −43, −44, −54, −61, −70, −72, −81, and −89) rarely elicit malignant carcinomas.⁵⁵ Skin lesions related to low-risk HPVs are typically self-limiting, non-life-threatening and can be eradicated by host immune responses.⁵⁵ However, a small number of susceptible individuals with congenital immunodeficiency, whose innate immune system cannot detect the invasion of low-risk HPVs and effectively eliminate them, can develop recurrent respiratory papillomatosis and epidermodysplasia verruciformis (EV), resulting in an increased risk of suffering from cancers.^55,56

HPV Genome and Functions

HPV is a non-enveloped small DNA virus (55 nm in diameter) containing approximately 8000 base pairs.^46,56,57 The HPV genome contains eight to ten open reading frames, which can be divided into three regions: early gene coding region (E), late gene coding region (L), and long control region (LCR) of the non-coding region (Figure 1).^46,58 Functionally, the early genes (E1–E2, E4–E7) are expressed at different stages of the HPV cycle and are responsible for viral replication and protein regulation; two of them (E6 and E7) are primary oncogenic factors.⁵⁹ The late genes encode the two late capsid proteins, L1 and L2.⁵⁹ Non-coding LCR is composed of the p97 core promoter and regulatory sequence elements (enhancer and silencer sequences) responsible for regulating genetic information transmission in open reading frames.^49,53

Figure 1 The HPV genome and gene functions. HPVs are typical non-enveloped double-stranded DNA viruses, with circular and approximately 8000 pairs in size. Most encode eight major genes, six genes located in the early regions and two in the late regions. The early genes can regulate the HPV genome replication and transcription, viral release, celling signaling and viral apoptosis, immune modulation, and structural modification of infected cells.60

The particle structure of the HPV virus is an icosahedral capsid composed of 72 capsomers (Figure 2A).⁵³ The late gene region encodes the viral capsid proteins, and each viral capsid is composed of five L1 and twelve L2 proteins.⁶¹ Capsid L1 protein contains five monomeric 55 kDa units, while minor capsid L2 protein contains 12 copies of 74 kDa units.⁶² L1, as the main capsid protein, can spontaneously associate with icosahedral capsid particles. As a secondary capsid protein, L2 can bind to a specific region of L1 and help the capsid assemble viral DNA to form a virion. However, L2 lacks the ability to form virus particles autonomously.⁶³

Early gene coding regions are responsible for DNA replication, transcription, and the carcinogenic transformation of HPV viruses.⁶¹ Briefly, the E1 and E2 genes promote viral replication by interacting with specific regions in the HPV genome replication origin and co-opting cellular DNA replication components.⁶⁴ E1 encodes virus-specific DNA helicase, which is highly conserved and directly activates the replication and amplification of HPV.⁴⁶ Transcription, replication, and genome portioning of HPV are controlled by E2.^49,64 The control of gene amplification, virus synthesis, release, and transmission has been associated with the E4 proteins.⁶⁵ Furthermore, high-risk E4 proteins form amyloid fibers, which are used as biomarkers to diagnose HPV infection.⁶⁵ The E5 gene product (about 80 amino acids) is present in all high-risk HPV types; it promotes tumor progression as an early oncoprotein. E5 is, therefore, considered to be a promising therapeutic target.⁶⁶ It is expressed in the early stage of replication, forming small, hydrophobic, single membrane-spanning proteins that bind with platelet-derived growth factor receptors and epidermal growth factor receptors, leading to apoptosis and proliferation of normal cells and evasion of host immune responses.^46,67 Research on the E5 protein has been challenging due to its small size, hydrophobicity, membrane localization, and low secretion level.⁶⁶

Finally, E6 and E7 are important oncoproteins necessary for malignant conversion. They undergo continuous expression and retention throughout the life cycle of HPV-positive cancer cells. Inhibition of E6 and E7 protein expression causes senescence and apoptosis in indefinitely proliferating cancer cells.⁶⁸ E6 and E7 proteins are responsible for a variety of hallmark cancer pathways, namely, evasion of growth suppressors, blocking of the normal cellular life cycle, promotion of tumor cell proliferation and differentiation, and resistance to cancer cell death.^3,59 Consequently, E6 and E7 are the main targets for therapeutic vaccine development.³

E6, as a major oncoprotein, is approximately 150–160 amino acids in length and around 18 kDa in mass.^3,69 E6 proteins contain four CXXC motifs, which can form two zinc fingers. Breaking any conserved regions can interfere with the activity of HPV E6 proteins.⁶⁹ All zinc-finger domains are preceded by one hydrophilic amino-terminal region, divided by a hydrophobic domain, and subsequently linked with a short carboxy-terminal domain.⁶⁹ High-risk HPV E6 proteins can degrade tumor suppressor p53 and PDZ proteins, which are the key regulatory factors in the E6 oncogenic pathway, augmenting the activation of telomerase and increasing tumor cell growth remarkably.^69,70 Without the secretion of p53 in vivo, tumor cells proliferate uncontrolled and invasion persists.⁷¹ The E6 gene can also methylate the promoter of interferon-kappa (IFN-κ) and inhibit IFN-κ and other cytokines participating in the regulation of immune response, thereby promoting persistent infection by the HPV virus.⁷²

E7 is a small phosphoprotein of about 100 amino acids. It contains three conserved regions (CR1, CR2, and CR3).^3,73 A small part of the sequence from CR1 and the entire CR2 region are similar to adenovirus E1A proteins and the large T antigen of simian virus 40 (SV40).³ Conserved sequences ("LXCXE" motifs) are located in CR2 and bind to retinoblastoma tumor suppressor protein (pRb). CR3 is located at the C-terminal region of E7 and consists of two CXXC zinc-binding motifs separated by 29–30 amino acid residues.⁷⁴ The conserved region is sufficient to control zinc-dependent dimerization, and it regulates the cell cycle and apoptosis.^75–77 The CR2 region is implicated in complex formation with pRb due to the existence of the paramount pRb-binding core sequence LXCEX and an enzymic site for casein kinase II (CKII).⁷⁴ By encoding oncoproteins, E7 causes controlled cell proliferation by binding and inactivating regulatory repressor pRb, which is the most significant target in the E7 regulatory pathway.⁶⁸ Specifically, the transcription factor E2F is an essential regulator of cell cycle entry into the S-phase, and is also a downstream target of pRb.^46,78 When cells are infected by HPV, the E7 gene product binds to pRb, producing the E7-pRb complex. This interferes with the normal function of pRb, which triggers the activation of E2F and prematurely initiates the S-phase of the cell life cycle, affecting HPV DNA synthesis and replication and the proliferation of cervical cancer cells.^68,79

Pathogenesis of HPV

HPVs infect epidermal or mucosal epithelial cells. Most infections are cleared by the host's immune system. However, persistent HPV infections not cleared by the immune system led to benign cervical lesions. Cervical intraepithelial neoplasia (CIN) is commonly classified into three stages: CIN1, CIN2, and CIN3. CIN1 is the low-grade lesion stage characteristic of a variety of HPV infections. More than 80% of CIN1 cases are cleared by the immune system within several months.^52,56 However, HPV infection not cleared by innate immune responses can develop into CIN2/3 as high-grade CIN lesions, eventually causing invasive cervical cancer (Figure 2B).^52,58

Figure 2 (A) Structure of HPV; (B) changes of epithelial cells in the cervix following HPV infection. (B): a. Viral infection and its early gene expression phase: HPV virus invade the basal layer of the stratified epithelium and initiates early gene (E1 and E2) expression. The oncogenic virus quickly amplifies into 50–100 copies per cell in E1- and E2-dependent manners, with a low copy number of HPV episomes maintained via replication with cellular DNA; b. Viral gene latter expression and amplification phase: one infected cell remains in the basal layer, while other cells continue to enter the suprabasal layer. The episomal DNA sequence of HPV diffuses into the nucleus of infected cells. There, it undergoes genetic replication and assembly; c. Viral particle assembly and release phase: the life cycle is directly controlled by differentiation of the host cell, where HPV viruses are released from keratinocytes.

Micro-wound is the primary pathway for HPVs to infect multi-layered stratified epithelium in the ectocervix, whereby the virus can enter the basal lamina.^48,80,81 Infected basal cells form a reservoir for cervical cancer infections.⁸¹ In the early stage of the HPV life cycle, the early promoter can be activated in the infected basal layer cell to initiate expression of the E1 viral helicase that interacts with E2. The viral episomes are then rapidly replicated and amplified.^48,81 At this stage, the viral genome maintains a low level of intact genome replication.^81,82 As infected basal cells begin to divide, their daughter cells progressively migrate to the upper layers, accompanied by epithelial differentiation. Subsequently, cell division accelerates bulk amplification of the viral genome, triggering late gene expression and completing virosome assembly and release.^81,83

Immune Response to HPV Infection

The human immune system has two fundamental responses: innate and adaptive (Figure 3), which protect the body from pathogens, as well as toxic and allergenic substances. The innate immune system is the first line of defense and provides a general protective response against microbial invaders and foreign proteins. It is non-specific, short-term, and non-inducible. This system includes physical barriers (skin or epithelial cell layer), mucosal layers, the gastrointestinal and genitourinary tracts, and epithelial cilia that prevent pathogens from entering the host.⁸⁴ In addition, it uses chemicals and non-specific immune cells, such as natural killer cells, phagocytes, dendritic cells (DCs), and mast cells from bone marrow progenitors, to attack and kill invading pathogens.⁸⁴

Sustained invasion by pathogens can trigger adaptive immune responses (Figure 3). The distinctive feature of the adaptive immune response is the establishment of a specific, long-term immune memory. Generally, an adaptive immune response needs around 7–14 days to establish. The adaptive immune response expresses a strong host defense for secondary infections due to the prolonged immune response. When the host is restimulated, memory cells trigger a faster and more effective immune response. The adaptive immune response also involves the secretion of specific cytokines (eg, interleukin (IL)-4, IL-10, tumor necrosis factor (TNF)-β, interferon (IFN)-γ) that bind to specific receptors on the surface of immune cells, triggering cellular signaling cascades that regulate immune defenses against antigens.^84,85

Figure 3 Human immune responses against invasive pathogens. The human immune system has been classified into two general groups, including innate and adaptive immune responses. Innate immune cells stimulate rapid reactions, whereas adaptive immune cells have a delayed response, producing immunological memory.86 Rapid responding innate cells include polymorphonuclear cells, mast cells, macrophages, and dendritic cells, which are capable of internalizing and destroying invading microbes as well as the secretion of cytokines and proinflammatory chemokines, inducing other immune cells to the site of infection. Macrophages and DCs, as antigen-presenting cells (APCs), is capacity to ingest pathogens and produce pathogen peptides on their cell surface, which can be recognized by major histocompatibility complex (MHC) class I and MHC-II, with the induction of cellular immune response and humoral immune response, respectively.

The innate immune response recognizes pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) on the surface of antigen-presenting cells (APCs). APCs take up endocytosed or phagocytized cells and cleave invading pathogens (or antigens) into small peptides. These are then presented by major histocompatibility complex (MHC)-I or MHC-II, which can be recognized by CD8 cells or CD4 cells, respectively, resulting in the stimulation of cellular and/or humoral immunity against invasive pathogens.

The MHC-I stimulation pathway activates cytotoxic T lymphocytes (CTLs, CD8⁺ T cells), which secrete cytokines to target and kill infected cells or tumor cells: this is the foremost immune pathway to eliminate tumor cells.⁸⁷ CTLs primarily recognize pathogenic antigens derived from intracellular proteins, while humoral immunity is associated with extracellular antigens and antibody-based immune responses.⁸⁷ However, cellular immunity against tumor invasion is stimulated by tumor antigens, which are specific antigen substances secreted in tumor cells. Typical tumor antigens are comprised of tumor-specific antigens (TSA) that only exist on tumor cells,^88–90 and tumor-associated antigens (TAA),^91–93 which can be expressed in high levels in cancer cells but may also be found in lower levels in healthy cells.

Both cytotoxic and humoral immune responses require activation of T helper cells (Th, CD4⁺ T cells).⁸⁷ CD4⁺ T helper 1 (Th1) cells can secrete different proinflammatory cytokines, including interleukin (IL)-2, IL-10, tumor necrosis factor (TNF)-α, and interferons, which are the hallmarks of mediated immune responses. Secreted proinflammatory cytokines can activate cytotoxicity against tumor pathogens, enhance anti-tumoral abilities of macrophages and natural killer cells, and increase tumor antigen presentation. CD4⁺ T helper 2 (Th2) cells can produce several anti-inflammatory cytokines, such as IL-4, IL-5, IL-6, IL-10, and IL-13, and promote B cells to antibodies against helminths, infections, and allergic disorders.⁹⁴

Under normal conditions, Th1 and Th2 immunity are balanced;⁹⁵ the existence of tumor cells can interfere with this balance. Following the downregulation of adaptive immunity, Th2 immunity is enhanced and Th1 immunity decreases, allowing tumor progression. A shift towards Th1 immunity, in contrast, can lead to tumor regression.⁹⁵

Cancer immune therapeutics and vaccines are designed to trigger cellular immune responses targeting intracellular antigens. Specifically, vaccine candidates need to be presented as endogenous antigens that are processed in the proteasome of the host cytoplasm and presented by MHC I to CD8⁺ T cells, generating a cell-mediated immune response. Cell-mediated immunity established by this pathway is essential for the elimination of HPV-infected cells.

Anticancer Vaccines

As the pivotal public health tool, the main principle of vaccination is the stimulation of adaptive immunity and immune memory to provide long-term protection against infections or diseases, reducing or eliminating the risk of invasive diseases or disease-related complications, without triggering any considerable side effects.⁶ As the second line of immune defense, the adaptive immune system recognizes antigens with high concentricity and specificity.⁶ Prophylactic vaccination aims to activate adaptive immunity and induce neutralizing antibodies prior to natural infection. On the other hand, therapeutic vaccines, which utilize the cellular arm of the immune system, seek to specifically target existing disease/infection.⁹⁶ However, triggering adaptive immunity against tumor cells is difficult, as these cells are similar to normal human cells and, therefore, are often tolerated by the immune system. Fortunately, in the case of cervical cancer, non-human proteins (derived from the virus) can be targeted by tumor-specific cell immune responses, as they are independent of central tolerance mechanisms.⁹⁷

Over the past decades, various types of vaccines have been designed to enhance CTL-based immune responses against cervical cancer, including vaccines based on peptides, proteins, nucleic acids (DNA and RNA), whole cells, and vectors (bacterial and viral) (Table 1).^10,33 Among them, peptide-based vaccines generate the most controlled immune responses as they only use minimal antigen components. They are developed through meticulous antigen selection and the use of purpose-designed adjuvants, which have allowed remarkable breakthroughs in preclinical and clinical development of therapeutic cervical cancer vaccines.^6,13,98

Table 1 Features of the Most Common Therapeutic Vaccine Candidates Against Cervical Cancers6,99,100

Development of Peptide-Based Therapeutic HPV Vaccine

Peptide-based vaccines utilize the minimal components (epitopes) from pathogenic or oncoproteins to induce a specific immune response against infections and cancers.^15,16,98,101 Peptide vaccines can be commonly produced at a large scale and in stable and water-soluble form.¹⁵ Compared to new types of vaccines (including live vector-based vaccines, DNA vaccines, and RNA vaccines), peptide-based vaccines are easily and exclusively produced by chemical synthetic approaches, directly avoiding all biological contaminations of the antigens without the induction of allergic and autoimmune responses.^102,103 Furthermore, synthesized peptides can be fully and precisely characterized as a chemical entity, with the utilization of classical analysis methods (including mass spectrometry and high-performance liquid chromatography). Peptide sequences can be simply produced and easily reproduced as well as fast and cost-effective due to the use of solid-phase peptide systems with automatic synthesizers and the application of microwave techniques, with higher purity and yield, in comparison to new types of vaccines.¹⁵ Peptides can be designed to bind specific objectives based on the selection of one or multiple epitopes against invasive pathogens.¹⁵

Unfortunately, peptide vaccine antigens bearing B cell and/or CD8⁺ epitopes often lack Th epitopes, which significantly reduces vaccine efficacy.¹⁷ Furthermore, peptides can be easily cleaved by peptidases.^29,78,80 Finally, peptides are not promptly recognized by APCs, so do not have the ability to stimulate significant immune response on their own. As a result, peptides must be administered with strong adjuvant, an appropriate delivery system and multiple dosing is usually required.^17,99 One of the most promising strategies to overcome these shortcomings is the use of nanoparticle-based delivery systems/adjuvants.^17,101 Therefore, the selection of antigenic epitopes, immunostimulatory adjuvants, and delivery systems (or vaccine formulation) are all crucial elements in the development of effective anticancer vaccine (Figure 4).¹⁵

Figure 4 Schematic process of the development of peptide-based vaccines. The whole and successful process of developing peptide-based vaccines is classified into six main steps, including the identification of pathogens and target proteins, selection of antigen epitopes, vaccine formulation to build vaccine candidates, in vivo trials of peptide-based vaccine candidates, clinical trials of vaccine candidates, and final vaccination and immunization in humans against infectious diseases.

Selection of Antigenic Epitopes

The first prerequisite for the development of peptide-based vaccines is the determination of antigenic proteins from which appropriate epitopes can be selected.¹¹ To achieve broad coverage of a wide range of pathogen subtypes, peptide-based vaccine must contain highly conserved epitopes or a combination of diverse, variable epitopes.¹⁵ Generally, antigen epitopes can be classified as B or T cell (CD4⁺ or CD8⁺) epitopes that can trigger humoral and cellular immune responses. HPV therapeutic peptide-based vaccines have to trigger cellular immunity and, therefore, CD8⁺ and CD4⁺ epitopes need to be incorporated in vaccine formulations.

The most important factor in antigen selection is how epitopes bind to the MHC molecule.¹⁰⁴ MHC I and MHC II molecules have similar 3D-structures with bound peptides located in a groove formed by two α-helices. The type of bond between peptide residues and the MHC molecule is affected by the charge distribution, shape, and hydrophobicity of the binding groove. The binding site for MHC class I molecules is a groove that can only hold about 8–11 amino acids with its C-terminal end binding to the F-pocket due to closure of the binding groove at both ends by conserved tyrosine residues.^15,104,105 In contrast, the binding grooves of MHC class II molecules are open and can bind peptide ligands of 11–20 amino acids in length as the N-terminal ends can extend from the P1 pocket.¹⁰⁴ The number of distinct short peptides that exist creates a massive T cell epitope space to be explored.¹⁰⁵ In each MHC class molecule, the promotion of peptide exchange for certain peptides can be achieved via specific "catalysts", including tapasin for MHC class I and HLA-DM for MHC classes II. These catalysts are similar to enzymes that can reduce the energy barrier for peptide exchange without breaking formed covalent bonds during the exchange reaction.¹⁰⁶ Additionally, human leukocyte antigen (HLA) genes encode peptide-presenting MHC molecules, which are polygenic and strongly polyallelic. Different HLA alleles encode MHC mutants with different peptide binding and T-cell receptor binding priorities.¹⁰⁵ Thus, selection of CTL epitopes must consider both immunogenicity and compatibility with a wide range of HLA alleles.

The current reliance on computational methods to predict epitopes has opened up a new mindset in vaccine design, introducing the concept of reverse vaccinology, which involves the process of vaccine development through immunoinformatics to evaluate multiple proteomes, identify and select target epitopes, and overlap epitope combinations.¹⁰⁷ A variety of antigen prediction tools exist, which can be broadly classified as position specific scoring matrix (PSSM)-based, artificial intelligence (AI)-based, structure-based methods, and meta-analysis studies (mixture of other methods).^108,109 Among them, MAPP, NetCTL, NetCTLpan, MHCpathway and PickPocket are the most popular data-driven methods.^104,110 A systematic and detailed review of MHC I/II epitope design tools for peptide-based HPV vaccines has been previously presented by Kaliamurthi et al.¹⁰⁷ The group also reported the identification of HPV-45 and HPV-58 genotypic epitopes based on computational immunology and structural vaccinology approaches.^111,112

Application of Antigenic Epitopes in Therapeutic HPV Peptide-Based Vaccines

E6 and E7 proteins are the two major oncogenes of HPV-associated cancers. These proteins regulate the life cycle of host cells through multiple pathways, and they are excellent targets for cervical cancer therapeutic peptide-based vaccines.^100,113 However, this implies that immunization against specific epitopes of E6 and E7 can precisely induce and target CTL responses (Table 2).¹¹³ Most therapeutic peptide-based vaccine candidates against HPV have used E7 epitopes as the antigen because of its high affinity to retinoblastoma protein (pRb), effective control of the early HPV life cycle, elimination of tumor immortalization, and its capacity to produce a more effective cellular immune response compared to E6.⁹⁶

HPV-16 E7 protein epitope, E7_43-77 (GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR) comprises one CD8⁺ T cell epitope (E7_49-57, RAHYNIVTF) and two CD4⁺ T cell epitopes (E7_48-54, DRAHYNI; E7_50-62, AHYNIVTFCCKCD).¹¹⁴ Its shorter version, 8Q epitope (E7_44-62, QAEPDRAHYNIVTFCCKCD), was also widely studied.¹¹⁵ Tindle's team demonstrated that E7_48-54-activated cells from mouse lymph nodes produced cytotoxin against tumor cells.¹¹⁶ Furthermore, E7_44-62 epitope was able to induce antibody production (as it is carrying B-cell epitope, as well).¹¹⁶ HPV-16 E7_7-15 (TLHEYMLDL), E7_11-20 (YMLDLQPETT), E7_82-90 (LLMGTLGIV), E_86-93 (TLGIVCPI), E7_67-75 (LCVQSTHVD) were also confirmed to be CD8⁺ T cell epitopes, differentially restricted to HLA alleles.^117,118

Modification of epitope structure may significantly alter immune responses. When 8Q C-terminus pentapeptide fragment (QAEPDRAHYNIVTFCCKCD) was altered to avoid disulphide bond formation, producing shortened 8Q_min (E7_44-57, QAEPDRAHYNIVTF), 8Q_Ser (QAEPDRAHYNIVTFSSKSD), and 8Q_Lys (QAEPDRAHYNIVTFSKKK), only 8Q_min maintained the ability to stimulate strong CTL responses.¹¹⁵ 8Q_Ser and 8Q_Lys were likely not processed appropriately in DCs and, thus, did not activate the MHC I pathway.¹¹⁵

HPV-16 E6_49-57 (VYDFAFRDL) was found to be a typical CTL immunodominant epitope, with restriction to the HLA-A24 allele.^119,120 E6_87-95 (CYGYTTL) and E6_98-106 (QYNKPLCDL) were similarly HLA-A24-restricted CTL epitopes, but their affinities to HLA molecules were not consistent.¹²⁰ As a CTL epitope, E6_29-38 (TIHDIILECV) was only effective in limited cervical carcinoma cell lines due to a low release of inflammatory cytotoxin by HPV 16 E6_29-38-specific T cells.¹²⁰ Multiepitope peptide-based vaccine, including a combination of HPV-16 E6_43-57 (QLLREVYDFAFRDL), E7 8Q_min,¹²¹ E6_43-57, E7 8Q,¹²² E6_50-57 (YDFAFRDL), and E7_49-57 epitopes, is one possible solution to overcome this issue.¹²³ An HPV-16 multiepitope vaccine candidate was designed to present multiple CTL epitopes of H2-D^b and H2-K^b restricted, E6_49-57, E6_127-135 (DKKQRFHNI), E7_49-57, and E7_67-75. It activated specific T cells and inhibited tumor growth.¹¹⁸ Moreover, vaccine candidates constructed of long, overlapping peptides are selected peptide sequences from E7/E6 oncogenic proteins overlapping with their neighbored peptides. As an HPV vaccine, the multiepitope candidate had a high binding affinity to MHC molecules, was directly taken up by APCs, facilitated the MHC I stimulation pathway to activate cellular immunity, secreted cytotoxin, and offered a wide range of allele coverage.^87,124

E5 protein can also be a potential target for therapeutic vaccine development, as it is expressed in the early stages of HPV infection and is responsible for both pro-apoptotic factors and tumor survival pathway modulation, the oncogenic cell cycle, and tumor proliferation.¹²⁵ Based on immunoinformatics analysis, Kumar et al identified the most potent MHC I and MHC II antigenic determinants in E5 to be E5_50-63 (SAFRCFIVYIIFVY) and E5_64-78 (IPLFLIHTHARFLIT), respectively.¹²⁵ In addition, reverse immunology confirmed that E5_25-33 (VCLLIRPLL) and E5_63-71 (YIIFVYIPL) had the ability to induce cell-mediated immune responses and affected tumor growth in the C3 cell line of a preclinical mouse model.¹²⁶

As mentioned above, CD4⁺ epitopes were identified in HPV proteins; however, universal T helper epitopes, unrelated to HPV, can also be applied in vaccine design. This is because, in contrast to CD8⁺ and B cell epitopes, CD4⁺ epitopes do not need to be pathogen-specific. Universal T helper epitopes are the MHC-II antigenic determinants that cover a broad range of HLA alleles in the global population and stimulate universal immune responses.^15,127 For example, Pan DR epitope peptide (PADRE; AKFVAAWTLKAAA) stimulates human peripheral blood mononuclear cells to produce more vital immune stimulation than natural Th epitopes.¹²⁸ This artificial epitope demonstrated its efficacy and safety in clinical trials.^129–131 When incorporated in HPV vaccine, PADRE induced specific proliferation responses and was well tolerated in patients with advanced cervical carcinoma.^130,131 The addition of influenza virus haemagglutinin-derived CD4⁺ peptide (HA_307-319, PKYVKQNTLKLAT-C) and tetanus toxin-derived CD4⁺ peptide (TT_830-844, CG-QYIKANSKFIGITEL) also increased HPV-associated specific antitumor activity in pre-clinical studies.¹³²

Adjuvants and Delivery Systems in Peptide-Based HPV Therapeutic Vaccines

To stimulate immune responses against a peptide and avoid immune tolerance in the tumor microenvironment, a suitable adjuvant is essential. Adjuvants serve mainly as Toll-like receptor (TLR) agonists.^15,133 TLRs commonly recognize a variety of PAMPs, activating signaling pathways leading to the production of inflammatory cytokines and inducing innate, or even adaptive immune responses.¹³⁴

In addition to aluminum salts (alum), the only widely approved adjuvant for use in human vaccines, other adjuvants, such as MF59, AS01, CpG-ODN, AF03, AS03, and AS04, have gradually been endorsed for human use in certain vaccines and countries over the past 20 years.¹³⁵ Aluminum-based adjuvants are poor stimulators of cellular immune responses, so they are frequently used in prophylactic HPV vaccines rather than therapeutic vaccines; for example, HPV-16/18 AS04 (monophosphoryl lipid A with aluminum hydroxide)-adjuvanted vaccine (Cervarix^®-GSK) had high, sustained IgG levels in clinical trials and was authorized in 2007.^136,137 Squalene-based emulsion adjuvants, such as MF59, AF03, and AS03, have been commonly used for vaccine candidates to promote strong humoral and cellular immune responses against invasive pathogens.¹³⁵ Other emulsion-based adjuvants, complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)-based Montanide ISA 51 and 720, were examined in HPV therapeutic vaccine formulations.^96,116,138 In addition, synthetic oligodeoxynucleotides (ODNs) containing CpG motifs form the important CpG-ODN adjuvants, which stimulate APCs and activate humoral and cellular immune responses. CpG-ODN has been broadly used in therapeutic cancer vaccines in both preclinical and clinical trials due to its high affinity with TLR-9, prolongation of CD8⁺ T cells in cellular immunity, and good safety profile.¹³⁹

A variety of experimental adjuvants and self-adjuvanting nanoparticle-based delivery systems have also been tested to stimulate stronger CTL responses against cervical cancer, especially for peptide-based antigens (Table 3). These include nanoparticle-based systems, such as polymeric nanostructures, liposomes, and virus-like particles.¹⁴⁰ Nanoparticles can mimic pathogens, are more readily taken up by APCs, are more likely to reach the lymphatic system, can accumulate in lymph nodes, and, most importantly, can induce CTL responses by loading internalized antigen on MHC I molecules via antigen cross-presentation.^15,141

Table 2 Epitope Sequences of Peptide-Based Therapeutic HPV Vaccines

Table 3 The Use of Immunological Adjuvants in Peptide-Based Nanovaccines Against Cervical Cancer

Emulsion-Based Adjuvants

Two types of emulsions: water-in-oil and oil-in-water, are utilized in vaccine delivery systems. Water-in-oil emulsions can directly prolong antigen presentation at the injection site as a result of the binding force between the antigen and droplet surface. The emulsion can also protect peptide antigens against degradation in the body fluids.^142,143 As early as 1991, Tindle et al demonstrated that emulsification of E7_48-54 in CFA stimulated T cell proliferation and cytokine production in mice.¹¹⁶ Despite the excellent immunostimulatory properties of CFA, it contains heat-inactivated Mycobacterium bovis that can trigger obvious side effects, such as inflammation.¹⁴⁴ Therefore, CFA is currently only used as a "gold standard" adjuvant in animal studies.

Montanide^TM oil-based adjuvants based on incomplete Freund's adjuvant, which does not have M. bovis components, were broadly utilized in the formulation of peptide-based vaccines against cancers.¹⁴⁵ For example, a vaccine formulation containing Montanide ISA 51, which was emulsified with PADRE Th epitope and the two CTL epitopes, HPV-16 E7_11-20 and E7_86-93, was tested in 19 HLA-A*0201-positive cervical cancer patients.¹³¹ The clinical Phase I–II trials showed no adverse effects of the formulation at three gradient doses. However, tumor regression was only observed in 4 of the 19 patients.¹³¹ Because all patients had advanced stages of cervical cancer, it was not possible to assess whether there was a specific immune response to the vaccine candidate.

Toll-Like Receptor (TLR) Agonists

Lipopeptides

In recent decades, peptide antigens have often been mixed with or conjugated to lipid moieties to produce lipopeptides, which are PRR agonists that can target TLR1/2 (eg, dipalmitoyl-S-glyceryl cysteine (Pam2Cys)), TLR2/6 (Pam3Cys), and TLR2 (Pam2CSK4).^132,142,146

As effective adjuvants, these lipopeptides were used in several studies to enhance the immunogenicity of peptide-based vaccines. For example, Moyle et al conducted tumor challenge studies on the synthetic bacterial lipopeptide adjuvant Pam2Cys conjugated to HPV-16-modified E7 protein (E7_m) to form a self-assembled vaccine candidate. Mice immunized with 25 µg of Pam2Cys-E7_m 3 days after TC-1 cell tumor challenge showed slow tumor growth rate over 24 days. Mice immunized 7 days after the same tumor challenge displayed a 10% (1/10) survival rate after 60 days.¹⁴⁷

Hussein et al developed lipoalkyne immunostimulant based on the Pam2Cys structure. Two epitopes derived from E6 and E7 proteins, E6_43-57 and E7_44-57, respectively, were conjugated together and acylated with a variety of double-chain lipid moieties.¹⁴⁸ In particular, the most promising self-adjuvanting lipopeptide candidate (450–750 nm) conjugated with multi-antigens (E6_43-57 and E7_44-57) was able to trigger eradication of 46% (6/13) of 3-day-old tumors in mice. The lipopeptide candidate induced a higher su...