HPV as a Model for the Development of Prophylactic and Therapeutic Cancer Vaccines

Abstract

HPV has been linked to many human malignancies and, as such, represents a major public health crisis. The understanding of HPV biology, however, has helped tremendously in developing prophylactic vaccines, which should help in decreasing mortality due to HPV infections. Understanding HPV biology has allowed researchers to use the virus as a model for the development of not only prophylactic vaccines, but also therapeutic ones. The advantages of HPV as a model stem from the limited number of proteins encoded by the HPV genome that can be targeted by vaccines, and also from the restricted expression of certain viral proteins during different stages of infection. In this review, we discuss how HPV can be used as a model for the development of both prophylactic and therapeutic vaccines.

INTRODUCTION

Epidemiological studies have demonstrated that the human papilloma virus (HPV) is linked to many human benign and malignant tumors, including esophageal squamous cell carcinoma [1], vulvar and vaginal cancers [2], skin and anogenital area [3], head and neck cancer [4], and cervical cancer [5]. Accordingly, HPV forms a major public health crisis in many developed and underdeveloped countries and poses a large burden on the health care system and economy of these countries.

Cervical cancer, for example, is the second most common malignancy in women worldwide. According to the World Health Organization (WHO), approximately 500,000 cases of cervical cancer are diagnosed and approximately 270,000 die worldwide because of the disease each year [6]. In the US and most of the developed countries, the disease is less prevalent because of aggressive screening programs, having said that, there are still around 3,500 yearly deaths from the disease in the US [6]. Studies show that HPV DNA has been identified in 99.7–100% of cervical cancer biopsies [7]. The bulk of the cases worldwide are in the developing world where the majority of the world’s high-risk populations do not have access or the financial means to consistent screening programs or follow-up therapy. Furthermore, the economic burden of HPV-related diseases in the US is high in both men and women with an average cost of $1709 for a treatment episode of CIN [8]. This can be compared to $1541 and $739 for the annual maintenance of chronic diabetes and hypertension, respectively [9].

Screening programs and treatment strategies of pre-invasive disease have had a significant impact on the incidence of invasive cervical cancer leading to an effective decrease in mortality from the disease. However, despite these efforts, mortality is still high, and such programs and strategies are very costly to individuals in developing countries. Therefore, since HPV has been shown to be a causative agent of invasive cervical cancer [7], mass vaccination represents an alternative and potentially a cost-effective approach to reduce mortality from cervical cancer.

In this review, we will discuss the scientific basis for the development of both HPV prophylactic and therapeutic vaccines. We will discuss the application of the vaccines in cervical cancer as a prototype and discuss their targets and limitations, and the current status of this art.

BIOLOGY OF HPV INFECTION

The human papilloma virus is a small, double stranded, DNA virus approximately 55 nm in diameter [10]. Its genome is 8 kb and contains eight genes: six early genes: E1, E2, E4, E5, E6, and E7, encode for early nonstructural proteins, and two genes, L1 and L2, encode late structural proteins [11] (Fig. 1).

Fig. (1).

Structure of the Human Papilloma virus (HPV). Upper panel represents the proteins encoded by the HPV genome. Lower panel represents HPV virions.

Strauss et al. identified viral particles of papilloma-viruses from skin papillomas in 1949 [12]. In 1974 Harold Zur Hausen identified the human papilloma virus in cervical cancer [13]. This has led him to receive the 2008 Nobel Prize for Medicine. Today, over 130 different HPV genotypes have been identified based on DNA sequence analysis [14]. About 25 of these types can infect the mucosa of the female lower genital tract. Of these, 13 are considered oncogenic or associated with the development of pre-invasive or invasive disease (high-risk HPV). Of the 13 oncogenic types, HPV 16, 18 and 45 account for 58%, 25% and 13% of cervical cancers in North America, respectively [15].

An understanding of HPV biology, the virus life cycle, and natural history of the infection is of crucial importance for the successful generation of effective vaccines.

Upon exposure to HPV, the virions pass through the upper layers of the cervix’ squamous epithelium to reach and infect the basal keratinocytes. Within the basal keratinocytes, the HPVs express the early genes responsible for the replication of viral DNA, E1and E2. Initiation of viral DNA replication is supported by the interaction of E1, the viral helicase gene, E2, the master regulator of viral gene expression, with the cell machinery. Infected keratinocytes then start dividing, and this division pushes cells away from the basement membrane causing their terminal epithelial differentiation. HPV viral gene expression has been shown to be confined to keratinocytes. Initially, the virus is maintained at a low copy number. A round of viral DNA replication is thought to cause the infected cells to enter the proliferative compartment of the epithelium. Here, the viral DNA is maintained as an episome and viral gene expression is minimal. When infected cells enter the differentiating compartment, viral gene expression gets upregulated and replication occurs. Virions get assembled as cells are differentiating. Shedding of virus particles occurs when infected cells terminally differentiate and die by natural causes [16].

The high-risk HPV continues to be episomal during the persistent infection and during the early phases of the part of the pre-invasive transformation. The HPV-DNA is usually integrated into the host’s genome sometime during the transformation of the low grade into high grade pre-malignant lesion. The integration of the HPV DNA into the host’s genome occurs in the area of the E2 sequences leading to the disruption of E2 expression. Accordingly, E2 protein is not found in some of the high grade lesions and in invasive cancers. The E2 protein is a major viral transcription and regulatory factor which has the ability to down-regulate both E6 and E7 proteins. Accordingly, the E2 protein is expressed in cells prior to the integration of the HPV genome, which is mainly in pre-malignant lesions. On the other hand, the integration of the DNA leads to up-regulation of both the E6 and E7 gene expression due to the loss of repression caused by E2 and the release of its repressive effect on the promoters of both of these genes [17, 18]. Accordingly, as shown in Fig. (2), the predominant proteins expressed in the persistent infection, cells undergoing terminal differentiation and early pre-invasive lesion phases are the L1 and L2, which assemble to form the viral capsid (virion) [19] and the E1 and E2 early proteins. Accordingly, they will not be expressed in cancer cells since the transformed cells do not undergo terminal differentiation. On the other hand, the E6 and E7 proteins are the dominant proteins in the later phases of the preinvasive lesion and in the invasive cancers.

Fig. (2).

Natural history and biology of HPV infection. A simplified diagram of HPV protein expression patterns during infection, HPV integration, and strategies to combat HPV infection and HPV-induced cancer.

The protein products of E6 and E7 in high-risk HPV types have been implicated in the oncogenic capability of the high risk HPV. E6 and E7 proteins exert their oncogenic activity by interacting with the biological functions of several cellular proteins, involved in the control of fundamental events, such as cell proliferation and apoptosis, particularly pRB and p53 proteins leading to their degradation losing cell-cycle control [20–22]. Therefore, the expression of E6 and E7 proteins is necessary for the initiation and maintenance of the malignant phenotype making these proteins attractive targets for therapy.

The natural history of HPV infections varies based on the individual’s immune system and the HPV type. However, it is estimated that about 80% of women in North America may be infected with at least one type of HPV in their lifetime. Most HPV infections of the cervix are asymptomatic and more than 90% of detected infections are spontaneously cleared within two years [23], with about 50–60% of women developing serum antibodies to HPV after natural infection [24]. Ten to 30 percent of the infected women, especially those infected with the oncogenic types may develop chronic infections which lead to persistent and recurrent episodes of CIN and an increased risk of invasive disease. More than 95% of human cervical carcinoma and 70–75% of CIN II-III harbor high risk HPV DNA, primarily of HPV16 and 18 [17, 25].

Cervical cancer is thought to originate from CIN lesions after long persistent infections. There are three stages of CIN: CINI, or mild dysplasia, CINII, or moderate dysplasia, and CINIII, or severe dysplasia (carcinoma in situ). These stages vary mainly in the type of viral DNA that is expressed. CINI and CINII lesions contain episomal viral DNA and have very low levels of E6 and E7 proteins. CINIII, on the other hand, contains integrated viral DNA and expresses high levels of E6 and E7 [26].

HPV IMMUNOLOGY

It has been known that the immune system plays an important role in determining the natural history of cancer. This is evident because in some anecdotes, tumors are known to regress spontaneously [27]; there is an increased incidence of malignancy with immune suppression, such as in post-transplant patients [28, 29]; and immune response modifiers have been shown to be effective in treating tumors [30, 31]. Therefore, this indicates that tumors are amenable for immune recognition, and hence, are able to present antigens that are recognized by the immune cells. These antigens are called tumor associated antigens “TAA”.

In cervical cancer, it has been shown, through epidemiologic evidence, that the immune system plays a major role in the natural history of this disease and its causative factor, HPV. In fact, many early premalignant lesions, including ASCUS and CINI can regress spontaneously (60% according to Ostor [32]); immunocompromised patients, such as renal transplant patients and patients infected with HIV, have an increased incidence of premalignant and malignant lesions [33]. Furthermore, patients with CIN lesions who were found to have lymphoproliferative responses to HPV E6 and/or E7 peptides were more likely to eliminate HPV/DNA and CIN lesions by their next clinic visit [34]. Therefore, HPV forms an attractive target for immune therapy.

HPV VACCINES

Vaccines, in general, are designed to induce either humoral or cellular immune responses. The humoral response produces neutralizing antibodies, which are most effective against extracellular organisms and pathogens. On the other hand, vaccines producing a cellular response may generate specific T lymphocytes that are capable of recognizing antigens produced by either intracellular pathogens or endogenous genetic changes and presented by MHC molecules.

Cervical cancer vaccines are administered either with a therapeutic or a prophylactic intent. Prophylactic vaccines are vaccines which are administered with the intent to prevent or eliminate an infection with a tumor-inducing pathogen. These vaccines are directed to generate a humoral immune response. Therapeutic vaccines, on the other hand, are vaccines which are given to patients with established disease with the intent of 1) prevention of progression of a precancerous lesion to an invasive tumor, or 2) prevention of recurrence of a completely regressed tumor, or 3) treatment of an established invasive or metastatic disease. The therapeutic vaccines are directed to generate a cellular immune response (Fig. 2).

PROPHYLACTIC VACCINES

Animal studies have shown that humoral immune responses can block papillomavirus infections, suggesting that these infections can be neutralized by antibodies thereby opening the door for the development of prophylactic vaccines [11]. The breakthrough in the development of such vaccines arrived when researchers demonstrated that highly immunogenic 3-D structures, subsequently called VLPs, can be produced in vitro by the expression of HPV viral capsid proteins L1 and L2 by recombinant technology [35, 36]. These two proteins assemble in vivo in the same way as the intact virus does, thereby, exactly mimicking the HPV capsid morphologically and immunologically. These VLPs are empty capsids and DNA-free, and contain no oncogenic or infectious material [37]. Prophylactic HPV vaccines are directed against the capsid proteins.

Substantial advances have been made in developing HPV L1 virus-like particles (VLP)-based prophylactic vaccines, and culminated in the FDA approval in June of 2006 of Gardasil, a quadrivalent prophylactic vaccine designed to protect young females from HPV infections and, subsequently, HPV-induced cervical malignancies. Another prophylactic vaccine, Cervarix, is a bivalent one and is nearing FDA approval, and has been approved in more than 60 countries around the world. Both vaccines provide protection by inducing neutralizing antibodies against HPV [38–40], and are designed to protect against high-risk HPV 16 and 18; Gardasil also protects against low-risk genotypes 6 and 11. Furthermore, both vaccines are able to cross-protect against HPV 31 and HPV45, which are closely related to HPV16 and HPV18, respectively [41, 42].

Results from randomized clinical trials using the VLP vaccines have shown significant efficacies. In the FUTURE I study, 5,455 women were randomized in a phase III study to receive either the Gardasil in a three-injection course or a placebo. For women who had not previously been exposed to HPV 16 or 18, the efficacy of the vaccine was 100 percent in preventing all premalignant lesions associated with the virus [43]. The second trial, FUTURE II, randomly assigned 12,167 women between the ages of 15 and 26 to receive either Gardasil, also in three separate injections, or placebo. In this trial, the vaccine was able to prevent 98 percent of high-grade cervical intraepithelial neoplasia (CIN2 or CIN3) related to those HPV types in women who had not previously been exposed to HPV 16 or 18 [44]. In both trials, when an intention-to-treat analysis was performed including women who had previously been exposed to HPV 16 or 18, the vaccine efficacy was much less; 73 percent protection against all grades of external anogenital or vaginal lesions and 55 percent against all grades of cervical lesions related to those HPV types in the FUTURE I study, and 44 percent against high-grade cervical disease caused by HPV types 16 or 18 in the FUTURE II trial.

In the PATRICIA study, 18,644 women aged 15–25 years were randomized in this phase III study to receive either Cervarix (n=9319) or hepatitis A vaccine (n=9325) in a three-injection course. Interim analysis revealed that vaccine efficacy was 90.4 percent on a modified intention-to-treat basis [45]. Furthermore, if infections with HPV types other than HPV16 or HPV18 were excluded, then vaccine efficacy would jump to 100 percent against CIN2+.

It is important to note that although both vaccines showed almost 100 percent prophylactic efficacy, vaccination with either vaccine did not appear to alter the course of cervical cancer if infection with HPV was present at the time of vaccination. As a result, women already infected with HPV may not benefit from either vaccine.

Although very efficacious, these prophylactic L1 VLP vaccines have limitations and drawbacks. First, due to the nature of HPV infection and the fact that these vaccines are only approved for young females, it would take more than 30 years to realize the full impact of a prophylactic vaccine if all the females in the world were to be vaccinated today [46]. Second, these prophylactic vaccines have the potential to prevent HPV infection but are ineffective against an established HPV infection. This inefficacy is because current prophylactic vaccines target capsid genes that are not expressed by cancer cells. Because of these limitations, many females with already established HPV infections may not benefit from these prophylactic vaccines. Third, the longevity of protection against HPV and the possible requirement for booster immunizations to maintain protection are crucial concerns [47], as subjects receiving these vaccines have been followed up for up to five years only. Fourth is the lack of cross-reactivity towards other HPV types. Currently, there is no data on the ability of Gardasil to cross-protect against other types of HPV. Cervarix, on the other hand, protects against HPV45 and HPV31, types that are very closely related to HPV16 and HPV18, but not against the more distantly related HPVs, like HPV33, HPV52, HPV58 [47].

One way to overcome the issue of restricted protection is to generate vaccines against the other capsid protein L2. Whereas L1 vaccines produce type-restricted antibodies, L2 vaccines have been shown to produce cross-neutralizing antibodies [48–52]. However, L2 vaccines have drawbacks as well. The L2 protein is poorly immunogenic, resulting in low titers of L2 neutralizing antibodies [52].

Accordingly, therapeutic vaccines are necessary to effectively treat women not benefiting from prophylactic vaccines, minimize the risk of persistent infection, to further exposure to the current therapeutic modalities, primarily surgical, which compromise fertility, and to overcome the limitations and drawbacks of prophylactic vaccines. Additionally, an immunologic approach to treatment would minimize the need for long-term surveillance for recurrent infection and further retreatment, thus making this form of therapy more cost-effective and appropriate for patients in low resource environments where access to care is compromised.

THERAPEUTIC VACCINES

The elimination of an established HPV induced lesion requires a therapeutic vaccine approach that induces T cell-mediated immunity against antigens of the early HPV genes. The design of such vaccines requires knowledge of the biology of HPV infections. Not all early genes make good antigen candidates. For instance, the E4 protein shows a pattern of expression similar to that of capsid proteins, and therefore, will not be expressed by malignant lesions. The E1 and E5 proteins are similar to E4 in that there is little evidence that they are expressed in cancers. On the other hand, the E2 protein is expressed in pre-malignant lesions, and E6 and E7 are expressed throughout the viral life cycle, making these three proteins ideal targets for therapeutic vaccines.

Therapeutic vaccines, in contrast to prophylactic ones, have not yet demonstrated any clinical efficacy against pre-malignant HPV-induced lesions, or cervical cancer. Encouraging results are being generated, however, in the clinic. Current therapeutic vaccines efforts are being directed to target the early genes of HPV E2, E6, and E7.

VACCINES TARGETING E6 AND E7

In a study examining the effects of vaccination with a protein encoding a bacterial heat shock protein fused to the oncogenic HPV-16 E7 protein in women with high-grade cervical intraepithelial neoplasia, seven of 20 women (35%) evaluable for response had complete regression of their intraepithelial neoplasia, 1 (5%) had regression to CIN I, 11 (55%) had stable disease and 1 (5%) had progression due to enlargement of lesion. Furthermore, immune responses were detected in 9 of the 17 women tested. Interestingly, 5 of the 7 complete responders had an immune response [53].

Another study assessed the safety and efficacy of ZYC101a for the treatment of women with histologically confirmed CIN 2/3. ZYC101a contains plasmid-DNA-encoding fragments from HPV16 and HPV18 E6 and E7 proteins, and is formulated within small biodegradable microparticles. Although the proportion of subjects who resolved their CIN2/3 was higher in the ZYC101a groups compared to placebo (43% versus 27%), this difference was not statistically significant (P =.12). However, in a prospectively defined population of women younger than 25 years (n = 43), resolution was significantly higher in the combined ZYC101a groups compared to placebo (70% versus 23%; P =.007). Moreover, ZYC101a activity was not HPV-16 or −18-restricted [54].

In a non-randomized phase I trial, Stellar et al. [55] tested the efficacy of multiple administrations of an HLA-A*0201-restricted HPV-16 E7_86–93 lipopeptide vaccine that is linked to PADRE, a non-specific helper peptide PADRE. Twelve women with refractory cervical or vaginal cancer were enrolled in this trial. Patients were vaccinated four times at 3-week intervals, and assessments included CTL and clinical responses. After 2 and 4 vaccinations, five of seven and two of three patients were evaluable, respectively. Although these patients demonstrated E7 specific CTL responses, no clinical responses were associated with vaccination in this trial.

Van Driel et al. [56] performed a phase I/II dose-escalation clinical trial testing the efficacy of a peptide vaccine. The vaccine consisted of 2 HPV16 peptides and the helper peptide PADRE emulsified in Montanide ISA 51 adjuvant. The trial enrolled 19 patients suffering from HPV16 positive cervical carcinoma, which was refractory to conventional therapy. Patients were vaccinated with 100 ug, 300 ug, or 1000 ug of each peptide every 3 weeks for 4 vaccinations. Two patients showed stable disease for 1 year after vaccination, 15 showed progressive disease, and 2 showed tumor regression after chemotherapy following vaccination. However, after analyzing specific immune responses against the vaccine components in peripheral blood samples, no CTL responses against the HPV16 E7 peptides were detectable [57].

Muderspach et al. [58] conducted a phase I clinical trial to test the efficacy of escalating doses of a vaccine consisting of the HPV16 E712–20 peptide emulsified in IFA. The trial enrolled 18 patients with high-grade cervical or vulvar intraepithelial neoplasia who were HPV16- and HLA-A2-positive. Patients received 4 immunizations of increasing doses of the vaccine each 3 weeks apart, followed by a colposcopy and definitive removal of dysplastic tissue 3 weeks after the fourth immunization. A total of 9 of 17 evaluable patients had partial or complete regression of their CIN lesions. Three had complete regression, and 6 had .50% shrinkage of their colposcopically measured disease.

In a placebo-controlled trial, Hallez et al. [59] tested the immunogenicity of a fusion protein (PD-E7) comprising a mutated HPV-16 E7 linked to the first 108 amino acids of Haemophilus influenzae protein D (PD-E7), formulated in the adjuvant AS02B, in patients with oncogenic HPV-positive cervical intraepithelial neoplasia (CIN). The trial enrolled ten patients, five with CIN3 and five with CIN1. CIN3 and two CIN1 patients received three intramuscular injections at 2-week intervals, while three CIN1 patients received a placebo. Conization was performed on CIN3 patients 8 weeks postvaccination. To monitor antigen-specific cellular and antibody responses, cytokine flow cytometry and ELISA were used. Their results indicate that all vaccinated patients, but not the placebo, made significant E7- and PD-specific IgG. Furthermore, their results show that systemic specific immune responses can be elicited in the majority of patients by vaccination, but not placebo injection. Significant increases in IFN-g CD8⁺ cell responses were observed in five vaccinated patients (71%), while two responding patients generated long-term T-cell immunity toward the vaccine antigen and E7.

Although therapeutic vaccinations have not produced very efficacious outcomes, results of clinical trials discussed above are promising. Therapeutic vaccination strategies have produced strong immune responses. However, these immune responses did not translate into strong clinical outcomes. Researchers now are exploring a wide range of strategies to improve clinical outcomes in patients receiving these vaccines such as the use of adjuvants and the inhibition of regulatory T cells. In addition, researchers started examining the effects of vaccination against the E2 protein as another strategy to fight HPV-induced malignancies.

VACCINES TARGETING E2

Targeting pre-malignant lesions as early as possible is another way to target the development of cervical cancer. Accordingly, targeting the E2 gene product, which is expressed in low grade non-invasive disease [60, 61], is another attractive strategy, which many investigators are starting to explore.

As outlined above, HPV E2 protein is a major viral transcription and regulation factor during the viral life cycle, and is required for papilloma formation. It has been found that the E2 protein from high risk HPV types 16 and 18 can induce apoptosis. In contrast, the E2 protein from low risk HPV types 6 and 11 does not [62]. It was reported that E2’s ability to induce apoptosis was linked to the intracellular location of the respective E2 rather than to inherent properties of the protein [62]. Therefore, artificially expressing E2 in cells has been a difficult task. Accordingly, while immune responses to E2 is an important factor in the biology of cervical and the development of vaccine against E has been an aim, it has been difficult to develop because of the difficulty in developing targets that express the E2 proteins to test immune efficacy.

Studies in rabbit and canine models have shown that immune response to E2 was correlated with papillomavirus clearance [63–65]. In a recent cross sectional study, T cell responses to E2 were inversely correlated with risk of cervical cancer [66,67], suggesting that E2 may be an important target for immune-mediated clearance of cervical infection in humans.

Garcia-Hernandez et al. [68] and Corona Gutierrez et al. [66] demonstrated the regression of high grade lesion, CIN 2 and 3, in patients by injecting a viral vector expressing HPV E2, MVA-E2, locally into the lesion, and compared this vaccination protocol to patients who underwent conization treatment without vaccination. They found that patients who received MVA-E2 generated a specific cytotoxic response against papilloma-transformed cells that correlated with the elimination of lesions and with a reduction of HPV viral load. In addition, some patients treated with conization were recurrent 1 year later.

Heinemann et al. immunized mice with E2 in the adjuvant MF 59 and found that T-cell responses are stimulated. Furthermore, they found that by reducing the time interval between the primary and secondary immunizations from 12 to 4 wk, the magnitude of the immune response was augmented [69].

Dillion et al. conducted a trial in patients with persistent cervical dysplasia to determine whether cell-mediated immunity (CMI) to the early protein E2 was associated with disease regression. They compared E2-specific responses in resolvers, persisters, and normal controls. Furthermore, they measured antibody responses L1 virus-like particles (VLPs). They found that significantly higher E2-specific responses were observed in resolvers, compared to persisters and normal controls. These responses were manifested by high levels of IFNg when T cells were stimulated with E2. Antibody responses, on the other hand, were associated with current or previous HPV infection, but not with disease regression [70].

We, at the NCI, have shown that vaccinating mice with constructs containing the full length HPV16 E2 protein can generate specific immune response. In addition, we identified strategies to enhance immune responses to E2 by using heterologous priming and boosting with chimeric VLPs containing E2 and E7 peptides with the combination of immune modulators. Furthermore, we have identified HLA-A2-restricted T cell epitopes from the E2 region of HPV 16, 18 and 45 that can induce cytolytic activity and can be used in the clinic [71]. Currently, we are in the process of developing a clinical study to assess a vaccine against E2 for the treatment of CIN1 and CIN2. The primary objective of this study is to determine whether a cellular immune response to HPV16 or 18 E2 can be generated, and to determine the clinical responses generated by HPV E2 vaccine as defined by the clearance of HPV 16.

CONCLUSION

This is a highly active area of research in cancer vaccine development. Many investigators and biotechnology companies are investing new and more innovative vaccine reagents that target HPV, including different forms of peptides, full HPV recombinant proteins, DNA and viral vectors containing partial sequences of the oncogenes. HPV E2, E6, and E7 are already being tested in clinical trials in different forms of antigen presentation for testing and some are expected to be starting soon.