Ali Mahdavi, Bradley J. Monk

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1 This material is protected by U.S. Copyright law. Unauthorized reproduction is prohibited. For reprints contact: Prevention Vaccines Against Human Papillomavirus and Cervical Cancer: Promises and Challenges Ali Mahdavi, Bradley J. Monk Learning Objectives Division of Gynecologic Oncology, Chao Family Comprehensive Cancer Center, University of California, Irvine, Orange, California, USA Key Words. Human papillomavirus Vaccine Cervical cancer Cervical dysplasia After completing this course, the reader will be able to: 1. Discuss the epidemiology and pathogenesis of HPV and HPV-associated diseases. 2. Explain the immune mechanisms relevant to the control of HPV infection. 3. Describe vaccine strategies for the prevention and therapy of HPV infection and cervical dysplasia and/or cancer. CME Access and take the CME test online and receive 1 hour of AMA PRA category 1 credit at CME.TheOncologist.com Abstract Cervical cancer and precancerous lesions of the genital tract are major threats to the health of women worldwide. The introduction of screening tests to detect cervical cancer precursor lesions has reduced cervical cancer rates in the developed world, but not in developing countries. Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer and dysplasia. Thus, cervical cancer and other HPV-associated malignancies might be prevented or treated by HPV vaccines. Two vaccine strategies have been developed. First, prevention of HPV infection through induction of capsid-specific neutralizing antibodies has been studied in clinical trials. However, because the capsid proteins are not expressed at detectable levels by infected basal keratinocytes or in HPV-transformed cells, a second approach of developing therapeutic vaccines by targeting nonstructural early viral antigens has also been developed. Because two HPV oncogenic proteins, E6 and E7, are critical to the induction and maintenance of cellular transformation and are coexpressed in the majority of HPV-containing carcinomas, most therapeutic vaccines target one or both of these gene products. A variety of approaches is being tested in therapeutic vaccine clinical trials, whereby E6 and/or E7 are administered in live vectors, as peptides or protein, in nucleic acid form, or in cell-based vaccines. The paradigm of preventing HPV infection through vaccination has been tested, and two vaccines are currently in phase III clinical trials. However, current therapeutic vaccine trials are less mature with respect to disease clearance. A number of approaches have shown significant therapeutic benefit in preclinical papillomavirus models and await testing in patient populations to determine the most effective curative strategy. The Oncologist 2005;10: Correspondence: Bradley J. Monk, M.D., Division of Gynecologic Oncology, Chao Family Comprehensive Cancer Center, University of California, Irvine, 101 The City Drive, Building 56, Room 262, Orange, California , USA. Telephone: ; Fax: ; bjmonk@uci.edu Received April 12, 2005; accepted for publication May 12, AlphaMed Press /2005/$12.00/0 The Oncologist 2005;10:

2 Mahdavi, Monk 529 Introduction Cervical cancer and precancerous cervical lesions constitute a major problem in women s health. Clinical, molecular, and epidemiological investigations have identified human papillomavirus (HPV) as the major cause of cervical cancer and cervical dysplasia [1]. Virtually all cervical cancers (99%) contain the genes of high-risk HPVs, most commonly types 16,18, 31, and 45 [1]. Every year, 470,000 cases of cervical cancer are diagnosed worldwide, and about half of the women afflicted will die. In the U.S. alone, 50 million Pap tests are performed each year, and they discover close to 1.2 million cases of low-grade dysplasia (cervical intraepithelial neoplasia [CIN]1), 300,000 cases of high-grade dysplasia (CIN2/3) and 10,000 cases of cervical cancer [2]. The total health care cost associated with the screening and treatment of cervical cancer in the U.S. is estimated to be $6 billion per year. Although screening has dramatically reduced the incidence of this disease in the developed world, it is still estimated that there will be 3,710 deaths from cervical cancer in the U.S. during In areas of the world where most women do not have access to regular gynecological care and screening, cervical cancer is second only to breast cancer as a cancer-related cause of death [2]. Current treatment of cervical dysplasia is limited to excisional or ablative procedures that remove or destroy cervical tissue. These procedures have efficacy rates of approximately 90% but are associated with morbidity and expense. Additionally, surgical treatments remove only the dysplastic tissue, leaving normal-appearing HPV-infected tissue untreated [3]. It is therefore desirable to eradicate this infection using a vaccine, as was done previously with great success with hepatitis B. Preventive vaccination of adolescents before they first encounter HPV aims at this target. However, already-infected women as well as patients suffering from advanced cervical cancer could also benefit from therapeutic vaccinations under development. This article reviews the available information on prophylactic and therapeutic vaccines for HPV infection, with an emphasis on recent clinical trials. HPV HPVs belong to a family of small (8-kb pairs) doublestranded circular DNA viruses that infect squamous epithelia of the genital tract, anal, and perianal areas, and mucosal epithelium of the larynx. HPV-associated genital tract disease is now the most commonly diagnosed sexually transmitted disease in the U.S. Approximately 20 million people in the U.S. at any given time are infected with HPV and are able to transmit it to others. Low-risk HPVs, such as HPV-6 and HPV-11, cause benign genital warts, whereas high-risk types, such as HPV-16 and HPV-18, are associated with the development of high-grade squamous intraepithelial lesions and cervical cancer. It is estimated that HPV-16 accounts for approximately 60% of cervical cancers, with HPV-18 adding another 10% 20%. Other high-risk types include types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73. Infection in both women and men is clearly related to sexual activity as well as environmental factors such as birth control pills and smoking. For women, the most striking risk factors for HPV infection and the development of detectable pathology are numerous lifetime sexual partners and early onset of sexual activity. Although HPV-related cancers are more common in women, increasing numbers of HPV-related carcinomas of the anal mucosa are being reported among men having sex with men [4]. A third group at risk for severe HPV infections is neonates. At birth, HPV can infect the mucosa of the pharynx and cause large wartlike lesions that can obstruct the airway. To date, more than 100 genotypes of HPV have been described. Genotyping of HPV is based on DNA sequences of the L1, E6, and E7 genes. A 10% difference in sequence with respect to previously established strains is sufficient to define a new type of virus. In general, HPV infects the basal cells of human epithelial surfaces. Infected basal cells divide; some progeny remain as infected basal cells, while others move away from the basement membrane, differentiate, and become epithelial cells. Virus replication and assembly is tightly linked to the differentiation program of epithelial cells. Infectious virions are produced only in the terminally differentiated cell and are shed as virus-laden squamous cells. This explains why HPV cannot grow in tissue culture. By infecting only the basal layer cells and executing viral replication and assembly only in a fully differentiated cell, HPV avoids the immune system of the host. The success of this strategy is documented by very poor immune response (humoral as well as cell-mediated) to HPV infection. The nature of this response is under investigation; nevertheless, most women infected spontaneously resolve their infection in less than 2 years. Infections that are not controlled and persist for prolonged periods can cause more severe pathologies and, ultimately, cancer [5]. The HPV genome codes for only eight proteins (open reading frames). The late L1 and L2 genes code for the viral capsid proteins, the early proteins E1 and E2 are responsible for viral replication and transcription, and E4 seems to aid virus release from infected cells [6]. The early genes of the high-risk HPV types (E6 and E7) encode the main transforming proteins. These genes are capable of immortalization of epithelial cells and are thought to play a role in

3 530 Human Papillomavirus Vaccines the initiation of the oncogenic process. The protein products of these early genes interfere with the normal function of tumor suppressor genes. HPV E6 is able to interact with p53, leading to its dysfunction, thereby impairing its ability to block the cell cycle when DNA errors occur. E6 also keeps the telomerase length above its critical point, protecting the cell from apoptosis. HPV E7 binds to retinoblastoma protein (prb) and activates genes that start the cell cycle, leading to tissue proliferation. E5 has also been implicated in cellular transformation. Thus, there is now ample evidence based on molecular data alone that HPV has the tools to cause cancer. Clinical, pathological, and virological studies have defined a progression of events in the development of cervical cancer. The pathogenesis of cervical cancer is initiated by HPV infection of the cervical epithelium during sexual intercourse. The initial cervical epithelial changes, pathologically classified as CIN1, are associated with continued viral replication and virus shedding. Progression to highgrade lesions (CIN2/3) and ultimately to invasive cancer is usually associated with conversion of the viral genome from an episomal form to an integrated form, along with deletion or inactivation of E2, a negative regulator of E6 and E7 expression. Development of invasive cancer requires additional genetic events facilitated by E6- and E7-mediated inactivation of the genome guardians p53 and prb, genomic instability, and suppression of apoptosis [7]. Several studies have demonstrated that virus-neutralizing antibodies mediate protection of animals from experimental papillomavirus infection. For example, passive transfer of sera from virus-like particle (VLP) vaccinated rabbits to naïve rabbits is sufficient for protection [8]. Similarly, vaccination with L2 peptides protects rabbits from papillomas resulting from viral but not from viral DNA challenge, consistent with the protection mediated by neutralizing antibodies [9]. Although antibody-mediated neutralizing of virus has important preventive utility, several lines of evidence suggest that cell-mediated immune responses are also important in controlling established HPV infections as well as HPV-associated neoplasms [10]: (a) The prevalence of HPV-related diseases (infections and neoplasms) is higher in patients with impaired cell-mediated immunity, including transplant recipients and HIV-infected patients; (b) animals immunized with nonstructural viral proteins are protected from papillomavirus infection and the development of neoplasia. Immunization also facilitates the regression of existing lesions; (c) Infiltrating CD4 + (T-helper cells) and CD8 + (cytotoxic T cells) cells have been observed in spontaneously regressing warts; (d) Warts in patients receiving immunosuppressive therapy often disappear when treatment is discontinued. The well-characterized foreign (viral) antigens and the well-defined virological, genetic, and pathological progression of HPV have provided a unique opportunity to evaluate interventions with antigen-specific immunotherapy. Conceptually, two different types of HPV vaccines can be designed: prophylactic (preventive) vaccines that prevent HPV infection and therapeutic (curative) vaccines that induce regression of established HPV infection and its sequelae. Vaccine Efficacy Traditionally, in etiological and cancer prevention studies, the measurable end point to determine efficacy of an intervention has been the incidence of cancer itself. But as some cancers take a long time to develop and are not common in a given population, trials with an end point of invasive cancer can be prohibitively large and lengthy. In the case of cervical cancer, a disease that can be prevented through proper detection and treatment, a study end point of cancer can be ethically impracticable. Recently, the U.S. Food and Drug Administration Vaccines and Related Biologicals Advisory Committee concluded that the primary end point for vaccine licensure in the U.S. should be CIN2/3 as well as cancer [11]. Because persistent infection with the same high-risk type is considered a predictor for high-grade cervical dysplasia and cancer, these surrogate end points might be useful in future vaccine efficacy studies. Indeed, if vaccines prove to be effective against transient or especially persistent HPV infections, it is likely that they will protect women against cervical cancers as well. An ideal prophylactic vaccine needs to possess several attributes [12]. It should be safe, because it would be given to young, normal individuals, the vast majority of whom, even without a vaccine, would not be expected to develop cancer from HPV infection. It should be able to be administered in settings with poor resources, be inexpensive, and be effective after a single dosage. Protection should last many years and the vaccine should confer a substantial reduction in the incidence of cervical cancer. However, it is difficult to define precisely what degree of reduction in cervical cancer would justify the widespread use of an HPV vaccine. Mammography is recommended for breast cancer screening, although it is believed to reduce breast cancer deaths by no more than one third. By comparison, an HPV vaccine that successfully targeted all cervical cancer cases attributable to HPV-16 alone would be expected to reduce the worldwide incidence of cervical cancer by more than half. In countries with effective population-wide cervical cancer screening programs, other considerations are whether the introduction of a vaccine might be able to reduce the physical, psychological, and financial costs

4 Mahdavi, Monk 531 associated with screening and follow-up through a reduction in the frequency of detection of cervical abnormalities and/or the frequency of screening. Prophylactic Vaccines Probably the most important contribution to the field of HPV vaccine development came in 1991, when Zhou et al. [13] showed that HPV-16 L1 capsid protein, when expressed in a recombinant system, formed virus-like particles (VLPs) that resembled native virions. These VLPs have been proven to be highly antigenic. Parenteral injection of these VLPs elicits high titers of serum-neutralizing antibodies and protection from experimental viral challenge with infectious virus in several animal papillomavirus models. Protection from experimental infection by cottontail rabbit papillomavirus (CRPV) or canine oral papillomavirus after passive transfer of IgG from VLP-immunized animals to naïve animals has been demonstrated in rabbits and dogs, respectively [8, 14]. The results from these protective vaccine studies indicate that neutralizing IgG provides protection from experimental infection. Although VLP vaccination provides immunity from experimental inoculation, protection against sexual transmission of HPV requires neutralizing antibodies acting on mucosal surfaces. Serum-neutralizing IgG induced by parenteral VLP vaccination may enter the genital tract via transudation and maintain a sufficient level to provide sterilizing immunity across the menstrual cycle. Neutralizing antibodies may either transudate from plasma into genital secretions or be synthesized by local plasma cells; induction of plasma cells requires direct immunization of mucosa-associated lymphoid tissue [15]. Recently, nasal instillation of VLPs has been found to be efficient in generating specific antibodies, including IgG in serum and IgA in mucosal secretions of mice [16]. Oral vaccination with HPV VLPs in mice has also been shown to induce systemic virus-neutralizing antibodies, suggesting that HPV VLPs may be antigenically stable in the environment of the gastrointestinal tract. Recently, Liu et al. [17] showed that HPV-16 L1 protein could be expressed in transgenic tobacco plants and that the expressed protein possessed the natural features of HPV-16 L1 protein, implying that the HPV-16 L1 transgenic plants could be potentially used as an edible vaccine. These studies provide the possibility of vaccinating large populations with HPV VLPs without using syringes. In recent clinical data, intramuscular vaccination of women with HPV-16 VLPs was found to induce significant antibody titers in cervical secretions [18]. A comparison of nasal spray versus aerosol for mucosal delivery of 50 μg of HPV-16 VLPs to humans revealed that aerosol was effective at inducing an antibody response, with IgG found predominantly in serum and both IgG and IgA found in cervical secretions. Phase I/II clinical trials using HPV L1 VLPs delivered intramuscularly have demonstrated the immunogenicity and safety of this vaccine. Koutsky et al. [19] recently reported data from a clinical trial of HPV-16 L1 VLPs, showing for the first time that a vaccine strategy could be implemented in humans to prevent HPV-16 infection and HPV-16 associated premalignant lesions. Young women (n = 2,392) were assigned to receive either placebo or yeast-derived HPV-16 L1 VLPs (40-μg dosage), formulated on 225 μg aluminum hydroxy phosphate sulfate adjuvant (Merck & Co., Inc., Whitehouse Station, NJ, at month 0, month 2, and month 6 by intramuscular injection (Table 1). Samples from the genital tract were obtained at enrollment, 1 month after the booster immunization, and every 6 months thereafter. In addition, the women underwent gynecological examinations and were referred for colposcopy according to the protocol. Biopsy tissue was evaluated for intraepithelial neoplasia and analyzed by polymerase chain reaction (PCR) for the presence of HPV-16 DNA. DNA was prepared from the specimen using routine methods. HPV-16 DNA was amplified by PCR using type- and gene-specific primers for the HPV-16 L1, E6, and E7 genes. PCR products were visualized by dot-blot hybridization using type- and gene-specific oligonucleotides. The assays were validated to have a 95% probability of detecting 13 copies of HPV-16 DNA per sample. The primary end point of the trial was persistent HPV-16 infection, defined as: (a) HPV- 16 DNA detected in samples obtained at two or more visits at least 4 months apart; (b) a cervical biopsy showing CIN or cancer and HPV-16 DNA in the biopsy and in a genital sample collected at the antecedent or subsequent visit; or (c) HPV-16 DNA detected in a sample collected during the last visit before being lost to follow-up. Women were followed for a median of 17.4 months after completion of the vaccination regimen, at which time, 41 cases of persistent HPV-16 infection were accrued. All 41 cases occurred in the placebo group, none occurred in the vaccine group. Of these 41 cases, 31 were persistent HPV- 16 infection, five were HPV-16 related CIN1, four were HPV-16 related CIN2, and one occurred in a woman who first tested positive for HPV-16 on the last visit before she was lost to follow-up. These results translate to a 100% efficacy (95% confidence interval [CI], 90% 100%; p <.001). Because all nine cases of HPV-16 related CIN were in the placebo group, there is great hope that an HPV-based vaccine may reduce the incidence of cervical cancer. In a fol-

5 532 Human Papillomavirus Vaccines low-up study, Ferris et al. [20] showed that none of the vaccinated women developed CIN caused by HPV-16 during 3.5 years of follow-up. The overall rates of CIN and particularly CIN2/3 were also lower in the vaccinated group. In another recent, randomized, double-blinded, controlled trial, Harper et al. [21] assessed the efficacy, safety, and immunogenicity of a bivalent HPV-16/18 L1 VLP vaccine for the prevention of incident and persistent infections with these two virus types, associated cervical cytological abnormalities, and precancerous lesions. They randomized 1,113 women between 15 and 25 years of age to receive three dosages of either the vaccine, formulated with 500 μg aluminum hydroxide and 50 μg 3-deacylated monophosphoryl lipid (AS04) adjuvant, or placebo at 0, 1, and 6 months in North America and Brazil (Table 1). Women were assessed for HPV infection by cervical cytology and self-obtained cervicovaginal samples for up to 27 months, and for vaccine safety and immunogenicity. In the according-to-protocol analyses, vaccine efficacy was 91.6% (95% CI, 64.5% 98.0%) against incident infection and 100% against persistent infection with HPV-16/18. In the intent-to-treat analyses, vaccine efficacy was 95.1% against persistent cervical infection with HPV- 16/18 and 92.9% against cytological abnormalities associated with HPV-16/18 infection. The vaccine was generally safe, well tolerated, and highly immunogenic. These two high-quality studies showed that HPV vaccines were not only safe and well tolerated but were also efficacious in the prevention of incident and persistent cervical HPV infections and associated cytological abnormalities and lesions. Vaccination against such infections could substantially reduce the incidence of cervical cancer. Merck and GlaxoSmithKline (Philadelphia, are currently conducting phase III clinical trials to assess the efficacy of quadrivalent HPV-6/11/16/18 and bivalent HPV-16/18 vaccines, respectively. From a technical perspective, vaccination with VLPs appears promising. Nevertheless, several practical issues must be addressed before these vaccines can be licensed and deployed in clinical practice and public health programs [6]. Regarding the optimal age for vaccination, it is estimated that an HPV-16/18 vaccine for 12-year-old girls would reduce cohort cancer cases by 61.8% with a costeffectiveness ratio of $14,583 per quality-adjusted life year (QALY) [22]. Including male participants in a vaccine rollout would further reduce cervical cancer cases by only 2.2% at an incremental cost-effectiveness ratio of $442,039/QALY compared with female-only vaccination. Vaccination against HPV-16 and HPV-18 can be cost-effective, although including male participants in a vaccination program is generally not cost-effective, compared with female-only vaccination. Even if HPV vaccines are shown to be safe and effective, marketing a vaccine against a sexually transmitted disease to the general public may be problematic. Parental resis- Table 1. Preventing HPV infection: comparison of monovalent (Merck) and bivalent (GlaxoSmithKline) L1 VLP prophylactic vaccines Study Koutsky et al. [19] (Merck study) Harper et al. [21] (GlaxoSmithKline study) Design Randomized double-blind controlled trial Randomized double-blind controlled trial Age (years) No. of enrollees 2,392 1,113 Location 16 sites in the U.S. 32 sites in North America and Brazil Antigen 40 μg HPV-16 L1 VLP 20 μg HPV-16 L1 VLP 20 μg HPV-18 L1 VLP Adjuvant 225 μg aluminum hydroxyphosphate sulfate 500 μg aluminum hydroxide and 50 μg 3-deacylated monophosphoryl lipid (ASO4) Vaccination schedule 0, 2, and 6 months 0, 1, and 6 months Follow-up Mean of 17.4 months Up to 27 months Specific titers compared with natural infection Clinical outcome 60 times greater 50 times greater for HPV-16; 80 times greater for HPV % efficacy preventing persistent HPV- 16 infection. No cytologic or histologic abnormalities. Adverse effects Nonsignificant Nonsignificant Abbreviations: HPV, human papillomavirus; VLP, virus-like particle. 100% efficacy preventing persistent HPV- 16/18 infection. 93% efficacy preventing cytological abnormalities.

6 Mahdavi, Monk 533 tance can easily be imagined. The most effective strategy is to maintain philosophical distance from sexual aspects of the question and focus on the prevention of a common cause of cancer. In a recent survey by Slomovitz et al. [23], 75% of women accepted a cervical cancer vaccine for themselves and 70% accepted such a vaccine for their children. Therapeutic Vaccines Even if prophylactic vaccination was instituted on a worldwide scale today, it would take decades (because of the long latency from HPV infection to the development of cervical cancer) to perceive the benefits; that is, lower incidences of cervical preinvasive and invasive disease. Therapeutic vaccines are a means of bridging the temporal deficit by attacking already-established HPV infections and HPV-related disease in the lower genital tract [24]. In contrast to preventive vaccines, HPV therapeutic vaccines need to include some antigenic determinants derived from the early HPV proteins (e.g., E2, E6, and E7) rather than the late proteins. This has proved to be a much more challenging task in terms of biodelivery and response. Whereas most tumor-specific antigens are derived from normal or mutated proteins, E6 and E7 are completely foreign viral proteins, and thus may harbor more antigenic peptides/epitopes than a mutant cellular protein. Furthermore, because E6 and E7 are required for the induction and maintenance of the malignant phenotype of cancer cells, cervical cancer cells are unlikely to evade an immune response through antigen loss. Thus, E6 and E7 proteins represent good targets for developing antigen-specific immunotherapies or vaccines for cervical cancer. It should be highlighted that cellular immune response appears to be the key component necessary for clearance of HPV infections and therefore would be the main target of any therapeutic HPV vaccine. Various forms of HPV vaccines have been described in experimental systems targeting HPV-16 E6 and E7 proteins (Table 2). Most studies have focused on E7, because it is more abundantly expressed and better characterized immunologically [25, 26]. Furthermore, its sequence is more conserved than that of the E6 gene. Viral Vector Vaccines Viral vector vaccines have the advantages of being highly immunogenic and having different immunogenic properties of viruses. The drawbacks include safety concerns and pre-existing viral immunity in the recipient (Table 2). Several studies have shown that immunotherapy targeting E6 and/or E7 using vaccinia vectors generates strong cytotoxic T-lymphocyte (CTL) activity and antitumor responses in preclinical studies. The advantage of these vaccines is that HPV proteins are endogenously synthesized from viral DNA by host cells, and an array of HPV peptides are produced, processed, and presented on the cell surface in conjunction with major histocompatibility complex (MHC) class I molecules. Such a system poses no restriction on patients HLA genotypes or CTL repertoire and allows several HPV types or antigens to be included in the vaccine. In a multicenter European Organisation for Research and Treatment of Cancer phase II trial, a live recombinant vaccinia virus encoding HPV-16 and HPV- 18 E6 and E7 (TA-HPV) was administered to early-stage cervical cancer patients, and immune responses were measured before and after immunization. That study established the safety and immunogenicity of the vaccine in at least a proportion of those patients vaccinated [27]. TA-HPV has also been used in the treatment of high-grade vulvar intraepithelial neoplasia (VIN), because this disease often presents as a chronic problem unresponsive to standard ablation treatments [28]. There was some, but not complete, correlation between HPV immunity and clinical response defined by lesion shrinkage at 24 weeks postvaccination. Importantly, the best correlation with response was local immune infiltration. Prior to vaccination, clinical responders had significantly higher levels of lesion-associated CD4 +, CD8 +, and CD1a + immune cells than nonresponders. It appears that local immune status may be a critical factor in potential responsiveness to vaccine therapy in HPV-associated neoplasia and should be carefully monitored in future placebo-controlled trials of immunotherapy for VIN [29]. Table 2. Characteristics of therapeutic HPV vaccines Advantages Drawbacks Vector-based: viral/bacterial Highly immunogenic Safety concerns, previous immunization Peptide-based Safety, easy to produce Weakly immunogenic, requires HLA compatibility Protein-based Safety, no HLA restriction Weak activator of cell-mediated immunity DNA Easy to produce, store, and transport; sustained antigenic expression Weakly immunogenic DC-based Highly immunogenic Difficult to produce and biodeliver Abbreviations: DC, dendritic cell; HPV, human papillomavirus.

7 534 Human Papillomavirus Vaccines Studies using modified adenovirus-expressing HPV- 16 E6 or E7 for vaccination [30] or for ex vivo preparation of dendritic cell (DC) based vaccines have demonstrated enhancement of antigen-specific T-cell immune responses and antitumor effects. Replication-defective adeno-associated virus encoding HPV-16 E7 fused to heat-shock protein 70 was found to induce CD4- and CD8-dependent CTL activity and antitumor effects in vitro [31]. Bacterial Vector Vaccines Bacterial vector vaccines are highly immunogenic and can deliver engineered plasmids or express proteins. As with viral vaccines, safety concerns, pre-existing immunity, and inhibited repeat immunization limit their clinical application. Attenuated bacteria (e.g., Listeria monocytogenes, Salmonella, Shigella, Escherichia coli) can serve as bacterial carriers to deliver either plasmids encoding genes of interest or proteins of interest to antigen-presenting cells (APCs). L. monocytogenes produces listeriolysin O, which allows escape into the cytoplasm after phagocytosis by macrophages and facilitates delivery of antigens into both MHC-I and MHC-II pathways. Recent studies have found that intraperitoneal or oral vaccination with recombinant L. monocytogenes secreting HPV-16 E7 can lead to regression of pre-existing E7-expressing murine tumors [32]. Attenuated Salmonella and Bacille Calmette-Guerin (Mycobacterium bovis) are safe bacterial vaccine vectors that have been used to develop vaccines encoding HPV-16 L1 and E7 and have been found to induce E7-specific antibody and cytotoxic immune responses [33]. Peptide Vaccines Peptide vaccines have the advantages of safety and ease of production; however, their weak immunogenic properties and the need for HLA matching must be overcome. Because 40% of Caucasians carry the HLA-A2 alleles, HPV-16 E7 peptides presented by this allele have been the immunogen in several phase I/II clinical trials [34]. Ressing et al. [35] performed a peptide-based phase I/II vaccination trial to induce antitumor immune responses in patients with recurrent or residual cervical carcinoma. Fifteen HLA-A*0201 positive patients with HPV-16 positive cervical carcinoma received vaccinations with synthetic peptides representing two HPV-16 E7 encoded, HLA-A*0201 restricted CTL epitopes and a pan-hla-dr binding T-helper epitope, in adjuvant. No signs of toxicity were observed. Two patients had stable disease for more than 1 year after vaccination, three patients died of the disease during or shortly after the vaccination period, and 10 patients maintained progressive cervical carcinoma [36]. In another study [37], 18 women with high-grade cervical or vulvar dysplasia who were positive for HPV-16 and HLA-A2 were treated with escalating dosages of a vaccine consisting of a nine amino acid peptide encoded by the E7 gene emulsified with incomplete Freund s adjuvant. They received four immunizations of the vaccine each, 3 weeks apart, followed by a repeat colposcopy and definitive removal of dysplastic tissue 3 weeks after the fourth immunization. Only three of the 18 patients were free of dysplasia after vaccination, but an increased S100 + DC infiltrate was observed in six of six patients tested. Virological assays showed that 12 of 18 patients showed no virus in cervical scrapings by the fourth vaccine injection, but all biopsy samples were still positive by in situ RNA hybridization after vaccination. In addition to the three complete responders, six patients had partial colposcopically measured regression of their CIN lesions. Protein Vaccines Whereas peptide vaccines exhibit MHC restriction, protein-based vaccines can bypass this restriction and thus are less dependent on the patient s HLA type (Table 2). TA-GW fusion protein, which consists of HPV-6 L2 fused to E7 protein, has been successfully tested for the clinical treatment of genital warts [38]. TA-CIN fusion protein, which consists of HPV-16 L2/E6/E7, can induce E7-specific CD8 + T-cell immune responses and tumor protection in mice. TA-CIN was well tolerated by patients and induced both humoral and T-cell mediated immune responses [39]. The potency of HPV-16 E7 peptide based vaccines may be further enhanced through the use of adjuvants, fusion proteins, or anchor-modified peptide epitopes. Chu et al. [40] demonstrated that adjuvant-free immunization of C57B1/6 mice with heat shock protein E7 (hspe7) protected the animals against challenge with an E7-expressing murine tumor cell line (TC-1) and also protected against rechallenge with higher doses of TC-1 cells. Similarly, immunization of tumor-bearing mice with hspe7 led to tumor regression, protection from rechallenge, and longterm survival. Tumor regression after hspe7 immunization appeared to be dependent on CD8 + T cells, but independent of CD4 + T cells. More recently, in a single-stage phase II study (Table 3), Einstein et al. [41] administered three monthly hspe7 vaccines to 31 women with biopsy-proven CIN3. At 4 months, all patients underwent a loop electrosurgical excision procedure or cone biopsy. A complete pathologic response was defined as no evidence of CIN or CIN1. A partial response was defined as colposcopic regression of the lesion by more than 50%. hspe7 vaccine treatment resulted in a significantly higher than expected pathologic response rate among women with CIN3: 48%

8 Mahdavi, Monk 535 had complete responses, 19% had partial responses, and 33% had stable disease. No patient had progressive disease. The responses were not significantly associated with HPV- 16 infection status or infection with multiple subtypes; however, the sample size was limited. hspe7 vaccine is now in clinical trials with Stressgen Biotechnologies (Victoria, British Columbia, Canada to treat asymptomatic HPV infections, HPV-associated anal dysplasia, and HPV-associated cervical cancer. A phase II evaluation of SGN (hspe7) fusion protein in women with CIN3 is under way by the Gynecologic Oncology Group (GOG protocol #197). DNA Vaccines DNA vaccines allow for sustained expression of antigen on MHC peptide complexes, compared with peptide or protein vaccines. Furthermore, the MHC restriction of peptide-based vaccines may be bypassed with approaches that directly transduce DNA coding for antigen to APCs so that the synthesized peptides can be presented by the patient s own HLA molecules [10]. Due to the weak intrinsic potency of naked DNA vaccines, various strategies have been developed to enhance their immunogenicity. Because DCs are the primary mediators of DNA vaccine induced immune responses, vaccines that modify intracellular or intercellular movement of antigen or other DC properties are able to enhance DNA vaccine potency. Because DCs have a limited lifespan, a method to prolong in vivo DC survival may help improve DNA potency. Coadministration of E7-containing DNA with DNA-encoding antiapoptotic proteins (Bcl-xl, Bcl-2) is able to enhance E7-specific immune responses, tumor treatment, and DC survival [42]. Another strategy to improve delivery and antigenicity of HPV DNA vaccines is the use of encapsulation. Garcia et al. [43] reported on the use of encapsulated plasmid DNAencoding fragments derived from E6 and E7 of HPV-16 and HPV-18 in biodegradable particles (ZYC101a). Their study population included women with biopsy-confirmed CIN2 or CIN3 (Table 3). The study design was double-blinded, randomized, and placebo-controlled and was carried out in multiple centers in the U.S. and Europe. A total of 161 patients was evaluated for efficacy and safety, but only 127 were evaluable for efficacy after pathologic review of the cases. Two dosages of the vaccine (100 μg and 200 μg) were evaluated against a saline placebo. Patients were injected at 0, 3, and 6 months. Approximately 6 months after study entry, all patients underwent an excisional procedure of the cervix, most often a loop electrosurgical excision procedure. Patients were monitored with periodic colposcopic evaluations, cytology, and HPV testing. There were statistically significant higher incidences of injection site pain, erythema, and induration in the treatment groups than in the placebo group, but no other adverse events differed significantly between the treatment and placebo groups. For analysis purposes, patients were divided into two age categories, defined as <25 years of age and >25 years of age. There was a statistically significant higher rate of CIN2 or CIN3 resolution in the <25 years of age group (placebo, 23%; 100 μg, 67%; and 200 μg, 72%). However, there was no difference in resolution rates between vaccine (either dosage) and placebo in the group >25 years of age. Neither Table 3. Treating high-grade CIN: comparison of ZYC101a a and hspe7 b therapeutic vaccines Study Garcia et al. [43] ZYC101 Einstein et al. [41] hspe7 Design Randomized double-blind controlled trial Single-stage phase II design Age (years) 18 or older Not specified No. of enrollees Delivery system Encapsulated polynucleotide Mycobacterium bovis BCG heat shock protein Antigen HPV-16 and HPV-18 E6/E7 HPV-16 E7 Disease group CIN2/3 CIN3 Vaccination schedule 0, 3, and 6 months 0, 1, and 2 months Follow-up 6 months 4 months Clinical outcome 67% 72% resolution of CIN2/3 in < 25- years-old age group (23% in placebo group) 48% resolution of CIN3; 19% partial response; 33% stable disease Adverse effects Injection site pain, erythema, and induration Not specified a Manufactured by MGI Pharmaceuticals. b Manufactured by Stressgen Biotechnologies. Abbreviations: BCG, Bacille Calmette-Guerin; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; hspe7, heat shock protein E7.

9 536 Human Papillomavirus Vaccines immune parameters nor other variables, such as tobacco use or infection with specific HPV types, correlated with response or lack thereof. This vaccine has been acquired by MGI Pharmaceuticals (Bloomington, MN, mgipharma.com) and further studies are planned. DC-Based Vaccines DCs are the most important APCs in the immune system. Techniques have been developed for isolation, culturing, and antigen loading. Santin et al. [44] demonstrated that recombinant, full-length, E7-pulsed, autologous DCs could elicit a specific CD8 + CTL response against autologous tumor target cells in three patients with HPV-16 or HPV- 18 positive cervical cancer. Their results demonstrated that full-length, E7-pulsed DCs could induce both E7-specific CD4 + T-cell proliferative responses and strong CD8 + CTL responses capable of lysing autologous HPV-infected cancer cells in patients with cervical cancer. In another study [45], 15 patients with stage IV cervical cancer were treated with autologous monocyte-derived DCs that had been pulsed with recombinant HPV-16 E7 or HPV-18 E7 oncoprotein. Specific cellular immune responses were detected in four of the 11 patients. A transient drop in a squamous cell carcinoma tumor marker was documented in five of nine patients but did not correlate with immune response. Tumor Cell Based Vaccines Transduction of tumor cells with genes encoding costimulatory molecules or cytokines may enhance immunogenicity, leading to T-cell activation and antitumor effects after vaccination. Vaccines using HPV-transformed tumor cells transduced with cytokine genes, such as interleukin-12, interleukin-2, or GM-CSF [46], can generate strong antitumor effects in mice. Combined Approaches With this approach, it is hoped that combining vaccine vehicles using a prime-boost strategy might allow priming the immune system with one vaccine technology, followed by augmenting and maintaining a long-term immune response with another boosting vaccine. Indeed, Chen et al. [47] have suggested that priming with a DNA vaccine followed by a recombinant vaccinia booster might provide the most potent anti-hpv effects. Another combination approach involves the simultaneous administration of DNA vaccines and other antiviral or anticancer agents. Christensen et al. [48] topically administered the antiviral agent cidofovir (Vistide ; Gilead Sciences, Foster City, CA, and intracutaneously administered DNA vaccine encoding CRPV genes. Cures of large, established, CRPV-induced rabbit papillomas and a lower incidence of lesion recurrence were observed, suggesting that this combination may also be useful in treating persistent HPV infections. Combined Prophylactic and Therapeutic Vaccines Therapeutic vaccination may be useful in the treatment of premalignant lesions in conjunction with prophylactic strategies. It has been shown that VLPs can activate DCs, and HPV-16 VLP-E7 chimera vaccines can generate useful T-cell responses to E7 [49], as well as neutralizing antibodies to viral capsids. This approach could provide a means to effectively treat incident HPV infection. The risk for cancer development from long-term HPV infection could be managed with local treatment and immunization in these patients, but as is presumably the case now, most individuals would develop effective natural immunity. Conclusion and Future Directions HPV is a ubiquitous virus. However, the infection is often transient and self-limiting. Several studies have suggested that HPV infection and cervical dysplasia can be prevented by HPV L1 VLP vaccines. It is anticipated that second-generation prophylactic vaccines that target neutralizing epitopes from both L1 and L2 as well as therapeutic antigens will increase the spectrum of covered oncogenic genotypes. Therapeutic vaccines present far more challenges than prophylactic vaccines. The challenges include the immunocompromised state of cancer patients, difficulty in stimulating the immune system, immune escape mechanisms used by tumors and virally infected cells, and safety concerns. Despite these challenges, there are many encouraging results suggesting that T-cell responses to the E6/E7 proteins can be generated in animal models as well as in humans. Much still needs to be investigated regarding local immune responses in the lower genital tract, longevity of immune responses, and alternative delivery routes, such as intravaginal, intranasal, and oral administration. This is an exciting time for HPV vaccine research; HPV vaccine strategies have the potential to eradicate a major cancer and source of morbidity around the world. Disclosure of Potential Conflicts of Interest Dr. Monk receives research support from MGI, Digene, and Cyto.

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