Vaccination to prevent and treat cervical cancer

Vaccination to prevent and treat cervical cancer

Perspectives in Pathology Vaccination to Prevent and Treat Cervical Cancer RICHARD B. S. RODEN, PHD, MORRIS LING, BS, AND T.-C. WU, MD, PHD Human papi...

188KB Sizes 2 Downloads 33 Views

Perspectives in Pathology Vaccination to Prevent and Treat Cervical Cancer RICHARD B. S. RODEN, PHD, MORRIS LING, BS, AND T.-C. WU, MD, PHD Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer. Thus, cervical cancer and other HPVassociated malignancies might be prevented or treated by HPV vaccines. Transmission of papillomavirus may be prevented by the generation of antibodies to capsid proteins L1 and L2 that neutralize viral infection. However, because the capsid proteins are not expressed at detectable levels by infected basal keratinocytes or in HPV-transformed cells, therapeutic vaccines generally target nonstructural early viral antigens. 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. Thus, therapeutic vaccines targeting E6 and E7 may provide the best option for controlling HPV-associated malignancies. Various candidate therapeutic HPV vaccines are currently being tested whereby E6 and/or E7 are administered in live vectors, as peptides or protein, in nucleic acid form, as components of chimeric virus-like

particles, or in cell-based vaccines. Encouraging results from experimental vaccination systems in animal models have led to several prophylactic and therapeutic vaccine clinical trials. If these preventive and therapeutic HPV vaccines prove successful in patients, as they have in animal models, then oncogenic HPV infection and its associated malignancies may be controllable by vaccination. HUM PATHOL 35:971-982. © 2004 Elsevier Inc. All rights reserved. Key words: cervical cancer, human papillomavirus, vaccine, viruslike particle, L2, immunotherapy, tumor-specific antigen, cytotoxic T lymphocyte and tumor immunology. Abbreviations: APC, antigen-presenting cell; BCG, bacille Calmette-Guerin; COPV, canine oral papillomavirus; CRPV, cottontail rabbit papillomavirus; CTL, cytotoxic T lymphocyte; DC, dendritic cell; HPV, human papillomavirus; HSP, heat-shock protein; HSV, herpes simplex virus; MHC, major histocompatibility complex; SIL, squamous intraepithelial lesion; VLP, virus-like particle.

Approximately 500,000 women worldwide develop cervical cancer yearly, and cervical cancer is the thirdleading cause of cancer death in women. Over the past 20 years, epidemiologic and virological data have identified a clear and consistent association of HPV infection with the development of cervical cancer. The evidence linking human papillomaviruses (HPVs) to cervical cancer comes from a wide variety of epidemiologic and laboratory studies. More than 99% of cervical cancers and their precursor lesions, squamous intraepithelial lesions (SILs), contain HPV DNA.1 Furthermore, molecular, biochemical, and cellular studies have unequivocally demonstrated that E6 and E7, HPV gene products that are consistently expressed in SILs and cervical cancer, lead to malignant transformation of epithelial cells.2 Although more than 100 HPV genotypes have been identified, about 80% of cervical cancer is associated with 4 “high-risk” types of HPV (types 16, 18, 31, and 45), and the remaining cases are associated with a dozen other oncogenic types.3,4 HPV-16 is present in approximately 50% of all cervical

cancers3 and consequently has been the focus of many recent HPV vaccine developments. 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. Most genital HPV infections are transient.5 However, a fraction of infections persist and initiate transformation events within cervical epithelium. The cervical epithelial changes associated with this initial set of events, pathologically classified as lowgrade SILs (LSILs; cervical intraepithelial neoplasia [CIN] 1), are associated with continued viral replication and virus shedding. In a study of 241 cytologically normal women recruited in a sexually transmitted disease clinic, the cumulative incidence of high-grade SILs (HSILs; CIN 2-3) at 2 years was 28% in HPV-positive women, compared with 3% in HPV-negative women.6 The progression from HSIL to invasive cancer occurs at high frequency. In the majority of cases, progression is 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.2 Several studies have demonstrated that virus-neutralizing antibodies mediate protection of animals from experimental papillomavirus infection.7-9 For example, passive transfer of sera from virus-like particle (VLP)-

From the Departments of Pathology, Oncology, Obstetrics and Gynecology, and Molecular Microbiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, MD. Accepted for publication April 7, 2004. Address correspondence and reprint requests to T.-C. Wu, MD, PHD, Professor, Department of Pathology, Johns Hopkins University, 512H Ross Building, 720 Rutland Avenue, Baltimore, MD 21205. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2004.04.007



Volume 35, No. 8 (August 2004)

Table 1. Summary of Published L1-Based Prophylactic Vaccine Studies in Animal Models and Humans Vaccination schedule

Study (et al)

Virus and animal

Dose and adjuvant

Jarrett Bell Donnelly Stanley Lin Breitburd Kirnbauer Suzich Christensen

BPV1,2,4,5,6: cow COPV: dog CRPV: rabbit COPV: dog CRPV: rabbit CRPV: rabbit BPV4: cow COPV: dog CRPV: rabbit

Virus 2⫻ inactivated virus 1 mg L1 DNA 9␮g L1 DNA Vaccinia L1 106 pfu 3⫻ 50 ␮g; VLP: Alum 2⫻ 150 ␮g; VLP: alum 2⫻ 20␮g; VLP: None 3⫻ 50␮g; VLP: None

0, 0, 0, 0, 0, 0, 0, 0, 0,

Jansen Yuan

CRPV: rabbit COPV: dog


HPV16: human


HPV11: human

Zhang Koutsky Dubin*

HPV6b: human HPV16: human HPV16 and 18: human

3⫻ 1␮g; VLP: none 0.4␮g L1; Pentamers: none 3 ⫻ 10 or 50 ␮g; VLP: none, alum, or MF59 3 ⫻ 3, 9, 30, 100 ␮g; VLP: Alum 3⫹⫻1, 5, 10 ␮g VLP 3 ⫻ 40 ␮g; VLP: alum 3 ⫻ HPV16/18: VLP: 3deacylated monophosphoryl lipid A



0, 4, 8 weeks 0, 2 weeks

⬎1000 N/D 3000 320 810–1080 5000 1000 ⬍1000 10,000 100 10,000 ⬎100

⫹4 weeks ⫹4 weeks ⫹3 weeks ⫹5 weeks ⫹1 week ⫹2 weeks ⫹2 weeks ⫹2 weeks ⫹12 months ⫹2 weeks ⫹2 weeks

0, 1, 4 months



0, 1, 4 months



2 ⫻ month 0, 2, 6 months 0, 1, 6 months

⬎100 1510 ␮molU/mL

N/A Natural transmission Natural transmission

3 weeks 2 weeks 3 weeks 6, 12 weeks 4 weeks 2, 4 weeks 4 weeks 2 weeks 4, 8 weeks

*21st International Papillomavirus Conference, Mexico City, 2004; Abstract #412.

vaccinated rabbits to naive rabbits is sufficient for protection.7,8 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 neutralization of virus has important preventive utility, several lines of evidence suggest that cell-mediated immune responses are important in controlling established HPV infections as well as HPV-associated neoplasms:10 1. The prevalence of HPV-related diseases (infections and neoplasms) is increased in patients with impaired cell-mediated immunity, including transplant recipients11 and human immunodeficiency virus–infected patients.12-14 2. Animals immunized with nonstructural viral proteins are protected from papillomavirus infection or the development of neoplasia. Immunization also facilitates the regression of existing lesions.15,16 3. Infiltrating CD4⫹ (T-helper cells) and CD8⫹ (cytotoxic T cells) cells have been observed in spontaneously regressing warts.17 4. Warts in patients receiving immunosuppressive therapy often disappear when treatment is discontinued.18 The well-characterized foreign (viral) antigens and the well-defined virological, genetic, and pathological progression of HPV—from initial infection to lesion formation to malignant tumor formation— have provided a unique opportunity to evaluate interventions with antigen-specific immunotherapy. Conceptually, 2 different types of HPV vaccines can be designed: prophylactic (preventive) vaccines that prevent HPV infec-

tion, and therapeutic (curative) vaccines that induce regression of established HPV infection and its sequelae. PREVENTIVE HPV VACCINES Preventive HPV vaccine development has been complicated by a lack of animal models for the genital mucosatropic HPV types and by difficulty in propagating the virus in culture. These problems have been partially overcome using cutaneous and mucosal animal papillomaviruses as models and by the development of VLPs and pseudovirions. The papillomavirus major capsid protein L1— overexpressed in mammalian,19,20 insect,21 yeast,22 and bacterial cells23—spontaneously assembles to form VLPs that are devoid of the oncogenic viral genome. Parenteral injection of these VLPs, or even the pentameric L1 capsomer,24 elicits high titers of serum-neutralizing antibodies and protection from experimental challenge with infectious virus in several animal papillomavirus models. 7,8,25 Genetic vaccination with an L1-expression vector also protects from experimental viral challenge. 26-28 A summary of published L1-based prophylactic vaccine studies in animal models and humans is presented in Table 1. Protection from experimental infection by cottontail rabbit papillomavirus (CRPV) or canine oral papillomavirus (COPV) after passive transfer of IgG from VLP-immunized animals to naive animals has been demonstrated in rabbits and dogs, respectively.7,8 The results from these protective vaccine studies indicate that neutralizing IgG provides protection from experimental infection.


HPV VACCINES (Roden et al)

Although VLP vaccination provides immunity from experimental inoculation, protection against sexual transmission of HPV requires neutralizing antibodies acting at mucosal surfaces. Serum-neutralizing IgG induced by parenteral VLP vaccination may enter via transudation into the genital tract and maintain a sufficient level to provide sterilizing immunity across the menstrual cycle.29 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 the mucosa-associated lymphoid tissue,30,31 but nasal instillation of VLPs was found to be efficient in generating specific antibodies, including IgG in serum and IgG and IgA in mucosal secretions of mice.23,32 More recently, oral vaccination with HPV VLPs in mice has been shown to induce systemic virus-neutralizing antibodies, suggesting that HPV VLPs may be antigenically stable in the environment of the gastrointestinal tract. These studies provide the possibility of vaccinating large populations with HPV VLP without using syringes.33 In recent clinical data, intramuscular vaccination of women with HPV-16 VLPs was found to induce significant antibody titers in cervical secretions.34 A comparison by the same researchers of nasal spray or aerosol for mucosal delivery of 50 ␮g 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.35 Early phase I/II clinical trials using HPV L1 VLP delivered intramuscularly have demonstrated the immunogenicity and safety of this vaccine. 34,36,37 Importantly, Koutsky et al38 recently reported data from a clinical trial of HPV 16 L1 VLPs indicating for the first time that a vaccine strategy can be implemented in humans to prevent HPV-16 infections and HPV-16 – associated premalignant lesions. A similar vaccine strategy is being tested with HPV-11 VLPs,39 and VLP vaccination also may be extended to other oncogenic HPV types. Vaccination of a single animal with multiple HPV VLP types does not detract from the response to the individual types.40 A quadrivalent HPV VLP vaccine (types 6, 11, 16, and 18) from Merck is currently in a clinical trial; preliminary results have found that the vaccine is well tolerated and generates neutralizing antibody titers.41 These HPV VLP vaccines may be useful in the prevention not only of cervical cancer, but also of other HPV-associated malignancies, including a subset of head and neck squamous cell carcinoma, vulvar cancers, and other anogenital cancers. A clearer picture of the long-term effects, including duration of antibody titers and side effects, will likely emerge several years after this study and other parallel studies of HPV VLP vaccines have concluded. Concern has been raised that widespread HPV-16 vaccination may eventually exert evolutionary pressure, selecting outgrowths of other types of HPV and/or some HPV-16 variants that carry mutation(s) on the L1 gene and thus are unaffected by the vaccine. But this is

improbable, given the slow evolution of HPV and evidence of cross-neutralization of variants by immune sera.42,43 Developing nations tend to have the highest prevalence of HPV-associated lesions and cervical cancer–related cancer death.44 Although the VLP vaccine shows great promise for HPV control in industrialized nations, it is probably not ready for use in developing countries due to its cost. Many developing countries do not have sufficient resources for routine Pap smear testing, let alone preparation and parenteral administration of VLPs. Another difficulty is the instability of the preventive HPV VLP vaccine at room temperature over long periods. The VLP vaccine requires sophisticated equipment for production and refrigerated storage facilities. Future vaccine research should work toward a new generation of HPV vaccines that are cheaper, more easily administered, and more stable to be applicable worldwide.45 Naked DNA vaccines that express L1 are effective in animals and may be cheaper than using purified VLP, particularly in the formulation of polymeric vaccines based on multiple HPV genotypes. There are at least 15 oncogenic HPV genotypes, and in vitro neutralization studies suggest that they may represent an equivalent number of serotypes,40,42,46 although limited cross-reactivity can occur between L1s of different genotypes with ⬎85% sequence identity.40 The multitude of distinct HPV serotypes has probably evolved from evolutionary pressure to evade neutralizing antibodies. Indeed, limited animal studies suggest that vaccination with L1 VLP of a heterologous genotype is not protective,7 although this has not been tested for more closely related genotypes. Therefore, protection against infection by all of these types with a VLP-based vaccine would likely require a highly polymeric vaccine that would be difficult to generate. Another potential alternative for comprehensive protection is to vaccinate against L2. L2 is immunologically subdominant to L1, because immunization with virions or capsids composed of L1 and L2 elicits almost exclusively L1-specific antibody.47 However, induction of in vitro neutralizing antibody47,48 and protection of animals from experimental infection by vaccination with recombinant L2 alone49-51 has demonstrated the promise of L2 as a prophylactic vaccine (Table 2).51a,51b,51c Indeed, L2-specific cross-neutralizing polyclonal sera and monoclonal antibodies have been demonstrated.47,52,53 A recent study by Embers et al9 indicated that protection by L2 peptide vaccination is likely mediated by neutralizing antibody. Vaccination of patients with fusion proteins containing L2 has proven safe. As seen in animal models, the humoral immune responses of patients to L2 are much weaker than the responses to L1 VLP.54,55-58 Therefore, if the relatively weak immunogenicity of L2 can be overcome, then vaccination against L2 holds great potential for prophylaxis against infection by a broad range of HPV types and their associated cancers.



Volume 35, No. 8 (August 2004)

Table 2. Summary of Published L2 Vaccine Studies in Animal Models and Humans

Study (et al)

Virus and host

Christensen 1991 Lin 1992 Embers 2002

CRPV: rabbit CRPV: rabbit ROPV, CRPV: rabbit

Campo 1993; Chandrachud 1995; Gaukroger 1996 Jarret 1991 Thompson 1999; Lacey 1999 Kanda§

BPV4: cattle

BPV2: cattle HPV6: human HPV, human

Vaccination schedule (weeks)

Dose and adjuvant 4⫻ gelpure: CFA 4⫻ 250 ␮g: RIBI 3⫻ 500 ␮g peptide/KLH: -CFA/IFA 2⫻ 100 ␮g: alum 2⫻ 50 ␮g: none 2⫻ 1 mg: alum 2⫻ 500 ␮g: IFA 3⫻ 300 ␮g: alum L2–E7 fusion HPV-16 L2 peptide amino acids 108–120 500 mg, 100 mg

Challenge (weeks)*

Neutralizing (ELISA) titer


0, 4, 8, 12 0, 3, 6, 9 0, 4, 8

⫹2 ⫹2 ⫹4–12

32 10 (1000) 1000

Yes Yes Yes

0, 0, 0, 0, 0,

4 4 4 2 1, 4

⫹2 ⫹2 ⫹2 ⫹2 ND

? (100) 5† Below detection (?) (⬃1004)


0, 4, 12



*Experimental challenge with the homologous papillomavirus type. †A 5-fold dilution of serum was the only serum concentration tested. ‡Cattle were not protected, but warts were eventually cleared in the vaccine group. This may reflect reduced inoculum due to partial neutralization or clearance, perhaps as a bystander effect. This enhanced clearance was not described in any other L2 vaccine study. §19th Papillomavirus Meeting, 2001; Abstract #0103. Abbreviation: ND, not done.

THERAPEUTIC HPV VACCINES Although vaccination with preventive HPV vaccines is able to generate high titers of serum-neutralizing antibodies in animals and in humans, such immunization may not be able to generate significant therapeutic effects for established or breakthrough HPV infections that have escaped antibody-mediated neutralization. Preexisting HPV infection is highly prevalent and is responsible for considerable morbidity and mortality. Therapeutic vaccines should induce specific cell-mediated immunity that prevents the development of lesions and eliminates preexisting lesions or even malignant tumors. Early viral antigens (E1, E2, E5, E6, and E7) have potential as therapeutic vaccine antigens. However, unlike E6 and E7, neither E1 nor E2 is consistently expressed in carcinoma. E5 shows limited immunogenicity and has not been extensively studied as a vaccine antigen. Likewise, E4 and the L1 and L2 capsid proteins are unlikely to be suitable targets for therapeutic vaccine development, because these proteins are not detectably expressed in basal epithelial cells of benign lesions or in abnormal proliferative cells of premalignant and malignant lesions.59,60 Although recent studies indicate that vaccination with VLPs may generate a capsid protein–specific cytotoxic T lymphocyte (CTL) response,61,62 such a response against L1 or L1/L2 VLPs alone may not be able to generate a significant therapeutic effect. Whereas most tumor-specific antigens are derived from normal or mutated proteins, E6 and E7 are completely foreign viral proteins, and thus they 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 pheno-

type of cancer cells,63 cervical cancer cells are unlikely to evade an immune response through antigen loss. Thus, although care must be taken, given the oncogenic nature of these genes, E6 and E7 proteins represent good targets for developing antigen-specific immunotherapies or vaccines for cervical cancer. Various forms of HPV vaccines, including vectorbased vaccines, peptide-based vaccines, protein-based vaccines, DNA-based vaccines, chimeric VLP-based vaccines, and cell-based vaccines, have been described in experimental systems targeting HPV-16 E6 and E7 proteins. Most studies focus on E7, because it is more abundantly expressed and better characterized immunologically. Furthermore, its sequence is more conserved than that of the E6 gene.64 A summary of therapeutic HPV vaccines, their advantages, and their disadvantages is provided in Table 3. The following sections describe each of the therapeutic HPV vaccines outlined in Table 3 (or combined approaches). Live Vector Vaccines Viral Vector Vaccines

Viral vectors, such as vaccinia virus and adenovirus, have been evaluated as tools for HPV vaccine development. Modified or replication-deficient forms of viruses, such as adeno-associated virus, alphavirus replicon particles, and HPV pseudovirions, have also been used to generate a potent immunogenic response with minimal toxicity. Several studies have shown that immunotherapy targeting E6 and/or E7 using vaccinia vectors generates strong CTL activity65,66 and antitumor responses in preclinical studies.57,67 Strategies that affect the intracellular


HPV VACCINES (Roden et al)

Table 3. Characteristics of HPV Vaccines Used to Treat Cervical Cancer Advantages Vector-based: Viral (e.g., vaccinia, Adv, AAV, alphavirus) Bacterial (e.g., Listeria, Salmonella, BCG) HPV pseudovirions Peptide-based Protein-based (including VLPs) DNA

RNA DC-based

Tumor cell–based

Highly immunogenic; different immunologic properties of viruses; versatility of construction Highly immunogenic; can deliver engineered plasmids or express protein Highly immunogenic; can induce DC maturation Ease of production; safety Ease of production; multiple known adjuvants; no HLA restriction Ease of production, storage, and transport; versatility in ability to add targeting and/or costimulatory genes; capable of multiple immunizations No risks of integration or cellular transformation; capable of multiple immunizations Highly immunogenic; multiple methods of antigen loading; gene transduction or cytokine treatment to enhance potency Useful if tumor antigen is unknown; gene transduction or cytokine treatment to enhance potency

sorting or localization of antigen may be able to improve the in vivo therapeutic potency of recombinant vaccinia vaccines against cervical cancer.68-71 Results of phase I/II clinical trials using recombinant vaccinia virus encoding HPV-16 and 18 E6/E7 (also called TA-HPV) indicated that some patients with advanced cervical cancer, CIN 3, or early invasive cervical cancer developed T-cell immune responses after vaccination.72-74 Studies using modified adenovirus (Adv)-expressing HPV-16 E6 or E7 for vaccination75,76 or for ex vivo preparation of dendritic cell (DC)-based vaccines (Adv-E7 targeted to CD40 using bispecific Abs77) have demonstrated enhancement of antigen-specific T-cell immune responses and antitumor effects. Replicationdefective adeno-associated virus, a parvovirus that is nonpathogenic in humans, encoding HPV-16 E7 fused to heat-shock protein 70 (HSP70) was found to induce CD4- and CD8-dependent CTL activity and antitumor effects in vitro.78 Alphavirus replicon packaging cell lines (ie, from Sindbis or Venezuelan equine encephalitis virus) can be used to produce replication-defective alphavirus replicon particles that are free of detectable replicationcompetent virus yet can efficiently deliver the antigen.79,80 Vaccination of mice with a replicationdefective Venezuelan equine encephalitis virus replicon particle vector containing HPV16 E7 RNA can enhance E7-specific CD8⫹ T-cell immune responses and eliminate established tumors.80,81 Another study found that replication-defective Sindbis virus replicon particles encoding herpes simplex virus type 1 (HSV-1) VP22, linked to HPV-16 E7-generated improved E7specific CD8⫹ T-cell immune responses and a potent antitumor effect in vaccinated mice.82 Nonreplicative pseudovirions are formed from na-

Drawbacks Safety; preexisting immunity; inhibited repeat immunization Safety; preexisting immunity; inhibited repeat immunization Cumbersome to prepare; inhibited repeat immunization Weakly immunogenic; must match patient’s HLA Usually better induction of antibody response than CTL response Intrinsically weak immunogen; concern of integration or cellular transformation Low transfection efficiency; unstable to store and handle Labor-intensive individualized cell processing; variable quality control; do not home to draining lymph nodes Safety; labor-intensive procedure; most suited for refractory advanced cancers

ked DNA encapsulated in papillomavirus capsids using various expression systems, including vaccinia virus,83 Semliki Forest virus,40 and baculovirus,84 or Saccharomyces cerevisiae85 systems. Pseudovirions can protect encapsulated DNA from nuclease activity, efficiently deliver the DNA, act as an adjuvant, and induce DC maturation.86,87 Studies have demonstrated higher frequency of gene transfer with HPV pseudovirions than with DNA alone or with liposome84 and induction of mucosal and systemic E7-specific CTL responses.88 Vaccine efficacy may be further improved by inclusion of L2.89 Bacterial Vector Vaccines

Attenuated bacteria (eg, 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 macrophages90 and facilitates delivery of antigens into both the major histocompatibility complex (MHC)-I and MHC-II pathways. Recent studies have found that intraperitoneal or oral vaccination with recombinant L. moncytogenes secreting HPV-16 E7 can lead to regression of preexisting E7-expressing murine tumors.91,92 Attenuated Salmonella has been used to deliver HPV-16 E793 or E7 epitopes harbored in hepatitis B virus core antigen particles94 or HPV-16 VLPs95 as therapeutic HPV vaccines. Bacille Calmette-Guerin (BCG; Mycobacterium bovis) is a safe bacterial vaccine vector (ie, for tuberculosis vaccination to induce prolonged immune responses) that has been used to develop a vaccine encoding HPV-16 L1 and E7 and has been found



Volume 35, No. 8 (August 2004)

to induce E7-specific antibody and cytotoxic immune responses.96 The future of live viral or bacterial vectors for HPV vaccine delivery is likely in the development and refinement of modified, replication-deficient, or otherwise attenuated forms of these vectors with the goal of boosting immunogenicity while minimizing toxicity. Peptide/Protein Vaccines Peptide Vaccines

The identification and characterization of murine (H-2Db)97 and human (HLA-A.2)98 CTL epitopes for HPV-16 has promoted the development of peptide vaccines against cervical cancer.99,100 Peptides relevant to other HPV types (ie, HPV-18)101 and other HLA backgrounds (ie, HLA- B18),102 as well as human MHC-II–restricted T-helper cell epitopes,103 have also been investigated. Vaccination with lipidated HPV-16 E7 peptide was found to generate CTL responses in some patients with HPV-associated cancer.104 Clinical data from another study demonstrate T-helper immune responses in humans in response to peptide vaccination105 with minimal adverse side effects.106 In another study, 10 of 16 patients with HPV-16 – and HLA-A.2–positive high-grade cervical or vulvar intraepithelial neoplasia vaccinated with an E7-specific peptide vaccine exhibited measurable enhancement in cytokine release and cytolysis mediated by CTLs derived from peripheral blood mononuclear cells, and some even demonstrated partial clearance of virus and regression of lesions.107 Protein Vaccines

Protein-based HPV vaccines have been tested in humans.108 Whereas peptide vaccines exhibit MHC restriction, protein-based vaccines can bypass this restriction and thus are less dependent on the patient’s HLA type. TA-GW fusion protein, which consists of HPV-6 L2 fused to E7 protein, has been tested for clinical treatment of genital warts,54,56 and TACIN fusion protein, which consists of HPV-16 L2/E6/ E7, can induce E7-specific CD8⫹ T-cell immune responses and tumor protection in mice.57 TA-CIN was well tolerated by patients and induced both humoral and T-cell–mediated immune responses.58 The potency of HPV-16 E7 peptide-based vaccines may be further enhanced through the use of adjuvants,109 fusion proteins (ie, HSPs), or anchor-modified peptide epitopes.110 Vaccination with HSP65 fused to E7 protein led to CD8-dependent and CD4-independent regression of HPV-16 E7-expressing tumors in mice,111 and this vaccine is now in clinical trials with Stressgen Biotechnologies for HPV-associated anal dysplasia and the National Cancer Institute for HPV-associated cervical cancer.112 In another fusion approach, vaccination of women with E7 fused to Hemophilus influenzae protein D (D16E7) and administered in GlaxoSmithKline adjuvant AS02B was well tolerated, leading

to enhanced T-cell activity and regression of lesions in some patients.113 Chimeric HPV VLPs containing E2 and/or E7 antigen can induce high-titer neutralizing antibodies in the serum, activate DCs, and prime T-cell–mediated immune responses.87,114-117 Currently, clinical grade HPV-16 L1/L2-E2-E7 chimeric VLPs, which contain 4 HPV-encoded proteins (L1, L2, E2, and E7) as target antigens, are being prepared for a phase I clinical trial (D. Lowy and J. Schiller, personal communication). Likewise, chimeras of L1 VLP or pentamers and E7 will soon be tested in the clinic.62,118 Nucleic Acid Vaccines 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 synthesized peptides can be presented by the patient’s own HLA molecules. DNA vaccines can be administered to the host by intramuscular injection, intradermal injection via hypodermic needle or gene gun (a ballistic device for delivering DNA-coated gold particles into the epidermis), intravenous injection, intranasal delivery, or biojector delivery.119,120 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.121 Intracellular targeting strategies are able to enhance MHC-I and/or -II presentation of antigen. Enhancement of MHC-I presentation of E7 can be achieved by linkage of E7 to M. tuberculosis HSP70,122 calreticulin,123 the translocation domain (domain II) of Pseudomonas aeruginosa exotoxin A,124 or ␥-tubulin125 in the context of a DNA vaccine, leading to potent CD8-dependent antitumor immune responses. Enhanced MHC-II presentation of E7 and antitumor immunity has been demonstrated by linkage of E7 to a signal sequence (Sig, at the N-terminus of E7) and lysosomeassociated membrane protein (LAMP-1, at the C-terminus),126 which routes antigen to the endosome/lysosomal compartments. Intercellular spreading of E7 is facilitated by linking E7 to HSV-1 VP22127 or one of its homologues (bovine herpesvirus VP22128 or Marek’s disease virus VP22129), unique viral tegument proteins that can mobilize antigen for intercellular transport to neighboring cells. Mice vaccinated with HVP22/E7 DNA generate a significantly greater number of E7-specific CD8⫹ T-cell precursors130-132 and a stronger antitumor effect than wild-type E7 DNA.131 BVP22/E7 DNA (Hung, unpublished data) and MVP22/E7 DNA133 are similarly potent. Because DCs have a limited lifespan, a method to


HPV VACCINES (Roden et al)

prolong in vivo DC survival may help improve DNA potency. Coadministration of E7-containing DNA with DNA-encoding antiapoptotic proteins (Bcl-xL, Bcl-2, X-linked inhibitor of apoptosis protein, and dominant negative caspase-9 or dominant negative caspase-8) is able to enhance E7-specific immune responses, tumor treatment, and DC survival.134 Other DNA-based strategies for improving E7-specific CD8⫹ T-cell immune responses include coadministration of E7 DNA with cytokines or costimulatory molecules,135,136 enhancing intracellular degradation of E7 protein,137-139 and codon optimization to enhance E7 antigen presentation.140,141 Currently, a plasmid DNA vaccine encoding multiple HLA-A2–restricted epitopes derived from the HPV-16 E7 protein has been tested in a phase I clinical trial in patients with high-grade anal intraepithelial lesions. The vaccine (ZYC101) is composed of plasmid DNA encapsulated in biodegradable polymer microparticles. The initial results of the trial suggest that the DNA vaccine was well tolerated in all subjects at all dosage levels tested. Furthermore, 10 of 12 subjects demonstrated increased immune response to the peptide epitopes encoded within the DNA vaccine.142 RNA Replicon Vaccines

Although RNA is typically less stable than DNA and often has lower transfection efficiency, self-replicating RNA replicon vectors may have improved immunogenicity. These noninfectious, self-replicating, and selflimiting RNA can be launched in RNA or DNA form, followed by transcription into RNA replicons and expression of the antigen of interest at high levels for an extended period.143-145 Studies have demonstrated that the potency of HPV-16 E7–specific self-replicating RNA vaccines can be enhanced by applying the LAMP-1 targeting strategy,146 the M. tuberculosis HSP70 strategy,147 or the HSV-1 VP22 strategy.148 Hsu et al149 recently used DNA-launched RNA replicons for the development of HPV vaccines and demonstrated significant E7-specific CTL activity and antitumor effects. Cell-Based Vaccines Dendritic Cell–Based Vaccines

Greater understanding about the origin of DCs, their antigen-uptake mechanisms, and the signals that stimulate their migration and maturation into immunostimulatory APCs150 has aided the development of DC-based vaccines, which include DCs pulsed with E6 and/or E7 peptides/proteins and DCs transduced with DNA, RNA, or viral vectors encoding E6 and/or E7. Presentation of HPV E6 and/or E7 peptides to the immune system by DCs is a promising method for circumventing tumor-mediated immunosuppression.151-155 HPV-18 E7-pulsed DCs have also been used in a human cervical cancer case.156 Transfer of the E7 gene into DCs can be accomplished by various methods, including DNA,157 RNA,158 and viral vectors.76,77,159,160 The route of administration

may be important for the efficacy of DC-based vaccines. Wang et al161 transduced the HPV-16 E7 gene into a DC line by electroporation using an E7-expressing DNA plasmid and demonstrated that intramuscular administration of DC-E7– generated greater antitumor immunity than subcutaneous and intravenous administration, eliciting the highest levels of E7-specific antibody and greatest numbers of E7-specific CD4⫹ T-helper and CD8⫹ T-cell precursors. Tumor Cell–Based Vaccines

Due to patient perceptions of safety, tumor cell– based vaccination may be a feasible treatment strategy only for advanced HPV-associated cancer. Transduction of tumor cells with genes encoding costimulatory molecules or cytokines may enhance immunogenicity, leading to T-cell activation and antitumor effects after vaccination.162 Vaccines using HPV-transformed tumor cells transduced with cytokine genes, such as interleukin-12,163 interleukin-2,164 or GM-CSF,165 can generate strong antitumor effects in mice. Combined Approaches Combining vaccine vehicles using a prime-boost regimen primes the immune system, then (ideally) augments and maintains a long-term immune response, making it an attractive approach for cancer immunotherapy. A study conducted by Chen et al166 to test various combinations of viral vectors and nucleic acids in a prime-boost regimen concluded that priming with a DNA vaccine followed by a recombinant vaccinia booster provided the most potent antitumor effects. Kowalczyk et al167 found that priming with intramuscularly delivered L1-expressing DNA and boosting with adenoviral HPV-16 L1 induces antibodies in vaccinated mice, with Ig2a and Ig2b isotypes predominating in sera. Another combinational approach involves the simultaneous administration of DNA vaccines and other antiviral or anticancer agents. Combined administration of intralesional antiviral treatment and immune stimulation may be a feasible means of providing a long-lasting cure to persistent HPV infections.168 Christensen et al169 topically administered the antiviral agent cidofovir and intracutaneously administered (via a gene gun) a DNA vaccine encoding CRPV genes. Cures of large established CRPV-induced rabbit papillomas and reduced incidence of lesion recurrence were observed, suggesting that this combination may also be useful in treating persistent HPV infections. Summary The next 5 years in the field of HPV vaccinology will be very exciting. During this period, we should gain a clearer understanding of the protective efficacy of HPV VLP-based vaccines. We also anticipate preliminary results from trials of “second-generation” prophylactic vaccines that target neutralizing epitopes in both L1 and L2, as well as therapeutic



Volume 35, No. 8 (August 2004)

antigens. They will be designed for simplicity of preparation and administration, and, importantly, for increasing the spectrum of oncogenic HPV genotypes covered. Our understanding of the adaptive immune system and vaccines continues to improve at a dramatic pace, as does the number of novel strategies for enhancing the immunogenicity of HPV antigens. As vaccinology becomes less empirical and the multitude of new therapeutic HPV vaccines are tested in the clinic, great strides will be made toward the elimination of HPV-associated malignancy. We are particularly excited about the prospect of human trials of DNA-based vaccines in both the therapeutic and prophylactic settings. The development of better, standardized assays for cellular immunity and surrogates for clinical efficacy will be critical to the rational development of vaccines targeting HPV, and these lessons will be translated to other viral systems. CONCLUSION In the past decade, dramatic progress has been made in the field of HPV vaccine development. The recognition of high-risk HPV as the primary etiologic agent for cervical cancer and its precursor lesions has paved the way for the development of preventive and therapeutic HPV vaccines that may lead to the control of cervical cancer and other HPV-associated malignancies. HPV VLPs show promise as protective vaccines capable of generating neutralizing antibodies to prevent HPV infection in patients. A quarter of a century after the development of the hepatitis B vaccine, these prophylactic vaccines could prove to be the second major vaccine for the prevention of a human cancer. However, such a preventative vaccine would not benefit those with established oncogenic HPV infections. Given the prevalence of HPV infection, it is therefore critical to continue in parallel the development of therapeutic vaccine approaches. Many experimental HPV vaccine strategies including vector-based vaccines, peptide-based vaccines, protein-based vaccines, nucleic acid-based vaccines, chimeric VLP-based vaccines, cell-based vaccines, pseudovirions, and RNA replicons have been shown to enhance virus-specific immune cell activity and antitumor responses in murine tumor systems. Several clinical trials are currently underway, based on encouraging preclinical results from these therapeutic HPV vaccines. A head-to-head comparison of these vaccines will help identify the most potent therapeutic HPV vaccine with minimal negative side effects. Clinical HPV vaccine trials provide a unique opportunity to identify the characteristics and mechanisms of the immune response that best correlate with clinical vaccine potency. Such immunologic parameters will help define protective and therapeutic immune mechanisms for controlling HPV infections and HPV-related disease. Rational development of more effective vaccines for HPV infections would be

greatly facilitated by comprehensive information on these protective immune mechanisms in humans. With continued endeavors in HPV vaccine development, we may soon be able to implement a variety of safe and effective strategies for the eventual control of HPV-associated cervical cancer. Acknowledgment. This review is not intended to be an encyclopedic one, and we apologize to any authors not cited. We thank Drs. Keerti V. Shah, Michelle Moniz, and Robert J. Kurman for their helpful discussions. We also thank Chien-Fu Hung for his critical review of the manuscript.

REFERENCES 1. Walboomers JM, Jacobs MV, Manos MM, et al: Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189:12-19, 1999 2. zur Hausen H: Papillomaviruses and cancer: From basic studies to clinical application. Nat Rev Cancer 2:342-350, 2002 3. Bosch FX, Manos MM, Munoz N, et al: Prevalence of human papillomavirus in cervical cancer: A worldwide perspective. International Biological Study on Cervical Cancer (IBSCC) Study Group. J Natl Cancer Inst 87:796-802, 1995 4. Munoz N, Bosch FX, de Sanjose S, et al: Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348:518-527, 2003 5. Moscicki AB, Shiboski S, Broering J, et al: The natural history of human papillomavirus infection as measured by repeated DNA testing in adolescent and young women. J Pediatr 132:277-284, 1998 6. Koutsky LA, Holmes KK, Critchlow CW, et al: A cohort study of the risk of cervical intraepithelial neoplasia grade 2 or 3 in relation to papillomavirus infection. N Engl J Med 327:1272-1278, 1992 7. Breitburd F, Kirnbauer R, Hubbert NL, et al: Immunization with viruslike particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection. J Virol 69:39593963, 1995 8. Suzich JA, Ghim SJ, Palmer-Hill FJ, et al: Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas. Proc Natl Acad Sci U S A 92:11553-11557, 1995 9. Embers ME, Budgeon LR, Pickel M, et al: Protective immunity to rabbit oral and cutaneous papillomaviruses by immunization with short peptides of L2, the minor capsid protein. J Virol 76:9798-9805, 2002 10. Wu TC: Immunology of the human papilloma virus in relation to cancer. Curr Opin Immunol 6:746-754, 1994 11. Halpert R, Fruchter RG, Sedlis A, et al: Human papillomavirus and lower genital neoplasia in renal transplant patients. Obstet Gynecol 68:251-258, 1986 12. Laga M, Icenogle JP, Marsella R, et al: Genital papillomavirus infection and cervical dysplasia: Opportunistic complications of HIV infection. Int J Cancer 50:45-48, 1992 13. Schafer A, Friedmann W, Mielke M, et al: The increased frequency of cervical dysplasia-neoplasia in women infected with the human immunodeficiency virus is related to the degree of immunosuppression. Am J Obstet Gynecol 164:593-599, 1991 14. Sun XW, Kuhn L, Ellerbrock TV, et al: Human papillomavirus infection in women infected with the human immunodeficiency virus. N Engl J Med 337:1343-1349, 1997 15. Brandsma JL: Animal models for HPV vaccine development. Papillomavirus Report 105-111, 1994 16. Selvakumar R, Borenstein LA, Lin YL, et al: Immunization with nonstructural proteins E1 and E2 of cottontail rabbit papillomavirus stimulates regression of virus-induced papillomas. J Virol 69:602605, 1995 17. Tagami H: Regression phenomenon of numerous flat warts: An experiment on the nature of tumor immunity in man. Int J Dermatol 22:570-571, 1983


HPV VACCINES (Roden et al) 18. Benton C, Shahidullah H, Hunter JAA: Human papillomavirus in the immunosuppressed. Papillomavirus Report 23-26, 1992 19. Hagensee ME, Yaegashi N, Galloway DA: Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins. J Virol 67:315-322, 1993 20. Zhou J, Sun XY, Davies H, et al: Definition of linear antigenic regions of the HPV16 L1 capsid protein using synthetic virionlike particles. Virology 189:592-599, 1992 21. Kirnbauer R, Booy F, Cheng N, et al: Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci U S A 89:12180-12184, 1992 22. Sasagawa T, Pushko P, Steers G, et al: Synthesis and assembly of virus-like particles of human papillomaviruses type 6 and type 16 in fission yeast Schizosaccharomyces pombe. Virology 206:126-135, 1995 23. Nardelli-Haefliger D, Roden RB, Benyacoub J, et al: Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal- and systemic-neutralizing antibodies in mice. Infect Immun 65:3328-3336, 1997 24. Yuan H, Estes PA, Chen Y, et al: Immunization with a pentameric L1 fusion protein protects against papillomavirus infection. J Virol 75:7848-7853, 2001 25. Kirnbauer R, Chandrachud LM, O’Neil BW, et al: Virus-like particles of bovine papillomavirus type 4 in prophylactic and therapeutic immunization. Virology 219:37-44, 1996 26. Donnelly JJ, Martinez D, Jansen KU, et al: Protection against papillomavirus with a polynucleotide vaccine. J Infect Dis 173:314320, 1996 27. Sundaram P, Tigelaar RE, Brandsma JL: Intracutaneous vaccination of rabbits with the cottontail rabbit papillomavirus (CRPV) L1 gene protects against virus challenge. Vaccine 15:664-671, 1997 28. Stanley MA, Moore RA, Nicholls PK, et al: Intra-epithelial vaccination with COPV L1 DNA by particle-mediated DNA delivery protects against mucosal challenge with infectious COPV in beagle dogs. Vaccine 19:2783-2792, 2001 29. Robbins JB, Schneerson R, Szu SC: Perspective. Hypothesis: serum IgG antibody is sufficient to confer protection against infectious diseases by inactivating the inoculum. J Infect Dis 171:13871398, 1995 30. Russell MW, Moldoveanu Z, White PL, et al: Salivary, nasal, genital, and systemic antibody responses in monkeys immunized intranasally with a bacterial protein antigen and the cholera toxin B subunit. Infect Immun 64:1272-1283, 1996 31. Lowe RS, Brown DR, Bryan JT, et al: Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus–like particles expressed in yeast. J Infect Dis 176:11411145, 1997 32. Balmelli C, Roden R, Potts A, et al: Nasal immunization of mice with human papillomavirus type 16 virus-like particles elicits neutralizing antibodies in mucosal secretions. J Virol 72:8220-8229, 1998 33. Rose RC, Lane C, Wilson S, et al: Oral vaccination of mice with human papillomavirus virus-like particles induces systemic virusneutralizing antibodies. Vaccine 17:2129-2135, 1999 34. Nardelli-Haefliger D, Wirthner D, Schiller J, et al: Antibody responses in cervical secretions of female volunteers after intramuscular vaccination with purified HPV 16 VLPs. Proceedings of the 19th International Papillomavirus Conference, September 1-7, 2001, pp 1-52 35. Nardelli-Haefliger D, Lurati F, Wirthner D, et al: Mucosal vaccination of female volunteers with purified HPV 16 VLPs. Proceedings of the 19th International Papillomavirus Conference, September 1-7, 2001, p 103 36. Harro CD, Pang YY, Roden RB, et al: Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus–like particle vaccine. J Natl Cancer Inst 93:284-292, 2001 37. Evans TG, Bonnez W, Rose RC, et al: A phase 1 study of a recombinant viruslike particle vaccine against human papillomavirus type 11 in healthy adult volunteers. J Infect Dis 183:1485-1493, 2001 38. Koutsky LA, Ault KA, Wheeler CM, et al: A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 347:16451651, 2002 39. Wheeler C, Brown D, Fife K, et al: A phase I study of the

safety/tolerability and immnogenicity profiles of research lot HPV 11 L1 virus-like partcle (VLP) vaccine: Persistence of anti-HPV 11 responses. Proceedings of the 19th International Papillomavirus Conference, September 1-7, 2001, p 90 40. Roden RB, Hubbert NL, Kirnbauer R, et al: Assessment of the serological relatedness of genital human papillomaviruses by hemagglutination inhibition. J Virol 70:3298-3301, 1996 41. Brown DR, Bryan JT, Schroeder JM, et al: Neutralization of human papillomavirus type 11 (HPV-11) by serum from women vaccinated with yeast-derived HPV-11 L1 virus-like particles: Correlation with competitive radioimmunoassay titer. J Infect Dis 184:11831186, 2001 42. Roden RB, Greenstone HL, Kirnbauer R, et al: In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype. J Virol 70:5875-5883, 1996 43. Pastrana DV, Vass WC, Lowy DR, et al: NHPV16 VLP vaccine induces human antibodies that neutralize divergent variants of HPV16. Virology 279:361-369, 2001 44. Plummer M, Franceschi S: Strategies for HPV prevention. Virus Res 89:285-293, 2002 45. Lowy DR, Frazer IH: Prophylactic human papillomavirus vaccines. J Natl Cancer Inst Monogr Issue 31:111-116, 2003 46. White WI, Wilson SD, Bonnez W, et al: In vitro infection- and type-restricted antibody-mediated neutralization of authentic human papillomavirus type 16. J Virol 72:959-964, 1998 47. Roden RB, Yutzy WH, Fallon R, et al: Minor capsid protein of human genital papillomaviruses contains subdominant, cross-neutralizing epitopes. Virology 270:254-257, 2000 48. Roden RB, Weissinger EM, Henderson DW, et al: Neutralization of bovine papillomavirus by antibodies to L1 and L2 capsid proteins. J Virol 68:7570-7574, 1994 49. Campo MS, Grindlay GJ, O’Neil BW, et al: Prophylactic and therapeutic vaccination against a mucosal papillomavirus. J Gen Virol 74:945-953, 1993 50. Christensen ND, Kreider JW, Kan NC, et al: The open reading frame L2 of cottontail rabbit papillomavirus contains antibodyinducing neutralizing epitopes. Virology 181:572-579, 1991 51. Lin YL, Borenstein LA, Selvakumar R, et al: Effective vaccination against papilloma development by immunization with L1 or L2 structural protein of cottontail rabbit papillomavirus. Virology 187:612-619, 1992 51a. Chandrachud LM, Grindlay GJ, McGarvie GM, et al: Vaccination of cattle with the N-terminus of L2 is necessary and sufficient for preventing infection by bovine papillomavirus-4. Virology 211: 204-208, 1995 51b. Gaukroger JM, Chandrachud BW, O’Neil GJ, et al: Vaccination of cattle with bovine papillomavirus type 4 L2 elicits the production of virus-neutralizing antibodies. J Gen Virol 77(Pt 7): 1577-1583, 1996 51c. Jarrett WF, Smith KT, O’Neil JM, et al: Studies on vaccination against papillomaviruses: prophylactic and therapeutic vaccination with recombinant structural proteins. Virology 184:33-42, 1991 52. Kawana K, Yoshikawa H, Taketani Y, et al: Common neutralization epitope in minor capsid protein L2 of human papillomavirus types 16 and 6. J Virol 73:6188-6190, 1999 53. Kawana K, Kawana Y, Yoshikawa H, et al: Nasal immunization of mice with peptide having a cross-neutralization epitope on minor capsid protein L2 of human papillomavirus type 16 elicits systemic and mucosal antibodies. Vaccine 19:1496-1502, 2001 54. Lacey CJ, Thompson HS, Monteiro EF, et al: Phase IIa safety and immunogenicity of a therapeutic vaccine, TA-GW, in persons with genital warts. J Infect Dis 179:612-618, 1999 55. Thompson HS, Davies ML, Watts MJ, et al: Enhanced immunogenicity of a recombinant genital wart vaccine adjuvanted with monophosphoryl lipid A. Vaccine 16:1993-1999, 1998 56. Thompson HS, Davies ML, Holding FP, et al: Phase I safety and antigenicity of TA-GW: A recombinant HPV6 L2E7 vaccine for the treatment of genital warts. Vaccine 17:40-49, 1999 57. van der Burg SH, Kwappenberg KM, O’Neill T, et al: Preclinical safety and efficacy of TA-CIN, a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and heterologous prime-boost regimens. Vaccine 19:3652-3660, 2001 58. de Jong A, O’Neill T, Khan AY, et al: Enhancement of human papillomavirus (HPV) type 16 E6 and E7–specific T-cell im-



Volume 35, No. 8 (August 2004)

munity in healthy volunteers through vaccination with TA-CIN, an HPV16 L2E7E6 fusion protein vaccine. Vaccine 20:3456-3464, 2002 59. Stoler MH, Rhodes CR, Whitbeck A, et al: Human papillomavirus type 16 and 18 gene expression in cervical neoplasias. Hum Pathol 23:117-128, 1992 60. Stoler MH, Wolinsky SM, Whitbeck A, et al: Differentiationlinked human papillomavirus types 6 and 11 transcription in genital condylomata revealed by in situ hybridization with message-specific RNA probes. Virology 172:331-340, 1989 61. Rudolf MP, Nieland JD, DaSilva DM, et al: Induction of HPV16 capsid protein-specific human T cell responses by virus-like particles. Biol Chem 380:335-340, 1999 62. Ohlschlager P, Osen W, Dell K, et al: Human papillomavirus type 16 l1 capsomeres induce l1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice. J Virol 77:4635-4645, 2003 63. Crook T, Morgenstern JP, Crawford L, et al: Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV-16 plus EJ-ras. EMBO J 8:513-519, 1989 64. Zehbe I, Wilander E, Delius H, et al: Human papillomavirus 16 E6 variants are more prevalent in invasive cervical carcinoma than the prototype. Cancer Res 58:829-833, 1998 65. Gao L, Chain B, Sinclair C, et al: Immune response to human papillomavirus type 16 E6 gene in a live vaccinia vector. J Gen Virol 75:157-164, 1994 66. Boursnell ME, Rutherford E, Hickling JK, et al: Construction and characterization of a recombinant vaccinia virus expressing human papillomavirus proteins for immunotherapy of cervical cancer. Vaccine 14:1485-1494, 1996 67. Meneguzzi G, Cerni C, Kieny MP, et al: Immunization against human papillomavirus type 16 tumor cells with recombinant vaccinia viruses expressing E6 and E7. Virology 181:62-69, 1991 68. Lin K-Y, Guarnieri FG, Staveley-O’Carroll KF, et al: Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res 56:21-26, 1996 69. Ji H, Chang EY, Lin KY, et al: Antigen-specific immunotherapy for murine lung metastatic tumors expressing human papillomavirus type 16 E7 oncoprotein. Int J Cancer 78:41-45, 1998 70. Chen CH, Suh KW, Ji H, et al: Antigen-specific immunotherapy for HPV-16 E7– expressing tumors grown in liver. J Hepatol 33:91-98, 2000 71. Lamikanra A, Pan ZK, Isaacs SN, et al: Regression of established human papillomavirus type 16 (HPV-16)-immortalized tumors in vivo by vaccinia viruses expressing different forms of HPV-16 E7 correlates with enhanced CD8(⫹) T-cell responses that home to the tumor site. J Virol 75:9654-9664, 2001 72. Borysiewicz LK, Fiander A, Nimako M, et al: A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer. Lancet 347: 1523-1527, 1996 73. Adams M, Borysiewicz L, Fiander A, et al: Clinical studies of human papilloma vaccines in pre-invasive and invasive cancer. Vaccine 19:2549-2556, 2001 74. Kaufmann AM, Stern PL, Rankin EM, et al: Safety and immunogenicity of TA-HPV, a recombinant vaccinia virus expressing modified human papillomavirus (HPV)-16 and HPV-18 E6 and E7 genes, in women with progressive cervical cancer. Clin Cancer Res 8:3676-3685, 2002 75. He Z, Wlazlo AP, Kowalczyk DW, et al: Viral recombinant vaccines to the E6 and E7 antigens of HPV-16. Virology 270:146-161, 2000 76. Li J, Sun Y, Garen A: Immunization and immunotherapy for cancers involving infection by a human papillomavirus in a mouse model. Proc Natl Acad Sci U S A 99:16232-16236, 2002 77. Tillman BW, Hayes TL, DeGruijl TD, et al: Adenoviral vectors targeted to CD40 enhance the efficacy of dendritic cell–-based vaccination against human papillomavirus 16 –induced tumor cells in a murine model. Cancer Res 60:5456-5463, 2000 78. Liu DW, Tsao YP, Kung JT, et al: Recombinant adeno-associated virus expressing human papillomavirus type 16 E7 peptide DNA fused with heat shock protein DNA as a potential vaccine for cervical cancer. J Virol 74:2888-2894, 2000 79. Polo JM, Belli BA, Driver DA, et al: Stable alphavirus pack-

aging cell lines for Sindbis virus and Semliki Forest virus– derived vectors. Proc Natl Acad Sci U S A 96:4598-4603, 1999 80. Velders MP, McElhiney S, Cassetti MC, et al: Eradication of established tumors by vaccination with Venezuelan equine encephalitis virus replicon particles delivering human papillomavirus 16 E7 RNA. Cancer Res 61:7861-7867, 2001 81. Eiben GL, Velders MP, Schreiber H, et al: Establishment of an HLA-A*0201 human papillomavirus type 16 tumor model to determine the efficacy of vaccination strategies in HLA-A*0201 transgenic mice. Cancer Res 62:5792-5799, 2002 82. Cheng WF, Hung CF, Hsu KF, et al: Cancer immunotherapy using Sindbis virus replicon particles encoding a VP22–antigen fusion. Hum Gene Ther 13:553-568, 2002 83. Zhao KN, Sun XY, Frazer IH, et al: DNA packaging by L1 and L2 capsid proteins of bovine papillomavirus type 1. Virology 243:482491, 1998 84. Touze A, Coursaget P: In vitro gene transfer using human papillomavirus–like particles. Nucleic Acids Res 26:1317-1323, 1998 85. Rossi JL, Gissman L, Jansen K, et al: Assembly of infectious human papillomavirus type 16 virus–like particle in Saccharomyces cerevisae. Hum Gene Ther 11:1165-1176, 2000 86. Lenz P, Day PM, Pang YY, et al: Papillomavirus-like particles induce acute activation of dendritic cells. J Immunol 166:5346-5355, 2001 87. Rudolf MP, Fausch SC, Da Silva DM, et al: Human dendritic cells are activated by chimeric human papillomavirus type-16 virus– like particles and induce epitope-specific human T cell responses in vitro. J Immunol 166:5917-5924, 2001 88. Shi W, Liu J, Huang Y, et al: Papillomavirus pseudovirus: A novel vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses. J Virol 75:10139-10148, 2001 89. Roden RB, Day PM, Bronzo BK, et al: Positively charged termini of the L2 minor capsid protein are necessary for papillomavirus infection. J Virol 75:10493-10497, 2001 90. Villanueva MS, Sijts AJ, Pamer EG: Listeriolysin is processed efficiently into an MHC class I–associated epitope in Listeria monocytogenes–infected cells. J Immunol 155:5227-5233, 1995 91. Gunn GR, Zubair A, Peters C, et al: Two Listeria monocytogenes vaccine vectors that express different molecular forms of human papilloma virus-16 (HPV-16) E7 induce qualitatively different T cell immunity that correlates with their ability to induce regression of established tumors immortalized by HPV-16. J Immunol 167:64716479, 2001 92. Lin CW, Lee JY, Tsao YP, et al: Oral vaccination with recombinant Listeria monocytogenes expressing human papillomavirus type 16 E7 can cause tumor growth in mice to regress. Int J Cancer 102:629637, 2002 93. Krul MR, Tijhaar EJ, Kleijne JA, et al: Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains. Cancer Immunol Immunother 43:44-48, 1996 94. Londono LP, Chatfield S, Tindle RW, et al: Immunization of mice using Salmonella typhimurium expressing human papillomavirus type 16 E7 epitopes inserted into hepatitis B virus core antigen. Vaccine 14:545-552, 1996 95. Revaz V, Benyacoub J, Kast WM, et al: Mucosal vaccination with a recombinant Salmonella typhimurium expressing human papillomavirus type 16 (HPV16) L1 virus–like particles (VLPs) or HPV16 VLPs purified from insect cells inhibits the growth of HPV16-expressing tumor cells in mice. Virology 279:354-360, 2001 96. Jabbar IA, Fernando GJ, Saunders N, et al: Immune responses induced by BCG recombinant for human papillomavirus L1 and E7 proteins. Vaccine 18:2444-2453, 2000 97. Tindle RW, Fernando GJ, Sterling JC, et al: A “public” Thelper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes. Proc Natl Acad Sci U S A 88:5887-5891, 1991 98. Kast WM, Brandt RM, Drijfhout JW, et al: Human leukocyte antigen-A2.1–restricted candidate cytotoxic T lymphocyte epitopes of human papillomavirus type 16 E6 and E7 proteins identified by using the processing-defective human cell line T2. J Immunother 14:115120, 1993 99. Feltkamp MC, Smits HL, Vierboom MP, et al: Vaccination


HPV VACCINES (Roden et al) with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16 –transformed cells. Eur J Immunol 23:2242-2249, 1993 100. Ressing ME, Sette A, Brandt RM, et al: Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLAA*0201– binding peptides. J Immunol 154:5934-5943, 1995 101. Rudolf MP, Man S, Melief CJ, et al: Human T-cell responses to HLA-A–restricted high binding affinity peptides of human papillomavirus type 18 proteins E6 and E7. Clin Cancer Res 7:788s-795s, 2001 102. Bourgault Villada I, Beneton N, Bony C, et al: Identification in humans of HPV-16 E6 and E7 protein epitopes recognized by cytolytic T lymphocytes in association with HLA-B18 and determination of the HLA-B18 –specific binding motif. Eur J Immunol 30:22812289, 2000 103. van der Burg SH, Ressing ME, Kwappenberg KM, et al: Natural T-helper immunity against human papillomavirus type 16 (HPV16) E7– derived peptide epitopes in patients with HPV16-positive cervical lesions: Identification of 3 human leukocyte antigen class II–restricted epitopes. Int J Cancer 91:612-618, 2001 104. Steller MA, Gurski KJ, Murakami M, et al: Cell-mediated immunological responses in cervical and vaginal cancer patients immunized with a lipidated epitope of human papillomavirus type 16 E7. Clin Cancer Res 4:2103-2109, 1998 105. Ressing ME, van Driel WJ, Brandt RM, et al: Detection of T helper responses, but not of human papillomavirus–specific cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma. J Immunother 23:255-266, 2000 106. van Driel WJ, Ressing ME, Kenter GG, et al: Vaccination with HPV16 peptides of patients with advanced cervical carcinoma: Clinical evaluation of a phase I–II trial. Eur J Cancer 35:946-952, 1999 107. Muderspach L, Wilczynski S, Roman L, et al: A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are HPV-16 positive. Clin Cancer Res 6:3406-3416, 2000 108. Berry JM, Palefsky JM: A review of human papillomavirus vaccines: From basic science to clinical trials. Front Biosci 8:S333-345, 2003 109. Gerard CM, Baudson N, Kraemer K, et al: Therapeutic potential of protein and adjuvant vaccinations on tumour growth. Vaccine 19:2583-2589, 2001 110. Fausch SC, Da Silva DM, Eiben GL, et al: HPV protein/ peptide vaccines: From animal models to clinical trials. Front Biosci 8:S81-91, 2003 111. Chu NR, Wu HB, Wu T, et al: Immunotherapy of a human papillomavirus (HPV) type 16 E7– expressing tumour by administration of fusion protein comprising Mycobacterium bovis bacille Calmette-Guerin (BCG) hsp65 and HPV16 E7. Clin Exp Immunol 121:216-225, 2000 112. Hunt S: Technology evaluation: HspE7, StressGen Biotechnologies Corp. Curr Opin Mol Ther 3:413-417, 2001 113. Hallez S, Maudoux F, Buxant F, et al: Use of cytokine flow cytometry to monitor cellular immune response to an HPV 16 E7derived vaccine: Phase I/II trial in women with HPV 16 positive grade 3 cervical intraepithelial neoplasia. Proceedings of the 19th International Papillomavirus Conference, September 1-7, 2001, p 98 114. Greenstone HL, Nieland JD, de Visser KE, et al: Chimeric papillomavirus virus-like particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model. Proc Natl Acad Sci U S A 95:1800-1805, 1998 115. Peng S, Frazer IH, Fernando GJ, et al: Papillomavirus virus– like particles can deliver defined CTL epitopes to the MHC class I pathway. Virology 240:147-157, 1998 116. Schafer K, Muller M, Faath S, et al: Immune response to human papillomavirus 16 L1E7 chimeric virus-like particles: Induction of cytotoxic T cells and specific tumor protection. Int J Cancer 81:881-888, 1999 117. Jochmus I, Schafer K, Faath S, et al: Chimeric virus-like particles of the human papillomavirus type 16 (HPV 16) as a prophylactic and therapeutic vaccine. Arch Med Res 30:269-274, 1999 118. Muller M, Zhou J, Reed TD, et al: Chimeric papillomaviruslike particles. Virology 234:93-111, 1997

119. Robinson HL, Torres CA: DNA vaccines. Semin Immunol 9:271-283, 1997 120. Donnelly JJ, Ulmer JB, Shiver JW, et al: DNA vaccines. Annu Rev Immunol 15:617-648, 1997 121. Hung CF, Wu TC: Improving DNA vaccine potency via modification of professional antigen presenting cells. Curr Opin Mol Ther 5:20-24, 2003 122. Chen C-H, Wang T-L, Hung C-F, et al: Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene. Cancer Res 60:1035-1042, 2000 123. Cheng WF, Hung CF, Chai CY, et al: Tumor-specific immunity and antiangiogenesis generated by a DNA vaccine encoding calreticulin linked to a tumor antigen. J Clin Invest 108:669-678, 2001 124. Hung CF, Cheng WF, Hsu KF, et al: Cancer immunotherapy using a DNA vaccine encoding the translocation domain of a bacterial toxin linked to a tumor antigen. Cancer Res 61:3698-3703, 2001 125. Hung CF, Cheng WF, He L, et al: Enhancing major histocompatibility complex class I antigen presentation by targeting antigen to centrosomes. Cancer Res 63:2393-2398, 2003 126. Ji H, Wang T-L, Chen C-H, et al: Targeting HPV-16 E7 to the endosomal/lysosomal compartment enhances the antitumor immunity of DNA vaccines against murine HPV-16 E7– expressing tumors. Hum Gene Ther 10:2727-2740, 1999 127. Elliott G, O’Hare P: Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell 88:223-233, 1997 128. Harms JS, Ren X, Oliveira SC, et al: Distinctions between bovine herpesvirus 1 and herpes simplex virus type 1 VP22 tegument protein subcellular associations. J Virol 74:3301-3312, 2000 129. Koptidesova D, Kopacek J, Zelnik V, et al: Identification and characterization of a cDNA clone derived from the Marek’s disease tumour cell line RPL1 encoding a homologue of alphatransinducing factor (VP16) of HSV-1. Arch Virol 140:355-362, 1995 130. Osen W, Peiler T, Ohlschlager P, et al: A DNA vaccine based on a shuffled E7 oncogene of the human papillomavirus type 16 (HPV 16) induces E7-specific cytotoxic T cells but lacks transforming activity. Vaccine 19:4276-4286, 2001 131. Hung CF, Cheng WF, Chai CY, et al: Improving vaccine potency through intercellular spreading and enhanced MHC class I presentation of antigen. J Immunol 166:5733-5740, 2001 132. Michel N, Osen W, Gissmann L, et al: Enhanced immunogenicity of HPV 16 E7 fusion proteins in DNA vaccination. Virology 294:47-59, 2002 133. Hung CF, He L, Juang J, et al: Improving DNA vaccine potency by linking Marek’s disease virus type 1 VP22 to an antigen. J Virol 76:2676-2682, 2002 134. Kim TW, Hung CF, Ling M, et al: Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins. J Clin Invest 112:109-117, 2003 135. Leachman SA, Tigelaar RE, Shlyankevich M, et al: Granulocyte-macrophage colony–stimulating factor priming plus papillomavirus E6 DNA vaccination: Effects on papilloma formation and regression in the cottontail rabbit papillomavirus–rabbit model. J Virol 74:8700-8708, 2000 136. Tan J, Yang NS, Turner JG, et al: Interleukin-12 cDNA skin transfection potentiates human papillomavirus E6 DNA vaccine–induced antitumor immune response. Cancer Gene Ther 6:331-339, 1999 137. Velders MP, Weijzen S, Eiben GL, et al: Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine. J Immunol 166:53665373, 2001 138. Liu WJ, Zhao KN, Gao FG, et al: Polynucleotide viral vaccines: Codon optimization and ubiquitin conjugation enhances prophylactic and therapeutic efficacy. Vaccine 20:862-869, 2001 139. Shi W, Bu P, Liu J, et al: Human papillomavirus type 16 E7 DNA vaccine: Mutation in the open reading frame of E7 enhances specific cytotoxic T-lymphocyte induction and antitumor activity. J Virol 73:7877-7881, 1999 140. Liu WJ, Gao F, Zhao KN, et al: Codon-modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity. Virology 301:43-52, 2002 141. Cid-Arregui A, Juarez V, zur Hausen H: A synthetic E7 gene of human papillomavirus type 16 that yields enhanced expression of



Volume 35, No. 8 (August 2004)

the protein in mammalian cells and is useful for DNA immunization studies. J Virol 77:4928-4937, 2003 142. Klencke B, Matijevic M, Urban RG, et al: Encapsulated plasmid DNA treatment for human papillomavirus 16-associated anal dysplasia: A phase I study of ZYC101. Clin Cancer Res 8:1028-1037, 2002 143. Berglund P, Smerdou C, Fleeton MN, et al: Enhancing immune responses using suicidal DNA vaccines. Nat Biotechnol 16: 562-565, 1998 144. Ying H, Zaks TZ, Wang RF, et al: Cancer therapy using a self-replicating RNA vaccine. Nat Med 5:823-827, 1999 145. Leitner WW, Ying H, Driver DA, et al: Enhancement of tumor-specific immune response with plasmid DNA replicon vectors. Cancer Res 60:51-55, 2000 146. Cheng WF, Hung CF, Hsu KF, et al: Enhancement of Sindbis virus self-replicating RNA vaccine potency by targeting antigen to endosomal/lysosomal compartments. Hum Gene Ther 12:235252, 2001 147. Cheng WF, Hung CF, Chai CY, et al: Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene. J Immunol 166:6218-6226, 2001 148. Cheng WF, Hung CF, Chai CY, et al: Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen. J Virol 75:23682376, 2001 149. Hsu KF, Hung KF, Cheng WF, et al: Enhancement of suicidal DNA vaccine potency by linkage of antigen gene to an HSP70 gene. Manuscript in preparation, 2000 150. Lanzavecchia A, Sallusto F: Regulation of T cell immunity by dendritic cells. Cell 106:263-266, 2001 151. Ossevoort MA, Feltkamp MC, van Veen KJ, et al: Dendritic cells as carriers for a cytotoxic T-lymphocyte epitope– based peptide vaccine in protection against a human papillomavirus type 16 –induced tumor. J Immunother Emphasis Tumor Immunol 18:86-94, 1995 152. Mayordomo JI, Zorina T, Storkus WJ, et al: Bone marrow– derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nat Med 1:12971302, 1995 153. De Bruijn ML, Schuurhuis DH, Vierboom MP, et al: Immunization with human papillomavirus type 16 (HPV16) oncoprotein– loaded dendritic cells as well as protein in adjuvant induces MHC class I–restricted protection to HPV16-induced tumor cells. Cancer Res 58:724-731, 1998 154. Murakami M, Gurski KJ, Marincola FM, et al: Induction of specific CD8⫹ T-lymphocyte responses using a human papillomavirus-16 E6/E7 fusion protein and autologous dendritic cells. Cancer Res 59:1184-1187, 1999 155. Doan T, Herd KA, Lambert PF, et al: Peripheral tolerance to human papillomavirus E7 oncoprotein occurs by cross-tolerization, is largely Th-2 independent, and is broken by dendritic cell immunization. Cancer Res 60:2810-2815, 2000

156. Santin AD, Bellone S, Gokden M, et al: Vaccination with HPV-18 E7–pulsed dendritic cells in a patient with metastatic cervical cancer. N Engl J Med 346:1752-1753, 2002 157. Tuting T, DeLeo AB, Lotze MT, et al: Genetically modified bone marrow-derived dendritic cells expressing tumor-associated viral or “self” antigens induce antitumor immunity in vivo. Eur J Immunol 27:2702-2707, 1997 158. Adams M, Navabi H, Jasani B, et al: Dendritic cell (DC) based therapy for cervical cancer: Use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic doublestranded RNA analogue poly [I]:poly [C(12)U](Ampligen((R))). Vaccine 21:787-790, 2003 159. Liu Y, Chiriva-Internati M, Grizzi F, et al: Rapid induction of cytotoxic T-cell response against cervical cancer cells by human papillomavirus type 16 E6 antigen gene delivery into human dendritic cells by an adeno-associated virus vector. Cancer Gene Ther 8:948-957, 2001 160. Chiriva-Internati M, Liu Y, Salati E, et al: Efficient generation of cytotoxic T lymphocytes against cervical cancer cells by adenoassociated virus/human papillomavirus type 16 E7 antigen gene transduction into dendritic cells. Eur J Immunol 32:30-38, 2002 161. Wang T-L, Ling M, Shih I-M, et al: Intramuscular administration of E7-transfected dendritic cells generates the most potent E7-specific anti-tumor immunity. Gene Ther 7:726-733, 2000 162. Chen CH, Wu TC: Experimental vaccine strategies for cancer immunotherapy. J Biomed Sci 5:231-252, 1998 163. Hallez S, Detremmerie O, Giannouli C, et al: Interleukin12–secreting human papillomavirus type 16 –transformed cells provide a potent cancer vaccine that generates E7-directed immunity. Int J Cancer 81:428-437, 1999 164. Bubenik J, Simova J, Hajkova R, et al: Interleukin 2 gene therapy of residual disease in mice carrying tumours induced by HPV 16. Int J Oncol 14:593-597, 1999 165. Chang EY, Chen CH, Ji H, et al: Antigen-specific cancer immunotherapy using a GM-CSF–secreting allogeneic tumor cell– based vaccine. Int J Cancer 86:725-730, 2000 166. Chen CH, Wang TL, Hung CF, et al: Boosting with recombinant vaccinia increases HPV-16 E7–specific T cell precursor frequencies of HPV-16 E7– expressing DNA vaccines. Vaccine 18:20152022, 2000 167. Kowalczyk DW, Wlazlo AP, Shane S, et al: Vaccine regimen for prevention of sexually transmitted infections with human papillomavirus type 16. Vaccine 19:3583-3590, 2001 168. Christensen ND, Han R, Cladel NM, et al: Combination treatment with intralesional cidofovir and viral-DNA vaccination cures large cottontail rabbit papillomavirus–induced papillomas and reduces recurrences. Antimicrob Agents Chemother 45:1201-1209, 2001 169. Christensen ND, Pickel MD, Budgeon LR, et al: In vivo anti-papillomavirus activity of nucleoside analogues including cidofovir on CRPV-induced rabbit papillomas. Antiviral Res 48:131-142, 2000