Prophylactic Human Papillomavirus Vaccine ...

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nature publishing group

GARDASILs: Prophylactic Human Papillomavirus Vaccine Development – From Bench Top to Bed-side L Shi1, HL Sings1, JT Bryan1, B Wang1, Y Wang1, H Mach1, M Kosinski1, MW Washabaugh1, R Sitrin1 and E Barr1 GARDASILs (Merck, Whitehouse Station, NJ) is a noninfectious recombinant, quadrivalent vaccine prepared from the highly purified virus-like particles (VLPs) of the major capsid proteins of human papillomavirus (HPV) types 6, 11, 16, and 18. GARDASILs is the first vaccine approved for use in women aged 9–26 years for the prevention of cervical cancer and genital warts, as well as vulvar and vaginal precancerous lesions. This report describes some of the key preclinical efforts, achievements in pharmaceutical development, in vivo animal evaluation, and clinical trial data.

RESULTS Pharmaceutical development

The pharmaceutical development of GARDASILs was carried out with the goal of designing a dosage form that is stable, efficacious, and scaleable for commercial use. Using the monovalent human papillomavirus (HPV) vaccine formulated with HPV type 16 virus-like particles (VLPs) and Merck aluminum adjuvant (MAA) as an example, Figure 1 shows the stability evaluation of various HPV vaccine formulations and highlights the role of pharmaceutical research and development in designing a stable dosage form. Formulation no. 001 was the initial HPV vaccine formulation with VLP antigen directly purified from yeast and adsorbed on MAA in physiological salt and pH solution. This formulation was unstable even at low temperature (41C). An updated formulation (no. 002) demonstrated relatively improved stability at 41C and was adequate to support the early-phase clinical studies. However, Formulation no. 002 could not be handled at room temperature for long periods, as it rapidly lost activity at 251C. Further development resulted in Formulation no. 003 with additionally enhanced stability at 251C. Although Formulation no.

003 could be processed and handled at room temperature without significant loss in antigenicity, additional stability studies with increased stress at 371C showed that it was unable to tolerate the challenge of excursion to higher temperatures. As the short- or long-term stability of a vaccine product at 371C or higher temperatures (40–451C) is beneficial to vaccine distribution in developing countries or where cold-chain protection cannot be ensured, additional efforts in formulation development were made. By treating the HPV VLPs through a process of disassembly and reassembly,1 both the stability and in vitro potency of the vaccine were enhanced significantly (Formulation no. 004). In addition, the in vivo immunogenicity of the vaccine was also improved by as much as approximately 10-fold, as shown by mouse potency studies.1 Figure 2 shows the representative atomic force images and transmission electronic micrographs of HPV VLPs pre- and post-disassembly/reassembly treatment, which significantly improved the structure and morphology of the HPV VLPs. This observation was confirmed by extensive biophysical characterization, including analytical ultracentrifugation, dynamic light scattering, and size exclusion high-performance liquid chromatography analyses.1 Furthermore, improvement in HPV VLP biophysical properties directly benefited the performance of the purification process and helped ensure process consistency. In vivo animal evaluation

The immunogenicity of the quadrivalent HPV vaccine was examined in African Green monkeys. HPV type-specific neutralizing antibody responses were measured using either a competitive radioimmunoassay for HPV types 11 and 16 or a competitive enzyme-linked immunosorbent assay for HPV types 6 and 18.2 Geometric mean titers are presented in Figure 3. Because different assays were used to evaluate the different HPV types, the level of immune response elicited by

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Merck Research Laboratories, West Point, Pennsylvania, USA. Correspondence: Li Shi ([email protected])

doi:10.1038/sj.clpt.6100055 CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 81 NUMBER 2 | FEBRUARY 2007

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Figure 1 Stability studies in support of optimal dosage form development step by step to achieve enhanced stability as well as improved antigenicity. The compositions of monovalent HPV16 vaccine formulations used in this study are as follows: no. 001: 0.15 M NaCl; no. 002: 0.32 M NaCl, 0.01% polysorbate 80, 10 mM histidine, pH 6.2; no. 003: no. 002 þ 0.1% polyanions; no. 004: no. 002 with disassembled and reassembled HPV VLPs. All formulations contain 450 mg/ml MAA.

Figure 2 The morphology of representative HPV VLPs (left) before and (right) after disassembly and reassembly treatment as determined by (top) atomic force imaging and (bottom) transmission electron microscopy. The two pictures in each pair of micrographs are shown in the same scale.

one HPV type cannot be directly compared to the titers of another HPV type. In addition, all the assays are competitive assays; therefore, the affinity of the neutralizing monoclonal antibody used plays a role in the range of titers seen for a given type. Nevertheless, the preclinical study demonstrated that the general tolerability and robust antibody response to HPV L1 VLPs can be achieved with the final quadrivalent formulation (Figure 3). Clinical trials: published results to date

The clinical trial program for GARDASILs included four randomized, placebo-controlled (1:1 vaccine:placebo recipi260

ent), multinational, multicenter studies that were similar in design.3–6 In each study, the primary analysis of vaccine efficacy was per-protocol: subjects must have received three doses, have no major protocol violations, and be both vaccine-type HPV sero-negative at day 1 and vaccine-type HPV DNA-negative through the completion of the vaccination regimen. The first was a proof-of-principle study (V501005) of the monovalent HPV 16 vaccine component conducted in 2,391 American women 16–23 years of age.3 The primary efficacy end points included persistent HPV 16 infection and HPV 16-related cervical intraepithelial neoplasia of any grade severity (CIN 1–3). After a median follow-up of 40 months post dose three, vaccine efficacy was 100% with regard to confirmed persistent HPV 16 infection, and 100% for HPV 16-related CIN 1–3. These results were the first to confirm that an HPV L1 VLP-based vaccine was not only effective for preventing persistent infection, but also for preventing precancerous lesions. The prototype monovalent vaccine trial was followed by a Phase II dose-ranging study (V501-007) that included an evaluation of the efficacy of the final GARDASILs formulation versus placebo, conducted in 552 women aged 16–23.4,7 Through 5 years, the overall vaccine efficacy was 100% for preventing HPV 6-, 11-, 16-, or 18-related disease, representing the longest-term efficacy evaluation of an HPV vaccine to date. The two pivotal Phase III studies of GARDASILs included 418,000 young women.5 Protocol V501-013 was designed to include an intensive visit schedule (every 6 months) with aggressive regimens for external genital inspection, cervical cytology, colposcopy, and biopsy to ensure complete ascertainment of HPV-related lesions. The co-primary end points were HPV 6-, 11-, 16-, or 18-related (a) CIN 1–3 or adenocarcinoma in situ (AIS), and cervical cancer, and (b) genital warts, vulvar/vaginal intraepithelial neoplasia or cancer. In the per-protocol population, 37 cases of HPV 6-, 11-, 16-, or 18-related CIN 1–3 or AIS were observed.5 There were no cases in the group that received GARDASILs. VOLUME 81 NUMBER 2 | FEBRUARY 2007 | www.nature.com/cpt

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Figure 3 Quadrivalent HPV vaccine in vivo immunogenicity study in African Green monkeys with vaccination at day 1, week 8, and week 24. HPV typespecific geometric mean titers were determined by competitive immunoassays: competitive radioimmunoassay for HPV 16 and HPV 11, and competitive enzyme-linked immunosorbent assay for HPV 18 and HPV 6. In each case, a type-specific neutralizing monoclonal antibody was displaced by HPV typespecific vaccine-induced serum antibodies. HPV 16 and 11 were evaluated through 52 weeks, whereas HPV 18 and 6 were evaluated only through week 26.

The vaccine was equally effective against genital warts, vulvar, and vaginal lesions.5 The second phase III study (V501-015)6 focused on obligate precancerous lesions (CIN 2/3 and AIS) due to the oncogenic HPV types 16 and 18. In contrast to the intensive visit schedule in the first study, this study allowed for a ‘‘real world’’ picture of cervical cancer screening as women underwent cervical screening on a yearly basis. In both trials, high efficacy against the primary endpoints was observed within an average of 17 months post-dose three, in the per-protocol population, the vaccine was 100% effective against HPV 16- and 18-related CIN 2/3 and AIS (data submitted for publication). GARDASILs is highly immunogenic. In adult women, vaccine-induced anti-HPV responses were detected in 99.5% of subjects 1 month post-dose three.4 Through at least 5 years, anti-HPV geometric mean titers remain at or above the geometric mean titers observed in placebo recipients who have serologic evidence of natural HPV infection.4,7 In addition, immunization of 10–15-year-old girls and boys resulted in robust anti-HPV type-specific virus-neutralizing antibody responses that were statistically non-inferior and observationally higher (1.6–2.6-fold) than those observed in 16–23-year-old women.8 DISCUSSION

Over the last three decades, it has become increasingly evident that infectious agents, particularly viruses, play a role in human cancers. Recently, Parkin estimated that the total of CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 81 NUMBER 2 | FEBRUARY 2007

infection-attributable cancer in the year 2002 was 1.9 million cases, or 17.8% of the global cancer burden.9 The principal agents were the bacterium Helicobacter pylori (5.5% of all cancer), the hepatitis B and C viruses (4.9%), Epstein–Barr virus (1%), HIV together with herpes virus (0.9%), and HPV (5.2%). The successes that prophylactic vaccines have had in preventing infectious diseases highlight the paradigm shift that is offered by vaccines, which may prevent cancer by protecting the host against the offending pathogen. In the past decade, it has been definitively established that HPV is the single most important risk factor for the development of cervical cancer. The causal association with cervical cancer, the high prevalence of HPV in the general population,10 and the lack of effective means to prevent the spread of HPV have led to the development of GARDASILs, a highly effective vaccine that protects against four HPV types responsible for a majority of HPV-related diseases. HPV infection is common, with a lifetime risk exceeding 50% for sexually active male and female subjects.11 Cervical cancer is the second leading cause of cancer death among women worldwide.12 Even in developed countries with organized cervical cancer screening programs, cervical cancer remains the second leading cancer among women under the age of 45.12 The most important public health impact of a prophylactic HPV vaccine is cervical cancer prevention; however, clinical trials to demonstrate prevention of cancer are neither feasible nor ethical. The World Health 261

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Organization and Vaccines and Related Biologic Products Advisory Committee to the US Food and Drug Administration have recommended that clinical trials for HPV vaccines be designed to demonstrate efficacy against CIN2/3 and AIS, as these lesions are the obligate and immediate precursors to invasive cancer.13,14 The VRBPAC also recommended that the vaccine trials should enroll subjects without consideration of baseline HPV status so that trial results may reflect the efficacy and safety of the vaccine in a general population. The GARDASILs clinical trial program was designed to accommodate these requirements. The confidence with which the efficacy results can be extrapolated to cervical cancer prevention is a function of the robustness of the surrogate end point for cervical cancer and the precision with which efficacy is demonstrated. The HPV VLP antigens used for GARDASILs are independently produced by expressing the type-specific major capsid protein, L1, in yeast. However, the development of an effective vaccine against HPV was initially hampered by the difficulty in propagating HPV in vitro and the lack of a suitable expression system. In the past decade, sufficient quantities of HPV VLPs have been successfully produced for research purposes using baculovirus vectors, vaccinia virus, yeast, and Escherichia coli.1,15–17 The expression of HPV VLP L1 proteins in yeast cells with appreciable yield has significantly benefited both clinical studies and the manufacturing of HPV vaccine for commercial use as it offers the advantages of being cost-effective and easily adaptable to large-scale fermentation.17,18 The in vivo VLP assembly during recombinant expression may differ from the capsid assembly during actual infection owing to the environment specific to the host system. The HPV type 16 major 55 kDa capsid protein, L1, when produced in certain recombinant expression systems such as Saccharomyces cerevisiae can form irregularly shaped VLPs with a broad size distribution.1 Although these HPV VLPs were shown to be immunogenic in a human efficacy study,17 they have relatively short shelf life and lose in vitro measured antigenicity at elevated temperatures.1,19 These HPV VLPs are inherently unstable and tend to aggregate in solution.19 Concentrating these HPV VLPs during bioprocessing can aggravate the aggregation problem even further. Specifically, at lower ionic strengths such as physiological levels of NaCl, these HPV VLPs tend to aggregate, at times, to the point of precipitating out of solution during process and storage.19 The primary challenge of HPV vaccine dosage form development was the preparation of aqueous HPV VLP solutions that are stable under a variety of purification, processing, and storage conditions. Furthermore, as HPV VLPs are formulated into a vaccine product with adjuvant, it is critical that HPV VLP antigens in the vaccine product are stable at physiologically acceptable salt concentrations that are compatible with parenteral administration. An extensive bench top formulation research and process development was carried out to obtain a vaccine dosage form 262

with enhanced stability and potency to support sample handling, bioprocess development, and clinical supply preparations. This was achieved by novel formulation design with the introduction of stabilizing non-ionic surfactants and adjusting salt concentration19 (Figure 1). This effort successfully stabilized the HPV VLPs against surface adsorption, conformational change, inducible antigen aggregation, and loss of antigenicity. The enhancement of the vaccine dosage form in both stability and potency was achieved with multiple formulation research contributions, including the precise control of pH and the addition of MAA.1,19,20 We further developed the critical formulation through disassembly and reassembly of HPV VLPs during the purification of the antigen, resulting in significant improvement in the biophysical and bioanalytical properties of the VLPs.1 The reassembled VLPs possess an architecture very similar to that of natural HPV virion particles, suggesting that what has been achieved naturally through cell entry and virus replication can also be achieved through an optimal in vitro assembly process for yeast-expressed VLPs (Figure 2). The direct benefit of this development was the dramatic enhancement of the overall storage and accelerated stabilities, in addition to increased potency (Figure 1). The stability enhancement allowed for process convenience (at room temperature), significantly improved process consistency (data not shown), and provided wider tolerance for potential temperature excursions, thus ensuring the success of a stable vaccine development. The pre-clinical studies (Figure 3) proved the concept of the vaccine in stimulating an anti-HPV immune response. A previous comparison study also demonstrated the effectiveness of MAA in improving the immunogenicity of the HPV vaccine as compared with the vaccine without aluminum adjuvant.21 In addition to its adjuvant function, the MAA also significantly enhanced accelerated stability of the vaccine against heat stress-induced degradation.20 The apparent stabilization of the adjuvant-adsorbed HPV vaccine is believed to be due to the physical properties of the adjuvant, which serves as a physical barrier, preventing intermolecular collisions and thus minimizing aggregation of HPV VLPs. The storage stability of GARDASILs has been evaluated under both long-term and accelerated storage conditions.20 GARDASILs showed no detectable degradation during the entire 36 months storage stability study at 41C, although the accelerated stability study did show different levels of antigenicity loss as a function of elevated storage temperatures such as 25, 37, and 421C. Using the accelerated stability data collected at various temperatures, the half-life of GARDASILs at temperatures up to 251C is predicted to be 130 months or more. At 37 and 421C, the degradation rates increased significantly, but even at these temperatures, the estimated half-lives are approximately 18 and 3 months, respectively.20 In summary, the key achievements in the pre-clinical development of GARDASILs include the stabilization of the VLP antigens via their disassembly and reassembly and by VOLUME 81 NUMBER 2 | FEBRUARY 2007 | www.nature.com/cpt

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adsorption to the MAA. The resulting quadrivalent HPV vaccine is highly immunogenic, both in initial pre-clinical and extensive clinical studies. The clinical studies have clearly demonstrated the high efficacy of GARDASILs in the prevention of cervical cancer, genital warts, and vulvar and vaginal precancerous lesions. GARDASILs is expected to significantly reduce the morbidity, mortality, and health-care costs associated with HPV-related diseases. METHODS HPV VLPs. Recombinant HPV L1 major capsid protein type 6, 11,

16, and 18 VLPs were independently produced intracellularly in a Saccharomyces cerevisiae expression system. The cells were harvested and lysed, and the self-assembled L1 protein VLPs were purified chromatographically to 495% purity as described previously.1,18 The purified VLPs were used directly for stability studies (see Figure 1) during early pre-clinical development without further treatment. The purified VLPs were later treated with dithiotheritolcontaining buffer to disassemble the particles and subsequently allowed to reassemble.1 The disassembled and reassembled HPV VLPs were used for updated HPV vaccine pre-clinical and clinical development. GARDASILs vaccine. For clinical studies, each type of aqueous

HPV VLPs was adsorbed on 450 mg/ml of MAA separately. The monovalent vaccine components were formulated in 10 mM histidine, 0.01% polysorbate 80, and 0.33 M NaCl. The four individual monovalent (adjuvant adsorbed) vaccines were blended at protein concentrations of 40, 80, 80, and 40 mg/ml for HPV types 6, 11, 16, and 18, respectively, to form the quadrivalent dosage form of GARDASILs. The vaccine was used in storage stability studies and clinical trials in which the patients were vaccinated with three 0.5 ml doses following a 0-, 2-, and 6-month schedule. The quadrivalent HPV vaccine used for animal studies was prepared in the same way, but diluted with blank MAA to achieve the desired dose.

collected with Digital Nanoscope III MultiMode AFM system (Digital Instrument, Santa Barbara, CA). Electron microscopy. Transmission electron microscopy was per-

formed using negative staining. Samples were fixed on a 300-mesh copper grid, stained with phosphotungstic acid, and examined in a JEOL 1200 EX transmission electron microscope. Micrographs were taken of random areas with samples prepared multiple times at a magnification between 30,000 and 40,000. An additional three-fold magnification was introduced in developing the prints from the negatives. Clinical studies. The general methodology for the clinical trials has been described.3–6 All subjects or parents/legal guardians signed informed consents following review of the individual protocol procedures. The studies were conducted in conformance with applicable country or local requirements regarding ethical committee review, informed consent, and other statutes or regulations regarding the protection of the rights and welfare of human subjects participating in biomedical research. Phase III trials are registered as ClinicalTrials.gov number NCT00092521, NCT00092534, and NCT00092495. ACKNOWLEDGMENTS Merck Research Laboratories, a division of Merck, funded this work in its entirety. We thank Merck colleagues in Basic Research, Pharmaceutical R&D, and Bioprocess R&D, who provided the HPV VLP materials and characterization supports, the clinical study investigators and coordinators, and study participants. Special thanks to Drs Peter Honig, Kathrin Jansen, David B Volkin, Ann Lee, and Kathryn Hoffmann for their support and helpful discussions.

CONFLICT OF INTEREST The authors are Merck employees and potentially own stock and/or hold stock options in the company. Merck is developing the quardrivalent HPV vaccine. & 2007 American Society for Clinical Pharmacology and Therapeutics

In vitro antigenicity and in vivo immunogenicity. The in vitro

antigenicity of HPV VLP samples was measured by a surface plasmon resonance instrument (Biacore, Piscataway, NJ) utilizing HPV VLP type-specific neutralizing antibodies (Chemicon, Temecula, CA). In vivo immunogenicity of the quadrivalent HPV vaccine was assessed by vaccination of six African Green monkeys. The quadrivalent vaccine contained 4 mg/ml of each HPV VLPs formulated with MAA. The vaccine was administered at 0.5 ml i.m./dose with dosing at day 1, week 8, and week 24. Immunogenicity was assessed by competitive radioimmunoassay2 or competitive enzyme-linked immunosorbent assay on sera from blood samples collected at day 1, and weeks 8, 10, 24, 26, and 52. All studies involving animals were approved by the Merck Institutional Animal Care and Use Committee. Stability studies. Storage stability studies of HPV VLPs, prepared in formulations with an aluminum adjuvant, were carried out generally at 4, 25, and 371C. The in vitro antigenicity of each sample was assayed at desired time intervals by Biacore analysis after the HPV antigens were released from the aluminum adjuvant particles using a citrate buffer. Atomic force microscopy. Atomic force microscopy studies were performed in aqueous media. Mica discs used as the samplesupporting substrate were freshly cleaved just before sample preparation. Aqueous HPV VLP sample was deposited on the freshly cleaved mica surface. Atomic force microscopy images were

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