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Three groups, Merck, MedImmune, and the U.S. National Cancer Institute independently moved forward with GMP production of VLPs for phase I clinical trials. The MedImmune and the NCI (under a contract to Novavax) chose to continue producing their VLPs in recombinant baculovirus infected insect cells. However,

Merck decided to manufacture their vaccine in Saccharomyces cerevisiae, presumably because they had extensive experience in yeast production of their HBV vaccine. GMP process development and scale-up proved to be challenging. For example, the VLPs have a propensity to interact with solid surfaces, leading to aggregation and flocculent precipitation. However, Merck scientists determined that this problem could be overcome by the addition of small amounts of certain nonionic detergents [47]. Both companies eventually settled on purification schemes involving the dissociation of the VLPs into pentameric capsomeric subunits followed by in vitro reassembly into VLPs [48, 49]. The conformation dependency of the neutralizing epitopes also presented difficulties. For instance, MedImmune/GSK added thimero-sal to their phase I product as a preservative. This formulation retained the VLP structure and induced VLP-reactive antibodies in vaccinees, but none of the antibodies were neutralizing [50]. Although the problem was solved by formulating the VLPs without thimerosal, this experience proved to be a substantial set back in GSK's development program. Additional insights into the commercial product development of these vaccines are provided in a recent review [50].

Results of the phase I clinical trials of HPV VLP vaccines were first published in 2001. The NCI collaborated with Johns Hopkins University to demonstrate that their HPV16 L1 VLPs were safe and highly immunogenic after three intramuscular doses of 10 or 50 mg when formulated in alum, MF59 or unadjuvanted [51]. At the higher dose, there was no significant difference in the titers induced by the three formulations. VLP binding and HPV16 pseudovirus neutralizing titers were both high and highly correlated. Encouragingly, the mean antibody titers in vaccinees were approximately 40-fold higher than those seen after natural HPV16 infection and similar to the titers that induced strong protection from experimental infection in the rabbit and bovine challenge models. MedImmune collaborated with the University of Rochester to conduct a trial of their HPV11 L1 VLPs. HPV11 was chosen for the initial study because the investigators had access to a recently developed semiquantitative in vitro HPV11 neutralizing assay. It was based on infection of an immortalized human keratinocyte line with SCID mouse-derived HPV11 virions and monitoring infection by production of HPV-specific mRNA [52]. Three intramuscular doses of 3, 9, 30, or 100 mg doses in aluminum hydroxide adjuvant were well tolerated and induced high titers of HPV11 VLP binding and HPV11 neutralizing antibodies [53]. Similar responses were seen with the three highest doses. The encouraging results of these trials provided the impetus for sponsorship of the larger phase II and III trials by GSK.

In 2002, the results of the first proof of concept efficacy trial of an HPV VLP vaccine were published. Three 40 mg doses of Merck's HPV16 VLP in an aluminum adjuvant were administered over a 6-month period. Over the course of 1.5 years of follow up, 41 of the approximately 1,000 placebo controls developed persistent HPV16 infection, while none of the 1,000 VLP vaccines became persistently infected. Laura Koutsky's unannounced presentation of these results at the 2002 International Papillomavirus conference is etched in our memories. It was electrifying to hear that simple intramuscular injection of a VLP vaccine could induce 100% protection against persistent cervical infection, even in the short term.

Table 2 Comparison of commercial HPV VLP vaccines

Gardasil

Cervarix

Manufacturer VLP types L1 protein dose Production

Merck & Co. 6/11/16/18 20/40/40/20 mg Saccharomyces cerevisiae

GlaxoSmithKline 16/18 20/20 mg

L1 recombinant baculovirus expressing L1

infection of Trichoplusia ni

(Hi 5) insect cells

Adjuvant

225 mg aluminum hydroxyphosphate sulfate

500 mg aluminum hydroxide, 50 mg 3-O-deacylated-4'-monophosphoryl lipid A

Injection schedule 0, 2, 6 months

Two important decisions needed to be made before proceeding to large-scale phase III studies aimed at generating the data required for licensure. The first was the composition of the vaccine. In this instance, the development paths diverged for Merck and GSK, which, as noted above, had taken over the vaccine development from MedImmune after their phase I trial. GSK decided to concentrate exclusively on cancer prevention and therefore included L1 VLPs of HPV16 and HPV18, the two types responsible for approximately 70% of cancer worldwide. They chose to use their proprietary adjuvant, AS04, which, in addition to an aluminum salt contains monophosphoryl lipid A (a detoxified form of LPS). AS04 tends to induce a Th1 type T helper response, compared to the Th2 type response generally associated with aluminum salt vaccines. Merck decided to target genital warts, in addition to cervical cancer, and therefore included VLPs of types 6 and 11, which induce about 90% of genital warts, in addition to VLPs of types 16 and 18. Merck chose to use a conventional aluminum salt adjuvant (Table 2).

The second critical decision was what primary endpoint to use in the pivotal efficacy trials. Although the main goal of both vaccines is to prevent cervical cancer, there was consensus that cancer was not a reasonable clinical trial endpoint. First, it would take an extremely large trial, several decades in duration, to accumulate a sufficient number of cervical cancer cases from incident HPV infection. Second, the trial would be unethical, because careful active follow up of participating women would identify virtually all high grade precancerous lesions, and standard of care would require their excision before they progressed to invasive cancer. At the opposite end of the spectrum, there was general agreement that incident HPV infection was not a sufficiently stringent endpoint. Most genital HPV infections regress spontaneously and so are not on the causal pathway to cancer. Also, HPV infection is identified by the presence of HPV DNA in genital swabs or scrapes and so transient infection cannot be unequivocally distinguished from contamination, as might be acquired during sexual activity with an infected partner. A strong argument was put forth that persistent HPV infection was an appropriate endpoint for efficacy trials. Essentially all cervical cancers arise from persistent infection and HPV DNA measurements using PCR-based technologies are sensitive and reproducible. However, objections were raised that persistent

HPV infection by itself is not an indication for therapeutic intervention. In addition, the duration of infection that distinguishes harmless transient infections from persistent infections with a high probability of progression had not been established. Therefore, advisory groups to national regulatory agencies, such as the U.S. FDA, recommended a histologically confirmed neoplastic disease endpoint, in particular intermediate or high grade cervical intraepithelial neoplasia (CIN2/3). These dysplasias are routinely treated by ablative therapy when they are diagnosed in Pap screening programs, and at least CIN3 is widely accepted as an obligate cervical cancer precursor. Low grade cervical dysplasia (CIN1) was not considered an appropriate endpoint, since it is a normal manifestation of productive HPV infection and in most cases spontaneously regresses.

The selection of CIN2/3 from incident infection by the vaccine-targeted types as the primary endpoint meant that the pivotal phase III trials had to be large and of relatively long duration. Merck, GSK, and the NCI independently initiated randomized, controlled and double-blind phase III trials of 5,000-19,000 women, with a duration of 4 years (Table 3). The NCI chose to test the GSK bivalent vaccine, because of difficulties in generating sufficient quantities of GMP vaccine in a timely fashion under contract. The Merck and GSK trials were multinational involving more than 100 individual sites each in Europe, North America, South America, Asia, and Australia. In contrast, the NCI chose to conduct, in collaboration with the Costa Rican government, a community-based study centered in the Guanacaste province of Costa Rica, in part because of the infrastructure established during a long-standing natural history study of HPV and cervical cancer in the province [54]. The trials enrolled nonpregnant young women, ages 15-26, generally with fewer than five or seven lifetime sexual partners. This exclusion criterion was used to limit the number of women with prior exposure to the vaccine types of HPV, since the primarily intent of the trials was to test prophylactic efficacy.

In 2004 and 2005 publications, phase IIb trials of the GSK bivalent vaccine, designated Cervarix, and the Merck quadrivalent vaccine, designated Gardasil, provided encouraging preliminary results [55, 56]. They demonstrated high levels of protection against persistent infection and cervical dysplasia (of any grade) by the vaccine targeted types. These publications were followed, in 2007, by the publication of interim analyses of the phase III trials, which were triggered 1.5-3.0 years postvaccination by the accumulation of a predetermined number of disease events [57, 58]. The according to protocol (ATP) analysis revealed that, in women without infection by the vaccine types at enrollment, three doses of either vaccines induced virtually complete protection from incident CIN2/3 lesions in which the vaccine types were detected (Table 3). Gardasil also demonstrated close to 100% protection from external genital lesions, including genital warts, induced by the vaccine types. As expected from the understanding of HPV carcinogenesis obtained in the prospective epidemiology studies, high levels of protection against persistent infection and CIN1 by the vaccine-targeted types were also observed for both vaccines. The high type-specific efficacy of these vaccines certainly exceeded our expectations and those of others in the field. However, their efficacy was clearly limited in that they did not induce clearance of preexisting infections or prevent

Table 3 VLP vaccine efficacy trials in young women: ATP analyses for vaccine-specific HPV types in women negative for vaccine type infections at enrollment

Characteristic

GSK 001/07

Merck 007

Patricia

Future I

Future II

Costa rica

Vaccine

Cervarix

Gardasil

Cervarix

Gardasil

Gardasil

Cervarix

Phase

II

II

III

III

III

III

Control

500 ng

225 ng

Hepatitis A vaccine

225 (ig aluminum

225 (ig aluminum

Hepatitis

aluminum

aluminum

hydroxy-

hydroxy-phosphate

A vaccine

phosphate sulfate

phosphate sulfate

sulfate

No. participants

1,113

552

18,644

5,455

12,167

7,466

Age range

15-25

16-23

15-25

16-24

15-26

18-25

Lifetime no. of sex

<6

<4

<6

<4

<4

Any no.

partners

Screening frequency

6 months

6 months

12 months

6 months

12 months

12 months

Mean duration of

48 months

60 months

15 months3

36 months3

36 months3

48 months1'

follow-up

Endpoint: vaccine

Persistent HPV

Persistent HPV

CIN2+: 90 (53-99)°

CIN1+, AIS: 100

CIN2+, AIS:

NA

efficacy (95% CI)

DNA: 96 (75-100)

DNA: 96 (83-100) CIN1+, AIS: 100 (cO-lOO)

(94-100) EGL: 100 (94-100)

98 (92-100)

ATP according to protocol; CIN1+ cervical intraepithelial neoplasia grade 1 or worse; CIN2+ cervical intraepithelial neoplasia grade 2 or worse; AIS adenocarcinoma in situ; EGL external genital lesions; NA not available " Interim analysis of 4-year trial bIn progress

0 Modified intention to treat analysis: received at least one dose, case counting started at first dose. Confidence intervals are 97.9%. A post hoc analysis including HPV-specific causal attribution of CIN2+ with multiple type infections generated efficacy estimates of 100% (97.5% CI: 74.2-100)

their progression [57, 59]. Also, the two vaccines have limited ability to prevent infections by other high-risk genital HPV types, as predicted by the in vitro neutralization studies. Submission of these interim analyses led to regulatory approval of Gardasil in the United States, European Union, and elsewhere, starting in mid-2006. Cervarix was approved in the European Union and elsewhere (in 2010 in the United States), starting in mid-2007, making it the first licensed produced to be produced via recombinant baculoviruses. Analysis of the full data sets accumulated over the complete 4 years of these trials, and also of the Costa Rican trial, will likely be published soon.

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