Expanded strain coverage for a highly successful public health tool: Prophylactic 9-valent human papillomavirus vaccine

ABSTRACT

Human papillomavirus is considered the causative factor for cervical cancer, which accounts for approximately 5% of the global cancer burden and more than 600,000 new cases annually that are attributable to HPV infection worldwide. The first-generation prophylactic HPV vaccines, Gardasil® and Cervarix®, were licensed approximately a decade ago. Both vaccines contain the most prevalent high-risk types, HPV16 and 18, which are associated with 70% of cervical cancer. To further increase the type coverage, 5 additional oncogenic HPV types (31, 33, 45, 52 and 58) were added to the existing Gardasil-4 to develop a 9-valent HPV vaccine (9vHPV), Gardasil 9®, increasing the potential level of protection from ∼70% to ∼90%. The efficacy of the vaccine lies primarily in its ability to elicit type-specific and neutralizing antibodies to fend off the viral infection. Therefore, type-specific and neutralizing murine monoclonal antibodies (mAbs) were used to quantitate the antigenicity of the individual vaccine antigens and to measure the antibody levels in the serum samples from vaccinees in a type- and epitope-specific manner in a competitive immunoassay. Assays for 9vHPV are extended from the proven platform used for 4vHPV by developing and adding new mAbs against the additional types. In Phase III clinical trials, comparable safety profile and immunogenicity against the original 4 types were demonstrated for the 9vHPV vaccine, and these were comparable to the 4vHPV vaccine. The efficacy of the 9vHPV vaccine was established in trials with young women. Immunobridging for younger boys and girls was performed, and the results showed higher immunogenicity in the younger age group. In a subsequent clinical trial, the 2-dose regimen of the 9vHPV vaccine used among girls and boys aged 9–14 y showed non-inferior immunogenicity to the regular 3-dose regimen for young women (aged 16–26 years). Overall, the clinical data and cost-effectiveness analysis for the 9vHPV vaccine support its widespread use to maximize the impact of this important, life-saving vaccine.

KEYWORDS:

• 2-dose vaccination
• comparable immunogenicity
• human papillomavirus
• immuno bridging
• neutralizing antibodies
• prophylactic vaccine
• vaccine efficacy

Introduction

Human papillomavirus (HPV) is a small, non-enveloped capsid virus that has double-stranded circular DNA genomes. ^1,2 In the 1970s, Harald zur Hausen and his colleagues first introduced the concept that the precancerous lesions and cancer were caused by persistent infection with HPV. ^3,4 It was well known that HPV is a causative factor in cervical cancer, which is the fourth most frequent cancer in women, ^5,6 and accounts for approximately 5% of the global cancer burden worldwide. ⁷

Cervical cancer is one of the few cancers that can be prevented using prophylactic vaccines. The first-generation vaccines, 4-valent HPV vaccine (referred to as 4vHPV vaccine or Gardasil®, Merck and Co., Inc.) and bivalent HPV vaccine (referred to as 2vHPV vaccine or Cervarix®, GlaxoSmithKline), were licensed in 2006 (in the US) and 2007 (in the EU), respectively. ⁸ Both vaccines were shown to effectively protect against infection from 2 high-risk HPV types (HPV16 and 18), which are responsible for causing approximately 70% of cervical cancers. ^9,10 The 4vHPV vaccine can also prevent against infection with 2 low-risk HPV genotypes (HPV6 and 11), which are responsible for approximately 90% of genital warts. ^11,12 Beyond the HPV types covered in the first generation vaccines, oncogenic HPV types 31, 33, 45, 52 and 58 comprise the next group of types with clinical significance because they account for nearly an additional 20% of cervical cancers worldwide (Fig. 1). ^13,14 Both HPV vaccines are shown to have some cross-protection against HPV types that are not included in the vaccines. In a meta-analysis, ¹⁵ cross-protection was shown that in the 2vHPV vaccine trial was higher than it was in the 4vHPV vaccine trial against persistent infections with HPV31 (77.1% vs 46.2%) and HPV45 (79.0% vs 7.8%), and against high-grade cervical intraepithelial neoplasia (CIN2/3) associated with HPV33 (82.3% vs 24.0%) and HPV45 (100% vs −51.9%). However, for both vaccines, there was very little evidence of cross-protection against HPV52 and 58. ¹⁶ Furthermore, antibody titers remain generally high for HPV16 and 18, but levels for HPV31, 33, and 45 reach much lower titers after 2vHPV or 4vHPV vaccination and decline within 2 y to the levels seen with natural infection or lower than the limit of detection, ^17,18 which suggests a potential for waning of cross-protection. Therefore, a multivalent HPV vaccine, including HPV types 16 and 18 plus these 5 additional HPV types, has the potential to prevent approximately 90% of cervical cancers or genital warts.

Figure 1. Percentages of HPV-related genital warts or cervical cancer attributed to different HPV genotypes. The 2 most prevalent high-risk HPV genotypes (HPV16 and 18) and 2 low-risk genotypes (HPV6 and 11) are included in the 4vHPV vaccine (Gardasil®) licensed in 2006. In addition to the above 4 genotypes, 5 common high-risk genotypes (HPV31, 33, 45, 52 and 58) are contained in the 9vHPV vaccine (Gardasil 9®) that was more recently licensed in 2014. The data were collected from Mariani et al. ⁷

To broaden protection against HPV genotypes beyond HPV types 6, 11, 16 and 18, 3 clinical trials ^19-21 (Study V502–001, V503–001, V504–001) were subsequently initiated to evaluate the multivalent HPV vaccine formulations (8-valent, 9-valent, and 4-valent combination with 5-valent) for selecting the optimal formulations and further evaluation. Based on the results of 3 studies, each 0.5 mL dose of the formulation of the 9-valent HPV (9vHPV) vaccine containing 30, 40, 60, 40, 20, 20, 20 and 20 μg of HPV6, 11, 16, 18, 31, 33, 45, 52 and 58 VLPs, respectively, was eventually selected. ²²

After more than a decade for the developmental cycle, the 9vHPV vaccine was licensed in the US in December 2014 with the trade name Gardasil 9® (Table 1). ²³ The milestones in the late stage of clinical development and licensure of prophylactic HPV vaccines are shown in Fig. 2. Because the same technology was used for developing the VLPs for the additional HPV types, the 9vHPV vaccine, like the 4vHPV vaccine, is a non-infectious recombinant VLP-based vaccine. The 9vHPV vaccine is prepared from the highly-purified virus-like particles (VLPs) of the major capsid L1 protein including the same 4 HPV types in the 4vHPV vaccine and 5 additional oncogenic high-risk HPV types. ²⁴ VLPs from 9 different types were purified and adjuvant-adsorbed separately and then mixed to formulate the final product. Basic information about 3 currently licensed prophylactic HPV vaccines by the Food and Drug Administration (FDA) is shown in Table 1. By preventing HPV persistent infection and diseases from the additional HPV types, the 9vHPV vaccine has a potential to increase the prevention rate of cervical cancer from ∼70% to ∼90% ²⁵ while providing additional protection against low/high-grade cervical intraepithelial neoplasia (CIN1/CIN2/3) and other HPV-related cancers (Table 2). ^26-31

Table 1. Basic information on the globally licensed human papillomavirus (HPV) vaccines.

	2vHPV (Cervarix®)	4vHPV (Gardasil®)	9vHPV (Gardasil 9®)
Vaccine vial image
Manufacturer	GlaxoSmithKline	Merck and Co., Inc.	Merck and Co., Inc.
Expression system	Baculovirus	Yeast	Yeast
	(Trichoplusia ni insect cell)	(Saccharomyces cerevisiae)	(Saccharomyces cerevisiae)
HPV types	16/18	6/11/16/18	6/11/16/18/31/33/45/52/58
VLPs protein (μg)	20/20	20/40/40/20	30/40/60/40/20/20/20/20/20
Adjuvants	500 μg aluminum hydroxide, 50 μg	225 μg amorphous aluminum	500 μg amorphous aluminum
	3-O-desacyl-4’ monophosphoryl lipid A	hydroxyphosphate sulfate	hydroxyphosphate sulfate
Volume per dose	0.5 mL	0.5 mL	0.5 mL
Dosing regime	0, 1, 6 month	0, 2, 6 month	0, 2, 6 month
Route of administration	Intramuscular injection	Intramuscular injection	Intramuscular injection
Approval time by FDA	October, 2009	June, 2006	December, 2014

1. The information of Cervarix® is available from http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM186981.pdf;

2. The information of Gardasil® is available from http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM111263.pdf;

3. The information of Gardasil 9® is available from http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM426457.pdf;

(Accessed on 20 April 2017)

Figure 2. Milestones and timelines in the late stage clinical development and licensure of HPV vaccines. The upper part of the timeline is related to the information on the 9vHPV vaccine, with the 2vHPV and 4vHPV vaccines in the lower part. The 5 important clinical trials for HPV vaccines in the last decade with a red frame are shown in the illustration. The specific information for these 5 clinical trials can be found in the Supplemental Material (Table S2).

Table 2. Estimated contribution from different types covered in the 4vHPV and 9vHPV vaccine for HPV-related cancers and diseases worldwide.

	4 HPV types (6/11/16/18)	5 HPV types (31/33/45/52/58)	9 HPV types (6/11/16/18/31/ 33/45/52/58)
	cause:	cause an additional:	cause a total of	References
Cervical cancer	70%	20%	90%	26
High-grade cervical lesions	50%	30%	80%	27
Low-grade cervical lesions	25%	25%	50%	28
Vaginal cancer	65%	20%	85%	28
Vulvar cancer	80%	10%	90%	29
Anal cancer	85%	5–10%	90–95%	30
Genital warts	90%	No contribution	90%	31

Prevention of cervical cancer and other genital cancers by vaccination against oncogenic high-risk types has been a pivotal public health strategy in many countries and regions. ³² Due to the broader protection coverage, the 9vHPV vaccine has the potential to address an unmet medical need to prevent infection of the additional HPV types and associated cancers or pre-cancerous lesions. In this review, we introduced the bioanalytics of the 9vHPV vaccine to support product quality analysis and serological testing in clinical trials. The results on the 9vHPV vaccine safety and efficacy will be reviewed. The latest recommendation for the 9vHPV vaccination schedule (i.e., 2 doses for boys and girls aged 9–14 years) by the FDA will be discussed, which should improve the uptake of HPV vaccination with the 2-dose regimen.

Bioanalytics in support of vaccine development and commercialization

Since the integrity of the functional epitopes on the recombinant VLP surface is the basis for its function as an efficacious vaccine, it is essential to monitor HPV vaccine antigen type-specific conformational epitopes during vaccine processing and in the final formulation. The in vitro relative potency (IVRP) assay is considered a suitable replacement for the classical mouse potency assay and is an appropriate method for product lot-releasing and stability testing. ^33-35 Because most of the clinically relevant epitopes are type specific, an IVRP assay needs to be developed for each antigen. Most of the monoclonal antibodies (mAbs) used in IVRP assays are type specific and have neutralizing activity based on the pseudovirion-based neutralization assay. ³⁶ The pair choices (normally chosen from a panel of mAbs) for each HPV vaccine type are listed in the Table 3. For a given HPV type, one mAb is used to capture the VLP on the ELISA plate, and another mAb is used for detection in a sandwich format (Fig. 3A) to yield quantitative information on the antigenicity of the VLPs by probing 2 different epitopes in the same assay.

Table 3. The type-specific and neutralizing monoclonal antibodies used for product potency analysis and for serological assay.

	mAb used in IVRP
Types	Capture	Detection	mAb used in cLIA	Notes
HPV 6	H6.M48	H6.B10.5	H6.M48
HPV 11	K11.B2	H11.B2	K11.B2	HPV11 was chosen as the calibrator in HPV-9 cLIA.
HPV 16	H16.J4	H16.V5	H16.V5
HPV 18	H18.J4	H18.R5	H18.J4
HPV 31	H31.5F12	H31.5D10	H31.5D10 *	H31.5D10 appeared cross reactivity with HPV58 VLP in cLIA.
HPV 33	H33.5D4	H33.6G9	H33.6G9
HPV 45	H45.6G6	H45.10B4	H45.10B4*	H45.6G6 was shown to be cross-reactive with HPV18 VLP.
HPV 52	H52.8D11	H52.9F7	H52.9F7*
HPV 58	H58.2C3	H58.6E11	H58.6E11*

Abbreviations: mAb, monoclonal antibody; IVRP, in vitro relative potency; cLIA, competitive Luminex immunoassay; EC₅₀, half maximal effective concentration.

1. The type- specific mAbs used in cLIA were all matched with detection mAbs in the IVRP assay except H6.M48, K11.B2, H18.J5, which is capture antibody of HPV6, 11, 18 in the IVRP assay, respectively. In cLIA, H31.5D10 *, H45.10B4*, H52.9F7*, H58.6E11* were also named with suffices as H31.5D10.E4, H45.10B4.H4, H52.9F7.E1, H58.6E11.F4.

2. HPV11 was chosen as the calibrator for the additional HPV types as it had been shown to have a positive correlation between the level of HPV11 L1 VLP-specific IgG in animals immunized with HPV 11 virions and neutralization of HPV 11 in the athymic mouse xenograft model.

3. HPV31.5D10 showed cross reactivity with HPV58 VLP in the assay of the specificity evaluation of the mAbs used in cLIA.

4. H45.6G6 was shown to be cross-reactive with HPV18 VLP in the assay of quantifying the binding affinity of the mAbs to the specific HPV VLPs.

5. The information of the capture and detection mAbs of HPV6, 11, 16 and 18 used in IVRP collected from Christensen ND, et al. ^37–39 The information of mAbs of HPV31, 33, 45, 52 and 58 used in IVRP was obtained from Martha J. Brown, et al. ⁴⁰ The information of the type-specific mAbs used in the HPV-9 cLIA was obtained from Christine Roberts, et al; ⁴¹

Figure 3. Schematic illustrations of IVRP for vaccine antigenicity on product quality and for serological assay for type-specific and epitope-focused determination of antibody titers elicited by vaccination. (A) IVRP for antigenicity. The IVRP assay is used for monitoring vaccine product quality during lot-release and stability testing. One monoclonal antibody is used to capture the type-specific VLP on the microplate, while the other monoclonal antibody is used for detection in a sandwich format. The final readout is performed with a horseradish peroxidase (HRP) or alkaline phosphatase (AP)-labeled secondary antibody that is specific to the subclass of the detection mAb. (B) Serological assay. The competitive Luminex immunoassay evaluates the level of functional antibody titers in vaccinees. The immunoassay quantitatively measures the ability of the type-specific antibody in serum to compete with a phycoerythrin (PE) labeled, HPV type-specific mAb for a given type (Table 3) for binding to the same epitope. The Luminex microspheres were coupled with a given VLP type via covalent bonds. Owing to the competition with the labeled detection Ab, the fluorescent signals binding to the Luminex beads decrease if there are L1-specific neutralizing antibodies in serum samples. Abbreviations: mAb, monoclonal antibody; VLP, virus like particle; PE, phycoerythrin.

To support the clinical trials on the 9vHPV vaccine and evaluate the vaccine-induced immune responses in vaccinees, a 9-plexed HPV competitive Luminex immunoassay (HPV-9 cLIA) was developed. ⁴¹ The assay is based on the previous HPV-4 cLIA, which was used to measure the level of antibody response after administration of the 4vHPV vaccine. ^42,43 The HPV-9 cLIA uses VLPs that have been coupled to a set of 9 different florescent Luminex microspheres (or “barcoded” beads with a unique address for each bead set). Each type-specific antibody that binds to a specific Luminex microsphere is identified by distinct fluorescent dye spectral properties on the Luminex. The type-specific, conformational antibodies used in HPV-9 cLIA (of the 2 mAbs used in the IVRP assay for the same type) are shown in Table 3. ^41,44 To correlate the assay on vaccine antigenicity and the immune response elicited by vaccine administration, each of the HPV type-specific mAbs used in cLIA is one of the corresponding mAbs used in the IVRP assay for a given type. Because a detection Ab is more sensitively reflected in a sandwich assay than a capture Ab, most of the type-specific antibodies used in cLIA are matched to the detection antibodies in the IVRP assay except H6.M48, K11.B2 and H18.J5 (Table 3).

The antibody levels against vaccine targeting HPV types were measured in a competitive format on the Luminex platform (Fig. 3B). The type-specific monoclonal antibody that labeled phycoerythrin competed with specific antibodies in individual serum samples for binding to conformational epitopes on the viral capsid protein. ⁴² The labeled fluorescent signals from the HPV type-specific monoclonal antibody that labeled phycoerythrin were inversely proportional to the antibody titers in vaccinees.

For the antigenicity measurement of the vaccine product, these conformational, type-specific mAbs were used to develop sandwich-format immunoassays for analyzing VLP antigens in aqueous solution or formulated antigens after dissolution treatment. The pairs of monoclonal antibodies of 9 different HPV types (listed in Table 3) have been used in the assays for supporting vaccine product lot-releasing and stability testing. ^40,42 The bioanalytics tools described here have played critical roles in the licensure of 4vHPV and 9vHPV vaccines as well as the post licensure life cycle management.

Safety profile of the 9vHPV vaccine

Adding more adjuvant and antigen components to an existing vaccine can potentially impact its safety. In one preclinical toxicity study of the 9vHPV vaccine, 0.5 mL of vaccine formulation containing between 1.0- and −1.5-fold of each of 9vHPV antigen type was administered to female rats before mating and during gestation. In another toxicity study, rats were injected with a single human dose (0.5 mL) of the 9vHPV vaccine before mating and then during gestation and lactation. Both animal studies showed no evidence of fetus harm from the injection of the 9vHPV vaccine. ⁴⁵

Clinically, the overall safety profile of the 9vHPV vaccine was assessed through 7 Phase III clinical trials (Study V503–001/002/003/005/006/007/009) ^20,46-51 that were conducted in adolescents and younger men and women aged 9–26 y (Table 4). Overall, more than 15,000 subjects received at least one dose of the 9vHPV vaccine, and over 7,000 control subjects received the 4vHPV vaccine. The vaccination schedule was administered as a 3-dose regimen at Day 1, Month 2 and Month 6. Each subject in the studies obtained a report card to record non-serious or serious adverse events (AE or SAEs) and other medical conditions.

Table 4. The basic information of the 7 Phase III clinical trials of the 9vHPV vaccine.

Study	Subjects	Enrollment	Key objective	Comparator	Primary endpoints	Secondary endpoints	References
V503–001	Women aged 16–26 years	N = 14840	Immunogenicity and safety of 9vHPV vs 4vHPV	Gardasil	1. CIN2+, invasive cervical cancer	Persistent infection	20
					2. Adenocarcinoma in situ	GMTs and seroconversion
					3. VIN2+, vulvar cancer
					4. VaIN2+, vaginal cancer
V503–002	Boys and girls aged 9–15 years; women aged 16–26 years	N = 3074	Immuno bridging for adults to adolescents	Women	—	GMTs and seroconversion	46
V503–003	Women and men aged 16–26 years	N = 2520	Immuno bridging for women to men	Women	—	GMTs and seroconversion	47
V503–005	Girls and boys aged 11–15 years	N = 1241	Concomitant use with Menactra™ /Adacel™	Menactra™ /Adacel™	—	GMTs and seroconversion	48
V503–006	Girls aged 12–15 years; women aged 16–26 years	N = 924	Evaluation of recipients previously vaccinated 4vHPV vaccine	Placebo	—	GMTs and seroconversion	49
V503–007	Girls and boys aged 11–15 years	N = 1054	Concomitant use with Repevax™	Repevax™	—	GMTs and seroconversion	50
V503–009	Girls aged 9–15 years	N = 600	Immuno bridging for 9vHPV to 4vHPV	Gardasil	—	GMTs and seroconversion	51

CIN2+, high-grade cervical intraepithelial neoplasia (CIN2/3); VIN2+, high-grade vulvar intraepithelial neoplasia (VIN2/3); VaIN2+, high-grade vaginal intraepithelial neoplasia; Persistent infection, infection lasting ≥ 6 months; GMTs, geometric mean titers

The 9vHPV vaccine was generally well tolerated in adolescents (aged 9–15 years) and younger women (aged 16–26 years). ⁵² The AEs were largely injection-site AEs, such as local pain, swelling and erythema, but most of them were mild to moderate in intensity. Headache and pyrexia were the most frequent systemic AEs after vaccination with the 9vHPV vaccine. Rarely did subjects not complete the studies due to AEs and vaccine-related SAEs. Compared to the 4vHPV vaccine group, a slightly higher incidence of injection-site AEs was noted for the 9vHPV vaccine (90.7%) than for the 4vHPV vaccine (84.9%). ⁵³

The most common SAEs in the 7 studies were elective or spontaneous abortions and appendicitis. ⁵² Among the subjects who received the 9vHPV vaccine, only 7 subjects reported vaccine-related SAEs. Seven subjects died during the studies. None of the deaths were considered vaccine-related. Therefore, the 9vHPV vaccine is generally well-tolerated in recipients who are 9–26 y of age, and the overall safety profile is comparable to the 4vHPV vaccine. Additionally, co-administration with other vaccines also slightly increases the incidence of local adverse reactions. ⁵⁴ Due to a higher content of proteins and adjuvants in the 9vHPV vaccine, these results are consistent with the expected results. The favorable safety profile of the 9vHPV vaccine will also support widespread vaccination programs.

Efficacy and immunogenicity of the 9vHPV vaccine

With the availability of prophylactic HPV vaccines (4vHPV and 2vHPV vaccines) in many countries and regions, it is unacceptable to evaluate the efficacy and immunogenicity of the 9vHPV vaccine compared with placebo. ⁵⁵ Therefore, the strategy of evaluation was implemented as described in a subsequent section.

Efficacy and immunogenicity against the original 4 types

Immunogenicity against the original 4 types (HPV6, 11, 16 and 18) was assessed in 3 clinical studies of the non-inferior immunogenicity of the 9vHPV compared with the 4vHPV vaccine in girls and boys aged 9–15 years, ^56-58 young women aged 16–26 years, ⁵⁹ and men aged 16–26 y. ^60,61 No significant difference in the geometric mean titers (GMTs) of anti-HPV6, 11, 16 and 18 has been demonstrated in 3 clinical studies between the 9vHPV and clinically proven 4vHPV vaccine groups. The seroconversion rates of 4 HPV types at one month after the administration of the 3^rd dose in the 9vHPV vaccine were comparable to those for the 4vHPV vaccine recipients.

Efficacy and immunogenicity against 5 additional types

A total of 14,204 women (9vHPV vaccine group, n = 7,099; 4vHPV vaccine group, n = 7,105) were followed up in a randomized, double-blind clinical trial. ⁶² The per-protocol efficacy (PPE) subjects included women who were seronegative on Day 1 and negative on polymerase chain reaction assays for the relevant HPV types from Day 1 to Month 7 during the study. The efficacy of the 9vHPV vaccine against a mixed end point of HPV 31, 33, 45, 52 and 58-related high-grade diseases in the PPE was 96.7% (1 case in the 9vHPV vaccine group versus 30 cases in the 4vHPV vaccine group). The efficacy against diseases, of any grade, related to the 5 new types was 97.1% (3 cases vs. 103 cases). The efficacy against 6-month persistent infection related to the 5 new types was 96.0% (35 cases vs. 810 cases). ⁶² Therefore, the 9vHPV vaccine is highly efficacious in preventing HPV31, 33, 45, 52 and 58-related persistent infection and diseases.

Immunobridging for different populations

In comparison to young girls and women aged 16–26 years, the immune response to the 9vHPV vaccine was assessed in adolescents aged 9–15 y and men aged 16–26 y. The seroconversion rates at Month 1 after the last dose were nearly 100% in all age groups. The GMTs to the 5 new types included in the 9vHPV vaccine is comparable in both sexes, while they are slightly higher in the younger age groups. ⁵⁶ Based on the immune-bridging evaluation model, the efficacy of the 9vHPV vaccine against the new vaccine types is inferred from women aged 16–26 years, men aged 16–26 y and the younger age groups.

Overall, the 9vHPV vaccine induced a robust immune response against all related HPV types with the seroconversion rates close to 100% for all types. In addition, the antibody levels, reflected by GMTs determined by the competitive immunoassay with neutralizing mAbs as reporting molecules, were also higher in the younger groups (9–15 y of age) than in the older groups (16–26 y of age).

Immunogenicity of the 9vHPV vaccine with 2-dose vs. 3-dose regimen

Because the vaccine elicited a higher antibody titer in the younger population, they wondered whether a 2-dose regimen can elicit a sufficient immune response in the younger population to confer comparable immunity. They evaluated the immunogenicity of the 9vHPV vaccine among younger adolescents aged 9–14 y after receiving the 2-dose regimen compared with girls and young women aged 16–26 y receiving the 3-dose regimen. The clinical trial conducted at 52 sites in 15 countries was initiated in December 2013. ⁶³

Immunogenicity was evaluated in a younger group of girls and boys aged 9–14 y who received the 2-dose schedule (Month 0 and 6 or 12) and an older group of women aged 16–26 y who received the 3-dose schedule (Month 0, 2 and 6) (Table 5). ⁶⁴ Four weeks after the last dose of the 2-dose regimen, the seroconversion rates for all covered HPV types were between 99.3% and 100% in all groups. The GMTs for each type were higher in the younger group that received the 2-dose regimen than in women aged 16–26 y who received the 3-dose regimen. In the same bridging study, exploratory analysis in the group of girls aged 9–14 y indicated that the GMTs of the 2-dose schedule were lower than the 3-dose schedule at one month after the last dose for some vaccine types (e.g., HPV types 18, 31, 45 and 52 after Month 6 and HPV type 45 after Month 12). However, the clinical relevance of these findings remains unclear because the minimal antibody level required for protection is uncertain for each HPV type.

Table 5. Immunogenicity of the 9vHPV vaccine using 2-dose regimen in girls and boys vs. 3-dose in girls and young women.

	2 doses (0, 6 month) in girls aged 9–14 ( n = 301 )		2 doses (0, 6 month) in boys aged 9–14 ( n = 301 )		2 doses (0, 12 month) in girls and boys aged 9–14 ( n = 300 )		3 doses (0, 2, 6 month) in girls and women aged 16–26 ( n = 314 )
Types	Seroconversion, %	GMTs	Seroconversion, %	GMTs	Seroconversion, %	GMTs	Seroconversion, %	GMTs
6	99.6	1657.9	100	1557.4	100	2678.8	99.6	770.9
11	100	1388.9	100	1423.9	100	2941.8	99.6	580.5
16	100	8004.9	100	8474.8	100	14329.3	99.6	3154.0
18	100	1872.8	100	1860.9	100	2810.4	98.5	761.5
31	99.6	1436.3	100	1498.2	100	2117.5	99.6	572.1
33	99.6	1030.0	100	1040.0	100	2197.5	99.6	348.1
45	99.3	357.6	99.3	352.3	100	417.7	97.9	213.6
52	99.6	581.1	100	640.4	100	1123.4	99.6	364.2
58	100	1251.2	100	1325.7	100	2444.5	99.6	491.1

Abbreviations: cLIA, competitive Luminex immunoassay; GMTs, geometric mean titers

1. The primary end point was predefined as evaluation of the GMTs against 9 HPV types in one month after the last dose using HPV-9 cLIA. The secondary end point was predefined as the percentage of subjects with seroconversion to 9 HPV types in one month after the last dose of the given regimen.

2. Seroconversion was defined as the serostatus from seronegative to seropositive in one month after the last dose. The positive serum level of anti-HPV6, 11, 16, 18, 31, 33, 45, 52, 58 was defined as ≥ 30, 16, 20, 24, 10, 8, 8, 8, 8 mMU/mL, respectively.

3. The unit of GMTs was mMU/mL (mili-Merck units per milliliter).

4. The data was obtained from Ole-Eric Iversen, et al⁶⁴ (Study V503–010).

In conclusion, among adolescents aged 9–14 y who received the 2-dose regimen of the 9vHPV vaccine on a schedule of Month 0 and Month 6 or 12, their immunogenicity was non-inferior to a 3-dose regimen in young women aged 16–26 y at 4 weeks after the last dose. However, further study is still needed to evaluate the persistent antibody immune responses and effectiveness in clinical trials.

Use of the 9vHPV vaccine in subjects previously vaccinated with the 4vHPV vaccine

The 4vHPV vaccine, a highly successful prophylactic vaccine, ^65-67 was licensed more than 8 y ahead of the 9vHPV vaccine. Therefore, the initial one or 2 doses (or even the full course of the 3-dose immunization) of the 4vHPV vaccine may have been administered before the availability of the 9vHPV vaccine. A Phase III, double-blind clinical study ⁴⁹ was initiated to assess whether the 3 doses of the 9vHPV vaccine is well tolerated in girls and women aged 12–26 y who had previously been vaccinated with the 4vHPV vaccine. The results showed that 98.3–100% of the subjects previously vaccinated with the 4vHPV vaccine seroconverted to the new types covered in the 9vHPV vaccine. Likely due to a boosting effect, the GMTs of the original 4 types after vaccination with the 9vHPV vaccine were higher in women aged 12–26 y who were vaccinated with the 4vHPV vaccine than in those who received the 9vHPV vaccine alone. ⁶⁸ Interestingly, the GMTs to the 5 new types were lower in those who were previously vaccinated with the 4vHPV vaccine. However, it is difficult to evaluate the significance of the findings because there is no categorical threshold for protection in terms of the minimum antibody titers.

Concomitant administration with other vaccines

To improve the uptake and reduce the cost of vaccination, concomitant administration with routine vaccines has been considered as an alternative in vaccination for pre-adolescents and adolescents. Previous data have showed that Gardasil® co-administered with Menactra™ (meningococcal vaccine) or Adacel™ (tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine) was well tolerated and did not interfere with the immune response to the respective vaccines. ^69-72 Both clinical studies (Studies V503–005 and V503–007) evaluated the tolerability and immunogenicity of administration of the first 9vHPV vaccine dose at the same time as Menactra™ and Adacel™ vs. administration of the 9vHPV vaccine one month before the administration of Menactra™ and Adacel™ in adolescents aged 9–14 y. ^73-76 The result demonstrated that both safety and the immune response to all components of the administered vaccines were non-inferior compared with non-concomitant administration (Table 1S).

Co-administration would reduce the number of visits required to deliver each dose of the vaccine. Additionally, it is estimated that co-administration of the 9vHPV vaccine with current routine vaccines could increase its uptake.

Recommendations for the 9vHPV vaccine

Since its licensure in the US in December 2014 (Canada in February 2014, the EU and Australia in June 2015), the 9vHPV vaccine has been used to prevent and decrease the incidence of HPV-related cancers and diseases in over 80 countries worldwide. However, the upper age range of recommendation differs among these countries. For instance, the vaccine is licensed in the US in women up to age 26 years, ⁷⁷ in Australia and Canada up to age 45 years, ⁷⁸ and in the EU without an upper age limit. In February 2015, the Advisory Committee on Immunization Practices (ACIP) included the 9vHPV vaccine in its recommendation for routine HPV vaccination of pre-adolescents aged 11 or 12 years, females aged 13–26 y and males aged 13–21 y who had not previously received 2vHPV or 4vHPV vaccination, including men who have sex with men, immunocompromised persons and those with HIV infection through the age of 26 y. ⁷⁷

In April 2016, the European Medicines Agency approved the 2-dose regimen of the 9vHPV vaccine for young adolescents at the age of 9–14 years, and recommended that the second dose should be followed within Months 5 to 13 after the first dose. ⁷⁹ In October 2016, the ACIP also changed the dosing schedule and recommended the HPV vaccine 2-dose regimen for individuals starting vaccination at the age of 9–14 y and recommending that the second dose be administered within Months 6 to 12 after the first dose. ⁸⁰

Cost-effectiveness of the 9vHPV vaccination

At present, the social-economic burden of HPV-related infection and diseases is substantial in both developing and developed countries. However, most developed countries are gradually alleviating this burden with the introduction of Pap smear screening and national HPV vaccination programs. ^81-84 Some pharmacoeconomic analysis models have been used to evaluate whether the 9vHPV vaccine is likely to be cost-effective compared with the current vaccination strategies by providing broader protection against HPV infection. ^85-91

Cost-effectiveness analyses in several countries (such as the US, Germany and Austria) demonstrated that vaccination programs with the 9vHPV vaccine are likely to be cost-effective compared with the current national vaccination programs using the 2vHPV or 4vHPV vaccine. In the cost-effectiveness analysis of HPV vaccination programs in the US, ^85-87 the results indicated that switching to the 9vHPV vaccine was cost-effective for any age groups (pre-adolescents, adolescents or young women) despite the higher cost of the new vaccine. The 9vHPV vaccination yielded the same public health benefit as covering an additional 11% of adolescents with the 2vHPV or 4vHPV vaccination and it had a cost saving of $2.7 billion in the model. In Germany, ⁸⁸ a dynamic transmission model was used to estimate the costs and quality adjusted life years (QALY) associated with vaccination strategies. Switching from the 2vHPV or 4vHPV vaccine to the 9vHPV vaccination program is highly cost-effective with an incremental cost-effectiveness ratio of €329 per QALY gained. Compared to the current strategy (4vHPV vaccination program), evaluation of the HPV vaccination program in Austria showed that vaccinating 60% of girls and 40% of boys at the age of 9-year old administered with one dose of the 9vHPV vaccine would substantially reduce the incidence of cervical cancer by additional 20%. ⁸⁹ The vaccination strategy performed with the 9vHPV vaccine in the study was estimated to have an incremental cost-effectiveness ratio of €16,441per QALY gained compared with the 4vHPV vaccination program by considering the cost-effectiveness willingness-to-pay threshold of €30,000 per QALY gained.

Compared to the 4vHPV vaccination program, the 9vHPV vaccination was shown to be cost-effective and should significantly enhance public health benefits. Additionally, inclusion of boys in the 9vHPV vaccination program would be an efficient, cost-effective strategy to further reduce HPV-related cancers and diseases worldwide. ^88,89

Beyond the 9vHPV vaccine

There are still some oncogenic high-risk HPV types that are not covered by the 9vHPV vaccine, and these are responsible for another 10% of cervical cancer worldwide (Fig. 1). ^92-94 Compared with the normal population, some of uncommon high-risk HPV types are seen more frequently in the immunocompromised patients and HIV patients. ⁹⁵ There is no doubt that cervical cancer and other HPV-related cancers screenings have to be continued in the general population, especially in high-risk groups. Unfortunately, these recommendations have not been well implemented in developing countries.

There are still significant populations with risks of HPV infection. In 2013, the Centers for Disease Control and Prevention in the US declared that only an average of 25.8% of teenagers aged 13–17 y completely received 3 doses of HPV vaccine, and 47.5% were vaccinated with at least one dose. ⁹⁶ Such a low uptake of vaccination may be due to the inconvenience caused by needing 3 separate visits to receive all of the doses. This phenomenon also indirectly reflects the need to reduce the dose regimen of the vaccination. ^97-101 Furthermore, the 9vHPV vaccine is more expensive, costing roughly $325 to $533 for 3 doses in the United States. ¹⁰² Further reducing the costs of vaccine production could enhance the vaccine accessibility.

Currently, no HPV vaccine has been licensed with therapeutic efficacy, although there are some promising clinical data from some early phase studies using vectored DNA vaccine candidates. ^103,104 One clinical study conducted with the 9vHPV and 4vHPV vaccines showed that, compared with 0.87% of the recipients who were naive before vaccination, more than 7.9% of modified intention-to-treat recipients (HPV positive during the study) vaccinated with 9vHPV or 4vHPV still had HPV-related diseases and carcinoma in situ. ¹⁰⁵ Therefore, the vaccine is only effective when administered before sexual debut or HPV infection. Considering the need for a broader coverage of the HPV vaccine, some researchers have been focused on developing a new generation HPV vaccine with a focus on the minor capsid protein, L2. ^106-109 Highly conserved peptides of the L2 protein was shown to induce cross-neutralizing and cross-protective antibody responses against multiple high-risk HPV types. ^110-112 It is promising that the HPV vaccine based on the L2 protein could be an effective new generation vaccine.

Conclusion

Over the last decade, the 4vHPV (and 2vHPV in some cases) vaccine has been successfully implemented across many countries for widespread vaccination among pre-adolescents and adolescent populations. This has likely prevented approximately 70% of cervical cancer cases that would occur in the future. The mechanism for protection is through the generation of type-specific neutralizing antibodies. By including an additional 5 oncogenic HPV types (31, 33, 45, 52 and 58) in the 2^nd generation vaccine, another ∼20% of cervical cancer could be prevented with the 9vHPV vaccine.

The safety and efficacy of the 9vHPV vaccine was shown to be comparable to that of 4vHPV vaccine across multiple clinical trials in different populations. For the simplified dosing regimen of the 9vHPV vaccine, 2-dose administered in pre-adolescents and adolescents instead of 3-dose in young women is not only being cost-saving but also can improve the vaccine uptake. Because of the additional coverage, the widespread 9vHPV vaccination would improve health outcomes and be used worldwide.

The 9vHPV vaccine will play a pivotal role in worldwide public health efforts to prevent HPV-related cancers and diseases in the coming decades as the 2vHPV and 4vHPV vaccines having been withdrawn from the major markets like in the US. Further expanding the type coverage of a future generation vaccine would further increase the effectiveness of this powerful and life-saving public health tool. However, a creative approach may have to be taken other than simply adding more types of the L1-based antigen as the total antigen content in the 9vHPV vaccine is already at 270 μg per dose. Accessibility and vaccine uptake should be further improved collectively by the cooperation among vaccine manufacturers, law makers, governments and public health organizations.

Abbreviations

ACIP	=	advisory committee on immunization practices
AEs	=	adverse events
CIN	=	cervical intraepithelial neoplasia
cLIA	=	competitive Luminex immunoassay
FDA	=	food and drug administration
GMTs	=	geometric mean titers
HPVs	=	human papillomaviruses
IVRP	=	in vitro relative potency
mAbs	=	monoclonal antibodies
NCT	=	national clinical trial
PE	=	phycoerythrin
QALY	=	quality adjusted life years
SAEs	=	serious adverse events
VIN	=	vulvar intraepithelial neoplasia
VLPs	=	virus like-particles
WHO	=	world health organization

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgment

We thank Mr. Xin Wang of Xiamen University for critical reading of the manuscript.

Funding

The writing was enabled with the supports from the National Natural Science Foundation of China (No.31670939 and No.81471934), Science and Technology Major Project of Fujian province (No.2015YZ0002) and the Scientific Research Foundation of State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics (Grant No. 2016ZY005).