Development and Validation of Two Optimized Multiplexed Serologic Assays for the 9-Valent Human Papillomavirus Vaccine Types.

Two multiplex immunoassays are routinely used to assess antibody responses in clinical trials of the 9-valent human papillomavirus (9vHPV) vaccine. The HPV6/11/16/18/31/33/45/52/58 competitive Luminex immunoassay (HPV-9 cLIA) and HPV6/11/16/18/31/33/45/52/58 total immunoglobulin G Luminex immunoassay are used for measurements of immunogenicity. Following their initial validation in 2010, both assays were redeveloped, and several parameters were optimized, including the coating concentration of virus-like particles, type of Luminex microspheres, serum sample and reference standard diluent, reference standard starting dilution and titration series, and vendor and concentration of the phycoerythrin-labeled antibodies. Validation studies evaluated the assay performance parameters, including the intra-assay precision (repeatability), intermediate precision, linearity, relative accuracy, and limits of quantitation. In addition, since maintaining a link to the original assays that were used in trials supporting vaccine licensure is critical, the assays were formally bridged to the previous assay versions by using individual patient sera from a 9vHPV vaccine clinical trial (n = 150 day 1 [prevaccination] samples; n = 100 month 7 [1 month post-last vaccine dose] and n = 100 month 36 [30 months post-last vaccine dose; antibody persistence] samples). The results of the validation studies indicate that both optimized assays are accurate, specific, and precise over their respective quantifiable ranges. There was a strong linear association between the new and previous versions of both assays. Assay serostatus cutoffs for the redeveloped assays were established based on the bridging studies and, for the HPV-9 cLIA, further refined, based on additional data from HPV vaccine clinical studies so as to align the seropositivity rates between assay versions. IMPORTANCE Assay modernization is a key aspect of vaccine life cycle management. Thus, new, reoptimized versions of two 9vHPV immunoassays have been developed and validated for use in ongoing and future HPV vaccine clinical trials. These assays are suitable for use in high-throughput testing for HPV antibodies in serum samples. Bridging to previous versions of the assays allows for the continuous monitoring of immune responses across assay versions, including in immunogenicity studies that involve new populations as well as long-term follow-up studies.

Immunogenicity and safety of quadrivalent and 9-valent human papillomavirus vaccines in Indian clinical trial participants.

The quadrivalent human papillomavirus (qHPV; HPV6/11/16/18) and 9-valent HPV (9vHPV; HPV6/11/16/18/31/33/45/52/58) vaccines have demonstrated efficacy, immunogenicity, and safety in international clinical trials. We report outcomes from three completed clinical trials in India: a single-arm study (V501-029 [NCT00380367]) in Indian girls (aged 9-15 years; N = 110) evaluating qHPV vaccine immunogenicity and safety; a subgroup analysis (n = 225) of Indian girls/boys (9-15 years) and women (16-26 years) from a global study (V503-002 [NCT00943722]) evaluating 9vHPV vaccine immunogenicity and safety; and a qHPV vaccine post-marketing safety surveillance study (V501-125) in Indian females (aged 9-45 years; N = 188) vaccinated during routine care. In V501-029 and V503-002, HPV vaccines were administered as 3 doses (Day 1, Month 2, Month 6). Serum HPV antibodies were evaluated by competitive Luminex immunoassays at Day 1 and Month 7 (both studies) and Months 12, 24, and 36 (V503-002 only). Adverse events (AEs) were collected by Vaccination Report Card. In V501-125, participants were actively surveilled for serious AEs (SAEs) within 30 days post-qHPV vaccination. In per-protocol analyses, qHPV and 9vHPV vaccines induced robust anti-HPV6/11/16/18 (V501-029) and HPV6/11/16/18/31/33/45/52/58 (V503-002) responses, respectively; ≥97% of participants seroconverted at Month 7 for each vaccine HPV type in both studies, and antibody responses persisted through 36 months in V503-002. The most common AEs were injection-site-associated. Most AEs were mild/moderate; no deaths, vaccine-related SAEs, or discontinuations due to AEs were reported. In V501-125, no SAE was reported. Overall, the qHPV and 9vHPV vaccines elicited robust antibody responses and were generally well tolerated in Indian participants.

Effectiveness, immunogenicity, and safety of the quadrivalent HPV vaccine in women and men aged 27-45 years.

Among women aged 27-45 years, the quadrivalent human papillomavirus (qHPV; HPV6/11/16/18) vaccine was generally well tolerated, efficacious, and immunogenic in the placebo-controlled FUTURE III study (NCT00090220; n = 3253). The qHPV vaccine was also generally well tolerated and highly immunogenic in men aged 27-45 years who participated in the single-cohort mid-adult male (MAM) study (NCT01432574; n = 150). Here, we report results of a long-term follow up (LTFU) extension of FUTURE III with up to 10 years follow-up. To understand the relevance of the mid-adult women LTFU study in the context of mid-adult men vaccination, we report results from post-hoc, cross-study immunogenicity analyses conducted to compare immunogenicity (geometric mean titers; GMTs) at 1-month post-qHPV vaccine dose 3 in women and men aged 27-45 years versus women and men aged 16-26 years from prior efficacy studies. The qHPV vaccine demonstrated durable protection against the combined endpoint of HPV6/11/16/18-related high-grade cervical dysplasia and genital warts up to 10 years (median 8.9) post-dose 3 and sustained HPV6/11/16/18 antibody responses through approximately 10 years in women aged 27-45 years. Efficacy of qHPV vaccine in men aged 27-45 years was inferred based on the cross-study analysis of qHPV vaccine immunogenicity demonstrating non-inferior HPV6/11/16/18 antibody responses in men aged 27-45 years versus 16-26 years. In conclusion, durable effectiveness of the qHPV vaccine was demonstrated in women 27-45 years of age, and vaccine efficacy was inferred in men 27-45 years of age based on the serological results.

Immunogenicity and safety of the 9-valent human papillomavirus vaccine in Chinese females 9-45 years of age: A phase 3 open-label study.

BACKGROUND: The 9-valent human papillomavirus (9vHPV; HPV6/11/16/18/31/33/45/52/58) vaccine was approved for use in Chinese women aged 16-26 years in 2018. This phase 3, open-label study (NCT03903562) compared 9vHPV vaccine immunogenicity and safety in Chinese females aged 9-19 years and 27-45 years with Chinese females aged 20-26 years; we report results from day 1 through 1 month post-Dose 3. The study will continue through 54 months post-Dose 3 to assess antibody persistence in Chinese girls aged 9-19 years. METHODS: Participants aged 9-45 years received three doses of the 9vHPV vaccine. Geometric mean titers (GMTs) and seroconversion percentages for anti-HPV6/11/16/18/31/33/45/52/58 antibodies were determined by competitive Luminex immunoassay in serum samples obtained at day 1 and 1 month post-Dose 3. Adverse events (AEs) within 30 days post-vaccination and serious AEs (SAEs) occurring at any time were recorded. RESULTS: In total, 1990 participants (690 aged 9-19 years; 650 aged 20-26 years; 650 aged 27-45 years) were enrolled. At 1 month post-Dose 3, >99% of participants in the per-protocol immunogenicity population seroconverted to each vaccine HPV type. Anti-HPV6/11/16/18/31/33/45/52/58 antibody GMTs in the 9-19-year age group were non-inferior to those in participants aged 20-26 years. Anti-HPV6/11/16/18/31/33/45/52/58 seroconversion percentages in the 27-45-year age group were non-inferior to those in participants aged 20-26 years. Injection-site and systemic AEs were reported by 43.3% and 50.9%, 50.5% and 57.1%, and 43.8% and 43.4% of participants aged 9-19, 20-26, and 27-45 years, respectively. There were no vaccine-related SAEs, discontinuations due to AEs, and deaths. CONCLUSION: Antibody responses induced by 9vHPV vaccination in Chinese females aged 9-19 years and 27-45 years were non-inferior to those in Chinese females aged 20-26 years. The vaccine was generally well tolerated. CLINICALTRIALS: gov Identifier: NCT03903562.

Efficacy, immunogenicity, and safety of a quadrivalent HPV vaccine in men: results of an open-label, long-term extension of a randomised, placebo-controlled, phase 3 trial.

BACKGROUND: The quadrivalent human papillomavirus (HPV) vaccine was shown to prevent infections and lesions related to HPV6, 11, 16, and 18 in a randomised, placebo-controlled study in men aged 16-26 years. We assessed the incidences of external genital warts related to HPV6 or 11, and external genital lesions and anal dysplasia related to HPV6, 11, 16, or 18, over 10 years of follow-up. METHODS: The 3-year base study was an international, multicentre, double-blind, randomised, placebo-controlled trial done at 71 sites in 18 countries. Eligible participants were heterosexual men (aged 16-23 years) or men who have sex with men (MSM; aged 16-26 years). Men who had clinically detectable anogenital warts or genital lesions at screening that were suggestive of infection with non-HPV sexually transmitted diseases, or who had a history of such findings, were excluded. Eligible participants were randomly assigned (1:1) to receive three doses of either quadrivalent HPV vaccine or placebo on day 1, month 2, and month 6, administered as a 0·5-mL injection into the deltoid muscle. The 7-year, open-label, long-term follow-up extension study was done at 46 centres in 16 countries. Participants who received one or more doses of the quadrivalent HPV vaccine in the base study were eligible for enrolment into the long-term follow-up study (early vaccination group). Placebo recipients were offered the three-dose quadrivalent HPV vaccine at the end of the base study; those who received one or more quadrivalent HPV vaccine doses were eligible for enrolment into the long-term follow-up study (catch-up vaccination group). The primary efficacy endpoints were the incidence of external genital warts related to HPV6 or 11 and the incidence of external genital lesions related to HPV6, 11, 16, or 18 in all participants and the incidence of anal intraepithelial neoplasia (including anal warts and flat lesions) or anal cancer related to HPV6, 11, 16, or 18 in MSM only. The primary efficacy analysis was done in the per-protocol population for the early vaccination group, which included participants who received all three vaccine doses, were seronegative at day 1 and PCR-negative from day 1 through month 7 of the base study for the HPV type being analysed, had no protocol violations that could affect evaluation of vaccine efficacy, and had attended at least one visit during the long-term follow-up study. For the catch-up vaccination group, efficacy was assessed in the modified intention-to-treat population, which included participants who had received at least one vaccine dose, were seronegative and PCR-negative for HPV types analysed from day 1 of the base study to the final follow-up visit before receiving the quadrivalent HPV vaccine, and had at least one long-term follow-up visit. Safety was assessed in all randomised participants who received at least one vaccine dose. This study is registered with ClinicalTrials.gov, NCT00090285. FINDINGS: Between Aug 10, 2010, and April 3, 2017, 1803 participants were enrolled in the long-term follow-up study, of whom 936 (827 heterosexual men and 109 MSM) were included in the early vaccination group and 867 (739 heterosexual men and 128 MSM) were included in the catch-up vaccination group. Participants in the early vaccination group were followed up for a median of 9·5 years (range 0·1-11·5) after receiving the third dose of the quadrivalent HPV vaccine, and participants in the catch-up vaccination group were followed up for a median of 4·7 years (0·0-6·6) after receiving the third dose. In early vaccine group participants during long-term follow-up compared with the placebo group in the base study, the incidence per 10 000 person-years of external genital warts related to HPV6 or 11 was 0·0 (95% CI 0·0-8·7) versus 137·3 (83·9-212·1), of external genital lesions related to HPV6, 11, 16, or 18 was 0·0 (0·0-7·7) versus 140·4 (89·0-210·7), and of anal intraepithelial neoplasia or anal cancer related to HPV6, 11, 16, or 18 in MSM only was 20·5 (0·5-114·4) versus 906·2 (553·5-1399·5). Compared with during the base study (ie, before quadrivalent HPV vaccine administration), during the long-term follow-up period, participants in the catch-up vaccination group had no new reported cases of external genital warts related to HPV6 or 11 (149·6 cases per 10 000 person-years [95% CI 101·6-212·3] vs 0 cases per 10 000 person-years [0·0-13·5]) or external genital lesions related to HPV6, 11, 16, or 18 (155·1 cases per 10 000 person-years [108·0-215·7] vs 0 cases per 10 000 person-years [0·0-10·2]), and a lower incidence of anal intraepithelial neoplasia or anal cancer related to HPV6, 11, 16, or 18 (886·0 cases per 10 000 person-years [583·9-1289·1] vs 101·3 cases per 10 000 person-years [32·9-236·3]). No vaccine-related serious adverse events were reported. INTERPRETATION: The quadrivalent HPV vaccine provides durable protection against anogenital disease related to HPV6, 11, 16, and 18. The results support quadrivalent HPV vaccination in men, including catch-up vaccination. FUNDING: Merck Sharp & Dohme.

Immunogenicity and safety of a nine-valent human papillomavirus vaccine in Vietnamese males and females (9 to 26 years of age): an open-label, phase 3 trial.

This open-label, single-center, Phase 3 study (NCT03546842) assessed the immunogenicity and safety of the nine-valent human papillomavirus (9vHPV; HPV6/11/16/18/31/33/45/52/58) vaccine in Vietnamese males and females, with the aim to support 9vHPV vaccine licensure in Vietnam. Participants aged 9-26 years received three 9vHPV vaccine doses (Day 1, Month 2, Month 6). Serum samples were obtained on Day 1 (pre-vaccination) and at Month 7 (one month post-Dose 3) for the measurement of anti-HPV antibodies. Geometric mean titers (GMTs) and seroconversion percentages were obtained using the HPV-9 competitive Luminex immunoassay. Injection-site adverse events (AEs), systemic AEs, serious AEs (SAEs), and study discontinuations due to AEs were recorded. Of 201 participants enrolled, 200 (99.5%) received ≥1 vaccine dose. All participants who received the three-dose regimen (198/200, 98.5%) seroconverted for all 9vHPV vaccine types by Month 7. Robust anti-HPV GMT responses were also observed. Half of participants (50.5%) reported ≥1 AE; the majority were injection-site-related (45.0%) and mild (43.0%). There were no deaths, vaccine-related SAEs, or discontinuations due to AEs. Administration of three 9vHPV vaccine doses was highly immunogenic and resulted in acceptable seropositivity percentages for all vaccine HPV types. The 9vHPV vaccine was generally well tolerated among this study population.Region of origin: VietnamTrial registration: clinicaltrials.gov Identifier NCT03546842.

Long-term effectiveness of the nine-valent human papillomavirus vaccine in Scandinavian women: interim analysis after 8 years of follow-up.

A long-term follow-up (LTFU) of the nine-valent human papillomavirus (9vHPV) vaccine efficacy study in young women aged 16-26 years was initiated to evaluate if vaccine effectiveness for up to 14 years post-vaccination will remain above 90%. Vaccine effectiveness is measured as percent reduction in the incidence of HPV16/18/31/33/45/52/58-related high-grade cervical dysplasia in the LTFU cohort relative to expected incidence in a similar unvaccinated cohort. We report an interim analysis 8 years post-vaccination. Overall, 2029 participants from Denmark, Norway, and Sweden who received the 9vHPV vaccine during the clinical efficacy study continued into the LTFU study. National health registries were used to identify screening attendance and cervical pre-cancer/cancer diagnoses. Tissue samples were retrieved for HPV testing by PCR and pathology diagnosis adjudication. A control chart method was used to detect signals indicative of vaccine effectiveness waning below 90%. No new cases of HPV16/18/31/33/45/52/58-related high-grade cervical dysplasia were observed during the LTFU study period over 4084.2 person-years' follow-up (per-protocol effectiveness population; n = 1448). Thus, there were no signals indicative of vaccine effectiveness waning below 90%. These observations show that the 9vHPV vaccine provides continued statistically significant protection through at least 6 years, with indications of continued effectiveness through 8 years. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00543543, NCT02653118.

Immunogenicity and safety of the quadrivalent human papillomavirus vaccine in Chinese females aged 9 to 26 years: A phase 3, open-label, immunobridging study.

BACKGROUND: The quadrivalent human papillomavirus (qHPV; HPV6/11/16/18) vaccine was approved for use in Chinese women aged 20-45 years in 2017. This Phase 3, open-label study (NCT03493542) aimed to assess immunogenicity and safety of the qHPV vaccine in Chinese girls aged 9-19 years versus Chinese young women aged 20-26 years; we report results from Day 1 through Month 7. The study will continue through Month 60 to assess antibody persistence in Chinese girls aged 9-19 years. METHODS: Participants aged 9-26 years received three doses of the qHPV vaccine (Day 1, Month 2, Month 6). Geometric mean titers (GMTs) and seroconversion percentages for anti-HPV6/11/16/18 antibodies were determined by competitive Luminex immunoassay (cLIA) in serum samples obtained on Day 1 and at Month 7. Injection-site adverse events (AEs) and systemic AEs within 30 days post-vaccination, and serious AEs (SAEs) occurring at any time during the study, were recorded. RESULTS: In total, 766 participants (383 aged 9-19 years; 383 aged 20-26 years) were enrolled and received ≥1 vaccine dose. All participants in the per-protocol immunogenicity population of both age groups seroconverted to each of the vaccine HPV types at Month 7. Anti-HPV6/11/16/18 antibody GMTs at Month 7 in participants aged 9-19 years were non-inferior to those in participants aged 20-26 years. Injection-site AEs and systemic AEs were reported by 36.6% and 49.3% of 9-19-year-olds, and 40.7% and 54.8% of 20-26-year-olds, respectively. There were no vaccine-related SAEs. No participants discontinued the vaccine due to an AE and no deaths were reported. CONCLUSION: Antibody responses induced by the 3-dose qHPV vaccination regimen in Chinese girls aged 9-19 years were non-inferior to those in Chinese young women aged 20-26 years. The vaccine was generally well tolerated in the study population. ClinicalTrials.gov Identifier: NCT03493542.

Final analysis of a 14-year long-term follow-up study of the effectiveness and immunogenicity of the quadrivalent human papillomavirus vaccine in women from four nordic countries.

BACKGROUND: The quadrivalent human papillomavirus (qHPV) vaccine prevented vaccine HPV type-related infection and disease in young women in the 4-year FUTURE II efficacy study (NCT00092534). We report long-term effectiveness and immunogenicity at the end of 14 years of follow-up after enrollment in FUTURE II. METHODS: Young women (16-23 years of age) from Denmark, Iceland, Norway, and Sweden who received three qHPV vaccine doses during the randomized, double-blind, placebo-controlled FUTURE II base study were followed for effectiveness for an additional ≥10 years through national registries. Tissue samples including but not limited to those collected during organized cervical cancer screening programs were obtained from regional biobanks to be adjudicated for histopathology diagnosis and tested for HPV DNA. The observed incidence of HPV16/18-related high-grade cervical dysplasia (primary outcome) was compared with recent historical background incidence rates in an unvaccinated population. Serum was collected at years 9 and 14 to assess antibody responses. FINDINGS: No cases of HPV16/18-related high-grade cervical dysplasia were observed in the per-protocol effectiveness population (N = 2121; 24,099·0 person-years of follow-up) during the entire study. Vaccine effectiveness of 100% (95% CI 94·7-100) was demonstrated for ≥12 years, with a trend toward continued protection through 14 years post-vaccination. Seropositivity rates at study conclusion were >90% (HPV6/11/16) and 52% (HPV18) using competitive Luminex immunoassay, and >90% (all four HPV types) using the more sensitive IgG Luminex immunoassay. INTERPRETATION: Vaccination of young women with qHPV vaccine offers durable protection against HPV16/18-related high-grade cervical dysplasia for ≥12 years, with a trend toward continued protection through 14 years post-vaccination, and induces sustained HPV6/11/16/18 antibody responses for up to 14 years post-vaccination. There was no evidence of waning immunity, suggesting no need for a booster dose during that period. FUNDING: Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.

Design of a long-term follow-up effectiveness, immunogenicity and safety study of women who received the 9-valent human papillomavirus vaccine.

The 9-valent human papillomavirus (HPV) (9vHPV) vaccine targets four HPV types (6/11/16/18) also covered by the quadrivalent HPV (qHPV) vaccine and five additional types (31/33/45/52/58). Vaccine efficacy to prevent HPV infection and disease was established in a Phase III clinical study in women 16-26years of age. A long-term follow-up (LTFU) study has been initiated as an extension of the Phase III clinical study to assess effectiveness of the 9vHPV vaccine up to at least 14years after the start of vaccination. It includes participants from Denmark, Norway and Sweden and uses national health registries from these countries to assess incidence of cervical pre-cancers and cancers due to the 7 oncogenic types in the vaccine (HPV 16/18/31/33/45/52/58). Incidences will be compared to the estimated incidence rate in an unvaccinated cohort of similar age and risk level. This LTFU study uses a unique design: it is an extension of a Phase III clinical study and also has elements of an epidemiological study (i.e., endpoints based on standard clinical practice; surveillance using searches from health registries); it uses a control chart method to determine whether vaccine effectiveness may be waning. Control chart methods which were developed in industrial and manufacturing settings for process and production monitoring, can be used to monitor disease incidence in real-time and promptly detect a decrease in vaccine effectiveness. Experience from this innovative study design may be applicable to other medicinal products when long-term outcomes need to be assessed, there is no control group, or outcomes are rare.

The puromycin-sensitive aminopeptidase PAM-1 is required for meiotic exit and anteroposterior polarity in the one-cell Caenorhabditis elegans embryo.

In the nematode Caenorhabditis elegans, sperm entry into the oocyte triggers the completion of meiosis and the establishment of the embryonic anteroposterior (AP) axis. How the early embryo makes the transition from a meiotic to a mitotic zygote and coordinates cell cycle changes with axis formation remains unclear. We have discovered roles for the C. elegans puromycin-sensitive aminopeptidase PAM-1 in both cell cycle progression and AP axis formation, further implicating proteolytic regulation in these processes. pam-1 mutant embryos exhibit a delay in exit from meiosis: thus, this peptidase is required for progression to mitotic interphase. In addition, the centrosomes associated with the sperm pronucleus fail to closely associate with the posterior cortex in pam-1 mutants, and the AP axis is not specified. The meiotic exit and polarity defects are separable, as inactivation of the B-type cyclin CYB-3 in pam-1 mutants rescues the meiotic exit delay but not the polarity defects. Thus PAM-1 may regulate CYB-3 during meiotic exit but presumably targets other protein(s) to regulate polarity. We also show that the pam-1 gene is expressed both maternally and paternally, providing additional evidence that sperm-donated gene products have important roles during early embryogenesis in C. elegans. The degradation of proteins through ubiquitin-mediated proteolysis has been previously shown to regulate the cell cycle and AP axis formation in the C. elegans zygote. Our analysis of PAM-1 requirements shows that a puromycin-sensitive aminopeptidase is also required for proteolytic regulation of the oocyte to embryo transition.