Rationale and design of a double-blind randomized non-inferiority clinical trial to evaluate one or two doses of vaccine against human papillomavirus including an epidemiologic survey to estimate vaccine efficacy: The Costa Rica ESCUDDO trial.

HPV vaccination of adolescent girls is the most effective measure to prevent cervical cancer. The World Health Organization recommends that adolescent girls receive two doses of vaccine but only a small proportion of girls from regions with the highest disease burden are vaccinated because of cost and logistical considerations. Our Costa Rica HPV Vaccine trial suggested that one dose of the bivalent HPV vaccine provides robust and lasting protection against persistent HPV infections for over a decade. Data from a post-licensure trial of the quadrivalent vaccine in India also suggested that a single dose may be effective in reducing cervical cancer risk. To formally compare one versus two doses of the bivalent and nonavalent HPV vaccines, we implemented a large, randomized, double-blind trial to investigate the non-inferiority of one compared to two vaccine doses in the prevention of new HPV16/18 infections that persist 6 or more months. Bivalent and nonavalent vaccines will be evaluated separately. The trial enrolled and randomized (1:1:1:1 to 1- and 2-dose arms of the bivalent and nonavalent vaccines) 20,330 girls 12 to 16 years old residing in Costa Rica. Trial participants are followed every 6 months for up to 5 years. We also aim to estimate vaccine efficacy by comparing the rates of 6 month persistent infection in unvaccinated women with the rates in the follow-up visits of trial participants. We included one survey of unvaccinated women at the start of the study (N = 4452) and will include another survey concomitant with follow up visits of trial participants at year 4.5 (planned N = 3000). Survey participants attend two visits 6 months appart. Herein, we present the rationale, design, and enrolled study population of the ESCUDDO trial. ClinicalTrials.gov Identifier: NCT03180034.

Multiplex immunoassay to measure antibody response to nine HPV vaccine types.

Well-characterized HPV serology assays are required to evaluate performance of biosimilar candidate vaccines, reduced dosing schedules and novel administration methods. We report characterization of an expanded assay, M9ELISA, that detects antibodies to HPV virus-like particles (VLP) of nine types using direct IgG ELISA on the Meso Scale Discovery (MSD) electrochemiluminescence platform. The method is based on the previously published M4ELISA which detects antibodies to HPV6,11,16, and 18. It has been modified to add detection of antibodies to HPV31,33,45,52 and 58, and to streamline assay and reduce background. The M9ELISA plates were prepared with purified type specific L1 + L2 VLPs coated on 10-spot/well standard MSD microplates. Results of ELISA on three serial dilutions of serum were read on MSD imager, and titers calculated using the parallel line method. Evaluations included dynamic range, assay reproducibility, and stability over time. We compared M9ELISA results to those from a pseudovirion-based neutralization assay in sera from a mixed cohort of unvaccinated and vaccinated individuals (n = ~116) and to competitive Luminex immunoassay (cLIA) results in sera from a predominantly unvaccinated cohort (n = 4426). The linear range of the assay extended over 5 logs, with inter-assay reproducibility coefficient of variation ≤25% for all types. The pre-coated plates were stable for at least 2 years. Spearman correlation of antibody titers showed excellent correlation with PBNA (r = 0.86-0.97) and moderate correlation (r = 0.52-0.68) with cLIA. Thus, the M9ELISA can serve as a useful platform for high-throughput, sensitive and simultaneous quantitation of the antibody responses to nine HPV vaccine types.

Serum HPV16 E7 Oncoprotein Is a Recurrence Marker of Oropharyngeal Squamous Cell Carcinomas.

Despite improved prognosis for many HPV-positive head and neck squamous cell carcinomas (HNSCCs), some cases are still marked by recurrence and metastasis. Our study aimed to identify novel biomarkers for patient stratification. Classical HPV markers: HPV-DNA, p16 and HPV mRNA expression were studied in HNSCC (n = 67) and controls (n = 58) by qPCR. Subsequently, ELISA tests were used for HPV16 L1 antibody and HPV16 E7 oncoprotein detection in serum at diagnosis and follow-up. All markers were correlated to relapse-free survival (RFS) and overall survival (OS). HPV-DNA was found in HNSCCs (29.85%), HPV16-DNA in 95% of cases, HPV16 E7 mRNA was revealed in 93.75%. p16 was overexpressed in 75% of HPV-positive HNSCC compared to negative samples and controls (p < 0.001). Classical markers correlated with improved OS (p < 0.05). Serological studies showed similar proportions of HPV16 L1 antibodies in all HNSCCs (p > 0.05). Serum E7 oncoprotein was present in 30% HPV-positive patients at diagnosis (p > 0.05) and correlated to HNSCC HPV16 E7 mRNA (p < 0.01), whereas it was associated to worse RFS and OS, especially for oropharyngeal squamous cell carcinoma (OPSCC) (p < 0.01). Detection of circulating HPV16 E7 oncoprotein at diagnosis may be useful for stratifying and monitoring HPV-positive HNSCC patients for worse prognosis, providing clinicians a tool for selecting patients for treatment de-escalation.

Non-invasive Assessment of Vaccine-Induced HPV Antibodies via First-Void Urine.

The potential of first-void (FV) urine as a non-invasive method to monitor human papillomavirus (HPV) vaccination has been reported, mainly focusing on urine as a sample to assess HPV DNA. Besides HPV DNA, vaccine-induced HPV antibodies originating from cervicovaginal secretions were recently shown to be detectable in FV urine as well. This presents a novel opportunity for non-invasive sampling to monitor HPV antibody status in women participating in large epidemiological studies and HPV vaccine trials. The simultaneous assessment of both HPV infection and immunogenicity on a non-invasive, readily obtained sample is particularly attractive.

Comparison of a VLP-based and GST-L1-based multiplex immunoassay to detect vaccine-induced HPV-specific antibodies in first-void urine.

Vaccine-induced human papillomavirus (HPV) antibodies originating from cervicovaginal secretions were recently shown to be detectable in first-void (FV) urine. This presents a novel opportunity for noninvasive sampling to monitor HPV antibody status in women participating in large epidemiological studies and HPV vaccine trials. With a view towards method optimization, this study compared the measurement of HPV antibodies in FV urine using a multiplex L1/L2 virus-like particles (VLP)-based ELISA (M4ELISA) with previously reported results using a glutathione S-transferase (GST)-L1-based immunoassay (GST-L1-MIA). We tested 53 paired FV urine and serum samples from 19- to 26-year-old healthy women, unvaccinated (n = 17) or vaccinated with either the bivalent or quadrivalent HPV-vaccine during adolescence (n = 36). HPV6/11/16/18 antibodies were measured using M4ELISA and compared with GST-L1-MIA results. Inter-assay and inter-specimen correlations were examined using the Spearman's rank test (rs). As expected, lower HPV antibody concentrations were found in FV urine than in serum. Vaccinated women had significantly higher HPV6/11/16/18 antibody levels in both FV urine and serum compared with those unvaccinated (M4ELISA; FV urine P = .0003; serum P ≤ .0001). HPV antibody levels in FV urine and serum showed a significant positive correlation (M4ELISA anti-HPV6/11/16/18, rs = 0.85/0.86/0.91/0.79, P ≤ .001). Despite assay differences, there was moderate to good correlation between M4ELISA and GST-L1-MIA (FV urine anti-HPV6/11/16/18, rs = 0.86/0.83/0.89/0.53, P ≤ .0001; serum anti-HPV6/11/16/18, rs = 0.93/0.89/0.94/0.75, P ≤ .0001). FV urine HPV antibody detection is comparable with both assays, further supporting this noninvasive sampling method as a possible option for HPV vaccine assessment. Approaches to improve the sensitivity and larger studies are warranted to determine the feasibility of FV urine for vaccine-induced HPV antibody detection.

Prevalence and Risk Factors for Oral HPV in Healthy Population, in Central Europe.

Background/Aim: The incidence of oropharyngeal tumours induced by human papillomaviruses (HPV) is ever increasing. Information about oral HPV prevalence and its risk factors are very important for future screening and early diagnosis of the disease. The present study aimed to assess oral HPV prevalence in healthy population and risk factors for HPV infection, since this data is scarce or even missing in Central Europe. Patients and Methods: HPV prevalence in oral rinse and HPV-specific antibodies in peripheral blood were investigated in two groups of healthy participants. Group I consisted of 294 students who reached sexual maturity after the HPV vaccine had been licensed with mean age 23.2 years, and Group II of 215 unvaccinated participants with the mean age 55.7 years. Additionally, the risk factors were evaluated. Results: In Group I, 2% of participants were positive for oral HPV DNA. A statistically significantly higher rate (8.8%) was found in Group II. The seropositivity rates for anamnestic HPV antibodies were comparable in both groups. None of the analysed risk factors was significantly associated with oral HPV positivity. Conclusion: The lower prevalence of oral HPV DNA in younger participants suggests the positive influence of vaccination against oral HPV.

Human Papillomavirus Serologic Profiles of Selected Filipinos with Head and Neck Squamous Cell Carcinoma.

BACKGROUND: The low prevalence of human papillomavirus (HPV) DNA and mRNA in biopsy samples of Filipinos with head and neck squamous cell carcinoma (HNSCC) has been reported previously. Here, the HPV serologic profiles of HNSCC cases were analyzed and associated with life-style and sexual practices. METHODS: Serum samples were collected between May 2012 and September 2013 from HNSCC patients (n = 22) in the northwest region of the Philippines, and age- and sex-matched clinically healthy controls. Antibodies to capsid and early oncoproteins of HPV16, 18, 31, 33, 45, 52, 58, 6, and 11 were analyzed using multiplex serology. RESULTS: Most of the cases were males with tumors of the oral cavity or larynx. Two of the cases tested positive for at least one of the early oncoproteins (E6, E7, E1, and/or E2) of HPV16, and 11 did not display reactivity to any HPV early or late oncoproteins. Of the controls, four tested positive for at least one of the HPV16 early oncoproteins, and 10 were non-reactive to all HPV types. Titers to HPV16 E6 or E7 of the seropositive cases and controls were considerably lower than those typically observed in economically developed countries. CONCLUSIONS: The low HPV titers seen here are consistent with the results of molecular analyses for this population. Hence, the seropositivity of some of the HNSCC cases is likely an indication of prior exposure to the virus and not the presence of HPV-driven tumors.

Human Papillomavirus Serologic Profiles of Selected Filipinos with Head and Neck Squamous Cell Carcinoma

BACKGROUND: The low prevalence of human papillomavirus (HPV) DNA and mRNA in biopsy samples of Filipinos with head and neck squamous cell carcinoma (HNSCC) has been reported previously. Here, the HPV serologic profiles of HNSCC cases were analyzed and associated with lifestyle and sexual practices. METHODS: Serum samples were collected between May 2012 and September 2013 from HNSCC patients (n = 22) in the northwest region of the Philippines, and age- and sex-matched clinically healthy controls. Antibodies to capsid and early oncoproteins of HPV16, 18, 31, 33, 45, 52, 58, 6, and 11 were analyzed using multiplex serology. RESULTS: Most of the cases were males with tumors of the oral cavity or larynx. Two of the cases tested positive for at least one of the early oncoproteins (E6, E7, E1, and/or E2) of HPV16, and 11 did not display reactivity to any HPV early or late oncoproteins. Of the controls, four tested positive for at least one of the HPV16 early oncoproteins, and 10 were non-reactive to all HPV types. Titers to HPV16 E6 or E7 of the seropositive cases and controls were considerably lower than those typically observed in economically developed countries. CONCLUSIONS: The low HPV titers seen here are consistent with the results of molecular analyses for this population. Hence, the seropositivity of some of the HNSCC cases is likely an indication of prior exposure to the virus and not the presence of HPV-driven tumors.

Description of a novel multiplex avidity assay for evaluating HPV antibodies.

Limited data exists regarding antibody avidity for human papillomavirus (HPV). We describe development of a multiplex electrochemiluminescent avidity ELISA for four HPV types (HPV 6, 11, 16, 18) by adding a dissociating step to our established multiplex HPV VLP ELISA. Initial experiments exploring ammonium thiocyanate, sodium thiocyanate and guanidine hydrochloride (GuHCl) as dissociating agents identified GuHCl as most promising. Dissociation conditions with GuHCl were varied (concentration, incubation time, temperature) to select conditions with minimal impact on VLP integrity as measured with monoclonal antibodies to conformational epitopes. Avidity index (AI) was calculated based on a standard curve as ratio of bound IgG in GuHCl treated versus untreated sample. To evaluate our assay we determined AI in sera with known HPV titers. We selected 32 residual anonymized sera from individuals with a wide range of titers for HPV6, 11, 16, and 18. AIs were similar across multiple dilutions of serum within the assay's dynamic range and were reproducible with two plate lots. This assay will aid in understanding HPV antibody avidity and maturation in response to natural infection and varying vaccine schedules. This is the first report of a VLP-based multiplexed avidity ELISA that evaluates assay parameters for all nine HPV vaccine types.

Characterization of serum antibodies from women immunized with Gardasil: A study of HPV-18 infection of primary human keratinocytes.

The prevalent human papillomaviruses (HPVs) infect human epithelial tissues. Infections by the mucosotropic HPV genotypes cause hyperproliferative ano-genital lesions. Persistent infections by high-risk (HR) HPVs such as HPV-16, HPV-18 and related types can progress to high grade intraepithelial neoplasias and cancers. Prophylactic HPV vaccines are based on DNA-free virus-like particles (VLPs) composed of the major capsid protein L1 of HPV-16, -18, -6 and -11 (Gardasil) or HPV-16 and -18 (Cervarix). Sera from vaccinated animals effectively prevent HPV pseudovirions to infect cell lines and mouse cervical epithelia. Both vaccines have proven to be highly protective in people. HPV pseudovirions are assembled in HEK293TT cells from matched L1 and L2 capsid proteins to encapsidate a reporter gene. Pseudovirions and genuine virions have structural differences and they infect cell lines or primary human keratinocytes (PHKs) with different efficiencies. In this study, we show that sera and isolated IgG from women immunized with Gardasil prevent authentic HPV-18 virions from infecting PHKs, whereas non-immune sera and purified IgG thereof are uniformly ineffective. Using early passage PHKs, neutralization is achieved only if immune sera are added within 2-4h of infection. We attribute the timing effect to a conformational change in HPV virions, thought to occur upon initial binding to heparan sulfate proteoglycans (HSPG) on the cell surface. This interpretation is consistent with the inability of immune IgG bound to or taken up by PHKs to neutralize the virus. Interestingly, the window of neutralization increases to 12-16h in slow growing, late passage PHKs, suggestive of altered cell surface molecules. In vivo, this window might be further lengthened by the time required to activate the normally quiescent basal cells to become susceptible to infection. Our observations help explain the high efficacy of HPV vaccines.

Characterization of human papillomavirus antibodies in individuals with head and neck cancer.

BACKGROUND: Human papillomavirus type 16 (HPV16) E6 antibodies are a promising biomarker of oropharyngeal cancer (OPC); however, seropositivity among non-OPC cases is not well characterized. METHODS: Pre-treatment sera from 260 (38 OPC, 222 non-OPC) incident head and neck cancers diagnosed at the University of Pittsburgh between 2003 and 2006 were tested for HPV16 (L1,E1,E2,E4,E6,E7) and non-HPV16 E6 (HPV6,11,18,33) antibodies. Sensitivity and specificity of HPV16 E6 antibodies for HPV-driven tumors was evaluated among tumors with known HPV status (n=25). RESULTS: 63.2% of OPC versus 27.5% of non-OPC cases were HPV16 seropositive; HPV16 E6 seroprevalence was 60.5% and 6.3% respectively, odds ratio 22.8 (95% confidence interval [CI] 9.8-53.1). Sensitivity and specificity of HPV16 E6 antibodies for HPV-driven OPC was 100% [95% CI: 50-100%; n=6] and 100% [95% CI: 60-100%, n=4] compared to 0% (n=2) and 0% (n=13) for non-OPC cases. CONCLUSIONS: HPV16 antibodies were significantly more common in OPC versus non-OPC cases, particularly HPV16 E6 antibodies.

Low prevalence of HPV-driven head and neck squamous cell carcinoma in North-East Italy.

OBJECTIVES: To investigate the frequency of Human papillomavirus (HPV)-driven head and neck squamous cell carcinoma (HNSCC) among patients living in North-East Italy, by assessing HPV-DNA positivity in all tumors and additional markers whenever possible. MATERIAL AND METHODS: HPV types, viral load, viral RNA, HPV16/18 E6 protein and p16INK4a and pRb expression were determined in primary tumor tissues from 247 HNSCC patients. Tumor-specific HPV seropositivity was analyzed in 102 patients. RESULTS: Tumor HPV-DNA prevalence was 8.5% overall (21/247) and 27% in oropharynx (17/63). HPV16 accounted for 95% of all HPV types found. Among HPV-DNA+ tumors, type-concordant HPV E6*I RNA prevalence was 79%. HPV DNA+ RNA+ tumors showed high viral load, up-regulated p16INK4a, down-regulated pRb and presence of HPV16 E6 protein. Eight cases showed tumor-specific HPV seropositivity, all type-concordant with the tumor. Tumors were defined as HPV-driven when positive for HPV-DNA plus 2 additional HPV transformation-related markers. CONCLUSION: Relative prevalence of HPV-driven tumors (14 HPV16, 1 HPV58) was 6% overall and 20% among oropharyngeal cancers. In the oropharynx the HPV-driven group showed a trend for better survival versus the HPV-negative group. The relative prevalence of HPV-driven oropharyngeal cancer is low in North-East Italy as compared to Western and Northern Europe.

Seroconversion Following Anal and Genital HPV Infection in Men: The HIM Study.

BACKGROUND: Protection from naturally acquired human papillomavirus (HPV) antibodies may influence HPV infection across the lifespan. This study describes seroconversion rates following genital, anal, and oral HPV 6/11/16/18 infections in men and examines differences by HPV type and anatomic site. METHODS: Men with HPV 6/11/16/18 infections who were seronegative for those genotypes at the time of DNA detection were selected from the HPV Infection in Men (HIM) Study. Sera specimens collected ≤36 months after detection were analyzed for HPV 6/11/16/18 antibodies using a virus-like particle-based ELISA. Time to seroconversion was separately assessed for each anatomic site, stratified by HPV type. RESULTS: Seroconversion to ≥1 HPV type (6/11/16/18) in this sub-cohort (N=384) varied by anatomic site, with 6.3, 18.9, and 0.0% seroconverting following anal, genital, and oral HPV infection, respectively. Regardless of anatomic site, seroconversion was highest for HPV 6 (19.3%). Overall, seroconversion was highest following anal HPV 6 infection (69.2%). HPV persistence was the only factor found to influence seroconversion. CONCLUSIONS: Low seroconversion rates following HPV infection leave men susceptible to recurrent infections that can progress to HPV-related cancers. This emphasizes the need for HPV vaccination in men to ensure immune protection against new HPV infections and subsequent disease.

Development and evaluation of multiplexed immunoassay for detection of antibodies to HPV vaccine types.

Reliable antibody based-assays are needed to evaluate the immunogenicity of current vaccines, impact of altered dosing schemes or of new vaccine formulations. An ideal assay platform would allow multiplex type-specific detection with minimal sample requirement. We used the Meso Scale Discovery (MSD) electrochemiluminescence based detection platform to develop a multiplex direct virus-like particle (VLP) ELISA to detect antibodies to HPV 6, 11, 16, and 18 with a protocol developed for detection using the SI 6000 imager (M4ELISA). MSD prepared the plates in the 7-spot/well format, using the purified VLPs (4 spots) and PBS+BSA pH7.4 (3 blank spots). Three-point titrations and the parallel line method were used to calculate antibody levels. Dynamic range, precision, and stability of pre-printed plates were determined using a panel of previously characterized sera. Cut-off values using children's sera were established using 99% RLU limits based on the 4-parameter Johnson Su best fit curve. Results of the M4ELISA were compared to competitive Luminex Immunoassay (cLIA) on n = 4454 sera from a predominantly unvaccinated cohort. Using a VLP coating concentration of 80 µg/ml with BSA provided the most robust RLU signal for all types. The dynamic range of the assay was about 1000 fold, with assay variability under 25% for each of the four vaccine types. Long-term stability of the plates extended to about 7 months from the time plates was received in the laboratory after printing. There was moderate agreement (κ = 0.38-0.54) between M4ELISA and cLIA, with antibody detection for each of the 4 types more frequent with M4ELISA. Quantitative analysis however showed a good correlation between concordant samples by both assays (ρ ≥ 0.6). The MSD platform shows promise for simultaneous quantitation of the antibody responses to four HPV vaccine types in a high-throughput manner.

Route of sexual exposure is independently associated with seropositivity to HPV-16 and HPV-18 among clients of an STI clinic in the Netherlands.

We investigated the route of sexual exposure as a determinant for human papillomavirus (HPV)-16 and HPV-18 seropositivity. At the Amsterdam sexually transmitted infections clinic we recruited 4 risk groups: (1) men who have sex with women only (MSW; n = 751); (2) women who have sex with men (WSM; n = 749); (3) men who have sex with men (MSM) reporting insertive anal sex only (insMSM; n = 156); and (4) MSM reporting receptive anal sex (recMSM; n = 415). In multivariable analyses, HPV-16 seropositivity was significantly more common in WSM vs MSW, recMSM vs MSW, and recMSM vs insMSM. HPV-18 results were similar. Route of sexual exposure is independently associated with HPV seropositivity.