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.

Human papillomavirus (HPV) types among Alaska native women attending a colposcopy clinic in Anchorage, Alaska, 2009-2011.

BACKGROUND: The first HPV vaccines licensed targeted two HPV types responsible for most cervical cancers. A 9-valent vaccine (9vHPV), targeting 5 additional types, was introduced in 2016 and is currently the only HPV vaccine available in the United States. Previous studies demonstrated high rates of HPV infection in Alaska Native (AN) women. We sought to measure prevalence of high risk HPV types in AN women undergoing colposcopy and to determine those preventable by vaccination. METHODS: For this cross-sectional study, we recruited women who were undergoing colposcopy for clinical indications at Alaska Native Medical Center to obtain cervical brush biopsy samples. Specimens were shipped to Atlanta, Georgia for DNA extraction, HPV detection, and typing using L1 PCR with type-specific hybridization to detect 37 HPV types. RESULTS: Four hundred eighty eight specimens from 489 women were tested. At least one HPV type was found in 458 (94%) specimens. Of 458 participants who were HPV positive, 332 (72%) had two or more types. At least one type targeted by 9vHPV was detected in 95% of participants with CIN 3 (21/22), 82% with CIN 2 (37/45), and 65% with CIN 1 (119/184). (p < 0.001) HPV 16 or 18 were detected in 77% (17/22) with CIN 3, 53% (24/45) with CIN 2, and 36% (67/184) with CIN 1. (p < 0.001). CONCLUSIONS: A substantial proportion of AN women attending colposcopy clinic had evidence of HPV 16/18 infection, as well as other high risk types targeted by 9vHPV. At least one 9vHPV type was detected in 62% of the participants overall, and 95% of participants with CIN3. AN women are expected to benefit from vaccination against HPV 16/18, and will have greater benefit from 9vHPV. Information from this study could be used to develop public health strategies to increase vaccine uptake, or to track HPV genotype prevalence over time.

Seroprevalence of HPV 6, 11, 16 and 18 and correlates of exposure in unvaccinated women aged 16-64 years in Puerto Rico.

BACKGROUND: To understand risk factors for HPV exposure in Puerto Rican women, we evaluated HPV 6, 11, 16, and 18 serology in women aged living in the San Juan metropolitan area. METHODS: As part of a cross-sectional study, a population-based sample of 524 HPV unvaccinated Hispanic women ages 16-64 years completed face-to-face and computer assisted interviews and provided blood and self-collected anal and cervical specimens. Serology used multiplex virus-like particle based-IgG ELISA and HPV DNA was detected with L1-consensus PCR. RESULTS: 32% and 47% were seropositive to HPV types included in the bivalent (16/18) and quadrivalent (6/11/16/18) vaccines, respectively. Type-specific seroprevalence was HPV6 - 29%, HPV11 - 18%, HPV16 - 23%, and HPV18 - 17%; seroprevalence was high in the youngest age-group (16-19: 26-37%). HPV seropositivity was associated with having ≥ 3 lifetime sexual partners (OR=2.5, 95% CI=1.7-3.9) and detection of anogenital HPV DNA (OR=1.8, 95% CI=1.2-2.6). CONCLUSIONS: The high cumulative exposure of HPV vaccine types 6/11/16/18 in this Hispanic population was influenced by factors related to HPV exposure through sexual behavior. High seroprevalence in the youngest age-group indicates early age of exposure to HPV in Puerto Rico, highlighting the need for HPV vaccination starting prior to age 16.

Methods of applying the 1994 case definition of chronic fatigue syndrome - impact on classification and observed illness characteristics.

BACKGROUND: Multiple case definitions are in use to identify chronic fatigue syndrome (CFS). Even when using the same definition, methods used to apply definitional criteria may affect results. The Centers for Disease Control and Prevention (CDC) conducted two population-based studies estimating CFS prevalence using the 1994 case definition; one relied on direct questions for criteria of fatigue, functional impairment and symptoms (1997 Wichita; Method 1), and the other used subscale score thresholds of standardized questionnaires for criteria (2004 Georgia; Method 2). Compared to previous reports the 2004 CFS prevalence estimate was higher, raising questions about whether changes in the method of operationalizing affected this and illness characteristics. METHODS: The follow-up of the Georgia cohort allowed direct comparison of both methods of applying the 1994 case definition. Of 1961 participants (53 % of eligible) who completed the detailed telephone interview, 919 (47 %) were eligible for and 751 (81 %) underwent clinical evaluation including medical/psychiatric evaluations. Data from the 499 individuals with complete data and without exclusionary conditions was available for this analysis. RESULTS: A total of 86 participants were classified as CFS by one or both methods; 44 cases identified by both methods, 15 only identified by Method 1, and 27 only identified by Method 2 (Kappa 0.63; 95 % confidence interval [CI]: 0.53, 0.73 and concordance 91.59 %). The CFS group identified by both methods were more fatigued, had worse functioning, and more symptoms than those identified by only one method. Moderate to severe depression was noted in only one individual who was classified as CFS by both methods. When comparing the CFS groups identified by only one method, those only identified by Method 2 were either similar to or more severely affected in fatigue, function, and symptoms than those only identified by Method 1. CONCLUSIONS: The two methods demonstrated substantial concordance. While Method 2 classified more participants as CFS, there was no indication that they were less severely ill or more depressed. The classification differences do not fully explain the prevalence increase noted in the 2004 Georgia study. Use of standardized instruments for the major CFS domains provides advantages for disease stratification and comparing CFS patients to other illnesses.

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.

Cross-sectional study of HPV-16 infection in a population-based subsample of Hispanic adults.

OBJECTIVE: This study aimed to estimate the prevalence and correlates of seropositivity to human papillomavirus (HPV)-16 in a subsample of adults who participated in the parent study Epidemiology of Hepatitis C in the adult population of Puerto Rico (PR). SETTING: The parent study was a population-based household survey aimed to estimate the seroprevalence of hepatitis C and other viral infections (hepatitis A, hepatitis B, HIV, and herpes simplex type 2) in PR (n=1654) between 2005 and 2008. PARTICIPANTS: A subsample of the last 450 consecutive adults aged 21-64 years, recruited between February 2007 and January 2008, who participated in the parent study and agreed to participate in HPV testing. PRIMARY AND SECONDARY OUTCOME MEASURES: The samples were tested by ELISA for HPV-16 viral-like particle-specific immunoglobulin G. Information on sociodemographic, health, and lifestyle characteristics was collected. Logistic regression modelling was used to estimate the prevalence odds ratio (POR) to assess factors associated to HPV-16 seropositivity. RESULTS: Prevalence of seropositivity to HPV-16 was 11.3%. Seroprevalence was higher in women (15.8%) than men (5.6%; p=0.001). After adjusting for age and sex, ever smokers (POR 2.06, 95% CI 1.08 to 3.92) and participants with at least five lifetime sexual partners (POR 2.91, 95% CI 1.24 to 6.81) were more likely to be HPV-16 seropositive. CONCLUSIONS: HPV-16 seropositivity is similar to that reported in the USA (10.4%) for NHANES 2003-2004 participants, although different assays were used in these studies. While future studies should evaluate HPV seroprevalence using a larger population-based sample, our results highlight the need to further understand the burden of HPV infection and HPV-related malignancies in PR, population with a low vaccine uptake.

Immunogenicity of quadrivalent human papillomavirus vaccine in organ transplant recipients.

Solid organ transplant recipients are at risk of morbidity from human papillomavirus (HPV)-related diseases. Quadrivalent HPV vaccine is recommended for posttransplant patients but there are no data on vaccine immunogenicity. We determined the immunogenicity of HPV vaccine in a cohort of young adult transplant patients. Patients were immunized with three doses of quadrivalent HPV vaccine containing viral types 6, 11, 16 and 18. Immunogenicity was determined by type-specific viral-like protein ELISA. Four weeks after the last dose of vaccine, a vaccine response was seen in 63.2%, 68.4%, 63.2% and 52.6% for HPV 6, 11, 16 and 18, respectively. Factors that led to reduced immunogenicity were vaccination early after transplant (p = 0.019), having a lung transplant (p = 0.007) and having higher tacrolimus levels (p = 0.048). At 12 months, there were significant declines in antibody titer for all HPV types although the number of patients who remained seropositive did not significantly differ. The vaccine was safe and well tolerated. We show suboptimal immunogenicity of HPV vaccine in transplant patients. This is important for counseling patients who choose to receive this vaccine. Further studies are needed to determine an optimal HPV vaccine type and schedule for this population.

Monitoring HPV type-specific prevalence over time through clinic-based surveillance: a perspective on vaccine effectiveness.

We investigated the feasibility of monitoring trends in prevalence of vaccine-preventable human papillomavirus (HPV) types in different clinic populations. We collected cervical specimens from women presenting to family planning, primary care, and sexually transmitted disease (STD) clinics for routine pap smears in five US cities during 2003-2005. We performed HPV genotyping and calculated annual type-specific prevalences; pre-vaccine era prevalence was highest for HPV 16 (6.0; 95% confidence interval [CI] 5.5-6.6%) and annual prevalences for vaccine-preventable types were stable, with few exceptions, after controlling for clinic type, age group, and city. With sufficient sample size and stable population characteristics, clinic-based surveillance systems can contribute to monitoring HPV vaccine impact in the cervical screening population.

Factors associated with prevalent abnormal anal cytology in a large cohort of HIV-infected adults in the United States.

BACKGROUND: The prevalence of and risk factors for abnormal anal cytology among men and women with human immunodeficiency virus (HIV) infection have not been extensively investigated. METHODS: The Study to Understand the Natural History of HIV and AIDS in the Era of Effective Therapy (SUN study) is a prospective cohort study of HIV-infected patients in 4 US cities. Baseline questionnaires were administered and anal samples for cytology and human papillomavirus (HPV) detection and genotyping were collected. RESULTS: Among 471 men and 150 women (median age, 41 years), 78% of participants were receiving combination antiretroviral therapy, 41% had a CD4(+) cell count of ≥500 cells/µL, and 71% had an HIV RNA viral load of <400 copies/mL. The anal cytology results were as follows: 336 participants (54%) had negative results, 96 participants (15%) had atypical squamous cells, 149 participants (24%) had low-grade squamous intraepithelial lesions, and 40 participants (6%) had high-grade squamous intraepithelial lesions. In a multivariate analysis, abnormal anal cytology was associated with number of high-risk and low-risk HPV types (adjusted odds ratio [AOR] for both, 1.28; P < .001), nadir CD4(+) cell count of <50 cells/µL (AOR, 2.38; P = .001), baseline CD4(+) cell count of <500 cells/µL (AOR, 1.75; P = .004), and ever having receptive anal intercourse (AOR, 2.51; P < .001). CONCLUSION: HIV-infected persons with multiple anal HPV types or a nadir CD4(+) cell count of <50 cells/µL have an increased risk for abnormal anal cytology.

Glucocorticoid receptor polymorphisms and haplotypes associated with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a significant public health problem of unknown etiology, the pathophysiology has not been elucidated, and there are no characteristic physical signs or laboratory abnormalities. Some studies have indicated an association of CFS with deregulation of immune functions and hypothalamic-pituitary-adrenal (HPA) axis activity. In this study, we examined the association of sequence variations in the glucocorticoid receptor gene (NR3C1) with CFS because NR3C1 is a major effector of the HPA axis. There were 137 study participants (40 with CFS, 55 with insufficient symptoms or fatigue, termed as ISF, and 42 non-fatigued controls) who were clinically evaluated and identified from the general population of Wichita, KS. Nine single nucleotide polymorphisms (SNPs) in NR3C1 were tested for association of polymorphisms and haplotypes with CFS. We observed an association of multiple SNPs with chronic fatigue compared to non-fatigued (NF) subjects (P < 0.05) and found similar associations with quantitative assessments of functional impairment (by the SF-36), with fatigue (by the Multidimensional Fatigue Inventory) and with symptoms (assessed by the Centers for Disease Control Symptom Inventory). Subjects homozygous for the major allele of all associated SNPs were at increased risk for CFS with odds ratios ranging from 2.61 (CI 1.05-6.45) to 3.00 (CI 1.12-8.05). Five SNPs, covering a region of approximately 80 kb, demonstrated high linkage disequilibrium (LD) in CFS, but LD gradually declined in ISF to NF subjects. Furthermore, haplotype analysis of the region in LD identified two associated haplotypes with opposite alleles: one protective and the other conferring risk of CFS. These results demonstrate NR3C1 as a potential mediator of chronic fatigue, and implicate variations in the 5' region of NR3C1 as a possible mechanism through which the alterations in HPA axis regulation and behavioural characteristics of CFS may manifest.

Improved detection of viral RNA isolated from liquid-based cytology samples.

BACKGROUND: Molecular diagnosis requires the ability to obtain high-quality nucleic acids that are representative of the disease state. We evaluated the recovery and detection of limiting amounts of viral oncogenic RNA from cells fixed in liquid-based cytology media. METHODS AND RESULTS: Serial dilutions of a human papillomavirus (HPV)-positive cell line fixed in a liquid media was used as a model system. Total nucleic acid (TNA) extraction produced RNA with clearly visible ribosomal bands even after one year of storage. These TNA extracts, treated with DNase-I, were used in an RT-PCR assay for HPV-16 E6-E7 oncogenic transcripts. With chemiluminscent Southern blot detection, samples with one HPV-positive cell in 30,000 were consistently detected. CONCLUSION: PreservCyt-fixed cells can yield RNA suitable for molecular assays even after one year of storage.

Interphase cytogenetic analysis of 1q12 satellite III DNA in melanocytic lesions: increased aneuploidy with malignant histology.

To examine the relationship of chromosome 1 copy number to melanocytic tumorigenesis, interphase cytogenetic analysis of 1q12 satellite III DNA was performed on the spectrum of melanocytic lesions comprising Clark's tumor progression model. Results showed increased copy number in a "step off" pattern between melanoma in-situ and the intraepidermal component of invasive melanoma rather than a progression between each lesional group. These findings support Clark's concept of independent clonal expansion of a cell population giving rise to the vertical growth phase and further demonstrates increased chromosome 1 copy number as a late event in melanoma tumor progression.

Real-time PCR-based fluorescent assay for quantitation of human papillomavirus types 6, 11, 16, and 18.

BACKGROUND: Quantitation of human papillomavirus (HPV) DNA in clinical samples may yield important clinical information. METHODS AND RESULTS: We developed a 5' exonuclease fluorescent probe assay for HPV quantitation that uses real-time PCR. The assay was optimized for HPV types 6 (HPV-6), -11, -16, and -18. A multiplex format was developed to quantify a cellular target of known iteration simultaneously with HPV quantitation, which controls for the amount of input DNA. Dilution series of target and heterologous templates were used to verify the assay. The assay was successfully used on fresh and PreservCyt-fixed cell lines, as well as cervical samples. The linear range of the assay is from 10 to 10 million copies. Intraclass correlations for HPV, actin, and globin assays ranged from 0.95 to 0.99, indicating the analytic precision of repeated measures. CONCLUSION: The method is accurate over a large copy number range, reproducible, type specific, normalized for input DNA quantity, and applicable to PreservCyt-fixed material.

Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR.

We evaluated real-time (kinetic) reverse transcription-polymerase chain reaction (RT-PCR) to validate differentially expressed genes identified by DNA arrays. Gene expression of two keratinocyte subclones differing in the physical state of human papillomavirus (episomal or integrated) was used as a model system. High-density filter arrays identified 444 of 588 genes as either negative or expressed with less than twofold difference, and the other 144 genes as expressed uniquely or with more than twofold difference between the two subclones. Real-time RT-PCR used LightCycler-based SYBR Green I dye detection and melting curve analysis to validate the relative change in gene expression. Real-time RT-PCR confirmed the change in expression of 17 of 24 (71%) genes identified by high-density filter arrays. Genes with strong hybridization signals and at least twofold difference were likely to be validated by real-time RT-PCR. This data suggests that (i) both hybridization intensity and the level of differential expression determine the likelihood of validating high-density filter array results and (ii) genes identified by DNA arrays with a two- to fourfold difference in expression cannot be eliminated as false nor be accepted as true without validation. Real-time RT-PCR based on LightCycler technology is well-suited to validate DNA array results because it is quantitative, rapid, and requires 1000-fold less RNA than conventional assays.

Human papillomaviruses: into the new millennium.

Human papillomaviruses (HPV) are newsworthy in this new millennium. Numerous articles have appeared in the lay press ranging in style and quality from informative essays to sensationalized exposes. Women, sensitized by confusing information, are asking obstetricians hard questions about HPV transmission and prevention, partner notification, the need for HPV testing, and methods of treatment. These questions are difficult because none of the answers are clear cut. This article provides the practicing gynecologist and obstetrician a concise and accurate summary of clinically important issues surrounding HPV. Current knowledge about HPV virology, epidemiology, testing, and the prospects for vaccination and other prevention measures is summarized.

Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies.

Real-time reverse transcription polymerase chain reaction (RT-PCR) methods that monitor product accumulation were adapted for the validation of differentially expressed genes. We describe a real-time quantitative PCR assay that uses SYBR Green I dye-based detection and product melting curve analysis to validate differentially expressed genes identified by gene expression profiling technologies. Since SYBR Green I dye is a nonspecific intercalating dye, the reaction is made specific by using "hot-start" PCR and empirically determined annealing and signal acquisition temperatures for each gene-specific primer. Relative expression levels were quantified by constructing a standard curve using cDNA dilutions of a highly expressed gene. Using this approach, real-time PCR validated 17 of 21 (71%) genes identified by DNA arrays, and all but 1 of 13 (91%) genes identified by differential display PCR (DD-PCR). Validation of differentially expressed genes detected by array analysis was related to hybridization intensity. Real-time RT-PCR results suggest that genes identified by DNA arrays with a two to fourfold difference in expression cannot be accepted as true or false without validation. Validation of differentially expressed genes detected by DD-PCR was not affected by band intensities. Regardless of the gene expression profiling technology (microarrays, DD-PCR, serial analysis of gene expression and subtraction hybridization), once the sequence of gene of interest is known, the real-time RT-PCR approach is well suited for validation of differential expression since it is quantitative and rapid and requires 1000-fold less RNA than conventional assays.

Detection of human papillomaviruses by polymerase chain reaction and in situ hybridization.

The clinical utility of human papillomavirus (HPV) testing continues to be the focus of much debate. The clear epidemiologic link of HPV infection with the development of cervical intraepithelial neoplasia and invasive cervical cancers.

In situ diagnosis of human papillomaviruses.

In situ hybridization (ISH) is the demonstration of specific genetic information within a morphologic context. For HPV, colorimetric ISH has the advantage that it can be applied to routine formalin-fixed paraffin embedded tissues. This conserves patient material and permits histologic selection of optimal material for testing. ISH allows for precise spatial localization of viral sequences within tissues. ISH also allows the integration status of HPV to be determined. The major limitations of the method are the potential for error in HPV typing because of probe cross-hybridization and relatively low sensitivity if the method is not fully optimized.

Comparison of human papillomavirus detection and typing by cycle sequencing, line blotting, and hybrid capture.

We compared the results of human papillomavirus (HPV) detection and typing from 781 cervical samples assayed by three methods: L1 consensus PCR followed by cycle sequencing, L1 consensus PCR with biotinylated primers followed by hybridization to a line blot, and Hybrid Capture assay. Both PCR assays used L1 consensus PCR with primers MY09 and MY11. We evaluated the amplification efficiencies of both PCR assays and also compared the specific HPV types detected by each method. The samples positive by the Hybrid Capture assay were compared to the specific types detected by the PCR-based assays. The concordance between the two PCR assays in producing an HPV amplicon visible by gel electrophoresis or in detecting any HPV type was moderate: kappa values were 0.61 (95% confidence interval [CI] = 0.56 to 0.67) and 0.51 (95% CI = 0.46 to 0.58), respectively. The McNemar test for correlated proportions indicated that biotinylated PCR was less likely to produce a band (P = 0.001) and to detect an HPV type (P = 0.001) than the other PCR assay. In comparing the Hybrid Capture assay results with the HPV types detected by the PCR-based assays, we found that positivity by the Hybrid Capture assay for a number of samples may be due to cross-hybridization with HPV types not included in the Hybrid Capture assay probe cocktails.