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Serial serum samples from 27 patients who underwent double umbilical cord blood transplantation (dUCBT) were analyzed for BK polyomavirus (BKPyV) DNA by real-time PCR and BKPyV-specific immune globulin by ELISA. Clinical data were collected on all patients. All pre-transplant sera had detectable anti-BKPyV IgG. Fifteen patients (56%) had detectable serum BKPyV DNA (median 8.9 × 10(4) copies/ml; range 4.1 × 10(3)-7.9 × 10(6) copies/ml) a median of 40 days (range, 27-733 days) after dUCBT, with highest viral loads on Day 100 assessment. The cumulative probability of developing BKPyV viremia by Day 100 was 0.52 (95% CI, 0.33-0.71). Six of 15 patients with BKPyV viremia experienced hemorrhagic cystitis by Day 100. By Day 100, there was a trend towards higher BKPyV viral loads in sera of patients with hemorrhagic cystitis than in those BKPyV viremic patients without hemorrhagic cystitis (p = 0.06). BKPyV viremia was associated with significantly higher anti-BKPyV IgM values at 6 months post-dUCBT (P = 0.003). BKPyV viremia occurs early after dUBCT and is associated with a detectable humoral immune response by 6 months post-dUBCT., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Rituximab has been successfully used as an experimental therapy in different autoimmune diseases. Recently, a double-blind placebo-controlled phase-2 study in early onset type 1 diabetes showed that rituximab delayed progression of the disease. However, like with any immunosuppressive therapy, there is a concern of opportunistic viral reactivations with the use of rituximab, including herpes and polyomaviruses., Objectives: To study the incidence of new infections and reactivations with BK, JC, Epstein-Barr and cytomegalovirus (BKV, JCV, EBV and CMV) in T1D participants in the phase-2 rituximab study., Study Design: Subjects received 4 weekly doses of rituximab (N = 57) or placebo (N = 30) during the first month of study. Blood samples obtained at weeks 0, 12, 26, 56 and 78 were assayed for CMV, EBV, BKV and JCV by real-time DNA PCR and serology., Results: EBV reactivations were diagnosed by PCR in 25% of placebo, but none of rituximab recipients (p < 0.01). There were no episodes of CMV viremia in either treatment group. BKV viremias were significantly more common in the rituximab recipients (9%) compared with placebo controls (0, p < 0.01). No JCV reactivations were detected in this study, but among 6 rituximab and 2 placebo recipients who seroconverted for JCV during the study, only one rituximab recipient had detectable viremia. All infections were asymptomatic., Conclusions: Four doses of rituximab administered to individuals with early onset T1D decreased the incidence of asymptomatic EBV reactivations, as predicted by the rituximab-mediated elimination of memory B-cells, but increased the frequency of asymptomatic viremias caused by polyomaviruses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Vertical transmission of JC virus and BK virus has been investigated by few authors, with conflicting results. We performed a combined serological and genomic study of 19 unselected pregnant women and their newborns. Blood and urine samples were collected during each gestational trimester from the pregnant women. Umbilical cord blood, peripheral blood, urine and nasopharyngeal secretion samples were taken from newborns at delivery and after 1 week and 1 month of life. Polyomavirus DNA was detected by nested PCR. Polyomavirus IgG-, IgM- and IgA-specific antibodies were measured in maternal and newborn serum samples using a virus-like-particle-based ELISA method. BKV and JCV DNA were detected in urine from 4 (21 %) and 5 (26 %) women, respectively. BKV and JCV seroprevalences in the pregnant women were 84 % and 42 %, respectively. Using a rise in the IgG level or the transient appearance of an IgA or IgM response as evidence of infection in the newborn, we detected BKV and JCV infections in four (21 %) and three (16 %) newborns, respectively. Three infants had serological evidence of infection with both BKV and JCV. In two of the four possible BKV-infected newborns, the mothers seroconverted during pregnancy, while another mother was viruric and IgA seropositive. The mother of one of the three possible JCV-infected newborns was viruric and IgA seropositive; another mother was viruric. These results suggest JC virus and BK virus can be transmitted from mother to newborn during pregnancy or soon after birth.

Polyomavirus BK (BKV) infections are increasingly recognized. The development of immune-monitoring strategies against BKV requires definition of antigenic epitopes. Bioinformatic algorithms were used to identify 60 BKV large T-antigen (LT-Ag) peptides predicted to bind HLA class I alleles. In vitro peptide binding was used to select a subset of 19 peptides for interferon (IFN)-gamma ELISPOT assays in 13 healthy subjects and 12 kidney transplant recipients. Four A01-, nine A03-, and five A24-binding immunogenic peptides were identified in 1 to 3 (14-67%) tested subjects in each group. BKV epitope sequences were identical to homologous JC virus sequences for 3 of 19 peptides and homologous SV40 sequences for 5 of 19 peptides. Homology modeling localized these epitopes to the helicase, origin of DNA binding, or J domains, respectively. In conclusion, we have identified multiple 9-mer BKV LT-Ag-derived immunogenic epitopes that bind HLA-A01, -A03, or -A24 molecules. Sequence alignments indicate that two epitopes, FLICKGVNK and RYWLFKGPI, are common to BKV, JC virus, and SV40 virus.

This study sought to evaluate serology and PCR as tools for measuring BK virus (BKV) replication. Levels of immunoglobulin G (IgG), IgM, and IgA against BKV capsids were measured at five time points for 535 serial samples from 107 patients by using a virus-like particle-based enzyme-linked immunosorbent assay. Viral DNA in urine and plasma samples was quantitated. The seroconversion rate was 87.5% (14/16); 78.6% (11/14) and 14.3% (2/14) of patients who seroconverted developed viruria and viremia, respectively. Transient seroreversion was observed in 18.7% of patients at 17.4 +/- 11.9 weeks posttransplant and was not attributable to loss of antigenic stimulation, changes in immunosuppression, or antiviral treatment. Titers for anti-BK IgG, IgA, and IgM were higher in patients with BKV replication than in those without BKV replication. A rise in the optical density (OD) of anti-BK IgA (0.19), IgM (0.04), or IgG (0.38) had a sensitivity of 76.6 to 88.0% and a specificity of 71.7 to 76.1% for detection of viruria. An anti-BK IgG- and IgA-positive phenotype at week 1 was less frequent in patients who subsequently developed viremia (14.3%) than in those who subsequently developed viruria (42.2%) (P = 0.04). Anti-BK IgG OD at week 1 showed a weak negative correlation with peak urine viral load (r = -0.25; P = 0.05). In summary, serial measurements of anti-BKV immunoglobulin class (i) detect onset of viral replication, (ii) document episodes of seroreversion, and (iii) can potentially provide prognostic information.

Background: The elevated risk for incident head and neck cancer among human papillomavirus (HPV)-16-seropositive individuals has substantiated a role for HPV in the etiology of head and neck cancers. The relationship between HPV seroreactivity and prevalent oral HPV infection in men and women without cancer has yet to be investigated., Goal: The goal of this study was to evaluate a possible association between oral HPV infection and HPV seroreactivity after adjustment for gender, sexual behaviors, and sexually transmitted disease., Study Design: A cross-sectional study of factors associated with HPV-16, -18, and -33 seroreactivity was performed in a population of 586 men and women with and without HIV infection. Antibodies in sera were measured by use of a virus-like protein (VLP)-based enzyme-linked immunosorbent assay. Exfoliated cells from the tonsillar and oral mucosa were analyzed for the presence of 38 mucosal HPV types by polymerase chain reaction., Results: Women had significantly greater seroreactivity for all HPV types investigated when compared with men (odds ratio, 4.3; 95% confidence interval, 3.0-6.0). Seroprevalence was greatest in men and women aged 35 to 45 years. Tonsillar HPV infection, oral sex with men, and HIV infection were independently associated with HPV seroreactivity in men after adjustment for age and number of sexual partners. In women, HSV-2 seropositivity and a history of sexually transmitted diseases were similarly important. Oral and tonsillar HPV infection were not associated with HPV seroreactivity in women., Conclusion: HPV seropositivity is associated with sexually transmitted diseases among women and possibly mucosal HPV exposures in men. Tonsillar HPV infection could impact seroprevalence, particularly in men.

The prevalence and risk factors for oral human papillomavirus (HPV) infection are unknown, despite evidence for an etiological role for HPV in oral cancers. Oral samples from human immunodeficiency virus (HIV)-seronegative (n=396) and HIV-seropositive (n=190) adults were tested for HPV DNA. High-risk HPV infections were present in 2.1% of tonsil and 6.3% of oral-rinse specimens. The prevalence of oral high-risk HPV infection was greater in HIV-seropositive individuals (13.7% vs. 4.5%; P<.001). In multiple logistic regression, odds of oral HPV infection increased with age, male sex, and herpes simplex virus (HSV)-2 seropositivity in HIV-seronegative individuals and with CD4 cell count <200 cells/mL, HSV-2 seropositivity, oral mucosal abnormalities, and >1 oral sex partner during the previous year (odds ratio, 12.8; 95% confidence interval, 3.1-52.7) among HIV-seropositive individuals. HPV type 16, which is present in most HPV-associated tonsillar cancers, was the most prevalent high-risk oral HPV infection.

Background: Human papillomavirus (HPV), the causal agent of cervical cancer, appears to be involved in the etiology of cancer of the oral cavity and oropharynx. To investigate these associations, we conducted a multicenter case-control study of cancer of the oral cavity and oropharynx in nine countries., Methods: We recruited 1670 case patients (1415 with cancer of the oral cavity and 255 with cancer of the oropharynx) and 1732 control subjects and obtained an interview, oral exfoliated cells, and blood from all participants and fresh biopsy specimens from case patients. HPV DNA was detected by polymerase chain reaction (PCR). Antibodies against HPV16 L1, E6, and E7 proteins in plasma were detected with enzyme-linked immunosorbent assays. Multivariable models were used for case-control and case-case comparisons., Results: HPV DNA was detected in biopsy specimens of 3.9% (95% confidence interval [CI] = 2.5% to 5.3%) of 766 cancers of the oral cavity with valid PCR results and 18.3% (95% CI = 12.0% to 24.7%) of 142 cancers of the oropharynx (oropharynx and tonsil combined) with valid PCR results. HPV DNA in cancer biopsy specimens was detected less frequently among tobacco smokers and paan chewers and more frequently among subjects who reported more than one sexual partner or who practiced oral sex. HPV16 DNA was found in 94.7% of HPV DNA-positive case patients. HPV DNA in exfoliated cells was not associated with cancer risk or with HPV DNA detection in biopsy specimens. Antibodies against HPV16 L1 were associated with risk for cancers of the oral cavity (odds ratio [OR] = 1.5, 95% CI = 1.1 to 2.1) and the oropharynx (OR = 3.5, 95% CI = 2.1 to 5.9). Antibodies against HPV16 E6 or E7 were also associated with risk for cancers of the oral cavity (OR = 2.9, 95% CI = 1.7 to 4.8) and the oropharynx (OR = 9.2, 95% CI = 4.8 to 17.7)., Conclusions: HPV appears to play an etiologic role in many cancers of the oropharynx and possibly a small subgroup of cancers of the oral cavity. The most common HPV type in genital cancers (HPV16) was also the most common in these tumors. The mechanism of transmission of HPV to the oral cavity warrants further investigation.

Serum samples from 194 cases and 217 controls participating in a case-control study of invasive cervical cancer in Brazil were examined for antibodies to human papillomavirus (HPV) 16 virus-like particles (VLPs) by ELISA. The prevalence of antibody in cases and controls was 47.4 versus 24.4% (P < 0.001). The prevalence was higher in women who had HPV-16 DNA in the genital tract (54.2%) than in those with other HPVs (36.8%) or no HPVs (44.8%), but the differences were not statistically significant. Among cases and controls, HPV-16 VLP antibodies were associated with a greater number of lifetime sexual partners (chi2 for trend, P < 0.001). Among controls, age was inversely associated with HPV-16 VLP seroreactivity (chi2 for trend, P = 0.019). The sera were previously tested for antibodies to HPV-16 E6 and E7 oncoproteins; there was no correlation between antibody titers to HPV-16 E6 or E7 and VLPs. The HPV-16 serological assays were compared as screening tests for invasive cervical cancer. The sensitivity and specificity estimates were 47.4 and 75.6% for HPV-16 VLP serology, 63.4 and 89.9% for either HPV-16 E6 or E7 serology, and 53.6 and 93.6% for high titers of either HPV-16 E6 or E7 or VLP antibodies. The utility of HPV-16 VLP ELISA as a screening test for invasive cervical cancer is limited by a high seroprevalence in women with probable prior exposure to HVP 16 but without disease.

Background: The presence of human papillomavirus (HPV) in the prostate and its role in prostate carcinoma are in dispute. To address these issues, two laboratories with extensive HPV experience were selected to test specimens from two populations at different risk for prostate carcinoma, using three different polymerase chain reaction (PCR) assays and two serologic assays for HPV., Methods: The cases were comprised of 51 African-American (men at high risk for prostate carcinoma) and 15 Italian (men at intermediate risk for prostate carcinoma) men with prostate carcinoma. Controls were 108 African-American men and 40 Italian men with histologically proven benign prostate hypertrophy (BPH). Prostate tissue was obtained from each patient at surgery and immediately frozen in liquid nitrogen. The PCR primer sets included two (MY09/MY11 and GP5+/ GP6+) that amplify different regions of L1 and a third (WD66,67,154/WD72,76) targeted to E6. Sensitivity in the 2 L1 PCR assays was shown to be 1 HPV DNA genome per 100 cells. Serum antibodies to HPV-16 and HPV-11 virus-like particles (VLPs) were detected using enzyme-linked immunosorbent assays., Results: All available prostate carcinoma tissue specimens (n = 63) and BPH specimens from selected controls (n = 61) were tested by PCR. Human beta-globin DNA could be amplified from all specimens except three carcinomas, but no HPV DNA was detected in any case or control specimens by MY09/MY11 or E6 PCR. Microdissection of 27 carcinoma specimens was conducted to minimize nontumor DNA, but results remained negative by MY09/MY11 and GP5+/GP6+ PCR. In addition, serum specimens in cases (n = 63) and controls (n = 144) showed no differences in their responses against HPV-16 (P = 0.54) or HPV-11 VLPs (P = 0.64)., Conclusions: The findings suggest that HPV is not associated with prostate carcinoma, and that HPV DNA is not at all common in the prostate glands of older men.

Serological assays for measuring antibodies to human papillomavirus type 16 (HPV-16) virus-like particles (VLPs) have become important epidemiologic tools in recent years. However, the interlaboratory replicability of these assays has not been assessed. In this investigation, three laboratories tested a panel of specimens obtained from two different groups: 265 subjects in a vulvar cancer case-control study and 107 healthy volunteer blood donors. Each laboratory used an enzyme-linked immunosorbent assay (ELISA), but no attempt was made to standardize assay procedures among the three laboratories. The data showed good day-to-day intralaboratory replicability in laboratory 1 (correlation coefficient, > or = 0.88) and good intra-assay variability in laboratory 3 (correlation coefficient, > or = 0.93). Interlaboratory correlations, likewise, ranged between 0.61 and 0.80 in both case-control study subjects and healthy blood donors, indicating that ELISA optical density (OD) values between laboratories were linearly related regardless of the population. Kappa coefficients (kappa), based on each laboratory's categorical interpretation of its results (as positive or negative), showed good agreement (kappa, > 0.6) in case-control study subjects and moderate agreement (kappa, > or = 0.4) in blood donors, a population that had few strongly positive sera. When OD values near seropositive cutoffs were treated as indeterminates, there was little discordance between laboratories in either population. The data suggest that each laboratory measured the same humoral immune response and that their HPV-16 VLP ELISAs performed similarly (Pearson correlations). Interlaboratory differences, however, probably due to reagents and procedures, were considerably greater than intralaboratory day-to-day variability. Interlaboratory agreement in determining seropositivity (kappa) could be improved by sharing positive and negative serum controls and by treating marginal results as indeterminate. As part of continuing cooperation to improve interlaboratory agreement, we are preparing bulk serum control specimens to be shared and made available to interested researchers.

Our objective was to examine whether past infection with human papillomavirus (HPV)-16, as determined by an antibody assay, is a risk factor for subsequent cervical cancer. Incident cases of in situ or invasive cervical cancer occurring between 1975 and 1990 in a cohort of over 11,000 healthy women in Washington County, MD, were identified. The baseline sera of cases and of matched controls, collected in 1974, were examined for IgG antibodies reactive with virus-like particles of HPV-16, a cancer-associated HPV, and HPV-6, a low-risk HPV. Postdiagnosis sera of 11 cases were also assessed similarly. Fourteen cases of invasive and 28 cases of in situ cervical cancer and 83 matched controls were evaluated. The main outcome measure was the risk of cervical cancer in women who had HPV-16 or HPV-6 antibodies in prediagnostic sera. Antibodies to HPV-16 but not to HPV-6 were a marker for subsequent occurrence of cervical cancer. Case sera were reactive more often and more strongly with HPV-16 virus-like particles than were sera of matched controls. The presence of antibodies to HPV-16 was significantly associated with an increased risk of cervical cancer (odds ratio, 3.9; 95% confidence limits, 1.4, 10.7); high antibody levels to HPV-16 were associated with an even greater risk of cervical cancer (odds ratio = 7.5, 95% confidence limits 1.5, 36.3). The association with cervical cancer was strengthened after adjustment for smoking and years of education. In tests of 11 pairs of pre- and postdiagnostic sera, HPV-16 antibodies did not decline markedly over a 7-13-year time period, and seroconversion to HPV-16 appeared to have occurred in 2 cases. The serological data indicate that HPV-16 infection is associated with future risk of cervical cancer and strengthen the evidence for the etiological role of HPVs in cervical cancer.

A human papillomavirus type 16 (HPV-16) virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA) was used to measure serum antibody to capsid proteins in 376 sexually active college women who were also screened for the presence of genital HPVs by PCR and interviewed for demographic and behavioral risk factors for HPV infection. The seroprevalence was 46% in women with HPV-16 DNA in the genital tract. The corresponding values for women who harbored other HPV types or no HPV in the genital tract were 30 and 19%, respectively (HPV-16 group versus no-HPV group; odds ratio [OR], 3.7; 95% confidence interval [CI], 1.5 to 8.9). The antibody response was significantly higher among women with a high viral load than among those with a low viral load (median optical density value, 0.838 versus 0.137, P = 0.009). Comparable levels of seroreactivity were observed among women infected with HPV types distantly or closely related genetically to HPV-16. Seroreactivity was significantly associated with an age of 25 to 30 years (OR, 2.3; 95% CI, 1.2 to 4.4), three or more lifetime sexual partners (OR, 2.9; 95% CI, 1.1 to 10), and history of a sexually transmitted disease other than HPV (OR, 3.1; 95% CI, 1.5 to 6.3). The percent seropositivity increased linearly with number of lifetime sexual partners until reaching a plateau at 35% for women with more than six partners (chi for linear trend, P < 0.001). The low sensitivity of HPV-16 VLP-based ELISA may limit the usefulness of the assay as a diagnostic test for HPV-16 infection. However, the assay appears to have adequate specificity and should be useful as an epidemiological marker of HPV-16 infection and sexual behavior.

Epidemiological and virological evidence suggests that invasive squamous cell carcinoma (SCC) of the vulva is etiologically heterogeneous and that basaloid or warty SCC (BWSCC) and vulvar intraepithelial neoplasia (VIN) are linked to human papillomavirus (HPV) infections while keratinizing SCC (KSCC) is a non-HPV-associated malignancy. In the present study, HPV-specific antibodies in sera of patients with BWSCC, VIN, and KSCC and of controls were examined by ELISA for antibodies reactive to HPV-16 virus-like particles (VLP) and in radioimmunoprecipitation assays for antibodies to HPV-16 E6 and E7 proteins expressed by in vitro transcription and translation. The prevalences of antibodies to HPV-16 VLPs were significantly higher in HPV-associated VIN (59.1%) and BWSCC (50.0%) than in KSCC (22.2%) and controls (18.2%). Antibodies to E6 and E7 proteins were more prevalent in BWSCC than in any other groups. Prevalence of serum antibodies to any one of the antigen preparations was significantly higher in BWSCC (64.3%) and VIN (59.1%) than in KSCC (27.8%) and controls (22.2%). Also, sera with high antibody titers were found more frequently in BWSCC and VIN cases than in controls. These data provide immunological evidence in support of the observation that VIN and BWSCC, but not KSCC, are associated with HPV infections.

In a cross-sectional study, titers of antibodies to the E4 and E7 proteins of human papillomavirus (HPV) type 16 were measured by peptide-based enzyme-linked immunosorbent assay in 1707 sera. Sera were obtained from healthy individuals (ages 1 to 95 years), from patients with HPV-associated infection (cervical intraepithelial neoplasia and cervical cancer), and from patients who were at high risk for HPV infection (attending a sexually transmitted disease clinic or referred to a colposcopist because of an abnormal Papanicolaou smear). The prevalence of anti-E7 antibodies increased with age, although the overall prevalence in the adult population was low (10.36%) compared to the frequent detection of HPV 16 DNA in the population. This suggests that only a fraction of patients infected with HPV 16 develop an anti-E7 response. The age distribution of anti-E4 antibodies showed a different pattern, i.e., the prevalence was low in the adult population (1.14%) but exceeded 20% in children and teenagers. As the specificity of the anti-E4 reaction was supported by a highly significant association with anti-E6 positivity in children's sera (p = 0.002), it was assumed that infection with HPV 16 can occur frequently early in life. As compared to healthy controls, patients at high risk for HPV infection had a significantly higher frequency (p < 0.001) of antibodies to the HPV 16 E4 protein (but not to the E6 or the E7 protein) in their sera. Therefore, we conclude that in adults E4-specific antibodies may be a marker for virus replication.

Antibodies to human papilloma virus (HPV) type 16 (HPV-16) E6 and E7 proteins in serum are markers for HPV-associated invasive cervical carcinoma. We compared two assays, a radioimmunoprecipitation assay with in vitro-translated HPV-16 E6 and E7 proteins and an enzyme-linked immunosorbent assay (ELISA) with E6 and E7 synthetic peptides, for their abilities to discriminate serologically between patients with invasive cervical cancer and controls. Among the patients, antibody prevalences were higher by the E6 radioimmunoprecipitation assay (55.7%) than by the E6 peptide ELISA (15.5%), but among the controls, they were lower by the radioimmunoprecipitation assay (1.7%) than by the E6 peptide ELISA (5%). For E7, antibody prevalences among the patients were comparable by the radioimmunoprecipitation assay (43%) and the peptide ELISA (41%), but among the controls they were higher by the E7 peptide ELISA (17.4%) than by the radioimmunoprecipitation assay (4.1%). There was good agreement between the E7 radioimmunoprecipitation assay and the E7 peptide ELISA among patients but not among controls. In tests with representative sera, heat denaturation of the translated proteins resulted in a complete loss of reactivity to the E6 protein and a marked decrease in reactivity to the E7 protein. Our study showed that the radioimmunoprecipitation assay discriminates better than the peptide ELISA between patients with invasive cervical cancer and controls and that this is related to the ability of the radioimmunoprecipitation assay to detect conformational epitopes.

Transforming proteins E6 and E7 of human papillomaviruses (HPVs) are consistently expressed in HPV-associated cervical cancers. In ELISA with four HPV-16 E6-E7 peptides, patients with HPV-16-associated invasive cervical cancer (group 1) had a greater seroreactivity than all other groups, which included patients with HPV-16-associated cervical intraepithelial neoplasia, invasive cervical cancer patients without HPVs, and unaffected controls. A larger proportion of group 1 sera, as compared to sera of all other groups, was reactive with at least one peptide (49% vs 17-27%), and with two or more peptides (22% vs 0-6%). A clear difference between group 1 and all other groups was also found for high ELISA absorbance values to at least one peptide (22% vs 0-8%). This high seroreactivity of group 1 sera was confirmed by a radioimmunoprecipitation assay with in vitro transcribed and translated HPV-16 E7 protein. Sera from 50% of group 1 but only 3% of controls were reactive in this test. Antibodies to HPV-16 E6 and E7 proteins appear to be virus-specific and disease state-specific markers of HPV-associated cervical cancer.