Tim Waterboer

BACKGROUND: More than 100 different human papillomaviruses (HPVs) can cause proliferative diseases, many of which are malignant, such as cervical cancer. HPV serology is complex because infection and disease lead to distinct type-specific antibody responses. Using bead-based technology, we have developed an assay platform that allows the simultaneous detection of antibodies against up to 100 in situ affinity-purified recombinant HPV proteins. METHODS: Twenty-seven HPV proteins were expressed as glutathione S-transferase fusion proteins and affinity-purified in one step by incubation of glutathione-displaying beads in bacterial lysate. Spectrally distinct bead sets, each carrying one particular antigen, were mixed, incubated with serum, and differentiated in a flow cytometer-like analyzer (xMAP; Luminex Corp). Antibodies bound to the antigens were detected via fluorescent secondary reagents. We studied 756 sera from 2 case-control studies of cervical cancer. RESULTS: Glutathione S-transferase fusion proteins bound with high affinity to glutathione-displaying beads (Kd = 6.9 x 10(-9) mol/L). The dynamic range of multiplex serology covered 1.5 orders of magnitude, and antibodies were detected at serum dilutions >1:1,000,000. Imprecision (median CV) was < or = 5.4%, and assay reproducibility was high (R2 = 0.97). Results on clinical samples showed high concordance with ELISA (kappa = 0.846), but multiplex serology exhibited increased detection of weak antibody responses. Antibodies to the E6 oncoproteins of the rare HPV types 52 and 58 were associated with cervical cancer (P < 0.001). CONCLUSION: Multiplex serology enables antibody analyses of large numbers of sera against up to 100 antigens in parallel and has the potential to replace ELISA technology.

BACKGROUND: Long COVID is defined as the persistence of symptoms beyond 3 months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To better understand the long-term course and etiology of symptoms we analyzed a cohort of patients with COVID-19 prospectively. METHODS: Patients were included at 5 months after acute COVID-19 in this prospective, noninterventional, follow-up study. Patients followed until 12 months after COVID-19 symptom onset (n = 96; 32.3% hospitalized, 55.2% females) were included in this analysis of symptoms, quality of life (based on an SF-12 survey), laboratory parameters including antinuclear antibodies (ANAs), and SARS-CoV-2 antibody levels. RESULTS: At month 12, only 22.9% of patients were completely free of symptoms and the most frequent symptoms were reduced exercise capacity (56.3%), fatigue (53.1%), dyspnea (37.5%), and problems with concentration (39.6%), finding words (32.3%), and sleeping (26.0%). Females showed significantly more neurocognitive symptoms than males. ANA titers were ≥1:160 in 43.6% of patients at 12 months post-COVID-19 symptom onset, and neurocognitive symptom frequency was significantly higher in the group with an ANA titer ≥1:160 versus <1:160. Compared with patients without symptoms, patients with ≥1 long-COVID symptom at 12 months did not differ significantly with respect to their SARS-CoV-2 antibody levels but had a significantly reduced physical and mental life quality compared with patients without symptoms. CONCLUSIONS: Neurocognitive long-COVID symptoms can persist ≥1 year after COVID-19 symptom onset and reduce life quality significantly. Several neurocognitive symptoms were associated with ANA titer elevations. This may indicate autoimmunity as a cofactor in etiology of long COVID.

Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.

PURPOSE: Human papillomavirus type 16 (HPV16) infection is causing an increasing number of oropharyngeal cancers in the United States and Europe. The aim of our study was to investigate whether HPV antibodies are associated with head and neck cancer risk when measured in prediagnostic sera. METHODS: We identified 638 participants with incident head and neck cancers (patients; 180 oral cancers, 135 oropharynx cancers, and 247 hypopharynx/larynx cancers) and 300 patients with esophageal cancers as well as 1,599 comparable controls from within the European Prospective Investigation Into Cancer and Nutrition cohort. Prediagnostic plasma samples from patients (collected, on average, 6 years before diagnosis) and control participants were analyzed for antibodies against multiple proteins of HPV16 as well as HPV6, HPV11, HPV18, HPV31, HPV33, HPV45, and HPV52. Odds ratios (ORs) of cancer and 95% CIs were calculated, adjusting for potential confounders. All-cause mortality was evaluated among patients using Cox proportional hazards regression. RESULTS: HPV16 E6 seropositivity was present in prediagnostic samples for 34.8% of patients with oropharyngeal cancer and 0.6% of controls (OR, 274; 95% CI, 110 to 681) but was not associated with other cancer sites. The increased risk of oropharyngeal cancer among HPV16 E6 seropositive participants was independent of time between blood collection and diagnosis and was observed more than 10 years before diagnosis. The all-cause mortality ratio among patients with oropharyngeal cancer was 0.30 (95% CI, 0.13 to 0.67), for patients who were HPV16 E6 seropositive compared with seronegative. CONCLUSION: HPV16 E6 seropositivity was present more than 10 years before diagnosis of oropharyngeal cancers.

A total of 308,036 women were selected from the European Prospective Investigation into Cancer and Nutrition (EPIC) study to evaluate the association between tobacco smoking and the risk of cervical intraepithelial neoplasia of grade 3 (CIN3)/carcinoma in situ (CIS) and invasive cervical cancer (ICC). At baseline, participants completed a questionnaire and provided blood samples. During a mean follow-up time of 9 years, 261 ICC cases and 804 CIN3/CIS cases were reported. In a nested case-control study, the baseline sera from 609 cases and 1,218 matched controls were tested for L1 antibodies against HPV types 11, 16, 18, 31, 33, 35, 45, 52, 58, and antibodies against Chlamydia trachomatis (CT), and Human Herpes Virus 2 (HHV-2). Cervical samples were not available for HPV-DNA analysis in this study. Multivariate analyses were used to estimate associations between smoking and risk of CIN3/CIS and ICC in the cohort and the case-control studies. In the cohort analyses smoking status, duration and intensity showed a two-fold increased risk of CIN3/CIS and ICC, while time since quitting was associated with a two-fold reduced risk. In the nested case-control study, consistent associations were observed after adjustment for HPV, CT and HHV-2 serostatus, in both HPV seronegative and seropositive women. Results from this large prospective study confirm the role of tobacco smoking as an important risk factor for both CIN3/CIS and ICC, even after taking into account HPV exposure as determined by HPV serology. The strong beneficial effect of quitting smoking is an important finding that will further support public health policies for smoking cessation.