Binding of human papillomavirus type 16 to heparan sulfate is inhibited by mucosal antibodies from patients with low-grade squamous intraepithelial lesions but not from cervical cancer patients.

Mucosal antibodies against human papillomavirus type 16 (HPV16) capsids have been detected in infected women. To determine whether these antibodies recognize and block the receptor site mediating attachment of HPV16 to heparan sulfate, mucus samples from 126 HPV16-associated low-grade squamous intraepithelial lesion (LSIL) and 85 cervical cancer patients, previously found to react to HPV16 virus-like particles (VLP), and 101 normal controls were tested in an inhibition assay, using HPV16 VLP and heparan sulfate proteoglycan-coated plates. Inhibition levels of 9.3-67.2% were mediated by type-specific antibodies in 94.4% of LSIL patients. Cervical cancer cases showed significantly lower levels of inhibition than LSIL samples (P < 0.0001). The potential of antibodies to inhibit infection was explored in a pseudoinfection system using HPV16 pseudovirions. Inhibition of pseudoinfection by LSIL samples was significantly higher than that observed in the controls (P < 0.001) and cervical cancer cases (P < 0.005). These results indicate that mucosal antibodies inhibiting binding of VLP to heparan sulfate are developed in most LSIL patients, but are hardly present in cervical cancer patients.

Construction of an internal RT-PCR standard control for the detection of human caliciviruses in stool.

RT-PCR is the most sensitive assay for the detection of human caliciviruses (HuCV) in stool and environmental samples. However, false negative results are commonly obtained due to the presence of RT-PCR inhibitors. In order to exclude such false negative results, an internal control (IC) was developed for the assay by cloning a 319 nt sequence of the Norwalk virus (NV) polymerase containing a 156 nt cDNA insert. The RT-PCR assay was carried out using RNA derived from the constructed plasmid and a primer set previously described for calicivirus detection, resulting in a 475 nt product. Distinct bands of the internal control and the viral specific RT-PCR products (319 nt) were obtained when the internal control was added to the samples. Similar results were also obtained when both the control RNA and viral RNA were seeded into stool samples from asymptomatic volunteers, or when the internal control was included into positive samples. Since the primer set used in the assays can detect a wide range of strains in both norovirus and sapovirus genera, this internal control should have a broad application for the diagnosis of human caliciviruses diagnosis in both clinical and environmental samples.