Abstract
The involvement of human papilloma virus (HPV) in the aetiology and progression of cervical intraepithelial neoplasia (CIN) is still unresolved. This study was designed to assess the immunological responses to HPV types in patients presenting with varying degrees of CIN and in control groups, using in vitro measures of cellular and humoral responses.
Lymphocyte proliferation assays (LPA) were performed using peripheral blood mononuclear cells (PBM) and various papillomavirus (PV) antigens. Twenty -five per cent (23/92) of patients with CIN responded to antigens derived from purified BPV, HPV -1 or HPV -2 with or without detergent disruption. The responses correlated with a past history of skin warts rather than cervical abnormalities, and the percentage of responders was similar to that in laboratory personnel (30 %) and lower than that in a group with recalcitrant common warts (50 %). Antigens specific to HPV -16 and HPV -18, in the form of bacterially expressed fusion proteins derived by the transcription and translation of the E6 and E4 open reading frames (ORF), occasionally produced specific positive responses, provided contaminating E.coli B galactosidase sequences had been removed during purification. Responses were low and suggested that the numbers of memory T cells specific to PV antigens were low and at the lower limit of detection of LPA. An indirect ELISA was developed to detect circulating IgG to PV antigens in colposcopy patients. Fifty per cent of patients had antibodies to disrupted HPV -1, HPV -2 or both, suggesting that a predominantly type- specific response was being detected. No correlation of immune responses with a degree of dysplasia or the presence of koilocytes in cervical biopsies was noted, but a high incidence of forgotten or inapparent past infection with cutaneous HPV types was found.
In situ hybridisation (ISH) methods using non -radioactively labelled, cloned probes and synthetic oligonucleotide probes were developed for use on paraffin sections. Synthetic probes allowed a quicker, less destructive hybridisation protocol, with the sensitivity of detection being
(y) improved by an anti -biotin -immunogold conjugated immunoglobulins -silver enhancement (IGSS) detection system. Double staining of PV antigen and nucleic acid on the same section was achieved. Synthetic oligonucleotides offer an exciting new tool for diagnostic virology, worthy of exploitation in many systems.
Implantation of human foreskin infected with HPV -11 was shown to provide an animal model, albeit technically difficult, in which HPV could be produced, but a more practical technique of productive HPV infection in vitro is still required if the biology and pathogenesis of HPV infections is to be clarified further.
