Development of a first-void urine based highly sensitive competitive HPV immunoassay.

Detection of transudated antibodies in female genital secretions, washed away with the first fraction of urine – i.e. first-void (FV) urine –, has been confirmed. In addition, vaccine-induced HPV antibodies have been detected in FV urine of women using different immunoassays. Although good correlations between paired FV urine and serum samples have been observed, urinary antibody titres are at least 1000-fold lower than serum antibody titres. To be able to distinguish between both vaccinated vs. not-vaccinated and seroconverted unvaccinated vs. non-seroconverted unvaccinated women using FV urine, antibody yield and assay sensitivity need to be increased. The first step in our process to upgrade the detection of HPV-specific antibodies in FV urine, will be the production of HPV pseudovirions (PsV) for the quadrivalent vaccine types (HPV6, 11, 16, 18) to be used in a highly sensitive immunoassay (WP1). These PsV will be used to create HPV conformational monoclonal antibodies (mAbs) in mice using the hybridoma technology (WP2.1). These mAbs will be made type-specific by desensitisation for all other included HPV PsV types before immunisation with the HPV PsV type of interest. To evaluate the quality of the produced mAbs, a DELFIA time-resolved fluorescence (TRF) assay will be developed using the created PsV. In addition, the neutralizing abilities of the generated mAbs will be assessed using our in-house pseudovirion based neutralisation assay (WP2.2). The produced PsV and mAbs will then be used in a multiplex competitive highly sensitive ELISA using the TRF technology (WP3). It is clear that monitoring neutralizing HPV antibodies non-invasively by using FV urine samples has major advantages since it (i) is an easy to collect non-invasive sample, (ii) can also provide information about the infection by applying parallel DNA testing and, (iii) is suitability for at-home sampling (currently boosted by the COVID-19 pandemic). If successful, the created assay provides a very useful and almost unique tool to monitor neutralizing HPV antibodies. With only limited adaptations, the developed assay will be compatible with serum samples as well. Since there are limited HPV immunoassays available overall, and currently only one other assay (cLIA) detects specifically neutralizing antibodies, this assay will be of great value. In addition, the validated pseudovirions and HPV type-specific neutralizing mAbs will generate substantial interest and wide applications in the field.

Keywords:HPV, HPV ANTIBODIES, URINE, SAMPLING

Disciplines:Vaccinology, Infectious diseases, Virology, Diagnostics