Development of a human papillomavirus competitive luminex immunoassay for 9 HPV types

Abstract

In the clinical trials of the quadrivalent human papillomavirus (qHPV) vaccine, antibodies were measured by a competitive Luminex immunoassay (HPV-4 cLIA). A nine-valent HPV (9vHPV) vaccine targeting the types in the qHPV vaccine (HPV6/11/16/18), as well as 5 of the next most frequent HPV types found in cervical cancers worldwide (HPV31/33/45/52/58) is under development. To support the 9vHPV vaccine program, a nine-multiplexed cLIA (HPV-9 cLIA) was developed. Antibody titers were determined in a competitive format, where type-specific phycoerythrin (PE)-labeled, neutralizing mAbs (mAbs-PE) compete with an individual's serum antibodies for binding to conformationally sensitive, neutralizing epitopes on the VLPs. Neutralizing antibody levels were quantitated against a reference standard - a pool of sera from 6 Rhesus macaques that were immunized with the 9vHPV vaccine. Specificity of the mAbs was assessed by measuring their individual binding capacities to the type-specific and non-type-specific VLPs at alternative concentrations of the mAbs. Antibody assignments to the HPV-9 cLIA reference standard for HPV6/11/16/18 were determined to provide for a measure of consistency in serostatus assignment between the HPV-4 and HPV-9 cLIAs. Antibody assignments to the HPV-9 reference standard for HPV31/33/45/52/58 were obtained by calibration to HPV11 using a direct binding IgG assay. For each HPV VLP type, the cross-reactivity of the mAb-PEs in the HPV-9 cLIA was <1% (i.e., the mAb-PEs result in <1% non-specific binding). The antibody concentrations assigned to the HPV-9 cLIA reference standard for types 6/11/16/18/31/33/45/52/58 were 3,817, 2,889, 23,061, 5,271, 3,942, 2,672, 1,489, 1274, and 2263 mMU/mL, respectively.

Keywords: Luminex; assay; human papillomavirus; vaccine.

Figures

Figure 1. (A) HPV-9 cLIA measures antibody concentration in a competitive format. Antibodies in test sera compete with type-specific mAbs for binding to neutralizing epitopes on each type-specific VLP coupled to Luminex microspheres. (B) Representative reference standard curve for HPV type 31 illustrating the competitive format of the HPV-9 cLIA. The circles denote the observed MFIs; the solid line shows the fitted curve; and the vertical dashed lines indicate the HPV type 31 lower and upper limits of quantitation. Similar curves are obtained for the other 8 HPV types.

Figure 2. Comparison of HPV-4 cLIA and HPV-9 cLIA antibody titers to assign antibody values to the HPV-9 reference standard for HPV6, 11, 16, and 18.

Figure 3. Cross-tabulation of test sample serostatus assignment between the HPV-4 and HPV-9 cLIAs and percent difference estimates for specific titer levels within the quantifiable range of the HPV-4 cLIA. The purpose of the evaluation was to determine the most appropriate assignment of antibody values for the HPV-9 cLIA reference standard. *Serostatus cutoff for the HPV-4 cLIA.