Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18

Abstract

More than 170 types of human papilloma viruses (HPV) exist with many causing proliferative diseases linked to malignancy in indications such as cervical cancer and head and neck squamous cell carcinoma. Characterization of antibody levels toward HPV serology is challenging due to complex biology of oncoproteins, pre-existing titers to multiple HPV types, cross-reactivity, and low affinity, polyclonal responses. Using multiplex technology from MSD, we have developed an assay that simultaneously characterizes antibodies against E6 and E7 oncoproteins of HPV16 and 18, the primary drivers of HPV-associated oncogenesis. We fusion tagged our E6 and E7 proteins with MBP via two-step purification, spot-printed an optimized concentration of protein into wells of MSD 96-well plates, and assayed various cynomolgus monkey, human and HPV+ cervical cancer patient serum to validate the assay. The dynamic range of the assay covered 4-orders of magnitude and antibodies were detected in serum at a dilution up to 100,000-fold. The assay was very precise (n = 5 assay runs) with median CV of human serum samples~5.3% and inter-run variability of 11.4%. The multiplex serology method has strong cross-reactivity between E6 oncoproteins from human serum samples as HPV18 E6 antigens neutralized 5 of 6 serum samples as strongly as HPV16 E6. Moderate concordance (Spearman's Rank = 0.775) was found between antibody responses against HPV16 E7 in the multiplex assay compared to standard ELISA serology methods. These results demonstrate the development of a high-throughput, multiplex assay that requires lower sample quantity input with greater dynamic range to detect type-specific anti-HPV concentrations to E6 and E7 oncoproteins of HPV16 and 18.

Immunodiagnosis and Immunotherapeutics Based on Human Papillomavirus for HPV-Induced Cancers

“…An assay based on electrochemiluminescence (ECL) was developed by using multiplex technology from Meso Scale Diagnostics (MSD, Rockville, MD) with maltose-binding protein (MBP)-tagged E6 and E7 oncoproteins. This is a high-throughput method, but it requires lower sample quantity input with greater dynamic range to detect type-specific anti-HPV E6 and E7 oncoproteins ( 87 ).…”

Association of high-risk human papillomavirus infection duration and cervical lesions with vaginal microbiota composition

“…FASTQ files were obtained by running the sequencing machine according to the manufacturer's instructions, and the overlapping paired-end FASTQ files were processed in a data curation pipeline implemented in QIIME 2, version 2017. 7…”

“…Generally, as a non-lytic infection, this process does not result in a strong immune response or inflammation by activating Toll-like receptors and natural killer cells (6). Currently, more than 170 types of HPV have been discovered, and >40 of these are associated with female reproductive tract infections and cervical lesions (7). HPV types can be broadly divided into high-and low-risk groups according to their differences in pathogenicity.…”

Production of recombinant HPV11/16 E6/E7-MBP-His6 fusion proteins and their potential to induce cytokine secretion by immune cells in peripheral blood

“…Although MBP could increase the solubility of HPV16 E6 protein, some proteins still inevitably aggregate [16,17]. Moreover, the hydrophobic nature of E6 proteins can lead to protein aggregation and loss during chromatography steps [15,18].…”