Over 600 000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers (OPC). A key challenge in understanding HPV immunobiology is the diversity of oncogenic HPV types and the need for multiplexed display of HPV antigens to measure antibody responses. We have generated custom HPV protein microarrays displaying 98 proteins as C-terminal GST fusion proteins, representing eight antigens of two low-risk HPV types (HPV6 and 11) and ten oncogenic high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58). We demonstrate robust and reproducible protein expression of 96/98 of the antigens using a human cell lysate expression system. The target epitopes and specificities of four monoclonal antibodies were identified. Using sera from ten patients with newly diagnosed OPC and ten controls, we demonstrate specific IgG seroreactivity to HPV16 E1, E2, and E7 (a fold increase of 1.52, 2.19 and 1.35 in cases vs. controls, respectively, all p < 0.005), confirming our prior data on an ELISA platform. We also detect HPV52 E7 Abs in serum from a patient with cervical cancer. The HPV protein array has potential for rapid identification of serologic responses to 12 HPV types.

DOI: HPV proteome arrays for immunoprofiling of HPV‐associated cancers. Left: Genomes of 12 HPV types were printed on the microarrays and picogreen was used to detect DNA content. Middle: Detection of GST‐tagged HPV proteins expressed in situ on the arrays. Right: Profiling of HPV‐specific IgG in human serum from patients with oropharyngeal cancer. For more details, see the article by Radwa Ewaisha et al. on page 1215.

Introduction: More than 260,000 women die of cervical cancer every year. Screening methods have reduced the incidence of cervical cancer in high-income countries, but detection continues to lag in low and middle income countries (LMICs). IgG antibody (Ab) immunity to early (E) HPV antigens (Ags) are potential biomarkers of disease progression. Since HPV16 accounts for only ~50% of invasive cervical cancers, we developed protein microarrays expressing the proteomes of 12 HPV types to detect host IgG Abs to a broad spectrum of viral Ags to detect pre-invasive and invasive cervical disease. Methods: We developed custom HPV protein microarrays displaying the proteomes of two low-risk HPV types (HPV6 and 11) and ten oncogenic high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58). Arrays were probed with serum samples obtained from women with invasive cervical cancer (ICC; n=80), no or low-grade cervical disease (CIN 0/I; n=60), and high-grade cervical dysplasia (CIN II/III; n=60). To identify positive serologic responses, arrays were scanned and the signal intensity of each protein spot was quantified and normalized. Visual examination of diffused signal (ring) around each spot was performed and the Ab response to each protein was scored on a scale from 0 to 5. Results: To verify array quality and reproducibility, we confirmed high protein expression levels for 98% (96/98) of the antigens printed and high correlation (R ≥ 0.90) of protein expression signals between different randomly selected arrays. Epitope expression was confirmed using four commercial monoclonal Abs raised against HPV16 Ags. Host Abs to at least one early antigen (E1, E2, E4, E6, or E7) were detected in the sera of 31.2% and 43.3% of ICC and CINII/III patients, respectively, compared with 11.7% of women with CIN 0/I. 73.1% of CINII/III cases detected had Abs only to non-HPV16 Ags. Abs to E1, E2, E4, E6, and E7 Ags were detected in 3.8%, 7.7%, 54%, 19%, and 31% of CINII/III versus 16%, 16%, 32%, 32%, and 40% of ICC cases that were positive. The immunodominant Ags in women with CINII/III and ICC were E4 (54%) and E7 (40%), respectively. These results are consistent with the difference in tissue expression levels of these proteins in these two disease stages. This emphasizes the importance of broadening the scope of serological detection to include non-HPV16 proteomes. Conclusions: This study demonstrates that Abs against high risk HPV16 and non-HPV16 serotypes are potential biomarkers for both the diagnosis of cervical cancer and the differentiation between high grade and low-grade pre-invasive cervical lesions. These results suggest that serology is a potential minimally invasive tool for early detection of cervical cancer. High-throughput immunoprofiling of HPV-associated cancers and pre-invasive lesions may be informative for understanding the diversity of host immunity and viral progression. Citation Format: Radwa Ewaisha, Ian Meshay, Jack Resnik, Tirinder Bharaj, Karen S. Anderson. Detection of circulating immune biomarkers of cervical disease using proteome arrays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2217. doi:10.1158/1538-7445.AM2017-2217