Rapid Point of Care Detection of HPV-Associated Malignancies

项目摘要。尽管开发出了有效的人类乳头瘤病毒(HPV)疫苗，但它一直是 预计到2020年，宫颈癌新增病例将超过20万例，死亡人数将超过10万人 在印度，该国的病例负担占全球的25%。巴氏涂片和人乳头瘤病毒联合筛查宫颈癌 DNA检测在美国和欧洲已经成为医疗保健的标准，但过于昂贵和后勤成本太高 在中低收入国家(LMIC)具有挑战性。一种简单的护理点(POC)生物标记面板 高度不典型增生和宫颈癌可以为宫颈疾病的分流提供一种经济有效的手段 这些国家。人乳头瘤病毒感染诱导具有抗人乳头瘤病毒抗体的体液免疫应答 衍生蛋白质。我们之前已经确定了针对五种HPV抗原的抗体，这些抗体可以检测到 88%的HPV阳性口咽癌患者，但不到4%的健康对照。使用扩展的 ，我们在45%的宫颈CIN II/III患者的血清中检测到至少一种HPV抗体。 发育不良。这些结果支持发展一种快速、定量和多重的检测方法。 HPV相关宫颈疾病的检测。我们利用我们的蛋白质微阵列技术在 荧光技术使HPV的荧光、可编程、多重ELISA(血清学)检测成为可能- 紧凑的一次性配置的特异性免疫球蛋白抗体具有高分析灵敏度和快速， 量化产出。我们在这方面的初步工作证明可以检测到HPV的Ig G抗体 检测下限在10pg/ml范围内的抗原，比现有的提高了100倍 比色法。在我们提议的努力下，我们的目标是开发和演示一个原型 同时具有高分析灵敏度的多重荧光可编程照射点分析 从单根手指大小的患者血液中检测16种HPV特异性Ig G血清生物标志物 每个患者样本的试剂总成本不到1美元。我们将利用我们的 擅长免疫分析开发，具有微流体、电子工程、光学、生物标记物方面的专业知识 针对LMIC临床环境的设备和分析进行分析和临床验证。我们还将 提供临床平台和生物信息库，以快速评估宫颈筛查的新兴技术。 这项提议将使工程和生物化学适用于开发强大的PoC设备 专门针对低收入和中等收入国家的HPV血清学检测。我们将对此进行转移和评估 技术，在印度筛查13,000名患者，与全印度医学研究所的一个合作者团队 科学学院(AIIMS)，印度新德里。

Project Summary. Despite the development of effective human papillomavirus (HPV) vaccines, it has been estimated that there will be over 200,000 new cases and over 100,000 deaths due to cervical cancer by 2020 in India, which has 25% of the global burden of cases. Cervical cancer screening by Pap smears and HPV DNA testing has become standard of care in the US and Europe, but has been too expensive and logistically challenging in low and middle income countries (LMICs). A simple, point-of-care (POC) biomarker panel for high-grade dysplasia and cervical cancer could provide a cost-effective means for triage of cervical disease in these countries. HPV infection induces systemic humoral immune responses with IgG antibodies to HPV- derived proteins. We have previously identified antibodies to a panel of five HPV antigens that detect up to 88% of patients with HPV+ oropharyngeal cancer, but less than 4% of healthy controls. Using an expanded panel of 16 HPV antigens, we have detected at least one HPV antibody in the sera of 45% of CIN II/III cervical dysplasia. These results support the development of a rapid, quantitative, and multiplexed assay for the detection of HPV-related cervical disease. We leverage our protein microarray technology with advances in fluorescent technologies to enable a fluorescent, programmable, multiplexed ELISA (serologic) assay for HPV- specific IgG antibodies in a compact and disposable configuration with high analytic sensitivity and rapid, quantitative output. Our preliminary work in this area has demonstrated detection of IgG antibodies to HPV antigens with lower limits of detection in the 10pg/mL range, which is a 100-fold improvement over existing colorimetric approaches. Under our proposed effort, we aim to develop and demonstrate a prototype multiplexed fluorescent programmable point-of-care assay with high analytical sensitivity for the simultaneous detection of 16 individual HPV-specific IgG serologic biomarkers from a single finger stick-sized patient blood sample which can be manufactured for a total reagent cost of less than $1/patient sample. We will leverage our expertise in immunoassay development with expertise in microfluidics, electrical engineering, optics, biomarker analytical and clinical validation to target the device and assays for the LMIC clinical setting. We will also provide a clinical platform and biorepository to rapidly evaluate emerging technologies for cervical screening. This proposal will adapt the engineering and biochemistry for the development of robust POC devices for detection of HPV serology specifically for low and middle-income countries. We will transfer and evaluate this technology, screening 13,000 patients in India, with a team of collaborators in the All India Institute of Medical Sciences (AIIMS) in New Delhi, India.