Understanding oncogenic human papillomavirus persistence and immune modulation in tonsil epithelia

病毒是专性细胞内寄生虫，其已经进化为操纵靶细胞和局部组织环境以促进复制。一些病毒可以建立靶细胞的持续性亚临床感染，这些感染可以逃避宿主免疫检测并存在数十年。人乳头瘤病毒（HPV）是一种感染人类皮肤潮湿和干燥表面的病毒家族。感染的靶细胞是一种特殊类型的皮肤细胞，称为角质形成细胞。许多不同类型的HPV感染导致疣的形成，但持续感染HPV类型的子集，所谓的高危HPV，是身体肛门和生殖器区域（包括子宫颈）癌症的原因，以及头颈部癌症，特别是扁桃体和舌根，统称为口咽。虽然存在通过疫苗接种和国家宫颈筛查方案预防高危HPV感染和检测癌前病变的强有力战略，但并非所有国家都有这些措施。事实上，尽管进行了筛查和疫苗接种，但全球每年HPV相关癌症的数量仍保持在60万例左右。此外，HPV驱动的口咽癌的发病率在年轻，主要是男性和非吸烟者的患者中正在增加。口咽部HPV感染的主要部位是扁桃体，扁桃体是一种富含免疫细胞的器官，可以检测入侵的病原体并预防呼吸道和胃肠道感染。因此，在扁桃体中建立持续的高危HPV感染必须强烈抑制局部免疫细胞活化。大多数（超过90%）HPV阳性口咽癌是由HPV 16型（HPV 16）引起的。虽然HPV16也是宫颈癌中最常见的HPV（约60%），但其负担远低于口咽癌。另一种高风险类型，HPV18占宫颈癌的约15%，但很少在口咽癌中发现。不同身体部位疾病相关性差异的原因尚不清楚。我们的初步数据表明，HPV16，而不是HPV18，是能够通过重编程宿主扁桃体角质形成细胞的基因表达来抑制免疫激活分子的产生。使用最先进的HPV感染的原代细胞模型和尖端的DNA和基因测序方法，我们将研究扁桃体角质形成细胞中HPV16和HPV18生命周期的相似性和差异。我们将确定HPV16如何通过抑制激活和吸引局部免疫细胞到感染部位的关键途径来抑制宿主免疫检测。然后，我们将利用我们在原代细胞培养方面的专业知识，将联合收割机原代角质形成细胞与供体匹配的淋巴样组织（包含扁桃体免疫细胞）结合，建立体外扁桃体器官样培养物，用于研究免疫细胞活化和迁移对HPV感染的反应。该项目的结果将揭示扁桃体中HPV持续存在的新机制。我们还将确定致癌HPV 16和HPV 18在免疫抑制方面的关键差异，以及这些差异如何有助于局部免疫细胞激活和病毒清除。这一点很重要，因为迫切需要了解HPV 16如何在免疫丰富的口咽中持续存在并诱导癌症，以开发新的（免疫）疗法来对抗HPV驱动的癌症。

Viruses are obligate intracellular parasites that have evolved to manipulate the target cell and local tissue environment to facilitate replication. Some viruses can establish persistent, sub-clinical infection of target cells and these infections can evade host immune detection and exist for decades. Human papillomaviruses (HPV) are a family of viruses that infect the moist and dry surfaces of human skin. The target cell of infection is a specific type of skin cell called a keratinocyte. Infection with many different types of HPV results in the formation of warts but persistent infection with a subset of HPV types, so called high-risk HPV, is the cause of cancers of anal and genital regions of the body (including the uterine cervix), and cancers of the head and neck, specifically in the tonsils and base of tongue, collectively known as the oropharynx. Although robust strategies exist to prevent high-risk HPV infection and to detect pre-cancerous lesions through vaccination and national cervical screening programmes, these measures are not available in all countries. Indeed, the number of HPV-associated cancers has remained at around 600,000 cases per year worldwide despite screening and vaccination. Furthermore, the incidence of HPV-driven oropharyngeal cancer is increasing in patients that are younger, predominantly male and non-smokers. The primary site of oropharyngeal HPV infection is the tonsil, a organ rich in immune cells that serve to detect invading pathogens and prevent respiratory and gastrointestinal infection. Establishment of persistent high-risk HPV infection in the tonsil must therefore require strong suppression of local immune cell activation. The majority (greater than 90%) of HPV-positive oropharyngeal cancers are caused by HPV type 16 (HPV16). While HPV16 is also the most common HPV found in cervical cancers (~60%) the burden is much less than for oropharyngeal cancers. Another high-risk type, HPV18 accounts for ~15% of cervical cancers but is rarely found in oropharyngeal cancers. The reasons for this disparity in disease association at different body sites are not known. Our preliminary data suggest that HPV16, but not HPV18, is able to suppress the production of immune activating molecules by reprogramming gene expression of the host tonsil keratinocyte. Using state-of-the-art primary cell-based models of HPV infection and cutting-edge DNA and gene sequencing methods, we will investigate the similarities and differences in HPV16 and HPV18 life cycles in tonsil keratinocytes. We will determine how HPV16 suppresses host immune detection through repression of key pathways that activate and attract local immune cells to the site of infection. We will then use our expertise in primary cell culture to combine primary keratinocytes with donor-matched lymphoidal tissue, which contains to tonsillar immune cells, to establish in vitro tonsil organ-like cultures that will be used to study immune cell activation and migration in response to HPV infection. The results of this project will uncover novel mechanisms of HPV persistence in the tonsil. We will also determine the key differences in immune suppression by cancer-causing HPV16 and HPV18 and how these differences contribute to local immune cell activation and viral clearance. This is important as there is a critical need to understand how HPV16 can persist and induce cancer within the immune-rich oropharynx to enable the development of novel (immuno-) therapies to combat HPV-driven cancers.