Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒（pv）感染动物和人的上皮，它们通常在感染部位诱导良性增殖。然而，人类生殖器病变的恶性进展与某些人类乳头瘤病毒（HPV）类型，最常见的是HPV 16之间有很强的联系。我们的研究涉及针对HPV和其他目标的疫苗的开发以及PV生命周期的阐明。我们已经开发了一种简单而有效的策略来产生高滴度的感染性乳头瘤病毒颗粒，这些颗粒可以转导封装的标记质粒，下文称为假病毒粒子。该方法包括：1)对L1主和L2次衣壳蛋白进行广泛的密码子修饰，以去除复制哺乳动物细胞中限制病毒粒子蛋白表达的负调控元件；2)在过表达SV40大T抗原的细胞中，通过携带SV40起源的质粒复制产生高拷贝数的假基因组；3)有效的感染性颗粒提取和纯化方法。这种方法代表了乳头瘤病毒研究的技术突破，我们已经以几种方式利用了这一发展，如下所述。假病毒生产技术被用于探索乳头瘤病毒病毒粒子组装的基本特征。我们已经确定，双链环状基因组的封装是大小依赖的，但令人惊讶的是，序列无关。没有乳头瘤病毒序列的质粒被有效地包装，使这些假病毒粒子非常灵活的基因转移载体。我们还确定衣壳通过有序的二硫键形成序列成熟，这导致衣壳稳定并使其抵抗蛋白质水解。在其他基础病毒学研究中，我们检查了乳头瘤病毒感染的早期事件。利用假病毒方法，我们确定乳头瘤病毒在早期内体脱壳，病毒基因组随后被L2引导到称为ND10小体或pod的亚核结构域。我们进一步确定，病毒基因组在nd10位点的共定位是在感染BPV1病毒粒子后有效转录病毒基因组所必需的。其他DNA病毒在感染后早期与nd10相互作用，但这些相互作用不涉及衣壳蛋白，导致结构破坏。因此，人们普遍发现nd10在病毒感染早期具有拮抗剂功能。我们的发现是第一个明确的证据，在某些情况下，nd10可以促进病毒感染的建立。我们开发了一种新的基于假病毒技术的乳头瘤病毒中和试验。这是第一个高通量乳头瘤病毒中性化试验，也是第一个不受感染性衣壳可用性严重限制的试验。它是敏感的，类型特异性，并在96孔微量滴度板格式高度重现。我们期望该分析能够极大地促进免疫反应监测和评估目前由NCI、默克和葛兰素史克公司进行的III期VLP疫苗试验中的免疫保护相关因素。该检测还应有助于第二代HPV预防疫苗的开发和血清流行病学研究的开展。我们之前已经证明，在VLP表面以有序阵列展示目标自多肽可以有效地打破b细胞的耐受性，并诱导对自蛋白的有效抗体反应。在小鼠中，与与T辅助表位简单连接的标准方法相比，在VLPs上显示自身肽可使IgG滴度增加1000倍。高密度的自体抗原展示似乎是消除B细胞耐受性的关键决定因素。我们现在已经完成了一项旨在诱导HIV共受体CCR5自身抗体的初步研究。用含有猕猴CCR5 n末端的VLPs接种的猕猴产生高滴度的自身抗体，这些抗体结合细胞表面CCR5，阻断培养细胞的HIV感染，并在CCR5-向性SHIV攻击后提高病毒清除率。在病毒攻击前的两年观察期中，未发现接种疫苗的猕猴产生CCR5自身抗体的不良后果。我们还确定，在转淀粉样蛋白前体蛋白的小鼠中，在VLPs上显示淀粉样蛋白肽可以打破B细胞对淀粉样蛋白的耐受性。与最近在临床试验中测试的疫苗相比，基于vlp的β淀粉样蛋白疫苗可能是一种更好的阿尔茨海默病候选疫苗。首先，它有望诱导更高滴度的β淀粉样蛋白抗体，这被认为是关键的效应分子。其次，在先前疫苗的少数接受者中检测到的神经病变被认为是由于T细胞对淀粉样蛋白c端部分的反应，而在淀粉样蛋白清除中活跃的抗体主要针对蛋白质的n端。因此，在基于vlp的疫苗中，帮助生成β淀粉样蛋白IgG的T细胞可以直接针对相关的vlp，而不是针对β淀粉样蛋白本身，从而避免与诱导β淀粉样蛋白T细胞反应相关的不良事件。

Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is concerned with development of vaccines against HPV and other targets and elucidation of the PV life cycle. We have developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, referred to hereafter as pseudovirions. The approach involves: 1) extensive codon modification of the L1 major and L2 minor capsid proteins to remove negative regulatory elements that limit virion protein expression in replicating mammalian cells; 2) production of high copy number pseudogenomes through replication of a plasmid carrying an SV40 origin of replication in cells overexpressing SV40 Large T Antigen; and 3) efficient methods for extraction and purification of infectious particles. This methodology represents a technical breakthrough in papillomavirus research, and we have exploited this development in several ways, as outlined below. Pseudovirus production technology is being used to explore the basic features of papillomavirus virion assembly. We have determined that encapsidation of the double stranded circular genome is size-dependent, but surprisingly sequence-independent. Plasmids with no papillomavirus sequences are efficiently packaged, making these pseudovirions exceptionally flexible gene transfer vehicles. We have also determined that the capsids mature through an ordered sequence of disulfide bond formation that results in stabilization of the capsids and makes them resistant to proteolysis. In other basic virologic studies, we examined the early events in papillomavirus infection. Taking advantage of the pseudovirus approach, we determined that papillomaviruses uncoat in early endosomes and that the viral genome is subsequently directed by L2 to subnuclear domains called ND10 bodies or PODs. We further determined that co-localization of the viral genome at ND10s is required for efficient transcription of the viral genome after infection by authentic BPV1 virions. Other DNA viruses interact with ND10s early after infections, but these interactions, which do not involve capsid proteins, lead to disruption of the structures. Therefore, it has generally been found that ND10s have an antagonist function early in viral infections. Our findings are the first clear evidence that in some instances, ND10s can promote establishment of viral infection. We have developed a new papillomavirus neutralization assay based on the pseudovirus technology. This is the first high throughput papillomavirus neturalization assay and the first assay that is not severely limited by the availability of infectious capsids. It is sensitive, type-specific, and highly reproducible in a 96-well microtiter plate format. We expect this assay to greatly facilitate immune response monitoring and assessment of immune correlates of protection in the phase III VLP vaccine trials currently being conducted by the NCI, Merck, and GlaxoSmithKline. The assay should also aid in the development of second generation HPV prophylactic vaccines, and in the conduct of seroepidemiological studies. We have previously shown that display of target self-polypeptides in an ordered array on a VLP surface is effective at breaking B-cell tolerance and inducing potent antibody responses to a self-protein. In mice, display of a self-peptide on the VLPs increased IgG titers 1000-fold, compared to standard approaches involving simple linkage to T helper epitopes. High density of self-antigen display appears to be the critical determinant for abrogating B cell tolerance. We have now completed an initial study aimed at induction of auto-antibodies to the HIV co-receptor CCR5. Macaques vaccinated with VLPs displaying the N-terminus of macaque CCR5 produced high titers of auto-antibodies that bound cell surface CCR5, blocked HIV infection of cultured cells, and increased the rate of viral clearance after challenge with a CCR5-tropic SHIV. No adverse consequences of CCR5 auto-antibody production were detected in the vaccinated macaques in a two-year observation period prior to viral challenge. We have also determined that display of an amyloid beta peptide on VLPs can break B cell tolerance to amyloid beta in mice transgenic for amyloid precursor protein. A VLP-based amyloid beta vaccine might be a better candidate vaccine against Alzheimer's Disease than the vaccine recently testing in clinical trials. First, it would be expected to induce higher titer of amyloid beta antibodies, which are thought to be critical effector molecules. Second, the neural pathologies detected in a minority of recipients of the previous vaccine are thought to be result from T cell responses to the C-terminal portion of amyloid beta, while the antibodies active in amyloid beta clearance are mainly directed against the N-terminus of the protein. Therefore, in a VLP-based vaccine, the T cell help for generating the amyloid beta IgG can be directed against the linked VLPs, rather than against amyloid beta itself, thereby avoiding the adverse events associated with induction of amyloid beta T cell responses.