Papillomavirus Virion Proteins and Vaccines

乳头瘤病毒（PV）感染动物和人的上皮细胞，其中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乳头瘤病毒（HPV）类型（最常见的是HPV 16）之间存在很强的相关性。我们的研究涉及针对HPV和其他靶点的疫苗的开发以及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，用于产生高滴度的感染性乳头瘤病毒颗粒，其包被标记质粒，下文称为假病毒体。该办法包括：1）L1主要和L2次要衣壳蛋白的广泛密码子修饰，以去除限制复制哺乳动物细胞中病毒体蛋白表达的负调控元件; 2）通过在过表达SV 40大T抗原的细胞中复制携带SV 40复制起点的质粒来产生高拷贝数假基因组;和3）用于提取和纯化感染性颗粒的有效方法。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。假病毒生产技术正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，双链环状基因组的双糖苷化是大小依赖性的，但令人惊讶的是序列无关的。没有乳头瘤病毒序列的质粒被有效地包装，使得这些假病毒体异常灵活的基因转移载体。我们还确定了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对蛋白水解具有抗性。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中脱壳，并且病毒基因组随后由L2引导至称为ND 10体或POD的亚核结构域。我们进一步确定，ND 10 s的病毒基因组的共定位是由真实的BPV 1病毒粒子感染后的病毒基因组的有效转录所必需的。其他DNA病毒在感染后早期与ND 10相互作用，但这些不涉及衣壳蛋白的相互作用导致结构破坏。因此，通常发现ND 10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，表明在某些情况下，ND 10可以促进病毒感染的建立。我们已经开发了一种新的乳头瘤病毒中和试验的基础上假病毒技术。这是第一个高通量乳头瘤病毒中和试验，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孔微量滴定板格式高度重现。我们希望这种检测方法能极大地促进目前由NCI、Merck和GlaxoSmithKline进行的III期VLP疫苗试验中的免疫应答监测和免疫保护相关性评估。该检测还有助于开发第二代HPV预防性疫苗，并进行血清流行病学研究。我们先前已经表明，在VLP表面上以有序阵列展示靶自身多肽在打破B细胞耐受性和诱导对自身蛋白的有效抗体应答方面是有效的。在小鼠中，与涉及与T辅助表位的简单连接的标准方法相比，在VLP上展示自身肽使IgG滴度增加了1000倍。高密度的自身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辅助受体CCR 5自身抗体的初步研究。用展示猕猴CCR 5的N-末端的VLP接种的猕猴产生高滴度的自身抗体，其结合细胞表面CCR 5，阻断培养细胞的HIV感染，并且在用嗜CCR 5的SHIV攻击后增加病毒清除率。在病毒攻击前的两年观察期内，在接种疫苗的猕猴中未检测到CCR 5自身抗体产生的不良后果。我们还确定在VLP上展示淀粉样β肽可以破坏淀粉样前体蛋白转基因小鼠中B细胞对淀粉样β蛋白的耐受性。一种基于VLP的β淀粉样蛋白疫苗可能是一种比最近在临床试验中测试的疫苗更好的对抗阿尔茨海默病的候选疫苗。首先，预期它将诱导更高滴度的淀粉样蛋白β抗体，其被认为是关键的效应分子。其次，在少数先前疫苗的接受者中检测到的神经病理被认为是由T细胞对淀粉样蛋白β的C末端部分的反应引起的，而在淀粉样蛋白β清除中有活性的抗体主要针对蛋白质的N末端。因此，在基于VLP的疫苗中，用于产生淀粉样蛋白β IgG的T细胞帮助可以针对连接的VLP，而不是针对淀粉样蛋白β本身，从而避免与诱导淀粉样蛋白β T细胞应答相关的不良事件。

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.