L1 capsomeres as a next generation preventive HPV vaccine

预防人类乳头瘤病毒(HPV)的最新发展是医学上的一个里程碑。目前的疫苗是由主要衣壳蛋白L1的重组病毒样颗粒(VLP)组成，包括高危HPV16型和18型。11/11期临床试验表明，这些VLP制剂在预防HPV16、18型感染方面有95%的有效性，从而有望阻止与这些类型相关的约70%的宫颈癌的最终发展。尽管 VLP疫苗的这一成功具有成本高(在美国最初的三次免疫接种为360美元)和需要冷藏(即冷链)的不利属性。我们之前已经确定了VLP亚单位，五聚体L1囊膜的免疫原性，并在犬感染模型中将这些“亚单位”与VLP进行了比较。在该模型中，在诱导抗感染方面，胶囊似乎与VLP几乎等同。在大肠杆菌中重组表达L1作为GST融合蛋白后，衣壳粒得到纯化，这表明制造成本显著降低。 该蛋白易于纯化，可以沉淀、再悬浮并在室温下储存，而不会失去免疫原性。在NCI正在进行的快速奖的背景下，GST-L1胶囊已被提议作为“下一代”HPV疫苗，该疫苗可能非常适合在宫颈癌特别流行的欠发达国家生产和使用。我们建议使用GMP(由Shantha BioTechnica生产)、瓶装和毒理筛选的GST-L1蛋白 在第一阶段人体试验研究的快速奖的赞助下。在目标1和目标2中，我们提出了三个剂量水平(10、50、500微克和安慰剂)各15名受试者的剂量递增方案，重复三次，同时分析毒性、中和抗体的产生、细胞毒性T细胞反应以及GST抗体产生的可能不良影响。在第三个目标中，临床标本和动物实验的实验室研究将测试新的佐剂策略，包括Toll-like 受体激活，并通过比较衣壳、交联衣壳和VLP来评价抗原间隔和连接对体液免疫的影响。将利用L1衣壳结构和与天然免疫系统的相互作用的知识，以更低和更少的剂量产生更长时间和更广泛的免疫。该项目的结果将决定GST-LI制剂是否可以进行进一步的第二阶段测试。

The recent development of prophylactic vaccines protective against "high risk" human papillomaviruses (HPV) is a landmark in medicine. The current vaccine is composed of recombinant virus-like particles (VLPs) of the major capsid protein, L1, including the high risk HPV types 16 and 18. Phase ll/lll clinical trials have shown >95% efficacy of these VLP preparations in preventing HPV 16, 18 infection, and thus hopefully preventing the eventual development of about 70% of cervical cancers associated with these types. Despite this success the VLP vaccines have the adverse attributes of high cost ($360 for the initial three immunizations In the USA) and a requirement for refrigeration {i.e., cold chain). We have previously characterized the immunogenic properties of VLP subunits, pentameric L1 capsomeres, and compared these "subunits" to VLPs in a canine infection model. In this model capsomeres appeared near equivalent to VLPs in inducing protection against infection. Capsomeres are purified after recombinant expression of L1 in E. coli as a GST-fusion protein, at levels suggesting a significant reduction in manufacturing expense. The protein is readily purified, and can be precipitated, resuspended, and stored at room temperature without loss of immunogenicity. In the context of an ongoing NCI RAPID award GST-L1 capsomeres have been proposed as a "next generation" HPV vaccine that might be ideally suited for production and use in underdeveloped countries of the world where cervical cancer is particularly prevalent. We propose to use the GST-L1 protein that has been GMP produced (by Shantha Biotechnica), vialed, and toxicology screened under the auspices of a RAPID award for study in a phase I human trial. In aims 1 and 2 we propose a dose escalation scheme of 15 subjects at each of three dose levels (10, 50, 500 micrograms and placebo), repeated three times, with concomitant analysis of toxicity, development of neutralizing antibodies, cytotoxic T-cell responses, and possible adverse effects of GST antibody development. In the third aim, laboratory studies with clinical specimens and animal experiments will test new adjuvant strategies including Toll-like receptor activation, and evaluate the effects on humoral immunity of antigen spacing and linking by comparing capsomeres, cross-linked capsomeres, and VLPs. Knowledge of L1 capsomere structure and interaction with the innate immune system will be utilized to generate longer-lived and broader immunity with lower and fewer doses. The results of this project will determine whether the GST-LI preparation can proceed to further phase II testing.