Development of a novel and broadly applicable thermostable bacteriophage VLPs platforms for vaccine design, drug delivery, and imaging

项目摘要 病毒样颗粒（VLP）在大小、结构和免疫原性方面与病毒相似， 包膜蛋白是来源于除了它们缺乏病毒基因组的事实; VLP是非感染性的， 是安全的这些特征已被用于开发针对人乳头瘤病毒的基于VLP的疫苗 和B型肝炎病毒;此外，目前正在探索来自约70种病毒的外壳蛋白以开发VLP- 针对这些病毒的疫苗。来自这些病毒中的一些的VLP也被用作展示 平台来开发展示来自其他感染因子、肿瘤因子、肿瘤抗原和肿瘤抗原的异源肽的嵌合VLP。 相关抗原和其他代谢疾病。这些嵌合VLP的目的是诱导抗体 针对平台上展示的异源抗原而不是平台。除了担任 作为展示平台，VLP也已用于将药物或货物靶向递送至特定癌细胞。 虽然展示异源肽的候选基于VLP的疫苗在动物中非常有效， 在大多数研究中，VLP平台（包括腺病毒VLP或十二面体）是衍生的。 从感染人类的病毒中分离出来，在某些情况下，研究使用了以前 用于对一般人群进行免疫接种;一个很好的例子是HBV疫苗，全球婴儿接种 2015年覆盖率达84%。基于其中一些平台的疫苗，在疫苗中预先存在抗体， 一般人群中，可能在人类中免疫原性较低。此外，还有一个限制的大小 可以在一些VLP平台上遗传展示的异源抗原， 在相同VLP上展示具有多个表位的单个肽。此外，大多数VLP平台 这些产品对温度敏感，因此不太适合制冷设施较差的发展中国家。 在本提案中，PI将使用以下方法开发和表征新型热稳定噬菌体VLP平台： 来自嗜热病毒P23-77和Φ IN 93的外壳蛋白。P23-77和Φ IN 93分离自 在70-75 °C下生长。因此，来源于这些病毒的VLP可能在室温（RT）下稳定，或 这些VLP平台的额外益处是，因为这些病毒不感染人类， 人类群体缺乏针对VLP平台的预先存在的中和抗体;因此， 平台的免疫原性不会受到预先存在的抗体的损害。许多表面- 衣壳上暴露的环可耐受异源抗原的较大插入。PI将共同表达 三种外壳蛋白来自P23-77，两种外壳蛋白来自Φ IN 93。PI将评估外套是否 蛋白质可以组装成VLP，如果它们是热稳定的，可以耐受异源肽插入 来自人乳头瘤病毒，以及VLP与病毒相比是否具有免疫原性。

PROJECT SUMMARY Virus-like particles (VLPs) resemble - in size, structure, and immunogenicity - the virus from which the coat or envelope protein(s) are derived from except for the fact they lack a viral genome; VLPs are non-infectious and are safe. These features have been exploited to develop VLP-based vaccines against human papillomaviruses and hepatitis B virus; furthermore, coat proteins from ~70 viruses are currently being explored to develop VLP- based vaccines against these viruses. VLPs from some of these viruses have also been used as display platforms to develop chimeric VLPs displaying heterologous peptides from other infectious agents, tumor- associated antigens and other metabolic diseases. The goal of these chimeric VLPs is to induce antibodies against the heterologous antigen displayed on the platforms and not the platforms. In addition to serving as display platforms, VLPs have also been used for targeted delivery of drugs or cargo to specific cancer cells. While the candidate VLPs-based vaccines displaying heterologous peptides are very effective in animal studies, in the majority of studies, VLPs platforms (including adenoviral VLPs or dodecahedron) are derived from viruses that infect humans and in some cases, studies used VLPs platforms that had previously been used to immunize the general population; a good example is HBV vaccine, with a global infant vaccination coverage of 84% in 2015. Vaccines based on some of these platforms, with pre-existing antibodies in the general population, are likely to be less immunogenic in humans. Additionally, there is a limitation on the size of heterologous antigens that can be genetically displayed on some VLPs platforms making it challenging to display a single peptide with multiple epitopes on the same VLPs. Moreover, most of the VLPs platforms are temperature-sensitive making them less suitable in developing countries with poor refrigeration facilities. In this proposal, the PI will develop and characterize novel thermostable bacteriophage VLPs platforms using coat proteins from thermophilic viruses P23-77 and ΦIN93. P23-77 and ΦIN93 was isolated from bacteria that grow at 70-75 °C. Thus VLPs derived from these viruses are likely to be stable at room temperature (RT) or above RT. Additional benefit of these VLPs platforms is that because these viruses do not infect humans, the human population lacks pre-existing neutralizing antibodies against the VLPs platforms; thus, the immunogenicity of the platforms cannot be compromised by pre-existing antibodies. Also, many surface- exposed loops on the capsid may tolerate larger insertions of heterologous antigens. The PI will co-express three coat proteins from P23-77 and two coat proteins from ΦIN93. The PI will assess whether the coat proteins can assemble into VLPs, if they VLPs are thermostable, can tolerate heterologous peptide insertions from human papillomaviruses, and if VLPs are immunogenic in comparison to the virus(es).