Synethic protein and peptide assemblies as novel adjuvants and vaccines

描述（由申请人提供）：全细胞疫苗通常是不添加佐剂的免疫原性疫苗，但生产成本和安全性问题促使了基于蛋白质的替代品的发展。然而，基于蛋白质的疫苗设计受到有限的免疫原性和佐剂的必要性的挑战，其中很少有佐剂目前可供人类使用。我们已经开发了一种基于肽的自组装平台，当这些肽与短肽抗原融合时，它们在小鼠中引起对短肽抗原的大量抗体反应，而不需要额外的佐剂(1)。抗体反应广泛，包括所有IgG亚类和IgM，并且在小鼠中强烈持续超过36周。这些发现表明，这些自组装材料是疫苗开发的有吸引力的新候选材料。我们建议扩展这些材料显示全长蛋白的能力，并测试它们作为临床重要的社区相关耐甲氧西林金黄色葡萄球菌（CA-MRSA）的保护模式。针对金黄色葡萄球菌α溶血素（Hla）的抗体（Abs），一种成孔细胞毒素，已被证明可以保护小鼠免受致命性肺炎和皮肤感染（2-6）。在第一个特定目标中，我们将评估Hla偶联到自组装肽平台上引发抗Hla Ab产生的能力。为此，我们将利用一种基于真菌角质酶突变体的新型蛋白质偶联技术，将角质酶- hla融合蛋白与自组装肽平台上的“自杀”膦酸盐配体共价结合(7)。免疫电镜和分析性超离心将用于验证原纤维组装和表位可用性，我们将评估Hla组装刺激小鼠抗Hla Ab产生的能力。这些研究旨在提供原理证明，这些自组装肽可以以高度免疫原性的方式显示全长蛋白。在第二个特定目标中，我们将评估Hla自组装对CA- MRSA感染的保护作用。我们将使用两种CA-MRSA感染小鼠模型，肺部和皮肤感染模型。我们的合作团队已经开发并广泛使用了这两种模型。保护措施包括存活、从肺部和皮肤恢复的金黄色葡萄球菌CFU、组织病理学和真皮坏死皮肤病变的大小。我们将通过将免疫小鼠的血清转移到感染CA-MRSA的幼稚小鼠中，进一步确定hla肽疫苗接种引发的hla特异性抗体在多大程度上是保护的基础。这些研究旨在提供原理证明，这些自组装肽呈递Hla可以引起抗体反应，对CA-MRSA具有保护作用。这些研究的成功将使我们深入了解这些肽组合作为高度工程化的疫苗的效用，用于预防性控制由病原体引起的疾病，并作为设计复杂免疫调节试剂的模块化平台。

DESCRIPTION (provided by applicant): Whole cell vaccines are often immunogenic without added adjuvants, but production costs and safety issues have prompted the development of protein-based alternatives. However, protein-based vaccine design is challenged by limited immunogenicity and the necessity of adjuvants, few of which are currently available for human use. We have developed a peptide-based platform that self-assembles, and when these peptides are fused to short peptide antigens, they elicit substantial antibody responses in mice to the short peptide antigens without the need for additional adjuvant (1). Antibody responses have been broad, including all IgG subclasses and IgM, and they have been strongly persistent for over 36 weeks in mice. These findings suggest these self-assembling materials as attractive novel candidates for vaccine development. We propose to expand on the capability of these materials to display full-length proteins, and to test them as a protective modality for clinically important community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). Antibodies (Abs) against the S. aureus alpha-hemolysin (Hla), a pore-forming cytotoxin, have been shown to protect against lethal pneumonia and skin infection in mice (2-6). In the first specific aim, we will assess the ability of the Hla conjugated to the self-assembled peptide platform to elicit anti-Hla Ab production. To this end, we will utilize a novel protein conjugation technique based on a mutant version of the fungal cutinase enzyme to covalently bond cutinase-Hla fusion proteins to "suicide" phosphonate ligands on the self- assembling peptide platform (7). Immunoelectron microscopy and analytical ultracentrifugation will be used to validate fibrillar assembly and epitope availability, and we will assess the ability of the Hla assemblies to stimulate anti-Hla Ab production in mice. These studies are designed to provide proof-of-principle that these self-assembling peptides can display full-length proteins in a highly immunogenic manner. In the second specific aim, we will assess protection afforded by Hla self-assemblies against CA- MRSA infection. We will utilize two CA-MRSA infection models in mice, a pulmonary and a skin infection model. Both models have been developed and utilized extensively by our collaborative team. Measures of protection include survival, S. aureus CFU recovered from the lung and skin, histopathology, and size of dermonecrotic skin lesions. We will further determine the extent to which Hla-specific antibodies elicited by the Hla-peptide vaccination are the basis for protection, by transferring sera from immunized mice into naive mice infected with CA-MRSA. These studies are designed to provide proof-of-principle that these self-assembling peptides presenting Hla can elicit antibody responses that are protective against CA-MRSA. Success in these studies will provide us with insights into the utility of these peptide assemblies as highly engineered vaccines for the prophylactic control of diseases caused by pathogens, and as a modular platform for designing sophisticated immune-modulating reagents. PUBLIC HEALTH RELEVANCE: Vaccination is the most effective means for the prevention of disease from multiple pathogens, but numerous challenges are faced by both whole cell vaccines and protein-based alternatives, which require adjuvants to enhance their efficacy. We have developed a self-assembling peptide system that, when coupled to peptide antigens, raises strong antibody responses to the peptide antigens in mice, without the need for additional adjuvant. In this proposal we seek to expand the capability of these materials to display clinically important full-length proteins and to conduct mechanistic studies to understand the basis of their immunogenicity, with the anticipation that success in these goals will provide us with critical insights into utility of these peptide systems for vaccination against pathogens.