Synethic protein and peptide assemblies as novel adjuvants and vaccines

描述（由申请人提供）：全细胞疫苗通常具有免疫原性，无需添加佐剂，但生产成本和安全性问题促使开发基于蛋白质的替代品。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，目前很少有佐剂可用于人类使用。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，它们在小鼠中引发对短肽抗原的实质性抗体应答，而不需要额外的佐剂（1）。抗体应答广泛，包括所有IgG亚类和IgM，并且它们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。我们建议扩大这些材料展示全长蛋白质的能力，并将其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）的保护模式进行测试。 抗S.金黄色葡萄球菌α-溶血素（Hla），一种成孔细胞毒素，已经显示在小鼠中保护免于致死性肺炎和皮肤感染（2-6）。在第一个具体目标中，我们将评估与自组装肽平台缀合的Hla引发抗Hla Ab产生的能力。为此，我们将利用基于真菌角质酶的突变体形式的新型蛋白质缀合技术，以将角质酶-Hla融合蛋白共价键合至自组装肽平台上的“自杀”膦酸酯配体（7）。免疫电子显微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组装体刺激小鼠中抗Hla Ab产生的能力。这些研究旨在提供这些自组装肽可以以高度免疫原性方式展示全长蛋白质的原理证明。 在第二个具体目标中，我们将评估Hla自组装体针对CA-MRSA感染提供的保护。我们将在小鼠中使用两种CA-MRSA感染模型，即肺部和皮肤感染模型。这两种模型都是由我们的合作团队开发和广泛使用的。保护措施包括生存，S。从肺和皮肤回收的金黄色葡萄球菌CFU、组织病理学和皮肤坏死性皮肤病变的大小。我们将通过将来自免疫小鼠的血清转移到感染CA-MRSA的幼稚小鼠中，进一步确定由Hla肽疫苗接种引发的Hla特异性抗体是保护基础的程度。这些研究旨在提供这些呈递Hla的自组装肽可以引发针对CA-MRSA的保护性抗体应答的原理证明。 这些研究的成功将使我们深入了解这些肽组件作为高度工程化疫苗用于预防性控制病原体引起的疾病的效用，以及作为设计复杂免疫调节试剂的模块化平台。 公共卫生相关性：疫苗接种是预防多种病原体引起的疾病的最有效手段，但全细胞疫苗和基于蛋白质的替代品都面临着许多挑战，这需要佐剂来增强其功效。我们已经开发了一种自组装肽系统，当与肽抗原偶联时，在小鼠中产生对肽抗原的强烈抗体应答，而不需要额外的佐剂。在这项提案中，我们寻求扩大这些材料的能力，以展示临床上重要的全长蛋白质，并进行机理研究，以了解其免疫原性的基础，预期这些目标的成功将为我们提供关键的见解，这些肽系统的效用，用于接种疫苗对抗病原体。

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.