Synethic protein and peptide assemblies as novel adjuvants and vaccines

描述(申请人提供)：全细胞疫苗通常是免疫原性的，无需添加佐剂，但生产成本和安全问题已促使基于蛋白质的替代品的开发。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，目前几乎没有佐剂可供人类使用。我们已经开发了一种基于多肽的自组装平台，当这些多肽与短肽抗原融合时，它们在小鼠中引起对短肽抗原的实质性抗体反应，而不需要额外的佐剂(1)。抗体反应广泛，包括所有的免疫球蛋白亚类和免疫球蛋白M，它们在小鼠体内持续了36周以上。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)的保护方式。抗金黄色葡萄球菌α-溶血素(Hla)的抗体(Hla)是一种形成毛孔的细胞毒素，已被证明对小鼠的致死性肺炎和皮肤感染具有保护作用(2-6)。在第一个特定目标中，我们将评估连接到自组装肽平台上的人类白细胞抗原(Hla)诱导抗人类白细胞抗原抗体(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版本的新的蛋白质连接技术，将角质酶-人类白细胞抗原融合蛋白共价结合到自组装肽平台上的“自杀”磷酸配体上(7)。免疫电子显微镜和分析超速离心法将被用来验证纤维组装和表位的可用性，我们将评估这些HLA组装的能力，以刺激小鼠产生抗HL A抗体。这些研究旨在提供原理证据，证明这些自组装肽可以以高度免疫的方式展示全长蛋白质。在第二个具体目标中，我们将评估人类白细胞抗原自组装对CA-MRSA感染的保护作用。我们将利用两种CA-MRSA感染小鼠模型，一种是肺部感染模型，一种是皮肤感染模型。这两种模型都由我们的协作团队开发和广泛使用。保护措施包括存活率、从肺和皮肤中恢复的金黄色葡萄球菌CFU、组织病理学和皮肤坏死性皮损的大小。我们将通过将免疫小鼠的血清转移到感染CA-MRSA的幼小鼠体内，进一步确定通过接种HL A多肽疫苗而产生的HL A特异性抗体是保护的基础的程度。这些研究的目的是提供原理证据，证明这些呈现人类白细胞抗原的自组装肽可以诱导抗体反应，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多肽组件作为高度工程疫苗的用途，以预防由病原体引起的疾病，并作为设计复杂的免疫调节试剂的模块化平台。 公共卫生相关性：接种疫苗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，需要佐剂来提高其效力。我们已经开发出一种自组装的多肽系统，当与多肽抗原偶联时，可以在小鼠体内引发对多肽抗原的强烈抗体反应，而不需要额外的佐剂。在这项提案中，我们试图扩大这些材料展示临床重要全长蛋白质的能力，并进行机制研究以了解它们的免疫原性基础，并期望这些目标的成功将为我们提供对这些多肽系统用于接种病原体疫苗的关键见解。

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.