A novel Structure Based Vaccine for staphylococcal alpha hemolysin

描述（由申请人提供）：金黄色葡萄球菌是一种革兰氏阳性人类病原体，可引起广泛的感染，从皮肤和软组织感染到脓毒症和肺炎等危及生命的疾病。众所周知，金黄色葡萄球菌具有获得并进化出对抗菌素耐药机制的能力，同时，它对临床实践中可用的所有抗生素都具有耐药性，因此非常需要新的方法来对抗这些病原体。目前还没有疫苗或治疗方法可用于预防或治疗金黄色葡萄球菌引起的疾病。金黄色葡萄球菌是医院获得性感染的最普遍原因，近年来其在社区中的传播速度也已上升到令人震惊的水平。肺炎是金黄色葡萄球菌感染最严重和最突出的并发症之一，仅在美国每年就有50,000例。传统上，金黄色葡萄球菌肺炎与呼吸机有关，但近年来，它也被认为是社区获得性肺炎的主要原因，主要发生在健康的儿童和年轻人中。金黄色葡萄球菌产生多种具有免疫逃避和免疫调节功能的分泌毒素。其中，溶血素是已知最有效的毒素之一，包括我们在内的多个小组的研究表明，在反映人类疾病的动物模型中进行测试时，α -溶血素（Hla）也称为α毒素（AT）似乎是金黄色葡萄球菌肺炎和皮肤感染的关键毒力因子。由于其毒性作用，野生型Hla不能用作疫苗。该I期SBIR的主要目标是完成基于晶体结构设计的新型Hla疫苗的概念验证研究。已经设计了两种候选疫苗，它们代表Hla的N端参与寡聚化的结构域：i) at的N端62个氨基酸（at -62aa）和ii)包括氨基酸1-62和223-236的扩展结构域，称为at -79aa。本研究提出的初步数据表明，AT-62aa在金黄色葡萄球菌感染的肺炎模型中具有中和抗体的诱导和保护作用，而先前描述的AT-50aa（破坏结构域结构）仅能轻微延长寿命，但与醛水凝胶佐剂一起使用时没有保护作用（与先前使用IFA的报道相反）。在目标1中，我们将根据his标记的AT-62aa蛋白的免疫原性研究，使用三种不同的佐剂(许可产品（明矾）或用于人类临床试验（CpG和IDC-1001）确定最佳佐剂，并确定最佳佐剂。在特定的Aim 2中，我们将为Aim 1中选择的疫苗构建体开发一个生产和纯化无标签蛋白的初步程序。我们还将通过生物物理方法表征蛋白质，并进行桥接研究以确认其体内免疫原性。在特定的Aim 3中，我们将进行一整套概念验证研究，以显示最佳候选疫苗在四种不同感染小鼠模型中的保护功效：肺炎、代表SSTI的皮下脓肿模型、菌血症/败血症和手术伤口感染。这项提案是IBT和布里格姆妇女医院Jean Lee博士实验室的合作成果。在I期完成后，我们设想II期SBIR侧重于完成临床前开发，从而向食品和药物管理局提交研究新药（IND）申请。

DESCRIPTION (provided by applicant): Staphylococcus aureus is a gram positive human pathogen that causes a wide range of infections ranging from skin and soft tissue infections to life threatening diseases like sepsis and pneumonia. S. aureus is well known for its ability to acquire and evolve resistance mechanisms towards antimicrobial agents and meanwhile there are resistances towards all antibiotics available for clinical practice emphasizing a great need fo novel approaches to combat these pathogens. Currently there are no vaccines or therapeutics available for prevention or treatment of diseases caused by S. aureus. S. aureus is the most prevalent cause of hospital acquired infections and in recent years its rate of spread through the community has also been rising to an alarming level. Pneumonia is one of the most severe and prominent complications of S. aureus infection leading with 50,000 cases per year in the US alone. S. aureus pneumonia has been traditionally ventilator associated but in recent years it has been recognized also as a major cause of community acquired pneumonia primarily in otherwise healthy children and young individuals. S. aureus produces a wide range of secreted toxins with immune evasion and immune modulatory functions. Among these alpha hemolysin is known to be one of the most potent toxins and studies from multiple groups including ours show that alpha- hemolysin (Hla) also known as alpha toxin (AT) seems to be a a key virulence factor for S. aureus pneumonia and skin infection when tested in animal models reflecting human diseases. Hla cannot be used as a vaccine in its wild type form due to its toxic effect. The main goal of this Phase I SBIR is to complete proof of concept studies on a novel Hla vaccine designed based on crystallographic structure. Two vaccine candidates have been designed that represent a structural domain at the N terminus of Hla involved in oligomerization: i) the N-terminal 62 amino acids of AT (AT-62aa) and ii) an extended construct including amino acids 1-62 and 223-236 denoted as AT-79aa. Preliminary data presented in this proposal indicate induction of neutralizing antibodies and protective efficacy of AT-62aa in a pneumonia model of S. aureus infection, while a previously described AT-50aa (which disrupts the domain structure) only afforded a slight extension of life but was not protective when used with Alhydrogel adjuvant (in contrast to previous reports using IFA). This proposal is outlined in three specific Aims: In Aim 1, we will identify the best vaccine candidate based on immunogenicity studies of His-tagged AT-62aa protein using three different adjuvants that are either in licensed products (alum) or in clinical trial for human use (CpG and IDC-1001) and identify the optimal adjuvant. In specific Aim 2, we will develop a preliminary procedure for production and purification of a tag-free protein for the vaccine construct selected in Aim 1. We will also characterize the protein by biophysical methods, and perform a bridging study to confirm its immunogenicity in vivo. In specific Aim 3, we will perform a full set of proof of concept studies to show the protective efficacy of the optimal vaccine candidate in four different mouse models of infection: pneumonia, subcutaneous abscess model representing SSTI, bacteremia/sepsis, and surgical wound infection. This proposal is a collaborative effort between IBT and Dr. Jean Lee's laboratory at Brigham Women Hospital. Upon completion of the Phase I we envision a Phase II SBIR focused on completing preclinical development leading to submission of an Investigational New Drug (IND) application to the Food and Drug Administration.