Oral Delivery of protein antigens and their stability by Alginate Microspheres

描述(由申请人提供)：成功和有效的粘膜蛋白输送需要被包裹蛋白的保护、持续释放和构象维持。聚丙交酯共乙交酯(PLG)、聚丙交酯和聚酸酐已被用来生成微粒基质1。不幸的是，这些聚合物的使用会导致这些微球的形成和生物降解过程中的酸性微环境，并且聚合物或其降解产物与包裹的多肽/蛋白质之间的共价反应也对蛋白质的完整性构成潜在威胁。考虑到这些观点，本研究描述了通过一种新的聚合物分散技术制备微球(1-10微米)，并用壳聚糖和聚己内酯(PCL)包覆，以防止与海藻酸盐基质的相互作用，防止肠道降解，并延长重组蛋白的释放。我们的初步研究表明，海藻酸盐微球(PACEA)在90天内释放其包裹的蛋白质，并证明了海藻酸盐微球用于口服疫苗递送破伤风类毒素(TT)。单剂量口服包裹TT的海藻酸盐微球后，小鼠在第7、30和90天释放内容物，形成保护性血清抗体效价。这些研究为我们的工作假设提供了理论基础，即包裹的蛋白质可以受到保护并保持天然蛋白质构象，这将影响其治疗效果。口服藻酸盐微球包裹的PSAA可诱导保护性黏膜和系统适应性免疫，以对抗肺炎球菌携带者和肺炎。我们已经强调了使用携带肺炎链球菌肺炎的小鼠模型的体外和体内方法来验证这一假设。目的通过不同的工艺方法对海藻酸微球包裹的PSAA疫苗的稳定性进行评价。目的二研究GFP微球在黏膜和全身适应器官中的摄取及其稳定性。目的用EF3030确定海藻酸微球pSAA疫苗诱导小鼠对肺炎链球菌攻击的保护性免疫的能力。本研究将为藻酸盐微球口服抗肺炎球菌蛋白和疫苗的细胞和分子机制提供重要的新信息。以前已经开发出针对各种感染性病原体的口服有效疫苗，例如脊髓灰质炎。最近，通过口服和鼻腔途径接种的疫苗在预防肠道和呼吸道感染方面显示出巨大的前景。这项研究解决了一个重要的健康问题，因为鼻咽部携带的肺炎球菌被认为是这种潜在致命生物的主要人类宿主。了解粘膜肺炎球菌免疫的细胞和分子机制对于了解和设计预防携带的方法非常重要，携带被认为是对肺炎链球菌的群体免疫所必需的。这种革兰氏阳性细菌是急性中耳炎、肺炎、菌血症和脑膜炎的主要原因。肺炎球菌肺炎是老年人群的前十大死因之一，因此，迫切需要接种肺炎球菌感染疫苗。因此，选择合适的肺炎球菌抗原和粘膜佐剂将大大提高未来疫苗的效力。此外，单次(或多次)口服疫苗接种将同时促进粘膜免疫和系统免疫，并无疑提高依从性。此外，由于依从性的提高和给药的简便性，单剂疫苗将导致免疫力增强。本应用提供了一种用于疫苗递送的聚己内酯(PCL)和壳聚糖包覆的环氧氯丙烷-交联型海藻酸盐(PACE-A)微球的新方法。

DESCRIPTION (provided by applicant): Successful and efficacious mucosal protein delivery requires protection, sustained release, and conformational maintenance of encapsulated proteins. Poly (lactide coglycolite) (PLG), poly (lactide), and poly (anhydride), have been used to generate a matrix for microparticulates1. Unfortunately, the use of these polymers can result in an acidic microenvironment during formulation and biodegradation of these microspheres, and co-valent reactions between the polymer or its degradation products with encapsulated peptides/proteins also a potential threat to protein integrity. Keeping these points in perspective, the present investigation describes the preparation of microspheres (1 to 10 microns) that were prepared by a novel polymer dispersion technique and coated with chitosan and poly caprolactone (PCL) to prevent interaction with the alginate matrix, protect against enteric degradation and extend the release of recombinant proteins. Our preliminary study shows that Alginate microspheres (PACEA) release their entrapped proteins over 90 days and also demonstrated the use of alginate microspheres for oral vaccine delivery of tetanus toxoid (TT). After a single oral dose of alginate microsphere encapsulated TT, formulated to release contents at 7, 30, and 90 days, mice developed protective serum Ab titers against TT. These studies provide the rationale for our working hypothesis that entrapped proteins can be protected and maintain the native protein conformation which would affect their therapeutic efficacy. Protective mucosal and systemic adaptive immunity against pneumococcal carriage and pneumonia can be induced by oral delivery of alginate microsphere- encapsulated PsaA. We have emphasized in-vitro as well as in vivo approaches using mouse models of pneumococcal -carriage pneumonia to test this hypothesis. Aim One will assess the stability of alginate microsphere-encapsulated PsaA vaccines by the various process methods. Aim Two will characterize the particle uptake by mucosal and systemic adaptive organs and their stability to alginate microsphere encapsulated GFP. Aim Three will ascertain the ability of alginate microsphere encapsulated PsaA vaccines to induce protective immunity against S. pneumoniae challenge, using EF3030 in mice. This study will provide important and new information regarding the cellular and molecular mechanisms of oral delivery of proteins and vaccines by alginate microspheres against pneumococci. Orally effective vaccines have been previously developed against various infective agents, e.g., polio. More recently, vaccines administered by the oral and intranasal routes have shown great promise against both intestinal and respiratory tract infections. This study addresses an important health problem since pneumococci in nasopharyngeal carriage are thought to be the main human reservoir for this potential-lethal organism. Understanding the cellular and molecular mechanisms of mucosal pneumococcal immunity is important for understanding and devising ways to protect against carriage, which is considered to be essential for herd immunity to Streptococcus pneumoniae. This Gram-positive bacterium is a major cause of acute otitis media, pneumonia, bacteremia, and meningitis. Pneumococcal pneumonia is among the top ten causes of death in aged populations hence, vaccination against pneumococcal infections is greatly needed. Therefore, selection of appropriate pneumococcal antigens and mucosal adjuvants will greatly improve the efficacy of future vaccines. Moreover, single (or multiple) oral vaccination would promote both mucosal and systemic immunity and no doubt improve compliance. Further, single-dose vaccines would result in enhanced immunity due to increases in compliance and the ease of administration. This application presents a novel method of polycaprolactone (PCL)- and chitosan-coated epichlorohydrin-crosslinked alginate (PACE-A) microspheres for vaccine delivery.