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递送需要保护、持续释放和胶囊化蛋白的构象维持。聚（丙交酯-共-乙交酯）（PLG）、聚（丙交酯）和聚（酸酐）已用于生成微粒基质1。不幸的是，这些聚合物的使用可导致在这些微球的配制和生物降解期间的酸性微环境，并且聚合物或其降解产物与包封的肽/蛋白质之间的共价反应也是对蛋白质完整性的潜在威胁。保持这些观点的角度来看，本调查描述了制备微球（1至10微米），制备了一种新的聚合物分散技术，并与壳聚糖和聚己内酯（PCL），以防止相互作用的藻酸盐基质，防止肠降解和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）释放超过90天的包埋蛋白，也证明了使用海藻酸盐微球口服疫苗破伤风类毒素（TT）。在单次口服剂量的藻酸盐微球包封的TT后，配制成在7、30和90天释放内容物，小鼠产生了针对TT的保护性血清Ab滴度。这些研究为我们的工作假设提供了理论基础，即包埋的蛋白质可以被保护并保持天然蛋白质构象，这将影响其治疗效果。口服海藻酸盐微球包封的PsaA可以诱导针对肺炎球菌携带和肺炎的保护性粘膜和全身适应性免疫。我们已经强调了在体外以及在体内的方法，使用小鼠模型的肺炎球菌携带肺炎来测试这一假设。目的考察海藻酸钠微球包封的PsaA疫苗在不同工艺条件下的稳定性。目的研究海藻酸钠微球包裹绿色荧光蛋白（GFP）后，对GFP的吸收和稳定性的影响。目的探讨海藻酸钠微球包裹的PsaA疫苗诱导抗沙门氏菌保护性免疫的能力。pneumoniae攻击，在小鼠中使用EF 3030。本研究将为海藻酸钠微球口服抗肺炎球菌蛋白质和疫苗的细胞和分子机制提供重要的新信息。先前已经开发了针对各种感染因子的口服有效疫苗，小儿麻痹症最近，通过口服和鼻内途径施用的疫苗已经显示出对抗肠道和呼吸道感染的巨大希望。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微生物的主要人类储存库。了解粘膜肺炎球菌免疫的细胞和分子机制对于理解和设计防止携带的方法是重要的，这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死亡的十大原因之一，因此，非常需要接种肺炎球菌感染的疫苗。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）口服疫苗接种将促进粘膜和全身免疫，无疑提高依从性。此外，单剂量疫苗由于增加了依从性和易于给药，将导致免疫力增强。本申请提出了一种用于疫苗递送的聚己内酯（PCL）和壳聚糖涂覆的表氯醇交联藻酸盐（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.