Peptide-Protein Conjugate Vaccines

肽-蛋白结合疫苗

• 批准号: 7334147
• 负责人: rachel schneerson
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7334147
• 关键词: Peptide; Protein; Conjugate; Vaccines

BACILLUS ANTHRACIS, a potential cause of lethal human infection, has 2 essential virulence factors without either of which it is not pathogenic for humans: the anthrax toxin and the capsule. The toxin is composed of 3 peptides: Lethal Factor, Edema Factor, and Protective Antigen (PA). PA is the toxin part that binds to mammalian cells. A 20 KDa peptide must be hydrolyzed off it exposing a site to which LF or EF may bind, rendering toxins that enzymatically modify substrates in the mammalian cell cytosol. The capsule is composed of poly-D-gamma-glutamic acid (PGA).It is non-immunogenic and its protective effect not clear. The licensed vaccine, PA based, is safe and protective but has limitations that justify development of improved vaccines. A recombinant PA was isolated from an uncapsulated strain. Several formulations with formaldehyde treated and alum adsorbed materials were found to be immunogenic in mice. Clinical evaluation of these formulations is underway. The capsule has been isolated from a non toxic strain and it or corresponding synthetic peptides were bound to BSA, rEPA, rPA or tetanus toxoid. Thioether, hydrazone and oxime linkages between the gamma D-PGA and the proteins, with active groups at the C or N termini yielded conjugates immunogenic in mice, with no statistical difference between them. These antibodies were opsonophagocytic. Peptides 10 to 20-mers long, and 10-15 mole gamma D-PGA per mole protein were the most immunogenic. Dose response experiments of an rPA-PGA conjugate, with doses between 0.31 and 20 mcg/mouse showed 1.25 mcg to be the optimal dose for a PGA response, while PA antibody levels increased with higher immunizing doses. The use of alum adjuvant increased PA antibody levels while having little effect upon anti PGA levels. Serum IgG anti PA was measured in 246 sera of recruits injected with the Anthrax Vaccine Adsorbed (AVA ) stored at the Department of Defense Serum Repository. Paired sera were analyzed by ELISA. Serum conversion rates of !Y 4-fold increase in antibody levels were: pre-post 3rd 85.3%, pre 4th-post 4th 67.9% and pre 6th-post 6th 45%. Geometric mean levels of all individuals were 59.9 microgr/mL following the 3rd injection, 157.4 microgr/mL following the 4th and 277 microgram/mL following the 6th. PLASMODIUM FALCIPARUM: Malaria is a leading cause of morbidity and mortality globally, especially in children, estimated to cause over a million childhood deaths annually. P. falciparum causes the most severe form of disease. Experimental vaccines have been described and some tested clinically but no licensed vaccine is available. The most studied is the circumsporozoite protein (CS), expressed extracellularly on the sporozoite, and various forms of its synthesized repeat unit, NANP. These vaccines were safe and immunogenic but poorly protective, even when administered with adjuvants. Based on our studies with peptides of the B. anthracis capsule, peptides of 4 repeat units of NANP were synthesized and bound to carrier proteins and their immunogenicity studied in general purpose mice without adjuvants and by a scheme suitable for humans. High levels of antibodies were induced, with circumsporozoite neutralizing activity roughly correlated to levels measured by ELISA. In another approach to provide a transmission blocking vaccine, Pfs25, a low molecular weight protein, non immunogenic by itself, was bound to itself or to carrier proteins by several methods: amide, hydrazone or thioether linkages and injected into mice to evaluate their antibody responses. All conjugates were immunogenic with booster responses upon reinjection. The best immunogens were created using Adipic acid dihydrazide as the linker.

炭疽杆菌是人类致命感染的潜在原因，它具有 2 种基本毒力因子，如果没有这两种因子，它对人类就不具有致病性：炭疽毒素和荚膜。该毒素由 3 种肽组成：致死因子、水肿因子和保护性抗原 (PA)。 PA是与哺乳动物细胞结合的毒素部分。 20 KDa 的肽必须被水解，暴露出 LF 或 EF 可能结合的位点，产生毒素，通过酶促修饰哺乳动物细胞胞浆中的底物。该胶囊由聚-D-γ-谷氨酸(PGA)组成，无免疫原性，其保护作用尚不明确。获得许可的基于 PA 的疫苗具有安全性和保护性，但也存在局限性，因此需要开发改进的疫苗。从无荚膜菌株中分离出重组 PA。发现几种含有甲醛处理和明矾吸附材料的制剂在小鼠中具有免疫原性。这些制剂的临床评估正在进行中。该胶囊是从无毒菌株中分离出来的，它或相应的合成肽与 BSA、rEPA、rPA 或破伤风类毒素结合。 γ-D-PGA 和蛋白质之间的硫醚、腙和肟键，在 C 或 N 末端具有活性基团，在小鼠中产生具有免疫原性的缀合物，它们之间没有统计差异。这些抗体具有调理吞噬作用。 10 至 20 聚体长的肽以及每摩尔蛋白质 10-15 摩尔 γ D-PGA 的免疫原性最强。 rPA-PGA 缀合物的剂量反应实验显示，剂量在 0.31 至 20 mcg/小鼠之间，1.25 mcg 是 PGA 反应的最佳剂量，而 PA 抗体水平随着免疫剂量的增加而增加。明矾佐剂的使用增加了PA抗体水平，而对抗PGA水平几乎没有影响。对注射了吸附炭疽疫苗 (AVA) 的 246 名新兵血清中的血清 IgG 抗 PA 进行了测量，这些血清储存在国防部血清库中。通过 ELISA 分析配对血清。抗体水平增加4倍的血清转化率为：第3天前-第4天后85.3%，第4天前-第4天后67.9%，以及第6天前-第6天后45%。第三次注射后所有个体的几何平均水平为 59.9 微克/毫升，第四次注射后为 157.4 微克/毫升，第六次注射后为 277 微克/毫升。 恶性疟原虫：疟疾是全球发病和死亡的主要原因，尤其是儿童，估计每年导致超过一百万儿童死亡。恶性疟原虫引起最严重的疾病。已经描述了实验性疫苗，其中一些已经进行了临床测试，但还没有获得许可的疫苗。研究最多的是在子孢子细胞外表达的环子孢子蛋白（CS），及其合成的重复单元 NANP 的各种形式。这些疫苗安全且具有免疫原性，但保护性较差，即使与佐剂一起施用也是如此。根据我们对炭疽杆菌荚膜肽的研究，合成了 4 个 NANP 重复单元的肽，并将其与载体蛋白结合，并在没有佐剂的通用小鼠中通过适合人类的方案研究了它们的免疫原性。诱导了高水平的抗体，环子孢子中和活性与 ELISA 测量的水平大致相关。在提供传播阻断疫苗的另一种方法中，Pfs25（一种本身不具有免疫原性的低分子量蛋白质）通过多种方法（酰胺、腙或硫醚键）与其自身或载体蛋白结合，并注射到小鼠体内以评估其抗体反应。所有缀合物均具有免疫原性，重新注射后可产生加强反应。使用己二酸二酰肼作为连接体创建了最好的免疫原。