Peptide-Protein Conjugate Vaccines

肽-蛋白结合疫苗

• 批准号: 7212387
• 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/7212387
• 关键词: Bacillus anthracis; Plasmodium falciparum; anthrax toxin; anthrax vaccines; bacterial capsules; bacterial proteins; bioterrorism /chemical warfare; circumsporozoite protein; enzyme activity; enzyme linked immunosorbent assay; human subject; immunoconjugates; immunoglobulin G; immunologic assay /test; laboratory mouse; malaria vaccines; microorganism antigen; microorganism immunology; neutralizing antibody; serum; statistics /biometry; vaccine development; vaccine evaluation

BACILLUS ANTHRACIS, a cause of lethal human infection and a bioterrorist weapon, has 2 essential virulence factors without either of which it is not pathogenic for humans. These factors are the anthrax toxin, and the capsule. The toxin is composed of 3 proteins: Lethal Factor (LF), Edema Factor (EF) and Protective Antigen (PA), each by itself non toxic. PA is the toxin part that binds to mammalian cells. A 20 KDa peptide must be hydrolyzed off PA, 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 unknown. The licensed vaccine 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 has begun. The capsule has been isolated from a non toxic strain and it or corresponding synthetic peptides were bound to BSA, rEPA or rPA. To identify the optimal construct peptides of varying lengths: 5, 10 and 20-mers, of D or L configuration with active groups at the C or N terminus were conjugated. The conjugates were characterized by physico-chemical and immunological assays and immunogenicity in 5-6 week old mice. Opposed to PGA alone all conjugates were immunogenic and the PGA induced antibodies were opsonophagocytic. rPA was the most effective carrier. Additional conjugation methods yielded conjugates immunogenic in mice, inducing levels not statistically different from those described in our publication. Dose response experiments of an rPA-PGA conjugate, with doses between 0.31 and 20 mcg/mouse showed 1.25-2.5 mcg to be optimal 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. SEROSURVEY OF ARMED FORCES PERSONNEL IMMUNIZED WITH THE LICENSED ANTHRAX VACCINE: A survey was made of IgG antibodies in 246 sera sets at the U.S. Armed Forces Repository, taken from Armed Forces Personnel immunized with the FDA-licensed Anthrax Vaccine Adsorbed (AVA). Paired sera from HIV-negative personnel before and after the 3rd, 4th, and 6th injections were assayed by ELISA using purified Protective Antigen (PA) from Bacillus anthracis. The personnel were stratified according to sex and age (18-24 years and >24 years). Serum conversion rates of >= 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 mcg/mL following the 3rd injection, 157.4 mcg/mL following the 4th, and 277 mcg/mL following the 6th. The levels were similar between the males and females but the GMC was statistically significantly higher for the younger vs. the older group. These values will be used to evaluate the IgG anti-PA levels induced by our investigational B. anthracis vaccines. 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 relevant for humans. Preliminary results showed high levels of antibodies, 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, was bound to carrier proteins and injected into mice to evaluate their antibody responses.

炭疽杆菌是一种致命的人类感染原因和生物恐怖武器，具有两种基本毒力因子，没有其中任何一种，它对人类都没有致病性。这些因素是炭疽毒素和胶囊。该毒素由3种蛋白质组成：致死因子（LF）、水肿因子（EF）和保护性抗原（PA），每种蛋白质本身都是无毒的。PA是与哺乳动物细胞结合的毒素部分。20 KDa的肽必须从PA水解，暴露LF或EF可能结合的位点，从而产生酶促修饰哺乳动物细胞胞质溶胶中底物的毒素。该胶囊由聚-D-γ-谷氨酸（PGA）组成，无免疫原性，其保护作用未知。许可的疫苗是安全和保护性的，但有限制，证明改进疫苗的发展。从未包囊化的菌株中分离重组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。这些制剂的临床评价已经开始。该胶囊已从无毒菌株中分离，并且其或相应的合成肽与BSA、rEPA或rPA结合。为了鉴定不同长度的最佳构建体肽：缀合在C或N末端具有活性基团的D或L构型的5、10和20聚体。在5-6周龄小鼠中通过物理化学和免疫学测定以及免疫原性来表征缀合物。与单独的PGA相反，所有缀合物都是免疫原性的，并且PGA诱导的抗体是调理吞噬的。rPA是最有效的载体。另外的缀合方法在小鼠中产生免疫原性缀合物，诱导水平与我们的出版物中描述的水平没有统计学差异。rPA-PGA偶联物的剂量反应实验（剂量在0.31和20 mcg/小鼠之间）显示1.25-2.5 mcg对于PGA反应是最佳的，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。 用许可的炭疽疫苗免疫的武装部队人员的血清调查：对美国武装部队储存库的246个血清组中的IgG抗体进行了调查，这些血清组取自用FDA许可的吸附炭疽疫苗（AVA）免疫的武装部队人员。使用纯化的炭疽杆菌保护性抗原（PA），通过ELISA检测HIV阴性人员在第3次、第4次和第6次注射前后的配对血清。根据性别和年龄（18-24岁和>24岁）对人员进行分层。抗体水平增加>= 4倍的血清转换率为：第3次治疗前-治疗后，85.3%;第4次治疗前-治疗后，67.9%;第6次治疗前-治疗后，45%。第3次注射后，所有个体的几何平均水平为59.9 mcg/mL，第4次注射后为157.4 mcg/mL，第6次注射后为277 mcg/mL。男性和女性之间的水平相似，但年轻组的GMC在统计学上显著高于老年组。这些值将用于评价我们的试验用B诱导的IgG抗PA水平。炭疽疫苗 疟原虫：疟疾是全球发病率和死亡率的主要原因，特别是在儿童中，估计每年造成100多万儿童死亡。恶性疟原虫引起最严重的疾病形式。实验性疫苗已经描述，一些临床测试，但没有许可的疫苗。研究最多的是环子孢子蛋白（CS），在孢子上细胞外表达，以及各种形式的合成重复单元NANP。这些疫苗是安全和免疫原性的，但保护性差，即使与佐剂一起施用。基于我们对B肽的研究。炭疽胶囊，NANP的4个重复单元的肽的合成和结合到载体蛋白和它们的免疫原性在没有佐剂的通用小鼠中研究，并通过与人类相关的方案。初步结果显示抗体水平较高，环子孢子中和活性与ELISA测量的水平大致相关。在提供传递阻断疫苗的另一种方法中，将低分子量蛋白PFS 25与载体蛋白结合并注射到小鼠中以评估其抗体应答。