Peptide-Protein Conjugate Vaccines

炭疽芽孢杆菌是人类致命感染的潜在原因，它有两个基本的毒力因素，如果没有这两个因素，它对人类就不是致病的：炭疽毒素和胶囊。该毒素由致死因子、水肿素和保护性抗原(PA)3种多肽组成。PA是与哺乳动物细胞结合的毒素部分。一个20 KDa的多肽必须被水解，暴露出一个Lf或EF可能结合的部位，从而产生毒素，对哺乳动物细胞胞浆中的底物进行酶促修饰。该胶囊由聚D-γ-谷氨酸(PGA)组成，无免疫原性，其保护作用尚不清楚。以PA为基础的许可疫苗是安全和保护的，但也有局限性，证明有理由开发改进的疫苗。从一株未被包裹的菌株中分离到一株重组PA。几种经甲醛处理和明矾吸附材料的配方在小鼠身上被发现具有免疫原性。对这些配方的临床评估正在进行中。该胶囊是从一株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。在小鼠体内，D-PGA与蛋白质之间的硫醚、硫酮和肟键，在C端或N端有活性基团，可产生免疫原性的偶联物，它们之间没有统计学差异。这些抗体是吞噬细胞的。免疫原性最强的是10-20-MERS多肽和10-15摩尔D-PGA/摩尔蛋白。RPA-PGA结合物的剂量反应实验表明，1.25微克是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。明胶佐剂的使用提高了PA抗体水平，而对抗PGA水平影响不大。对246名新兵注射国防部血清库保存的炭疽吸附疫苗(AVA)后的血清进行了血清免疫球蛋白Ig G、抗PA抗体的检测。双份血清用酶联免疫吸附试验进行分析。抗体水平提高4倍的血清阳转率分别为：术后第3天85.3%，第4天67.9%，第6天45%。所有个体的几何平均水平在第三次注射后为59.9微克/毫升，在第四次注射后为157.4微克/毫升，在第六次注射后为277微克/毫升。 恶性疟原虫：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。恶性疟原虫是最严重的疾病。已经描述了实验性疫苗，并对一些疫苗进行了临床测试，但没有获得许可的疫苗。目前研究最多的是胞外表达的环子孢子蛋白(CS)及其合成重复单位NANP。这些疫苗是安全的和免疫原性的，但即使与佐剂一起接种，保护作用也很差。在炭疽杆菌胶囊肽研究的基础上，合成了NANP的4个重复单元的多肽，并与载体蛋白结合，在无佐剂的普通小鼠和适合人类的方案下研究了它们的免疫原性。诱导了高水平的抗体，环子孢子的中和活性与酶联免疫吸附试验测定的水平大致相关。在另一种提供传播阻断疫苗的方法中，Pfs25是一种本身不具有免疫原性的低分子蛋白，通过几种方法结合到自身或载体蛋白上，并注射到小鼠体内，以评估它们的抗体反应。所有的结合物都是免疫原性的，在再次注射时具有增强反应。以己二酸二肼为连接物制备的免疫原性最好。

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.