Peptide-Protein Conjugate Vaccines

炭疽芽孢杆菌： 从一株未被包裹的菌株中分离到一株重组PA。几种甲醛处理和或明矾吸附的制剂对小鼠具有免疫原性。对这些配方的临床评估正在进行中。150名18-45岁的志愿者共注射4次，未见严重不良反应。局部和全身反应罕见且轻微。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倍的血清转换率分别为：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，第四次后为157.4微克/毫升，第六次后为277微克/毫升。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PGA和蛋白质之间使用硫醚、肼和肟键，在C端或N端带有活性基团的其他偶联方法在小鼠中产生免疫原性的偶联物，它们之间没有统计学差异。诱导产生的抗体为吞噬细胞抗体。10~20聚体、10~15摩尔PGA/摩尔蛋白的多肽免疫原性最强。RPA-PGA结合物的剂量反应实验表明，1.25微克的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。明矾的使用提高了PA抗体水平，而对抗PGA水平影响不大。 用10只PGA/动物皮下注射免疫黑猩猩，1只用PA-PGA免疫，1只用TT-PGA免疫，目的是制备人源化的抗PGA的单抗。两只黑猩猩都对这两种疫苗成分产生了抗体。注射TT-PGA的黑猩猩体内抗PGA抗体水平较高。制备PGS特异性IgG1和IgG3。 恶性疟原虫： 研究最多的实验性疟疾疫苗候选蛋白是环子孢子蛋白(CSP)，它在子孢子上胞外表达，以及各种形式的合成重复单位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，采用适合人类的方案，在无佐剂的普通小鼠身上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。在另一种方法中，为了提供传播阻断疫苗，Pfs25是一种本身不具有免疫原性的低分子蛋白质，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。值得注意的是，免疫后3个月和7个月的血清抗体水平均高于末次免疫后1周。以己二酸二肼为连接物制备的免疫原性最好。偶联物在明矾上的吸附进一步增加了抗体水平。免疫血清的传播阻断活性与ELISA检测的抗体水平相关。 Pfs25结合物还与CSP的重复单位的合成(NANP)5结合。两次注射后，Pfs25和CsP的吸附量分别从0.8到87g/mL和3.3到53EU/mL。第2次免疫后3个月，抗Pfs25抗体升高至524微克/毫升，抗CSP抗体升高至120 EU/毫升。

Bacillus anthracis: A recombinant PA was isolated from an uncapsulated strain. Several formaldehyde treated and or alum adsorbed formulations were immunogenic in mice. Clinical evaluation of these formulations is underway. 150 18-45 year old volunteers have been injected up to 4 times with no serious adverse effects. Local and systemic reactions were rare and minor. 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 ≥ 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 277microgram/mL following the 6th. 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. Additional conjugating methods using thioether, hydrazone and oxime linkages between the PGA and the proteins, with active groups at the C or N termini yielded conjugates immunogenic in mice, with no statistical difference between them. The induced antibodies were opsonophagocytic. Peptides 10 to 20-mer long, and 10-15 mole PGA per mole protein were the most immunogenic. Dose response experiments of an rPA-PGA conjugate, using between 0.31 to 20 mcg PGA/mouse showed 1.25 mcg to be optimal for a PGA response, while PA antibody levels increased with higher immunizing dosages. The use of alum increased PA antibody levels while having little effect upon anti PGA levels. Chimpanzees were immunized, 10 mcg PGA/animal, sc, one with PA-PGA another with TT-PGA, with the goal of preparing humanized monoclonal antibodies to PGA. Both chimps responded with antibodies to both vaccine components. Higher anti PGA levels were obtained in the TT-PGA injected chimp. PGS specific IgG1 and IgG3 were prepared. Plasmodium falciparum: The most studied experimental malaria vaccine candidate is the circumsporozoite protein (CSP), 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 by a scheme suitable for humans. High levels of antibodies were induced, with circumsporozoite neutralizing activity correlated roughly 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. Remarkably, the serum antibody levels 3 and 7 months after immunization were higher than 1 week after the last injection. The best immunogens were created using adipic acid dihydrazide as the linker. Adsorption of the conjugates onto alum increased further the antibody levels. Transmission blocking activity of immune sera correlated with antibody levels measured by ELISA. The Pfs25 conjugate was also bound to a synthetic (NANP)5 of the repeat unit of the CSP. This conjugate induced IgG antibodies to both its components and adsorption onto alum increased further both levels: from 0.8 to 87g/mL for Pfs25 and from 3.3 to 53 EU/mL for CSP after 2 injections. Three months after the 2nd injection IgG anti Pfs25 increased to 524 mcg/mL and anti CSP to 120 EU/mL.