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。这些制剂在成年志愿者中是安全的。局部和全身反应罕见且轻微。他们的血清免疫球蛋白、抗PA水平正在进行分析。 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。抗体水平增加4倍以上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3次注射后为59.9微克/毫升，第4次后为157.4微克/毫升，第6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。PGA与蛋白质之间的硫醚、硫酮和肟键，在C端或N端带有活性基团，在小鼠体内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。 Bcla的糖基部分是一种由2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4，6-dideoxy-D-glucose和三个鼠李糖残基组成的寡糖。与人类相似的结构，在 (1)希万氏菌(Shewanella spp.)MR-4：4-(3-hydroxy-3-methylbutanamido)-4，6-dideoxy-D-glucose.在一定的生长条件下，细菌会产生一种在羟丁酸酯上缺少一个甲基的变异CP：4-(3-hydroxybutanamido)-4，6-dideoxy-D-glucose.与人类相反，两个希瓦尼拉CPS都不是2-O-甲基化的。两种希瓦氏菌CPS变异体均与抗B抗体反应。炭疽孢子血清。 (2)紫丁香假单胞菌的鞭毛，据报道被类似的末端糖4-(3-hydroxybutanamido)-4，6-dideoxy-2-O-methyl-D-glucose.糖基化在荧光显微镜分析中，用与炭疽杆菌孢子结合但不与蜡状芽胞结合的希瓦氏菌或丁香疫霉菌细胞免疫产生的血清。 这两种变异型希瓦氏菌CP的蛋白结合物诱导的抗体结合到这两个变异型的希瓦纳氏菌变异体，通过酶联免疫吸附试验，并结合到炭疽杆菌孢子，通过荧光显微镜检测。我们建议使用希瓦氏菌CP结合物作为炭疽疫苗的一个组成部分。 阐明了希瓦氏菌MR-4脂多糖骨架的结构。没有发现类似人类的糖，这只证实了CP中存在这种糖。 恶性疟原虫： 研究最多的实验性疟疾疫苗是环子孢子蛋白(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、免疫原性的，但保护性差，有效期有限，即使在给予佐剂的情况下也是如此。我们使用了两种方法来提供实验性疟疾疫苗： (1)针对寄生虫的无性、人类阶段；将CSP克隆到大肠杆菌中，分离并在幼年杂交小鼠身上评估了几种制剂，包括对明胶的吸附。在炭疽杆菌胶囊多肽研究的基础上，合成了NANP的4-5个重复单元的多肽，并以不同的摩尔比和末端基团与载体蛋白结合。注射到一般用途的小鼠中，所有测试的制剂都能诱导高水平的抗体，这些抗体与IFA中的环子孢子结合。NANP-Pfs25结合物诱导对这两种疫苗成分产生持久抗体；最后一次注射后3个月血清水平高于1周。明矾吸附CSP诱导的抗体水平高于未吸附的蛋白。CSP衍生肽的末端氨基酸是一个重要的决定因素，其中NPNA蛋白是最好的免疫原。 (2)针对有性、蚊虫寄生期，提供传播阻断疫苗。Pfs25是一种本身不具有免疫原性的小分子蛋白质，它通过酰胺、肼或硫醚键与自身或载体蛋白结合。注射到小鼠体内，所有的结合物都是免疫原性的，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的免疫原效果最好。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。

Bacillus anthracis: A recombinant PA was isolated from an uncapsulated strain. Several formaldehyde treated and/or alum adsorbed formulations were immunogenic in mice. These formulations were safe in adult volunteers. Local and systemic reactions were rare and minor. Their serum IgG anti PA levels are being analyzed. 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 greater than or equal to a 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 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 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. The glycosyl part of BclA is an oligosaccharide composed of 2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-D-glucose referred to as anthrose, and three rhamnose residues. Similar structures to anthrose, were found in (1) the side-chain of the capsular polysaccharide (CP) of Shewanella spp. MR-4: 4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-D-glucose. Under certain growth conditions the bacteria produce a variant CP lacking one methyl group on the hydroxybutyrate: 4-(3-hydroxybutanamido)-4,6-dideoxy-D-glucose. Contrary to anthrose, neither of the Shewanella CPs is 2-O-methylated. Both Shewanella CPS variants reacted with anti-B. anthracis spore sera. (2) flagellae of Pseudomonas syringae, reported to be glycosylated with a similar terminal saccharide, 4-(3-hydroxybutanamido)-4,6-dideoxy-2-O-methyl-D-glucose. Sera produced by immunization with Shewanella or P. syringae cells bound to B. anthracis spores but not to B. cereus spores in a fluorescent microscopy assay. Protein conjugates of the two variants of Shewanella CP induced antibodies that bound to both Shewanella CP variants, by ELISA, and to B. anthracis spores, detected by fluorescent microscopy. We propose the use of Shewanella CP conjugates as a component of an anthrax vaccine. The structure of Shewanella MR-4 LPS carbohydrate backbone was elucidated. No anthrose-like sugar was found, confirming the presence of such sugar in the CP only. Plasmodium falciparum: The most studied experimental malaria vaccine is the circumsporozoite protein (CSP), expressed extracellularly on the sporozoite, and various forms of its synthesized repeat unit, NANP. These vaccines were safe, immunogenic but poorly protective and of limited duration, even when administered with adjuvants. We used two approaches to provide experimental malaria vaccines: (1) directed to the asexual, human stage of the parasite; CSP was cloned into e.coli, isolated and several formulations, including adsorption onto alum, were evaluated in young outbred mice. Based on our studies with peptides of the B. anthracis capsule, peptides of 4-5 repeat units of NANP were synthesized and boundnanp- to carrier proteins at different molar ratios and end groups. Injected into general purpose mice, all tested preparations induced high levels of antibodies that bound to circumsporozoites in IFA. NANP-Pfs25 conjugates induced long lasting antibodies to both vaccine components; 3 months serum levels higher than 1 week after the last injection. Alum adsorbed CSP induced higher antibody levels than the non-adsorbed protein. The terminal amino acid of the CSP-derived peptides was shown to be an important determinant, with NPNA-protein being the best immunogen. (2) directed to the sexual, mosquito parasite stage, to provide a transmission blocking vaccine. Pfs25, a low molecular mass protein, non immunogenic by itself, was bound onto itself or to carrier proteins by amide, hydrazone or thioether linkages. Injected into mice, 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 used 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.