Protein and Polysaccharide Conjugate Vaccines to enteric diseases

革兰氏阴性菌表面多糖，即衣壳或脂多糖，是重要的毒力因子和保护性抗原。通过与载体蛋白的共价结合，提高了多糖的免疫原性。作为疫苗，必须对内毒素进行解毒，并分离其O-特异性多糖(O-SP)并与蛋白质结合。细菌毒素或类毒素和病毒衣壳蛋白可能是保护性抗原，并作为载体蛋白。 伤寒沙门氏菌Vi衣壳多糖(Vi)是一种许可疫苗，对5岁以下儿童的疗效有限。为给幼儿提供疫苗，将Vi与重组铜绿假单胞菌外源蛋白A(REPA)偶联。在11600名越南2至5岁儿童中进行的Vi-REPA第三阶段试验显示，47个月时有效率为89%。在第二阶段试验中，301名婴儿在2个月、4个月、6个月和12个月时同时注射Vi-REPA和DPT。对照组接受Hib-TT+DTP或DTP治疗。未见严重不良反应。将检测Ig G抗Vi抗体水平，并与2至5岁儿童进行比较。 评估成人和儿童接种Vi-REPA后的抗体持久性。单次注射10年后，18名成人的GM.Ig G抗Vi水平比建议的保护水平(3.52 EU)高出20倍。9年后，97名2-5岁儿童两次注射的转基因抗体水平仅比建议的保护水平高2.2倍(7.62EU比3.52EU；P&lt；0.01)。本组Vi抗体水平与单次注射(7.04EU)或不注射(8.14EU；8.14vs7.62或7.04，P=NS)之间无显著差异，一方面反映了幼儿抗Vi抗体的减弱，另一方面反映了天然抗体的产生。在对5至8岁的儿童进行单次注射四年后，G.M抗体水平为15.9EU，显著高于年龄匹配的对照组。 甲型副伤寒沙门氏菌(SPA)是发展中国家引起肠热病的第二大常见原因。我们的1期和2期研究表明，与TT结合的SPA O-SP对成人、青少年和幼儿是安全的和免疫原性的。为了提高结合免疫原性，将SPA内毒素链长调节因子WZZ基因替换为E.coliK12(D.Kopecko)的基因。与野生型O-SP相比，该细长O-SP的几种TT结合物可诱导更高水平的抗内毒素。 产肠毒素大肠杆菌(ETEC)是发展中国家和前往这些地区的旅行者腹泻的最常见原因。ETEC疾病由两种外毒素介导：热不稳定毒素(LT)和热稳定毒素(ST)。它对人类有免疫原性。ST是一种由19个氨基酸组成的多肽；由于其体积较小，不具有免疫原性。它们的抗感染效果尚未在人类身上得到证明。LT192是一种毒性降低的突变体，(来自J.Clements)在小鼠中诱导高水平的免疫球蛋白G抗LT。然而，即使在福尔马林脱毒后，注射部位仍有剂量依赖性的肿胀。当使用双突变LT(第192和211位氨基酸突变)作为免疫原时，这种不良反应被减少。野生型LT和双突变株在小鼠体内产生相似的抗LT抗体水平。临床研究也在计划之中。由于二硫键的数量较多(3对)，化学合成ST的几次尝试都以失败告终。在与唐纳德·罗伯逊的合作下，从大肠杆菌中提纯了ST。制备了一种牛血清白蛋白偶联物，并对其免疫原性进行了评价。 O1霍乱弧菌仍然是印度次大陆和非洲的一个主要健康问题。现场研究表明，血清杀灭弧菌活性是针对其内毒素的。在一期试验中，O-SP偶联产生具有杀灭弧菌活性的抗脂多糖抗体。为了提高O-SP的免疫原性，我们设计了一种新的偶联方案，将载体蛋白和O-SP用异双功能试剂硫基化。这些结合物在小鼠中诱导的抗体水平显著高于我们第一阶段研究中使用的结合物诱导的抗体水平。我们还合成了霍乱弧菌O-SP的六聚体。该六聚体与TT结合，具有抗原性。将对新的细菌O-SP结合物和合成的糖结合物的免疫原性进行评估。 O157大肠杆菌是溶血性尿毒症综合征的主要原因，尤其是在幼儿中。对55名2~5岁儿童注射O157O-SP-REPA结合物1~2次。该疫苗安全，具有免疫原性；注射后6个月，98%的儿童血清中的免疫球蛋白抗体水平比免疫前增加了4倍以上。第二次注射没有引起增强反应。美国疾病控制与预防中心肠道疾病流行病学部的帕特里夏·格里芬和明尼苏达大学传染病中心的迈克尔·奥斯特霍尔姆共同策划了一项计划，在O157大肠杆菌感染率最高的州为2至10岁的儿童接种疫苗。 轮状病毒是全球婴儿腹泻的最常见原因。两种轮状病毒疫苗获得许可，一种是减毒活疫苗，另一种是重组疫苗，均为口服疫苗。我们正在设计一种基于衣壳蛋白VP8和VP7的非肠道疫苗。从杆状病毒感染的昆虫细胞中纯化出VP8，注射的小鼠产生了针对同源(P4)和异源(P8)两种血清型轮状病毒的中和抗体。为了提高VP8的产量，在大肠杆菌中表达了VP8的基因。重组VP8能诱导出抗P4和P8轮状病毒的中和抗体。

Surface polysaccharides of Gram-negative pathogens, capsules or lipopolysaccharides (LPS), are essential virulence factors and protective antigens. The immunogenicity of polysaccharides is enhanced by covalent binding to carrier proteins. To serve as vaccines, LPS must be detoxified and their O-specific polysaccharides (O-SP) isolated and conjugated to proteins. Bacterial toxins or toxoids and viral capsid proteins may be protective antigens and serve as carrier proteins. The Vi capsular polysaccharide of Salmonella typhi (Vi) is a licensed vaccine with limited efficacy in children less than 5 years old. To provide a vaccine for younger children Vi was conjugated to recombinant exoprotein A of Pseudomonas aeruginosa (rEPA). A phase 3 trial of Vi-rEPA in 11,600 Vietnamese 2-to-5-year olds showed an efficacy of 89% at 47 months. In a phase 2 trial, 301 infants were injected with Vi-rEPA concurrently with DPT at 2, 4, 6 and 12 months. Controls received Hib-TT +DTP or DTP. There were no serious adverse reactions. IgG anti-Vi levels will be assayed and compared with those elicited in 2-to-5 year old children. Antibody persistence after vaccination with Vi-rEPA was evaluated in adults and in children. The G.M. IgG anti-Vi level of 18 adults 10 years after a single injection was 20 fold higher than the proposed protective level (3.52 EU). The G.M. antibody level of 97 children injected twice at 2-5 years of age 9 years later was only 2.2 fold higher than the proposed protective level (7.62 EU vs 3.52 EU; P<0.01). There was no significant difference between Vi antibody levels in this group and in those that had only one injection (7.04 EU) or no injection (8.14 EU; 8.14 vs 7.62 or 7.04, P=NS), reflecting waning of anti-Vi in young children on one hand and the development of natural antibodies on the other. Four years after a single injection into 5-to-8-year olds the G.M antibody level was 15.9 EU, significantly higher than of age-matched controls. Salmonella paratyphi A (SPA) is the second most common cause of enteric fever in developing countries. Our phase 1 and 2 studies showed that SPA O-SP conjugated to TT was safe and immunogenic in adults, teenagers, and toddlers. To improve conjugate immunogenicity, SPA LPS chain length elongation regulator wzz gene was replaced with that of E. coli K12 (from D. Kopecko). Several TT conjugates of this elongated O-SP elicited significantly higher levels of anti-LPS than those prepared with O-SP from the wild type. Enterotoxigenic E. coli (ETEC) are the most common cause of diarrhea in developing countries and in travelers to these areas. ETEC disease is mediated by two exotoxins: heat-labile toxin (LT) and heat stable toxin (ST). LT is immunogenic in humans. ST is a polypeptide of 19 amino acids; not immunogenic due to its small size. Their efficacy against infection has not been demonstrated in humans. A mutant of reduced toxicity, LT192 (from J. Clements) elicited high levels of IgG anti-LT in mice. However, there was a dosage dependent swelling at the injection site even after formalin detoxification. This adverse reaction was reduced when a double mutant LT (mutation at amino acids 192 and 211) was used as an immunogen. The wild type LT and the double mutant elicited similar IgG anti-LT levels in mice. Clinical studies are planed. Due to the high number of disulfide bonds (3 pairs), several attempts of chemical synthesis of ST have failed. In collaboration with Donald Robertson, ST was purified from E. coli. A conjugate of ST linked to bovine serum albumin was prepared and its immunogenicity will be evaluated in mice. Vibrio cholerae O1 remains a major health problem in the Indian subcontinent and in Africa. Field studies showed that serum vibriocidal activity is directed toward its LPS. In a phase 1 trial, O-SP conjugates elicited IgG anti-LPS with vibriocidal activity. To enhance its immunogenicity, a new scheme of conjugation was devised by thiolating both the carrier protein and the O-SP with heterobifunctional reagents. Antibody levels induced in mice by these conjugates were significantly higher than of those induced by the conjugate used in our phase 1 study. We also synthesized a hexamer of V. cholerae O-SP. Conjugated to TT, the hexamer was antigenic. The immunogenicity of the new bacterial O-SP conjugates and of the synthetic saccharide conjugates will be evaluated. E. coli O157 is a major cause of hemolytic uremic syndrome, especially in young children. An O157 O-SP-rEPA conjugate was injected into 55 2-to-5 years old children once or twice. The vaccine was safe and immunogenic; 6 months after injection, 98% of the children had a greater than 4 fold increase of IgG anti-LPS over their levels before immunization. The second injection did not induce a booster response. A proposal to immunize children between 2-to-10 years old in states having the highest incidence rates of E. coli O157 infection was planed in collaboration with Patricia Griffin of the Division of Enteric Disease Epidemiology, CDC and Michael Osterholm of the Center for Infectious Diseases, University of Minnesota. Rotavirus is the most common cause of infantile diarrhea worldwide. Two rotavirus vaccines are licensed, a live attenuated and a reassortant, both orally administered. We are designing a parenteral vaccine based on capsid proteins VP8 and VP7. VP8 was purified from bacular-virus infected insect cells (from T. Hoshino) and mice injected produced neutralizing antibodies against rotavirus of both homologous (P4) and heterologous (P8) serotypes. To increase the yield of VP8, its cDNA was expressed in E. coli. The recombinant VP8 elicited neutralizing antibodies to both P4 and P8 rotavirus.