Protein and Polysaccharide Conjugate Vaccines to enteric diseases

革兰氏阴性病原体的表面多糖，即荚膜或脂多糖，是重要的毒力因子和保护性抗原。脂多糖需要被解毒并分离其O-特异性多糖（O-SP）。多糖的免疫原性可以通过与载体蛋白结合来提高。细菌毒素或类毒素和病毒衣壳蛋白可以是保护性抗原，也可以作为载体蛋白。 伤寒沙门氏菌。（共同主要研究者，林凤英）。对S.伤寒是一种已获批准的伤寒疫苗。为了提高其在幼儿中的免疫原性，将Vi与铜绿假单胞菌的重组外蛋白A（rEPA）缀合。Vi-rEPA在11，600名越南2-5岁奥尔兹中进行的3期试验显示，47个月的疗效为89%。一项针对2至5岁奥尔兹的不同剂量的研究表明，较高的剂量更具免疫原性。2006年开始在301名婴儿中进行第二阶段试验。Vi-rEPA在2、4、6和12个月时与DPT同时注射。对照品接受Hib-TT +DTP或DTP。将IgG抗Vi水平与III期试验中的水平进行比较。 在第一阶段研究中，对注射过一次Vi-rEPA的成年人进行了10年的随访，结果显示，IgG抗Vi水平比建议保护水平高20倍（P<0.0001）。 正在分析8年前用Vi-rEPA免疫的儿童血清抗Vi IgG的持续时间，并将其与4年前的水平和另一个村庄未免疫对照的水平进行比较。 甲型副伤寒沙门氏菌（SPA）是发展中国家第二大常见的肠热病病因。在我们的1期和2期研究中，S.与TT结合的甲型副伤寒O-SP在成人、青少年和幼儿中是安全的和免疫原性的。突变型S.用大肠杆菌的LPS延长链长度调节子wzz基因替换大肠杆菌的LPS延长链长度调节子wzz基因，构建了甲型副伤寒大肠杆菌。coli K12。制备了这种延长的O-SP的缀合物疫苗，并将在小鼠中评价其免疫原性。 E.大肠杆菌O 157是溶血性尿毒综合征的主要原因，尤其是在幼儿中。在我们在2-5岁儿童中进行的O-SP-rEPA缀合物的2期试验中（在卡罗莱纳医学中心），疫苗显示出安全性、免疫原性并引发杀菌抗体; 98%的人在6个月时IgG抗LPS升高4倍。E. coli O 157为滋贺毒素II。由Alison OBrien构建的突变型类毒素与O-SP缀合以增强保护作用正在研究中。 产肠毒素大肠大肠杆菌（ETEC）：ETEC是发展中国家腹泻的最常见原因。ETEC分泌两种外毒素：热不稳定毒素（LT）和热稳定毒素（ST），一种由19个氨基酸组成的多肽。LT在人体内具有免疫原性。然而，其抗感染的功效尚未得到证实。ST体积小，无免疫原性，难以纯化。 一个无毒的突变体，rLT，从约翰克莱门茨福尔马林处理，并在小鼠中诱导高水平的IgG抗LT。有一个剂量依赖性肿胀在注射部位。与Donald Robertson合作，ST被纯化，以适合与LT或其他蛋白质缀合。 霍乱弧菌O 1仍然是印度次大陆和非洲的一个主要健康问题。田间研究表明，抗血清的杀弧菌活性针对LPS。在我们的1期试验中，O-SP缀合物引发高水平的具有杀弧菌活性的IgG抗LPS。O 1霍乱弧菌O-SP的化学合成正在进行中。 空肠弯曲菌感染是常见的，并可能导致严重的并发症，如格林-巴利综合征，可能是由于神经节苷脂和脂寡糖（LOS）的结构相似的C。空肠。2型LOS或去酰化LOS的蛋白质缀合物引起抗LOS IgG和杀菌抗体。28株C.分析了以色列儿科患者空肠分离株的血清型分布，发现16株分离株中约40%在LOS中含有唾液酸，并与人神经节苷脂抗血清结合。 轮状病毒感染是世界范围内婴儿腹泻的最常见原因。目前的疫苗是reflectant全细胞口服疫苗。其中一种疫苗Rotashield在接种者肠套叠疑似增加后退出市场。我们设计了基于衣壳蛋白VP 8和VP 7的肠胃外疫苗。注射缀合衣壳蛋白的小鼠具有针对同源（P4）和异源（P8）血清型的轮状病毒的中和抗体。

Surface polysaccharides of Gram-negative pathogens, capsules or lipopolysaccharides, are essential virulence factors and protective antigens. Lipopolysaccharides need to be detoxified and their O-specific polysaccharides (O-SP) isolated. Immunogenicity of polysaccharides can be improved by binding to carrier proteins. Bacterial toxins or toxoids and viral capsid proteins may be protective antigens and can also serve as carrier proteins. Salmonella typhi. (Co-principal Investigator, Feng-Ying Lin). The Vi capsular polysaccharide of S. typhi is a licensed typhoid vaccine. To improve its immunogenicity in young children, Vi was conjugated to a recombinant exoprotein A of Pseudomonas aeruginosa (rEPA). A phase 3 trial of the Vi-rEPA in 11,600 Vietnamese 2-5-year olds showed an efficacy of 89% at 47 months. A study of various dosages for 2-to-5-year olds showed a higher dosage was more immunogenic. A phase 2 trial in 301 infants started in 2006. Vi-rEPA is injected concurrently with DPT at 2, 4, 6 and 12 months. Controls receive Hib-TT +DTP or DTP. IgG anti-Vi levels will be compared with those elicited in the phase 3 trial. A 10 years follow up of adults injected once with Vi-rEPA in the phase 1 study, showed a G.M. IgG anti-Vi level20 fold higher than the proposed protective level (P<0.0001). The duration of serum anti-Vi IgG in children immunized with Vi-rEPA 8 years ago is under analysis and will be compared with levels 4 years ago and of those of unimmunized controls in another village. Salmonella paratyphi A (SPA) is the second most common cause of enteric fever in developing countries. In our phase 1 and 2 studies, S. paratyphi A O-SP conjugated to TT was safe and immunogenic in adults, teenagers, and toddlers. A mutant S. paratyphi A was constructed by replacing the LPS elongation chain length regulator wzz gene with that of E. coli K12. A conjugate vaccine of this elongated O-SP was prepared and its immunogenicity will be evaluated in mice. E. coli O157 is a major cause of hemolytic uremic syndrome, especially in young children. In our phase 2 trial of O-SP-rEPA conjugate in 2-5 year-olds (at Carolina Medical Center), the vaccine was shown to be safe, immunogenic and elicited bactericidal antibodies; 98% had 4 fold rise of IgG anti-LPS at 6 months. A major virulence factor of E. coli O157 is the Shiga toxin II. A mutant toxoid constructed by Alison OBrien conjugated with O-SP for enhanced protection is under study. Enterotoxigenic E. coli (ETEC): ETEC is the most common cause of diarrhea in developing countries. ETEC secretes two exotoxins: heat-labile toxin (LT) and heat stable toxin (ST), a polypeptide of 19 amino acids. LT is immunogenic in humans. However its efficacy against infection has not been demonstrated. ST is small, non-immunogenic and difficult to purify. A non-toxic mutant, rLT, from John Clements was formalin-treated and in mice it induced high levels of IgG anti-LT. There was a dose dependent swelling at the injection site. In collaboration with Donald Robertson, the ST was purified, to be suitable for conjugation with LT or other proteins. Vibrio cholerae O1 remains a major health problem in the Indian subcontinent and in Africa. Field studies showed that the vibriocidal activity of antisera is directed towards the LPS. In our Phase 1 trial the O-SP conjugates elicited high levels of IgG anti-LPS with vibriocidal activity. Chemical synthesis of O1 V. cholera O-SP is underway. Campylobacter jejuni infection is common and may cause serious complications such as Guillain- Barre syndrome; possibly due to the structural similarity between gangliosides and lipooligosaccharides (LOS) of C. jejuni. Protein conjugates of type 2 LOS or of the de-acylated LOS elicited anti-LOS IgG and bactericidal antibodies. 28 isolates of C. jejuni from pediatric patients in Israel were analyzed for serotype distribution; a diverse serotype distribution was found. Approximately 40% of the 16 isolates contain sialic acid in their LOS and bind to human ganglioside antisera. Rotavirus infection is the most common cause of infantile diarrhea worldwide. Current vaccines are reassortant whole-cell oral vaccines. One such vaccine, Rotashield, was withdrawn from the market after a suspected increase in intussusception in its recipients. We designed parenteral vaccines based on capsid proteins VP8 and VP7. Mice injected with conjugate capsid proteins had neutralizing antibodies against rotavirus of homologous (P4) and heterologous (P8) serotypes.