Protein and Polysaccharide Conjugate Vaccines to enteric diseases

伤寒沙门氏菌荚膜多糖(Vi)是一种获得许可的疫苗，但对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。在注射Vi-REPA的2-5岁儿童中，47个月时的有效率为89%。在接种后2、4、6和12个月时，Vi-REPA与扩大免疫计划(EPI)常规疫苗同时接种时，Vi-REPA是安全的，与EPI免疫相容，95%的婴儿末次免疫后抗体水平高于估计保护性抗体水平，因此Vi-REPA适合于婴儿常规免疫。在与韩国国际疫苗研究所和意大利诺华全球卫生研究所的合作下，Vi与获得许可的疫苗结合，如白喉类毒素或其突变体CRM197。在小鼠中，这些结合物诱导的抗Vi抗体水平与Vi-REPA相似。与兰州生物研究所中国合作，纯化了Vi-REPA免疫志愿者的高滴度人血浆池，并测定了单位重量的Ig G抗Vi水平。它将作为人类免疫球蛋白抗体Vi的参考标准在全球范围内分发。与哈佛大学儿童医院的科学家(Lu博士、Malley博士和Anderson博士)合作，我们探索了由Vi和肺炎球菌蛋白组成的二价儿科疫苗的潜力。 与伤寒沙门氏菌不同的是，没有针对非伤寒沙门氏菌的许可疫苗。沙门氏菌D群和B群是发达国家最常见的肠道感染。在非洲，这两个血清群也是导致菌血症的最常见的沙门氏菌。中国常规接种Vi疫苗后甲型副伤寒沙门氏菌感染较伤寒沙门氏菌多见。我们在越南证明了基于内毒素的甲型副伤寒沙门氏菌结合物对2-5岁的儿童是安全的和免疫原性的。D组和B组沙门氏菌的相似结构在小鼠中也显示出免疫原性。这些项目正在我们实验室进行调查。 O1霍乱弧菌仍然是发展中国家的一个主要健康问题；特别是在印度次大陆和非洲。2010年10月，海地爆发霍乱疫情。截至2012年7月，有60多万例病例和8000人死亡。目前还没有疫苗可以控制这种传播。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，霍乱弧菌O-SP与具有杀灭弧菌活性的抗体结合。化学合成与破伤风类毒素(TT)相对应的六聚体、八聚体和十聚体。在小鼠中，这些结合物比我们第一阶段研究中使用的天然O-SP结合物具有更高的杀弧菌活性。由十聚体形成的结合物比六聚体或八聚体结合物产生更高的抗体水平。对各种连接子进行了研究。与非十八聚体接头合成的结合物相比，具有更强的免疫原性。比较了具有不同碳氢或羟基链的连接体的效率、稳定性和溶解性。在与诺华全球健康基金会的合作下，合成的六和十糖将被连接到临床批次TT进行I期研究。 轮状病毒是全球婴儿腹泻的最常见原因。两种获得许可的口服轮状病毒疫苗提供了有限的保护，继续显示肠套叠严重不良反应的病例，一些批次被猪圆环病毒1型和2型DNA污染。也有报道称，接种疫苗后，婴儿体内的基因回复到强毒株。与NIAID的Kapikian、Hoshino和Win博士合作，我们开发了一种基于衣壳蛋白的非肠道疫苗。在大肠杆菌中表达截短C或N端的重组衣壳蛋白，并诱导小鼠和豚鼠产生中和抗体。将重组蛋白质连接到多糖疫苗上，改善了蛋白质的溶解性。衣壳蛋白的双端截短(C端和N端)核心区提供了一种高产量的抗原，在空斑形成试验中可诱导针对P4、P6和P8血清型的中和抗体。为了提高免疫原性，将制备衣壳蛋白和基于VP7的多肽的结合物。 肠出血性大肠杆菌(EHEC)感染是发达国家与大肠杆菌相关的死亡的主要原因。在美国，流行的血清型是O157H：7。2011年在德国和邻国爆发的疫情是由大肠杆菌O104引起的。这两种EHEC菌株都含有志贺毒素(STX)基因，释放的毒素可导致严重的症状；从腹泻、出血性结肠炎到溶血性尿毒症综合征(HUS)和血栓性血小板减少性紫癜。HUS是幼儿急性和慢性肾脏损害的主要原因，并可能导致死亡。在美国一项针对2-5岁儿童的第二阶段研究中，一种基于O157内毒素的结合疫苗在98%的儿童中诱导了具有杀菌活性的抗体增加了10倍。大多数EHEC感染是由分泌Stx2的菌株引起的。通过改进的重组克隆，高效纯化了Stx2的无毒B亚基。Stx2B可作为O157OSP的载体蛋白，为非O157EHEC感染提供更广泛的覆盖范围。

The capsular polysaccharide of S. typhi (Vi) is a licensed vaccine but 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). An efficacy of 89% at 47 months was shown in 2-to-5-year olds injected with Vi-rEPA. When administered concurrently with routine vaccines in the Expanded Program of Immunization (EPI) at 2, 4, 6 and 12 months, Vi-rEPA showed to be safe, compatible with EPI immunization and 95% infants had higher than the estimated protective antibody level after the last injection; thus Vi-rEPA is suitable for routine immunization of infants. In collaboration with the International Vaccine Institute, Korea and the Novartis Institute for Global Health, Italy, Vi was conjugated to licensed vaccines such as diphtheria toxoid or its mutant CRM197. In mice, these conjugates elicited similar IgG anti-Vi levels to those induced by Vi-rEPA. In collaboration with the Lanzhou Institute of Biologics, China, a high tittered human plasma pool from volunteers immunized with Vi-rEPA was purified and the level of IgG anti-Vi determined in weight units. It will be available for distribution globally as a human IgG anti-Vi reference standard. In collaboration with scientists at Children's Hospital, Harvard University (Drs. Lu, Malley and Anderson), we explore the potential of a bivalent pediatric vaccine composed of Vi and a pneumococcal protein. In contrast to S. typhi, there is no licensed vaccine for non-typhoidal Salmonella. Salmonella group D and B are the most common enteric infections in developed countries. In African both sero groups are also the most common Salmonella causing bacterimia. Salmonella paratyphi A infection is more common than S. typhi in China after the routine Vi vaccination. We demonstrated in Vietnam that LPS based S. paratyphi A conjugate was safe and immunogenic in children 2-5 years old. Similar constructs for group D and B Salmonella also showed to be immunogenic in mice. These projects are under investigation in our laboratory. Vibrio cholerae O1 remains a major health problem in developing countries; especially in the Indian subcontinent and in Africa. A cholera outbreak started in Haiti in October 2010. Up to July 2012, there were more than 600,000 cases and 8,000 deaths. No vaccine is available to control the spread. Field studies showed that serum vibriocidal activity is directed toward its LPS. In a phase 1 trial, V. cholerae O-SP conjugates elicited IgG anti-LPS with vibriocidal activity. A hexamer, octamer and decamer corresponding to the O-SP were chemically synthesized and conjugated to tetanus toxoid (TT). In mice these conjugates elicited higher vibriocidal activities than the native O-SP conjugate used in our Phase I study. Conjugates formed by decamer elicited highest antibody levels than hexamer or octamer conjugates. Various linkers were studied. A conjugate synthesized with a heptadecamer linker was more immunogenic than the one with a nonamer linker. Linkers with various hydrocarbon or hydroxyl chains were compared for efficiency, stability, and solubility. In collaboration with Novartis Foundation for Global Health, the synthetic hexa and deca saccharide will be linked to clinical lot TT for a phase I study. Rotavirus is the most common cause of infantile diarrhea worldwide. Two licensed oral rotavirus vaccines conferred limited protection, continued showing cases of the serious adverse reaction of intussusception, and some lots were contaminated with porcine circovirus 1 and 2 DNA. There was also reports on in vivo genetic reversion to virulent strains in infants after vaccination. In collaboration with Drs. Kapikian, Hoshino and Wen at NIAID, we developed a parenteral vaccine based on capsid proteins. Recombinant capsid proteins with truncated C or N termini were expressed in E. coli and elicited neutralizing antibodies in mice and guinea pigs. Conjugation of the recombinant proteins to a polysaccharide vaccine improved protein solubility. The double truncated (both C and N terminals) core region of the capsid protein provided a high yield antigen that elicited neutralizing antibody to the P4, P6 and P8 serotypes in the plaque-forming assay. To improve immunogenicity, conjugates of capsid proteins and VP7 based peptides will be prepared. Enterohemorrahagic E. coli (EHEC) infections are the leading cause of E. coli-related deaths in developed countries. In the US, the prevalent serotype is O157H:7. An outbreak in Germany and neighboring countries in 2011 was caused by E. coli O104. Both of these EHEC strains contain the Shiga toxin (Stx) gene and the released toxin can cause serious manifestations; from diarrhea, hemorrhagic colitis, to hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura. HUS is a major cause of acute and chronic kidney damage in young children and could lead to death. In a phase II study in 2-5 years old children in the US, an O157 LPS based conjugate vaccine elicited a >10 fold rise of antibodies with bactericidal activity in 98% of children. Most EHEC infections are caused by strains secreting Stx2. The non-toxic B-subunit of Stx2 was purified in high yield by an improved recombinant clone. Stx2B could serve as a carrier protein for the E. coli O157 O-SP and provide a broader coverage of non-O157 EHEC infections.