CONTINUED DEVELOPMENT OF RIVAX VACCINE FOR RICIN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 蓖麻毒素是已知的最有效的生物毒素之一，被美国疾病控制与预防中心列为B类生物毒素。最近，许多注意力集中在实际使用蓖麻毒素的潜在威胁上。由于暴露后治疗无效，除非在很短的时间内进行，接种疫苗可能是预防蓖麻毒素致死和对组织造成损害的唯一方法。我们已经开发出一种安全有效的疫苗(RiVax(Tm))，它基于一种能消除A链毒性的重组突变体。已经实现了一种健壮、高产和可扩展的疫苗生产工艺。基于临床前的安全性和有效性数据，启动了一项小型I期试验，以测试疫苗在人类志愿者身上的耐受性和免疫原性。我们已经确定了疫苗的佐剂配方，接下来将在志愿者身上进行测试。该疫苗已经通过了最初的开发障碍。这个项目的目的是继续发展这一既定的候选人。具体来说，我们将对蛋白质在溶液中的长期稳定性进行研究，并将其吸附到铝盐佐剂上。我们将评估蛋白质的构象方面，并将它们与效价联系起来。其次，我们的目标是证明该疫苗将在兔子和人类身上产生抗体，这些抗体可以在雾化或口服蓖麻毒素后被动地为兔子提供保护。使用除小鼠以外的另一种动物将为关键的动物功效试验奠定基础，这些试验必须取代人体试验(根据FDA的动物规则)。第三，我们将在小鼠和兔身上进行GLP临床前毒理学和有效性试验，以支持佐剂疫苗的临床评估。我们的目标是获得几千剂经过稳定性评估的释放疫苗。最后，我们打算为IND提交进行必要的监管工作。这项建议代表着RiVax进一步发展的关键一步，朝着更多的临床试验以及最终的注册和营销。在生物恐怖主义中使用蓖麻毒素是一个非常现实的全球威胁。军事人员迫切需要一种安全有效的FDA批准的疫苗，如果发生国内袭击，急救人员甚至可能是普通公众都需要这种疫苗。 我们已经完成了候选RiVax疫苗在非人类灵长类动物身上的疗效试验的第一阶段。将100ug疫苗吸附于琼脂糖凝胶上免疫动物(n=6)或仅用佐剂进行假免疫(n=3)。用ELISA法测定血清抗体(α-蓖麻毒素免疫球蛋白G)，用蓖麻毒素细胞毒试验测定抗体的中和能力。然后，所有动物用气雾剂攻击致死剂量(1LD/50)的蓖麻毒素。结果表明，免疫组存活率较低(1/6，16%)，而假免疫对照组存活率为100%(0/3)。这项研究的结果表明，尽管免疫动物的α-蓖麻毒素免疫终点滴度相对较高(2.0E+04)，但与抗体ELISA联合进行的体外中和试验显示，对蓖麻毒素全毒素的中和能力相对较低。此外，抗体的产生和动力学显示在基础免疫后+14天达到峰值，在两次免疫增强时都没有明确的记忆反应。这一结果表明，只有当抗体的质量与体内产生的数量相匹配时，才能获得保护。这项研究的第二阶段正在进行中，将纳入RiVax免疫剂量和时间表的变化，这可能会刺激免疫动物的记忆反应，并增加候选疫苗的整体保护能力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ricin is one of the most potent biological toxins known, and is classified by the CDC as a category B biothreat. Much attention has been recently focused on the potential threat of actual ricin use. Since post exposure treatment is ineffective unless administered within a narrow window of time, vaccination may be the only ways to prevent lethality and damage to tissue caused by ricin. We have developed a safe and effective vaccine (RiVax(tm)) based on a recombinant mutant that eliminates the toxicities of the A chain. A robust, high yield and scalable process for manufacturing the vaccine has been achieved. Based on preclinical safety and efficacy data, a small Phase I trial was initiated to test the tolerability and immunogenicity of the vaccine in human volunteers. We have characterized adjuvant formulations of the vaccine which will be tested next in volunteers. The vaccine has passed the initial development hurdles. The purpose of this project is to continue development of this established candidate. Specifically, we will conduct long term stability studies of the protein in solution and adsorbed to aluminum salts adjuvant. We will assess the conformational aspects of the protein and relate them to potency. Secondly, we aim to demonstrate that the vaccine will generate antibodies in rabbits and humans that can passively confer protection to rabbits after aerosol or oral ricin exposure. The use of an additional animal species other than mice will lay the groundwork for pivotal animal efficacy trials which must be conducted in place of human trials (under the FDA animal rule). Thirdly, we will conduct GLP preclinical toxicology and efficacy trials in mice and rabbits to support the clinical evaluation an adjuvanted vaccine. Our goal is to obtain several thousand doses of released vaccine that has been evaluated for stability. And finally, we intend to perform the regulatory work necessary for IND submission. This proposal represents a critical step in the further development of Rivax towards additional clinical trials and ultimately registration and marketing. There is a very real worldwide threat for the use of ricin in bioterrorism. A safe and effective FDA-approved vaccine is urgently needed for military personnel and, in the event of a domestic attack, for first responders and perhaps for the general public. We have completed Phase I of an efficacy trial with the candidate Rivax vaccine in nonhuman primates. Animals (n=6) were immunized (prime, two boosts) with 100 ug of the vaccine adsorbed to alhydrogel or sham-vaccinated with adjuvant only (n=3). Serum antibodies (alpha ricin IgG) and neutralizing capacity of antibodies were performed by ELISA and ricin cytotoxicity assay, respectively. All animals were then challenged by aerosol to a lethal dose (1 LD/50) of ricin toxin. Results indicated poor survival in the immunized group (1/6, 16%) and 100% lethality in the sham-immunized controls (0/3). The results of this study suggest that, although alpha ricin IgG endpoint titers were relatively high in immunized animals (2.0E+04), the neutralizing capacity to ricin holotoxin was relatively low as shown in the in vitro neutralization assay performed in conjunction with the antibody ELISAs. In addition, antibody production and kinetics showed a peak +14 days post prime immunization, with no definable memory response at either of the immunizing boosts. This result suggests that protection may only be conferred when the quality of the antibodies match the quantity produced in vivo. The second phase of this study is ongoing and will incorporate a variation of RiVax immunizing doses and schedule which may stimulate a memory response in the immunized animals and increase the overall protective capacity of the candidate vaccine.