CONTINUED DEVELOPMENT OF RIVAX VACCINE FOR RICIN

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 蓖麻毒素是已知的最有效的生物毒素之一，被CDC列为B类生物威胁。最近，许多注意力集中在实际使用蓖麻毒素的潜在威胁上。由于暴露后的治疗是无效的，除非在一个狭窄的时间窗口内，接种疫苗可能是唯一的方法，以防止致命性和组织损伤所造成的蓖麻毒素。我们已经开发了一种安全有效的疫苗（RiVax（tm）），该疫苗基于消除A链毒性的重组突变体。已经实现了用于制造疫苗的稳健、高产率和可扩展的工艺。基于临床前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中的耐受性和免疫原性。我们已经确定了疫苗的佐剂配方，接下来将在志愿者中进行测试。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个既定的候选人。具体而言，我们将对溶液中的蛋白质和吸附至铝盐佐剂的蛋白质进行长期稳定性研究。我们将评估蛋白质的构象方面，并将其与效价相关。其次，我们的目标是证明疫苗将在兔和人体内产生抗体，这些抗体可以在气雾剂或口服蓖麻毒素暴露后被动地为兔提供保护。使用小鼠以外的其他动物物种将为关键动物疗效试验奠定基础，这些试验必须代替人体试验进行（根据FDA动物规则）。第三，我们将在小鼠和家兔中进行GLP临床前毒理学和有效性试验，以支持佐剂疫苗的临床评价。我们的目标是获得数千剂已发布的疫苗，并对其进行稳定性评估。最后，我们打算执行IND提交所需的监管工作。该提案代表了Rivax进一步发展的关键一步，以进行更多的临床试验并最终注册和上市。在生物恐怖主义中使用蓖麻毒素是一个非常真实的世界性的威胁。一种安全有效的FDA批准的疫苗是迫切需要的军事人员，并在发生国内攻击，为第一反应，也许为广大公众。 我们已经完成了候选Rivax疫苗在非人灵长类动物中的I期有效性试验。 用吸附在铝胶上的100 μ g疫苗免疫动物（n=6）（初免，两次加强）或仅用佐剂假免疫（n=3）。 血清抗体（α蓖麻毒素IgG）和抗体的中和能力分别通过ELISA和蓖麻毒素细胞毒性试验。 然后通过气雾剂以致死剂量（1 LD/50）的蓖麻毒素攻击所有动物。 结果表明，免疫组的存活率很低（16，16%），假免疫对照组的死亡率为100%（0/3）。 本研究的结果表明，尽管在免疫动物中α蓖麻毒素IgG终点滴度相对较高（2.0E+04），但如结合抗体ELISA进行的体外中和试验所示，对蓖麻毒素全毒素的中和能力相对较低。 此外，抗体产生和动力学在初免后+14天显示出峰值，在任一免疫加强时均没有可定义的记忆应答。 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，才能提供保护。 本研究的第二阶段正在进行中，并将纳入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，并增加候选疫苗的总体保护能力。

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.