EVALUATION OF A PRIME-BOOST VACCINE FOR AEROSOL RICIN EXPOSURE IN NHPS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 摘要：蓖麻毒素是一种高效、快速的生物毒素，已被用于生物战。保护受威胁人群免受蓖麻毒素生物攻击的有效策略是生产含有改良蓖麻毒素的疫苗。RiVax是一种新的转基因蓖麻毒素候选疫苗，在非人类灵长类动物模型中非肠道注射时显示出有希望但不是最佳的结果。通过改变给药地点和方式，RiVax的疗效可能会显著提高。首先，鼻腔接种提供了一种无针免疫方法，有可能诱导抗原特异性的全身免疫球蛋白和粘膜免疫球蛋白A。第二，异种Prime/Boost免疫方案诱导的免疫反应通常优于同源Prime/Boost免疫方案所诱导的免疫反应。我们推测，一种新型佐剂(Mastoparan-7；MP-7)和一种新的鼻腔给药方法的使用将增强RiVax鼻腔注射疫苗的免疫原性，并提供一种无针免疫方法，当与RiVax注射免疫相结合时，将在接种动物中诱导抗体。我们的具体目的是确定使用肠外疫苗和鼻腔注射RiVax的异种Prime/Boost免疫策略是否优于肠外免疫，因为它能够诱导血清蓖麻毒素中和抗体。对6只恒河猴(M.mulatta)(n=6)进行异种免疫，先用RiVax肌肉注射(IM)，然后用蓖麻毒素疫苗(仅蛋白质)与Mastoparan-7混合免疫2次，间隔30天鼻腔免疫(IN)。其他组按相同的计划使用掺有氢氧化铝的RiVax进行预接种和加强接种，只使用IM注射作为疫苗接种途径，或假接种生理盐水。不同途径接种疫苗的动物血清免疫应答相似，注射异种疫苗和单独注射疫苗的动物，终点抗体效价分别为1：4,500~1：10,500。通过体外细胞毒性试验测定的血清抗体的中和能力，在接受异种疫苗的动物中几乎没有活性，而接受疫苗IM的组导致中和效价高达1：400。对支气管灌洗液和口腔拭子的分泌性IgA分析正在进行中。在第二次免疫后大约45天，所有动物都被雾化吸入剂量相当于多次(2-5)LD^50的蓖麻毒素。假疫苗接种组的所有动物在暴露后大约+45-50小时死于中毒。IM免疫组的6只动物中有1只(1/6；16%)在攻击后存活。异种免疫组6只动物中有4只存活(4/6；66%)。结果表明，结合MP-7作为粘膜佐剂的异种疫苗给药途径被证明比传统的疫苗给药更有效，尽管血清a-蓖麻毒素中和抗体的免疫球蛋白水平不能预测这种反应。IN疫苗组存活率的提高可能是由于分泌的IgA在进入动物的部位而不是在动物的外围中和了毒素，从而减少了导致血管渗漏综合征的毒性效应。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Abstract: Ricin is a highly potent and rapidly acting biological toxin which has been employed for biologic warfare. An effective strategy to protect threatened populations against biological attack by ricin is to generate vaccines comprising a modified ricin. RiVax is a novel genetically modified ricin vaccine candidate that has shown promising but not optimal results in nonhuman primate models when parenterally administered. The efficacy of RiVax may be significantly improved by altering the site and manner of its delivery. First, nasal vaccination provides a needle-free method of immunization that has the potential to induce antigen-specific systemic IgG as well as mucosal IgA. Second, heterologous prime/boost immunization regimens often induce immune responses that are superior to those induced by homologous prime/boost immunization. We hypothesized that the use of a novel adjuvant (Mastoparan-7; MP-7) and a novel nasal delivery method will enhance the immunogenicity of the nasally delivered RiVax vaccine and provide a needle-free method of immunization that when combined with a parenteral RiVax priming immunization, will induce antibodies in vaccinated animals. Our specific aim is to determine if a heterologous prime/boost immunization strategy utilizing a parenteral prime and a nasal boost with RiVax is superior to parenteral immunization for its ability to induce serum ricin-neutralizing antibodies. A group of rhesus macaques (M. mulatta) (n=6) were heterologously vaccinated by prime immunizing intramuscularly (IM) with RiVax and then boosted twice with ricin vaccine (protein only) admixed with Mastoparan-7 and administered intranasally (IN) 30 days apart. Other groups were primed and boosted with RiVax admixed with aluminum hydroxide by the same schedule using only IM injection as the vaccination route or sham vaccinated with saline. Serum immune response among the groups receiving vaccine were comparable regardless of route, with endpoint IgG titer ranging from 1:4,500 to 1:10,500 in the animals receiving vaccine heterologously or solely by IM injection, respectively. Neutralizing capability of serum antibodies, as determined by an in vitro cytotoxicity assay, showed little to no activity in the animals receiving the vaccine heterologously, whereas the group receiving the vaccine IM resulted in neutralizing titers up to 1:400. Secretory IgA analysis of bronchoalevolar lavage and buccal swabs is in process. Approximately 45 days after the 2nd boost, all animals were challenged with ricin toxin by aerosol at a dose equivalent to multiple (2-5) LD^50s. All animals in the sham-vaccinated group succumbed to intoxication approximately +45-50 hours postexposure. One of the six animals (1/6; 16%) in the IM-vaccinated group survived challenge. Four of the six animals in the heterologous vaccinated group survived challenge (4/6; 66%). Results indicate that the heterologous route of vaccine administration paired with the use of MP-7 as a mucosal adjuvant proved more efficacious than traditional vaccine administration, although serum IgG levels of a-ricin neutralizing antibodies was not predictive of this response. Increased survival in the IN vaccinated group may be due to generation of secretory IgA that neutralized toxin at the site of entry rather than in the periphery of the animal, thereby reducing the toxic effects that lead to vascular leak syndrome.