Second-generation Gene Transfer Vector-based Anti-cocaine Vaccines

描述(申请人提供)：治疗可卡因成瘾的一种方法是接种可卡因疫苗，防止可卡因到达大脑。挑战在于，可卡因和大多数小分子一样，是一种糟糕的免疫原。根据我们在动物和人类中对腺病毒(Ad)基因转移载体的经验，以及对Ad是激活免疫系统的有效佐剂的认识，我们假设与Ad衣壳蛋白相连的可卡因类似物可以诱导足以对Trat可卡因上瘾的高水平、高亲和力的可卡因特异性抗体。我们的第一个方法是将可卡因类似物GNC或GNE与中断的E1-E3-5型Ad(DAd5)联系起来。DAd5GNC疫苗可诱导小鼠产生高亲和力(Kd45nM)的抗可卡因抗体，免疫小鼠不再出现反复静脉注射50g可卡因后的多动行为。DAd5GNE诱导的效价高于dAd5GNC，接种dAd5GNE的大鼠表现出抑制的可卡因奖赏，未接种的大鼠没有看到活动的“突然”消失，也没有在可卡因激发后恢复寻找可卡因。这项提案的三个目标的重点是开发第二代反可卡因疫苗，该疫苗建立在dAd5GNC和dAd5GNE成功的基础上，使较低剂量能够产生更高滴度、更高亲和力的抗可卡因抗体，从而消除与可卡因给药相关的活性和自我给药表型。目的1.衣壳蛋白、六邻体蛋白和纤维蛋白是免疫原性最强的Ad蛋白。我们假设，可卡因类似物GNE直接连接到纯化的六邻体和/或纤维上，将产生比GNE连接到整个被破坏的Ad的更有效的疫苗。此外，将对六邻体和纤维序列进行基因改造，以包括更多的赖氨酸残基，增加GNE的共价结合位点。目的2.针对Ad衣壳蛋白的免疫作用限制了Ad疫苗的效力。由于不同的Ad血清型对免疫的刺激作用不同，我们推测，重复交替给药与破坏的Ad5和sAd36(一种高度免疫原性的非人类灵长类Ad)或交替给药与每种血清型的纯化六角体/纤维相结合，将引起比单一血清型更高的滴度。目的3.我们还利用编码抗可卡因单抗(AAVRh.10antiCoc)的腺相关病毒r.10型，开发了一种“持续被动抗可卡因免疫”策略，其产生的持久抗可卡因抗体足以抑制可卡因诱导的小鼠多动。我们推测，AAVRh.10antiCoc单独或与AIMS 1和2最好的活性Ad疫苗一起，都将为中枢神经系统提供高效的反可卡因保护。还将对鼻腔注射抗可卡因的AAV编码，以确定进入部位的高滴度抗可卡因抗体是否有助于第一次抗可卡因防御。

DESCRIPTION (provided by applicant): One approach to treating cocaine addiction is to vaccinate against cocaine, preventing it from reaching the brain. The challenge is that cocaine, like most small molecules, is a poor immunogen. Based on our experience with adenovirus (Ad) gene transfer vectors in animals and humans, and the recognition that Ad is a potent adjuvant that activates the immune system, we hypothesized that cocaine analogs linked to Ad capsid proteins would elicit high level, high affinity cocaine specific antibodies sufficient to trat cocaine addiction. Our 1st approach was to link the cocaine analogs GNC or GNE to a disrupted E1-E3- serotype 5 Ad (dAd5). The dAd5GNC vaccine elicited high affinity (Kd 45 nM) IgG anti-cocaine titers in mice, and vaccinated mice no longer responded with hyperactive behavior following repetitive intravenous doses of 50 ?g cocaine. dAd5GNE evoked higher titers than dAd5GNC, and dAd5GNE-vaccinated rats exhibited suppressed cocaine reward, no extinction "burst" of activity seen in non-vaccinated rats, and did not reinstate cocaine seeking following a cocaine prime. The focus of the 3 aims of this proposal is to develop 2nd generation anti-cocaine vaccines that build on the success of dAd5GNC and dAd5GNE, enabling lower doses to generate higher titer, higher affinity anti-cocaine antibodies that abrogate the activity and self-administration phenotypes associated with cocaine administration. Aim 1. The capsid hexon and fiber are the most immunogenic Ad proteins. We hypothesize that the cocaine analog GNE coupled directly to purified hexon and/or fiber will generate a more potent vaccine than GNE coupled to the entire disrupted Ad. Further, the hexon and fiber sequences will be genetically modified to include additional lysine residues, increasing covalent attachment sites for GNE. Aim 2. Immunity against Ad capsid proteins limit efficacy of Ad vaccines. Since different Ad serotypes stimulate immunity differently, we hypothesize that repeated alternating administration of GNE coupled to disrupted Ad5 and sAd36 (a highly immunogenic nonhuman primate Ad) or alternating administration of GNE coupled to purified hexons/fibers of each serotype, will evoke higher titers than with a single serotype. Aim 3. We have also developed a "persistent passive anti-cocaine immunity" strategy using an adeno-associated virus serotype rh.10 coding for an anti-cocaine monoclonal antibody (AAVrh.10antiCoc) that generates persistent anti-cocaine antibodies sufficient to suppress cocaine induced hyperactivity in mice. We hypothesize that AAVrh.10antiCoc either alone, or together with the best active Ad vaccines from aims 1 and 2, will provide highly effective anti-cocaine protection to the CNS. An AAV coding for anti-cocaine will also be administered to the nose to determine if high titer anti-cocaine antibodies at the site of entry can aid as a 1st anti-cocaine defense.