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

描述（由申请人提供）：治疗可卡因成瘾的一种方法是注射抗可卡因疫苗，防止可卡因进入大脑。挑战在于可卡因和大多数小分子一样，是一种很差的免疫原。基于我们在动物和人类中腺病毒（Ad）基因转移载体的经验，以及Ad是激活免疫系统的有效佐剂的认识，我们假设与Ad衣壳蛋白相关的可卡因类似物会引发高水平、高亲和力的可卡因特异性抗体，足以治疗可卡因成瘾。我们的第一种方法是将可卡因类似物GNC或GNE与中断的E1-E3-血清型5ad （dAd5）联系起来。dAd5GNC疫苗在小鼠中引发了高亲和力（Kd为45 nM）的抗可卡因IgG滴度，并且接种疫苗的小鼠在重复静脉注射50 ？克可卡因。dAd5GNE诱导的滴度高于dAd5GNC，接种dAd5GNE的大鼠表现出抑制的可卡因奖励，没有像未接种dAd5GNE的大鼠那样出现灭绝“爆发”的活动，并且在可卡因启动后没有恢复可卡因寻求。本提案的3个目标的重点是在dAd5GNC和dAd5GNE成功的基础上开发第二代抗可卡因疫苗，使低剂量产生高滴度、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。衣壳六邻体和纤维是最具免疫原性的Ad蛋白。我们假设可卡因类似物GNE直接偶联到纯化的六邻体和/或纤维将产生比GNE偶联到整个中断的Ad更有效的疫苗。此外，六邻体和纤维序列将进行基因修饰，以包括额外的赖氨酸残基，增加GNE的共价附着位点。目标2。对Ad衣壳蛋白的免疫限制了Ad疫苗的效力。由于不同的Ad血清型对免疫的刺激不同，我们假设反复交替给药，将GNE偶联到中断的Ad5和sAd36（一种高度免疫原性的非人灵长类Ad），或交替给药，将GNE偶联到纯化的六分体/纤维的每种血清型，将产生比单一血清型更高的滴度。目标3。我们还开发了一种使用腺相关病毒rh血清型的“持久被动抗可卡因免疫”策略。10编码抗可卡因单克隆抗体（AAVrh.10antiCoc），产生持久的抗可卡因抗体，足以抑制小鼠可卡因诱导的过度活跃。我们假设。抗coc单独使用或与目标1和目标2中最好的活性Ad疫苗一起使用，将为中枢神经系统提供非常有效的抗可卡因保护。抗可卡因的AAV编码也将被施用于鼻腔，以确定进入部位的高滴度抗可卡因抗体是否可以作为第一道抗可卡因防御。

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.