C3 chemical conjugation to improve the anti-cocaine immune response

描述（由申请人提供）：PA-10 - 069下的R21提案是免疫药物治疗和NIDA滥用药物疫苗计划的一个飞跃，并且“测试了一种新的重要假设，如果通过实验证实，将对关于小分子疫苗的思考产生重大影响”。核心平台利用PI研究中新发现的用功能性酰肼分子化学修饰天然状态下补体蛋白C3的能力。该小组以小分子药物滥用分子为基础，作为与C3复合的免疫原，生产革命性的疫苗。疫苗开发的四步理论方法见图1。这一概念既简单，又适用于全球，而且可能具有开创性。免疫原是宿主蛋白（不是外来的），不需要外部佐剂。该新免疫原平台的开发和应用将在以下两个目标的范围内进行测试：目标1线性B细胞表位-小分子酰肼与鼠C3的设计、合成和连接（图1的步骤1 - 3）。将研究这种新的免疫药物治疗平台在小鼠中主要应用于可卡因（COC），然后应用于苯环己哌啶（PCP）和甲基苯丙胺（MET）。因此，将使用PI实验室的常规技术分离和纯化小鼠C3（非人）用于工艺步骤1。对于第2步，将在PI实验室合成两个含有门戈病毒VP 1259 - 277线性B细胞表位的酰肼炔，并且这些B细胞表位酰肼炔将使用PI实验室已经开发的条件与天然鼠C3反应。对于步骤3，可卡因、PCP和甲基苯丙胺的叠氮化物类似物将在PI的实验室中合成，并将使用标准"点击"化学条件与步骤2的C3-炔缀合物反应。目标2.用C3-药物生物缀合物主动免疫小鼠并定量免疫应答（图1的步骤4）。在存在和不存在外部佐剂（RIBI）的情况下，来自目标1的C3-生物缀合物将用于免疫雌性Balb/c小鼠（每组3 - 5只）。抗药物血清滴度、多克隆IgG结合亲和力、结合特异性（药物相对于B细胞表位）和IgG持久性都被测量。用药物的KLH-缀合物进行比较性免疫接种作为对照。 公共卫生相关性：生产允许免疫系统识别小分子的有效疫苗的能力是目前药物滥用科学中未满足的需求。该提议通过化学合成与补体蛋白C3偶联的小分子疫苗来利用先天免疫系统的能力，以产生新型小分子疫苗。

DESCRIPTION (provided by applicant): This R21 proposal under PA-10-069 offers to be a leap-forward in immunopharmacotherapy and NIDA's vaccine program for drugs of abuse and 'tests a novel and significant hypothesis which if confirmed by experiment would have a substantial impact on thinking regarding vaccines against small molecules'. The core platform exploits the newly discovered ability to chemically modify complement protein C3 in its native state with functional hydrazide molecules that was made in the PI's research. group to generate revolutionary vaccines based on small molecule drug of abuse molecules as immunogens complexed to C3. The four step theoretical approach to vaccine development is shown in Figure 1. The concept is at once simple, globally applicable and potentially ground breaking. The immunogen is a host protein (not foreign) and should not require an external adjuvant. The development and application of this new immunogen platform will be tested under the remit of the following two aims: Aim 1 Design, synthesis and attachment of linear B-cell epitope-small molecules hydrazides to murine C3 (STEPS 1-3 of Figure 1). The aplication of this new immunopharmacotherapy platform primarily to cocaine (COC), and then to phencyclidine (PCP) and methamphetamine (MET) in mice will be investigated. Thus, mouse C3 (not human) will be isolated and purified for Step 1 of the process using a technique routine in the PI's lab. For Step 2, two hydrazide alkynes, incorporating the Mengo virus VP1259-277 linear B-cell epitope, will be synthesized in the PI's laboratory and these B-cell epitope hydrazide alkynes will be reacted with native murine C3 using conditions already developed in the PI's laboratory. For Step 3, azide analogs of cocaine, PCP and methamphetamine will be synthesized in the PI's laboratory and will be reacted with the C3-alkyne conjugates from Step 2 using standard 'click' chemistry conditions. Aim 2. Active immunization of mice with C3-drug bioconjugates and quantification of the immune response (STEP 4 of Figure 1). The C3-bioconjugates from Aim 1 will be used to immunize female Balb/c mice (3-5 per group), in the presence and absence of external adjuvant (RIBIs). Anti-drug serum titers, polyclonal IgG binding affinity, binding specificity (drug versus B-cell epitope), and IgG persistence all being measured. Comparative immunizations with KLH-conjugates of the drugs will be performed as a control. PUBLIC HEALTH RELEVANCE: The ability to produce effective vaccines that allow immune system recognition of small molecules is a current unmet need in dug abuse sciences. This proposal harnesses the power of the innate immune system by chemical synthesis of small molecule vaccines coupled to complement protein C3 to generate novel small molecule vaccines.