C3 chemical conjugation to improve the anti-cocaine immune response

描述（由申请人提供）：PA-10-069下的R21提案提供了免疫药物治疗和NIDA滥用药物疫苗计划的飞跃，并“测试了一个新的重要假设，如果通过实验证实，将对小分子疫苗的思考产生重大影响”。核心平台利用了PI研究中新发现的用功能性肼分子化学修饰天然状态下补体蛋白C3的能力。基于小分子药物滥用分子与C3络合的免疫原，产生革命性的疫苗。疫苗研制的四步理论方法如图1所示。这个概念既简单，又适用于全球，而且可能具有开创性。免疫原是一种宿主蛋白（不是外来的），不需要外部佐剂。这种新的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：设计、合成线性b细胞表位小分子肼并将其附着在小鼠C3上（图1的步骤1-3）。我们将首先研究这种新的免疫药物治疗平台在可卡因（COC）、苯环利定（PCP）和甲基苯丙胺（MET）小鼠中的应用。因此，小鼠C3（非人类）将被分离和纯化，用于该过程的第一步，使用PI实验室的常规技术。对于第二步，将在PI实验室合成两个含Mengo病毒VP1259-277线性b细胞表位的肼酰炔烃，这些b细胞表位肼酰炔烃将在PI实验室已经开发的条件下与天然小鼠C3反应。在步骤3中，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，并将在标准的“点击”化学条件下与步骤2中的c3 -炔偶联物反应。目标2。c3 -药物生物偶联物小鼠主动免疫及免疫应答定量（图1步骤4）。来自Aim 1的c3生物偶联物将用于免疫雌性Balb/c小鼠（每组3-5只），无论是否存在外部佐剂（RIBIs）。检测抗药血清滴度、多克隆IgG结合亲和力、结合特异性（药物对b细胞表位）和IgG持久性。与药物的klh偶联物进行比较免疫接种将作为对照。

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.