C3 chemical conjugation to improve the anti-cocaine immune response

描述(由申请人提供)：根据PA-10-069的这项R21提案将是免疫药物疗法和NIDA滥用药物疫苗计划的一次飞跃，并“测试一个新的和重要的假设，如果实验得到证实，将对考虑针对小分子的疫苗产生重大影响”。核心平台利用了新发现的能力，用PI的研究中制造的功能性肼分子对天然状态的补体蛋白C3进行化学修饰。该小组将基于滥用小分子药物作为免疫原与C3复合而产生革命性的疫苗。疫苗开发的四步理论方法如图1所示。这一概念既简单，又在全球范围内适用，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。这一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：目的1设计、合成线性B细胞表位-小分子肼并将其附着到小鼠C3上(图1的步骤1-3)。这一新的免疫药物治疗平台主要应用于可卡因(COC)，然后是苯环利定(PCP)和甲基苯丙胺(MET)，将被研究。因此，小鼠C3(非人类)将被分离和提纯，用于该过程的第一步，使用PI实验室的技术程序。对于步骤2，将在PI的实验室合成两个并入Mengo病毒VP1259-277线性B细胞表位的联氨基炔，这些B细胞表位将在PI的实验室已开发的条件下与天然小鼠C3反应。对于步骤3，叠氮类似物可卡因、五氯苯酚和甲基苯丙胺将在PI的实验室中合成，并将在标准的化学条件下与步骤2中的C3-炔烃偶联物反应。目的2.用C3药物生物偶联物对小鼠进行主动免疫和免疫反应的量化(图1的步骤4)。来自AIM 1的C3生物结合物将用于免疫雌性Balb/c小鼠(每组3-5只)，在存在和不存在外部佐剂(Ribis)的情况下。检测抗药物血清效价、多克隆免疫球蛋白结合亲和力、结合特异性(药物与B细胞表位)和免疫球蛋白持久性。作为对照，将进行药物的KLH结合物的比较免疫。 与公共卫生相关：能够生产有效的疫苗，使免疫系统能够识别小分子，这是目前挖掘滥用科学中尚未满足的需求。这一建议利用先天免疫系统的力量，通过化学合成与补体蛋白C3结合的小分子疫苗来产生新的小分子疫苗。

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.