CORE--ADDITIONAL IN VITRO EFFICACY AND TOXICITY TESTING

Core C的长期目标是评估氰韦林(由乳杆菌产生)以及自然产生的抗菌肽Retroclin的体外疗效和毒性。通过为杀微生物剂提供一种自然的传递途径并维持阴道的酸性环境，这些杀菌剂为预防艾滋病毒提供了一种独特的方法。所提出的体外测试算法将有助于将安全、新颖的杀微生物剂组合策略从临床前研究推进到临床研究。每种杀微生物剂的配方将在项目1(氰韦林)和项目2(逆转录环素)的各个实验室进行测试，并且仅 那些具有最高抗艾滋病毒活性的人将被送往Core C进行检测。预计每个项目每年将用以下体外算法测试一到两种化合物：1)项目1和项目2的杀菌剂最初将在依赖于CD4的细胞-细胞传播试验中进行测试，以评估这些化合物抑制CCR5嗜性HIV-1毒株(JR-CSF分子克隆)细胞对细胞感染的能力。2)如果在该检测中观察到阳性活性，则在粘蛋白存在或pH转变的情况下，在该检测中将重复检测每种活性化合物。这种二次测试将有助于确定化合物与粘蛋白或酸性环境的相互作用，类似于阴道中可能发生的情况，可能会影响 杀微生物剂抑制细胞间HIV-1传播的能力。3)如果在重复试验中观察到阳性活性，则每种活性化合物都将在使用嗜CXCR4的HIV-1毒株(SK-1临床分离株)的CD4依赖的细胞-细胞传递试验中进行试验；然而，不会对该试验进行进一步的粘蛋白或pH变化试验。4)与算法的步骤1-3并行，所有发送到该核心的化合物都将通过评估它们对卷叶乳杆菌和简氏乳杆菌生长的影响并确定抑制细菌生长的浓度来进行毒性测试。这些数据与组织外植体和动物研究相结合，将有助于确定临床试验的最佳候选者。

The long-term objective of Core C is to assess the in vitro efficacy and toxicity of Cyanovirin (generated by Lactobacilli) as well as the naturally occurring antimicrobial peptide, Retrocyclin. By providing a natural route of delivery for the microbicides and maintaining the acidic environment of the vagina, these microbicides offer a unique approach to HIV prevention. The proposed in vitro testing algorithm will aid in advancing safe, novel microbicide combination strategies from preclinical to clinical studies. Formulations of each microbicide will be tested in the individual laboratories for Project 1 (Cyanovirin) and Project 2 (Retrocyclin), and only those with the highest anti-HIV activity will be sent to Core C for testing. It is anticipated that one to two compounds per year from each project will be tested in the following in vitro algorithm: 1) Microbicides from Projects 1 and 2 will initially be tested in a CD4-dependent, cell-cell transmission assay to assess the ability of these compounds to inhibit cell to cell infection with a CCR5-tropic HIV-1 strain (JR-CSF molecular clone). 2) If positive activity is observed in this assay, testing of each active compound will be repeated in this assay in the presence of mucin or a pH transition. This secondary testing will help determine if interaction of the compound with mucin or an acidic environment, similar to what may occur in the vagina, might affect the ability of the microbicide to inihibit cell to cell HIV-1 transmission. 3) If positive activity is observed in the repeat testing, each active compound will be tested in a CD4-dependent, cell-cell transmission assay that utilizes a CXCR4-tropic HIV-1 strain (SK-1 clinical isolate); however, further testing with mucin or a pH transition will not be done for this assay. 4) In parallel to steps 1-3 of the algorithm, all compounds sent to this Core will be tested for toxicity by assessing their effect on L. crispatus and L. jensenii growth and determining those concentrations that inhibit bacterial growth. These data, in conjunction with tissue explant and animal studies, will help determine the best candidates for clinical trials.