Liu Xiaowen, Lagenaur Laurel A, Simpson David A, Essenmacher Kirsten P, Frazier-Parker Courtney L, Liu Yang, Tsai Daniel, Rao Srinivas S, Hamer Dean H, Parks Thomas P, Lee Peter P, Xu Qiang
Osel, Inc, Santa Clara, CA 95054, USA.
Antimicrob Agents Chemother. 2006 Oct;50(10):3250-9. doi: 10.1128/AAC.00493-06.
Women are at significant risk of human immunodeficiency virus (HIV) infection, with the cervicovaginal mucosa serving as a major portal for virus entry. Female-initiated preventatives, including topical microbicides, are urgently needed to help curtail the HIV/AIDS pandemic. Here we report on the development of a novel, live microbicide that employs a natural vaginal strain of Lactobacillus jensenii engineered to deliver the potent HIV inhibitor cyanovirin-N (CV-N). To facilitate efficient expression of CV-N by this bacterium, the L. jensenii 1153 genome was sequenced, allowing identification of native regulatory elements and sites for the chromosomal integration of heterologous genes. A CV-N expression cassette was optimized and shown to produce high levels of structurally intact CV-N when expressed in L. jensenii. Lactobacillus-derived CV-N was capable of inhibiting CCR5-tropic HIV(BaL) infectivity in vitro with a 50% inhibitory concentration of 0.3 nM. The CV-N expression cassette was stably integrated as a single copy into the bacterial chromosome and resolved from extraneous plasmid DNA without adversely affecting the bacterial phenotype. This bacterial strain was capable of colonizing the vagina and producing full-length CV-N when administered intravaginally to mice during estrus phase. The CV-N-producing Lactobacillus was genetically stable when propagated in vitro and in vivo. This work represents a major step towards the development of an inexpensive yet durable protein-based microbicide to block the heterosexual transmission of HIV in women.
女性面临着感染人类免疫缺陷病毒(HIV)的重大风险,宫颈阴道黏膜是病毒进入的主要门户。迫切需要女性主导的预防措施,包括局部用杀菌剂,以帮助遏制HIV/艾滋病的流行。在此,我们报告了一种新型活杀菌剂的研发情况,该杀菌剂采用经基因工程改造的詹氏乳杆菌天然阴道菌株来递送强效HIV抑制剂蓝藻素-N(CV-N)。为便于该细菌高效表达CV-N,对詹氏乳杆菌1153的基因组进行了测序,从而能够鉴定天然调控元件以及异源基因的染色体整合位点。对CV-N表达盒进行了优化,结果表明在詹氏乳杆菌中表达时可产生高水平的结构完整的CV-N。源自乳杆菌的CV-N能够在体外抑制CCR5嗜性HIV(BaL)的感染性,其50%抑制浓度为0.3 nM。CV-N表达盒作为单拷贝稳定整合到细菌染色体中,并与外源质粒DNA分离,且未对细菌表型产生不利影响。在发情期将该细菌菌株经阴道给予小鼠时,它能够在阴道定植并产生全长CV-N。产生CV-N的乳杆菌在体外和体内繁殖时遗传稳定。这项工作朝着开发一种廉价且持久的基于蛋白质的杀菌剂迈出了重要一步,该杀菌剂可阻断HIV在女性中的异性传播。