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Engineered Vaginal Lactobacillus Strain for Mucosal Delivery of the Human Immunodeficiency Virus Inhibitor Cyanovirin-N

Osel, Inc., 4008 Burton Drive, Santa Clara, California 95054,¹ Laboratory Animal Medicine, Vaccine Research Center, National Institutes of Health, Bethesda, Maryland 20892,² Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892,³ Department of Medicine, Stanford University, Stanford, California 94305⁴

Received 20 April 2006/ Returned for modification 7 June 2006/ Accepted 31 July 2006

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.