Development of an HIV-1 Microbicide Based on Caulobacter crescentus: Blocking Infection by High- Density Display of Virus Entry Inhibitors Christina Farr 1 , John F. Nomellini 1 , Evan Ailon 1 , Iryna Shanina 1 , Sassan Sangsari 1 , Lisa A. Cavacini 2 , John Smit 1. , Marc S. Horwitz 1 * . 1 Department of Microbiology and Immunology, The University of British Columbia, Vancouver, British Columbia, Canada, 2 Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America Abstract The HIV/AIDS pandemic remains an enormous global health concern. Despite effective prevention options, 2.6 million new infections occur annually, with women in developing countries accounting for more than half of these infections. New prevention strategies that can be used by women are urgently needed. Topical microbicides specific for HIV-1 represent a promising prevention strategy. Conceptually, using harmless bacteria to display peptides or proteins capable of blocking entry provides an inexpensive approach to microbicide development. To avoid the potential pitfalls of engineering commensal bacteria, our strategy is to genetically display infection inhibitors on a non-native bacterium and rely on topical application of stabilized bacteria before potential virus exposure. Due to the high density cell-surface display capabilities and the inherent low toxicity of the bacterium, the S-layer mediated protein display capabilities of the non-pathogenic bacterium Caulobacter crescentus has been exploited for this approach. We have demonstrated that C. crescentus displaying MIP1a or CD4 interfered with the virus entry pathway and provided significant protection from HIV-1 pseudovirus representing clade B in a standard single cycle infection assay. Here we have expanded our C. crescentus based microbicide approach with additional and diverse classes of natural and synthetic inhibitors of the HIV-1 entry pathway. All display constructs provided variable but significant protection from HIV-1 infection; some with protection as high as 70%. Further, we describe protection from infection with additional viral clades. These findings indicate the significant potential for engineering C. crescentus to be an effective and readily adaptable HIV-1 microbicide platform. Citation: Farr C, Nomellini JF, Ailon E, Shanina I, Sangsari S, et al. (2013) Development of an HIV-1 Microbicide Based on Caulobacter crescentus: Blocking Infection by High-Density Display of Virus Entry Inhibitors. PLoS ONE 8(6): e65965. doi:10.1371/journal.pone.0065965 Editor: Philippe Gallay, Scripps Research Institute, United States of America Received October 31, 2012; Accepted May 2, 2013; Published June 19, 2013 Copyright: ß 2013 Farr et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Canadian Institute for Health Research. The funders had no role in study design, data collection and analysis. decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mhorwitz@mail.ubc.ca . These authors contributed equally to this work. Introduction The HIV/AIDS pandemic is one of the largest global health concerns, with over 32 million people worldwide living with an HIV infection [1]. The majority of new infections occur through sexual transmission, with 2.6 million new infections annually [1,2]. Sexual transmission of HIV can be prevented by the use of condoms, but women in developing countries do not always have the option to insist on condom use, often due to cultural or religious practices [2]. As such, women over the age of 15 in developing countries account for the majority of new HIV-1 infections [1,2]. The development of prevention strategies that can be used by women is urgently needed. With the difficulties in developing an efficacious vaccine, alternative prevention options are required. Microbicides are drug products that are topically applied to mucosal surfaces to prevent infection [3]. They are able to fill the need for female controlled prevention, as they can be administered without partner knowledge, and are able to maintain efficacy for extended periods of time. Currently, no microbicides for HIV are on the market, but approximately 50 candidates are under development [3–5]. Many of these are non-specific and/or expensive to produce, which may limit the clinical effectiveness and practicality for delivery to third world populations [3,6]. To counteract these difficulties, some efforts have focussed on engineering bacteria to display HIV blocking agents [6–8]. While most of these approaches have used commensal vaginal bacteria, such as Lactobacillus, use of these recombinant bacteria would rely on their ability to colonize the vaginal tract and maintain expression of the protein for extended periods of time, which has shown limited success [6–8]. Issues with expression and secretion of larger proteins, as well as concerns about eradication of the bacteria should unwanted side effects occur, suggest the use of Lactobacillus species in microbicide formulations may be problematic [6–8]. In contrast, we have engineered an alternative bacterium-based microbicide strategy that does not require colonization or maintenance of the bacteria within the individual. Using the non-pathogenic, freshwater bacterium Caulobacter crescentus [9,10] we have generated a system to insert foreign protein sequences PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e65965