Background:  Heterosexual transmission accounts for the majority of new cases of HIV-1 infection each year. The increased incidence of HIV-1 infection in women underscores the urgent need for an effective preventative microbicide. Lactic acid bacteria (LAB) are a part of the normal vaginal microflora. Here, we demonstrate that LAB can be genetically engineered to secrete HIV-1 fusion inhibitors (FI). We hypothesize that vaginal application of recombinant LAB secreting HIV-1 FI could be an effective microbicide to prevent or reduce HIV-1 transmission in females.

Methods:  Lactococcus lactis and Lactobacillus plantarum were transformed with the pKS series of theta-replicating plasmids. Four different fusion inhibitors [C34, T20, T1249, and T45] were introduced in a secretion cassette containing the USP45 secretion signal. A propeptide sequence (DTNSD) was added to the N-terminus of each fusion-inhibitor peptide. FI sequences were codon-adjusted to the codon usage of Lb. plantarum. Bacterial supernatants (sup) were sampled in the stationary growth phase, sterile filtered, and pH adjusted. Antiviral activity of bacterial sup was tested in MT4 and TZM cells using a single-cycle luciferase reporter virus NL-Luc/HXB2 and the infectious molecular clone NL4.3, respectively.

Results:  HIV-1 fusion inhibitors can be efficiently expressed and secreted in L. lactis and Lb.plantarum. Inclusion of the DTNSD propeptide sequence increased FI secretion efficiency and enhanced antiviral activity. Sup containing the fusion inhibitor D-T45 reduced HIV-1 infection of MT4 cells with NL-Luc/HXB by > 95 % and infection of TZM cells with a primary patient-derived isolate by ~ 73 %. Sup containing the fusion inhibitors D-T45 and D-T1249 suppressed viral infection of TZM cells with the infectious molecular clone HIV-1_NL4.3 by > 95%.

Conclusion:  Inhibitors of HIV-1 fusion can be expressed by LAB and retain antiviral activity. These data form the basis for the potential intravaginal application of recombinant lactobacilli-secreting HIV-1 fusion inhibitors may be an effective strategy to prevent transmission of HIV-1 in women. Furthermore, lactobacterial expression of additional antiviral compounds such as cyanovirin, single-chain antibodies, and CCR5 antagonists merits further investigation.

Keywords: fusion inhibitors; lactic acid bacteria; HIV-1 infection