Anti-gp120 Minibody Gene Transfer to Female Genital Epithelial Cells Protects against HIV-1 Virus Challenge In Vitro

Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb) that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS.This study tested the hypothesis that adeno-associated virus (AAV)-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc), or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal) in an organotypic human vaginal epithelial cell (VEC) model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP.This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles

Inhibition of HIV-1 transfer and activity by b12 minibodies in the human VEC organotypic model tissues.

<p>After a 1 h pre-incubation of b12 minibodies or full-length b12 IgG (10 µg/ml) with HIV-1_bal (50 ng), medium from the basal chambers were collected at different time points and tested for inhibition of HIV-1_bal transfer by measuring p24 content by ELISA (A) and for inhibition of virus infectivity by incubation on TZM-bl target cells (B). Note that media collected at 3 and 6 h from tissue samples treated with HIV-1_bal and b12 IgG1 antibodies or with b12 minibodies had almost completely lost their ability to infect TZM-bl cells. Irrelevant 11 A minibodies served as negative controls.</p

Functional comparison of b12 minibodies and full-length b12 IgG.

<p>Both b12 minibodies and full-length b12 IgG proteins were tested at equimolar concentrations for their capacity to (A) bind to HIV-1 gp120 by ELISA, and (B) to neutralize HIV-1_bal virus.</p

Transduction of human genital epithelial stem cells by AAV-6-GFP. At 3 days after exposure to AAV-6-GFP, cells were stained with anti-Ck17 and anti-p63 antibodies.

<p>(A) The stained cells were analyzed by flow cytometry by gating first on CK17/p63 double positive cells (A) and then on the GFP-positive cells within this population (B). The AAV-6-GFP transduced cells were then sorted for further examination by fluorescence microscopy. A representative cluster of cells displaying all three distinct colors are shown: (C) blue (anti-ck17), (D) red (anti-p63) and (E) green (GFP). (F) Merged image of C, D and E. (G-I) Immunohistochemical staining of epithelial stem cells in vaginal, ectocervical and endocervical tissues. The p63 positively stained cells are mainly located in the basal epithelial cell layer. Note that in the endocervix (I), the epithelium is composed of a single cell thick layer under which the epithelial stem cells are located.</p

Transduction of human endocervical, ectocervical and vaginal epithelial cells by various AAV serotypes expressing GFP.

<p>(A) Expressions of GFP protein by transduced cells were detected by FACS and presented as percentages of GFP positive cells. Note that AAV-2 and AAV-6 yielded the highest transduction rates. (B) A dose dilution of AAV-6-GFP vector. (C) AAV-8-GFP and AAV-9-GFP transduction of COS-1 cells. (D) Visual assessment of AAV-6-GFP transduction by fluorescence microscopy of vaginal, ectocervical and endocervicel cell lines.</p

Inhibition of HIV-1 transfer and activity in the human VEC organotypic model tissues transduced with AAV-6 expressing b12 minibodies.

<p>AAV-6-b12 minibodies or AAV-6-11A minibodies (control) at 5×10¹⁰ particles was applied to the upper layers of the VEC tissues for 24 h for transduction. Four days after the transduction, HIV-1_bal (50 ng) was applied to the upper layers of the tissues, and medium from the basal chambers were collected at various timepoints and tested for inhibition of viral transfer (A) and infectivity (B) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026473#pone-0026473-g005" target="_blank">Fig. 5</a>.</p