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Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

Abdel-Motal UM, Sarkis PT, Han T, Pudney J, Anderson DJ, Zhu Q, Marasco WA - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: 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.

Methods and findings: 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.

Conclusion: 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.

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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.(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.
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pone-0026473-g003: 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.(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.

Mentions: Since terminally-differentiated apical vaginal epithelial cells continuously shed, targeting the genital epithelial stem cells for transduction by AAV would be ideal for stable and durable gene transfer in vivo. Accordingly, we examined whether human genital epithelial stem cells could be transduced by AAV-6-GFP in vitro. The isolated huPGECs were exposed to AAV-6-GFP overnight and then stained with antibodies against the epithelial stem cell markers, anti-CK17 and nuclear anti-p63 [25]. The stained cells were analyzed by flow cytometry by gating first on CK17/p63 double positive cells (4.47%) (Fig. 3A) and then on the GFP-positive cells within this population (Fig. 3B). The AAV-6-GFP transduced cells were then sorted for further examination by fluorescence microscopy (Fig. 3C-F). Representative images of cells displaying the epithelial cell marker CK17 (blue), stem cell marker p63 (red) and GFP (green) are shown in Figure 3C-E (individual signals) and Figure 3D (merged image). These data demonstrate that AAV-6-GFP can successfully transduce cells with reported epithelial stem cell characteristics.


Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

Abdel-Motal UM, Sarkis PT, Han T, Pudney J, Anderson DJ, Zhu Q, Marasco WA - PLoS ONE (2011)

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.(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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198777&req=5

pone-0026473-g003: 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.(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.
Mentions: Since terminally-differentiated apical vaginal epithelial cells continuously shed, targeting the genital epithelial stem cells for transduction by AAV would be ideal for stable and durable gene transfer in vivo. Accordingly, we examined whether human genital epithelial stem cells could be transduced by AAV-6-GFP in vitro. The isolated huPGECs were exposed to AAV-6-GFP overnight and then stained with antibodies against the epithelial stem cell markers, anti-CK17 and nuclear anti-p63 [25]. The stained cells were analyzed by flow cytometry by gating first on CK17/p63 double positive cells (4.47%) (Fig. 3A) and then on the GFP-positive cells within this population (Fig. 3B). The AAV-6-GFP transduced cells were then sorted for further examination by fluorescence microscopy (Fig. 3C-F). Representative images of cells displaying the epithelial cell marker CK17 (blue), stem cell marker p63 (red) and GFP (green) are shown in Figure 3C-E (individual signals) and Figure 3D (merged image). These data demonstrate that AAV-6-GFP can successfully transduce cells with reported epithelial stem cell characteristics.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: 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.

Methods and findings: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus