Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of HIV-1 transfer and activity in the human VEC organotypic model tissues transduced with AAV-6 expressing b12 minibodies.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-1bal (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 Fig. 5.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198777&req=5

pone-0026473-g006: Inhibition of HIV-1 transfer and activity in the human VEC organotypic model tissues transduced with AAV-6 expressing b12 minibodies.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-1bal (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 Fig. 5.

Mentions: In assessing the AAV-6 vectors, we first wanted to determine the levels of b12 minibodies that could be secreted from AAV transduced cells in the VEC model. For these studies, we obtained commercially produced and CsCl purified AAV-6-b12minibody and control AAV-6-11A minibody The human VEC tissues in transwells were transduced by applying AAV-6 encoding b12 minibody or the negative control 11A minibody (5×1010 genomic copies) to the apical surface of the tissues. Twenty-four hours later, the tissues were washed and cultured for four days before use in the blocking assay. Using a quantitative IgG ELISA, we confirmed that >12 µg/ml of b12 minibody was secreted into the upper chamber of the transwell at day 5 after transduction (data not shown). Cell-free virus transfer and in vitro infectivity after virus challenge were examined in the AAV transduced VEC tissue model. HIV-1bal virus (50 ng) was applied to the apical layer, and media from the bottom chamber was sampled at 1 h, 3 h, 6 h and overnight. The AAV-6-b12 minibody transduced tissue effectively blocked transfer of virus to the lower chamber, while the level of virus transferred in the AAV-6-negative control transduced tissues were not significantly different from that of the untreated control (no AAV transduction) (Fig. 6A). Importantly, the supernatants in the lower chamber of the tissues transduced with AAV-6- -b12 minibody contained little or no virus particles and were not infectious, while those of the tissues that were untreated or transduced with the negative control AAV had high levels of infectious virus in the lower chambers at the 3 and 6 h time points (Fig. 6B). This data demonstrates that recombinant AAV-6 can be used to deliver bNAb/minibodies to primary cervical and vaginal epithelial cells and protect against HIV-1 challenge in vitro.


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)

Inhibition of HIV-1 transfer and activity in the human VEC organotypic model tissues transduced with AAV-6 expressing b12 minibodies.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-1bal (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 Fig. 5.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026473-g006: Inhibition of HIV-1 transfer and activity in the human VEC organotypic model tissues transduced with AAV-6 expressing b12 minibodies.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-1bal (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 Fig. 5.
Mentions: In assessing the AAV-6 vectors, we first wanted to determine the levels of b12 minibodies that could be secreted from AAV transduced cells in the VEC model. For these studies, we obtained commercially produced and CsCl purified AAV-6-b12minibody and control AAV-6-11A minibody The human VEC tissues in transwells were transduced by applying AAV-6 encoding b12 minibody or the negative control 11A minibody (5×1010 genomic copies) to the apical surface of the tissues. Twenty-four hours later, the tissues were washed and cultured for four days before use in the blocking assay. Using a quantitative IgG ELISA, we confirmed that >12 µg/ml of b12 minibody was secreted into the upper chamber of the transwell at day 5 after transduction (data not shown). Cell-free virus transfer and in vitro infectivity after virus challenge were examined in the AAV transduced VEC tissue model. HIV-1bal virus (50 ng) was applied to the apical layer, and media from the bottom chamber was sampled at 1 h, 3 h, 6 h and overnight. The AAV-6-b12 minibody transduced tissue effectively blocked transfer of virus to the lower chamber, while the level of virus transferred in the AAV-6-negative control transduced tissues were not significantly different from that of the untreated control (no AAV transduction) (Fig. 6A). Importantly, the supernatants in the lower chamber of the tissues transduced with AAV-6- -b12 minibody contained little or no virus particles and were not infectious, while those of the tissues that were untreated or transduced with the negative control AAV had high levels of infectious virus in the lower chambers at the 3 and 6 h time points (Fig. 6B). This data demonstrates that recombinant AAV-6 can be used to deliver bNAb/minibodies to primary cervical and vaginal epithelial cells and protect against HIV-1 challenge in vitro.

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