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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 endocervical, ectocervical and vaginal epithelial cells by various AAV serotypes expressing GFP.(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.
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pone-0026473-g001: Transduction of human endocervical, ectocervical and vaginal epithelial cells by various AAV serotypes expressing GFP.(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.

Mentions: To identify the most optimal AAV serotype for transduction of female genital cells, the efficiencies of AAV 1, 2, 3, 4, 5, 6, 8 and 9 expressing GFP were evaluated by transducing immortalized human endocervical (Endl/E6E7), ectocervical (Ectl/E6E7) and vaginal (VK2/E6E7) epithelial cell lines. Relative transduction efficiencies were evaluated by flow cytometry (Fig. 1A) as well as visual assessment by fluorescence microscopy (Fig. 1D). AAV-1, AAV-2, AAV-5 and AAV-6 were found to transduce the endocervical, ectocervical and vaginal epithelial cell lines with AAV-2 and AAV6 being the most efficient. By contrast, the transduction efficiencies of AAV-3, AAV-4, AAV-8 and AAV-9 serotypes were low in these lines. In a dose dilution experiment using AAV-6-GFP vector, transduction efficiencies of the three genital epithelial cell lines were compared over a 100-fold range from 1×1010 to 1×108 genomic copies (Fig. 1B); 1×1010 genomic copies gave the highest gene transfer efficiency and showed no evidence of cytotoxicity (data not shown) and was chosen as the optimal transducing units for our study. To confirm that the inability of AAV-8 and AAV-9 to transduce the female genital cell lines was due to their tropism for the specific cell type and not to a defect in the vectors themselves, COS-1 cells were tested for transduction. As shown in Fig. 1C, AAV-8-GFP and AAV-9-GFP were able to transduce COS-1 cells effectively, indicating that both vectors were functional.


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 endocervical, ectocervical and vaginal epithelial cells by various AAV serotypes expressing GFP.(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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026473-g001: Transduction of human endocervical, ectocervical and vaginal epithelial cells by various AAV serotypes expressing GFP.(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.
Mentions: To identify the most optimal AAV serotype for transduction of female genital cells, the efficiencies of AAV 1, 2, 3, 4, 5, 6, 8 and 9 expressing GFP were evaluated by transducing immortalized human endocervical (Endl/E6E7), ectocervical (Ectl/E6E7) and vaginal (VK2/E6E7) epithelial cell lines. Relative transduction efficiencies were evaluated by flow cytometry (Fig. 1A) as well as visual assessment by fluorescence microscopy (Fig. 1D). AAV-1, AAV-2, AAV-5 and AAV-6 were found to transduce the endocervical, ectocervical and vaginal epithelial cell lines with AAV-2 and AAV6 being the most efficient. By contrast, the transduction efficiencies of AAV-3, AAV-4, AAV-8 and AAV-9 serotypes were low in these lines. In a dose dilution experiment using AAV-6-GFP vector, transduction efficiencies of the three genital epithelial cell lines were compared over a 100-fold range from 1×1010 to 1×108 genomic copies (Fig. 1B); 1×1010 genomic copies gave the highest gene transfer efficiency and showed no evidence of cytotoxicity (data not shown) and was chosen as the optimal transducing units for our study. To confirm that the inability of AAV-8 and AAV-9 to transduce the female genital cell lines was due to their tropism for the specific cell type and not to a defect in the vectors themselves, COS-1 cells were tested for transduction. As shown in Fig. 1C, AAV-8-GFP and AAV-9-GFP were able to transduce COS-1 cells effectively, indicating that both vectors were functional.

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