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Survival of the fittest: positive selection of CD4+ T cells expressing a membrane-bound fusion inhibitor following HIV-1 infection.

Kimpel J, Braun SE, Qiu G, Wong FE, Conolle M, Schmitz JE, Brendel C, Humeau LM, Dropulic B, Rossi JJ, Berger A, von Laer D, Johnson RP - PLoS ONE (2010)

Bottom Line: However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells.These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo.Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

View Article: PubMed Central - PubMed

Affiliation: Angewandte Virologie und Gentherapie, Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt, Germany.

ABSTRACT
Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

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Survival advantage of maC46-expressing human CD4+ T cells in HIV-1-infected humanized mice.Human T cells were transduced with a control vector (M420) expressing GFP alone (squares) or the M589 vector expressing maC46-GFP (circles) and then infected with the HIV strain D117/II. Infected and uninfected cells were injected intraperitoneally into adult NOG mice. Four infected and 5 uninfected mice were followed for 5 weeks for each vector. A. Relative numbers of human CD4+ T cells. The percentage of human CD4+ T cells as a fraction of total viable cells was calculated. Means and the SEM for each group are shown. B. HIV infection leads to a selective survival of maC46-GFP expressing human CD4+ T cells (red closed circles) in vivo. Means and the SEM for each group are shown. C. Viral load in the serum of individual infected mice is not altered by expression of maC46-GFP. Differences in viral loads between mice receiving CD4+ T cells transduced with the maC46-expressing vector and the GFP control vector were not significant (Mann-Whitney test).
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pone-0012357-g006: Survival advantage of maC46-expressing human CD4+ T cells in HIV-1-infected humanized mice.Human T cells were transduced with a control vector (M420) expressing GFP alone (squares) or the M589 vector expressing maC46-GFP (circles) and then infected with the HIV strain D117/II. Infected and uninfected cells were injected intraperitoneally into adult NOG mice. Four infected and 5 uninfected mice were followed for 5 weeks for each vector. A. Relative numbers of human CD4+ T cells. The percentage of human CD4+ T cells as a fraction of total viable cells was calculated. Means and the SEM for each group are shown. B. HIV infection leads to a selective survival of maC46-GFP expressing human CD4+ T cells (red closed circles) in vivo. Means and the SEM for each group are shown. C. Viral load in the serum of individual infected mice is not altered by expression of maC46-GFP. Differences in viral loads between mice receiving CD4+ T cells transduced with the maC46-expressing vector and the GFP control vector were not significant (Mann-Whitney test).

Mentions: Finally, we studied the ability of maC46 to provide a selective advantage in vivo in a human xenotransplant mouse model. Human primary CD4+ T cells were transduced with maC46 (M589) or the lentiviral GFP control vector, infected with HIV-1D177/II, and transplanted into NOG (NOD SCID gammac−/−) mice. Human CD4, human CD45, and GFP expression were monitored by flow cytometry. HIV-1 replication was analyzed by a commercial RT-PCR-based assay in mouse serum. When CD4+ T cell counts were examined as a percentage of all cells, CD4+ T cell counts in the blood of uninfected animals increased over time (Figure 6A), whereas in the HIV-1-infected animals the percentage of CD4+ T cells failed to expand in mice transplanted with CD4+ T cells expressing maC46 or in mice transplanted with cells transduced with the control vector. However, when the percentage of CD4+ cells was expressed as a percentage of human CD45+ cells, we observed a marked expansion of maC46-expressing cells from 18% up to 60% in the infected group during the first 20 days after transplantation (Figure 6B), while a stable level of transgene positive CD4+ T cells was seen in the uninfected mice and in the infected mice transplanted with cells expressing the control vector. Thus, maC46 is also able to confer a selective advantage in this in vivo setting. However, the percentage of maC46-expressing cells was not sustained and the level of transgene-positive cells declined again towards the end of follow-up. High levels of HIV-1 RNA of over 105 copies per ml were measured in mouse sera at day 14, but viral loads decreased by 30 to 100-fold at day 34 (Figure 6C). No significant difference in viral load was seen between the maC46- and control vector treated groups, perhaps because sustained regeneration of maC46-expressing cells was not achieved in this xenograft model. At day 35, all mice developed severe xenogenic graft versus host disease and had to be euthanized.


Survival of the fittest: positive selection of CD4+ T cells expressing a membrane-bound fusion inhibitor following HIV-1 infection.

Kimpel J, Braun SE, Qiu G, Wong FE, Conolle M, Schmitz JE, Brendel C, Humeau LM, Dropulic B, Rossi JJ, Berger A, von Laer D, Johnson RP - PLoS ONE (2010)

Survival advantage of maC46-expressing human CD4+ T cells in HIV-1-infected humanized mice.Human T cells were transduced with a control vector (M420) expressing GFP alone (squares) or the M589 vector expressing maC46-GFP (circles) and then infected with the HIV strain D117/II. Infected and uninfected cells were injected intraperitoneally into adult NOG mice. Four infected and 5 uninfected mice were followed for 5 weeks for each vector. A. Relative numbers of human CD4+ T cells. The percentage of human CD4+ T cells as a fraction of total viable cells was calculated. Means and the SEM for each group are shown. B. HIV infection leads to a selective survival of maC46-GFP expressing human CD4+ T cells (red closed circles) in vivo. Means and the SEM for each group are shown. C. Viral load in the serum of individual infected mice is not altered by expression of maC46-GFP. Differences in viral loads between mice receiving CD4+ T cells transduced with the maC46-expressing vector and the GFP control vector were not significant (Mann-Whitney test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2925957&req=5

pone-0012357-g006: Survival advantage of maC46-expressing human CD4+ T cells in HIV-1-infected humanized mice.Human T cells were transduced with a control vector (M420) expressing GFP alone (squares) or the M589 vector expressing maC46-GFP (circles) and then infected with the HIV strain D117/II. Infected and uninfected cells were injected intraperitoneally into adult NOG mice. Four infected and 5 uninfected mice were followed for 5 weeks for each vector. A. Relative numbers of human CD4+ T cells. The percentage of human CD4+ T cells as a fraction of total viable cells was calculated. Means and the SEM for each group are shown. B. HIV infection leads to a selective survival of maC46-GFP expressing human CD4+ T cells (red closed circles) in vivo. Means and the SEM for each group are shown. C. Viral load in the serum of individual infected mice is not altered by expression of maC46-GFP. Differences in viral loads between mice receiving CD4+ T cells transduced with the maC46-expressing vector and the GFP control vector were not significant (Mann-Whitney test).
Mentions: Finally, we studied the ability of maC46 to provide a selective advantage in vivo in a human xenotransplant mouse model. Human primary CD4+ T cells were transduced with maC46 (M589) or the lentiviral GFP control vector, infected with HIV-1D177/II, and transplanted into NOG (NOD SCID gammac−/−) mice. Human CD4, human CD45, and GFP expression were monitored by flow cytometry. HIV-1 replication was analyzed by a commercial RT-PCR-based assay in mouse serum. When CD4+ T cell counts were examined as a percentage of all cells, CD4+ T cell counts in the blood of uninfected animals increased over time (Figure 6A), whereas in the HIV-1-infected animals the percentage of CD4+ T cells failed to expand in mice transplanted with CD4+ T cells expressing maC46 or in mice transplanted with cells transduced with the control vector. However, when the percentage of CD4+ cells was expressed as a percentage of human CD45+ cells, we observed a marked expansion of maC46-expressing cells from 18% up to 60% in the infected group during the first 20 days after transplantation (Figure 6B), while a stable level of transgene positive CD4+ T cells was seen in the uninfected mice and in the infected mice transplanted with cells expressing the control vector. Thus, maC46 is also able to confer a selective advantage in this in vivo setting. However, the percentage of maC46-expressing cells was not sustained and the level of transgene-positive cells declined again towards the end of follow-up. High levels of HIV-1 RNA of over 105 copies per ml were measured in mouse sera at day 14, but viral loads decreased by 30 to 100-fold at day 34 (Figure 6C). No significant difference in viral load was seen between the maC46- and control vector treated groups, perhaps because sustained regeneration of maC46-expressing cells was not achieved in this xenograft model. At day 35, all mice developed severe xenogenic graft versus host disease and had to be euthanized.

Bottom Line: However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells.These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo.Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

View Article: PubMed Central - PubMed

Affiliation: Angewandte Virologie und Gentherapie, Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt, Germany.

ABSTRACT
Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

Show MeSH
Related in: MedlinePlus