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Inhibition of virus attachment to CD4+ target cells is a major mechanism of T cell line-adapted HIV-1 neutralization.

Ugolini S, Mondor I, Parren PW, Burton DR, Tilley SA, Klasse PJ, Sattentau QJ - J. Exp. Med. (1997)

Bottom Line: Here we show, by the use of a novel virus-cell binding assay, that soluble CD4 and monoclonal antibodies to all confirmed glycoprotein (gp)120 neutralizing epitopes, including the CD4 binding site and the V2 and V3 loops, inhibit the adsorption of two T cell line-adapted HIV-1 viruses to CD4+ cells.By contrast, antibodies specific for regions of gp120 other than the CD4 binding site showed little or no inhibition of either soluble gp120 binding to CD4+ cells or soluble CD4 binding to HIV-infected cells, implying that this effect is specific to the virion-cell interaction.However, inhibition of HIV-1 attachment to cells is not a universal mechanism of neutralization, since an anti-gp41 antibody did not inhibit virus-cell binding at neutralizing concentrations, implying activity after virus-cell binding.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, France.

ABSTRACT
Antibody-mediated neutralization of human immunodeficiency virus type-1 (HIV-1) is thought to function by at least two distinct mechanisms: inhibition of virus-receptor binding, and interference with events after binding, such as virus-cell membrane fusion. Here we show, by the use of a novel virus-cell binding assay, that soluble CD4 and monoclonal antibodies to all confirmed glycoprotein (gp)120 neutralizing epitopes, including the CD4 binding site and the V2 and V3 loops, inhibit the adsorption of two T cell line-adapted HIV-1 viruses to CD4+ cells. A correlation between the inhibition of virus binding and virus neutralization was observed for soluble CD4 and all anti-gp120 antibodies, indicating that this is a major mechanism of HIV neutralization. By contrast, antibodies specific for regions of gp120 other than the CD4 binding site showed little or no inhibition of either soluble gp120 binding to CD4+ cells or soluble CD4 binding to HIV-infected cells, implying that this effect is specific to the virion-cell interaction. However, inhibition of HIV-1 attachment to cells is not a universal mechanism of neutralization, since an anti-gp41 antibody did not inhibit virus-cell binding at neutralizing concentrations, implying activity after virus-cell binding.

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Binding of HLA-DR+ HIV-1 to HLA-DR−/CD4− and  CD4+ cells. Concentrated HIV-1 or mock virus was incubated either undiluted or at the dilutions shown for 30 min at 37°C with either A3.01 or  A2.01 cells, then cell-bound virus was detected by staining with anti– HLA-DR mAbs followed by indirect immunofluorescent staining and  analysis by flow cytometry. Each point represents the mean fluorescence  intensity of 10,000 accumulated events transformed into percentage of inhibition. A and B show flow cytometry profiles for Hx10 and MN, respectively; the left peak corresponds to the background staining with  anti–HLA-DR mAb alone on A3.01 cells in the absence of virus. C and  D show the concentration dependence of Hx10- and MN-cell binding  respectively as compared to controls.
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Figure 1: Binding of HLA-DR+ HIV-1 to HLA-DR−/CD4− and CD4+ cells. Concentrated HIV-1 or mock virus was incubated either undiluted or at the dilutions shown for 30 min at 37°C with either A3.01 or A2.01 cells, then cell-bound virus was detected by staining with anti– HLA-DR mAbs followed by indirect immunofluorescent staining and analysis by flow cytometry. Each point represents the mean fluorescence intensity of 10,000 accumulated events transformed into percentage of inhibition. A and B show flow cytometry profiles for Hx10 and MN, respectively; the left peak corresponds to the background staining with anti–HLA-DR mAb alone on A3.01 cells in the absence of virus. C and D show the concentration dependence of Hx10- and MN-cell binding respectively as compared to controls.

Mentions: The detection of HIV–cell binding by the use of anti– HIV-specific reagents (anti-gp120 mAbs or anti–HIV-specific antisera) has two major disadvantages. First, these reagents will detect cell-associated sgp120 which has been shed from the virus before or during the assay, and second, if the virus has been pretreated with HIV-specific antibodies, these may interfere with binding of the detection antibody. To eliminate these potential problems, we took advantage of the fact that during budding, HIV incorporates large numbers of HLA-DR molecules into its membrane. The binding of HLA-DR+ virus particles to CD4+/HLA-DR− cells allows detection of bound virions with anti– HLA-DR mAbs, followed by indirect immunofluorescent staining and flow cytometric analysis. Binding was carried out with CD4+/HLA-DR− A3.01 cells, and the sister cell line, A2.01 (CD4−/HLA-DR−), was used as a control for specificity. Moreover, we included mock-infected control preparations, since cell culture supernatants contain large numbers of membrane vesicles that carry molecules of cellular origin (59, 60). In preliminary experiments, we incubated concentrated Hx10 or MN virus or mock virus with A3.01 or A2.01 cells for different times at 37 and 4°C. Since the binding at 4°C was weak or undetectable and considered nonphysiological (results not shown), and we were concerned that incubation periods of >30 min at 37°C would lead to significant levels of virus–cell membrane fusion which might affect detection of cell-bound, virion-associated HLA-DR, we selected 30 min at 37°C for later experiments. The binding of 50 μl of an undiluted Hx10 preparation (Fig. 1 A) or 100 μl of MN (Fig. 1 B) to A3.01 and A2.01 cells are represented as flow cytometry histograms. Despite a significant signal obtained on A2.01 cells, which represents CD4-independent binding of virus and HLA-DR–containing vesicular material, the signal obtained on A3.01 cells was substantially greater with both viruses, and all of the cells bound virus as demonstrated by the increased fluorescence of the entire population. The binding of both Hx10 and MN to A3.01 cells was dose dependent, although nonsaturating under the conditions used (Fig. 1, C and D), suggesting a low-avidity interaction between virions and cell-associated CD4. By contrast, the signal obtained with mock virus on A3.01 cells or virus on A2.01 cells did not substantially decrease with increasing dilution of the preparation, suggesting a saturating low level of CD4-independent binding. Overall, these results suggest that CD4 is required for efficient virus–cell binding. To confirm this, we preincubated the A3.01 cells with CD4 mAbs at a saturating concentration under the experimental conditions used, before addition of virus. The mAb Q4120, which binds to domain 1 of CD4 and competes for gp120 binding, inhibited Hx10 virus binding by 95% (Fig. 2). By contrast, neither Q425, which binds to CD4 domain 3 and interferes with HIV–cell fusion but not sgp120–cell binding, nor L120, which binds CD4 domain 4 and has little effect on HIV infection, interfered with HIV-1 binding. The lack of effect of Q425 on the virion–cell binding signal implies that HIV–cell fusion has little effect on the assay readout. Since we were obliged to use a mixture of three (nonbiotinylated) murine anti–HLA-DR mAbs to detect MN virus bound to the A3.01 cells to increase sensitivity, we were unable to carry out inhibition experiments with antibodies of murine origin. For this reason we used a polyclonal sheep anti-sCD4 antiserum at a dilution of 1:90, controlled by normal sheep serum at the same dilution, to inhibit MN binding. Fig. 2 B shows that under these conditions, virus binding was completely inhibited by the antiserum, whereas the control serum had no obvious effect.


Inhibition of virus attachment to CD4+ target cells is a major mechanism of T cell line-adapted HIV-1 neutralization.

Ugolini S, Mondor I, Parren PW, Burton DR, Tilley SA, Klasse PJ, Sattentau QJ - J. Exp. Med. (1997)

Binding of HLA-DR+ HIV-1 to HLA-DR−/CD4− and  CD4+ cells. Concentrated HIV-1 or mock virus was incubated either undiluted or at the dilutions shown for 30 min at 37°C with either A3.01 or  A2.01 cells, then cell-bound virus was detected by staining with anti– HLA-DR mAbs followed by indirect immunofluorescent staining and  analysis by flow cytometry. Each point represents the mean fluorescence  intensity of 10,000 accumulated events transformed into percentage of inhibition. A and B show flow cytometry profiles for Hx10 and MN, respectively; the left peak corresponds to the background staining with  anti–HLA-DR mAb alone on A3.01 cells in the absence of virus. C and  D show the concentration dependence of Hx10- and MN-cell binding  respectively as compared to controls.
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Related In: Results  -  Collection

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Figure 1: Binding of HLA-DR+ HIV-1 to HLA-DR−/CD4− and CD4+ cells. Concentrated HIV-1 or mock virus was incubated either undiluted or at the dilutions shown for 30 min at 37°C with either A3.01 or A2.01 cells, then cell-bound virus was detected by staining with anti– HLA-DR mAbs followed by indirect immunofluorescent staining and analysis by flow cytometry. Each point represents the mean fluorescence intensity of 10,000 accumulated events transformed into percentage of inhibition. A and B show flow cytometry profiles for Hx10 and MN, respectively; the left peak corresponds to the background staining with anti–HLA-DR mAb alone on A3.01 cells in the absence of virus. C and D show the concentration dependence of Hx10- and MN-cell binding respectively as compared to controls.
Mentions: The detection of HIV–cell binding by the use of anti– HIV-specific reagents (anti-gp120 mAbs or anti–HIV-specific antisera) has two major disadvantages. First, these reagents will detect cell-associated sgp120 which has been shed from the virus before or during the assay, and second, if the virus has been pretreated with HIV-specific antibodies, these may interfere with binding of the detection antibody. To eliminate these potential problems, we took advantage of the fact that during budding, HIV incorporates large numbers of HLA-DR molecules into its membrane. The binding of HLA-DR+ virus particles to CD4+/HLA-DR− cells allows detection of bound virions with anti– HLA-DR mAbs, followed by indirect immunofluorescent staining and flow cytometric analysis. Binding was carried out with CD4+/HLA-DR− A3.01 cells, and the sister cell line, A2.01 (CD4−/HLA-DR−), was used as a control for specificity. Moreover, we included mock-infected control preparations, since cell culture supernatants contain large numbers of membrane vesicles that carry molecules of cellular origin (59, 60). In preliminary experiments, we incubated concentrated Hx10 or MN virus or mock virus with A3.01 or A2.01 cells for different times at 37 and 4°C. Since the binding at 4°C was weak or undetectable and considered nonphysiological (results not shown), and we were concerned that incubation periods of >30 min at 37°C would lead to significant levels of virus–cell membrane fusion which might affect detection of cell-bound, virion-associated HLA-DR, we selected 30 min at 37°C for later experiments. The binding of 50 μl of an undiluted Hx10 preparation (Fig. 1 A) or 100 μl of MN (Fig. 1 B) to A3.01 and A2.01 cells are represented as flow cytometry histograms. Despite a significant signal obtained on A2.01 cells, which represents CD4-independent binding of virus and HLA-DR–containing vesicular material, the signal obtained on A3.01 cells was substantially greater with both viruses, and all of the cells bound virus as demonstrated by the increased fluorescence of the entire population. The binding of both Hx10 and MN to A3.01 cells was dose dependent, although nonsaturating under the conditions used (Fig. 1, C and D), suggesting a low-avidity interaction between virions and cell-associated CD4. By contrast, the signal obtained with mock virus on A3.01 cells or virus on A2.01 cells did not substantially decrease with increasing dilution of the preparation, suggesting a saturating low level of CD4-independent binding. Overall, these results suggest that CD4 is required for efficient virus–cell binding. To confirm this, we preincubated the A3.01 cells with CD4 mAbs at a saturating concentration under the experimental conditions used, before addition of virus. The mAb Q4120, which binds to domain 1 of CD4 and competes for gp120 binding, inhibited Hx10 virus binding by 95% (Fig. 2). By contrast, neither Q425, which binds to CD4 domain 3 and interferes with HIV–cell fusion but not sgp120–cell binding, nor L120, which binds CD4 domain 4 and has little effect on HIV infection, interfered with HIV-1 binding. The lack of effect of Q425 on the virion–cell binding signal implies that HIV–cell fusion has little effect on the assay readout. Since we were obliged to use a mixture of three (nonbiotinylated) murine anti–HLA-DR mAbs to detect MN virus bound to the A3.01 cells to increase sensitivity, we were unable to carry out inhibition experiments with antibodies of murine origin. For this reason we used a polyclonal sheep anti-sCD4 antiserum at a dilution of 1:90, controlled by normal sheep serum at the same dilution, to inhibit MN binding. Fig. 2 B shows that under these conditions, virus binding was completely inhibited by the antiserum, whereas the control serum had no obvious effect.

Bottom Line: Here we show, by the use of a novel virus-cell binding assay, that soluble CD4 and monoclonal antibodies to all confirmed glycoprotein (gp)120 neutralizing epitopes, including the CD4 binding site and the V2 and V3 loops, inhibit the adsorption of two T cell line-adapted HIV-1 viruses to CD4+ cells.By contrast, antibodies specific for regions of gp120 other than the CD4 binding site showed little or no inhibition of either soluble gp120 binding to CD4+ cells or soluble CD4 binding to HIV-infected cells, implying that this effect is specific to the virion-cell interaction.However, inhibition of HIV-1 attachment to cells is not a universal mechanism of neutralization, since an anti-gp41 antibody did not inhibit virus-cell binding at neutralizing concentrations, implying activity after virus-cell binding.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, France.

ABSTRACT
Antibody-mediated neutralization of human immunodeficiency virus type-1 (HIV-1) is thought to function by at least two distinct mechanisms: inhibition of virus-receptor binding, and interference with events after binding, such as virus-cell membrane fusion. Here we show, by the use of a novel virus-cell binding assay, that soluble CD4 and monoclonal antibodies to all confirmed glycoprotein (gp)120 neutralizing epitopes, including the CD4 binding site and the V2 and V3 loops, inhibit the adsorption of two T cell line-adapted HIV-1 viruses to CD4+ cells. A correlation between the inhibition of virus binding and virus neutralization was observed for soluble CD4 and all anti-gp120 antibodies, indicating that this is a major mechanism of HIV neutralization. By contrast, antibodies specific for regions of gp120 other than the CD4 binding site showed little or no inhibition of either soluble gp120 binding to CD4+ cells or soluble CD4 binding to HIV-infected cells, implying that this effect is specific to the virion-cell interaction. However, inhibition of HIV-1 attachment to cells is not a universal mechanism of neutralization, since an anti-gp41 antibody did not inhibit virus-cell binding at neutralizing concentrations, implying activity after virus-cell binding.

Show MeSH
Related in: MedlinePlus