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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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Inhibition of HIV–cell binding by sCD4. HIV Hx10 (A) or  MN (B) virus was preincubated with increasing concentrations of sCD4  for 2 h at 37°C before addition of the A3.01 cells. The virus binding test  was carried out as described for Fig. 1, and data are represented as percentage of binding. To measure virus infectivity, serial dilutions of a sample of the same sCD4-treated virus used for the binding test were incubated with A3.01 cells for 2 h at 37°C before culture for 10 d, and the  viral titer calculated at peak p24 production in the culture supernatant.  Neutralization is represented as reduction in infectivity (TCID50).
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Figure 3: Inhibition of HIV–cell binding by sCD4. HIV Hx10 (A) or MN (B) virus was preincubated with increasing concentrations of sCD4 for 2 h at 37°C before addition of the A3.01 cells. The virus binding test was carried out as described for Fig. 1, and data are represented as percentage of binding. To measure virus infectivity, serial dilutions of a sample of the same sCD4-treated virus used for the binding test were incubated with A3.01 cells for 2 h at 37°C before culture for 10 d, and the viral titer calculated at peak p24 production in the culture supernatant. Neutralization is represented as reduction in infectivity (TCID50).

Mentions: The experiments with CD4 mAbs confirmed that HIV required CD4 on the target cell for specific binding. We next wished to investigate whether the binding of ligands to the virus was also able to inhibit HIV–cell binding in this assay. We initially tested sCD4 for its ability to interfere with virus binding and to neutralize virus infectivity, since it has previously been shown to compete with membrane CD4 for both sgp120 and virion binding. Fig. 3 shows the inhibition of HIV–cell binding and HIV infectivity for Hx10 and MN, respectively. The inhibition of virus binding paralleled that of neutralization with increasing sCD4 concentration, suggesting a relationship: an excellent and highly significant correlation was demonstrated after analysis of the results, and is summarized in Table 1 (r = 0.96 and 0.93 for Hx10 and MN, respectively). We next tested a panel of antibodies specific for the CD4 binding region of gp120 for their ability to neutralize and inhibit HIV–cell attachment. Fig. 4 shows the effect of Fab b12 and its IgG derivative, IgG1b12, mAb 21h on Hx10–cell binding and infectivity, and IgG1b12 on MN–cell binding and infectivity. The binding of both viruses to A3.01 cells was strongly inhibited by these anti–CD4 binding site–specific antibodies. Maximum inhibition of Hx10 binding was ∼93, 55, and 86% for IgG1b12, Fab b12 and 21h, respectively, at 200 nM, and maximum inhibition of MN binding was 89% for IgG1b12 at the same concentration. These data confirm that a large part of the neutralizing activity of these antibodies is likely to be competition for virus–CD4 binding. The finding that Fab b12 neutralized and inhibited virus attachment demonstrates that cross-linking of the viral glycoproteins is not required for these functions. Further analysis of the relationship between inhibition of virion– cell attachment and neutralization for these antibodies revealed a strong to excellent and highly significant correlation for both viruses (Table 1).


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)

Inhibition of HIV–cell binding by sCD4. HIV Hx10 (A) or  MN (B) virus was preincubated with increasing concentrations of sCD4  for 2 h at 37°C before addition of the A3.01 cells. The virus binding test  was carried out as described for Fig. 1, and data are represented as percentage of binding. To measure virus infectivity, serial dilutions of a sample of the same sCD4-treated virus used for the binding test were incubated with A3.01 cells for 2 h at 37°C before culture for 10 d, and the  viral titer calculated at peak p24 production in the culture supernatant.  Neutralization is represented as reduction in infectivity (TCID50).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199094&req=5

Figure 3: Inhibition of HIV–cell binding by sCD4. HIV Hx10 (A) or MN (B) virus was preincubated with increasing concentrations of sCD4 for 2 h at 37°C before addition of the A3.01 cells. The virus binding test was carried out as described for Fig. 1, and data are represented as percentage of binding. To measure virus infectivity, serial dilutions of a sample of the same sCD4-treated virus used for the binding test were incubated with A3.01 cells for 2 h at 37°C before culture for 10 d, and the viral titer calculated at peak p24 production in the culture supernatant. Neutralization is represented as reduction in infectivity (TCID50).
Mentions: The experiments with CD4 mAbs confirmed that HIV required CD4 on the target cell for specific binding. We next wished to investigate whether the binding of ligands to the virus was also able to inhibit HIV–cell binding in this assay. We initially tested sCD4 for its ability to interfere with virus binding and to neutralize virus infectivity, since it has previously been shown to compete with membrane CD4 for both sgp120 and virion binding. Fig. 3 shows the inhibition of HIV–cell binding and HIV infectivity for Hx10 and MN, respectively. The inhibition of virus binding paralleled that of neutralization with increasing sCD4 concentration, suggesting a relationship: an excellent and highly significant correlation was demonstrated after analysis of the results, and is summarized in Table 1 (r = 0.96 and 0.93 for Hx10 and MN, respectively). We next tested a panel of antibodies specific for the CD4 binding region of gp120 for their ability to neutralize and inhibit HIV–cell attachment. Fig. 4 shows the effect of Fab b12 and its IgG derivative, IgG1b12, mAb 21h on Hx10–cell binding and infectivity, and IgG1b12 on MN–cell binding and infectivity. The binding of both viruses to A3.01 cells was strongly inhibited by these anti–CD4 binding site–specific antibodies. Maximum inhibition of Hx10 binding was ∼93, 55, and 86% for IgG1b12, Fab b12 and 21h, respectively, at 200 nM, and maximum inhibition of MN binding was 89% for IgG1b12 at the same concentration. These data confirm that a large part of the neutralizing activity of these antibodies is likely to be competition for virus–CD4 binding. The finding that Fab b12 neutralized and inhibited virus attachment demonstrates that cross-linking of the viral glycoproteins is not required for these functions. Further analysis of the relationship between inhibition of virion– cell attachment and neutralization for these antibodies revealed a strong to excellent and highly significant correlation for both viruses (Table 1).

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