Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentration

Abstract

It has been observed that some antibodies, including the CD4-induced (CD4i) antibody IgG X5 and the gp41-specific antibody IgG 2F5, exhibit higher neutralizing activity in PBMC-based assays than in cell line based assays [J.M. Binley, T. Wrin, B. Korber, M.B. Zwick, M. Wang, C. Chappey, G. Stiegler, R. Kunert, S. Zolla-Pazner, H. Katinger, C.J. Petropoulos, D.R. Burton, Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies, J. Virol. 78 (2004) 13232-13252]. It has been hypothesized that the lower CCR5 concentration on the surface of the CD4 T lymphocytes compared to that on cell lines used for the neutralization assays could be a contributing factor to the observed differences in neutralizing activity. To test this hypothesis and to further elucidate the contribution of CCR5 concentration differences on antibody neutralizing activity, we used a panel of HeLa cell lines with well-defined and differential surface concentrations of CCR5 and CD4 in a pseudovirus-based assay. We observed that the CCR5 cell surface concentration but not the CD4 concentration had a significant effect on the inhibitory activity of X5 and several other CD4i antibodies including 17b and m9, as well as that of the gp41-specifc antibodies 2F5 and 4E10 but not on that of the CD4 binding site antibody (CD4bs), b12. The 50% inhibitory concentration (IC50) decreased up to two orders of magnitude in cell lines with low CCR5 concentration corresponding to that in CD4 T cells used in PBMC-based assays (about 10(3) per cell) compared to cell lines with high CCR5 concentration (about 10(4) or more). Our results suggest that the CCR5 cell surface concentration could be a contributing factor to the high neutralizing activities of some antibodies in PBMC-based-assays but other factors could also play an important role. These findings could have implications for development of vaccine immunogens based on the epitopes of X5 and other CD4i antibodies, for elucidation of the mechanisms of HIV-1 neutralization by antibodies, and for design of novel therapeutic approaches.

Figure 1. Cell surface expression of CD4 and CCR5 in the four cell lines used for measurement of antibody neutralization

The immunostaining of different HeLa and TZM-bl cell lines was performed with FITC-conjugated anti-CD4 and PE-conjugated anti-CCR5 antibodies by flow cytometry. The mean fluorescence values for each cell line are shown.

Figure 2. Comparison of the CCR5 expression levels on the surface of PBMCs and TZM bl cells

The cells were double stained with FITC-conjugated anti-CD4 (RPA-T4) and PE-conjugated anti-CCR5 (3A9) antibodies. The histograms show un-gated data for the uniform TZM-bl cells and T-cells gated population data for the PBMCs. (A) CCR5 cell surface expression (B) CD4 cell surface expression, staining of PBMCs and TZM-bl cells with isotype control antibodies have been shown in gray color.

Figure 2. Comparison of the CCR5 expression levels on the surface of PBMCs and TZM bl cells

The cells were double stained with FITC-conjugated anti-CD4 (RPA-T4) and PE-conjugated anti-CCR5 (3A9) antibodies. The histograms show un-gated data for the uniform TZM-bl cells and T-cells gated population data for the PBMCs. (A) CCR5 cell surface expression (B) CD4 cell surface expression, staining of PBMCs and TZM-bl cells with isotype control antibodies have been shown in gray color.

Figure 3. Neutralizing activity of CD4i antibodies against primary HIV-1 isolate from clade B (Bal) in a pseudovirus-based assay

Neutralization assays were carried out in triplicate wells by preincubation of serial dilutions of (A) scFv m9, (B) IgG X5, (C) scFv m16 and (D) IgG m16 with pseudotyped viruses for 30 minutes at 37 °C followed by infection of 1.5 × 10⁴ HeLa cell lines. Luminescence was measured after 3 days, the mean luminescence readings for triplicate wells and standard deviations were determined; the percentage inhibition of luciferase activity is presented as a measure of the antibody inhibitory activity.

Figure 4. Neutralizing activity of CD4bs and anti-gp41 antibodies against primary HIV-1 isolate from clade B (Bal) in a pseudovirus-based assay

The percentage inhibition of luciferase activity is presented as a measure of the antibody inhibitory activity (see legend of Figure 2) of gp41-specific antibodies, (A) IgG 2F5 and (B) IgG 4E10 and (C) for CD4bs antibody IgG b12.

Figure 5. Neutralization of HIV-1 Env (Bal) by antibodies

Inhibitory activity of CD4i (X5, m9, 17b, m16), CD4bs (b12) and gp41-specific antibodies (2F5 and 4E10) was determined by a pseudovirus-based assay for primary isolate of HIV-1 from clade B (Bal). IC₅₀ value (µg/ml) was assigned to the antibody concentration at which 50% neutralization was observed. * Indicate the highest tested concentration of IgG 17b and IgG m16 at which 50% inhibition could not be achieved. The mean of three measured IC₅₀s (in µg/ml) for each antibody and the standard deviations are presented.

Figure 6. Neutralization of HIV-1 isolates from different clades by IgG X5

The percentage inhibition of luciferase activity is presented as a measure of the IgG X5 neutralizing activity (see legend of Figure 2) against the indicated four isolates from four different clades.

Figure 7. Inhibition of cell-cell fusion by scFv m9

Inhibitory activity of scFv m9 was studied by incubating the serially diluted antibody with Env expressing cells for 30 minutes at 37°C before mixing with HeLa cells. The inhibitory effect of m9 was evaluated by mixing the Env expressing cells with m9 for 30 minutes at 37 °C and then performing the fusion assay. Percentage inhibition of β-Gal activity is presented as a measure of antibody inhibitory activity for (A) Env Bal and (B) Env AD8.