Erythrocyte preparations selectively adsorb most of the infectious HIV-1 virions.

<p>HIV-1 isolate 89BZ_167 (105,900 pg) was adsorbed with erythrocytes the indicated numbers of times by adding 3.5×10⁹ erythrocytes from the indicated donors. After removing the erythrocytes by centrifugation, non-adsorbed virus in the supernatant was assayed for the ability to infect CD4(+) TZM-bl cells <i>in trans</i>. The relative degree of infection, determined by the mean p24 (± SD of triplicate measurements), was compared with infection by the original free virus (shown as 100%).</p

Binding of a HIV-1 isolate to erythrocytes.

<p>(A) Increasing amounts of HIV-1 isolate 90US_873 (as quantified by p24) were incubated with 5×10⁷ erythrocytes (donor Q6), and binding of p24 to the cells was determined. (B) Dose-dependent binding of the HIV-1 isolate (8,486 pg p24) with increasing numbers of erythrocytes. The experiment shown is representative of 3 separate experiments. In each experiment HIV-1 was bound to erythrocytes in triplicate, washed, and the triplicates were pooled for p24 determination.</p

Binding of four HIV-1 isolates to erythrocytes derived from 30 different donors.

a<p>Values are from a single experiment except for means of ^btwo, ^cfour or ^dfive experiments.</p

HIV-1 binds to erythrocytes, erythrocytic ghosts, and leukocytes.

<p>(A) After incubation of 151,286 pg of HIV isolate 89BZ_167 with the indicated preparation containing 2.5×10⁹ erythrocytes, the mean p24 bound (± SD of triplicate measurements) was determined. The erythrocytes were then hemolyzed and the p24 bound to the ghosts and the leukocytes was separately measured. (B) After incubation of 105,900 pg of HIV-1 isolate 89BZ_167 with the indicated erythrocyte preparation (3.5×10⁹ erythrocytes), or with purified ghosts previously depleted of leukocytes, from each donor, the mean bound p24 (± SD of triplicate measurements) was determined.</p

Release of erythrocyte-bound HIV-1 by treatment with EDTA.

<p>(A) HIV-1 isolate 89BZ_167 (75,643 pg) was incubated with 5×10⁸ erythrocytes (donor S4) in RPMI, washed three times, and then treated with either EDTA or RPMI (no EDTA). The bound HIV-1 was completely removed by treatment with EDTA. The dashed line shows the limit of detection of the p24 assay. (B) After incubation of HIV-1 with erythrocytes from 3 different donors, following washing, the cells were incubated with different amounts of EDTA (or no EDTA), and washed either in the absence or presence (control) of Ca²⁺ and Mg²⁺.</p

NK cells participate in viral inhibition.

<p>PBMC from 4 donors were depleted of NK cells (white bars), and then compared with matched bulk PBMC as targets (black bars) in neutralization assays. The IC₈₀s (mAbs, panel A) or ID₈₀s (polyclonal plasma, panel B) are indicated. Each bar represents the average of 2 experiments and the error bars show the standard error of the mean.</p

Target PBMC from KIR3DS1+ and FcgRIIIa 158V+ donors show higher levels of neutralization than KIR3DS1− and FcgRIIIa 158V−.

<p>The IC₅₀s for the mAbs and sCD4, and the ID₅₀s for the polyclonal antibodies, are displayed for NL-LucR.T2A-BaL.ecto (A and C) and NL-LucR.T2A-SF162.ecto (B and D). Donors were separated by the KIR3DS genotype (A and B) and the FcγRIIIa 158V genotype (C and D). The inter-quartile ranges for the KIR3DS1− and FcγRIIIa 158V− donors are displayed in blue bars while the inter-quartile range for the KIR3DS1+ and FcγRIIIa 158V+ donors are displayed in red bars. The black lines within each group represent the median neutralization values, while the grey symbols represent individual neutralization titers. Each neutralization titer is an average of 2 experiments.</p

NK cell depletion results in loss of neutralization against a CRF01_AE virus when using a ICp24 assay format.

<p>Neutralization was calculated employing a subtype CRF01_AE IMC (virus CM235) without a reporter gene inserted and using a flow cytometric intracellular p24 endpoint (panels A–D). The ID₅₀s for 2 polyclonal antibody pools (A, C) or IC₅₀s for 4E10 (B, D) are displayed for PBMC from 2 donors that were used either in bulk (black bars) or after NK cell depletion (white bars).</p

Donor PBMC ranked by neutralization.

<p>PBMC from 19 donors were used as target cells for neutralization using 6 virus stocks and 7 neutralization reagents. The ranking and separation of donors into the upper two quartiles (“High neutralization") and the lower two quartiles (“Low neutralization") is indicated, along with the genotypes for KIR3D, HLA-B_80, and FcγRIIIa 158. Red text indicates 3DS1+, Valine (V) at FcγRIIIa position 158, or licensing through HLA-B with Bw4-80I or Bw4-80T.</p

Donors with more NK cells have decreased viral growth.

<p>Donors were stratified by viral growth (A, n=25) and neutralization (B, n=19) then separated into quartiles. The percentages of NK cells for each donor were determined from an average of 3 experiments using flow cytometry and plotted based on quartile. The bars represent median. The Kruskal-Wallis test was used to determine statistical significance.</p