Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies

Abstract

Induction of broadly cross-reactive neutralizing antibodies (NAb) is an important goal for a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. Some HIV-infected patients make a NAb response that reacts with diverse strains of HIV-1, but most candidate vaccines have induced NAb only against a subset of highly sensitive isolates. To better understand the nature of broad NAb responses that arise during natural infection, we screened patients for sera able to neutralize diverse HIV strains and explored the frequency and phenotype of their peripheral Envelope-specific B cells. We screened 113 HIV-infected patients of various clinical statuses for the prevalence of broad NAb. Sera able to neutralize at least four of five viral isolates were found in over one-third of progressors and slow progressors, but much less frequently in aviremic long-term nonprogressors. Most Env-specific antibody-secreting B cells were CD27(hi) CD38(hi) plasmablasts, and the total plasmablast frequency was higher in HIV-infected patients than in uninfected donors. We found that 0.0031% of B cells and 0.047% of plasmablasts secreted Env-specific immunoglobulin G (IgG) in an enzyme-linked immunospot (ELISPOT) assay. We developed a novel staining protocol to label HIV-specific B cells with Env gp140 protein. A total of 0.09% of B cells were found to be Env-specific by this method, a frequency far higher than that indicated by ELISPOT assay. gp140-labeled B cells were predominantly CD27(+) and surface IgG(+). These data describe the breadth and titer of serum NAb and the frequency and phenotype of HIV-specific B cells in a cohort of patients with broad cross-neutralizing antibody responses that are potential goals for vaccines for HIV.

FIG. 1.

Distribution of broadly cross-reactive sera. (A) Percentages of 113 sera that neutralize the indicated number of isolates. Sera that neutralize none or one of five are classified as not broad (blue), two or three of five as intermediate (white), and four or five of five as broad (green). (B) Percentages of broad, intermediate, and not-broad sera in LTNP, slow progressor, and progressor patient groups (colors are as defined in panel A).

FIG. 2.

Clinical characteristics and neutralization data for patients in B-cell analysis. Each column shows data from a single patient. In the top panel, each row shows the ID₅₀ of serum against the indicated virus isolate. ID₅₀ values: white, <100; yellow, 100 to 400; orange, 401 to 1,000; red, >1,000. In the bottom panel, abbreviations are defined as follows: VL, viral load in copies/ml; CD4, CD4⁺ T cells/μl; Yrs Since Dx, years since HIV diagnosis. Clinical data are from the same time point or within 3 months of the serum sample. Broad and not-broad categories are shown as classified for Fig. 1.

FIG. 3.

The plasmablast frequency is increased in HIV-infected patients. (A) Flow cytometric analysis of plasmablasts. A representative sample is shown. Plasmablasts are CD3⁻ CD19⁺ CD20^−/lo CD27^hi CD38^hi. The large gate in the forward-scatter/side-scatter plot was set to include plasmablasts based on backgating. (B) Plasmablasts as a percentage of CD19⁺ lymphocytes in HIV-positive patients and uninfected donors. Horizontal bar, median value.

FIG. 4.

gp120-specific ASC are plasmablasts. The top panels present representative flow cytometry data showing gating strategy for plasmablasts. The bottom panels show representative anti-gp120 IgG ELISPOT wells from the indicated populations. Input cell numbers: CD19⁺ well, 30,000; CD19⁺ CD20⁺ well, 200,000; CD27^−/lo CD38^−/lo, 25,000; plasmablasts, 4,000.

FIG. 5.

gp140 specifically stains Env-specific B cells. (A) PBMC were labeled with biotinylated gp140-F trimer (right panels) or buffer only (left panels) and later stained with Qdot605-streptavidin, along with antibodies for lymphocyte markers. Panels show CD3⁻ CD14⁻ CD19⁺ cells. The gate shows gp140-labeled cells and is set so that ∼0.01% of cells are positive in the streptavidin-only sample for each patient. Representative plots of an uninfected control (top) and an HIV-infected patient (bottom) are shown. (B) Percent of B cells that are labeled with gp140⁺ in HIV-infected patients and uninfected controls.

FIG. 6.

Frequencies of B cells and gp140-labeled B cells expressing surface markers. (A) Left panel, CD3⁻ CD19⁺ cells from a representative patient. Right panels, CD3⁻ CD19⁺ cells of the same sample stained for B-cell markers. gp140-labeled cells (blue) are shown on the background of all B cells (red). Totals of 1.8 × 10⁵ to 3.4 × 10⁵ gated events are shown. (B) Gating strategy for CD27 and CD38. Left panel, CD3⁻ CD19⁺ cells from a representative patient. Right panel, gp140-labeled cells (blue) on the background of total B cells (red). (C and D) Percent of cells of the indicated phenotype in total B cells or gp140-labeled B cells. Horizontal bars, medians. (E) Left panel, CD3⁻ CD19⁺ cells from a representative patient. Center panel, CD3⁻ CD14⁻ CD19⁺ CD20^−/lo cells of the same sample. The CD27^hi CD38^hi cells are plasmablasts. gp140-labeled cells (blue) are shown on the background of other B cells (red). A total of 4.7 × 10⁵ gated events are shown. Right panel, percentage of B cells or gp140 labeled B cells that are plasmablasts.