Antibody specificities associated with neutralization breadth in plasma from human immunodeficiency virus type 1 subtype C-infected blood donors

Abstract

Defining the specificities of the anti-human immunodeficiency virus type 1 (HIV-1) envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from chronically HIV-1-infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4 binding site (CD4bs) antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect on the primary virus, Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma, this activity was mapped to a site overlapping the CD4-induced (CD4i) epitope and CD4bs. Anti-membrane-proximal external region (MPER) (r = 0.69; P < 0.001) and anti-CD4i (r = 0.49; P < 0.001) antibody titers were found to be correlated with the neutralization breadth. These anti-MPER antibodies were not 4E10- or 2F5-like but spanned the 4E10 epitope. Furthermore, we found that anti-cardiolipin antibodies were correlated with the neutralization breadth (r = 0.67; P < 0.001) and anti-MPER antibodies (r = 0.6; P < 0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during natural HIV-1 infection, many of which are yet to be determined, and that polyreactive antibodies are possibly involved in this phenomenon.

FIG. 1.

Hierarchical clustering of sera and viruses according to the ID₅₀ neutralization titers. The neutralization titer of each of 70 plasma samples against 10 HIV-1 strains is shown in a heat map with the most potent plasmas shown in dark red and those with no activity in light yellow. The remaining values are binned into eight distinct colors from a palette ranging from pale yellow to dark red. Rows and columns are clustered using a complete linkage-clustering algorithm based on Euclidean distance between log₁₀ titer values (12). The resulting rows and columns are reordered, within the constraints of the respective dendrogram, using row and column means. The 16 samples with BCN antibodies are indicated with asterisks.

FIG. 2.

Neutralization analysis of plasmas adsorbed onto gp120-coated beads. The plasmas were adsorbed onto magnetic beads coated with Du151 wild-type (wt) gp120, D368R gp120, D368R/E370A gp120, or blank beads. The residual and eluted antibodies were tested for neutralization of various envelope-pseudotyped viruses. (A) Neutralization curves of adsorbed plasmas, BB55 (top row) and BB70 (bottom row), against four primary viruses. (B) Antibodies eluted from beads were tested for neutralization and standardized by their IgG concentrations. Shown are BB55 (top row) and BB70 (bottom row) against HxB2, Du151, Du156, and CAAN5342. (C) Neutralization curves of adsorbed plasma samples BB10 (left column), BB67 (middle column), and BB89 (right column) using viruses against which these plasmas were active.

FIG. 3.

Analysis of anti-CD4i epitope and anti-MPER antibodies in comparison to neutralization breadth. Shown are correlations of the number of viruses neutralized with anti-CD4i (A) and anti-MPER (B) antibody (Ab) titers. Spearman rank coefficients and P values are shown for each correlation. (C) A Kruskal-Wallis test indicated a significant difference between the median numbers of viruses neutralized by plasma segregated into four groups based on their antibody specificities (P = 0.0003). The median and interquartile range are indicated.

FIG. 4.

Contributions of anti-CD4i and anti-CD4bs antibodies to cross-neutralizing activity in BB55. (A) Control experiments showing neutralization of 7312A plus 9 nM sCD4 by BB55 plasma previously adsorbed with either wild-type or I420R gp120. No neutralization of 7312A was evident in the absence of sCD4. (B) Neutralization of Du151 by BB55 plasma after absorption with I420R gp120 alone or I420R gp120 followed by wild-type or D368R/E370A gp120. (C) Neutralization of Du151 by BB55 plasma after absorption with D368R/E370A gp120 alone or D368R/E370A gp120 followed by wild-type or I420R gp120. Wild-type gp120 and blank beads were included as controls. (D and E) BB55 eluates from wild-type, D368R, D368R/E370A, and I420R gp120s were assessed for neutralization of 7312A plus 9 nM sCD4 (D) and Du151 (E). (Figure 2B shows the D368R gp120 curve).

FIG. 5.

Analysis of reactivity to autoantigens in relation to neutralization breadth. (A to C) Correlations between antibody (Ab) titers to anti-dsDNA (A), anti-CL (B), and RF (C) and the number of viruses neutralized. (D) Correlation of anti-CL and anti-MPER antibody titers. Spearman rank coefficients and P values are shown for each correlation. OD, optical density; LAU, Luminex Athena units.